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Yadav KK, Boley PA, Khatiwada S, Lee CM, Bhandari M, Wood R, Hanson J, Kenney SP. The zoonotic LCK-3110 strain of Rocahepevirus ratti leads to mild infection in chickens after experimental inoculation. Virus Res 2024; 350:199477. [PMID: 39406033 PMCID: PMC11525770 DOI: 10.1016/j.virusres.2024.199477] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2024] [Revised: 09/23/2024] [Accepted: 09/29/2024] [Indexed: 11/03/2024]
Abstract
Rocahepevirus ratti [rat hepatitis E virus (HEV)] was originally isolated from rats and found to be non-infectious to nonhuman primates, suggesting humans were not a susceptible host. However, in 2018, rat HEV infections were identified in human patients. High seroprevalence for rat HEV in rats in many countries necessitates studying this emerging zoonotic outbreak. Lack of a human derived rat HEV infectious clone, cell culture systems, and animal models have hindered this effort. In response to the increase in human infection cases by rat HEV, we utilized an infectious clone of the zoonotic rat HEV LCK-3110 strain originally reported from human cases. Capped RNA transcripts of the rat HEV LCK-3110 strain were synthesized, and replication was assessed in both cell culture via transfection and chickens via intrahepatic inoculation. Naive chickens were cohoused together with inoculated chickens. Our results demonstrated that although chickens were susceptible, virus replication was inefficient with only a few of the chickens inoculated with rat HEV having low levels of viremia and fecal virus shedding. However, LCK-3110 HEV was able to transmit between chickens as several naive cohoused chickens became infected as evidenced by viremia, fecal shedding, and the presence of viral protein upon histopathology of the liver. Rat HEV is an emerging zoonotic virus with an ability to spillover across species. Chickens have potential to serve as intermediary hosts, possibly playing a role in rat HEV spread and exposure to humans.
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Affiliation(s)
- Kush Kumar Yadav
- Center for Food Animal Health, Department of Animal Sciences, The Ohio State University, Wooster, OH, USA; Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA
| | - Patricia A Boley
- Center for Food Animal Health, Department of Animal Sciences, The Ohio State University, Wooster, OH, USA
| | - Saroj Khatiwada
- Center for Food Animal Health, Department of Animal Sciences, The Ohio State University, Wooster, OH, USA
| | - Carolyn M Lee
- Center for Food Animal Health, Department of Animal Sciences, The Ohio State University, Wooster, OH, USA; Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA
| | - Menuka Bhandari
- Center for Food Animal Health, Department of Animal Sciences, The Ohio State University, Wooster, OH, USA
| | - Ronna Wood
- Center for Food Animal Health, Department of Animal Sciences, The Ohio State University, Wooster, OH, USA
| | - Juliette Hanson
- Center for Food Animal Health, Department of Animal Sciences, The Ohio State University, Wooster, OH, USA
| | - Scott P Kenney
- Center for Food Animal Health, Department of Animal Sciences, The Ohio State University, Wooster, OH, USA; Department of Veterinary Preventive Medicine, College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA.
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Hooda P, Al-Dosari M, Sinha N, Parvez MK, Sehgal D. Inhibition of HEV Replication by FDA-Approved RdRp Inhibitors. ACS OMEGA 2023; 8:41570-41578. [PMID: 37969986 PMCID: PMC10633873 DOI: 10.1021/acsomega.3c05637] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Revised: 10/11/2023] [Accepted: 10/12/2023] [Indexed: 11/17/2023]
Abstract
Hepatitis E virus (HEV) is primarily a hepatotropic virus that is responsible for acute hepatitis E in the general population and for chronic hepatitis in immunocompromised individuals. In the absence of a globally accessible vaccine, pegylated interferon-α and ribavirin are the only antiviral agents available for the treatment of chronic patients. As viral RNA-dependent RNA polymerases (RdRps) are indispensable for RNA replication, they are considered potential drug targets. In this study, we screened some well-known RdRp inhibitor molecules, notably, favipiravir, sofosbuvir, remdesivir, filibuvir, and tegobuvir. Of these, monotherapy with favipiravir and sofosbuvir inhibited the RdRp activity with an IC50 value of 10.2 ± 4.9 and 5.2 ± 2.9 μM, respectively, compared to the reference drug ribavirin (3.5 ± 1.6 μM). Further investigation of the combination therapy showed a reduction in viral RNA copy numbers by approximately 90%. Therefore, favipiravir has an additive effect when used with sofosbuvir. Therefore, we propose that favipiravir is a promising anti-HEV drug that can be used in combination with sofosbuvir.
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Affiliation(s)
- Preeti Hooda
- Virology
Lab, Department of Life Sciences, Shiv Nadar
Institute of Eminence, Gautam Budh
Nagar 201314, India
| | - Mohammed Al-Dosari
- Department
of Pharmacognosy, College of Pharmacy, King
Saud University, Riyadh 11451, Saudi Arabia
| | - Neha Sinha
- Department
of Infectious Diseases and Microbiology, School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, United States
| | - Mohammad K. Parvez
- Department
of Pharmacognosy, College of Pharmacy, King
Saud University, Riyadh 11451, Saudi Arabia
| | - Deepak Sehgal
- Virology
Lab, Department of Life Sciences, Shiv Nadar
Institute of Eminence, Gautam Budh
Nagar 201314, India
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3
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Siedlecka M, Kublicka A, Wieliczko A, Matczuk AK. Molecular detection of avian hepatitis E virus (Orthohepevirus B) in chickens, ducks, geese, and western capercaillies in Poland. PLoS One 2022; 17:e0269854. [PMID: 35737708 PMCID: PMC9223332 DOI: 10.1371/journal.pone.0269854] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2021] [Accepted: 05/28/2022] [Indexed: 12/02/2022] Open
Abstract
Orthohepevirus B, commonly known as avian hepatitis E virus (aHEV), causes big liver and spleen disease (BLS) or hepatitis-splenomegaly syndrome (HSS) in chickens. BLS is an emerging disease among chicken flocks in several countries around the world. In our previous studies, serology and molecular biology screening revealed that chicken flocks are widely affected by aHEV in Poland. The present study, which was conducted between 2019 and 2020, aimed to investigate the prevalence of aHEV in chicken flocks and other poultry, including ducks, geese, and turkeys. A total of 307 flocks were examined. In addition, 29 samples from captive wild birds (western capercaillies, Tetrao urogallus) were analyzed. In all the investigated poultry species, except turkeys, the nucleic acid sequence covering part of the ORF1 gene of the aHEV genome was detected (34/336 samples, 10.1%). The infection rate was found to be the highest in broiler breeder chicken flocks (14/40 samples; 35%). Phylogenetic analysis of partial ORF1 gene, which encodes helicase, revealed that the obtained sequences belonged to genotypes 2 and 4, while one belonged to genotype 3. Genotype 2 was detected for the first time in domestic geese and ducks, and genotype 4 was detected for the first time in Poland. The study demonstrated the presence of aHEV among the investigated western capercaillies, suggesting that this species is susceptible to aHEV infections and biosecurity is therefore required in western capercaillie breeding facilities.
