national congress of the italian society of virology

Transcript

7th NATIONAL CONGRESS OF THE
ITALIAN SOCIETY OF VIROLOGY
Orvieto (TR)
Palazzo del Capitano del Popolo
June 24 - 26, 2007
1
COMMITEES
President of the Congress
Palù Giorgio
Scientific Committee
Franco Buonaguro
Canio Buonavoglia
Gabriella Campadelli-Fiume
Francesco Maria Cancellotti
Maurizio Conti
Carlo De Giuli Morghen
Antonina Dolei
Giuseppe Gerna
Colomba Giorgi
Giorgio Gribaudo
Maria Paola Landini
Roberto Manservigi
Giorgio Palù
Maria Cristina Parolin
Carlo Federico Perno
Mauro Pistello
Luisa Rubino
Franco Maria Ruggeri
Paola Verani Borgucci
Maurizio Zazzi
Organizing Committee
Elisabetta Affabris
Alberta Azzi
Francesco Maria Cancellotti
Colomba Giorni
Anna Maria Iorio
Franco Maria Ruggeri
Cristiano Salata
Eurovirology Travel Grant and poster-prize Committee
Canio Buonavoglia
Gabriella Campadelli-Fiume
Antonina Dolei
Giovanni Martelli
Giorgio Palù
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SECRETARIATS
SCIENTIFIC SECRETARIAT
SIV - Italian Society of Virology
c/o Department of Histology, Microbiology and Medical Biotechnologies
University of Padova - Italy
www.siv-virologia.it
The Italian Society of Virology is supported by
ORGANIZING SECRETARIAT
MZ CONGRESSI
Member of the MZ International Group (Milano, Torino, Barcelona, London)
Via Carlo Farini, 81
20159 Milano - Italy
Phone +39 0266802323 - Fax +39 026686699
www.mzcongressi.com
- Speakers, chairs and companies: Patrizia Sirtori ([email protected])
- Registrations: ([email protected])
ACKNOWLEDGMENTS
We thank the ‘‘Comune di Orvieto’’ for its continuous support.
GENERAL INFORMATION
OFFICIAL LANGUAGE
The official language of the symposium is English
CME
Italian CME Credits have been assigned by the Italian Ministry of Health for the following
categories:
- Medical Doctor: 10 credits
(Specialist Areas: Microbiology and Virology, Infectious Diseases, Pharmacology and Clinical Toxicology)
- Biologist: 10 credits
(Specialist Areas: Microbiology and Virology)
- Biomedical Laboratory Technician: 11 credits
Italian CME certificates will be sent after the Congress directly to the address indicated on the
registration form.
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PROGRAMME
Sunday, 24 June 2007
16:00-17.30
Registration
17:30
Opening and welcome
Chair: Antonina Dolei (Sassari)
17:45
GB Rossi Lecture
New aspects of interferon systems, immune evasion and new potential applications
Filippo Belardelli (Roma)
GENERAL VIROLOGY AND VIRAL GENETICS
Chairs: Maurizio Conti (Torino), Maria Cristina Parolin (Padova)
18.20-20.00
Selected oral presentations
Increasing circulation and diversification of HIV-1 non-B subtypes in Italy over a 10year observation period
F. Razzolini
Detection and quantification of noroviruses in environmental samples by newly
designed nested PCR assays and Taqman Real-Time RT-PCR
M. Muscillo
UL131-128 products are required for the activation of HCMV fusion complex during
endothelial cell infection
M. Patrone
Prevalence and Persistence of human papillomavirus genotypes and their variants
in Human Immunodeficiency Virus (HIV) positive women from south-Italy
F.M. Buonaguro
Polyomavirus BK and anti-dsDNA antibodies in renal transplant recipients
C. Costa
Inconsistent responses of cytomegalovirus-specific T-cells to pp65 and IE-1 vs
infected dendritic cells in organ transplant recipients
D. Lilleri
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Monday, 25 June 2007
MEDICAL VIROLOGY AND ANTIVIRAL THERAPY
Chairs: Carlo Federico Perno (Roma), Maurizio Zazzi (Siena)
09.00-09.30
KeyNote Lecture
Antiviral strategies, mechanism, and clinical efficacy
Massimo Puti (Brescia)
09:30-10.45
Human respiratory syncytial virus (hRSV) RNA quantification in nasopharyngeal
secretions identifies the hRSV etiologic role in acute respiratory tract infections of
hospitalized infants
G. Campanini
The human bocavirus role in acute respiratory tract infections of pediatric patients as
defined by viral load quantification
G. Gerna
Antiviral activity of Retinoic Acid derivatives against HHV-8
E. Caselli
Pre-transplant analysis of preservation and washing solutions for rapid detection of
viruses in the kidney graft
M. Pacenti
Antiviral activity of WC5, a new 6-Aminoquinolone compound, against human
herpesviruses
B. Mercorelli
10.45-11.15
BREAK
VIRAL BIOTECHNOLOGIES AND GENE THERAPY
Chairs: Giorgio Gribaudo (Torino), Giorgio Palù (Padova)
11.15-12.45
Persistent viral detection in the kidney allograft is a risk factor for chronic allograft
injury
L. Barzon
Viral clearance evaluation model using 4 different viruses for GMP-Production of
biopharmaceutical drugs or vaccines
JL. McDermott
Effects of adenoviral vector infection on human adrenocortical cells
U. Matkovic
Role of recombinant poxvirus vectors expressing HIV/SIVgenes in modulating
Th1/Th2 cytokine profiles in human dendritic cells
P. Beggio
6
Human placenta derived mesenchymal stem cells are fully permissive to human
cytomegalovirus infection
L. Luconi
Expression of the HPV-16 L1 Antigen in Transplastomic Tobacco Plants
P. Lenzi
12:45-14.00
LUNCH
VIRAL ONCOGENESIS AND VACCINES
Chairs: Franco Buonaguro (Napoli), Colomba Giorgi (Roma)
14.00-14.30
KeyNote Lecture
New strategies in Vaccine Development – The HIV experience
Hans Wolf (Regensburg – Germany)
14.30-16.00
Anti-cancer activity of plant-produded HPV16 E7 vaccine
S. Massa
Development of a vaccine against Toscana Virus
G. Gori Savellini
Development of avipoxvirus recombinant vaccines for the control of HPV16-induced
cervical cancer
E. Pozzi
Impact of the seropositivity status on the pattern of DC activation by Virus-Like
Particles
L. Buonaguro
Distribution of human papillomavirus type 16 variants in penile keratinizing
squamous cell carcinoma
ML. Tornesello
Expression, processing and assembly of the HIV-1 Pr55gag protein in transgenic
tobacco chloroplasts
N. Scotti
16.00-16.30
BREAK
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HPV INFECTION AND PREVENTION
Chairs: Franco Buonaguro (Napoli), Colomba Giorni (Roma)
16.30-16.45
HPV and HPV-associated diseases worldwide burden
Kate Soldan (London – UK)
16.45-17.00
Characterisation of the interaction of HPV E6 with the Discs Large Tumour
Suppressor
Lawrence Banks (Trieste)
17.00-17.15
VLPs: past and future
Reinhard Kirnbauer (Vienna – Austria)
17.15-17.30
Gardasil
Franco Scaglione (Milano)
17.30-17.45
Long-term impact of prophylactic ant-HPV 16/18 vaccine
Davis Jenkins (Rixensart – Belgium)
17.45-18.00
New therapeutic strategies
Aldo Venuti (Roma)
18.00-19.00
Tavola rotonda
Panel discussion: issues and guidelines (in italiano)
Coordinator
Sergio Pecorelli (Brescia)
La vaccinazione in Italia
Maria Grazia Pompa (Roma)
Metodi diagnostici e screening nell'era del vaccino
Francesca Carozzi (Firenze)
Il punto di vista del virologo
Giorgio Palù (Padova), Carlo Federico Perno (Roma), MariaLina Tornesello (Napoli)
Il punto di vista del ginecologo
Aldo Vecchione (Napoli)
Come controllare l'efficacia del vaccino, chi lo farà e come lo farà
Marta Ciofi degli Atti (Roma)
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Tuesday, 26 June 2007
08.30-09.00
KeyNote Lecture
Virological monitoring of environmental matrices and food: importance for the risk
assessment
Annalaura Carducci (Pisa)
VIRUS HOST INTERACTIONS AND PATHOGENESIS
Chairs: Mauro Pistello (Pisa), Luisa Rubino (Bari)
09.00-10.45
Down-regulation of proteolytic complexes following Epstein Barr Virus activation in
Burkitt’s lymphoma cells
G. Matusali
Analysis of HCMV ppUL44 homodimerization, intra-nuclear mobility and
phosphorylation-regulated nuclear transport using live cells imaging
G. Alvisi
Human cytomegalovirus replicates in adrenocortical cells and stimulates steroid
hormone production
M. Trevisan
HSV-1 induces dysregulation of monocyte anticandida functions
C. Cermelli
What’s the clue in the progression of liver damage during HCV-infection?
Analysis of the role of HCV NS5A and Core proteins
M. Marcolongo
Involvement of the E62EEE65 Nef Acidic Domain and TRAF Family Members on
Signaling Events Induced by treatment of Monocytes/Macrophages with HIV-1 Nef
G. Mangino
HPV infection in HIV positive subjects: 10 years experience of follow-up
F. Lillo
10:45-11.15
BREAK
11:15-11.45
Guidelines for human Cytomegalovirus infections
Giuseppe Gerna (Pavia)
11:45 -12:45 Poster Session
12:45-14.00
LUNCH
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EMERGING AND ZOONOTIC VIRAL INFECTIONS
Chairs: Canio Buonavoglia (Bari), Franco Ruggeri (Roma)
14.00-14.30
KeyNote Lecture
An emerging zoonosis: European bat lyssaviruses
Franco Mutinelli (Legnaro – PD)
14:30- 15.30 Selected oral presentations
Emerging respiratory infections in hematopoietic stem cell-transplanted children in
Germany
V. Falcone
Characterization of the biological properties of human and rhesus VP8* proteins
F. Cappuccini
Detection of norovirus in a captive lion cub with haemorrhagic enteritis
(Panthera leo)
V. Martella
Virologic Characterization of a Poxvirus Zoonosis in Northern Italy
F. Carletti
15:30-17:00
SOCIETY MEETING
17:00
Best Poster Award and Travel Awards for Eurovirology 2007
Closing remarks
10
GB Rossi
&
Keynote Lectures
11
New aspects of interferon systems, immune evasion and new potential applications
Filippo Belardelli
Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.
Half a century has passed since the discovery of interferons (IFNs) as antiviral factors released by virus-infected cells.
The first two decades of IFN research were characterized by major difficulties in convincing the scientific community
about the specificity of the emerging biological effects. In the early 1980s, when recombinant IFNs became available,
it was recognized that this family of cytokines, comprising the type I or “viral” IFNs (mainly and ) and the type II
or “immune” IFN ( ), could also exert multiple activities on cell growth, differentiation and immune response. Today,
in revisiting 50 years of history of IFN research, the impact of early studies on the IFN system appears to be
impressive. IFNs- are the cytokines with a longest record of clinical use and are still used in patients with certain
malignancies and viral diseases, while IFN- is currently used in patients with multiple sclerosis. Notably, IFN research
has been instrumental for understanding cytokine signal transduction pathways and important mechanisms regulating
antiviral, antitumor and immune responses. Recently, multiple mechanisms by which viruses can undermine the potent
IFN-mediated host defence system have been described. These mechanisms are important for evading the direct IFNmediated control on viral replication as well as for allowing evasion from an effective immune response, which is also
regulated by the IFN system. Likewise, immune suppression, linked to impaired IFN production or defective cytokine
response, has been observed in cancer patients. These findings are consistent with data in mouse models, demonstrating:
i) the importance of the integrity of the IFN system in the host immune surveillance against tumors; ii) the role of
endogenous IFN in chemotherapy-induced reversion of immune suppression in tumor-bearing animals. Notably, recent
studies have underscored new effects of IFNs (especially type I IFN) on cells of the immune system (including T cells
and dendritic cells), which are important in linking innate and adaptive immunity. Thus, in mouse models, type I IFN
can act as a powerful vaccine adjuvant by acting on dendritic cells (DCs). Likewise, DCs rapidly generated from human
monocytes after exposure to IFN- (IFN-DCs) act as powerful cellular adjuvants for the generation of MHC class I
restricted CD8+ T cell responses against viral (HIV and EBV) and tumor (melanoma) antigens and efficiently crossprime CD8+ T cells against exogenous antigens. On the whole, the preclinical data suggest that these IFN-DCs exhibit
some unique characteristics particularly suitable for the generation of effective DC-based vaccines. New therapeutic
strategies can now be based on a novel rationale for using IFN- , including the direct in vivo use of these cytokines as
vaccine adjuvants and their in vitro use to generate highly active patient’s monocyte-derived DCs to be utilized in
therapeutic vaccination protocols. Such immunotherapy strategies are particularly suitable in patients showing immune
suppression features (induced either by virus infection of by tumor progression) which can be effectively restored by
IFN-induced immune interventions targeted to DCs and by additional combination treatments. Moreover, a detailed
knowledge on the IFN system in patients with cancer and HCV-infection would be instrumental in selecting categories
of patients responding to IFN therapy. Planned clinical studies are expected to provide evidence on the importance of
IFN-DC interactions in patients and on the perspectives of this novel use of IFN- in immunotherapy protocols.
13
Antiviral strategies, mechanism and clinical efficacy
Massimo Puoti
Le terapie antivirali negli ultimi tempi hanno assunto una crescente importanza in Clinica in virtù del loro impatto sulla
sopravvivenza di soggetti con malattie virali croniche legato all'immediato efetto sulle loro sequele. L'impato dei
farmaci antiretrovirali sulla sopravvivenza e sulla qualità di vita dei soggetti con AIDS è ben noto, ma recentemente
l'impiego delle terapie anti HBV ed anti HCV ha consentito di ridurre la mortalità per cirrosi ed epatocarcinoma e
l'impiego delle terapie anti CMV quella per le infezioni da CMV in soggetti immunodepressi.
NEW STRATEGIES IN VACCINE DEVELOPMENT: THE HIV EXPERIENCE
Prof. Dr. Hans Wolf
University of Regensburg, Institute for Medical Microbiology and Hygiene
In response to the growing problems related with HIV-infections in poor economics, industrialized countries responded
with strategies to intensify the development of HIV-vaccines. INCO-projects and the formation of sizeable research
clusters in the EU allowed strategies starting from molecular epidemiology to evaluation of vaccine candidates in
primates and humans. Together with the Chinese Center for Disease Control early at onset of the HIV-epidemic in
China relevant HIV-strains could be selected (B-clade RL42 and C-clade 97CN54, 97CN54 is a B’-C recombinant and
the most prevalent strain in Western and North-Western provinces of China).
Using sequence modified genes from 97CN54 a set of immunogens comprising gag pol nef and env was developed
which was found to be highly immunogenic but inactive in their original enzymatic functions. In context of the
EUROVAC-cluster and the INCO-programme different presentation systems based amongst others DNA-plasmids
(COBRA) and vaccinia viruses (NYVAC, MVA, TienTan) have been developed into GMP-manufactured products.
These have been evaluated in parallel to the human trials in Rhesus monkeys. The combination of DNA prime vaccinia
boost gave strong immune response in almost all animals in multiple HIV reading frames and on the basis of IFN , IL-2
and IL-4 Elispots. Parallel experiments using the SHIV 89.6p-model showed protection from disease and rapid
clearance of viremia to set point in challenge experiments.
Data from human trials with NYVAC-C alone looked encouraging with 45 % responders, DNA alone did, upon first
preliminary evaluation, not induce significant immune response jugged by CTL with about 10 % of the vaccinees. A
trial with the combination of DNA-C prime and NYVAC-C boost parallels data in primates and achieved rigorous
(Elispots above 400 up to 1200) broad (multiple antigens and epitopes), rigorous (multiple cytokines) and long lasting
immunogenicity on almost all vaccinees (above 90 %). Further trials are up-coming in Regensburg and other European
sites and in Beijing/China.
14
Virological monitoring of environmental matrices and food: importance for the risk
assessment
Annalaura Carducci, Dip. Biologia dell'Università di Pisa
The virological risk coming from the exposure to environmental contaminated matrices have characteristics
that impose its specific consideration: viruses cannot be efficacy represented by the classic microbial indicators (i.e. E.
Coli, enterococci and coliphages) because they have a lower infectious dose, multiple ways of transmission, different
ecology in comparison with bacteria and a higher genetic variability often associated with the possibility of
recombination. The recent examples of SARS and H5N1 avian influenza as well as studies on HEV, rotavirus and
calicivirus, showed the importance of animal viruses in human pathology and the possible role of foods and
environment as vehicle between infected and susceptible organisms: although viruses cannot multiply outside living
cells, they can persist in the environment for a very long time because of their high resistance to natural and artificial
disinfection agents and the protective action of environmental factors.
The role of viruses as environmental biological risk agents has been proven by epidemiological data for
different matrices: potable and recreational waters, food, arosol and surfaces. Unfortunately, in most cases, the
epidemiological evidence has not been confirmed by the virus detection in the suspected matrices owing to the
difficulties in applying to the environment analytical techniques developped for clinical materials. In fact,
environmental samples have a highly variable composition often interfeering with the detection procedures, viruses are
extremely diluted and multiple contamination of human and animal origin are frequent.
Nevertheless, progress in clinical virology, in particular biomolecular techniques, and the development of
methods for sample concentration and purification, have greatly improved the efficiency of virological analysis of
environmental matrices and increased the amount of data on dectected viruses in waters, foods, aerosol, surfaces, etc.
Although the majority of methods are still produced “in house” and need standardization, the virological environmental
and food monitoring is now a real possibility and it can be useful for the risk analysis (assessment, control and
communication) in different ways: to identify the routes of virus spreading, to verify the efficacy of control measures, to
assess the level of sanitation thus helping the operators education, to monitor the environmental pollution, to carry out
epidemiological surveillance of viral circulation, allowing molecular epidemiology and phylogenetic studies.
The virological monitoring for the routine risk assessment of food and enviroment needs, besides the
standardization of techniques, the selection of significant viral species and types or representative indicators and the
clarification of the real meaning of the nucleic acid detection in the environment. Furtherly, monitoring data should be
related to the clinical and surveillance ones to obtain quantitative information in terms of a dose-response relationship
and probability of adverse outcomes.
In conclusion, to use the environmental and food virology in the risk assessment, sharing knowledge and data
with clinical human and veterinarian virologists will be an essential evolution.
An emerging zoonosis: European bat lyssaviruses
Franco Mutinelli, NRL for Rabies, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro (PD)
In Europe, two bat lyssaviruses referred to as European bat lyssaviruses (EBLVs) types 1 and 2 (genotypes 5 and 6
respectively) which are closely related to classical rabies virus are responsible for an emerging zoonosis. EBLVs are
host restricted to bats, and have been known to infect not only their primary hosts but also in rare circumstances, induce
spillover infections to terrestrial mammals including domestic livestock, wildlife and man. Although spillover infections
have occurred, there has been no evidence that the virus adapted to a new host. Since 1977, five human deaths from
EBLVs have been reported. None of them had a record of prophylactic rabies immunization. Only fragmentary data
exist about the effectiveness of current vaccines in cross-protection against EBLVs. EBLV in bats cannot be eliminated
using conventional strategies similar to the control programmes based on vaccine baits used for fox rabies in Europe.
Due to the protected status of bats in Europe, our knowledge of EBLV prevalence and epidemiology is limited. It is
possible that EBLV is under-reported and that the recorded cases of EBLV represent only a small proportion of the
actual number of infected bats. Human exposure through biting incidents, especially unprovoked attacks, should be
treated immediately with rabies post-exposure treatment and the bat, where possible, retained for laboratory analysis
and species identification. Preventative measures include educating all bat handlers of the risks posed by rabies-infected
animals and advising them to be immunized.
15
General Virology and Viral Genetics
17
SELECTED ORAL COMMUNICATIONS
Increasing circulation and diversification of HIV-1 non-B subtypes in Italy over a 10-year
observation period
Razzolini F, Saladini F, Vicenti I, Marconi A, Balestrieri M, Romano L, Zazzi M
Department of Molecular Biology, University of Siena, Italy
HIV-1 isolates belonging to the M (major) group are classified into subtypes (A, B, C, D, F, G, H, J, K), sub-subtypes
(A1, A2, A3, F1, F2) and more than 30 circulating recombinant forms (CRFs). Geographic distribution of the different
clades is subject to evolution over time as a result of human migration and settlement as well as possible preferential
routes of transmission. HIV-1 pol sequence databases maintained at genotypic antiretroviral resistance testing reference
laboratories can provide useful insights into the temporal changes in the circulation of HIV-1 clades. We analyzed clade
assignment data stored at the HIV Monitoring Service of the University of Siena, Italy over a 10-year period (19972006). More than 8000 HIV-1 sequences obtained from a total of 3485 patients were available. The first sequence of
each patient was considered and assigned to the year of sampling. Subtyping was carried out by phylogenetic analysis
using the Kimura distance model and neighbor-joining method with 1000-replicate bootstrap analysis. The overall
prevalence of non-B subtypes in the data set was 10.4% and increased significantly over time reaching 29.8% in 2006
(p < 0.0001). A wide variety of subtypes and CRFs was represented, with subtypes A1, C, G, F1 and CRFs 01_AE and
02_AG each accounting for >5% of the pooled non-B sequences. Interestingly, as many as 9.4% (n = 34) of the non-B
viruses were unique recombinant forms (URFs), particularly involving recombination between B and F subtypes.
Significant association between individual highly represented subtypes and patient demographics were detected (e. g.
CRF02_AG and African origin, decreased male-to-female ratio and clades A1, C, e CRF02_AG). Sequencing of
additional HIV-1 regions (gag, env and in some cases full-length genome) for select strains allowed to characterize
several complex and novel recombinant forms. The diversity of the HIV-1 sequence pool has increased significantly in
recent years in Italy and will likely contribute to further exploration of HIV-1 genetic space at the population level.
Detection and quantification of noroviruses in environmental samples by newly designed
nested PCR assays and Taqman Real-Time RT-PCR.
Muscillo, M., Pourshaban M., Fontana S., Di Grazia A., Iaconelli M., and La Rosa, G.
Noroviruses have received increased attention in recent years because their role as etiologic agents in acute
gastroenteritis outbreaks is now clearly established.
In the present study, environmental samples from sea water, estuarine water, and effluents of sewage treatment plants
were analyzed in order to evaluate the role of environmental surface contamination as a possible vehicle for
transmission of norovirus genogroups I (GI) and II (GII). We used a previously published RT-PCR assay and also one
that was developed in this study. The two assays resulted in similar detection, however with the new assay longer
amplicons were obtained that provided more accurate phylogenetic assessment of the norovirus strains detected. To
complete qualitative PCR assays, we performed quantitative RT-PCR (RT-Q-PCR) assays to detect norovirus
concentrations in environmental samples by broadly reactive one-step TaqMan reverse transcription (RT)-PCR assays.
Transcribed norovirus RNA from GI and GII plasmids were used as calibration curves. Quantitative data on the
concentrations of noroviruses present in recreational water are indispensable for assessment of the public health risks
caused by norovirus infections.
While river, estuarine, and seawater samples were scarcely contaminated (0.9% of samples), all sewage samples were
positive for norovirus GII; in 60% of samples both genogroup I and II were detected.
This is the first study in Italy reporting the presence of Noroviruses in environmental samples not associated to evident
outbreaks and outline the possible role played by water as a vehicle for transmission.
19
UL131-128 products are required for the activation of HCMV fusion complex during
endothelial cell infection
PATRONE Marco, MILANESI Gabriele and GALLINA Andrea
University of Milano School of Medicine, Department of Medicine Surgery and Dentistry, Milano, Italy
UL131-128 locus products are determinants of HCMV tropism for a number of cell types –including leukocytes, DC
and endothelial cells (EC)– other than fibroblasts. The encoded proteins have been detected in the virion, complexed to
envelope glycoproteins gH-gL, which suggests that UL131-128 polypeptides play role in viral entry. We have directly
addressed this issue by comparing the fusion capability of an EC-tropic isolate (VR1814) with that of fibroblast-adapted
strains. Indeed, all non-EC-tropic strains failed to fuse. The role of UL131-128 proteins in fusion was further proven by
the ability of a recombinant pUL128 (rpUL128) to specifically inhibit VR1814 fusion with, and infection of, EC. Virion
fusion was executed at the plasma membrane, as only fusion-deficient viral particles were found to be endocytosed.
What is more, in VR1814 infections of EC we have characterized a post-attachment conformational intermediate of gB,
the appearance of which is followed by a transient gB-gH interaction, detected in proximity of fusion. Both the gB
intermediate and the gB-gH complex were inhibited by rpUL128, and were absent in infections with non-EC-tropic
strains. Altogether these findings indicate that UL131-128 products are required for the initiation of a chain of events
triggering HCMV multi-component fusion machinery.
