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P.312 Isolation and characterization of hepatitis E virus (HEV)-specific T cell clones from an asymptomatic HEV-exposed subject
$158
[P.3~
Journal of Clinical Virology 2006, Vol 36 (suppl 2) Isolation and characterization of hepatitis E virus (HEV)-specific T cell clones from an asymptomatic HEV-exposed subject
M.T. Shata 1 ', A. Barrett 1, S. Rouster 1, N.J. Shire 1, J.T. Blackard 1, M. Atiq 1, R. Engle 2, R. Purcell 2, S. Emerson 2, G.T. Strickland 3, K.E. Sherman 1. 1Internal Medicine, Division of Digestive Diseases,
Abstracts, 12th ISHVLD the water samples may suggest the maintenance of this virus in the environment and constitutes a public health risk.
[-~-]
Development of bicistronic selectable vector for non-radioactive antigen specific reporter release T cell cytotoxicity assay
University of Cincinnati, Cincinnati; 2Laboratory of Infectious Diseases, NIAID, NIH, Bethesda; 3Epidemiology and Preventive Medicine, University of Maryland, Baltimore, USA
R. Tayal 1 ', H. Durgapal 1, G. Satpathy Panda 2, S.K. Acharya 3, S.K. Panda 1. 1Department of Pathology, 2Department of Ocular
Background and Objectives: Hepatitis E virus (HEV) is a common
Microbiology, 3Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India
cause of acute viral hepatitis in many less-developed countries. The transmission route is fecal-oral, and water-borne outbreaks have been frequently reported. Studies have reported a high seroprevalence of anti-HEV antibody in both the USA and Egypt (20% and 70%, respectively). However, HEV has not been identified as a major cause of acute sporadic or epidemic hepatitis in either country, in contrast to many other parts of the world. Our objective is to determine why HEV morbidity manifests differently in hosts from various parts of the world. Methods: An HEY-specific IFN-gamma ELISPOT assay has been developed and evaluated in our laboratory to analyze cell-mediated immune responses among susceptible hosts. We used peripheral blood lymphocytes (PBLs) and sera from experimentally infected chimpanzees as well as from seroconverted subjects, and 16-mer overlapping ORF2 peptides. Results: HEV-specific IFN-gamma ELISPOT responses correlated strongly and significantly with anti-HEV ELISA positive/negative results (rho = 0.86, p < 0.001, Spearman rank correlation). Additionally, we have isolated an ORF2-specific T cell line and multiple clones from an asymptomatic seroconverted subject. Fine specificity of the ORF2-specific T cell clones identified a dominant epitope that is well conserved in distinct HEV genotypes. Conclusion: The HEV-specific IFN-gamma ELISPOT assay may be a useful surrogate marker for HEV exposure. Moreover, fine specificities of HEV-T cell clones isolated from an asymptomatic seroconverted subject suggest cross-reactivity and possible protection among different HEV genotypes. The significance of these findings in the endemic area will be discussed.