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Affiliation(s)
- Magdalena Siedlecka
- Department of Epizootiology with Clinic of Birds and Exotic Animals, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
| | - Agata Kublicka
- Department of Pathology, Division of Microbiology, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
| | - Alina Wieliczko
- Department of Epizootiology with Clinic of Birds and Exotic Animals, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
| | - Anna Karolina Matczuk
- Department of Pathology, Division of Microbiology, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
- * E-mail:
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Abstract
Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in humans. A convenient small mammalian model for basic research and antiviral testing is still greatly needed. Although a small rodent, the Mongolian gerbil, was reported to be susceptible to swine genotype-4 HEV infection, whether the previous results were reliable and consistent needs to be validated by using biologically pure HEV stocks or infectious RNA. In this study, we revisited this gerbil infection model for human HEV of genotype 1, 3, or 4 (G1, G3, or G4) by HEV reverse genetics. Gerbils inoculated intrahepatically with capped G3 HEV RNA transcripts or intraperitoneally with infectious G3 cloned HEV produced robust infection, as evidenced by presence of HEV in livers, spleens, and feces for up to 7 weeks post inoculation, seroconversion, and pathological liver lesions. Furthermore, the value of the gerbil model in antiviral testing and type I IFN in host defense was assessed. We demonstrated the effectiveness of peg-IFNα-2a and ribavirin in inhibiting HEV replication in gerbils. By treatment with two molecule inhibitors of TBK1, we also revealed a role of RIG-I like receptor-interferon regulatory factor 3 in host anti-HEV innate immune sensing in this in vivo model. Finally, susceptibility of G4 HEV was demonstrated in intrahepatically inoculated gerbils with infectious HEV RNA transcripts, whereas no evidence for G1 HEV susceptibility was found. The availability of the convenient gerbil model will greatly facilitate HEV-specific antiviral development and assess the mechanism of host immune response during HEV infection. IMPORTANCE HEV infects >20 million people annually, causing acute viral hepatitis as well as chronic hepatitis, neurological diseases, and pregnancy-associated high mortality, which require therapeutic intervention. The HEV antiviral research is largely limited by the lack of a convenient small animal model. Here we revisit the Mongolian gerbil model for three genotypes of human HEV by infectious HEV clones and recognized standards of experimental procedures. Fecal virus shedding, seroconversion, and pathological liver lesions could be detected in HEV-inoculated gerbils. We demonstrate the effectiveness and usefulness of this model in testing antiviral drugs, and in assessing the mechanism of host innate immune response upon HEV infection. This conventional rodent model will aid in future antiviral development and delineating mechanism of host immune response.
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Scholz J, Falkenhagen A, Bock CT, Johne R. Reverse genetics approaches for hepatitis E virus and related viruses. Curr Opin Virol 2020; 44:121-128. [PMID: 32818718 DOI: 10.1016/j.coviro.2020.07.004] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2020] [Revised: 06/05/2020] [Accepted: 07/06/2020] [Indexed: 12/19/2022]
Abstract
The hepatitis E virus (HEV) is the causative agent of acute and chronic hepatitis in humans. Related viruses have been found in several animal species. Reverse genetics systems (RGSs), which enable the generation of infectious virus from cloned cDNA by transfection of cultured cells or intrahepatic injection into laboratory animals, have been developed for HEV genotypes 1, 3, 4, 5 and 7 as well as for avian HEV and rat HEV. However, low virus recovery rates and slow replication in cell cultures are observed for most of the HEV types. Nevertheless, the RGSs enabled the site-directed mutagenesis of single nucleotides, deletion of genome fragments, insertion of sequence tags and a marker gene as well as the generation of chimeric viruses.
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Affiliation(s)
- Johannes Scholz
- Department Biological Safety, German Federal Institute for Risk Assessment, Berlin, Germany
| | - Alexander Falkenhagen
- Department Biological Safety, German Federal Institute for Risk Assessment, Berlin, Germany
| | - Claus-Thomas Bock
- Division of Viral Gastroenteritis and Hepatitis Pathogens and Enteroviruses, Department of Infectious Diseases, Robert Koch Institute, Berlin, Germany
| | - Reimar Johne
- Department Biological Safety, German Federal Institute for Risk Assessment, Berlin, Germany.
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Scholz J, Bächlein C, Gadicherla AK, Falkenhagen A, Tausch SH, Johne R. Establishment of a Plasmid-Based Reverse Genetics System for the Cell Culture-Adapted Hepatitis E Virus Genotype 3c Strain 47832c. Pathogens 2020; 9:pathogens9030157. [PMID: 32106549 PMCID: PMC7157446 DOI: 10.3390/pathogens9030157] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 02/20/2020] [Accepted: 02/21/2020] [Indexed: 12/13/2022] Open
Abstract
The hepatitis E virus (HEV) causes acute and chronic hepatitis in humans. Investigation of HEV replication is hampered by the lack of broadly applicable, efficient cell culture systems and tools for site-directed mutagenesis of HEV. The cell culture-adapted genotype 3c strain 47832c, which represents a typical genotype predominantly detected in Europe, has previously been used for several basic and applied research studies. Here, a plasmid-based reverse genetics system was developed for this strain, which efficiently rescued the infectious virus without the need for in vitro RNA transcription. The cotransfection of T7 RNA polymerase-expressing BSR/T7 cells with one plasmid encoding the full-length viral genome and two helper plasmids encoding vaccinia virus capping enzymes resulted in the production of infectious HEV, which could be serially passaged on A549/D3 cells. The parental and recombinant virus exhibited similar replication kinetics. A single point mutation creating an additional restriction enzyme site could be successfully introduced into the virus genome of progeny virus, indicating that the system is suitable for site-directed mutagenesis. This system is the first plasmid-based HEV reverse genetics system, as well as the first reverse genetics system for HEV genotype 3c, and should therefore be of broad use for basic and applied HEV research.
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Affiliation(s)
- Johannes Scholz
- Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany; (J.S.); (A.K.G.); (A.F.); (S.H.T.)
| | - Christine Bächlein
- Institute of Virology, Department of Infectious Diseases, University of Veterinary Medicine Hannover, Buenteweg 17, 30559 Hannover, Germany;
| | - Ashish K. Gadicherla
- Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany; (J.S.); (A.K.G.); (A.F.); (S.H.T.)
| | - Alexander Falkenhagen
- Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany; (J.S.); (A.K.G.); (A.F.); (S.H.T.)
| | - Simon H. Tausch
- Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany; (J.S.); (A.K.G.); (A.F.); (S.H.T.)
| | - Reimar Johne
- Department Biological Safety, German Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany; (J.S.); (A.K.G.); (A.F.); (S.H.T.)
- Correspondence:
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7
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Sayed IM, Elkhawaga AA, El-Mokhtar MA. In vivo models for studying Hepatitis E virus infection; Updates and applications. Virus Res 2019; 274:197765. [PMID: 31563457 DOI: 10.1016/j.virusres.2019.197765] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2019] [Revised: 09/17/2019] [Accepted: 09/20/2019] [Indexed: 02/08/2023]
Abstract
Hepatitis E virus (HEV) is the most common cause of acute viral hepatitis globally. HEV belongs to the Hepeviridae family and at least five genotypes (gt) infect humans. Several animal species are reservoirs for different HEV strains, and they are the source of infection for humans. Some HEV strains are species specific, but other strains could cross species and infect many hosts. The study of HEV infection and pathogenesis was hampered due to the lack of an in vitro and in vivo robust model system. The cell culture system has been established for certain HEV strains, especially gt3 and 4, but gt1 strains replicate poorly in vitro. To date, animal models are the best tool for studying HEV infection. Non-human primates (NHPs) and pigs are the main animal models used for studying HEV infection, but ethical and financial concerns restrict the use of NHPs in research. Therefore, new small animal models have been developed which help more progress in HEV research. In this review, we give updates on the animal models used for studying HEV infection, focusing on the applicability of each model in studying different HEV infections, cross-species infection, virus-host interaction, evaluation of anti-HEV therapies and testing potential HEV vaccines.
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Affiliation(s)
- Ibrahim M Sayed
- Department of Pathology, School of Medicine, University of California, San Diego, La Jolla, California, USA; Medical Microbiology and Immunology Department, Faculty of Medicine, Assiut University, Assiut, Egypt.
| | - Amal A Elkhawaga
- Medical Microbiology and Immunology Department, Faculty of Medicine, Assiut University, Assiut, Egypt
| | - Mohamed A El-Mokhtar
- Medical Microbiology and Immunology Department, Faculty of Medicine, Assiut University, Assiut, Egypt
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8
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Fu RM, Decker CC, Dao Thi VL. Cell Culture Models for Hepatitis E Virus. Viruses 2019; 11:E608. [PMID: 31277308 PMCID: PMC6669563 DOI: 10.3390/v11070608] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2019] [Revised: 06/24/2019] [Accepted: 06/29/2019] [Indexed: 12/14/2022] Open
Abstract
Despite a growing awareness, hepatitis E virus (HEV) remains understudied and investigations have been historically hampered by the absence of efficient cell culture systems. As a result, the pathogenesis of HEV infection and basic steps of the HEV life cycle are poorly understood. Major efforts have recently been made through the development of HEV infectious clones and cellular systems that significantly advanced HEV research. Here, we summarize these systems, discussing their advantages and disadvantages for HEV studies. We further capitalize on the need for HEV-permissive polarized cell models to better recapitulate the entire HEV life cycle and transmission.