Prevalence and Persistence of human papillomavirus genotypes and their variants in Human
Immunodeficiency Virus (HIV) positive women from south-Italy
Maria Lina Tornesello1, Maria Luisa Duraturo1, Matilde Sansone2, Roberto Piccoli2, Luigi Buonaguro1, Franco
Maria Buonaguro1,*,
1Ist. Naz. Tumori "Fond. G. Pascale", Cappella Cangiani, I-80131, Naples, Italy,
2Gynecology Department, University of Medicine, Naples, Italy
Human immunodeficiency virus (HIV) positive women have high rates of infection with a broad range of human
papillomavirus genotypes (HPV) whose oncogenic risk is not well known. Furthermore revalence and persistence of
mucosal HPV genotypes have been analysed in 112 HIV-positive and 115 HIV-negative women during a 3-year followup period. Compared with HIV-negative, HIV-positive women were at the higher risk of HPV infection with a
prevalence rate of 39.3% at the first visit and 43.7% after at least three visits. Among the twenty different viral
genotypes identified the high risk HPVs (16, 18, 31, 33, 35, 45, 52, 58, and 66) were detected in 33% of HIV-positive
and in 13% of HIV negative women, with the HPV16 as the most represented (16.1% and 6.9%) in both groups.
Persistent infections with less characterized HPVs (45, 53, 54, 55, 61, 62, 70, 72, 81 and 83) were frequently found in
HIV-positive women (within a range of 0.9%-16.3%) and significantly associated with low and high grade
intraepithelial lesions. Phylogenetic analysis of HPV16 isolates allowed the identification of HPV16 African 2 and
African 1 variants differently distributed in HIV-positive and HIV-negative women, suggesting different sexual mixing
behaviours. The association of uncommon genotypes and variants with cytologic abnormalities in HIV-positive women
underscores the need for targeting a wide range of HPV types in the screening analysis and in the design of future
vaccines.
20
Polyomavirus BK and anti-dsDNA antibodies in renal transplant recipients
1
Cristina Costa, 1Massimiliano Bergallo, 2Giovanni Antonio Touscoz, 1Francesca Sidoti, 1Maria Elena Terlizzi, 1Sara
Astegiano, 1Chiara Merlino, 3Giuseppe P. Segoloni, 1Rossana Cavallo
1
Dept. of Public Health and Microbiology, Virology Unit, University of Turin, Italy
2
Gastro-Hepatology Unity, Laboratory of Digestive and Hepatic Physiopathology, and
3
Dept. of Internal Medicine, Renal Transplant Unit, Molinette Hospital, Turin, Italy
Background. Polyomavirus BK reactivation is common in renal transplant recipients and may cause nephropathy with
significant graft dysfunction. The induction of anti-dsDNA antibodies by BKV has been described in experimental
animals and during primary infection, and a role in the pathogenesis of systemic lupus erythematosus has been
suggested. Methods. In this study we evaluated the occurrence of anti-dsDNA antibodies and non-organ-specific
autoantibodies by indirect immunofluorescence in 90 renal transplant recipients and the association with BKV
reactivation as determined by BKV-DNA positivity on urine and/or serum samples. Results. Forty-four of 90 patients
experienced a BKV reactivation; 17/90 developed anti-dsDNA antibodies. Considering the 44 patients with BK viruria
and/or viremia, anti-dsDNA antibodies were present in 15 patients vs 29 who did not developed anti-dsDNA antibodies,
while in BKV DNA-negative patients 2 were anti-dsDNA-positive vs 44 anti-dsDNA-negative (p < 0.001). Considering
the 22 patients with BK viremia (with or without viruria), anti-dsDNA antibodies were present in 9 patients vs 13 who
did not developed anti-dsDNA antibodies, while in BK viremia-negative patients 8 were anti-dsDNA positive vs 60
anti-dsDNA negative (p < 0.01). No significant difference in terms of clinical parameters of renal function was found
between anti-dsDNA positive and negative patients. Conclusions. There is a significant association between BKV and
production of anti-dsDNA antibodies in renal transplant patients, however there is no evidence that these virus-induced
autoreactive responses are themselves pathogenic.
Inconsistent responses of cytomegalovirus-specific T-cells to pp65 and IE-1 vs infected
dendritic cells in organ transplant recipients
Daniele Lilleri1, Paola Zelini1, Chiara Fornara1, Giuditta Comolli1,2, Giuseppe Gerna1
Servizio di Virologia, 2Laboratori Sperimentali di Ricerca-Area Biotecnologie, Fondazione IRCCS Policlinico San
Matteo, Pavia, Italy
1
CD4+ and CD8+ T-cells specific for human cytomegalovirus (HCMV) and two immunodominant HCMV antigens
(pp65 and IE-1) were monitored in 20 solid organ transplant recipients undergoing primary (n=4) or reactivated (n=16)
HCMV infection during the first year after transplantation by using as a stimulator either HCMV-infected autologous
dendritic cells (DCs) or pp65- or IE-1 peptide mixtures. Turnaround times for test performance were 7 days for infected
DCs and 24h for peptides. Using infected DCs, HCMV-specific T-cell restoration occurred in all patients for CD8+ and
in 18/20 (90%) for CD4+ T-cell subpopulations, resulting in virus clearance from blood. Using peptide mixtures, T-cell
responses were less frequently detected. In detail, 14 (70%) patients showed pp65-specific CD8+ T-cells and 10 (50%)
patients IE-1-specific CD8+ T-cells, whereas pp65-specific CD4+ T-cells were detected in 14 (70%) patients, and IE-1specific CD4+ T-cells in 3 (15%) patients only. Protection from HCMV infection was associated with the presence of a
HCMV-specific T-cell response directed against multiple viral proteins, but not against pp65 or IE-1 only. In
conclusion, the use of pp65 and IE-1 peptide mixtures for rapid monitoring of HCMV-specific T-cell responses in solid
organ transplant recipients underestimates the actual level of protection against HCMV.
21
POSTERS
1
Rotavirus strains circulating in Salento (Italy) during 2005: emergence of genotype G9
T GRASSI1, A IDOLO1, M. GUIDO1, A DE DONNO1
Department of Biological and Environmental Sciences and Technologies, Lab of Hygiene, University of
Salento, Italy
1
Rotaviruses have been recognized as the major cause of acute gastroenteritis in young children worldwide. G1, G2, G3
and G4 are the most important strains clinically and epidemiologically reported from all country. However, the pattern
of G-type distribution seems to be changing as some unusual types (G5, G8 and G9).
The purpose of this study was to determine the G-genotype of rotaviruses circulating in the Salento area, Italy.
During 2005, 73 stool samples were collected from subjects admitted to six Hospitals in the Salento with a positive
rotavirus screening test. The specimens were stored at –20°C until confirmation could take place by molecular biology
techniques. A reverse transcription-polymerase chain reaction (RT-PCR) assay for the amplification of gene VP7 of
rotavirus was developed and used; G-serotype was determined by sequencing and phylogenetic analysis.
A total of 69 (94.5%) stool samples were detected positive by RT-PCR and 35 were sequenced; the sequence profile
was compared to that of the VP7 genes of human rotaviruses from GenBank. The most prevalent G genotype was
G2(40%), followed by G1(31.4%), G9(22.9%), and G4(5.7%).
In conclusion, this study highlighted the presence of four different G-genotypes in the Salento. Sequencing results
showed G2 genotype be predominant. This doesn’t correspond to the usual distribution described in the literature where
G1 is described as the most common genotype.
The presence of G9 may be related to a local re-emergence of this genotype, detected in Italy in the early and mid1990s, or to the global emergence registered in recent years.
2
Detection of a Coronavirus strain in a quail farm: a novel coronavirus?
Camarda A.1, Circella E.1, Martella V.1, Bruni G.1, Lavazza A.2, Buonavoglia C.1
1
Dipartimento di Sanità e Benessere degli Animali, Facoltà di Medicina Veterinaria, Università degli Studi di Bari
2
Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Sezione di Brescia
Coronavirus (CoV) infections are of high relevance in poultry. Until recently, only a few species of birds, such as
chicken, turkey and pheasant, were believed to be susceptible to CoVs. In the last years, CoVs have been detected in the
gut of a number of avian species, including guinea fowl, graylag geese, mallard and feral pigeon (Jonassen et al. 2005),
domestic peafowl and teal (Liu et al. 2005).
In this report, CoV infection is described in quails (Coturnix coturnix) semi-intensively reared for restocking in Apulia
(South Italy). The affected birds, 3 weeks of age, displayed depression, weakness, severe diarrhoea, dehydration and
drop in feed consumption. At necropsy, the prominent lesion was enteritis.
A CoV strain was detected by electron microscopy and RT-PCR (Reverse Transcriptase – Polymerase Chain Reaction)
in the intestinal content of the dead quails. The virus was not cultivatable in chicken embryos.
By sequence analyses of a fragment of region 1b of the polymerase gene, the Quail CoV (QCoV) was grouped along
with CoVs representative of group 3, displaying 93% nucleotide identity to the other avian CoVs.
By analysis of the S1 portion of the spike protein-encoding gene, the virus displayed 16-18% amino acid (aa) identity to
infectious bronchitis virus and 79-81% aa to turkey coronavirus (TCoV). These findings suggest the existence of a
novel coronavirus genetically related to TCoV.
A specific pathogenic role of QCoV in the syndrome may be suggested.
22
3
CIRCULATION
OF
HUMAN
METAPNEUMOVIRUS-ASSOCIATED
RESPIRATORY TRACT INFECTIONS IN INPATIENTS CHILDREN.
LOWER
Sonia Caracciolo, Daniele Rossi, Chiara Minini, Manuela Avolio, Giorgio Tosti, Arnaldo Caruso and Simona
Fiorentini
Department of Experimental and Applied Medicine, Section of Microbiology, University of Brescia Medical School,
Brescia, Italy.
Acute respiratory tract infections (ARTIs) are a leading cause of morbidity and mortality in children, but the aetiology
of many ARTIs is still unknown. In 2001, the discovery of the human metapneumovirus (hMPV) was reported and,
since its initial description, hMPV has been associated with ARTI in individuals of all ages worldwide.
hMPV seems to play an important role as a cause of paediatric upper and lower ARTI with similar, but not identical,
epidemiological and clinical features to those of RSV and FLU viruses. Aim of this study was to estimate the incidence
of hMPV in hospitalized children with clinical symptoms of ARTI and to evaluate the molecular epidemiologic profile
of hMPV infection during winter season.
Our results show that hMPV is commonly found in children with ARTI (39 out of 157) and that a concurrent seasonal
circulation of all 4 hMPV subtypes occurs. Because this study evaluates a relative low number of hMPV infected
children, further researches are needed to elucidate the quantitative and qualitative importance of hMPV infection in the
pathogenesis of ARTI.
4
Typing and quantification of adenoviruses in estuarine, bathing waters and sewages.
La Rosa G., Pourshaban M., Iaconelli M., Fontana S., Di Grazia A., and Muscillo M.
Adenoviruses are emergent pathogens which may represent new indicators of microbial water quality.
A total of 33 samples, each of 10L, from estuarine and bathing waters were analyzed in order to evaluate the role of
recreational waters as a possible vehicle for transmission of adenoviruses. To reinforce epidemiological data, 10
sewage samples collected at a wastewater treatment plant in Rome, were also examined in 2006.
Firstly, samples were concentrated to 1 ml, then adenoviruses were identified by PCR and sequenced in the hexon
gene region. A new nested PCR on a fiber gene region was used for confirmation purposes. An Internal Amplification
Control (IAC) was introduced to check inhibitor interferences.
The yield of the concentration process was checked on 10 L of artificial seawater seeded with a total of 103 CCID50 of
an adenovirus 2 from our clinical collection. Subsequently, RealTime PCR (QPCR) assays based on TaqMan
technology were used to detect virus concentrations in environmental samples.
Results of this work showed a high diffusion of adenoviruses in water environments, even when bacteria counts were
low, sometime undetectable at all. Results from QPCR showed a gradient sewage>estuary>seawater of viral particles.
Adenovirus type 2 and 41 were detected but more information are needed on the epidemiology of adenoviruses to
evaluate their role as indicators of microbial water quality for human health risk assessment. The QPCR assay is a
suitable tool for quantitative determination of adenoviruses in water samples impacted by sewage contamination and
represents a considerable advancement in pathogen quantification in aquatic environments.
23
5
Quantitative detection of enteroviruses in bathing waters by TaqMan reverse transcription
(RT)-PCR assays.
Pourshaban M., La Rosa, G., Iaconelli M., and Muscillo, M.
Enteroviruses cause a wide range of diseases including poliomyelitis, aseptic meningitis, encephalitis, myocarditis,
pericarditis, upper respiratory disease, and others.
They are commonly found in human feces and represent a good indicator of viral contamination of water. The present
European directive 76/160/ECC contemplates monitoring for these viruses, stipulating a standard of 0 enteroviruses per
10 litres.
The traditional method of human enterovirus detection is cell culture assay. It is costly, time-consuming and impractical
for monitoring, because it requires days to weeks for results; therefore reliable, fast, sensitive and practical methods for
detecting enteroviruses in water samples are needed. In this study 125 seawater samples collected in bathing waters
during summer 2006 were analyzed for enteroviruses. Quantitative PCR assays based on TaqMan probe hydrolysis
technology were used to detect and enumerate viruses in environmental samples.
A 513 nt transcribed RNA from 536/pVR220/811 pGEM-4z plasmid containing the 5’non coding of Poliovirus 1
Mahoney was used to set up a calibration curve. The Echovirus 30 Bastianni, Echovirus 7 Wallace, Coxsackievirus B3
Nancy and Coxsackievirus A9 Griggs from ATCC were used as references.
The presented Q-RT-PCR approach is fast and sensitive, and is promising for monitoring viral contamination in
environmental water samples.
6
ROLE
OF
THE
CARBOSSI-TERMINAL
REGION
IMMUNODEFICIENCY VIRUS GAG IN VIRAL ASSEMBLY
OF
THE
FELINE
Arianna Calistri, Claudia Del Vecchio, Marta Celegato, Michele Celestino, Paola Sette, Giorgio Palù and Cristina
Parolin1
Department of Histology, Microbiology and Medical Biotechnologies and 1Department of Biology, University of
Padova, Italy
We previously characterized short proline rich domains present at the level of structural proteins in different family of
enveloped RNA viruses, which are essential in the late stages of viral life cycle. For this reason they have been named
late-domain (L-domain). In particular, we demonstrated that the release of retroviral particles from infected cells is
strictly dependent from the presence of functional L-domain at the level of the Gag protein. Recent studies have shown
that the L-domains interact with cellular factors, involved in the biogenesis of intraluminal vesicles at the level of late
endosomes known as multivesicular bodies. The generation of these vesicles is topologically identical to the budding of
viruses away from the cytosol. In the present study, we identified a putative L-domain in the Gag protein of the feline
immunodeficiency virus (FIV) and we addressed its role in the late stages of virus replication, by analyzing the
assembly phenotype of FIV mutant viruses. Our results suggest that assembly domains in the FIV carbossi-terminal
region may be functionally equivalent to those present in the Gag polyprotein of other retroviruses and in the structural
proteins of different RNA enveloped viruses. Indeed, we could replace the FIV late domain with those characteristic of
other viruses maintaining the ability of Gag to produce virus like particles. In addition, our data confirm that primate
and not primate lentiviruses exploit the same cellular pathway for their budding and release.
24
7
A split EGFP complementation assay detects complexes among the quartet of glycoproteins
required for HSV entry
Elisa Avitabile and Gabriella Campadelli-Fiume - Department of Experimental Pathology
Section on Microbiology and Virology Alma Mater Studiorum - University of Bologna
Entry of HSV requires a multipartite fusion system made of a quartet of glycoproteins, gD, gB, gH, gL. gD represents
the receptor-binding glycoprotein, and also triggers fusion. Fusion execution requires gB and gH.gL. The aim of this
work was to study the interactions that occur among the quartet of membrane glycoproteins required for HSV entry into
the cells. We developed a protein complementation assay applied to membrane-bound proteins. EGFP can be split into
two portions (N-terminal and C-terminal) that, if brought into sufficient closeness, interact with each other, refold and
reconstitute fluorescence emission. We fused the N- and C-portions of EGFP to the endodomains of HSV glycoproteins.
If the glycoproteins interact with each other through their ectodomains, they enable reconstitution of EGFP.
Formation of complexes was analyzed in whole transfected cells. We first searched evidence that it is suitable to the
study the of membrane-bound protein interactions. To this end, we first focused on a well known protein-protein
interaction system, that of gD and its receptor nectin1. Our results document complex formation between gD and
nectin1. They also document complex formation between gD and gH.gL and between gD and gB; the latter interaction
appears to be weaker than the former. Thus gD can recruit gH.gL and gB independently one of the other.
8
THE HCMV DNA POLYMERASE ACCESSORY SUBUNIT, UL44, FUNCTIONS AS A DIMER IN
VITRO AND IN CELLS
Elisa Sinigalia,1 Daniela Zanocco,1 Beatrice Mercorelli,1 Gualtiero Alvisi,2 Donald M. Coen3, Gregory S. Pari,4
Alessandro Ripalti,5 David A. Jans,6 Giorgio Palù,1 and Arianna Loregian1
1
Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, 35121 Padova, Italy;
2
Department of Experimental and Specialistic Medicine, University of Bologna, 40138 Bologna, Italy; 3Department of
Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA;
4
Department of Microbiology and Immunology and the Cell and Molecular Biology Graduate Program, University of
Nevada-Reno, Reno, Nevada 89557, USA; 5Microbiology Operative Unit, St. Orsola General Hospital, University of
Bologna, 40138 Bologna, Italy; 6Department of Biochemistry and Molecular Biology, Monash University, 3800 Clayton,
Victoria, Australia
The HCMV DNA polymerase includes an accessory protein, UL44, which acts as a processivity factor for the catalytic
subunit, UL54. Recently, the crystal structure of UL44 revealed that it forms homodimers. Substitution with alanine for
certain residues (L86, L87, and F121) impaired dimerization in vitro. Here we investigated the importance of
homodimerization of UL44 for its functions. We measured the binding of the UL44 L86A/L87A and F121A mutants to
an UL54 C-terminal peptide. The wild-type and mutant UL44 constructs displayed similar affinities for the peptide,
indicating that the mutants are not misfolded and that dimerization is not required to bind UL54. In addition, the ability
of the mutants to interact with UL54 was confirmed both by yeast two-hybrid assays and by confocal microscopy
experiments. We also assayed the ability of the mutants, which are impaired in binding to DNA, to stimulate long-chain
DNA synthesis by UL54 in vitro. Confocal microscopy experiments showed that residues L86, L87, and F121 of UL44
are also required for dimerization in live cells. Finally, the ability of the L86A/L87A and F121A mutants to support
HCMV oriLyt-dependent DNA replication in cells was assayed using a cotransfection-replication assay. Both mutants
failed to complement DNA replication in this assay. Control Western blot experiments showed that the mutant proteins
are expressed at levels similar to that of wild-type UL44. These data indicate that UL44 dimerization is essential for
oriLyt-dependent DNA replication and suggest that disruption of UL44 homodimers could provide a strategy for the
development of novel anti-HCMV compounds.
25
9
Human Metapneumovirus from Alto Adige and surrounding Italian and Austrian regions: a
comparison study
Elisabetta Pagani1, Christine Petters1, Daniela Secolo1, Patrizia Rossi1, Barbara Amato2, Lydia Pescollderungg3,
Giulia Campanini4, Elena Percivalle4, Hartwig P. Huemer5 and Clara Larcher1.
1
Laboratory of Microbiology and Virology, Azienda Sanitaria dell’Alto Adige, Comprensorio Sanitario di Bolzano,
Italy;
2
Department of Haematology and Bone Marrow Transplantation, Azienda Sanitaria dell’Alto Adige, Comprensorio
Sanitario di Bolzano, Italy;
3
Department of Pediatrics, Azienda Sanitaria dell’Alto Adige, Comprensorio Sanitario di Bolzano, Italy;
4
Servizio di Virologia, IRCCS Policlinico San Matteo, Pavia, Italy;
5
Department of Hygiene, Medical Microbiology and Social Medicine, Medical University of Innsbruck, Austria.
Background: Human Metapneumovirus (hMPV), in addition to paediatric patients, has been increasingly described in
adults and has been found to cause more severe infections in immunocompromised patients.
Methods: In our laboratory in Bolzano, serving a regional hospital, hMPV diagnosis is based on direct
immunofluorescence assay, using monoclonal antibodies, confirmed by reverse-transcriptase-PCR. From 2005 we
performed an epidemiological survey to compare the local strains to those of the surrounding regions, by doing
sequencing analysis (N-genes as well as L-genes) of the obtained isolates.
Results/discussion: By alignments with the established Dutch reference strains we identified three different genotypes
circulating in our region, with predominantly type B strains being detected. This is in contrast to published data from
nearby Italian regions from the 2003/2004 winter season, where predominantly A2 strains have been characterized, and
Austrian Tyrol region, where only A-type strains have been detected. Whether this represents a further indication for
similar strains cycling in a wider geographic area with the respective types replacing each other on a seasonal basis,
remains to be clarified, although absolute sequence identity due to the high variability of RNA viruses was rarely
detected. Similar hMPV strains circulated in both the paediatric and adult patients and, of interest, the two isolates
originating from bone marrow transplant recipients (BMT) were of different genotypes. Both findings rather exclude
hospital transmission in our BMT patients as has been suspected by a study in lung transplant patients (C. Larcher et al.,
J. Heart & Lung Transplant., 2005).
10
Activity of a synthetic microbicidal peptide against Influenza A virus multiplication
Conti G., Magliani V., Portincasa P., Conti S., Polonelli L.
Department of Pathology and Laboratory Medicine- Microbiology Section - University of Parma
A killer peptide (KP) engineered from the sequence of the variable region of a recombinant antiidiotypic antibody
representing the internal image of a killer toxin, is characterized by the wide spectrum of microbicidal activity against
pathogenic eukaryotic and prokaryotic microrganisms.
Based on the surprising sequence homology of KP with the light chain variable region of an antibody (HC63) that
prevents the haemagglutinin, HA, low pH fusogenic transition of Influenza A virus, we report the “in vitro” inhibition
of Influenza A virus multiplication in LLC-MK2 cells which is mediated by a marked reduction of glycosylation of HA,
as well as its synthesis.
The restriction of the production of mature virion particles could be the result of a defect involving the migration and /
or the association - insertion of virus haemagglutinin HA into the plasma membrane of infected LLC-MK2 cells treated
with KP.
Moreover, this hypothesis is also in good agreement with the haemadsorption as well as immunoprecipitation
experiment results.
26
Medical Virology and Antiviral Therapy
27
Human respiratory syncytial virus (hRSV) RNA quantification in nasopharyngeal secretions
identifies the hRSV etiologic role in acute respiratory tract infections of hospitalized infants
Giulia Campaninia, Elena Percivallea, Fausto Baldantia,b, Francesca Rovidaa, Alice Bertainac, Antonietta Marchic,
Mauro Stronatid, Giuseppe Gernaa
a
Servizio di Virologia, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, bLaboratori Sperimentali di Ricerca ,
Fondazione IRCCS Policlinico San Matteo, Pavia, Italy, cDipartimento di Pediatria, Università di Pavia, Pavia, Italy,,
d
Divisione di Patologia Neonatale, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy
Background: Human respiratory syncytial virus (hRSV) detection in nasopharyngeal aspirates (NPAs) from infants with
acute respiratory tract infection (ARTI) does not prove the hRSV etiology of the current ARTI episode. HRSV RNA
quantification may help in affording this issue.
Objectives: hRSV was detected by quantitative reverse-transcription-PCR in NPAs taken upon admission to hospital
and, whenever possible, at discharge and subsequent medical visits.
Study Design: Prospective study, including 63 infants affected by either hRSV upper or lower ARTI.
Results: Based on the kinetics of viral load, hRSV etiology was identified in 25 infants in whom hRSV load dropped
from 2.5x106 upon admission (presence of respiratory symptoms) to 7.5x102 RNA copies/ml NPA upon discharge
(absence of symptoms) after a median time of 5 days, and in 19 infants, in whom hRSV load was determined at
admission only, in association with clinical symptoms (2.4x106 copies/ml). Furthermore, low levels of hRSV RNA
(<1x105 copies/ml NPA) identified 14 patients with non-hRSV ARTI. Finally, in 14 infants with hRSV coinfections or
sequential infections, hRSV quantification defined the hRSV role in the current ARTI episode.
Conclusions: hRSV RNA quantification is critical in defining the hRSV role in respiratory infections.