rP-~
Hepatitis A virus in environmental water samples from Amazon Basin
M.S. De Paula 1 *, L. Diniz-Mendes 1, L.M. Villar 1, S.B. Luz 2, A.C. Gaspar 1. 1Virology, Oswaldo Cruz Foundation, Rio de Janeiro,
2Biodiversity, Oswaldo Cruz Foundation, Manaus, Brazil Background and Objectives: Water scarcity is leading to an intense discussion worldwide. Brazil holds 13% of the world potable water. Seventy percent of this amount is concentrated in the Amazon basin. Hepatitis A virus (HAV) is currently recognized as an important human water pathogen with regard to the number of outbreaks and sporadic cases. Although a progressive decline in hepatitis A morbidity rate could be seen in Brazil, in Amazon region this rate continues to be high. The aim of this work was to investigate the presence of HAV in environmental samples from Amazon Basin. Methods: Water samples were taken at thirteen small rivers, according to the pulse of inundation of the main river (ebb tide, low tide, flood tide and high tide). A total of fifty-two river water samples were analyzed and the sampling locations had their geological coordinates marked by a global positioning system (GPS). Viruses were attached to a negatively charged membrane and concentrated by centrifuging. Specific nested and real-time reverse transcriptionPCR were used to detect and quantify HAV in water. Results: When submitted to Real-time, 96% (50/52) of the samples were HAVRNA positive. Depending on the pulse of inundation and the sanitary conditions, the viral load in these samples varied from 60 to 5500 copies of HAV RNA/L. Using conventional Nested RT-PCR with primers based on the sequence at the VP1-2A, HAV RNA could be detected in 23% of the samples (11/52). After nucleotide sequencing, samples classified in genotype I, subgenotype IA and lB. The HAVRNA in samples with low virus concentration (<1200) only could be detected using Real-time PCR. Conclusion: Therefore, quantitative real-time RT-PCR is useful in monitoring HAV contamination in water. The detection of HAV in
Background and Objectives: Pathogenesis of Hepatitis E virus might be immunologically mediated, but there is no direct evidence for or against this hypothesis. It is important to determine if cellular immune responses occur in HEV infection and whether these produce cell-mediated cytotoxic damage to hepatocytes as do hepatitis B and C viruses. Most commonly used assay to detect cytotoxic T cells is Chromium-51 (51Cr) release assay, which has several disadvantages: radioactive, expensive, difficult to load and spontaneous release. This led us to develop a novel antigen specific non-radioactive reporter release T cell cytotoxicity assay. The assay provides a simple, sensitive, specific and responsive method for measuring T cell cytotoxicity. Also, the MHC restriction problem can be overcome in syngeneic mouse strains and the availability of knockout mice will help in dissecting the immune system. This is based on a bicistronic selectable vector with one cistron expressing the test antigen and the second expressing a reporter protein (EGFP/Firefly luciferase Fluc). The bicistronic vector permits the expression of two proteins simultaneously in the same cell using endogenously expressed antigen to present on the target cells and reporter protein, to be assayed easily in culture supernatant following lysis. Hepatitis B virus core antigen was used to validate the assay. Methods: HBV core was PCR amplified and cloned into eukaryotic and prokaryotic expression vectors. Bicistronic vector were constructed containing both HBV core/HEV proteins under strong CMV promoter and EMCV IRES with reporter proteins (EGFP/Fluc) under EMCV IRES and CMV promoter respectively. All the constructs were transiently transfected in human HepG2 and mouse A20 cell line and were analyzed by EGFP fluorescence, firefly luciferase assay and Immunoprecipitation/Immunofluorescence for the test antigen. Results: The bicistronic selectable vector expressing HBV core/HEV proteins (RdRp, ORF2 and ORF3) and reporter proteins (EGFP and Fluc) have been generated and were checked for protein expression in human HepG2 and mouse A20 cell line. Mouse A20 cell line showed low transfection efficiency thereby retroviral system was used further for the development of CTL assay. HBV core antigen has been expressed, analyzed by western blotting and purified by Ni-NTA column chromatography. Conclusion: This study focuses on the development of a novel bicistronic antigen-reporter expression vector for non-radioactive antigen specific reporter based cytotoxicity assay and to envisage the immunopathogenesis of HEV.
[-P-.~
Hepatitis A infection in Naples and Caserta areas during the period 2000-2004: is the virus normally circulating or it has been re-introduced?
D. Genovese 1, S. Taffon 1, C. Argentini 1, N. Coppola 2, E. Sagnelli 2, R Amoroso 3, E Faella 3, L. Croci 4, S. Salmaso 5, M. Rapicetta 1 '.
1Viral Hepatitis Unit, Dept. 2Dept. of Public Medicine, 3Infectious Diseases Unit, Health, CNRA, 5CNESPS,
MIPI, Istituto Superiore di Sanit& Rome; "San Sebastiano" Hospital, Caserta; "D. Cotugno" Hospital, Naples; 4public Istituto Superiore di San#& Rome, Italy
Background and Objectives: Italy is considered a country at low endemicity level for Hepatitis A infection that is characterized by low annual incidence rates (around 5 per 100,000 inhabitants) and a shift in the average age of infection towards adulthood. However, available data from the official notification system suggest that the epidemiological pattern is not homogenous across the country. Seroprevalence rates are still more than 30% among 20-30 years old individuals in several areas in Southern Italy, where also insurgence
[P.3~
Journal of Clinical Virology 2006, Vol 36 (suppl 2) Isolation and characterization of hepatitis E virus (HEV)-specific T cell clones from an asymptomatic HEV-exposed subject
M.T. Shata 1 ', A. Barrett 1, S. Rouster 1, N.J. Shire 1, J.T. Blackard 1, M. Atiq 1, R. Engle 2, R. Purcell 2, S. Emerson 2, G.T. Strickland 3, K.E. Sherman 1. 1Internal Medicine, Division of Digestive Diseases,
Abstracts, 12th ISHVLD the water samples may suggest the maintenance of this virus in the environment and constitutes a public health risk.