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Affiliation(s)
- Rebecca Menhua Fu
- Schaller Research Group at Department of Infectious Diseases and Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, 69120 Heidelberg, Germany
- Heidelberg Biosciences International Graduate School, Heidelberg University, 69120 Heidelberg, Germany
| | - Charlotte Caroline Decker
- Schaller Research Group at Department of Infectious Diseases and Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, 69120 Heidelberg, Germany
- Heidelberg Biosciences International Graduate School, Heidelberg University, 69120 Heidelberg, Germany
| | - Viet Loan Dao Thi
- Schaller Research Group at Department of Infectious Diseases and Virology, Heidelberg University Hospital, Cluster of Excellence CellNetworks, 69120 Heidelberg, Germany.
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The Current Host Range of Hepatitis E Viruses. Viruses 2019; 11:v11050452. [PMID: 31108942 PMCID: PMC6563279 DOI: 10.3390/v11050452] [Citation(s) in RCA: 68] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2019] [Revised: 05/08/2019] [Accepted: 05/14/2019] [Indexed: 01/01/2023] Open
Abstract
Hepatitis E virus (HEV) is an emerging zoonotic pathogen transmitting both human to human via the fecal oral route and from animals to humans through feces, direct contact, and consumption of contaminated meat products. Understanding the host range of the virus is critical for determining where potential threats to human health may be emerging from and where potential reservoirs for viral persistence in the environment may be hiding. Initially thought to be a human specific disease endemic to developing countries, the identification of swine as a primary host for genotypes 3 and 4 HEV in industrialized countries has begun a long journey of discovering novel strains of HEV and their animal hosts. As we continue identifying new strains of HEV in disparate animal species, it is becoming abundantly clear that HEV has a broad host range and many of these HEV strains can cross between differing animal species. These cross-species transmitting strains pose many unique challenges to human health as they are often unrecognized as sources of viral transmission.
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10
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Chicken Organic Anion-Transporting Polypeptide 1A2, a Novel Avian Hepatitis E Virus (HEV) ORF2-Interacting Protein, Is Involved in Avian HEV Infection. J Virol 2019; 93:JVI.02205-18. [PMID: 30894478 DOI: 10.1128/jvi.02205-18] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2018] [Accepted: 03/10/2019] [Indexed: 12/12/2022] Open
Abstract
Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S-transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells.IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro, there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells.
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Meister TL, Bruening J, Todt D, Steinmann E. Cell culture systems for the study of hepatitis E virus. Antiviral Res 2019; 163:34-49. [PMID: 30653997 DOI: 10.1016/j.antiviral.2019.01.007] [Citation(s) in RCA: 65] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2018] [Revised: 01/08/2019] [Accepted: 01/13/2019] [Indexed: 12/26/2022]
Abstract
Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and is the leading cause of enterically-transmitted viral hepatitis worldwide. Increasing numbers of HEV infections, together with no available specific anti-HEV treatment, contributes to the pathogen's major health burden. A robust cell culture system is required for virologic studies and the development of new antiviral drugs. Unfortunately, like other hepatitis viruses, HEV is difficult to propagate in conventional cell lines. Many different cell culture systems have been tested using various HEV strains, but viral replication usually progresses very slowly, and infection with low virion counts results in non-productive HEV replication. However, recent progress involving generation of cDNA clones and passaging primary patient isolates in distinct cell lines has improved in vitro HEV propagation. This review describes various approaches to cultivate HEV in cellular and animal models and how these systems are used to study HEV infections and evaluate anti-HEV drug candidates.
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Affiliation(s)
- Toni L Meister
- Ruhr-University Bochum, Faculty of Medicine, Department of Molecular and Medical Virology, Bochum, Germany
| | - Janina Bruening
- Ruhr-University Bochum, Faculty of Medicine, Department of Molecular and Medical Virology, Bochum, Germany
| | - Daniel Todt
- Ruhr-University Bochum, Faculty of Medicine, Department of Molecular and Medical Virology, Bochum, Germany.
| | - Eike Steinmann
- Ruhr-University Bochum, Faculty of Medicine, Department of Molecular and Medical Virology, Bochum, Germany.
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12
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Matczuk AK, Ćwiek K, Wieliczko A. Avian hepatitis E virus is widespread among chickens in Poland and belongs to genotype 2. Arch Virol 2018; 164:595-599. [PMID: 30392050 PMCID: PMC6373257 DOI: 10.1007/s00705-018-4089-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2018] [Accepted: 10/17/2018] [Indexed: 12/27/2022]
Abstract
Big liver and spleen disease, caused by avian hepatitis E virus, has been reported in Poland, but the prevalence of the virus has not yet been investigated. In this study, 1034 serum samples from 57 breeder broiler and laying hen flocks were screened for the presence of anti-aHEV antibodies. In a random serology study, 56.1% of flocks were positive. Seroprevalence was higher in laying hen flocks than in broiler breeder flocks. Phylogenetic analysis of partial ORF1 and ORF2 sequences revealed that all Polish isolates belonged to genotype 2. This is the first time this genotype has been detected in Central Europe.
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Affiliation(s)
- Anna Karolina Matczuk
- Department of Pathology, Division of Microbiology, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Norwida 31, 51-375, Wrocław, Poland.
| | - Katarzyna Ćwiek
- Department of Epizootiology with Clinic of Birds and Exotic Animals, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, pl. Grunwaldzki 45, 50-366, Wrocław, Poland
| | - Alina Wieliczko
- Department of Epizootiology with Clinic of Birds and Exotic Animals, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences, pl. Grunwaldzki 45, 50-366, Wrocław, Poland
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13
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Tanggis, Kobayashi T, Takahashi M, Jirintai S, Nishizawa T, Nagashima S, Nishiyama T, Kunita S, Hayama E, Tanaka T, Mulyanto, Okamoto H. An analysis of two open reading frames (ORF3 and ORF4) of rat hepatitis E virus genome using its infectious cDNA clones with mutations in ORF3 or ORF4. Virus Res 2018; 249:16-30. [DOI: 10.1016/j.virusres.2018.02.014] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2018] [Revised: 02/14/2018] [Accepted: 02/14/2018] [Indexed: 01/13/2023]
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14
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An ethanol extract of Lysimachia mauritiana exhibits inhibitory activity against hepatitis E virus genotype 3 replication. J Microbiol 2017; 55:984-988. [PMID: 29214492 DOI: 10.1007/s12275-017-7477-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2017] [Revised: 11/14/2017] [Accepted: 11/15/2017] [Indexed: 12/30/2022]
Abstract
Hepatitis E virus (HEV) is an etiological agent of acute hepatitis E, a self-limiting disease prevalent in developing countries. HEV can cause fulminant hepatic failure with high mortality rates in pregnant women, and genotype 3 is reported to trigger chronic hepatitis in immunocompromised individuals worldwide. Screening of plant extracts for compounds with potential anti-HEV effects led to the identification of a 70% ethanol extract of Lysimachia mauritiana (LME) that interferes with replication of the swine HEV genotype 3 replicon. Furthermore, LME significantly inhibited replication of HEV genotype 3 and expression of HEV ORF2 in infected cells without exerting cytotoxic effects. Collectively, our findings demonstrate the potential utility of LME in the development of novel antiviral drugs against HEV infection.