The human bocavirus role in acute respiratory tract infections of pediatric patients as defined
by viral load quantification
Giuseppe Gernaa,, Antonio Pirallaa, Giulia Campaninia, Antonietta Marchib, Mauro Stronatic, Francesca Rovidaa
Servizio di Virologia, Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy, bDipartimento di Pediatria,
Università di Pavia, 27100 Pavia, Italy, cDivisione di Patologia Neonatale, Fondazione IRCCS Policlinico San Matteo,
27100 Pavia, Italy
a
The major objective of this study was to investigate the pathogenic role of human bocavirus (hBoV) in patients
hospitalized with acute respiratory tract infection (ARTI). Overall, 685 respiratory samples from 426 patients were
examined by PCR for human bocavirus, as well as for other known human respiratory viruses. HBoV was quantified by
PCR. Forty/283 (14.1%) pediatric patients, and 2/143 (1.4%) adult patients were found to harbor hBoV for a total of 45
episodes (16 detected as single infection, and 29 as coinfection) of hBoV-associated respiratory infection. HBoV DNA
quantification revealed the presence of an NPA viral load >1.0x105 DNA copies/ml in respiratory secretions from 17/40
(42.5%) children and 0/2 adults. Below this cut-off, hBoV appeared to be an innocent bystander or a persistent virus.
Although hBoV may be frequently detected in children with upper or lower ARTI, in less than 50% young patients it
appears to be potentially pathogenic.
Antiviral activity of Retinoic Acid derivatives against HHV-8
Caselli E., Galvan M., Santoni F., Cassai E., Di Luca D.
Retinoic acid (RA) has been proposed as an antitumor drug for the treatment of HHV-8 associated Kaposi’s Sarcoma
(KS), based on several clinical trials showing that topical application of 9-cis-RA induces clinical responses in
approximately 40% of patients. The antineoplastic mechanism of RA in the context of KS is only partially understood.
RA is known to have antiproliferative effects on neoplastic cells, and RA treatment of KS is associated with inhibition
of angiogenesis and a decreased vascular response both in vitro and in vivo. However, it has never been determined
whether the biologic effects induced on KS cells are due only to the antiproliferative activity of RA, or are also related
to an inhibition of HHV-8 replication. A major obstacle in assessing the antiHHV-8 activity of RA is represented by the
lack of a cell system permitting exogenous viral infection. Here we describe productive infection with HHV-8 of human
epithelial and endothelial cells, and provide evidence that 9-cis-RA, as well as newly synthesized RA compounds, have
relevant antiviral effects on HHV-8-infected cells. In fact, RA inhibit HHV-8 replication and transcription, with a
significant down-modulation of viral promoters. RA antiviral activity is not due to IFN production or MCP-1 inhibition.
Nevertheless, RA treatment of HHV-8 infected endothelial cells prevents the virus-induced alterations of cell
physiology (i.e. acquisition of spindle cell phenotype) and blocks in vitro angiogenic ability.
29
Pre-transplant analysis of preservation and washing solutions for rapid detection of viruses in
the kidney graft
Monia Pacenti, Luisa Barzon, Valentina Militello, Manuela Della Vella1, Luisa Murer1, Giorgio Palù.
Department of Histology, Microbiology and Medical Biotechnologies and 1Department of Paediatrics, University of
Padova, Padova, Italy.
Viral infections, which may be achieved from the graft, are serious complications in kidney transplant recipients. In
order to set-up a sensitive and non-invasive test for diagnosis of viral infections of the graft before implantation, we
investigated whether viral sequences could be detected in kidney graft biopsies and in graft preservation (PS) and
washing solutions (WS). We analyzed 42 kidney grafts obtained from 41 cadaveric donors (median age, 12 years, range
1-45). Belzer solution was used for graft preservation and washing before implantation. For viral detection, total DNA
was purified from graft biopsies obtained at bench surgery, PS, WS, and the cell fraction of both solutions. The
presence of EBV, HCMV, BKV, and B19 DNA was determined by TaqMan real-time PCR. Out of 42 graft biopsies, 27
were negative for viral DNA and 15 positive (1 EBV-positive; 13 B19-positive, 1 B19+BKV-positive). Overall, viral
detection in the cell fraction of both solutions was more sensitive than in whole solutions. EBV DNA was detectable in
20% PS and WS (including the case with EBV-positive biopsy); HCMV DNA was identified in 10% and 6% of PS and
WS, respectively (all biopsies were negative); BKV DNA was demonstrated in 2 PS (with negative biopsy); B19 DNA
was positive in 26% and 32% of PS and WS, respectively (50% with B19-positive biopsies). Our results indicate that,
for optimal sensitivity of testing, EBV and HCMV DNA detection should be done in PS and WS, whereas BKV and
B19 DNA should be tested in both biopsies and solutions. This results are in agreement with the natural tropisms of
each virus.
ANTIVIRAL ACTIVITY OF WC5, A NEW 6-AMINOQUINOLONE COMPOUND,
AGAINST HUMAN HERPESVIRUSES
Beatrice Mercorelli1, Giulia Muratore1, Elisa Sinigalia1, Maria Angela Biasolo1, Stefano Sabatini2, Oriana Tabarrini2,
Arianna Loregian1, and Giorgio Palu1
1
Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Italy
2
Department of Chemistry and Technology of Drugs, University of Perugia, Italy
Among a series of 6-aminoquinolone compounds, a molecule - termed WC5 - was identified that inhibited the
replication of human cytomegalovirus (HCMV) AD169 in infected cell cultures with a selectivity index (SI) of 500
(ED50 = 0.9 µM; CC50 = 431 µM). WC5 also showed similar inhibitory activity against PFA- and GCV-resistant HCMV
strains, suggesting that it might act against a viral target(s) different from that of those DNA polymerase inhibitors.
Indeed, WC5 did not significantly inhibit either the activity of purified baculovirus-expressed DNA polymerase
catalytic subunit (UL54) or the activity of the UL54/UL44 complex in DNA polymerase assays in vitro. Moreover,
time-of-addition and time-of-removal experiments indicated that WC5 most likely affects an early stage of viral
replication that is located after virus adsorption and penetration but before viral DNA synthesis. In keeping with these
observations, the anti-HCMV effect of WC5 was MOI-dependent, as already shown for other anti-herpesvirus
compounds that act at early stages of the virus replication cycle. Antiviral activity of WC5 was also tested against other
human herpesviruses. WC5 did not significantly affect herpes simplex virus type 1 replication in infected cell cultures
(ED50 = 11 µM; CC50 =200 µM; SI = ~ 18). In addition, WC5 did not inhibit the replication of both human herpesvirus6 A and B variants in permissive T-lymphoblastoid cell lines, as evaluated by quantitative Real-Time PCR (qt-RT-PCR)
analysis (SI = 0.5 – 1). WC5 also did not interfere with lytic reactivation of human herpesvirus-8 in BC-3 cells, as
determined by qt-RT-PCR analysis. Studies to elucidate the mechanisms of action of WC5 are ongoing.
30
POSTERS
11
Monitoring the efficacy of preventive HPV vaccine: differences of different commercial
techniques in detecting and identifying HPV genotypes in clinical samples.
Brandi Rossella, Rollo Francesca, Quattrini Marco, Benevolo Maria, Marandino Ferdinando, Mariani Luciano,
Vocaturo Amina, and Venuti Aldo.
Translational Research Group in Gynaecological Oncology -Regina Elena Cancer Institute – Rome – Italy
Several commercial HPV tests have been introduced in the clinical practice and typing is expected to be more and more
utilized after the introduction of the preventive vaccines in order to monitor the efficacy of these treatments.
HPV analysis was performed in consecutive Thin-Prep samples. Testing for HPV DNA was performed with the
following commercial kit: the high risk Hybrid Capture II assay (Digene, Italy); PreTect-HPV-Proofer (Norchip,
Norway); and the Reverse Linear Array (Roche, Italy). To check the specificity and sensitivity of the methods the same
samples were also analyzed by home made PCR with MY or GP 5+/6+ or cluster specific primers followed by direct
sequencing or sub-cloning and sequencing.
The detection rate of HPV was similar with all the methods utilised with the highest percentage for the Roche (60%)
and the home made tests (53%) and the lowest for the Norchip assay (27%).
Comparison between HCII and Linear Array showed same results in 86,9% (K=0,75) of cases while HCII and PCR in
90% (K=0,8) of cases and HCII and Norchip only in 71% (K=0,45) of cases. PCR and Linear-Array gave concordant
results in 82% (K=0,65) of cases while PCR and Norchip in 84% (K=0,63). The concordance between Linear Array and
Norchip was 63% (K=0,33). Multiple infections were detected in 16 % of samples. The typing analysis showed
discordant results suggesting that the existing assays still fail to identify all of the possible HPV types involved. This
circumstance justifies the development of new and improved HPV typing assays.
12
Characterization of group A human rotaviruses detected in 2006 in Brescia, Italy
Lorusso Eleonora1, Colombrita Domenico 2, Draghin Emanuele2,
Cavalli Alessandra1, Buonavoglia Canio1, Caruso Arnaldo2 Martella Vito1
1
Department of Animal Health and Well-being, University of Bari, Valenzano, Bari, Italy
2
Department of Applied and Experimental Medicine, Section of Microbiology, University of Brescia Medical School,
Brescia, Italy
Group A rotaviruses are common agents of acute dehydrating diarrhoea in infants and young children. Classification of
HRV strains is based on the antigenic/genetic differences of the two outer capsid proteins VP4 (P-type) and VP7 (Gtype). Either monovalent (G1P[8]) or polyvalent (G1 to G4) vaccines have been developed for immunization of children,
since serotype-specific immunity is important for protection against infections. The pattern of distribution of human
rotavirus G and P types among children hospitalized with acute gastroenteritis in Brescia Hospital, Italy, was evaluated
from January 2006 to December 2006. HRV infection was detected in 192 (24.7%) of 775 feces of children examined.
Of these, 83 was further characterized to determine the G-types (VP7 genotypes) and P-types (VP4 genotypes) by RTPCR genotyping with multiple sets of primers. The predominant combination of genotypes was G1[P8] (51.8%; 43 of
83) followed by G9[P8] genotype (27.7%; 23 of 83), G4[P8] (3.6%; 3 of 83), G2[P4] (2.4%; 2 of 83), G3[P8] (1.2%; 1
of 83) and G3[P6] (1.2%; 1 of 83). Infection whit multiple rotavirus strains was detected in 12% of the samples (10 of
83). The present study confirms the role of G9 HRVs as agents of enteritis in the paediatric population in Brescia in the
year 2006. The G9 serotype is not included in the polyvalent rotavirus vaccines, although it appears to be common
worldwide. A continuous epidemiological surveillance for HRV is necessary to detect changes in the circulation of
HRV G and P types or the onset of novel strains.
31
13
High prevalence of Human papillomavirus infection in HIV-1 positive men
A. Amendola^, S. Bianchi^, G. Orlando*, R. Beretta*, MM. Fasolo*, E. Tanzi^.
^Dep. Public Health-Microbiology-Virology, University of Milan, Italy
*STD Unit, II Dept Infectious Diseases, L Sacco Hospital, Milan, Italy
INTRODUCTION:
Human papillomavirus (HPV) infection is estimated to be the most common sexually transmitted infection and HIV-1
positive men who have sex with men are at very high risk to acquire HPV infection and HPV-related anogenital cancer.
OBJECTIVE:
To evaluate the prevalence of HPV infection in a male HIV-1 positive population, to analyze the HPV high-risk (HR)
and low-risk (LR) types circulating both in single and in multiple infections.
MATHERIALS-METHODS:
From June 2003 to October 2006, 165 HIV-1 positive males(mean age 36yrs; range 21-69yrs)referring to the STD
Unit, Sacco Hospital, Milan (Italy) were involved in that study. Cytological brushing of the anal canal were analyzed
for HPV-DNA by PCR in L1 region, using MY09/MY11 primers. For HPV genotyping the 450bp fragments obtained
were tested by RFLP.
RESULTS-CONCLUSIONS:
HPV-DNA was detected in 90.3% (149/165) subjects, in 35.6% (47.1% HR and 52.9% LR) as a single infections and in
64.4% as multiple.
30 different genotypes were identified and HPV-16 was the prevalent HR-type, both in single (22.6%) and in multiple
(54.7%) infections, followed by HR-HPV-58 (5.7%) in single and HR-HPV-30 (14.6%) in multiple infections.
The prevalent LR-types were 6 and 11, both in single (24.5% and 22.6%) and in multiple infections (42.7% and 34.4%).
HPV prevalence was very high and a wide variety of HPV genotypes was observed in HIV-infected males. The high
presence of HR-HPV in these subjects underlines the need of preventive measures, like a screening program or the
introduction of a preventive vaccine in such high-risk population.
14
The use of complementary medical techniques for the optimisation of treatment of patients
with genital herpes
Igor G. Bondarenko, MD, PhD, Jukka Chydenius, DC
CHYDENIUS Clinic, Helsinki, Finland
Applied kinesiology is a modern neurophysiology-based area of complementary medicine that, through the use of a
standard muscle test (SMT), enables a practitioner to select a treatment (pharmaceutical, nutritional, homoeopathic etc.)
specific for each patient (substances, their doses and duration of treatment).
12 patients (both genders, 19-50 years) with clinically diagnosed genital herpes (GH) were enrolled in the study.
Previous studies have demonstrated that a frequency of GH relapses is linked to the expression of a point mutation
(T->G) leading to the synthesis of enzymes with decreased affinity to alpha-lipoic acid. With the SMT, we evaluated
the applicability of alpha-lipoic acid to the treatment of GH. Required dosage and the duration of supplementation were
selected for each patient individually with the use of the SMT.
Major symptoms of GH (tingling, itching, burning, tenderness, prickling, soreness, bump/swelling, small blisters,
oozing blisters) were daily monitored on a 10-point scale. Days 1, 3 and 7 were arbitrarily set as fixed time markers.
Alpha-lipoic acid produced a clinical cure (all CHS symptoms score of zero) in 1 patients (8%) on the day 2, in 8
patients (67%) on the day 3, in 3 patients (25%) – on the day 7. All lesions were completely cured on the day 10. Score
data showed a significant improvement on the day 7 compared to baseline and day 3. Biochemical mechanisms of
action of alpha-lipoic acid used and the possible role of applied kinesiology in the treatment of GH are discussed.
32
15
Clinical and Epidemiological Correlates of Antibody Response to Denatured Recombinant
Human Papillomavirus (HPV16) L and E Antigens as Measured by a Novel ELISA
Colomba Giorgi,1 Paola Di Bonito,1 Felicia Grasso,1 Stefania Mochi,1 Luisa Accardi,1 Maria Gabriella Donà,1
Margherita Branca,2 Silvano Costa,3 Luciano Mariani,4 Alberto Agarossi,5 Marco Ciotti6 and Kari Syrjänen7 on
behalf of the HPV-Pathogen ISS group*
1
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità. Rome, Italy
2
Unità Citoistopatologia, Centro Nazionale di Epidemiologia, Sorveglianza e Promozione della Salute, Istituto
Superiore di Sanità (ISS), 00161 Rome, Italy
3
Dipartimento di Ginecologia e Ostetricia, Azienda Ospedaliera S. Orsola Malpighi, Bologna, Italy
4
Ginecologia e Ostetricia, IFO, Istituto Regina Elena, Rome, Italy, Clinica Ostetrica e Ginecologica, 5Istituto Scienze
Biomediche, Ospedale Luigi Sacco, Milano, Italy
6
Laboratory of Clinical Microbiology and Virology, University Hospital "Policlinico Tor Vergata", Rome, Italy
7
Department of Oncology and Radiotherapy, Turku University Hospital, FIN-20521 Turku, Finland
At present, the diagnosis of HPV infections is not based on serology data but rather on viral DNA and virus-associated
lesion detection. HPV serology has developed slowly mainly for inability to obtain viral antigens in vitro.
We have recently developed an in-house ELISA based on denatured E. coli-expressed HPV16 proteins. This assay is
based on the idea that as viral proteins from different HPV types share high sequence identity the denatured HPV16
proteins could be used as antigens to capture the non-structural antibodies directed against other HPV types. An ELISA
based on denatured recombinant HPV16 L1, L2, E4, E6 and E7 antigens was used to study the antibody response in
sera of 96 women in a cohort of HIV-negative and HIV-positive women with abnormal pap smear. The results have
shown that this assay could really detect antibodies against HPV types different from 16.
The next objective was to correlate the HPV seroreactivity with all clinical and epidemiological data of this cohort of
women previously reported from a multi-centre Italian HPV-PathogenISS study. The results showed that HPV DNA
detection and seroreactivity did not have any correlation. Current smokers had significantly less seroreactivity to L
antigens as compared with the non-smokers. Particularly HIV+/current smokers showed the lowest seroprevalence
(33.3%) as compared to 75.9% among all other women (p=0.011).
In summary, it is tempting to speculate that HIV+ current smokers comprise a special high-risk group, with extremely
high probability of developing CIN3/CC, due to their failure to eradicate persistent HR-HPV infections.
16
Impact of Respiratory Syncytial Virus and human Metapneumovirus infections among
hospitalized children younger than 3 years of age
Zappa A, Perin S°, Pariani E, Amendola A, Ruzza ML*, Colzani D, Podestà A*, Farina C°, Tanzi E.
Department of Public Health-Microbiology-Virology, University of Milan (Italy)
°Microbiology Unit, AO Ospedale S. Carlo Borromeo, Milan (Italy)
*Pediatric Unit, AO Ospedale S. Carlo Borromeo, Milan (Italy)
Objectives: To determine the contribution in hospitalization of RSV and hMPV infections among children younger than
3 years of age in Milan (Italy) during 2004/05 and 2005/06 winter seasons (November-March).
Methods: Oropharyngeal Swabs (OS) were collected from 70 (39M, 31F, mean age: 9.1 months) and from 33 (21M,
12F, mean age: 5.9 months) children hospitalized during the 2004/05 and 2005/06 seasons, respectively. Viral RNA
was detected from OS samples by amplification of 218 bp of gene F of RSV and 151 bp of gene M of hMPV.
Results: Overall, RSV-RNA was detected in 23 out of 103 (22.3%) OS samples and hMPV-RNA in 8 out of 103 (7.8%)
OS samples (p<0.05). One children was co-infected with hMPV and RSV. Symptoms and clinical data were similar
among RSV and hMPV-infected children, even though 61% of RSV-positive children showed bronchiolitis. A
statistically variation of the impact of RSV among hospitalized children was observed during the two seasons: RSVRNA was detected in 10 out of 70 (14.3%) and in 13 out of 33 (39.4%) OS samples analysed during the 2004/05 and
2005/06 winter seasons, respectively. Frequency of hMPV was similar (7.1% vs 9%; p=n.s.) in the two periods.
Conclusions. Our data suggest that RSV and hMPV are associated with severe acute respiratory infections and involved
in episodes of hospitalization in children younger than 3 years of age. The impact of these viral infections may vary
seasonally.
33
17
Typing and distribution of HCV strains in South of Italy (Apulia region).
Paola Menegazzi, Luigi Tagliaferro, Oliviero E. Varnier1
Virology & Molecular biology section, Lab. “ Dr. P. Pignatelli”, Lecce, Italy
1
Institute of Microbiology Medical School, University and DiMMI, CBA, Genova, Italy
Typing and epidemiological distribution of HCV isolates in the population of a southern region of Italy (Apulia) have
been evaluated by direct sequencing of purified amplicons, using the OpenGene System (Visible Genetics, Siemens
MSD) after removal of FRET probes.
The HCV 5’ UTR region was amplified by an “in house” rapid, single tube LightCycler Real Time RT-PCR with
hybridization probes technology. The accuracy and the reproducibility of this test have been confirmed by the very low
crossing point coefficient of variation and standard deviation values of the used international WHO HCV human
reference standard plasma (HCV Accurun series, BBI Inc.), calculated on more than 5,000 samples tested in 409 runs.
A total of 469 HCV LightCycler amplicons were sequenced from October 2002 to February 2007. 5 of the 6 wide-world
known principal HCV genotypes were observed, identifying 16 types and subtypes, with a homology average of 99.9%.
The sequencer has been able to distinguish 58 different HCV isolates, while 176 of 469 samples (38%) have been typed
but not subtyped, because of the presence of some undistinguishable sequences, common to other strains, mainly
observing 1b (38%) and 2 (27%) genotypes, with a clear prevalence of the K0014, HCJ5 and S83 HCV isolates.
All samples sequences have been revised by a newer software version, with the aim to subtype most of strains.
18
Lack of selection of lamivudine-resistant mutants in HBV/HIV coinfection during lamivudineincluding antiretroviral therapy
Meini G., Zazzi M, *Toti M and Caudai C.
Department of Molecular Biology, Virology Section, University of Siena; * Division of Infectious Disease,
Misericordia Hospital, Grosseto, Italy.
Lamivudine (3TC) is effective against both HIV and HBV replication. Among HIV/HBV-coinfected patients,
lamivudine given for HIV infection as part of an antiretroviral regimen, promptly inhibits HBV replication.
To evaluate the impact of lamivudine-containing highly active antiretroviral therapy (HAART) on hepatitis B (HBV)
infection, we retrospectively evaluated the clinical and virological response in 12 HBV/HIV coinfected patients
followed for one to four years. HBV and HIV viral loads, serum alanine aminotransferase (ALT), CD4+ cell/mm3 and
HBeAg were determined at baseline and during the follow-up in plasma samples. Moreover YMDD mutants and HBV
subtype were determined by sequencing of the HBV polymerase region.
The results showed that HAART containing lamivudine effectively decreased plasma HBV-DNA by 4 to 6 log10,
leading to complete suppression in most HBeAg negative patients. By contrast, all but one HBsAg positive patients had
high HBV DNA levels at baseline and did not experience suppression of HBV viremia. Two of these non responders
showed a switch from D to A genotype, likely resulting from a D/A coinfection. Interestingly, none of the 12 patients
showed lamivudine-resistance mutations in HBV polymerase region. Administration of low doses of 3TC in the context
of the antiretroviral regimen and erratic adherence to treatment could explain the lack of selection of 3TC resistance.
Further studies are needed to investigate the relationship between efficacy of treatment and selection of drug-resistant
mutants with treatment of HBV/HIV coinfected patients with lamivudine.
34
Viral Biotechnologies and Gene Therapy
35
PERSISTENT VIRAL DETECTION IN THE KIDNEY ALLOGRAFT IS A RISK FACTOR
FOR CHRONIC ALLOGRAFT INJURY
Luisa Barzon, Monia Pacenti, Maria Angela Biasolo, Manuela Della Vella1, Luisa Murer1, Giorgio Palù.
Department of Histology, Microbiology and Medical Biotechnologies and Department of Paediatrics, University of
Padova, Padova, Italy.
BACKGROUND: Chronic allograft injury, which is the main cause of late renal allograft failure, has been associated
with both immune-dependent and immune-independent risk factors, such as viral infections. Aim of this study is the
investigation of the prevalence and persistence of viruses in transplanted kidneys and their association with renal
function and the risk to develop chronic allograft injury.
METHODS: Screening for the presence of all herpesviruses, polyomaviruses, and parvovirus B19 DNA in baseline and
follow-up renal allograft biopsies obtained from 69 transplanted children at 6, 12, and 24 months after transplantation.
Correlation of virological findings with renal function tests and with histological analysis according to Banff
classification, performed at the same time points.
RESULTS: Viral genome sequences were detected in 42% baseline allograft biopsies and in 70% protocol biopsies.
Parvovirus B19, HHV-6, BKV, and EBV were the most frequently detected viruses, often as coinfection. The presence
of viral genome sequences in the allograft and, in particular, their persistence, was associated with the risk to develop
acute rejection and chronic allograft injury. Among viruses, the presence of B19 in the renal graft was clearly associated
with the risk of both acute and chronic lesions and impairment of renal function, whereas persistent EBV infection was
associated with the risk of late acute rejection.
CONCLUSIONS: Persistent intrarenal viral detection is a risk factor for allograft lesions. Among viruses, B19 and
EBV are associated with the risk of chronic lesions and acute rejection, respectively.
VIRAL CLEARANCE EVALUATION MODEL USING 4 DIFFERENT VIRUSES FOR
GMP-PRODUCTION OF BIOPHARMACEUTICAL DRUGS OR VACCINES
McDermott JL1, Martini I1, *Giacomazzi C2, *Losada Cabruja E2, Bobbio V1 and *Varnier OE1,2.
Laboratory of Microbiology and Virology1, Advanced Biotechnology Center and *Institute of Microbiology2,
University Medical School, Genova, Italy.
The risk of virus contamination is a feature common to biopharmaceutical products such as recombinant monoclonal
antibodies, vaccines, cytokines and human-derived proteins or cells. Viral contaminant can arise from the source cell
line or from adventitious virus introduced during production. The capacity of the purification process to remove or
inactivate viral contaminants must be evaluated to assure the absence of these viruses in the final product. We
developed an analysis model of clearance evaluation for Herpes Simplex type 2, Simian Virus type 40 (SV40), Murine
Leukaemia Virus and Poliovirus type 3, selected for differences in physicochemical resistance. Each clearance involved
the spiking of a high titre of infectious virus before each downscaled purification step [i.e.: low pH, ion exchange
chromatography (IEC), affinity chromatography (AFC), nanofiltration (NFN), etc.]. At the end of each step, infectivity
and quantity of the challenging virus was determined using plaque or focus assays in permissive cell lines and
quantitative DNA or RNA PCRs, respectively. Removal and inactivation factors were calculated for each virus e.g. for
the SV40 clearance of the downscaled production for a monoclonal antibody now in clinical trails, a spike containing
1010.30 infectious viruses and 1010.37 virions per ml was used. Removal factors of 4.05, 1.89 and 5.10 Logs and
inactivation factors of 8.57, 7.30, and 7.50 Logs were observed for AFC, IEC, and NFN steps, respectively.