[-~-]
Development of bicistronic selectable vector for non-radioactive antigen specific reporter release T cell cytotoxicity assay
University of Cincinnati, Cincinnati; 2Laboratory of Infectious Diseases, NIAID, NIH, Bethesda; 3Epidemiology and Preventive Medicine, University of Maryland, Baltimore, USA
R. Tayal 1 ', H. Durgapal 1, G. Satpathy Panda 2, S.K. Acharya 3, S.K. Panda 1. 1Department of Pathology, 2Department of Ocular
Background and Objectives: Hepatitis E virus (HEV) is a common
Microbiology, 3Department of Gastroenterology, All India Institute of Medical Sciences, New Delhi, India
cause of acute viral hepatitis in many less-developed countries. The transmission route is fecal-oral, and water-borne outbreaks have been frequently reported. Studies have reported a high seroprevalence of anti-HEV antibody in both the USA and Egypt (20% and 70%, respectively). However, HEV has not been identified as a major cause of acute sporadic or epidemic hepatitis in either country, in contrast to many other parts of the world. Our objective is to determine why HEV morbidity manifests differently in hosts from various parts of the world. Methods: An HEY-specific IFN-gamma ELISPOT assay has been developed and evaluated in our laboratory to analyze cell-mediated immune responses among susceptible hosts. We used peripheral blood lymphocytes (PBLs) and sera from experimentally infected chimpanzees as well as from seroconverted subjects, and 16-mer overlapping ORF2 peptides. Results: HEV-specific IFN-gamma ELISPOT responses correlated strongly and significantly with anti-HEV ELISA positive/negative results (rho = 0.86, p < 0.001, Spearman rank correlation). Additionally, we have isolated an ORF2-specific T cell line and multiple clones from an asymptomatic seroconverted subject. Fine specificity of the ORF2-specific T cell clones identified a dominant epitope that is well conserved in distinct HEV genotypes. Conclusion: The HEV-specific IFN-gamma ELISPOT assay may be a useful surrogate marker for HEV exposure. Moreover, fine specificities of HEV-T cell clones isolated from an asymptomatic seroconverted subject suggest cross-reactivity and possible protection among different HEV genotypes. The significance of these findings in the endemic area will be discussed.
rP-~
Hepatitis A virus in environmental water samples from Amazon Basin
M.S. De Paula 1 *, L. Diniz-Mendes 1, L.M. Villar 1, S.B. Luz 2, A.C. Gaspar 1. 1Virology, Oswaldo Cruz Foundation, Rio de Janeiro,
2Biodiversity, Oswaldo Cruz Foundation, Manaus, Brazil Background and Objectives: Water scarcity is leading to an intense discussion worldwide. Brazil holds 13% of the world potable water. Seventy percent of this amount is concentrated in the Amazon basin. Hepatitis A virus (HAV) is currently recognized as an important human water pathogen with regard to the number of outbreaks and sporadic cases. Although a progressive decline in hepatitis A morbidity rate could be seen in Brazil, in Amazon region this rate continues to be high. The aim of this work was to investigate the presence of HAV in environmental samples from Amazon Basin. Methods: Water samples were taken at thirteen small rivers, according to the pulse of inundation of the main river (ebb tide, low tide, flood tide and high tide). A total of fifty-two river water samples were analyzed and the sampling locations had their geological coordinates marked by a global positioning system (GPS). Viruses were attached to a negatively charged membrane and concentrated by centrifuging. Specific nested and real-time reverse transcriptionPCR were used to detect and quantify HAV in water. Results: When submitted to Real-time, 96% (50/52) of the samples were HAVRNA positive. Depending on the pulse of inundation and the sanitary conditions, the viral load in these samples varied from 60 to 5500 copies of HAV RNA/L. Using conventional Nested RT-PCR with primers based on the sequence at the VP1-2A, HAV RNA could be detected in 23% of the samples (11/52). After nucleotide sequencing, samples classified in genotype I, subgenotype IA and lB. The HAVRNA in samples with low virus concentration (<1200) only could be detected using Real-time PCR. Conclusion: Therefore, quantitative real-time RT-PCR is useful in monitoring HAV contamination in water. The detection of HAV in