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15
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Abstract
Viral hepatitis in poultry is a complex disease syndrome caused by several viruses belonging to different families including avian hepatitis E virus (HEV), duck hepatitis B virus (DHBV), duck hepatitis A virus (DHAV-1, -2, -3), duck hepatitis virus Types 2 and 3, fowl adenoviruses (FAdV), and turkey hepatitis virus (THV). While these hepatitis viruses share the same target organ, the liver, they each possess unique clinical and biological features. In this article, we aim to review the common and unique features of major poultry hepatitis viruses in an effort to identify the knowledge gaps and aid the prevention and control of poultry viral hepatitis. Avian HEV is an Orthohepevirus B in the family Hepeviridae that naturally infects chickens and consists of three distinct genotypes worldwide. Avian HEV is associated with hepatitis-splenomegaly syndrome or big liver and spleen disease in chickens, although the majority of the infected birds are subclinical. Avihepadnaviruses in the family of Hepadnaviridae have been isolated from ducks, snow geese, white storks, grey herons, cranes, and parrots. DHBV evolved with the host as a noncytopathic form without clinical signs and rarely progressed to chronicity. The outcome for DHBV infection varies by the host's ability to elicit an immune response and is dose and age dependent in ducks, thus mimicking the pathogenesis of human hepatitis B virus (HBV) infections and providing an excellent animal model for human HBV. DHAV is a picornavirus that causes a highly contagious virus infection in ducks with up to 100% flock mortality in ducklings under 6 wk of age, while older birds remain unaffected. The high morbidity and mortality has an economic impact on intensive duck production farming. Duck hepatitis virus Types 2 and 3 are astroviruses in the family of Astroviridae with similarity phylogenetically to turkey astroviruses, implicating the potential for cross-species infections between strains. Duck astrovirus (DAstV) causes acute, fatal infections in ducklings with a rapid decline within 1-2 hr and clinical and pathologic signs virtually indistinguishable from DHAV. DAstV-1 has only been recognized in the United Kingdom and recently in China, while DAstV-2 has been reported in ducks in the United States. FAdV, the causative agent of inclusion body hepatitis, is a Group I avian adenovirus in the genus Aviadenovirus. The affected birds have a swollen, friable, and discolored liver, sometimes with necrotic or hemorrhagic foci. Histologic lesions include multifocal necrosis of hepatocytes and acute hepatitis with intranuclear inclusion bodies in the nuclei of the hepatocytes. THV is a picornavirus that is likely the causative agent of turkey viral hepatitis. Currently there are more questions than answers about THV, and the pathogenesis and clinical impacts remain largely unknown. Future research in viral hepatic diseases of poultry is warranted to develop specific diagnostic assays, identify suitable cell culture systems for virus propagation, and develop effective vaccines.
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Affiliation(s)
- Danielle M Yugo
- A Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1981 Kraft Drive, Blacksburg, VA 24061-0913
| | - Ruediger Hauck
- B Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA 95616
| | - H L Shivaprasad
- C California Animal Health and Food Safety Laboratory System, University of California-Davis, Tulare, CA 93274
| | - Xiang-Jin Meng
- A Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, 1981 Kraft Drive, Blacksburg, VA 24061-0913
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Production of infectious dromedary camel hepatitis E virus by a reverse genetic system: Potential for zoonotic infection. J Hepatol 2016; 65:1104-1111. [PMID: 27449916 DOI: 10.1016/j.jhep.2016.07.013] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/03/2016] [Revised: 07/03/2016] [Accepted: 07/07/2016] [Indexed: 01/20/2023]
Abstract
BACKGROUND & AIMS The pathogenicity, epidemiology and replication mechanism of dromedary camel hepatitis E virus (DcHEV), a novel hepatitis E virus (HEV), has been unclear. Here we used a reverse genetic system to produce DcHEV and examined the possibility of zoonotic infection. METHODS Capped genomic RNA derived from a synthetic DcHEV cDNA was transfected into human hepatocarcinoma cells PLC/PRF/5. The DcHEV capsid protein and RNA were detected by an enzyme-linked immunosorbent assay (ELISA) or RT-qPCR. A neutralization test for DcHEV was carried out by using antisera against HEV-like particles. DcHEV was used to inoculate two cynomolgus monkeys to examine the potential for cross-species infection. RESULTS The transfection of PLC/PRF/5 cells with capped DcHEV RNA resulted in the production of infectious DcHEV. The genome sequence analysis demonstrated that both nucleotide and amino acid changes accumulated during the passages in PLC/PRF/5 cells. The cynomolgus monkeys showed serological signs of infection when DcHEV was intravenously inoculated. DcHEV was neutralized by not only anti-DcHEV-LPs antibody, but also anti-genotype 1 (G1), G3 and G4 HEV-LPs antibodies. Moreover, the monkeys immunized with DcHEV escaped the G3 HEV challenge, indicating that the serotype of DcHEV is similar to those of other human HEVs. CONCLUSIONS Infectious DcHEV was produced using a reverse genetic system and propagated in PLC/PRF/5 cells. The antigenicity and immunogenicity of DcHEV are similar to those of G1, G3 and G4 HEV. DcHEV was experimentally transmitted to primates, demonstrating the possibility of a zoonotic infection by DcHEV. LAY SUMMARY Dromedary camel hepatitis E virus (DcHEV) was produced by a reverse genetic system and grows well in PLC/PRF/5 cells. Cynomolgus monkeys experimentally infected with DcHEV indicated serological signs of infection, suggesting that DcHEV has the potential to cause zoonotic HEV infection.
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Doceul V, Bagdassarian E, Demange A, Pavio N. Zoonotic Hepatitis E Virus: Classification, Animal Reservoirs and Transmission Routes. Viruses 2016; 8:v8100270. [PMID: 27706110 PMCID: PMC5086606 DOI: 10.3390/v8100270] [Citation(s) in RCA: 182] [Impact Index Per Article: 20.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2016] [Accepted: 09/22/2016] [Indexed: 12/11/2022] Open
Abstract
During the past ten years, several new hepatitis E viruses (HEVs) have been identified in various animal species. In parallel, the number of reports of autochthonous hepatitis E in Western countries has increased as well, raising the question of what role these possible animal reservoirs play in human infections. The aim of this review is to present the recent discoveries of animal HEVs and their classification within the Hepeviridae family, their zoonotic and species barrier crossing potential, and possible use as models to study hepatitis E pathogenesis. Lastly, this review describes the transmission pathways identified from animal sources.
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Affiliation(s)
- Virginie Doceul
- French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Animal Health Laboratory, UMR (joint research unit) 1161 Virology, 94701 Maisons-Alfort, France.
- French National Institute for Agricultural Research (INRA), UMR (joint research unit) 1161 Virology, 94700 Maisons-Alfort, France.
- Association of Universities and High Education Institutions (ComUE), Paris-Est Créteil Val-de-Marne University, National Veterinary School, UMR (joint research unit) 1161 Virology, 94700 Maisons-Alfort, France.
| | - Eugénie Bagdassarian
- French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Animal Health Laboratory, UMR (joint research unit) 1161 Virology, 94701 Maisons-Alfort, France.
- French National Institute for Agricultural Research (INRA), UMR (joint research unit) 1161 Virology, 94700 Maisons-Alfort, France.
- Association of Universities and High Education Institutions (ComUE), Paris-Est Créteil Val-de-Marne University, National Veterinary School, UMR (joint research unit) 1161 Virology, 94700 Maisons-Alfort, France.
| | - Antonin Demange
- French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Animal Health Laboratory, UMR (joint research unit) 1161 Virology, 94701 Maisons-Alfort, France.
- French National Institute for Agricultural Research (INRA), UMR (joint research unit) 1161 Virology, 94700 Maisons-Alfort, France.
- Association of Universities and High Education Institutions (ComUE), Paris-Est Créteil Val-de-Marne University, National Veterinary School, UMR (joint research unit) 1161 Virology, 94700 Maisons-Alfort, France.
| | - Nicole Pavio
- French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Animal Health Laboratory, UMR (joint research unit) 1161 Virology, 94701 Maisons-Alfort, France.
- French National Institute for Agricultural Research (INRA), UMR (joint research unit) 1161 Virology, 94700 Maisons-Alfort, France.
- Association of Universities and High Education Institutions (ComUE), Paris-Est Créteil Val-de-Marne University, National Veterinary School, UMR (joint research unit) 1161 Virology, 94700 Maisons-Alfort, France.
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Abstract
INTRODUCTION Infection with hepatitis E virus (HEV) is the commonest cause of acute hepatitis worldwide. HEV was discovered in 1980s and is known to have small non-enveloped virions with single-stranded RNA genome of positive polarity. In recent years. In recent years, availability of new information has changed our understanding of this virus and the pathogenesis of the related disease. AREAS COVERED This article reviews the current knowledge about structure, genomic organization, taxonomy, genetic epidemiology, host specificity and replication of the human HEV and of various closely-related viruses that infect other animals. In addition, the models available for the study of HEV infection, the available information on the pathogenesis of this infection and the techniques available for its diagnosis are also reviewed. Expert commentary: A circulating, enveloped form of the human HEV has been recently recognized. Originally believed to naturally infect only humans and possibly primates, HEV-like viruses are now known to infect several vertebrate animals. Based on this, phylogenetic classification of these viruses has recently been revised. In vitro replicons and infection systems have been developed, which have improved our understanding about the virus and the pathogenesis of infection with it. Recent development of mouse models with chimeric livers that contain human hepatocytes provides another avenue for further advancement of this knowledge.