The safety of human therapeutical compounds can be only be rationally guaranteed by the analysis of viral inactivation
and removal during the GMP production process.
37
EFFECTS OF ADENOVIRAL VECTOR INFECTION ON HUMAN ADRENOCORTICAL
CELLS
Urska Matkovic, Monia Pacenti, Enrico Lavezzo, Luisa Barzon, Giorgio Palù.
Department of Histology, Microbiology and Medical Biotechnologies, University of Padova, Italy.
Adrenocortical tropism of adenovirus has been reported, but the effects of adenoviral vector transduction on adrenal
gland has been poorly examined. In this study, we investigated the direct effects of first-generation adenoviral vector
infection on adrenocortical cell proliferation, cell cycle, cell death, and steroidogenesis. First, we demonstrated
expression of the adenoviral receptors CAR in human normal, benign and malignant adrenocortical tissues. Efficient
transduction with recombinant E1-/E3- adenoviral vector expressing green fluorescent protein (AdEGFP) was
demonstrated in human adrenocortical cell lines and primary cell cultures. No marked effect on adrenocortical cell
proliferation and cell cycle was found after adenoviral transduction with Adnull (Ad vector expressing no transgene),
AdEGFP, and AdHSV-TK (Ad vector expressing HSV thymidine kinase) at low MOI (2-50), whereas high MOI (100500) decreased cell proliferation of about 20% compared to uninfected control and increased G2/M phase, without a
significant induction of apoptosis or necrosis. Furthermore, adenoviral vectors induced cortisol, estradiol and
aldosterone production and, consistently, adenoviral vectors upregulated gene expression of the key activator of
steroidogenesis StAR and of the steroidogenic enzymes CYP11A1, CYP11B1, and CYP11B2. In conclusion,
adenoviral vectors demonstrated high transduction efficiency of human adrenocortical cells and enhanced steroid
hormone production. These effects should be evaluated in patients treated with adenoviral vectors for gene therapy.
Role of recombinant poxvirus vectors expressing HIV/SIVgenes in modulating Th1/Th2
cytokine profiles in human dendritic cells
Paola Beggioa, Carlo Zanottoa, Eleana Pozzia, Sole Pacchionia, Carlo De Giuli Morghena,c and Antonia Radaellib,c
a
Department of Medical Pharmacology, bDepartment of Pharmacological Sciences and CNR Institute of Neurosciences,
University of Milan, Italy
Vaccinia virus (VV) has been developed for use as a candidate vaccine vector and, in particular, for HIV. Fowlpox (FP)
and Canarypox (CP), although able to express foreign genes, cannot replicate in mammalian cells and represent
therefore safer vectors. In our study, we evaluated the modulation of cytokine expression after infection of human
dendritic cells (DCs) with either FP, CP or VV, engineered with HIV-1 env and/or gag-pol SIV genes. To analyze the
cytokine profile we used a protein microarray system that allows simultaneous detection of multiple secreted cytokines
(IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, GM-CSF, IFN- , and TNF- ). Cell culture supernatants were incubated over
chip glasses spotted with cytokine antibodies. Cytokine detection was performed after incubation with a cocktail of
biotinylated antibodies followed by Alexa 555-labeled streptavidin. Signals from the arrays were revealed by a laser
scanner. Our results showed that, after infection, VV drastically inhibited IL-4, IL-8 and TNF- cytokines production
by DCs, whereas FP and CP recombinants induced an increase in IL-6 and TNF- levels, thus suggesting a Th2-type
immune response. These results may contribute to better understand the different cellular host immune response after
immunization by FP, CP and VV recombinants.
38
Human placenta derived mesenchymal stem cells are fully permissive to human
cytomegalovirus infection
LUCONI Lucia*, CANTONI Silvia°, ALVISI Gualtiero*, BIANCHI Francesca°, Daniele Musiani*, CAVALLINI
Claudia°, VENTURA Carlo° and RIPALTI Alessandro*§
*Dipartimento di Medicina Clinica Specialistica e Sperimentale, Sez. di Microbiologia, °Istituto di Cardiologia,
Università di Bologna and §U.O. Microbiologia, A.O-U. S.Orsola-Malpighi, Bologna, Italy
Mesenchymal stem cells (MSCs) are characterized by a broad differentiation potential, a strong indication for clinical
applications. Adult BM is the common source of MSCs for clinical use, however, the frequency of MSCs in human
adult BM is relatively low, and availability is conditional to invasive procedures. Umbilical cord blood (UCB) and
second-trimester amniotic fluid (AF) are a poor source of MSC, while MSC collection from AF requires amniocentesis,
a risky procedure not amenable to routine clinical applications. The human placenta is an alternative, abundant and
easily available source of MSCs, and term placenta derived MSCs (PMSCs) are capable of differentiating into
neurogenic, chondrogenic, osteogenic, adipogenic, hepatogenic and myogenic lineages. PMSCs have a higher
expansion potential than adult BM-derived MSCs, they have been implicated in regenerative processes after foetal
lesions, and evidence from experiments in neurodegenerative disorders in adults suggests that transplantation of stem
cells could lead to regeneration of injured neural tissue. These characteristics together with a low infection rate and
young age of placenta compared with adult stem cells of other tissue origin make PMSCs an attractive target for cellbased therapy and a precious tool in regenerative medicine. The possibility that PMSCs can sustain viral infections is
relevant not only to purification, propagation, conservation and direct therapeutic use issues but also to their potential
viral vector-mediated genetic modification. We present here evidence that PMSCs are fully permissive for
Cytomegalovirus (CMV) infection, and data on the effect of CMV infection on their growth characteristics.
Expression of the HPV-16 L1 Antigen in Transplastomic Tobacco Plants
Paolo Lenzi1,4, Nunzia Scotti1, Fiammetta Alagna1, Maria L Tornesello2, Franco M Buonaguro2, Andrea Pompa3,
Alessandro Vitale3, Stefania Grillo1, Pal Maliga4, Teodoro Cardi1
1
CNR-IGV, Institute of Plant Genetics-Res. Div. Portici, Portici, Italy
Viral Oncogenesis and Immunotherapy, Dept. of Experimental Oncology, Ist. Naz. Tumori "Fondazione Senatore G.
Pascale”, Napoli, Italy
3
CNR-IBBA, Institute of Agriculture Biology and Biotechnology, Milano, Italy
4
Waksman Institute, Rutgers, The State
2
Plants are considered a promising production platform of therapeutic proteins, showing several advantages over
conventional systems. The objective of this project was the expression in tobacco plants of the L1 protein from the
Human Papillomavirus (HPV-16), a virus that causes cervical cancer. L1 gene codes for the major capsid protein, which
forms Virus Like Particles (VLPs). To express the viral antigen, we used plastid transformation, which allows
delivering of the transforming DNA in the plastid genome instead of the nucleus. Compared to conventional transgenic
technologies, plastid engineering generally shows higher protein expression levels, no gene silencing and transgene
containment. Several vectors containing the wild-type L1 sequence were constructed, in which L1 is regulated by strong
plastid promoters and different 5’-UTRs. The gene sequence was also changed according to the plastid codon usage and
vectors carrying the plastid-modified sequence were assembled. Transplastomic plants were obtained following biolistic
DNA delivery. Correct gene insertion and homoplasmy level were tested by PCR and Southern blot analyses. RNA gel
blot analyses demonstrated the presence of L1 transcripts in tobacco plastids. Recombinant L1 protein was detected by
western blots when translated plastid sequences were added upstream of the viral gene (Downstream Box – DB). L1
accumulated up to 2% total proteins when compared with known amounts of baculovirus-derived VLPs. Positive
capture ELISA assays, carried out with MAbs that recognize conformational epitopes, suggest the ability of plantderived L1 protein to assemble into structures (i.e. capsomers, VLPs). Pulse-chase labeling experiments demonstrated
that the protein is stable in the plastid environment.
39
POSTERS
19
Intrabody-based approach for the therapy of the HPV-associated cancer.
Luisa Accardi*, Maria Gabriella Donà*, Tamara Petrucci+, Paola Torreri+, Linda Chiappalupi*, Luca Spagnoli*,
Colomba Giorgi*
*Department of infectious, parasitic and immunomediated diseases, +Department of Cell Biology and Neurosciences ,
Istituto Superiore di Sanità, Roma.
Intrabodies directed against intracellular proteins involved in carcinogenesis can abrogate their activity. The HPV
tumour-specific antigens E6 and E7 are appropriate targets to develop an intrabody-based anti-cancer therapy.
Two interesting mutations with respect to the consensus of the germ-line genes were identified in the VH and VL
scaffold of an antibody in single-chain format (scFv43) able to inhibit cell proliferation when expressed in the nuclear
and secretory compartments of the HPV16-positive SiHa cells (Accardi et al. 2005). The resulting aminoacid changes at
positions VH34 and VL92 were reverted back by site-directed mutagenesis, obtaining scFv43M2 with improved
stability characteristics (Donà et al. 2007).
The most stable scFv43 M2 and 51 were provided with either nuclear or endoplasmic reticulum localisation signals and
expressed by retrovirus system at Dr Tommasino Laboratory. After transduction with the recombinant retroviruses, the
scFv-expressing SiHa cells were neomycin-selected. Evidence of antiproliferative effect of both scFvs in both nucleus
and endoplasmic reticulum was obtained by different assays.
In collaboration with Dr Bank’s Group, binding of three different scFvs on E7 was analyzed by ELISA, Western
blotting and BIAcore. Unlike scFv32 and 51, scFv43M2 bound to the pRb-binding region.
The E6 oncoprotein interacts with the p53 oncosuppressor and other proteins involved in apoptosis, growth arrest,
terminal differentiation and antiviral defence. Two stable anti-HPV16E6 scFvs were selected by Intracellular Antibody
Capture Technology at Prof Cattaneo Laboratory. The anti-HPV16E6 scFvs were purified from E. coli, characterized
and expressed in the SiHa cell nucleus for future evaluation of the scFv effect on E6.
20
LENTIVIRAL-MEDIATED DELIVERY OF A SHORT HAIRPIN RNA TARGETING
VEGF IN HUMAN RPE
Giulia Lombardi1, Elena Vendramini1, Arianna Calistri1, Cristina Parolin2, Giuseppe Lo Giudice3, Giovanni
Prosdocimo3, Giorgio Palù1
1
2
Department of Biology, University of Padova, Italy
3
Department of Ophthalmology, Conegliano Hospital, Italy
Choroidal NeoVascularization (CNV) is the leading cause of blindness in Age-related Macular Degeneration (AMD).
Several lines of evidence implicate increased levels of Vascular Endothelial Growth Factor (VEGF) in Retinal Pigment
Epithelium (RPE) from patients with AMD. Current approaches to attenuate VEGF or its receptors show significant
promises, but still require repeat administrations. The aim of this study was to develop a strategy for long-term
endogenous expression of a short hairpin RNA (shRNA) that would significantly attenuate VEGF. We employed a
Human Immunodeficiency Virus type 1 (HIV-1)-based vector in order to exploit the innate peculiarity of lentiviruses to
efficiently and stably infect post-mitotic target cells as RPE.
An shRNA against VEGF was cloned into HIV-1-derived Self-Inactivating (SIN) vector and packaged into VSVG
protein pseudotyped recombinant infectious particles (shRNA-VEGF-LVTHM). Lentiviral vectors were used to
transduce human RPE cell cultures in normoxic and in hypoxic condition in order to mimic AMD in vitro. The
efficiency of transduction of target cells was quantified thanks to the Green Fluorescent Protein (GFP) as reporter gene
in the lentiviral vector. At different times post-transduction VEGF expression levels were analyzed by Western Blotting.
In addition, the amount of released VEGF was determined by ELISA.
Our preliminary data indicate that more than 90% of human RPE cells were successfully transduced by shRNA-VEGFLVTHM with the GFP expression lasting for more than 10 days. A reduction in VEGF release, compared with nottransduced cells, both in normoxic and hypoxic conditions, was observed. Taking into account limitations of current
therapies for AMD, our approach seems a new potential anti-VEGF therapy.
40
Viral Oncogenesis and Vaccines
41
ANTI-CANCER ACTIVITY OF PLANT-PRODUCED HPV16 E7 VACCINE
Massa* Silvia, Franconi* Rosella, Brandi° Rossella, Muller° Antonio, Mett Vadim, Yusibov Vidadi and Venuti°
Aldo.
*ENEA, Italian National Agency for New Technologies, Energy and the Environment, BIOTEC GEN, C.R. Casaccia,
P.O. Box 2400 I-00100 Roma, Italy;
°
Laboratory of Virology, Regina Elena Cancer Institute, Via delle Messi d’Oro 156, Roma, Italy;
Fraunhofer USA Center for Molecular Biotechnology, 9 Innovation Way, Suite 200, Newark, DE 19711, USA
Plants represent a source of safe and inexpensive vaccines. The human papillomavirus 16 (HPV16) E7 oncoprotein is a
tumor-specific antigen, a key target for therapeutic vaccines against HPV-associated tumors. We have already produced
the HPV16 E7 protein in Nicotiana benthamiana (Nb) by transient expression mediated by a potato virus X-derived
vector, demonstrating that E7-containing plant extracts are able to trigger a strong cell-mediated immune response able
to protect immunized mice from artificial tumor growth. To improve the E7 oncoprotein yield by transient expression
and in view of its purification from the plant tissue, we engineered both the HPV16 E7 sequence and the mutagenised
sequence E7GGG as fusions to -1,3-1,4-bacterial glucanase (LicKM) with a KDEL signal and a His6-tag for
purification and we cloned the fusions in a TMV-derived vector for agro-infiltration. As a consequence, Nb expressed
lichenase-E7 and lichenase-E7GGG fusion proteins and E7 protein yields in Nb extracts were enhanced about 100-fold.
Fusion proteins were purified (about 400 µg lichenase-E7 / g leaf tissue) and evaluated in mice as vaccine candidates. Both
fusion proteins, administered with the adjuvant Quil A, induce high E7-specific IgG titres and stimulate E7-specific
cytotoxic and helper T-lymphocytes protecting mice against E7-expressing tumours (100% protection) after challenge.
Interestingly, the E7-derived fusion proteins, without adjuvant, protected 80% of the animals, suggesting possible
intrinsic adjuvating activities of the purified antigen. Our results might lead to phase I clinical trials with the first
purified plant-derived vaccine against HPV-associated cancer.
Development of a vaccine against Toscana Virus
Gianni Gori Savellini1, Giuseppa Di Genova1, Chiara Terrosi1, Paola Di Bonito2, Colomba Giorgi2 and Maria Grazia
Cusi1.
1
Department of Molecular Biology, Microbiology Section, University of Siena, Siena, Italy, 2 Istituto Superiore di
Sanità, Rome, Italy.
Toscana Virus (TOSV) is a Phlebovirus responsible for aseptic meningitis, meningoencephalitis and, rarely,
encephalitis. The aim of this study is to develop an efficient and safe recombinant vaccine against TOSV. In this study,
mice were immunized with single or different combinations of TOSV antigens in order to evaluate identify those
responsible for protection against the specific virus. A preliminary study was carried out on mice immunized with
plasmids encoding the nucleocapsid protein (N) and the two envelope glycoproteins: G1 and G2. The preliminary study
indicated that the N protein is able to induce a strong immune response which, however, is not sufficient to protect mice
from challenge with a neuroadapted strain of TOSV. The combination of the N and the G2 proteins gave the best
protection against viral challenge. Thus, N and G2 proteins could be potential candidates for a vaccine against TOSV.
For this purpose, mice were ip. immunized with a combination of the two antigens three times, then subjected to
intracanial challenge with TOSV 1812V (10 LD50) and monitored for survival. Another group of mice immunized
following the same protocol was sacrificed after the last immunization for immunological studies. Serum samples and
splenocytes drawn from these mice were tested for the presence of specific antibodies and cytokines such as interferon
gamma (IFN- ) and interleukin 5 (IL-5), respectively. The results of mice survival after virus challenge indicate that the
N and G2 proteins are able to protect animals by inducing an efficacious humoral and cell-mediated immune response
against TOSV.
43
DEVELOPMENT OF AVIPOXVIRUS RECOMBINANT VACCINES FOR THE CONTROL OF HPV16INDUCED CERVICAL CANCER
Eleana Pozzia, Carlo Zanottoa, Paola Beggioa, Sole Pacchionia, Carlo De Giuli Morghena,c and Antonia Radaellib
a
Department of Medical Pharmacology, University of Milan, Italy
b
Department of Pharmacological Sciences, University of Milan, Italy
c
CNR, Institute of Neurosciences
An attenuated strain of fowlpox virus (FPwt) was engineered by inserting the E6 (FPE6) or E7 (FPE7) oncogenes of the
Human Papilloma Virus 16 (HPV16). The transgene expression was evaluated by Western blotting, IP and IF. The
immune response was assayed in four groups of rabbits inoculated with either FPE6, FPE7 or FPE6+FPE7 (108 pfu/animal,
i.d.) or FPwt, used as a control. Priming with the recombinants was followed by three boosts with purified E6, E7 or
E6+E7 proteins (100 µg, i.d.) in Freund's incomplete adjuvant. Collected sera were tested for the specific antibody
response by Western blot and ELISA. No specific antibody response was detected after priming, whereas high antibody
levels were observed after boosting. Preliminary data also seem to show a specific CTL response by 51Cr release assay
on SV40-immortalized syngeneic target cells, suggesting the elicitation of a cellular immunity. The prophylactic
efficacy of the vaccine will be demonstrated by challenging rabbits with CRPV-transformed tumor cells.
Impact of the seropositivity status on the pattern of DC activation by Virus-Like Particles.
L. Buonaguro1, M.L. Tornesello1, M. Tagliamonte1, G.K. Lewis2,3, A. Monaco4, F. Marincola4 and F.M. Buonaguro1.
1
Lab. of Viral Oncology and AIDS Ref. Center, Ist. Naz. Tumori “Fond. G. Pascale”, Naples - Italy; 2Div. of Vaccine
Res., Inst. Human Virol., and 3Dept Microbiol. and Immunol., Univ. Maryland, Univ. Maryland Baltimore, Baltimore,
USA; 4Immunogenetics Lab., Dept. Transfusion Medicine, NIH, Bethesda, MD,USA.
Aim: The present study has been performed to evaluate the pattern of maturation and activation of dendritic cells (DCs)
induced by HIV-VLPAs, according to the donor’s seropositivity status, and to analyze the immunogenetic pathways
involved.
Results: The baculovirus-expressed HIV-VLPs has been previously shown to efficiently induce maturation and
activation of MDDCs. In the present study, the seropositivity status, and a different HIV-1 viremia level, does not
appear to influence the normal pattern of DC activation and maturation, although a different baseline pattern of cytokine
production is observed. This is further evident as consequence of in vitro stimulation with HIV-VLPs, with the absence
of a committed Th pathway. Furthermore, the baseline and VLP-induced PBMCs genomic transcriptional profiles show
a modulation of selective pathways according to the seropositivity status and the levels of viremia.
Conclusions: Our data show that a different seropositivity status and, within the seropositivity group, the levels of
viremia might not affect the activation and maturation of DCs. However, the difference in the modulation of
transcriptional profiles induced by an immunogen, as the VLPs, may results in a different activation of immune
response. These immunogenetic data give an insight into the mechanisms of the in vivo immune activation, also in
therapeutic vaccination strategies, with the possible identification of genetic predictors of individual response.
44
Distribution of human papillomavirus type 16 variants in penile keratinizing squamous cell
carcinoma
ML Tornesello1, ML Duraturo1, S Losito2, G Botti2, S Pilotti3, B Stefanon, G De Palo4, A Gallo5, L Buonaguro1, FM
Buonaguro1,*
1
Viral Oncology and AIDS Reference Centre, National Cancer Institute, "Fond. Pascale", Naples, Italy
2
Department of Pathology, National Cancer Institute, "Fond. Pascale", Naples, Italy;
3
Department of Pathology, National Cancer Institute, Milan, Italy;
4
Preventive and Predictive Medicine, National Cancer Institute, Milan, Italy;
5
Urology Unit, National Cancer Institute, "Fond. Pascale", Naples, Italy
The causative role of human papillomaviruses (HPV) and HPV16 variants has been extensively studied in uterine
cervix dysplastic lesions and invasive carcinoma; few such studies, however, have been performed in penile tumors. We
have investigated HPV genotype and HPV16 variant distribution on 27 penile cancer biopsies from Italian patients.
Cases were extracted from the respective pathology departments databases of National Cancer Institutes in Naples and
Milan. HPV sequences were detected by PCR and characterized by direct sequence analysis. Among the 14 HPVpositive cases (51.8%) two viral genotypes were identified (HPV16 and 18) with HPV16 accounting for 92.8% (13 out
of 14) of the infections. Sequence analysis of E6, E7 genes and long control region (LCR) of 13 HPV16 isolates
allowed the identification of European (E-G-350) and non-European (AA and Af-1) variants in 46.2% and in 53.8% of
the samples, respectively. The AA variant alone represented 38.5% of all HPV16 infections, a significant much higher
frequency of that observed in cervical carcinoma from Italian women (Tornesello et al., J Med Virol 2004). Our results
suggest that HPV16 has a very high prevalence among penile cancer patients in Italy and the increased frequency of
non-European HPV16 classes, particularly the AA, implies that their presence in penile tissue is associated with higher
risk of neoplastic progression in comparison to European variants.
Expression, processing and assembly of the HIV-1 Pr55gag protein in transgenic tobacco
chloroplasts
Scotti N.1, Alagna F.1, De Stradis A.2, Buonaguro L.3, Buonaguro F.3, Grillo S.1, Cardi T.1
1
CNR-IGV, Institute of Plant Genetics, Portici, Italy;
2
CNR-IVV, Institute of Plant Virology, Bari, Italy;
3
Viral Oncology, Cancer Institute “Fond. G. Pascale”, Naples, Italy
Gag, the major structural protein of HIV-1, is capable of assembling in Virus-Like Particles (VLPs), eliciting both
humoral and cellular immune response, and is therefore a primary candidate for the development of a preventive or
therapeutic HIV-1 vaccine. Plants show several advantages compared to other systems for the production of
pharmaceuticals. We investigated plastid transformation to express the Pr55gag protein in tobacco plants. Plastid
transformation allows transgene containment through maternal inheritance of cytoplasmic organelles, precise transgene
insertion by homologous recombination, as well as high gene expression and protein accumulation. The full gag gene
was cloned in vectors for plastid transformation. Transplastomic plants showed the correct integration of the transgene
and homoplasmy, and the presence of a stable gag transcript. Western analyses with anti-p24 and anti-p17 antibodies
detected the presence of a strong band of about 40 kDa consisting of the N-terminal matrix (p17) and the central capsid
(p24) domains. Hence, the Pr55gag polyprotein is processed by plastidial proteases similarly to what occurs with the
viral protease in human cells. Similar results were obtained by nuclear transient expression experiments in tobacco
plants agroinfiltrated with constructs containing either the full gag gene or its subunits. The plastid-expressed Gag
protein was also able to assemble into particles resembling VLPs produced in baculovirus and E. coli systems.
45
POSTERS
21
Detection of oncogenic viruses (SV40, BKV, JCV, HCMV, HPV) and p53 codon 72
polymorphism in lung carcinoma.
1
Laura Giuliani, 1Terese Jaxmar, 2Caterina Casadio , 2Marisa Gariglio, 3 Assunta Manna, 3Domenico D’Antonio, 4Kari
Syrjanen, 1Cartesio Favalli, 1Carlo Federico Perno and 1Marco Ciotti. 1Laboratory of Clinical Microbiology and
Virology, University Hospital Tor Vergata, Viale Oxford, 81-00133 Rome, Italy. 2University of Eastern Piedmont,
Novara, Italy. 3 Laboratory of Clinical Microbiology and Virology, Hospital Spirito Santo, Pescara, Italy. 4Department
of Oncology & Radiotherapy, Turku University Hospital, Turku, Finland.
As a part of our search for oncogenic viruses in bronchial cancer, we extended our HPV studies to SV40, BKV, JCV
and HCMV sequences and related these data with p53 codon 72 polymorphism. Fresh tumor samples from 78 patients
were analysed for SV40, BKV, JCV, HCMV and HPV sequences by PCR. HPV genotypes were determined using
reverse blot hybridization and sequencing. All HPV-positive tumors were tested for the presence of E6/E7 transcripts by
RT-PCR. All samples were analysed for p53 codon 72 polymorphism, by RFLP method.
Of the 78 cases studied, 11 (14.1%) were positive for T-Ag gene of SV40, while BKV and JCV sequences were both
amplified in 1 tumor only. Altogether, 10/78 lesions were HPV-positive; 6 HPV16, 1 HPV31, 2 HPV6/53, 1 HPV16/18.