Background and Objectives: Pathogenesis of Hepatitis E virus might be immunologically mediated, but there is no direct evidence for or against this hypothesis. It is important to determine if cellular immune responses occur in HEV infection and whether these produce cell-mediated cytotoxic damage to hepatocytes as do hepatitis B and C viruses. Most commonly used assay to detect cytotoxic T cells is Chromium-51 (51Cr) release assay, which has several disadvantages: radioactive, expensive, difficult to load and spontaneous release. This led us to develop a novel antigen specific non-radioactive reporter release T cell cytotoxicity assay. The assay provides a simple, sensitive, specific and responsive method for measuring T cell cytotoxicity. Also, the MHC restriction problem can be overcome in syngeneic mouse strains and the availability of knockout mice will help in dissecting the immune system. This is based on a bicistronic selectable vector with one cistron expressing the test antigen and the second expressing a reporter protein (EGFP/Firefly luciferase Fluc). The bicistronic vector permits the expression of two proteins simultaneously in the same cell using endogenously expressed antigen to present on the target cells and reporter protein, to be assayed easily in culture supernatant following lysis. Hepatitis B virus core antigen was used to validate the assay. Methods: HBV core was PCR amplified and cloned into eukaryotic and prokaryotic expression vectors. Bicistronic vector were constructed containing both HBV core/HEV proteins under strong CMV promoter and EMCV IRES with reporter proteins (EGFP/Fluc) under EMCV IRES and CMV promoter respectively. All the constructs were transiently transfected in human HepG2 and mouse A20 cell line and were analyzed by EGFP fluorescence, firefly luciferase assay and Immunoprecipitation/Immunofluorescence for the test antigen. Results: The bicistronic selectable vector expressing HBV core/HEV proteins (RdRp, ORF2 and ORF3) and reporter proteins (EGFP and Fluc) have been generated and were checked for protein expression in human HepG2 and mouse A20 cell line. Mouse A20 cell line showed low transfection efficiency thereby retroviral system was used further for the development of CTL assay. HBV core antigen has been expressed, analyzed by western blotting and purified by Ni-NTA column chromatography. Conclusion: This study focuses on the development of a novel bicistronic antigen-reporter expression vector for non-radioactive antigen specific reporter based cytotoxicity assay and to envisage the immunopathogenesis of HEV.
[-P-.~
Hepatitis A infection in Naples and Caserta areas during the period 2000-2004: is the virus normally circulating or it has been re-introduced?
D. Genovese 1, S. Taffon 1, C. Argentini 1, N. Coppola 2, E. Sagnelli 2, R Amoroso 3, E Faella 3, L. Croci 4, S. Salmaso 5, M. Rapicetta 1 '.
1Viral Hepatitis Unit, Dept. 2Dept. of Public Medicine, 3Infectious Diseases Unit, Health, CNRA, 5CNESPS,
MIPI, Istituto Superiore di Sanit& Rome; "San Sebastiano" Hospital, Caserta; "D. Cotugno" Hospital, Naples; 4public Istituto Superiore di San#& Rome, Italy
Background and Objectives: Italy is considered a country at low endemicity level for Hepatitis A infection that is characterized by low annual incidence rates (around 5 per 100,000 inhabitants) and a shift in the average age of infection towards adulthood. However, available data from the official notification system suggest that the epidemiological pattern is not homogenous across the country. Seroprevalence rates are still more than 30% among 20-30 years old individuals in several areas in Southern Italy, where also insurgence