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Affiliation(s)
- Rakesh Aggarwal
- a Department of Gastroenterology , Sanjay Gandhi Postgraduate Institute of Medical Sciences , Lucknow , India
| | - Amit Goel
- a Department of Gastroenterology , Sanjay Gandhi Postgraduate Institute of Medical Sciences , Lucknow , India
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Khuroo MS, Khuroo MS, Khuroo NS. Hepatitis E: Discovery, global impact, control and cure. World J Gastroenterol 2016; 22:7030-7045. [PMID: 27610014 PMCID: PMC4988308 DOI: 10.3748/wjg.v22.i31.7030] [Citation(s) in RCA: 114] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2016] [Revised: 06/10/2016] [Accepted: 07/06/2016] [Indexed: 02/06/2023] Open
Abstract
Hepatitis E was identified as an epidemic of non-A, non-B hepatitis from Kashmir, India in 1978. Hepatitis E virus (HEV), the etiological agent is the sole member of family Hepeviridae. The virus has marked heterogeneity and infects many animals like bats, camel, chicken, deer, boar, mongoose, pigs, rats, rabbit and cutthroat trout. Hepatitis E is a disease with a major global impact and has two distinct epidemiological patterns. Hepatitis E is an imperative health issue in developing nations, transmitted through sullied water and happens most every now in young adults. The disease is particularly severe during pregnancy and in people with underlying liver cirrhosis. Autochthonous hepatitis E is increasingly recognized in developed countries. The virus infects domestic pigs, wild boar and Sika deer in these countries. HEV infections in humans occur by eating the undercooked game flesh, raw liver from supermarkets and Figatelli sausages. Blood transfusion-associated HEV infections occur in many countries and screening of donors for HEV RNA is under consideration. Hepatitis E causes a number of extrahepatic diseases, including a wide spectrum of neurological syndromes. HEV genotype 3 causes prolonged viremia, chronic hepatitis, liver fibrosis and cirrhosis in organ transplant patients. The virus is amenable to ribavirin monotherapy and most patients clear the virus in a few weeks. Hepatitis E vaccine -239, marketed in China, has shown high efficacy with sustained protection for over four years.
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Zhang X, Bilic I, Marek A, Glösmann M, Hess M. C-Terminal Amino Acids 471-507 of Avian Hepatitis E Virus Capsid Protein Are Crucial for Binding to Avian and Human Cells. PLoS One 2016; 11:e0153723. [PMID: 27073893 PMCID: PMC4830555 DOI: 10.1371/journal.pone.0153723] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2016] [Accepted: 04/01/2016] [Indexed: 01/12/2023] Open
Abstract
The infection of chickens with avian Hepatitis E virus (avian HEV) can be asymptomatic or induces clinical signs characterized by increased mortality and decreased egg production in adult birds. Due to the lack of an efficient cell culture system for avian HEV, the interaction between virus and host cells is still barely understood. In this study, four truncated avian HEV capsid proteins (ORF2-1 – ORF2-4) with an identical 338aa deletion at the N-terminus and gradual deletions from 0, 42, 99 and 136aa at the C-terminus, respectively, were expressed and used to map the possible binding site within avian HEV capsid protein. Results from the binding assay showed that three truncated capsid proteins attached to avian LMH cells, but did not penetrate into cells. However, the shortest construct, ORF2-4, lost the capability of binding to cells suggesting that the presence of amino acids 471 to 507 of the capsid protein is crucial for the attachment. The construct ORF2-3 (aa339-507) was used to study the potential binding of avian HEV capsid protein to human and other avian species. It could be demonstrated that ORF2-3 was capable of binding to QT-35 cells from Japanese quail and human HepG2 cells but failed to bind to P815 cells. Additionally, chicken serum raised against ORF2-3 successfully blocked the binding to LMH cells. Treatment with heparin sodium salt or sodium chlorate significantly reduced binding of ORF2-3 to LMH cells. However, heparinase II treatment of LMH cells had no effect on binding of the ORF2-3 construct, suggesting a possible distinct attachment mechanism of avian as compared to human HEV. For the first time, interactions between avian HEV capsid protein and host cells were investigated demonstrating that aa471 to 507 of the capsid protein are needed to facilitate interaction with different kind of cells from different species.
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Affiliation(s)
- Xinquan Zhang
- Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria
| | - Ivana Bilic
- Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria
| | - Ana Marek
- Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria
| | - Martin Glösmann
- VetCore Facility for Research, University of Veterinary Medicine, Vienna, Austria
| | - Michael Hess
- Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine, Vienna, Austria
- * E-mail:
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21
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Li TC, Yang T, Yoshizaki S, Ami Y, Suzaki Y, Ishii K, Haga K, Nakamura T, Ochiai S, Takaji W, Johne R. Construction and characterization of an infectious cDNA clone of rat hepatitis E virus. J Gen Virol 2015; 96:1320-1327. [PMID: 25634930 DOI: 10.1099/vir.0.000072] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2014] [Accepted: 01/24/2015] [Indexed: 12/13/2022] Open
Abstract
Rat hepatitis E virus (HEV) is related to human HEV and has been detected in wild rats worldwide. Here, the complete genome of rat HEV strain R63/DEU/2009 was cloned downstream of the T7 RNA polymerase promoter and capped genomic RNA generated by in vitro transcription was injected into nude rats. Rat HEV RNA could be detected in serum and faeces of rats injected intrahepatically, but not in those injected intravenously. Rat HEV RNA-positive faecal suspension was intravenously inoculated into nude rats and Wistar rats leading to rat HEV RNA detection in serum and faeces of nude rats, and to seroconversion in Wistar rats. In addition, rat HEV was isolated in PLC/PRF/5 cells from the rat HEV RNA-positive faecal suspension of nude rats and then passaged. The cell culture supernatant was infectious for nude rats. Genome analysis identified nine point mutations of the cell-culture-passaged virus in comparison with the originally cloned rat HEV genome. The results indicated that infectious rat HEV could be generated from the cDNA clone. As rats are widely used and well-characterized laboratory animals, studies on genetically engineered rat HEV may provide novel insights into organ tropism, replication and excretion kinetics as well as immunological changes induced by hepeviruses.
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Affiliation(s)
- Tian-Cheng Li
- Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan
| | - Tingting Yang
- Department of Clinical Laboratory, Affiliated Hospital of Qingdao University Medical College, Jiangsu Road 16, Qingdao 266003, PR China
| | - Sayaka Yoshizaki
- Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan
| | - Yasushi Ami
- Division of Experimental Animals Research, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan
| | - Yuriko Suzaki
- Division of Experimental Animals Research, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan
| | - Koji Ishii
- Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan
| | - Kei Haga
- Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan
| | - Tomofumi Nakamura
- Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan
| | - Susumu Ochiai
- Research and Production Technology Department, The Research Foundation for Microbial Diseases of Osaka University, 5-34-4 Kumegawacho Higashimurayama-shi, Tokyo 189-0003, Japan
| | - Wakita Takaji
- Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashi-murayama, Tokyo 208-0011, Japan
| | - Reimar Johne
- Department Biological Safety, Federal Institute for Risk Assessment, Max-Dohrn-Straße 8-10, 10589 Berlin, Germany
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Park SJ, Lee BW, Moon HW, Sung HW, Yoon BI, Meng XJ, Kwon HM. Construction of an infectious cDNA clone of genotype 1 avian hepatitis E virus: characterization of its pathogenicity in broiler breeders and demonstration of its utility in studying the role of the hypervariable region in virus replication. J Gen Virol 2015; 96:1015-1026. [PMID: 25593160 DOI: 10.1099/vir.0.000045] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2014] [Accepted: 01/08/2015] [Indexed: 02/07/2023] Open
Abstract
A full-length infectious cDNA clone of the genotype 1 Korean avian hepatitis E virus (avian HEV) (pT11-aHEV-K) was constructed and its infectivity and pathogenicity were investigated in leghorn male hepatoma (LMH) chicken cells and broiler breeders. We demonstrated that capped RNA transcripts from the pT11-aHEV-K clone were translation competent when transfected into LMH cells and infectious when injected intrahepatically into the livers of chickens. Gross and microscopic pathological lesions underpinned the avian HEV infection and helped characterize its pathogenicity in broiler breeder chickens. The avian HEV genome contains a hypervariable region (HVR) in ORF1. To demonstrate the utility of the avian HEV infectious clone, several mutants with various deletions in and beyond the known HVR were derived from the pT11-aHEV-K clone. The HVR-deletion mutants were replication competent in LMH cells, although the deletion mutants extending beyond the known HVR were non-viable. By using the pT11-aHEV-K infectious clone as the backbone, an avian HEV luciferase reporter replicon and HVR-deletion mutant replicons were also generated. The luciferase assay results of the reporter replicon and its mutants support the data obtained from the infectious clone and its derived mutants. To further determine the effect of HVR deletion on virus replication, the capped RNA transcripts from the wild-type pT11-aHEV-K clone and its mutants were injected intrahepatically into chickens. The HVR-deletion mutants that were translation competent in LMH cells displayed in chickens an attenuation phenotype of avian HEV infectivity, suggesting that the avian HEV HVR is important in modulating the virus infectivity and pathogenicity.