All HPV DNA-positive samples except one also expressed E6 and E7 transcripts. HCMV was amplified in 18 (23%)
cases. RFLP analysis of p53 codon 72 revealed 32 homozygotes for arg/arg allele (50.8%), 26 heterozygotes for
arg/pro allele (41.3%), and 5 homozygotes for pro/pro allele (7.9%).P53 codon 72 polymorphism was not significantly
different between cases (n=63) and controls (n=50) (p=0.455), among virus positive and negative patients, nor was it
related to HPV genotypes (p=0.384), expression of E6 (p=0.384) and E7 oncogenes (p=0.293). Among the co-detected
viruses only SV40-HCMV association was statistically significant (OR=5.500, 95%CI 1.43-21.02; p=0.015). Taken
the known mechanisms of these individual viruses, there is a chance that these viruses could affect cell cycle control
and inhibit apoptosis potentially causing genetic instability and promote oncogenesis.
22
EFFECT OF AGE ON SERUM HETEROLOGOUS ANTIBODY RESPONSE ELICITED
BY INACTIVATED INFLUENZA VIRUS VACCINE
Camilloni B., Neri M., Lepri E., Iorio A.M.
University of Perugia, Dept. Med. Surg. Spec. and Public Health, Via del Giochetto, Perugia, Italy
The aim of the study was to evaluate the influence of ageing per se as well as of priming histories on the antibody
response to influenza vaccination. The study was carried out in the winter 2003/04 in two groups of volunteers with
different priming histories for A/H1N1 virus as a consequence of the different age: middle-aged, 26 volunteers, mean
age 41 years, birth cohorts 1958 to 1971, and elderly, 91 subjects, mean age 83 years, birth cohorts 1905 to 1940.
Following trivalent MF59-adjuvanted subunit influenza vaccine (FLUAD, Chiron) administration, the
haemagglutination inhibiting (HI) antibody response of old, compared with middle-aged, was equal for
A/Moscow/10/99/H3N2, slightly impaired for B/Hong Kong/330/01, significantly lower for A/New
Caledonia/20/99/H1N1. In order to understand if the lower response against the A/H1N1 strain might be due to the
“original antigen sin” mechanism, the vaccine induced HI antibody response against vaccine and earlier A/H1N1 strains
was compared. A distorsion of the specificity of the antibodies produced was seen in the elderly. Significantly higher
numbers of old people, as compared with middle-aged, not showing response against the homologous vaccine A/H1N1
antigen, produced antibody against earlier A/H1N1 viruses. The present data confirm that serological responses to
influenza vaccines might be reduced with aging. However, since elderly volunteers were capable of antibody responses
against A/H3N2 strain similar to those of middle-aged, they suggest that host factors other than age (priming
backgrounds, possibly differences in prior vaccination/infection history) might contribute to this phenomenon.
46
23
Large-scale synthesis of anionic core-shell microparticles for vaccine application: physicochemical and biological characterization
Francesca Bortolazzi¹, Arianna Castaldello¹, Egidio Brocca-Cofano¹, Rebecca Voltan¹, Michele Laus², Katia
Sparnacci², Barbara Ensoli³, Silvia Spaccasassi4, Marco Ballestri4, Luisa Tondelli4, Antonella Caputo¹
¹Department of Histology, Microbiology and Medical Biotechnology, University of Padova, Via A. Gabelli 63, 35122
Padova, Italy
²Department of Life and Ambient Sciences (DISAV), University of Piemonte Orientale and INSTM,
UdR Piemonte Orientale, Via Bellini 25/G, 15100 Alessandria, Italy
³National AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy
4
I.S.O.F., Consiglio Nazionale delle Ricerche, Via Piero Gobetti 101, 40129 Bologna, Italy
Large-scale preparations of novel anionic microparticles (H1D) were synthetized by dispersion polymerization. They
exhibit a core-shell structure, with a inner core constituted by polymethylmethacrylate (PMMA) and a highly
hydrophilic outer shell constituted by Eudragit L100/55 bearing carboxylic groups capable of interacting with basic
proteins. Their process reproducibility was evaluated in terms of physico-chemical properties (size, -potential, the
loading and release profiles of the Tat-protein onto the microparticles surface) and cell culture release profiles of the
protein. In addition, immunogenicity studies were carried out in mice with a selected H1D batch and the HIV-1 Tat
protein vaccine.
The results showed that the preparation method was both robust and reproducible, and that the H1D/Tat formulation is
safe and immunogenic.
24
Impact of the seropositivity status on the pattern of DC activation by Virus-Like Particles.
L. Buonaguro1, M.L. Tornesello1, M. Tagliamonte1, G.K. Lewis2,3, A. Monaco4, F. Marincola4 and F.M. Buonaguro1.
1
Lab. of Viral Oncology and AIDS Ref. Center, Ist. Naz. Tumori “Fond. G. Pascale”, Naples - Italy; 2Div. of Vaccine
Res., Inst. Human Virol., and 3Dept Microbiol. and Immunol., Univ. Maryland, Univ. Maryland Baltimore, Baltimore,
USA; 4Immunogenetics Lab., Dept. Transfusion Medicine, NIH, Bethesda, MD,USA.
Aim: The present study has been performed to evaluate the pattern of maturation and activation of dendritic cells (DCs)
induced by HIV-VLPAs, according to the donor’s seropositivity status, and to analyze the immunogenetic pathways
involved.
Results: The baculovirus-expressed HIV-VLPs has been previously shown to efficiently induce maturation and
activation of MDDCs. In the present study, the seropositivity status, and a different HIV-1 viremia level, does not
appear to influence the normal pattern of DC activation and maturation, although a different baseline pattern of cytokine
production is observed. This is further evident as consequence of in vitro stimulation with HIV-VLPs, with the absence
of a committed Th pathway. Furthermore, the baseline and VLP-induced PBMCs genomic transcriptional profiles show
a modulation of selective pathways according to the seropositivity status and the levels of viremia.
Conclusions: Our data show that a different seropositivity status and, within the seropositivity group, the levels of
viremia might not affect the activation and maturation of DCs. However, the difference in the modulation of
transcriptional profiles induced by an immunogen, as the VLPs, may results in a different activation of immune
response. These immunogenetic data give an insight into the mechanisms of the in vivo immune activation, also in
therapeutic vaccination strategies, with the possible identification of genetic predictors of individual response.
47
HPV Infection and Prevention
49
ORAL COMMUNICATIONS
Characterisation of the interaction of HPV E6 with the Discs Large Tumour Suppressor.
Lawrence Banks, Nisha Narayan, Paola Massimi and Miranda Thomas.
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy.
Continued expression of the HPV E6 oncoprotein is essential for the maintenance of the transformed phenotype.
Numerous studies have been performed to investigate the mechanism by which E6 contributes to malignant progression,
and interactions with p53 and a subset of PDZ domain-containing cellular proteins appear critical. However, the
association with the PDZ domain-containing proteins is a unique feature of those mucosal HPV E6 proteins associated
with the development of malignancy. Low risk HPV E6 proteins lack this capacity. The list of such proteins that are
targeted by E6 is continually growing and includes Discs Large (Dlg), Scribble, the MAGI family, PATJ, PSD95 and
PTPH1. Many of these proteins are critical for the regulation of normal cell growth and polarity; breakdowns in the
control of these processes are common features of malignant progression.
We have been particularly interested in dissecting the mechanism by which E6 targets the PDZ domain-containing
proteins for proteasome-mediated degradation, as well aiming to understand the relevance of these associations for
HPV-induced malignancy. Most of our work is currently focussed on the E6-Dlg interaction. Detailed mutational
analysis has determined the molecular basis underlying E6 targeting of this substrate. In addition, we also have
compelling evidence that E6 can target at least a subset of these PDZ proteins through an E6AP independent pathway.
Finally, we have also found evidence for E6 targeting only specific forms of Dlg. Interestingly, those forms of the
protein that are degraded appear to have growth suppressive activities. In contrast, those forms of Dlg that are not
targeted by E6 actually possess growth promoting functions. These results highlight the highly selective nature of E6
function and reveal unexpected activities for the Dlg tumour suppressor protein.
HPV Vaccines development (L1-based VLP) and evolution (L2-based)
Reinhard Kirnbauer
Laboratory of Viral Oncology (LVO), Department of Dermatology (DIAID), Medical University Vienna, Austria
A prophylactic human papillomavirus (HPV) vaccine based on virus-like particles (VLP) has become approved recently
following extensive clinical evaluation. HPV vaccines have demonstrated 100% efficacy in preventing persistent genital
infection and the development of type-specific cervical intraepithelial neoplasia (CIN), the precursor lesions of invasive
cervical cancer. HPV subunit vaccines consist of L1 major capsid proteins that self-assemble into VLP following
recombinant expression in yeast or insect cells. HPV VLP do not contain potentially oncogenic viral DNA and thus are
safe and well tolerated. Conceptually HPV vaccination is similar to Hepatitis B immunization that reduces the incidence
of hepatocellular carcinoma in countries with high-prevalence of Hepatits B infection.
Papillomaviruses and VLP present conformation-dependent neutralization epitopes on their surface in a repetitive and
quasi-crystalline manner. These highly immunogenic assemblies induce potent B-cell immune responses by directly
cross-linking the B-cell receptor and, in addition, stimulate maturation of antigen presenting cells (APC) and T-cell
responses. These highly immunogenic properties of HPV VLP are responsible for the >99% seroconversion rate
observed in animal and human vaccination studies. Importantly, a combination of several VLP types as in current HPV
vaccines does not impair the immune response against the individual types.
Two high-risk HPV types 16 and 18 account for more than 70% of cervical and other anogenital neoplasias. However,
30% of cervical cancers are caused by infection of about 12 additional high-risk types, which are not included in current
vaccine formulations. It is likely that vaccine manufacturers will include additional HPV VLP types into a second
generation HPV vaccine, although the higher costs of such a vaccine will result only in modest increase of protection
against cervical neoplasia. An alternative strategy to develop a ‘pan-HPV’ vaccine is based on the minor capsid protein
L2 that contains cross-neutralization epitopes. Immunization with bacterially expressed or synthetic L2 peptides can
induce a broadly cross-neutralizing immune response against genital and even skin-specific HPV types, although of
relatively low titer compared to L1 VLP vaccination. To further increase L2 immunogenicity, VLP that display L2
neutralization epitopes repetitively by immunogenic VLP surface loops have been genetically engineered, that are
currently being tested for their ability to induce cross-neutralizing immunity.
51
GARDASIL® : NUOVO VACCINO ANTI- HPV
F. Scaglione.
Dipartimento di Farmacologia ,Chemioterapia e Tossicologia medica.
Università degli studi di Milano
Gardasil® è il primo vaccino, sviluppato dalla Sanofi Pasteur MSD, approvato per prevenire il tumore della cervice, le
lesioni genitali precancerose e i condilomi genitali dovuti ai tipi 6, 11, 16 e 18 del papillomavirus umano ( HPV ). Il
vaccino è stato approvato per l’impiego in soggetti di sesso femminile tra 9 e 26 anni di età.
La produzione del vaccino è assolutamente innovativa ed è il risultato di anni di ricerca per ottenere un vaccino ad alta
induzione anticorpale e privo di rischi.
La metodica viene utilizzata per produrre separatamente, mediante tecniche di biologia molecolare, le
particelle virali (VLP) degli HPV tipo 6,11,16 e 18, le quali, dopo un attento e complesso processo di purificazione,
vengono, per ogni tipo, adsorbite su idrossifosfato di alluminio solfato che funziona da adiuvante. Le particelle dei
quattro virus, cosi prodotte, vengono unite nella preparazione iniettabile.
Numerosi studi controllati randomizzati in doppio cieco, hanno valutato l’immunogenicità del vaccino anti-HPV, basato
sulle VLP ottenute con la proteina L1, in giovani donne (età 16- 26 anni) .Alcuni di questi studi hanno usato vaccini
monovalenti mentre tre grossi studi hanno valutato il vaccino quadrivalente.
I risultati sono stati sorprendenti e molto gratificanti per tutti colori i quali hanno partecipato alla ricerca di questo
vaccino innovativo.
Il titolo anticorpale si è significativamente innalzato per tutti e 4 tipi e si è mantenuto relativamente alto per 5anni di
osservazione .Questi risultati sono stati ottenuti con il vaccino che conteneva la più bassa dose di VLP (HPV 6 [20 µg],
11[40 µg ], 16 [40 µg] e 18 [20 µg]), che è stato poi usato per lo sviluppo della fase III. Il livello anticorpale ottenuto
con questa dose è risultato del tutto simile a quello ottenuto con dosi più alte. (40, 40, 40 and 40 µg, rispettivamente) o
(80, 80, 40 and 80µg) dosi di VLP.
Il vaccino quadrivalente è stato ampiamente studiato clinicamente nelle fasi II e III che lo hanno portato
all’approvazione per l’uso clinico nella prevenzione delle infezioni da HPV e del cancro cervicale HPV-correlato.
I risultati sono stati estremamente positivi nessun caso di CIN o neoplasia si è verificato nei gruppi che avevano
completato la vaccinazione , mentre nei soggetti che avevano assunto placebo sono stati osservati 37 casi nello studio
FUTURE I e 21 nello studio FUTURE II. Il vaccino ha mostrato una protezione del 100%.
Un altro dato di grande interesse che è emerso dagli studi è che anche quando il virus invade la mucosa non è in grado
di dare la malattia nei soggetti vaccinati. Infatti, mentre il vaccino ha mostrato una protezione del 90% nel prevenire
l’infezione persistente ha dimostrato una protezione del 100% nel prevenire ogni forma di malattia.
In tutti gli studi effettuati la tollerabilità generale del vaccine è stata buona. Non risono verificati mai eventi avversi
gravi. La vaccinazione è stata sospesa in pochi soggetti (0.1%).
Gli effetti più frequenti sono stati dolore nella sede di inoculo ,eritema e febbre quest’ultima si è verificata in
circa il 10% dei casi.
New therapeutic strategies.
Aldo Venuti.
Laboratory of Virology Regina Elena Cancer Institute, Rome, Italy
At the present, a vaccine against the genital HPV has been licensed and it would be able to prevent primary viral
infection and, consequently, to reduce the incidence of cancer. However, due to the long latency period between
infection and cancer, the benefits of a prophylactic vaccination would be visible after decades. Moreover HPVs are also
associated with non-genital tumors (i.e. skin and head/neck cancers) involving males that are excluded from the
prophylactic vaccination, at least in this first phase. Thus, a real need exists to develop therapeutic strategies able to
hinder the progression of HPV associated tumors, or even to eliminate them. Two main routes can be followed: the
immunotherapy or the interaction with the virus-host cell interplay.
Preliminary promising results have been disclosed by immunotherapy but they still need further improvement by the
association with more appropriate adjuvants able to stimulate efficacious cell mediated immunity. The development of
‘second generation’ vaccines, that are more active and more accessible for developing countries, is an urgent issue. Data
will be presented on new therapeutic vaccines based on DNA immunizations and on plant-derived antigens, as well as
experimental data on compounds interacting with the virus-host cell interplay.
52
Metodi diagnostici e screening nell’era del vaccino.
Francesca Carozzi
U.O. Citologia Analitica e Molecolare
CSPO Firenze Istituto Scientifico della Regione Toscana
L’individuazione che l’infezione da PapillomaVirus Umano (HPV) è la causa principale del carcinoma cervicale ha
aperto nuovi fronti per la prevenzione mediante vaccinazione ed il miglioramento delle metodologie di screening.
La recente disponibilità del vaccino contro i tipi 16 e 18 del Papilloma Virus (HPV) rappresenta quindi una novità
importante nel mondo in rapido cambiamento della prevenzione del cervicocarcinoma. Fondamentale è capire come
questa pratica possa integrarsi con i programmi di screening per la prevenzione del cervicocarcinoma. Verosimilmente
saranno necessari molti anni per poter valutare pienamente l’impatto dei vaccini, ma screenare con l’attuale modalità
una popolazione vaccinata rischia di essere poco efficiente.
Alla stato attuale , le indicazioni d’uso del vaccino sono basate sulla dimostrazione di efficacia in donne di età
compresa tra 16 e 26 anni e le decisioni nazionali , al momento , sono quelle di offrire la vaccinazione gratuita a coorti
successive di ragazze a partire dai 12 di età. Dunque prima che le corti vaccinate arrivino nelle classi di età in cui
l’incidenza del cervicocarcinoma è più alta passeranno molti anni. Inoltre una valutazione di impatto è resa più
complicata dal fatto che i tassi di incidenza del carcinoma cervicale stanno diminuendo stabilmente nel nostro paese da
molti anni come conseguenza dell’estendersi dei programmi e della pratica spontanea del pap test. Dunque la possibilità
di valutare (e di scegliere conseguentemente) quale sarà il possibile impatto della vaccinazione si dovrà basare su una
serie di studi in corso ma anche su modelli di simulazione matematici.
Con la diminuzione della incidenza e della prevalenza della malattia sarà importante valutare l’impatto sul numero
totale di anormalità citologiche e individuare la migliore strategia di screening in una popolazione vaccinata (età di
intervento, tipo di test, intervalli tra i test, procedure di follow-up) per minimizzare procedure non necessarie e i costi.
Nelle donne immunizzate la sensibilità e specificità dei test di screening dovrà essere rivalutata. In una situazione di
bassa prevalenza diminuisce infatti il valore predittivo positivo del pap test per cui molte donne sarebbero inviate in
colposcopia con una limitata probabilità di avere una lesione. D’altra parte c’è stato negli ultimi dieci anni un grande
interesse sull’uso del test HPV sia come test primario di screening sia come triage di anormalità citologiche borderline.
L’HPV test ha mostrato una maggiore sensibilità (20-40%) , ma una specificità più bassa (5-10%) rispetto al Pap- test
nell’individuare lesioni di alto grado e cancro. Nell’epoca del vaccino, una diversa strategia di screening potrebbe
essere quella di utilizzare il test più sensibile per primo seguito da un triage con un test più specifico, cioè uno
screening con HPV e un triage, delle donne HPV positive, con la citologia.
Comunque, se da una parte il Servizio Sanitario Nazionale offrirà la vaccinazione a determinate classi di età è
ipotizzabile che anche le donne di altre classi di età si sottopongano a vaccinazione. Da qui scaturisce la necessità di
una sinergia tra i servizi di vaccinazione ed i servizi di screening per poter individuare tra le donne invitate allo
screening quelle che hanno effettuato la vaccinazione; questo link potrebbe permettere di valutare nelle donne
vaccinate eventuali modificazioni sulle abitudini allo screening, l’efficacia nella riduzione di malattia, la prevalenza
delle infezioni per evidenziare eventuali cambiamenti nella frequenza dei virus oncogeni minori nella popolazione
generale e nelle lesioni.
53
Virus Host Interactions and Pathogenesis
55
Down-regulation of proteolytic complexes following Epstein Barr Virus activation in Burkitt’s
lymphoma cells
Giulia Matusali , Alessandra De Leo , Laura Bertelli , Livia Di Renzo , Elena Mattia
Dept. of Public Health Sciences and Dept. of Experimental Medicine and Pathology, Sapienza Univ.,Rome, Italy
In latently-infected Burkitt’s lymphoma cells, Epstein Barr virus (EBV) proteins interact with the ubiquitin-proteasome
system to promote episomal maintenance and immunological evasion while the alternative protease tripeptidylpeptidase
II (TPPII) is highly expressed. In the present study, we have examined the activities and levels of the proteasome and
TPPII complex in Raji and in Akata cells after induction of EBV lytic cycle. The results show that the chymotrypsinlike and caspase-like activities of the proteasome were substantially reduced in Raji and Akata cells. Similarly, TPPII
activity was diminished in both cell lines but was recovered in Akata cells at longer time after induction. Protein levels
of the / subunits of the 20S proteasome and TPPII concentration decreased to different extents after EBV activation,
whereas the ubiquitin binding S6’ subunit of the 19S regulatory complex increased three to four-fold along with the
levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular
proteolytic systems at the onset of EBV lytic infection.
ANALYSIS OF HCMV PPUL44
HOMODIMERIZATION,
INTRA-NUCLEAR
MOBILITY AND PHOSPHORYLATION-REGULATED NUCLEAR TRANSPORT USING
LIVE CELLS IMAGING
Gualtiero Alvisi 1,2, David A. Jans2, Elisa Sinigalia3, Daniele Musiani1, Daniela M. Roth2, Arianna Loregian3, and
Alessandro Ripalti4,1Department of Clinical and Experimental Medicine, Microbiology Division, St. Orsola General
Hospital, University of Bologna, 40138 Bologna, Italy, 2Department of Biochemistry and Molecular Biology, Monash
University, 3800 Clayton, Victoria, Australia, 3Department of Histology, Microbiology and Medical Biotechnologies,
University of Padova, 35121 Padova, Italy, 4Department of Hematology, Oncology and Laboratory Medicine,
Microbiology Operative Unit, St. Orsola General Hospital, University of Bologna, 40138 Bologna, Italy
HCMV DNA polymerase processivty factor phosphoprotein ppUL44 is essential for viral replication and hence
represents an attractive therapeutic target. We have used live cells quantitative confocal laser scanning microscopy
(CLSM) and fluorescence recovery after photobleaching (FRAP) analysis applied to transiently expressed,
spontaneously fluorescent ppUL44 point mutant derivatives, to test the role of specific amino acids in several ppUL44
biochemical properties, including homodimerization, binding to DNA polymerase catalytic subunit, intranuclear
binding and phosphorylation-regulated nuclear transport. Mutation of hydrophobic residues important for dimerization
in vitro significantly decreased dimerization ability, as shown by our quantitative analysis performed using a nuclear
relocalization assay we recently described. We also show that ppUL44’s basic loop (PHTRVKRNVKKAP174), which
is extremely well conserved amongst all -herpesviruses, is involved in ppUL44 intranuclear binding and responsible
for ppUL44 limited intranuclear mobility, suggesting its possible role in dsDNA binding. Finally we identify residues
responsible for positive and negative modulation of nuclear transport by phosphorylation as shown by altered nuclear
transport of phosphomimetic mutant derivatives. Since ppUL44 has been implicated in nuclear transport of pUL54 and
ppUL114, regulation of its nuclear transport might affect the intracellular localization of those viral products. Moreover
the identification of sequences involved in binding of ppUL44 to DNA might facilitate the understanding of how
ppUL44 functions in conferring processivity to the DNA polymerase holoenzyme.
57
Human Cytomegalovirus Replicates in Adrenocortical Cells and Stimulates Steroid Hormone
Production
Marta Trevisan, Giulia Masi, Riccardo Cusinato, Alessandro Sinigaglia, Luisa Barzon, Giorgio Palù.
Dept Histology, Microbiology, and Medical Biotechnologies, University of Padova, Padova, Italy.
We recently detected HCMV genome and expression of both early and late HCMV genes in human adrenocortical
tumors and demonstrated that the presence of HCMV sequences was significantly more frequent and at higher titer in
functioning than in nonfunctioning tumors. Aim of this study was to investigate whether HCMV infects and replicates
in human adrenocortical cells in vitro and the effects of viral infection on steroidogenesis and adrenal function. By
using SW13 and H295R human adrenocortical carcinoma cells and primary adrenocortical cell cultures, we
demonstrated that HCMV AD169 established full productive infection in adrenocortical cells as demonstrated by pp72
and pp65 expression, a marked cytopathic effect, and efficient production of viral particles from infected cells. HCMV
infection of adrenocortical cells stimulated cortisol and estrogen production by modulation of steroidogenic enzyme
expression, with induction of CYP11B1 and CYP19 and downregulation of DAX-1. Treatment of adrenocortical cells
during infection with hydrocortisone and aminoglutethimide increased viral yield. Finally, evaluation of adrenal gene
expression profile by microarray analysis showed that AD169 infection induced genes promoting cell proliferation and
tumor invasiveness and repressed tumor suppressor genes, pro-apoptotic, and anti-angiogenetic genes, in agreement
with the oncomodulatory effects of HCMV infection. In conclusion, these results demonstrate for the first time that
HCMV productively infects human adrenocortical cells, stimulates cortisol and estrogen production by modulation of
steroidogenic enzyme expression, and changes cellular gene expression profile toward a condition of proliferation and
stress response.
HSV-1 INDUCES DYSREGULATION OF MONOCYTE ANTICANDIDA FUNCTIONS
C. Cermelli*, C. Orsi*, L. Fantoni**, E. Lugli**, E. Blasi*
Departments of: * Public Health Sciences and ** Biomedica Sciences, University of Modena and Reggio Emilia, Via
Campi 287, 41100 Modena, Italy
Clinical cases of double infections by fungi and viruses are increasing, especially in immunocompromised hosts. To
date, the biomolecular events characterizing the outcome of polymicrobic diseases remain poorly investigated: little is
known on the mutual interactions occurring between pathogens and on their concomitant, either synergistic or
antagonistic, effects. In order to investigate the interplay occurring between pathogens in the course of double infections,
we set up an in vitro model in which the monocytic cell line THP-1, was infected with HSV-1 and then exposed to
Candida albicans. The effects of HSV-1 infection on macrophages were measured as capability to alter macrophagemediated effector functions, namely phagocytosis and killing of Candida, and as gene and protein expression by FACS
and RNA microarrays.
Phagocytosis of Candida by THP-1 cells was significantly increased when macrophages were infected with HSV-1.
Conversely, antifungal activity was impaired in HSV-1 infected macrophages at 6 hours after infection with Candida.
FACS analysis of protein expression revealed a significant downregulation of TLR2 and TLR4, important molecules
involved in fungi recognition and increase in the number of apoptotic cells. Gene expression profile disclosed a huge
decrease in gene presence in HSV-1 infected macrophage (40% of gene presence in uninfected cells vs 20% in infected
cells). The analysis of gene clusters showed a downregulation of genes involved in opsonized phagocytosis and
intracellular killing, of many adhesion molecules and of TLR2; on the contrary, several lectin receptor genes (involved
in Candida adhesion and phagocytosis) and apoptosis genes were significantly up-regulated.
58
What’s the clue in the progression of liver damage during HCV-infection? Analysis of the
role of HCV NS5A and Core proteins.
M. Marcolongo1, S. Mirandola1, S. Realdon1, L. Franceschini1, C. Frezza1, F. dal Pero1, M. Gerotto1, D. Bison11, G.
Bortoletto, L. Scorrano1 and A. Alberti1,2
1
VIMM- Venetian Institute of Molecular Medicine, Padova, Italy;
2
Department of Clinical Experimental Medicine, University of Padova, Padova, Italy.
In patients with HCV infection, we recently demonstrated that development of liver steatosis is associated to inhibition
of the Microsomal-triglycerides-transfer-protein (MTP). Other studies, suggest that the expression of some HCV
proteins (including Core and NS5A) can affect the host lipid metabolism. The mechanisms causing fat accumulation
and progression of liver disease in HCV patients are poorly understood. Here, we studied in transiently transfected
human hepatoma cell lines, the localization and the association with the MTP of Core and NS5A proteins from HCV of
genotypes 1 and 3. Cells were transfected with plasmids coreG1, coreG3, NS5AG1, NS5AG3 and with h-MTP-flag and
treated for immunofluorescence using specific antibody for NS5A, Core and anti-flag.
Core proteins of both genotypes mainly localized on lipid droplets (showing frequently a macro-vescicular pattern in
cells with HCV-3). No co-localization was found between Core and hMTP. Both NS5A-1, 3 mainly localized in the
perinuclear space together with hMTP, exhibiting a net-like staining. Interestingly, NS5A-3 exhibited also
mitochondrial localization less visible in cells with NS5A-1.
The generation of macro-vescicular droplets by Core-3 and the association of NS5A with mithocondrial, suggest that
these proteins could have a role, in vivo, in promoting fibrogenesis by affecting apoptosis or by inducing lipid
accumulation in the infected cell.
Expression analysis of genes involved in lipid metabolism and MTP enzymatic assays in cells transfected with HCV
proteins of different genotypes will further elucidate the mechanisms at the basis of the progression of liver damage
related to steatosis.
Involvement of the E62EEE65 Nef Acidic Domain and TRAF Family Members on Signaling
Events Induced by treatment of Monocytes/Macrophages with HIV-1 Nef
Giorgio Mangino1, Zulema Percario1, Alessia Noto1, Giana Fiorucci2, Giovanna Romeo2,3, Matthias Geyer4, Elisabetta
Affabris1
1
Dept. of Biology, Univ. Roma Tre, 2Inst. of Mol. Biol. and Pathology, CNR, 3Dept. of Exp. Med. and Pathology, Univ.
La Sapienza, Rome, 4MPI für Mol. Physiologie, Abteilung Physikalische Biochemie, Dortmund
Nef is a virulence factor that plays multiple roles during the HIV replication. It regulates the cell surface expression of
critical proteins, TCR signaling and apoptosis, inducing proapoptotic effects in uninfected bystander cells and
antiapoptotic effects in infected cells. It has been proposed that Nef intersects the CD40 signaling pathway in
macrophages, leading to modification in the pattern of secreted factors that appear able to recruit and activate T
lymphocytes, rendering them susceptible to HIV infection. As TRAFs adaptor proteins mediate the CD40 signaling we
searched for TRAFs consensus binding sequences on Nef finding the A60QEEEE65 acidic sequence. Previously, we
reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces the rapid activation of
NF- B, MAPKs and IRF-3 as well as the synthesis and secretion of a set of cyto-/chemokines that activate STAT1, -2
and -3.Here we show that these effects depend on the integrity of the E62EEE65 acidic sequence of Nef. In addition,
silencing experiments performed using TRAF6 and TRAF2 siRNA suggest that the former and to a lesser extent the
latter are required for the Nef induced phosphorylation of STATs.
59
HPV infection in HIV positive subjects: 10 years experience of follow-up
Flavia Lillo1, Davide Ferrari2, Laura Galli3, Adriano Lazzarin3, Caterina Uberti Foppa3
servizio di Patologia Clinica e Microbiologia HSR G.Giglio Cefalù (PA)
2
Dept. Gynaecology and Obstetrics Università Vita Salute HSR, Milan
3
Dept. Infectious Diseases – Università Vita Salute HSR, Milan
1
At the gynaecologic service of the Infectious Diseases Department of HSR – Milan, from 1997 to date 580 HIV-1
positive women have been enrolled in a longitudinal study on HPV related cervical pathology. Women are monitored at
6-12 months intervals by gynaecologic visit, PAP test, cervical brush for HPV testing, and colposcopy with biopsy, if
needed. HIV related parameters are recorded (HIV-RNA load, CD4 cell count, ARV). Mean age at enrolment was 35.9
years (median 43.7, range 19-68). Baseline data are available for the whole population (N=580) while longitudinal a
mean follow of at list 5 yy (range 5-10.8) will be presented for 385 women. Baseline CD4 cell count was lower than
200/mm3 in 20% of women, between 200 and 500 in 45.5% and greater than 500 in 34.5%. Cytology was negative in
65.5%, LSIL in 23.5% and HSIL in 10.2%. Histology was negative in 61%, CIN-1 in 26.5% and CIN-2+ in 12.1%. On
890 couples of PAP+Histo samples the level of agreement was calculated obtaining a fair level of concordance
(K=0.475) for all level of lesion and high level of concordance (K=0.874) for high-grade lesions. Oncogenic HPV types
prevalence at baseline was 58.2%, either with (26.8%) or w/o low risk infections (31.4%). Overall, multiple infections
were 85.7%. A persistent HR-HPV infection was recorded in 39%, and was significantly related with low CD4 count at
baseline (p=0.037) and with lesion development (p<0.0001), independently from CD4 recovery due to ARV. HPV viral
load and mRNA expression have been evaluated to select the best predictive value for each parameter.
60
POSTERS
25
The effects of feline immunodeficiency virus on feline monocyte derived dendritic cells
infected by spinoculation
P.Mazzetti, G.Freer*, D.Matteucci, L.Bozzacco. F.Tarabella, V.Catalucci and M.Bendinelli.
Retrovirus centre and Virology section, Department of Experimental Pathology, University of Pisa
During HIV-1 infection, dendritic cells (DCs) can not only prime T cells against the virus but also transfer HIV-1 to T
cells. Feline AIDS is caused by feline immunodeficiency virus (FIV) and is considered a model for the human illness
because the two diseases have many features in common. Little is known the interaction of feline DCs with FIV,
therefore this study attempts to tackle such an issue. Infection of monocyte-derived DCs was attempted by spinoculation
with FIV strains Petaluma and M2. FIV Petaluma was rapidly released in the supernatants of both infected DCs and
activated T cells after spinoculation. We show that FIV Petaluma was produced by DCs by monitoring viral content in
supernatants of infected DCs, by intracellular staining for p25 and by showing its cytopathic effect. Although activated
T cells were better substrates for FIV replication, leading to prolonged viral shedding, both immature DCs and DCs
matured with lipopolysaccharide supported virus production, mostly during the first two days after infection. At later
times, FIV induced syncytium formation by DCs. As concerns the FIV receptors, feline DCs turned out to CD134negative and CXCR4 -positive, a phenotype compatible with permissiveness to FIV Petaluma.
Our results also suggest that maturation is not hampered by FIV infection and that virus infection per se does not induce
DC maturation. We also show that infected iDCs can efficiently transfer FIV to activated PBMCs. It is concluded that
feline DCs can be infected by FIV although infection does not appear to influence their functionality.
26
HSV-1 gH pre-transmembrane domain in Membrane Fusion and Virus Inhibition
Aikaterini Kampanaraki1, Mariateresa Vitiello1, Marina D’Isanto1, Annarita Falanga1, Stefania Galdiero2,
Massimiliano Galdiero1
1
Department of Experimental Medicine - II University of Naples Naples 80138, Italy; 2Department of Biological
Sciences, Division of Biostructures - University of Naples “Federico II”, and Istituto di Biostrutture e Bioimmagini –
CNR, Naples 80134, Italy
We have identified a putative membrane interacting domain preceding the transmembrane domain of Herpes simplex
virus type 1 (HSV-1) glycoprotein H (gH). Peptides derived from this region interact strongly with membranes, and
show a high tendency to partition at the interface. This region is predicted to bind the membrane interface adopting an
-helical structure. Peptides representing either the HSV-1 gH pre-transmembrane region with a different hydrophobic
at interface moment have been studied. The peptides derived from this domain of gH induce the fusion of liposomal
membranes, adopt an helical conformation in membrane mimetic environments and are able to inhibit HSV-1
infectivity. The pre-transmembrane region appears to be a common feature in viral fusion proteins of several virus
families, and such a feature might be related to their fusogenic function. The identification of membrane interacting
regions, which are capable of modifying the biophysical properties of phospholipids membranes, lends weight to the
view that such domains may function directly in the fusion process, and may facilitate the future development of HSV-1
entry inhibitors.
61
27
A Bipartite Nuclear Localization Signal Mediates Importin alfa/beta Targeting of the Human
Herpes Simplex Type 1 DNA Polymerase Catalytic Subunit pUL30 to the nucleus
Daniele Musiani‡, Gualtiero Alvisi‡§*, David A. Jans§ , and Alessandro Ripalti
From the ‡Dipartimento di Medicina Clinica Specialistica e Sperimentale, Divisione di Microbiologia, Universtià degli
Studi di Bologna, Bologna, Italia; the §Department of Biochemistry and Molecular Biology, Monash University,
Clayton (Vic), Australia; the ARC Centre of Excellence for Biotechnology and Development; and the Azienda
Ospedaliera Universitaria di Bologna Policlinico S. Orsola–Malpighi, Dipartimento di Ematologia, Oncologia e
Medicina di Laboratorio – Unità Operativa di Microbiologia, Bologna, Italia
Although the 1235 amino acid Human herpesvirus 1 (HSV-1) DNA polymerase catalytic subunit, pUL30, is essential
for HSV-1 replication in the nucleus of host cells, little information is available regarding its nuclear import mechanism.
The present study addresses this issue directly, characterizing pUL30’s nuclear import pathway for the first time using
quantitative confocal laser scanning microscopy (CLSM) on living cells, and fluorescent binding assays. In addition to
a previously described nuclear localization signal (NLS) located within pUL30 binding site for the polymerase
accessory protein (PAP) pUL42, that appears to be dispensable for nuclear targeting, pUL30 possesses three putative
basic NLSs. Intriguingly, the core of pUL30-NLS2 (residues 1114-1120) is highly homologous to that of the recently
described NLS, similarly located upstream of the PAP binding site, of the human cytomegalovirus (hCMV) DNA
polymerase catalytic subunit, pUL54. Here we show for the first time that pUL30-NLS2 itself is only partially
functional in terms of nuclear import due to residue P1118 present in position 3 of the NLS core. Intriguingly, pUL30NLS2 together with pUL30-NLS3 (residues 1133-1136) represents a fully functional bipartite NLS (pUL30-NLSbip),
able to confer nuclear localization on heterologous proteins by conferring high-affinity interaction with the importin
(IMP) a/b heterodimer. Since nuclear targeting of HSV-1 proteins forming the replication fork is crucial for viral
replication, the pUL30-NLSbip emerges for the first time as a viable therapeutic target.
28
HPV genotyping in women attending at a second level setting for cervical cancer prevention
Comar M., D’Agaro P., Dal Molin G., Zanotta N., Campello C.
Hygiene and Preventive Medicine Institute, Department of Public Medicine Sciences, University of Trieste-Burlo
Garofolo, Trieste, Italy
Sensitive and specific molecular techniques that detect HPV DNA and distinguish high-risk HPV (HR-HPV) from lowrisk HPV (LR-HPV) types have been adjuncted to cytology. Detection of HR-HPV types can improve triage and
follow-up of patients during the second level prevention for cervical cancer. The aim of this study was to evaluate the
prevalence of HPV infection and genotypes distribution in a series of women attending a second level gynecology
structure.
Cytological specimens from 356 women affected by cytological lesions of different grade of severity and classified by
the Bethesda system were investigated. The median age of the women was 44 years (range 17 to 79 years).
HPV detection and genotyping were performed by linear array HPV genotyping test (Roche) following manufacturer’s
instructions. The overall HPV prevalence was of 43,5%. HR-HPV represented the 82%. Among HR-HPV genotypes,
HPV 16 was the most frequent (50,4%), followed by HPV-18 (19,7%); multiple infections were detected in 16,4%.
Distribution of HPV genotypes according to cytological classification was available for 247 patients:
Citology
NEG
ASCUS
LSIL
HSIL
Ca
Totale
HPV pos %
42 (33,6)
5 (33,3)
31 (57,4)
41 (83,7)
3 (100)
122 (49,4)
HPV-LR %
1 (2,4)
10 (3,1)
11 (3,7)
22/122 (18)
HPV-HR %
41 (97,6)
5 (100)
21 (67,7)
30 (73))
3 (100)
104/122 (85,2)
HPV-16 %
26 (63,4)
4 (80)
6 (28,6)
22 (73,3)
1 (33,3)
58/104 (55,8)
In particular, in normal women the prevalence of HR-HPV was estimated to be 97,6% (41/42): 17 of them resulted
positive after a previous treatment and 31 out of 42 were more than 35 years old, suggesting a long lasting infection.
Our data outline the high presence HR-HPV in patients attending a second level clinical setting, identifying women at
risk for malignant lesions. Specifically, also in presence of a normal cytology, virological data could add useful
information for the clinical follow-up.
62
29
Implications of HIV-1 Tat and Nef in B cell functions and dysfunctions in a preclinical model
of HIV-1 infection
Hammer D., Maggiorella M.T., Federico M., Titti F. and Ensoli B.
National AIDS Center, Istituto Superiore di Sanità, Rome, Italy
HIV infection is associated with a number of B cell dysfunctions comprising increased cell activation, cell turnover and
the number of cells secreting Ig spontaneously, polyclonal expansion, production of IL-6 and poor proliferative
response.
Nef released by infected cells enters naïve B cells inducing activation, polyclonal expansion potentially associated with
lymphoproliferative disease. Tat promotes B cell activation and expansion through increased release of IL-6 and IL-10.
Both Tat and Nef induce apoptosis in bystander cells through FasL upregulation.
The aim of this study is to characterize the quantity and quality of B cell responses in the non human primate model
following SHIV infection and to evaluate the in vitro effects of Tat and Nef on primary and on in vitro established
HVFM-1-transformed simian B cell lines. Therefore the cell surface marker and cytokine/ chemokine expression are
determined, likewise the in vitro production of antibodies. The stimulation, clonal expansion and immortalization of B
cells is also part of this study, followed by the analyses of the Ig repertoire in the immortalized B cells. The effects of
Tat and Nef are evaluated on B cells, but also the effect of these HIV-1 derived proteins on the susceptibility of B cells
to HIV-1 infection.
The characterization of the Tat and Nef interactions with B cells and their effects on the dysregulation or diversion of
the immune system might play a major role in the generation of both a preventive and therapeutic intervention against
HIV/AIDS.
(Grant “National AIDS Programme” from Ministry of Health)
30
HIV-1 matrix protein p17 prevents loss of CD28 expression during IL-2-induced maturation
of naïve CD8+ T cells.
Manuela Avolio, Sonia Caracciolo, Luana Vollero, Giorgio Tosti, Simona Fiorentini, and Arnaldo Caruso.
Department of Experimental and Applied Medicine, Section of Microbiology, University of Brescia Medical School,
Brescia, Italy.
Naïve CD8+ T cells differentiate into effectors secreting various cytokines that modulate immune functions. A striking
finding for most HIV-1-infected patients, is that they accumulate CD8+ T cells belonging to early and intermediate
differentiated elements. Structural HIV-1 proteins and, among these, the matrix protein p17, have been associated with
loss of functional competence by different immune cells. We therefore evaluated the influence of p17 on naïve CD8+ T
cell activation and maturation. Anti-CD3 mAb preactivation and subsequent IL-2 stimulation are able to drive human
naïve CD8+ T cells to an effector phenotype characterized, among other features, by down-regulation of the
costimulatory molecule CD28. Strikingly, however, IL-2-induced down-modulation of CD28 was completely prevented
by p17 and cells derived from p17-stimulated cultures showed a strong Tc1 polarization which was four fold higher
than that observed in IL-2-stimulated cultures. Moreover, p17 preserved a markedly high proportion of CD8+ T cells
able to respond to a CD28 triggering with a proinflammatory cytokine storm. Our evidences suggest that p17 induces
important effects on cytokine polarization and phenotype of terminally differentiated CD8+ T cells, and that new p17based therapeutic approaches could control or prevent HIV-1-related immune disorders.
63
31
HIV-1 accessory protein Nef and cellular GTPase Dynamin 2 interaction sites
Ilaria Frasson1, Sara Richter1, Elena Calore1, Arianna Loregian1, Arianna Calistri1, Massimo Pizzato2, Cristina
Parolin1 and Giorgio Palù1
1
Dept. of Histology, Microbiology and Medical Biotechnologies, University of Padova, Italy and 2Virology/Molecular
Virology, Division of Medicine, Imperial College, London, UK
Nef is a virulence factor of HIV-1 and other primate lentiviruses that is crucial for rapid progression to AIDS. In cell
culture, Nef increases the infectivity of HIV-1 progeny virions by an unknown mechanism. Dynamin 2 (Dyn2) is a key
regulator of vesicular trafficking, clathrin mediated endocytosis, actin nucleation and cytokynesis. It is a 98 kDa
intracellular and ubiquitary GTPases protein belonging to the dynamin superfamily. It is present in four splice variants,
named AA, AB, BA and BB. It has recently been shown that Dyn2 is a binding partner of Nef that is required for its
ability to increase viral infectivity. Dominant-negative Dyn2 or the depletion of Dyn2 by small interfering RNA
potently inhibited the effect of Nef on HIV-1 infectivity. Furthermore, in Dyn2-depleted cells, this function of Nef
could be rescued by ectopically expressed Dyn2 but not by Dyn1, a closely related isoform that does not bind Nef. The
aim of this study was to identify the significant interaction sites on both Dyn2 and Nef proteins. This was achieved by
setting up a direct yeast two hybrid assay that visualized HIV-1 Nef/Dyn2 interaction. By performing targeted
mutations on the two proteins, we were able to identify the main domains of Dyn2 and specific aminoacids of Nef
relevant/irrrelevant to the interaction.
32
Analysis of Nef involvement in the development of HIV-1 Associated Dementia.
Valeria Bergonzini, Arianna Calistri, Cristiano Salata, Claudia Del Vecchio, Cristina Parolin1, Giorgio Palù
Department of Histology, Microbiology and Medical Biotechnologies and 1Department of Biology, University of
Padova
HIV-1 enters the brain at an early stage after systemic infection and resides primarly in macrophages/microglia and
astrocytes. At a late stage of the disease 15-20% of the patients develop the HIV-1 associated Dementia (HAD). A
variety of HIV-induced lesions of the central nervous system (CNS) have been described, including neuronal loss, but
very little is known about the mechanisms that lead to neuronal loss.
Among HIV-1 proteins, Nef is a 27kDa N-myristoylated accessory protein expressed early and abundantly during
infection, and, in particular, present in persistently HIV-1-infected astrocytes in vivo and in vitro in rapidly progressive
dementia. Furthermore, the neuropathogenicity of Nef has been demonstrated in in vivo models where HIV-1 strains
characterised by different Nef can penetrate the CNS and cause damage. We focused on the involvement of Nef in the
regulation of Matrix MetalloProteinases, which can contribute to explain the Blood-Brain Barrier (BBB) damage
present in HAD patients. In particular, we analised the cytokines/chemokines pattern expression that could contribute to
the inflammatory and damage process present during HAD. To elucidate the molecular basis that lead to this process,
we focused on the signaling pathways that Nef could alter, such as Stat-3 and MAP-K1/2, which are important in the
CNS for neuronal differentiation program and maintenance of the differentiated phenotype. Furthermore, we cloned Nef
in a lentiviral vector in order to transduce fully differentiated neuronal cells to evaluate the possible alterations induced
by Nef in the expression of neuronal markers. Our data could contribute to the dissection of molecular basis of HAD.
64
33
Patterns of gene expression exhibited by the vOX2 (orfK14) protein of human herpesvirus 8
in PBMC-derived macrophages
Matteo Curtarello, Cristiano Salata, Alessandra Comin, Arianna Calistri, Marta Trevisan, Luisa Barzon, Cristina
Parolin*, Giorgio Palù
Department of Histology, Microbiology and Medical Biotechnologies;* Department of Biology; University of Padova
Kaposi’s sarcoma is an inflammatory cytokine-mediated angioproliferative disease triggered by infection by human
herpesvirus 8 (HHV8). This virus is unique because of its extensive molecular piracy of critical cell regulatory and
immune modulatory genes. HHV8’s orfK14 encode a surface glycoprotein (vOX2) of the immunoglobulin superfamily,
significantly homologous with the broadly distributed cellular OX2. OX2 delivers an inhibitory signal for macrophages
through the binding to a specific receptor whose distribution is restricted to this cell type. OX2 acts locally by
downmodulating the inflammatory response. Several reports suggest an immunosuppressive activity of the vOX2 in
basophil and neutrophil cells while in macrophages, litically infected by HHV8 in vivo and infiltrating KS lesions, the
effect of vOX2 is still controversial.
In order to clarify the vOX2 function in viral biology and pathogenesis, expression profiling of cellular genes in PBMCderived macrophages was performed using human gene arrays. Primary coltures have been transduced by a lentiviral
vector expressing orfK14 and evaluated by FACS analysis for vOX2 detection. Preliminary results with microarray
analysis showed that a large number of genes involved in inflammation, host defense, signal transduction and cell
growth were up-regulated in vOX2-expressing macrophages. However, an opposite outcome was observed into IFN activated transduced cells. In addition, a real-time PCR analysis performed on a set of cytokines, critics in KS
development, showed a positive modulation in vOX2-expressing macrophages. Experiments to confirm these
observations are ongoing, as well as the evaluation of secreted cytokines by ELISA. Our results will provide valuable
insights into the molecular mechanism of KS tumorigenesis.
34
Use of the Murine Cell Line RAW 264.7 to Study the Effect on Cell Signaling Induced by
Treatment of Monocytes/Macrophages with Recombinant HIV-1 Nef
Valeria Serra1, Giorgio Mangino1, Zulema Percario1, Matthias Geyer2, Elisabetta Affabris1
Dept. of Biology, University Roma Tre, Rome, 2MPI für Molekulare Physiologie, Abteilung Physikalische Biochemie,
Dortmund
1
The viral protein Nef is a virulence factor that plays multiple roles during the HIV replication cycle. Nef expression
induces downregulation of CD4 and MHC-I, alteration of TCR signaling, proapoptotic effects in uninfected bystander
cells and antiapoptotic effects in infected cells. In cells of the monocytic/macrophage lineage Nef expression induces
the production of factors able to recruit T lymphocytes making them susceptible to HIV replication. We previously
reported that Nef treatment of primary human monocyte-derived macrophages (MDMs) induces a rapid activation of
NF- B, MAPKs and IRF-3 that is followed by the production of a set of cyto-/chemokines, including IL-1 , IL-6,
TNF , IFN , MIP1 /CCL3 and MIP1 /CCL4, that activate STAT1, -2 and -3. It is also well established that transgenic
Nef expression in mice leads to the development of an AIDS-like syndrome. For these reasons we sought to
characterize the Nef-induced effects also in monocytic/macrophage murine cell lines. Results obtained on RAW 264.7
show that also in murine macrophages recNef treatment allows the activation of NF- B, MAPKs and IRF-3, the release
of IFN and tyrosine phosphorylation of STAT1 and STAT2
65
35
Type III and I interferons increase HIV uptake and replication in human cells, that
overexpress CD4, CCR5 and CXCR4
Caterina Serraa, Adriana Biolchinia, Alessandra Meia, Sergei Kotenkob, Antonina Doleia
Section of Microbiology, Department of Biomedical Sciences, 1 Center of Excellence for Biotechnology
Development and Biodiversity Research, University of Sassari, Sassari, Italy;
b
Department of Biochemistry & Molecular Biology, New Jersey Medical School, Newark, NJ, USA.
a
Objective: The newly discovered type III interferon (IFN ) has antiviral activity against a broad spectrum of
viruses, and potent immune-related activities. Its major producers are peripheral blood mononuclear cells (PBMC)
and dendritic cells. The above functions and cells are deeply involved in AIDS pathogenesis, but there are no
informations so far on IFN effects on HIV. Therefore we addressed the sensitivity of HIV-1 replication to cell
exposure to human IFN 2.