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Affiliation(s)
- Soo-Jeong Park
- Department of Veterinary Microbiology, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea
| | - Byung-Woo Lee
- Department of Veterinary Microbiology, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea
| | - Hyun-Woo Moon
- Department of Veterinary Microbiology, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea
| | - Haan Woo Sung
- Department of Veterinary Microbiology, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea
| | - Byung-Il Yoon
- Department of Veterinary Microbiology, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea
| | - Xiang-Jin Meng
- Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0913, USA
| | - Hyuk Moo Kwon
- Department of Veterinary Microbiology, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea
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Johne R, Dremsek P, Reetz J, Heckel G, Hess M, Ulrich RG. Hepeviridae: an expanding family of vertebrate viruses. INFECTION GENETICS AND EVOLUTION 2014; 27:212-29. [PMID: 25050488 DOI: 10.1016/j.meegid.2014.06.024] [Citation(s) in RCA: 111] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/06/2014] [Revised: 06/25/2014] [Accepted: 06/26/2014] [Indexed: 12/15/2022]
Abstract
The hepatitis E virus (HEV) was first identified in 1990, although hepatitis E-like diseases in humans have been recorded for a long time dating back to the 18th century. The HEV genotypes 1-4 have been subsequently detected in human hepatitis E cases with different geographical distribution and different modes of transmission. Genotypes 3 and 4 have been identified in parallel in pigs, wild boars and other animal species and their zoonotic potential has been confirmed. Until 2010, these genotypes along with avian HEV strains infecting chicken were the only known representatives of the family Hepeviridae. Thereafter, additional HEV-related viruses have been detected in wild boars, distinct HEV-like viruses were identified in rats, rabbit, ferret, mink, fox, bats and moose, and a distantly related agent was described from closely related salmonid fish. This review summarizes the characteristics of the so far known HEV-like viruses, their phylogenetic relationship, host association and proposed involvement in diseases. Based on the reviewed knowledge, a suggestion for a new taxonomic grouping scheme of the viruses within the family Hepeviridae is presented.
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Affiliation(s)
- Reimar Johne
- Federal Institute for Risk Assessment, Berlin, Germany
| | - Paul Dremsek
- Friedrich-Loeffler-Institut, Institute for Novel and Emerging Infectious Diseases, Greifswald-Insel Riems, Germany
| | - Jochen Reetz
- Federal Institute for Risk Assessment, Berlin, Germany
| | - Gerald Heckel
- University of Bern, Institute of Ecology and Evolution, Bern, Switzerland; Swiss Institute of Bioinformatics, Genopode, Lausanne, Switzerland
| | - Michael Hess
- Clinic for Poultry and Fish Medicine, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine (Vetmeduni Vienna), Vienna, Austria
| | - Rainer G Ulrich
- Friedrich-Loeffler-Institut, Institute for Novel and Emerging Infectious Diseases, Greifswald-Insel Riems, Germany.
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Troxler S, Pać K, Prokofieva I, Liebhart D, Chodakowska B, Furmanek D, Hess M. Subclinical circulation of avian hepatitis E virus within a multiple-age rearing and broiler breeder farm indicates persistence and vertical transmission of the virus. Avian Pathol 2014; 43:310-8. [DOI: 10.1080/03079457.2014.924616] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
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Si F, Shi B, Wang X, Zhu Y, Liu X, Yang Q, Li Z. Construction of an infectious cDNA clone of a swine genotype 3 HEV strain isolated in Shanghai, China. Intervirology 2014; 57:74-82. [PMID: 24480875 DOI: 10.1159/000357192] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2012] [Accepted: 10/28/2013] [Indexed: 11/19/2022] Open
Abstract
OBJECTIVES Infectious cDNA clones are important tools for studying molecular mechanisms in RNA viruses. The aim of this study was to construct an infectious cDNA clone for SAAS-JDY5, which is a genotype 3 HEV strain of swine origin. METHODS Construction employed overlapping PCR and restriction analysis to ligate nine cDNA fragments into a full-length cDNA clone containing 14 mutations compared to the consensus HEV genome sequence. Megaprimer PCR-directed mutagenesis restored nine non-silent mutations back to the consensus sequence while the other five silent mutations were maintained as genetic markers. RESULTS HEV proteins were identified by an immunofluorescence assay in Huh7 cells infected with capped RNA transcripts of the full-length cDNA clone, while HEV viremia, fecal HEV RNA and seroconversion were recorded in inoculated Sprague-Dawley rats. CONCLUSIONS Our data confirmed the successful construction of an infectious cDNA clone of swine HEV strain pGEM4z-SAAS-JDY5, and support the use of rats as an HEV infectious model.
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Affiliation(s)
- Fusheng Si
- Institute of Animal Science and Veterinary Medicine and Shanghai Key Laboratory of Agricultural Genetics and Breeding, Shanghai Academy of Agricultural Sciences (SAAS), Shanghai, PR China
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Zhao Q, Li S, Yu H, Xia N, Modis Y. Virus-like particle-based human vaccines: quality assessment based on structural and functional properties. Trends Biotechnol 2013; 31:654-63. [PMID: 24125746 DOI: 10.1016/j.tibtech.2013.09.002] [Citation(s) in RCA: 147] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2013] [Revised: 08/20/2013] [Accepted: 09/03/2013] [Indexed: 12/18/2022]
Abstract
Human vaccines against three viruses use recombinant virus-like particles (VLPs) as the antigen: hepatitis B virus, human papillomavirus, and hepatitis E virus. VLPs are excellent prophylactic vaccine antigens because they are self-assembling bionanoparticles (20 to 60 nm in diameter) that expose multiple epitopes on their surface and faithfully mimic the native virions. Here we summarize the long journey of these vaccines from bench to patients. The physical properties and structural features of each recombinant VLP vaccine are described. With the recent licensure of Hecolin against hepatitis E virus adding a third disease indication to prophylactic VLP-based vaccines, we review how the crucial quality attributes of VLP-based human vaccines against all three disease indications were assessed, controlled, and improved during bioprocessing through an array of structural and functional analyses.
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Affiliation(s)
- Qinjian Zhao
- National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Public Health, Xiamen University, Xiamen, Fujian 361102, People's Republic of China.