Design and Methods: human PBMC and C8166 T cells were treated with human Type III or Type I IFNs. We
investigated HIV-1 ability to bind and replicate in cells pre-treated with IFN . Virus amounts were quantified by
infectivity and p24 assays. In parallel we evaluated possible antiproliferative effects of IFN 2 and the expression
of CD4, CXCR4 and CCR5 genes, whose transcripts were quantified by real time RT-PCR.
Results: Increased adsorption of HIV to IFN-treated cells was observed, in a dose-dependent fashion. Virus yields
increased accordingly. In both systems the accumulation of CD4, CXCR4 and CCR5 transcripts was increased,
particularly in PBMC. Antiproliferative activity and classical antiviral state were instead detected on PBMC, but
not on C8166 cells.
Conclusions: Pre-treatment of PBMC and C8166 cells with Type III and Type I IFNs causes increased HIV
binding and replication. These effects are likely to be due to increased expression of HIV receptors and co-receptors
on the plasmamembrane. These findings indicate another mechanism utilized by HIV for subversion of host
defences.
36
Longitudinal study of HERV-W/MSRV circulation in MS patients undergoing IFN therapy.
GIUSEPPE MAMELI1, MASSIMILIANO CASTELLAZZI2, VITO ASTONE1, CATERINA SERRA1, LUCIANA PODDIGHE1,
ENRICO GRANIERI2, AND ANTONINA DOLEI1
1
Section of Microbiology, Department of Biomedical Sciences, University of Sassari, Viale San Pietro 43/B, I-07100
Sassari; 2Institute of Clinical Neurology, University of Sassari, Viale San Pietro 10, I-07100 Sassari, Italy
2
Multiple Sclerosis Center, Department of Neurology, University of Ferrara,
Multiple sclerosis (MS) is associated with destruction of axons and consequent neuronal cell death that leads to
progressive clinical disability of patients. So far no prognostic marker have been identified, really useful for the
individual patient. The aetiopathogenesis of MS is complex and debated. Immunopathogenic phenomena are thought to
be triggered by environmental (viral?) factor(s) operating on a predisposing genetic background. Among the viruses
suggested as MS co-factors, is the MS-associated retrovirus (MSRV). It is a member of the HERV-W multi-copy family
of human endogenous retroviruses (HERV), whose links with some human diseases are increasingly observed.
Interferon-beta (IFN- ) is an immunomodulatory agent that has been shown to be beneficial in the therapy of patients
with relapsing-remitting MS (RRMS). We have shown in the past that IFN
is a powerful inhibitor of MSRV/HERVW release in culture (Serra et al. 2003).
Based on this premise, we made a longitudinal study to monitor the presence and viral load of extracellular MSRV in
blood of patients under IFN treatment, by using real time RT-PCR. The study has been conducted over a period of
12 months in group of eleven patients (6 females, 5 males, mean age 33.9± 8,6 years), who satisfied the diagnostic
criteria for RRMS. Every three months the patients were clinically evaluated and plasma samples were collected.
Our data show that at study entry (Time 0) MSRV positivity and viral load were related to the previous duration of
disease. After three months of treatment (Time 1), MSRV copy numbers in plasma were barely detected. This strong
inhibition of MSRV production remained stable throughout the twelve months of the observation period, with the
exception of one patient, who, from time Time 2 onward, showed increasing virus production up to his pre-therapy
values. Clinical evaluation indicated that he had an exacerbation and strong progression in the disability scale.
We concluded that MSRV detection can be considered as a marker of disease progression and sensitivity to therapy.
Work supported partly by grants from Fondazione Italiana Sclerosi Multipla Onlus (grant No. 2005/R/11),. G.M. was
supported by a training research No. 2005/B/2 fellowship FISM (Fondazione Italiana Sclerosi Multipla). Ministero
Università e Ricerca PRIN 2005-
66
37
Persitence and tissue distribution of parvovirus B19 genotypes
K.Zakrzewska, F. Corcioli, A.Rinieri, A.Azzi
Department of Public Health - University of Firenze
Parvovirus B19 persistent infections have been described in immunocompromised patients. In addition, parvovirus B19
seems to be able to persist also in immunocompetent individuals. However, the frequency, the mechanisms and the
clinical outcome of this fenomenon in immunocompetent subjects are unknown so far. The aim of this study was to
assess the prevalence of parvovirus B19 persistent infections in immunocompetent healthy individuals, or in
individuals without diseases attributable to parvovirus B19 infection. At this purpose, 117 tissue bioptic samples (38
bone marrow samples, 38 skin biopsies, 30 synovial biopsies, 11 cardiac autoptic samples), plus 97 sera obtained from
the same group of patients, have been examined for the presence of DNA sequences of the three genotypes of
parvovirus B19. Two consensus PCR were used in order to amplify sequences of all the three genotypes. For
genotyping, the restriction pattern of the consensus PCR products and two genotype specific PCRs have been employed,
followed by sequencing in doubtful cases. Altogether, B19 DNA sequences were present in 63 out of 117 tissue
samples and in 4 out of 97 sera. Genotype 2 was more frequently demonstrated than genotype 1 and genotype 3 was
rarely shown. The high prevalence of parvovirus B19 persistence in tissues from healthy individuals should be
considered when studies are performed in order to establish a new association of parvovirus B19 infection with a
disease of unknown aetiology.
38
HUMAN BOCAVIRUS IN ITALIAN PATIENTS WITH RESPIRATORY DISEASES
Fabrizio Maggi1, Elisabetta Andreoli1, Jara Rocchi1, Letizia Lanini1, Valentina Ricci1,Melania Albani1, Massimo
Pifferi2, Maria Linda Vatteroni1, Mauro Pistello1, Luca Ceccherini Nelli1, and Mauro Bendinelli1
1
Virology Section and Retrovirus Center, Department of Experimental Pathology, 2Department of Pediatrics, University
of Pisa, Pisa, Italy
This study examined for the presence of human bocavirus (hBoV) 335 respiratory specimens obtained in Pisa, Italy
over a period of 7 consecutive years. Two hundred specimens were nasal swabs from infants hospitalized for acute
respiratory tract infections. The overall rate of hBoV detection in these specimens was 4.5% and varied slightly from
year to year, except for the period 2000-2002 when none of the 43 nasal swabs examined was positive. At a difference
with other respiratory viruses, no seasonal variations in hBoV incidence were noted. The infants in whom hBoV was
detected had either bronchiolitis or bronchopneumonia and in 4/9 cases yielded no other viral pathogen. Eighty-four
specimens were nasal swabs or bronchoalveolar lavages from adults with pneumonia, bronchopneumonia, or asthma.
Only one of these samples tested hBoV positive. Finally, none of 51 nasal swabs obtained from healthy pediatric
subjects tested hBoV-positive. Collectively, these finding support the notion that hBoV is a significant cause of ARD in
infants.
67
39
Human Herpesvirus 8 acute infection of endothelial cells induces monocyte chemoattractant
protein 1-dependent capillary-like structure formation
Simona Fiorentini1, Elisabetta Caselli2, Carla Amici3, Luana Vollero1, M. Gabriella Santoro3, Dario Di Luca2, Arnaldo
Caruso1
1
Department of Experimental and Applied Medicine, Section of Microbiology, University of Brescia, Brescia, Italy;
2
Section of Microbiology, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy;
3
Department of Biology, University of Rome Tor Vergata, Rome, Italy.
Human herpesvirus 8 (HHV-8) is considered the causative agent of Kaposi’s sarcoma, a highly vascularized neoplasm
characterized by spindle-shape cells of endothelial origin and inflammatory cell infiltration. HHV-8 cell-transforming
ability has been associated with HHV-8 induction of angiogenesis but molecules involved in this phenomenon are not
yet well characterized. This study was undertaken to investigate the role played by Monocyte chemoattractant protein
1 (MCP-1) in mediating HHV-8-induced angiogenesis. Using a recently established in vitro model of HHV-8 infection
in endothelial cells (ECs), we have shown that HHV-8 infection rapidly and selectively trigger the production of high
levels of MCP-1 which is accompanied by a MCP-1-mediated enhancement of ECs ability to form capillary-like
structures. In fact, in a basement membrane extract morphogenesis assay, we observed that HHV-8 infection led to a
dramatic rearrangement of ECs as early as 3h after seeding that could be abolished by the addition of anti-MCP-1
antibody. HHV-8-induced MCP-1 expression was then efficiently (>80%) silenced by transfecting ECs with small
interference RNA. Again, when MCP-1-silenced ECs were used, the ability of infected ECs to form tubes was inhibited,
whereas the capacity of uninfected cells to form capillary-like structures was not altered. These results suggest that
HHV-8 make infected ECs more susceptible to the angiogenic activity of endothelial MCP-1. The HHV-8-induced
chemokine may therefore play a dual role in promoting inflammation and pathogenic angiogenesis typical of virusassociated lesions by recruiting macrophages and other effector cells to the site of infection as well as by directly
stimulating vascular remodeling.
68
Giudelines for human Cytomegalovirus
infections
SIV guidelines for the preemptive (presymptomatic) therapy of human cytomegalovirus
infections in transplant recipients
Giuseppe Gerna, Daniele Lilleri
Servizio di Virologia, Fondazione IRCCS Policlinico San Matteo, Pavia
Human cytomegalovirus (HCMV) infections are major infectious viral complications of the post-transplant period in
both solid organ (SOTR) and hematopoietic stem cell transplant recipients (HSCTR). Some HCMV infections progress
to HCMV disease, while some patients are able to resolve the infection spontaneously. The preemptive
(presymptomatic) therapy is aimed to treat only patients reaching a predetermined level of viral load in blood, while
patients resolving spontaneously the infection (and not reaching the predetermined level of viral load) are not treated
with antiviral drugs. In order to differentiate the 2 groups of patients, threshold or cutoff levels of viral load in blood
must be predetermined. Following a sustained period of preemptive therapy performed on the guidance of pp65
antigenemia assay, and a prospective study in which antigenemia and DNAemia cutoffs were compared in SOTR, it has
been shown that a cutoff of 300,000 HCMV DNA copies/ml whole blood, compared with antigenemia cutoff (100
pp65-positive cells/2x105 peripheral blood leukocytes): 1) significantly reduces the number of patients requiring
treatment; 2) may be safely adopted to guide preemptive therapy of both primary and reactivated HCMV infections; 3)
does not significantly modify the overall duration of antiviral treatment. In parallel, a prospective study conducted in
HSCTR (young patients), comparing use of a DNAemia cutoff of 10,000 DNA copies/ml with first confirmed
antigenemia positivity (qualitative antigenemia), has allowed to conclude that the number of patients treated was
significantly lower in the DNAemia arm, thus avoiding unnecessary antiviral treatment and permitting reduction in
treatment-related costs and toxicity. In both SOTR and HSCTR use of DNAemia cutoff did not interfere with patients
morbidity and mortality. In conclusion, following standardization of method for DNAemia quantification, use of the
above reported DNAemia cutoff could be extended (transferred) to all transplantation centers.
69
Emerging and Zoonotic Viral Infections
71
Emerging Respiratory Infections in Hematopoietic Stem Cell-Transplanted Children in
Germany
V. Falcone (1), B. Huck (1), G. Peyerl-Hoffman (2), W. Kern (2), T. Schenk (1), U. Kontny (3) and D. Neumann-Haefelin
(1).
(1) Department of Virology , (2) Department of Medicine, and (3) Center for Pediatrics and Adolescent Medicine,
Freiburg University Medical Center, Freiburg, Germany
Respiratory viruses represent a frequent cause of morbidity in immunocompromised patients. New viruses, such as
human metapneumovirus (HMPV), human bocavirus (HBoV) and human coronavirus NL-63 (HCoV-NL63), have
recently been suggested as etiology of acute respiratory tract infections. The aim of our study was to investigate the
relative contribution of these newly described agents to morbidity in haematopoietic stem cell transplant (HSCT)
recipients. 135 samples obtained from 69 symptomatic children (mean age 8.5 years; 53% presenting with symptoms of
upper respiratory tract infection, 19.6% with tracheobronchitis, 14 % with pneumonia and 7.1% requiring artificial
ventilation), were evaluated by real-time PCR for the presence of HMPV, respiratory syncytial virus (RSV), influenza
A and B (Flu A and B), rhinovirus (RV), parainfluenza 1-3 (PIV1-3), and adenovirus (ADV). Moreover, they were
retrospectively analysed for HBoV DNA and HCoV-NL63 RNA. A total of 66 (48.8%) of the analysed samples were
found positive for one of the viral agents. In particular, 16 (11.9%) ADV-, 15 (11.1%) RV-, 7 (5.2%) HBoV-, 4 (3%)
RSV-, 4 (3%) Flu A and B -, 4 (3%) PIV1-3-, 4 (3%) HCoV-NL63-, and 3 (2,2%) HMPV- positive samples were
detected. Pneumonia was diagnosed in 2 HBoV-, 1 HMPV-, 2 Adeno-, and 3 RV-positive children. Partial sequencing
and phylogenetic analysis of the HMPV, HBoV and NL-63 amplicons was also performed. Our results confirm the
occurrence of HMPV infection, although at low incidence, among HSCT recipients, as previously described (Huck et al.
J Clin Microb. 2006, 44:2300-2303). Moreover, they suggest a role for HBoV and HCoV-NL63 in the etiology of
respiratory tract infections in these patients.
Characterization of the biological properties of human and rhesus VP8* proteins
Cappuccini F., Monini M., and Ruggeri F.M.
Dipartimento di Sanità Alimentare e Animale
Istituto Superiore di Sanità V.le Regina Elena 299, 00161 Roma
Rotavirus is the most common cause of severe, dehydrating diarrhea in infants and young children worldwide,
producing high childhood mortality in developing countries. The VP8* subunit, corrisponding to the N-terminal trypsin
cleavage product of VP4, has been found to play a significant role in viral infectivity and neutralization of the virus.
Rotavirus VP4 has been shown to dimerize and to form spikes projecting from the viral surface. VP8* binds to sialic
acid on erythrocytes and is responsible for hemagglutination (HA). Although VP8* has been postulated to be one of the
virus binding proteins, a direct evidence of binding to cells is still missing.
We expressed VP8* from the human Wa and the rhesus RRV rotavirus strains, as fusion proteins with a polyhistidine
tag in Escherichia coli. This system provided an excellent tool to investigate VP8* properties. The biological
characteristics of the expressed peptides were compared to the native proteins assembled in the virus, using antigenbased assays with hyperimmune polyclonal sera and monoclonal antibodies. The soluble rVP8* proteins were tested for
their ability to bind to human 0 type erythrocytes, in HA and hemagglutination inhibition (HI) assays with specific
antisera raised against RRV and Wa viral particles. Moreover, to investigate possible binding of expressed VP8* to the
permissive MA-104 cells, nickel-coated magnetic beads were labelled with rVP8* peptides via their his tag. The ability
of rVP8*-beads to specifically bind eukaryotic cells in suspension was evaluated after magnetic separation of cell-beads
complexes, through observation and count of live cells by light microscopy.
73
Detection of norovirus in a captive lion cub with haemorrhagic enteritis (Panthera leo).
Martella Vito1, Campolo Marco1, Lorusso Eleonora1, Cavicchio Paolo2, Camero Michele1, Bellacicco Anna Lucia1,
Decaro Nicola1, Greco Grazia1, Corrente Marialaura1, Desario Costantina1, Arista Serenella3, Bányai Krisztián4,
Koopmans Marion5, Buonavoglia C1.
1
2
Giardino Zoologico di Pistoia, via Pieve a Celle 160, 51030 Pistoia
3
Department of Hygiene and Microbiology, University of Palermo, Palermo, Italy
4
Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Pécs, Hungary
5
Laboratory for Infectious Diseases and Screening, National Institute of Public Health and the Environment, Bilthoven,
The Netherlands
New pathogens may readily emerge via interspecies transmission and spread to other animal species and to humans.
These issues have prompted the attention of researchers worldwide to identify readily new biological risks for animal
and human health. Lions (Panthera leo) are susceptible to viral diseases of domestic carnivores, including feline
parvovirus, retroviruses, calicivirus and herpesvirus and canine distemper virus. Here we report the identification in
lions of a novel calicivirus, related genetically to human noroviruses (NoVs). The novel lion NoV was identified in a
cub died from severe hemorrhagic enteritis in the Zoo of Pistoia, Italy, in May 2006. By analysis of the capsid gene
(ORF2), the lion NoV, strain 387/06, appeared to be related genetically to human GGIV NoVs (69.3-70.1% aa identity
in the capsid protein). NoVs are considered the major cause of epidemic, non-bacterial gastroenteritis worldwide in
humans of all age groups. The viruses are highly contagious and are transmitted by direct contact or by contaminated
water and food. The identification of a GGIV strain of animal origin provides additional support to the evidence that the
evolution of human NoVs is intermingled with that of animal NoVs and warrants studies to investigate the ecology of
this novel virus in large felids, domestic carnivores and humans.
F. Carletti1,C. Castilletti1, L. Bordi1, C. Gioia1, M.S. Zaniratti1, L. Falasca1, L. Viale2, A. Beltrame2, M. Crapis2, G.
Ippolito1, M.R. Capobianchi1
1
National Institute for Infectious Diseases "L.Spallanzani", Rome, Italy, 2Clinic of Infectious Diseases, University of
Udine, Udine, Italy
Background: Poxviruses are a family of large, dsDNA viruses that recently have received increased attention, due to
fear of bioterrorist attacks and zoonotic transmission. Few data are available on European orthopoxvirus strains
involved in zoonotic events. We report a case of human orthopoxvirus zoonosis in Northern Italy.
The clinical case: In January 2006, an Italian male scratched by a diseased cat presented at the infectious disease clinic
with a suspected orthopoxvirus (OPV) lesion on the right hand, accompanied by systemic symptoms such as moderate
fever and malaise.
Methods: Viral DNA was detected by pan-orthopoxvirus-specific real-time and classical PCR, targeting CrmB and
HA, respectively. Further characterization was carried out by RFLP analysis of CrmB and sequencing of full length
HA gene. Humoral immune response was analyzed by IF and microneutralization assays; cellular immune response was
established by intracellular detection of IFN-gamma after in vitro PBMC stimulation with both patient's isolate and
reference vaccinia virus.
Results: Orthopoxvirus infection was identified by electron microscopy, PCR and virus isolation. The diagnosis was
confirmed by detection of both cellular and humoral specific immune response. RFLP of CrmB and phylogenetic
analysis of HA were unable to conclusively identify the poxvirus species. In particular, the HA sequence of the new
isolate showed significant similarity to ectromelia, cowpoxvirus and camelpox viruses.
Conclusion: Enhanced diagnostic capability for orthopoxvirus infections, resulting from global awareness of
bioterrorism menace, will be helpful to identify new orthopoxvirus infections in animals and humans, and to improve
the knowledge on ortopoxviruses circulation in Italy.
74
POSTERS
40
Genotyping of GII.4 and GIIb Norovirus strains by PCR restriction analysis.
Stefania Ramirez1, Giovanni M. Giammanco1, Simona De Grazia1, Claudia Colomba2, Vito Martella3, Serenella
Arista1*.
1. Dipartimento di Igiene e Microbiologia, Università di Palermo, Italy; 2. Istituto di Patologia Infettiva e Virologia,
Università di Palermo, Italy; 3. Dipartimento di Sanità e Benessere degli Animali, Università di Bari, Italy;
Human noroviruses (NoV) have been classified into 3 genogroups and at least 25 genotypes, with an impressive number
of variants, but only a few strains (GGII.b and GII.4) within genogroup GGII appear to predominate worldwide. The
NoV ORF2, coding for the RNA dependent RNA polymerase (RdRp), is highly conserved and broadly-reactive primers
have been designed at the 3’ end of this region to detect almost all human NoV genotypes. Based on virtual restriction
fragment length polymorphism (RA) analysis of this short RdRp fragment (330 bp), the endonucleases XmnI, AhDI,
BstXI, and AcuI appeared suitable for correct identification of GIIb and GII.4 NoV genotypes with 92,4% accuracy.
Virtual application of such enzymes on 806 sequences of GI and GII NoV genotypes retrieved from the databases
confirmed that the RA assay was applicable as a rapid and useful method for characterization of NoV GIIb and GII.4
strains, regardless of the temporal and geographical variability existing among the various strains. The RFLP protocol
was able to characterise correctly a collection of NoV strains detected in children with sporadic acute gastroenteritis in
Palermo in the years 2002-2005.
41
PREVALENCE OF TBE VIRUS IN TICKS FROM AN ALPINE AREA IN FRIULI
VENEZIA GIULIA REGION
Pierlanfranco D’Agaro1, E. Martinelli1, F. Nazzi2, A .Iob3, I. Bernardinelli2, S. Del Fabbro2, M. Ruscio4, C. Campello1
Dept of Public Health Sciences, University of Trieste; IRCCS “Burlo Garofolo”, Trieste, Italy
2
Dept of Biology and Plant Protection, University of Udine, Udine, Italy
3
Azienda Servizi Sanitari n. 3 Alto Friuli – Dept of Prevention – Gemona del Friuli, Udine, Italy
4
Diagnostic Dept, Hospital of San Daniele, San Daniele del Friuli, Italy
1
In the Friuli Venezia Giulia region, in north-eastern Italy the first autochthonous TBE case was reported in 2001.
Subsequently, 4 infections were reported in 2003, 7 in 2004 and 13 both in 2005 and 2006. In addition, serologic
investigations on a population from a high risk area could demonstrate a seroprevalence rate as high as 11,7 %.
In 2005 and 2006 a study was carried out to evaluate the distribution of ticks in the area and to analyze the prevalence
of TBEV. Sixteen sites, located in the main valleys of the mountain area of the region, were selected for sampling.
Ixodes ricinus were sampled by dragging monthly from April to November. Collected ticks were classified, grouped by
sampling site, by date and by stage and stored at -80°C. Five to ten ticks were homogenised in a glass micro tissue
grinder and DNA/RNA was extracted using the QIAmp Viral RNA Mini Kit (Quiagen). RT nested PCR amplification
was performed with two sets of primers pairs directed to a 5’NCR and NS5 region as described by Suss et al. and
Puchhammer-Stockl et al.. The amplicons were sequenced with the Big Dye Terminator Cycle sequencing kit. 582 and
1897 ticks were collected in 2005 and 2006, respectively. One adults and three the nymphs resulted positive at PCR
analysis with a prevalence rate of 5.9/1000 and 1.7/1000, respectively. One sequences were identical to the Neudoerfl
strain, two differed from the Neudoerfl strain for one mutation and one was similar to the finnish Isosaari strain.
75
42
Genetic heterogeneity in the VP7 of group C rotaviruses detected in piglets with enteritis in
Italy
Martella Vito1*, Lorusso Eleonora1, Bányai Krisztián2, Decaro Nicola1, Bellacicco Anna1, Desario Costantina1,
Corrente Marialaura1, Greco Grazia1, Moschidou Paschalina1, Tempesta Maria1, Arista Serenella3, Cavalli
Alessandra1, Lavazza Antonio4, Buonavoglia Canio1
1
2
Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Pécs, Hungary
3
Department of Hygiene and Microbiology, University of Palermo, Palermo, Italy
4
Istituto Zooprofilattico Sperimentale di Lombardia/Emilia Romagna, Brescia – Italy
Evidence for a possible zoonotic role of group C rotaviruses (GCRVs) has been recently provided. To gain information
on the genetic relationships between human and animal GCRVs, we sequenced the VP7 gene of 10 porcine strains
detected during a large surveillance study from different outbreaks of gastroenteritis in piglets. Four GCRV strains were
genetically related to the prototype GCRV porcine Cowden strain. A completely new VP7 genotype included 4 strains
(344/04-7-like) that shared 92.5% to 97.0% aa identity to each other, but <83% to human GCRVs and <79% to other
porcine and bovine GCRVs. A unique 4-aa insertion (SSSV or SSTI), within a variable region at the carboxy-terminus
of VP7, represented a distinctive feature for these 4 unique strains. An additional strain, 134/04-18, was clearly different
from all human and animal GCRVs (<85% aa identity) and likely accounts for a distinct VP7 genotype. The VP7 of a
unique strain, 42/05-21, shared similar ranges of aa sequence identities with porcine and human strains (88.0-90.7% to
porcine GCRVs and 85.2-88.2% to human GCRVs). Plotting the VP7 gene of strain 42/05-21 against the VP7 of human
and porcine strains revealed discontinuous evolution rates throughout the VP7 molecule, suggesting different
mutational pressure rather than a remote intragenic recombination event. These findings provide the need for future
epidemiological surveys and warrant studies to investigate the pathogenic potential of these novel GCRVs in pigs.