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The PSAP motif within the ORF3 protein of an avian strain of the hepatitis E virus is not critical for viral infectivity in vivo but plays a role in virus release. J Virol 2012; 86:5637-46. [PMID: 22438540 DOI: 10.1128/jvi.06711-11] [Citation(s) in RCA: 40] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
The ORF3 protein of hepatitis E virus (HEV) is a multifunctional protein important for virus replication. The ORF3 proteins from human, swine, and avian strains of HEV contain a conserved PXXP amino acid motif, resembling either Src homology 3 (SH3) cell signaling interaction motifs or "late domains" involved in host cell interactions aiding in particle release. Using an avian strain of HEV, we determined the roles of the conserved prolines within the PREPSAPP motif in HEV replication and infectivity in Leghorn male hepatoma (LMH) chicken liver cells and in chickens. Each proline was changed to alanine to produce 8 avian HEV mutants containing single mutations (P64, P67, P70, and P71 to A), double mutations (P64/67A, P64/70A, and P67/70A), and triple mutations (P64/67/70A). The results showed that avian HEV mutants are replication competent in vitro, and none of the prolines in the PXXPXXPP motif are essential for infectivity in vivo; however, the second and third prolines appear to aid in fecal virus shedding, suggesting that the PSAP motif, but not the PREP motif, is involved in virus release. We also showed that the PSAP motif interacts with the host protein tumor suppressor gene 101 (TSG101) and that altering any proline within the PSAP motif disrupts this interaction. However, we showed that the ORF2 protein expressed in LMH cells is efficiently released from the cells in the absence of ORF3 and that coexpression of ORF2 and ORF3 did not act synergistically in this release, suggesting that another factor(s) such as ORF1 or viral genomic RNA may be necessary for proper particle release.
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Pudupakam RS, Kenney SP, Córdoba L, Huang YW, Dryman BA, LeRoith T, Pierson FW, Meng XJ. Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication. J Virol 2011; 85:10031-40. [PMID: 21775444 PMCID: PMC3196386 DOI: 10.1128/jvi.00763-11] [Citation(s) in RCA: 59] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2011] [Accepted: 07/07/2011] [Indexed: 12/15/2022] Open
Abstract
The RNA genome of the hepatitis E virus (HEV) contains a hypervariable region (HVR) in ORF1 that tolerates small deletions with respect to infectivity. To further investigate the role of the HVR in HEV replication, we constructed a panel of mutants with overlapping deletions in the N-terminal, central, and C-terminal regions of the HVR by using a genotype 1 human HEV luciferase replicon and analyzed the effects of deletions on viral RNA replication in Huh7 cells. We found that the replication levels of the HVR deletion mutants were markedly reduced in Huh7 cells, suggesting a role of the HVR in viral replication efficiency. To further verify the results, we constructed HVR deletion mutants by using a genetically divergent, nonmammalian avian HEV, and similar effects on viral replication efficiency were observed when the avian HEV mutants were tested in LMH cells. Furthermore, the impact of complete HVR deletion on virus infectivity was tested in chickens, using an avian HEV mutant with a complete HVR deletion. Although the deletion mutant was still replication competent in LMH cells, the complete HVR deletion resulted in a loss of avian HEV infectivity in chickens. Since the HVR exhibits extensive variations in sequence and length among different HEV genotypes, we further examined the interchangeability of HVRs and demonstrated that HVR sequences are functionally exchangeable between HEV genotypes with regard to viral replication and infectivity in vitro, although genotype-specific HVR differences in replication efficiency were observed. The results showed that although the HVR tolerates small deletions with regard to infectivity, it may interact with viral and host factors to modulate the efficiency of HEV replication.
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Affiliation(s)
- R. S. Pudupakam
- Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
| | - Scott P. Kenney
- Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
| | - Laura Córdoba
- Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
| | - Yao-Wei Huang
- Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
| | - Barbara A. Dryman
- Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
| | - Tanya LeRoith
- Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
| | - F. William Pierson
- Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
| | - Xiang-Jin Meng
- Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia
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Suppiah S, Zhou Y, Frey TK. Lack of processing of the expressed ORF1 gene product of hepatitis E virus. Virol J 2011; 8:245. [PMID: 21595991 PMCID: PMC3116494 DOI: 10.1186/1743-422x-8-245] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/22/2011] [Accepted: 05/20/2011] [Indexed: 01/25/2023] Open
Abstract
Background Proteolytic processing is a common mechanism among plus strand RNA viruses and the replicases of all plus strand RNA viruses of animals thus far characterized undergo such processing. The replicase proteins of hepatitis E virus (HEV) are encoded by ORF1. A previous report published by our group [1] provided data that processing potentially occurred when ORF1 (Burma strain; genotype 1) was expressed using a vaccinia virus-based expression system. Findings To further test for processing and to rule out artifacts associated with the expression system, ORF1 was re-expressed using a plasmid-based expression vector with the result that the previous processing profile could not be confirmed. When ORF1 from an HEV infectious cDNA clone (US swine strain; genotype 3) was expressed using the plasmid-based system, the only species detected was the 185 kDa precursor of ORF1. A putative papain-like cysteine protease [2] had been predicted within ORF1 using the original HEV genomic sequence. However, analysis of subsequent ORF1 sequences from a large number of HEV isolates reveals that this protease motif is not conserved. Conclusions The expressed HEV ORF1 gene product does not undergo proteolytic processing, indicating that the replicase precursor of HEV is potentially unique in this regard.
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Affiliation(s)
- Suganthi Suppiah
- Department of Biology, Georgia State University, Atlanta, GA, USA
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TaqMan real-time reverse transcription-PCR assay for universal detection and quantification of avian hepatitis E virus from clinical samples in the presence of a heterologous internal control RNA. J Clin Microbiol 2011; 49:1339-46. [PMID: 21307216 DOI: 10.1128/jcm.01626-10] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
Avian hepatitis E virus (HEV) isolates could be separated into at least three genotypes. In this study, the development of the first duplex TaqMan real-time reverse transcription-PCR (RT-PCR) assay for detection and quantification of avian HEV is presented. Primers and probes binding within relatively conserved open reading frame 3 (ORF3) were designed. Tenfold dilution series of in vitro-transcribed avian HEV RNA were used as the standard for quantification. A 712-bp region of the green fluorescent protein gene was transcribed in vitro and used as a heterologous internal control for both RNA isolation and real-time RT-PCR. The duplex real-time RT-PCR for avian HEV had an efficiency of 1.04, a regression squared value of 0.996, and a sensitivity of approximately 3.6 × 10(3) copies per reaction mixture when in vitro-transcribed RNA was used as the template. The presence of in vitro-transcribed heterologous internal control RNA did not affect amplification of avian HEV RNA compared to that achieved by the single assay. The sensitivity of the real-time RT-PCR assay was comparable to that of conventional RT-PCR, and it was shown to be highly specific, as tissues from uninfected chickens, mammalian HEVs, and other viral genomes did not produce positive signals. All tested field samples with virus belonging to different avian HEV genotypes were successfully detected with this new duplex TaqMan real-time RT-PCR assay.
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The nucleotides on the stem-loop RNA structure in the junction region of the hepatitis E virus genome are critical for virus replication. J Virol 2010; 84:13040-4. [PMID: 20943962 DOI: 10.1128/jvi.01475-10] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The roles of conserved nucleotides on the stem-loop (SL) structure in the intergenic region of the hepatitis E virus (HEV) genome in virus replication were determined by using Huh7 cells transfected with HEV SL mutant replicons containing reporter genes. One or two nucleotide mutations of the AGA motif on the loop significantly reduced HEV replication, and three or more nucleotide mutations on the loop abolished HEV replication. Mutations on the stem and of the subgenome start sequence also significantly inhibited HEV replication. The results indicated that both the sequence and the SL structure in the junction region play important roles in HEV replication.
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Kwon HM, LeRoith T, Pudupakam RS, Pierson FW, Huang YW, Dryman BA, Meng XJ. Construction of an infectious cDNA clone of avian hepatitis E virus (avian HEV) recovered from a clinically healthy chicken in the United States and characterization of its pathogenicity in specific-pathogen-free chickens. Vet Microbiol 2010; 147:310-9. [PMID: 20708350 DOI: 10.1016/j.vetmic.2010.07.016] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2010] [Revised: 07/06/2010] [Accepted: 07/15/2010] [Indexed: 01/10/2023]
Abstract
A genetically distinct strain of avian hepatitis E virus (avian HEV-VA strain) was isolated from a healthy chicken in Virginia, and thus it is important to characterize and compare its pathogenicity with the prototype strain (avian HEV-prototype) isolated from a diseased chicken. Here we first constructed an infectious clone of the avian HEV-VA strain. Capped RNA transcripts from the avian HEV-VA clone were replication-competent after transfection of LMH chicken liver cells. Chickens inoculated intrahepatically with RNA transcripts of avian HEV-VA clone developed active infection as evidenced by fecal virus shedding, viremia, and seroconversion. To characterize the pathogenicity, RNA transcripts of both avian HEV-VA and avian HEV-prototype clones were intrahepatically inoculated into the livers of chickens. Avian HEV RNA was detected in feces, serum and bile samples from 10/10 avian HEV-VA-inoculated and 9/9 avian HEV-prototype-inoculated chickens although seroconversion occurred only in some chickens during the experimental period. The histopathological lesion scores were lower for avian HEV-VA group than avian HEV-prototype group in the liver at 3 and 5 weeks post-inoculation (wpi) and in the spleen at 3 wpi, although the differences were not statistically significant. The liver/body weight ratio, indicative of liver enlargement, of both avian HEV-VA and avian HEV-prototype groups were significantly higher than that of the control group at 5 wpi. Overall, the avian HEV-VA strain still induces histological liver lesions even though it was isolated from a healthy chicken. The results also showed that intrahepatic inoculation of chickens with RNA transcripts of avian HEV infectious clone may serve as an alternative for live virus in animal pathogenicity studies.