43
Surveillance for Canine Distemper Virus (CDV) in 2005-2006 in Italy reveals a change in the
epidemiology
Lucente Maria Stella1, Martella Vito1, Cirone Francesco1, Buonavoglia Domenico1, Lorusso Eleonora1, Lorusso
Alessio1, Elia Gabriella1, Buonavoglia Canio1
1
CDV is a highly contagious viral pathogen causing a lethal systemic disease in dogs and other carnivores. Molecular
epidemiology may help to understand the dynamics of evolution of CDV. Several lineages or genotypes of CDV exist
that are variously distributed throughout the continents but their distribution and the reason for this diversification are
unclear. During a surveillance program for viral infections in domestic carnivores, in the years 2005-2006, a total of 39
CDV strains were detected. By using lineage-specific primers targeting the H gene in a hemi-nested PCR assay, the
genotype of the CDV strains was characterized. Out of 14 strains detected in 2005, 7 were characterized as Arctic and 7
as European. In 2006, 14 out of 25 strains were characterized as Arctic, and 10 as European, while one strain was
characterized as a vaccine-like virus. In Italy and Europe, the Arctic CDVs were not reported before 2004. The
European CDVs appeared to be associated more frequently with enteric symptoms and ocular signs, while the Arctic
CDVs were associated more frequently with respiratory signs. By epidemiological tracing, the origin of one such Arctic
CDV strain was identified in an Hungarian breeding kennel. Legal or uncontrolled trading of animals may alter the
epidemiology of CDV, introducing novel strains in CDV-naïve areas or accounting for the resurgence of CDV in areas
where vaccine prophylaxis was effective and successful to control the disease.
76
44
Molecular analysis of porcine genogroup I picobirnaviruses
Bányai K 1,2 *, Bogdán Á 1, Martella V 3, Forgách P 4, Jakab F 1, Meleg E 1, Bíró H 5, Melegh B 6, Sz cs G 1,2
1 Regional Laboratory of Virology, Baranya County Institute of State Public Health Service, Szabadság út 7., H-7623
Pécs, Hungary
2 Department of Medical Microbiology and Immunology, Faculty of Medicine, University of Pécs, Szigeti út 12., H7624 Pécs, Hungary
3 Department of Animal Health and Well-Being, University of Bari, Sp Casamassima Km 3, 70010 Valenzano, Bari,
Italy
4
Department of Microbiology and Infectious Diseases, Faculty of Veterinary Science, Szent István University, István u.
2., H-1078 Budapest, Hungary
5
Department of Medical Genetics and Child Development, Faculty of Medicine, University of Pécs, Szigeti út 12., H7624 Pécs, Hungary
Picobirnaviruses (PBVs) are small, unclassified viruses with a bisegmented double-stranded RNA genome. Their
pathogenic potential, ecology, and evolutionary features are virtually unexplored. In this study we describe the
molecular analysis of porcine PBVs identified in intestinal content of dead pigs at weaning and post-weaning age. A
subset of positive samples (6 of 13) were cloned and then subjected to single-stranded conformational polymorphism
analysis and nucleotide sequencing. All clones belonged to genogroup I PBVs, and, with a single exception, all clones
clustered on separate branches from human strains. One strain shared the closest genetic relationship with a Hungarian
human PBV strain (89.9% nt identity). Genetic diversity was also observed among strains identified in mixed infections.
Single point mutations and deleterious mutations within highly related strains suggested that PBVs exist as quasispecies
in the swine alimentary tract. Clones with complete sequence identities originating from different animals suggested
effective animal-to-animal transmission of the virus. Our findings indicate that infection with genogroup I PBVs is
common in pigs.
45
Surveillance of human enteric viruses by monitoring of sweater
Carducci A., Verani M., Battistini R., Pizzi F., Rovini E.
Department of Biology – University of Pisa
The evaluation of environmental viral hazards is an emerging problem and many questions remain still unsolved.
Environmental monitoring through effective, standardised virus detection systems combined with clinical surveillance
could promote continuous, rapid exchange of information on the spread and distribution of the main enteric viral agents
and the incidence of correlated pathologies.
Since May 2004 an epidemiological surveillance of viral gastroenteritis diagnosed on faecal samples was carried out,
parallely with an environmental monthly monitoring of seawater receiving the effluent of treated wastewater.
Faecal samples analyzed with immunological technique and retested with PCR.
E. coli was isolated and counted by membrane filtration methods. For virological examination water samples were
concentrated using tangential-flow ultrafiltration and assayed with bimolecular tests For coliphages analysis the
concentrated samples were tested by plaque assay. Surveillance of cases (May 2004-March 2005) has revealed 15,3%,
positive on total faecal samples of which 4,3% rotavirus, 1,9% adenovirus, 2,3% astrovirus, 2,8% norovirus genI and
4,1% norovirus genII, without particular epidemic peaks.
E. coli was found in 21% samples and coliphage counts were variable probably due to seawater dilution. No correlation
between E. coli and somatic coliphages concentrations.
Seawater resulted contaminated for adenovirus (type 41) in April and October 2005, January, September (2 times) and
October 2006 corresponding 16% of samples; while Norovirus gen. 2 was presented only in August 2005.
Clinical samples showed a continuous circulation of enteric viruses. Environmental virological analysis frequently
resulted positive for adenovirus, E. coli and coliphages counts indicated a strong dilution in the sea. No correlation was
found between indicators and enteric virus presence. We cannot identify a predominant virus for risk assessment yet,
nor an viral pollution indicator.
77
46
Viability evaluation of long-term burial of Newcastle Disease Virus (NDV) vaccine strains.
S. Bianchi^, A. Amendola^, M. Canuti^, A. Zappa^, R. Koncan*, E. Tanzi^.
^ Dep. Public Health-Microbiology-Virology, University of Milan, Italy
*Dep. Of Pathology, University of Verona, Italy
Introduction. In the past Century biohazard waste-products, such as vaccines and other industrial products, had been
buried in the soil of a former pharmaceutical research institute in Milan (Italy). While reclaiming, numerous
hermetically sealed vials of Newcastle Disease (ND) vaccine were found and unearthed. NDV is considered to be a
biological agent that can cause human disease and may be hazard to employers.
The present study aims to investigate the residual viability of live NDV vaccine strains stored in sealed vials and
recovered after burial for 30 years.
Material and methods. Lyophilised vaccines against Newcastle Disease recovered from a wasteyard area were
analysed. The specimens were submitted to viability tests by inoculation into the allantoic cavity of embryonated
chicken eggs (the preferred substrate for NDV growth). To evaluate the presence of active replication ability of our
strains, both the freshly resuspended whole vaccine and the allantoic fluid, harvested after 48h of incubation, were
tested for haemoaglutination (HA) activity. As a positive control, a vaccine currently being used against ND (Izovac,
Brescia, Italy) containing live NDV strains (titre >106 EID50) was included.
Results and conclusions. The recovered viruses were endowed with replicative ability. In fact, an increase of HA titre
in the allantoic fluid after infection with our strains was observed: from 4 (whole vaccine) to 2.048 (allantoic fluid) HA
units.
This suggests that live NDV vaccine strains can preserve their replication ability in embryonated chicken eggs for
several years even under uncontrolled environmental conditions if preserved in sealed vials.
47
Presence of cutaneous HPVs, predominantly HPV types of the genus Beta-Papillomavirus, in
the oral cavity.
Francesca Paolini, Barbara Mafera1, Consuelo Rizzo, Siavash S. Rahimi2, Maurizio G. Vigili1, Gianna Badaracco and
Aldo Venuti.
Laboratory of Virology Regina Elena Cancer Institute, Rome, Italy
1
Division of Otorhinolaringology and 2Histophatology Service, San Carlo IDI-Hospital IRCCS, Rome, Italy.
A number of studies have reported the presence and expression of high-risk HPV viruses in the diseased oral cavity. Of
special significance is the association of HPV 16 with a subset of head and neck cancers localised in the tonsils and
oropharynx. However, most of these studies have concentrated on identification of specific high-risk viruses in head
and neck squamous cell carcinomas (HNSCC). The aim of our study was to identify a broad spectrum of HPV DNA
sequences in three different patient groups: a) patients with pre-neoplastic lesions (e.g. leukoplakia, erythroplakia), b)
patients with tumors of the oral cavity, and c) patients attending outpatients for dental diseases. Samples were collected
by mouth washes or brushes (groups a and c) or biopsies (group b). HPV DNA was identified by nested PCR with
consensus primer CP, MY, and GP+. The genotype was determined by direct sequencing of the amplified products and
the type identification was made on the basis of > 90% homology with HPV sequences deposited in Gen Bank with
BLAST Software. In the group c we detected the presence of alpha (mucosal/genital) HPV (in particular the HPV16)
but, surprisingly, in the other groups the predominant types were the beta (cutaneous) HPV. Our results indicate a much
wider spectrum of HPV types in the oral cavity than previously reported and stress the need to use technologies for the
detection of the majority of HPV in order to define the contribution of different HPVs to HNSCC.
78
48
Microglia cells are the replication site for some prion strains and can control amyloid plaques
toxicity in transmissible spongiform encephalopathies.
Enrico Cancellotti, Chris Plinston*, Ruth Hennion+, Elena Sartori, Arianna Calistri, Giorgio Palu’, Jean C. Manson*,
Rona M. Barron*
*Neuropathogenesis Unit, Roslin Istitute, Edinburgh, UK
+
Institute for Animal Health, Compton, UK
In transmissible spongiform encephalopathies (TSE) or prion diseases, neurons are supposed to be the main target for
different strains of the infectious agent. However, it is possible that some strains may replicate in different cell types
before spreading into neurons. To investigate this hypothesis, we have used a microglia-like cell culture model. These
cells responded differently to infection with a number of prion strains suggesting that strains may have different cellular
tropism within the brain. Surprisingly, persistently infected microglia cells were then able to kill PrP knock-out primary
neurons in co-culture showing how some strains may spread between different cells with PrP independent mechanisms.
Moreover an in vivo analysis carried out in mouse brains showed how microglia are able to surround amyloid plaques,
as it has been observed in Alzheimer’s disease, and prevent the outcome of TSE disease
The data presented here highlight a central role of microglia in preventing or facilitating the spreading of prion
infectivity according to different strains. Using our cell culture model, we are now investigating in more detail the
mechanisms behind the prion infectious proces in microglia. These results may also be important for future therapeutic
and diagnostic approaches for these infectious diseases.
49
F. Carletti1,C. Castilletti1, L. Bordi1, C. Gioia1, M.S. Zaniratti1, L. Falasca1, L. Viale2, A. Beltrame2, M. Crapis2, G.
Ippolito1, M.R. Capobianchi1
1
National Institute for Infectious Diseases "L.Spallanzani", Rome, Italy, 2Clinic of Infectious Diseases, University of
Udine, Udine, Italy
Background: Poxviruses are a family of large, dsDNA viruses that recently have received increased attention, due to
fear of bioterrorist attacks and zoonotic transmission. Few data are available on European orthopoxvirus strains
involved in zoonotic events. We report a case of human orthopoxvirus zoonosis in Northern Italy.
The clinical case: In January 2006, an Italian male scratched by a diseased cat presented at the infectious disease clinic
with a suspected orthopoxvirus (OPV) lesion on the right hand, accompanied by systemic symptoms such as moderate
fever and malaise.
Methods: Viral DNA was detected by pan-orthopoxvirus-specific real-time and classical PCR, targeting CrmB and
HA, respectively. Further characterization was carried out by RFLP analysis of CrmB and sequencing of full length
HA gene. Humoral immune response was analyzed by IF and microneutralization assays; cellular immune response was
established by intracellular detection of IFN-gamma after in vitro PBMC stimulation with both patient's isolate and
reference vaccinia virus.
Results: Orthopoxvirus infection was identified by electron microscopy, PCR and virus isolation. The diagnosis was
confirmed by detection of both cellular and humoral specific immune response. RFLP of CrmB and phylogenetic
analysis of HA were unable to conclusively identify the poxvirus species. In particular, the HA sequence of the new
isolate showed significant similarity to ectromelia, cowpoxvirus and camelpox viruses.
Conclusion: Enhanced diagnostic capability for orthopoxvirus infections, resulting from global awareness of
bioterrorism menace, will be helpful to identify new orthopoxvirus infections in animals and humans, and to improve
the knowledge on ortopoxviruses circulation in Italy.
79
50
A New Quantitative Real-Time PCR for Chikungunya Virus Detection
L. Bordi, C. Castilletti, M. Sciarrone, G. Ippolito, M.R.
National Institute for Infectious Diseases "L.Spallanzani", Rome, Italy
Capobianchi,
A.
Di
Caro
F.
Carletti,
Background: A large outbreak of a mosquito-borne viral disease, Chikungunya, has begun in 2005 in the Comoros
islands. In few months many other countries in Indian Ocean have experienced a dramatic increase of cases, and
diseased returning travellers are also appearing in Europe, raising concerns about the spread of the infection in South
Europe, where a suitable vector is present.
Chikungunya virus (CHIKV) is a mosquito-transmitted Alphavirus belonging to Togaviridae family. The clinical
manifestations of the infection may be confused with those caused by Dengue fever or Yellow fever, and laboratory
confirmation of suspected cases is mandatory to launch control measures during an outbreak.
Aim and Results: We describe a rapid, sensitive and specific method based on real-time RT-PCR targeting the nsP1
region for CHIKV, able to quantify viral genome concentrations over a wide dynamic range (along 7 Logs), with a
sensitivity of 20 copies. The quantitative assay was validated by in vitro experiments, were IFN- (a well known virus
inhibitor) exerted a dose-dependent inhibition of virus replication, assessed as both viral infectivity and viral RNA
production. By using the newly established method, CHIKV viremia was detected in acute phase sera from cases
recently imported to Italy, with viremia levels ranging from 1.3x105 to 6x108 copies/ml.
Conclusion: Our results indicate that the newly established method can be an useful tool for the rapid detection of
Chikungunya virus during natural infection and to monitor the extent of viral replication in patients as well as in vitro.
80
Index
A
Accardi L., 33, 40
Affabris E., 59, 65
Agarossi A., 33
Alagna F., 39, 45
Albani M., 67
Alberti A., 59
Alvisi G., 25, 39, 57, 62
Amato B., 26
Amendola A., 32, 33, 78
Amici C., 68
Andreoli E., 67
Arista S., 74, 75, 76
Astegiano S., 21
Astone V., 66
Avitabile E., 25
Avolio M., 23, 63
Azzi A., 67
B
Badaracco G., 78
Baldanti F., 29
Balestrieri M., 19
Ballestri M., 47
Banyai K., 74, 76, 77
Banks L., 51
Barzon L., 30, 37, 38, 58, 65
Barron R.M., 79
Battistini R., 77
Beggio P., 38, 44
Belardelli F, 13
Bellacicco A.L., 74, 76
Beltrame A., 74, 79
Bendinelli M., 61, 67
Benevolo M., 31
Beretta R., 32
Bergallo M., 21
Bergonzini V., 64
Bernardinelli I., 75
Bertaina A., 29
Bertelli L., 57
Bianchi F., 39
Bianchi S., 32, 78
Biasolo M.A., 30, 37
Biolchini A., 66
Biro H., 77
Bison D., 59
Blasi E., 58
Bobbio V., 37
Bogdan A., 77
Bondarenko I.G., 32
Bordi L., 74, 79, 80
Bortolazzi F., 47
Bortoletto G., 59
Botti G., 45
Bozzaco L., 61
Branca M., 33
Brandi R., 31
Brocca-Cofano E., 47
81
Bruni G., 22
Buonaguro F.M., 20, 39, 44, 45, 47
Buonaguro L., 20, 44, 45, 47
Buonavoglia C., 22, 74, 76
Buonavoglia D., 76
C
Calistri A., 24, 40, 64, 65, 79
Calore E., 64
Camarda A., 22
Camero M., 74
Camilloni B., 46
Campadelli-Fiume G., 25
Campanini G., 26, 29
Campello C., 62, 75
Campolo M., 74
Cancellotti E., 79
Cantoni S., 39
Canuti M., 78
Capobianchi M.R., 74, 79, 80
Cappuccini F., 73
Caputo A., 47
Caracciolo S., 23, 63
Cardi T., 39, 45
Carducci A., 15, 77
Carletti F., 74, 79, 80
Carozzi F., 53
Caruso A., 23, 63, 68
Casadio C., 46
Caselli E., 29, 68
Cassai E., 29
Castaldello A., 47
Castellazzi M., 66
Castilletti C., 74, 79, 80
Catalucci V., 61
Caudai C., 34
Cavalli A., 76
Cavallini C., 39
Cavallo R., 21
Cavicchio P., 74
Ceccherini Nelli L., 67
Celegato M., 24
Celestino M., 24
Cermelli C., 58
Chiappalupi L., 40
Ciotti M., 33, 46
Circella E., 22
Cirone F., 76
Coen D.M., 25
Colazani D., 33
Colomba C., 75
Colombrita D., 31
Comar M., 62
Comin A., 65
Comolli G., 21
Conti G., 26
Conti S., 26
Corcioli F., 67
Corrente M., 74, 76
Costa C., 21
Costa S., 33
Crapis M., 74, 79
82
Curtarello M., 65
Cusi M.G., 43
Cusinato R., 58
D
D’Agaro P., 62, 75
D’Antonio D., 46
D’Isanto M., 61
Dal Pero F., 59
Dal Molin G., 62
De Donno A., 22
De Giuli Morghen C., 38, 44
De Grazia S., 75
De Leo A., 57
De Palo G., 45
De Stradis A., 45
Decaro N., 74, 76
Del Fabbro S., 75
Del Vecchio C., 24, 64
Della Valle M., 30
Della Vella M., 37
Desario C., 74, 76
Di Bonito P., 33, 43
Di Caro A., 80
Di Genova G., 43
Di Grazia A., 19, 23
Di Luca D., 29, 68
Di Renzo L., 57
Dolei A., 66
Donà M.G., 33, 40
Draghin E., 31
Duraturo M.L., 20, 45
E
Elia G., 76
Ensoli B., 47, 63
F
Falanga A., 61
Falasca L., 74, 79
Falcone V., 73
Fantoni L., 58
Farina C., 33
Fasolo M.M., 32
Favalli C., 46
Federico M., 63
Ferrari D., 60
Fiorentini S., 23, 63, 68
Fiorucci G., 59
Fontana S., 19, 23
Forgach P., 77
Fornara C., 21
Franceschini L., 59
Franconi R., 43
Frasson I., 64
Freer G., 61
Frezza C., 59
83
G
Galdiero M., 61
Galdiero S., 61
Galli L., 60
Gallina A., 20
Gallo A., 45
Galvan M., 29
Gariglio M., 46
Gerna G., 21, 29, 69
Gerotto M., 59
Geyer M., 59, 65
Giacomazzi C., 37
Giammanco G.M., 75
Gioia C., 74, 79
Giorgi C., 33, 40, 43
Giuliani L., 46
Gori Savellini G., 43
Granirei E., 66
Grassi T., 22
Grasso F., 33
Greco G., 74, 76
Grillo S., 39, 45
Guido M., 22
H
Hammer D., 63
Hennion R., 79
Huck B., 73
I
Iaconelli M., 19, 23, 24
Idolo A., 22
Iob A., 75
Iorio A.M., 46
Ippolito G., 74, 79, 80
J
Jakab F., 77
Jans D.A., 57, 62
Jaxmar T., 46
K
Kampanaraki A., 61
Kern W., 73
Kirnbauer R., 51
Koncan R., 78
Kontny U., 73
Koopmans M., 74
Kotenko S., 66
L
La Rosa G., 19, 23, 24
Lanini L., 67
Larcher C., 26
Laus M., 47
Lavazza A., 22, 76
Lavezzo E., 38
Lazzarin A., 60
84
Lenzi P., 39
Lepri E., 46
Lewis G.K., 44, 47
Lilleri D., 21, 69
Lillo F., 60
Lo Giudice G., 40
Lombardi G., 40
Loregian A., 25, 30, 57, 64
Lorusso A., 76
Lorusso E., 31, 74, 76
Losada Cabruja E., 37
Losito S., 45
Lucani L., 39
Lucente M.S., 76
Lugli E., 58
M
Mafera B., 78
Maggi F., 67
Maggiorella M.T., 63
Magliani V., 26
Maliga P., 39
Mameli G., 66
Mangino G., 59, 65
Manna A., 46
Manson J.C., 79
Marandino F., 31
Marchi A., 29
Marcolongo M., 59
Marconi A., 19
Mariani L., 31, 33
Marincola F., 44, 47
Martella V., 22, 74, 75, 76, 77
Martinelli E., 75
Martini I., 37
Masi G., 58
Massa S., 43
Massimi P., 51
Matkovic U., 38
Matteucci D., 61
Mattia E., 57
Matusali G., 57
Mazzetti P., 61
McDermott J.L., 37
Mei A., 66
Meini G., 34
Meleg E., 77
Melegh B., 77
Menegazzi P., 34
Mercorelli B., 25, 30
Merlino C., 21
Mett V., 43
Milanese G., 20
Militello V., 30
Minini C., 23
Mirandola S., 59
Mochi S., 33
Monaco A., 44, 47
Monini M., 73
Moschidou P., 76
Muller A., 43
Muratore G., 30
85
Murer L., 30, 37
Muscillo M., 19, 23, 24
Musiani D., 39, 57, 62
Mutinelli F., 15
N
Narayan N., 51
Nazzi F., 75
Neri M., 46
Neumann-Haefelin D., 73
Noto A., 59
O
Orlando G., 32
P
Pacenti M., 30, 37, 38
Pacchioni S., 38, 44
Paganelli A.,
Pagani E., 26
Palù G., 24, 25, 30, 37, 38, 40, 58, 64, 65, 79
Paolini F., 78
Pari G.S., 25
Pariani E., 33
Parolin C., 24, 40, 64, 65
Patrone M., 20
Percario Z.A., 59, 65,
Percivalle E., 26
Perno C.F., 46
Pescollderungg L., 26
Petrucci T., 40
Petters C., 26
Peyerl-Hoffman G., 73
Piccoli R., 20
Pifferi M., 67
Pilotti S., 45
Piralla A., 29
Pistello M., 67
Pizzato M., 64
Pizzi F., 77
Pliston C., 79
Podestà A., 33
Poddighe L., 66
Polonelli L., 26
Pompa A., 39
Portincasa P., 26
Pourshaban M., 19, 23, 24
Pozzi E., 38, 44
Prosdocimo G., 40
Puoti M., 14
Q
Quattrini M., 31
R
Radaelli A., 38, 44
Rahimi S.S., 78
Ramirez S., 75
86
Razzolini F., 19
Realdon S., 59
Ricci V., 67
Richter S., 64
Ripalti A., 25, 39, 57, 62
Rinieri A., 67
Rizzo C., 78
Rocchi J., 67
Rollo F., 31
Romano L., 19
Romeo G., 59
Rossella B., 43
Rossi D., 23
Rossi P., 26
Roth D.M., 57
Rovida F., 29
Rovini E. 77,
Ruggeri F.M., 73
Ruscio M., 75
Ruzza M.L., 33
S
Sabatini S., 30
Saladini F., 19
Salata C., 64, 65
Sansone M., 20
Santoni F., 29
Santoro M.G., 68
Sartori E., 79
Savini G., 20,
Scaglione F., 52
Schenk T., 73
Sciarrone M., 80
Scorrano L., 59
Scotti N., 39, 45
Secolo D., 26
Segoloni G.P., 21
Serra C., 66
Serra V., 65
Sette P., 24
Sidoti F., 21
Sinigaglia A., 58
Sinigalia E., 25, 30, 57
Spaccasassi S., 47
Spagnoli L., 40
Sparnacci K., 47
Stefanon B., 45
Stronati M., 29
Syrjanen K., 33, 46
Szucs G., 77
T
Tabarrini O., 30
Tagliaferro L., 34
Tagliamone M., 44, 47
Tanzi E., 32, 33, 78
Tarabella F., 61
Tempesta M., 76
Terlizzi M.E., 21
Terrosi C., 43
Thomas M., 51
87
Titti F., 63
Tondelli L., 47
Tornesello M.L., 20, 39, 44, 45, 47
Torreri P., 40
Tosti G., 23, 63
Toti M., 34
Touscoz G.A., 21
Trevisan M., 58, 65
U
Uberti Foppa C., 60
V
Varnier O.E., 34, 37
Vatteroni M.L., 67
Vendramini L., 40
Ventura C., 39
Venuti A., 31, 43, 52, 78
Verani M., 77
Viale L., 74, 79
Vicenti I., 19
Vigili M.G., 78
Vitale A., 39
Vitiello M., 61
Vocaturo A., 31
Vollero L., 63, 68
Voltan R., 47
W
Wolf H., 14
Y
Yusibov V., 43
Z
Zakrzewska K., 67
Zaniratti M.S., 74, 79
Zanocco D., 25
Zanotta N., 62
Zanotto C., 38, 44
Zappa A., 33, 78
Zazzi M., 19, 34
88

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