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Affiliation(s)
- Hyuk Moo Kwon
- Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0913, USA
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Deletions of the hypervariable region (HVR) in open reading frame 1 of hepatitis E virus do not abolish virus infectivity: evidence for attenuation of HVR deletion mutants in vivo. J Virol 2008; 83:384-95. [PMID: 18945785 DOI: 10.1128/jvi.01854-08] [Citation(s) in RCA: 70] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Hepatitis E virus (HEV) is an important human pathogen, although little is known about its biology and replication. Comparative sequence analysis revealed a hypervariable region (HVR) with extensive sequence variations in open reading frame 1 of HEV. To elucidate the role of the HVR in HEV replication, we first constructed two HVR deletion mutants, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a genotype 1 human HEV replicon. Evidence of HEV replication was detected in Huh7 cells transfected with RNA transcripts from mutant hHVRd2, as evidenced by expression of enhanced green fluorescent protein. To confirm the in vitro results, we constructed three avian HEV mutants with various HVR deletions: mutants aHVRd1, with deletion of aa 557 to 585 (Delta557-585); aHVRd2 (Delta612-641); and aHVRd3 (Delta557-641). Chickens intrahepatically inoculated with capped RNA transcripts from mutants aHVRd1 and aHVRd2 developed active viral infection, as evidenced by seroconversion, viremia, and fecal virus shedding, although mutant aHVRd3, with complete HVR deletion, was apparently attenuated in chickens. To further verify the results, we constructed four additional HVR deletion mutants using the genotype 3 swine HEV as the backbone. Mutants sHVRd2 (Delta722-781), sHVRd3 (Delta735-765), and sHVRd4 (Delta712-765) were shown to tolerate deletions and were infectious in pigs intrahepatically inoculated with capped RNA transcripts from the mutants, whereas mutant sHVRd1 (Delta712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype in infected pigs. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion.
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Morrow CJ, Samu G, Mátrai E, Klausz Á, Wood AM, Richter S, Jaskulska B, Hess M. Avian hepatitis E virus infection and possible associated clinical disease in broiler breeder flocks in Hungary. Avian Pathol 2008; 37:527-35. [DOI: 10.1080/03079450802356946] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Affiliation(s)
- Chris J. Morrow
- a Bioproperties Ltd , 36 Charter St, Ringwood , Victoria , Australia , 3134
| | - Győző Samu
- b Bábolna Breeding Farms KFT , H-2943 , Meszaros u. 1 , Hungary
| | - Eszter Mátrai
- b Bábolna Breeding Farms KFT , H-2943 , Meszaros u. 1 , Hungary
| | - Ákos Klausz
- c Aviagen Kft, Győr, Fehérvári u. 75 , H-9028 , Hungary
| | - Alasdair M. Wood
- d Veterinary Laboratories Agency, Pentlands Science Park, Bush Loan , Penicuik , Midlothian , EH26 0PZ , UK
| | - Susanne Richter
- e Department for Electronmicroscopy , Institute for Veterinary Disease Control , Robert Kochgasse 17 , A-2340 , Mödling , Austria
| | - Barbara Jaskulska
- f Clinic for Avian, Reptile and Fish Medicine, Veterinärplatz 1 , University of Veterinary Medicine , A-1210 , Vienna , Austria
| | - Michael Hess
- f Clinic for Avian, Reptile and Fish Medicine, Veterinärplatz 1 , University of Veterinary Medicine , A-1210 , Vienna , Austria
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Abstract
Hepatitis E virus (HEV) is the aetiological agent of non-HAV enterically transmitted hepatitis. It is the major cause of sporadic as well as epidemic hepatitis, which is no longer confined to Asia and developing countries but has also become a concern of the developed nations. In the Indian subcontinent, it accounts for 30-60% of sporadic hepatitis. It is generally accepted that hepatitis E is mostly self-limited and never progresses to chronicity. It has a higher mortality in pregnant women where the disease condition is accentuated with the development of fulminant liver disease. Currently, no antiviral drug or vaccine is licensed for HEV, although a vaccine candidate is in clinical trials. HEV genome is 7.2kb in size with three open reading frames (ORFs) and 5' and 3' cis acting elements, which have important roles to play in HEV replication and transcription. ORF1 codes for methyl transferase, protease, helicase and replicase; ORF2 codes for the capsid protein and ORF3 for a protein of undefined function. HEV has recently been classified in the genus Hepevirus of the family Hepeviridae. There are four major recognised genotypes with a single known serotype. The absence of a reliable in vitro propagation system is an obstacle to deciphering HEV biology. The genome of HEV has been cloned, sequenced and the infectious nature of these replicons has been established. However, questions related to replication, transcription, virus-host interactions and pathogenesis remain to be answered. This comprehensive review summarises the progress made so far in HEV research, and addresses some of the unanswered questions.
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Affiliation(s)
- Subrat Kumar Panda
- Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India.
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Huang YW, Opriessnig T, Halbur PG, Meng XJ. Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo. J Virol 2007; 81:3018-26. [PMID: 17202216 PMCID: PMC1866010 DOI: 10.1128/jvi.02259-06] [Citation(s) in RCA: 128] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023] Open
Abstract
To determine the initiation strategy of the hepatitis E virus (HEV) open reading frame 3 (ORF3), we constructed five HEV mutants with desired mutations in the ORF1 and ORF2 junction region and tested their levels of in vivo infectivity in pigs. A mutant with a C-terminally truncated ORF3 is noninfectious in pigs, indicating that an intact ORF3 is required for in vivo infectivity. Mutations with substitutions in the first in-frame AUG in the junction region or with the same T insertion at the corresponding position of HEV genotype 4 did not affect the virus infectivity or rescue, although mutations with combinations of the two affected virus recovery efficiency, and a single mutation at the third in-frame AUG completely abolished virus infectivity in vivo, indicating that the third in-frame AUG in the junction region is required for virus infection and is likely the authentic initiation site for ORF3. A conserved double stem-loop RNA structure, which may be important for HEV replication, was identified in the junction region. This represents the first report of using a unique homologous pig model system to study the molecular mechanism of HEV replication and to systematically and definitively identify the authentic ORF3 initiation site.
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Affiliation(s)
- Y W Huang
- Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia Polytechnic Institute and State University, 1410 Price's Fork Road, Blacksburg, VA 24601, USA
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Li X, Kamili S, Krawczynski K. Quantitative detection of hepatitis E virus RNA and dynamics of viral replication in experimental infection. J Viral Hepat 2006; 13:835-9. [PMID: 17109683 DOI: 10.1111/j.1365-2893.2006.00754.x] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/09/2022]
Abstract
Hepatitis E virus (HEV) RNA has been detected in the stool and serum of patients with HEV infection and experimentally infected nonhuman primates. However, dynamics of HEV levels in the stool and serum during clinical and subclinical infections have not been determined. A real-time reverse transcription polymerase chain reaction assay, using SYBR Green I in a LightCycler, was developed and optimized to allow quantification of HEV RNA in the stool and serum of both genotype 1 and 2 isolates. The specificity of the assay was confirmed by testing known HEV-RNA-positive and -negative stool and serum specimens and the sensitivity was evaluated using a synthetic HEV RNA standard. Profiles of viraemia and faecal shedding in two chimpanzees inoculated with an isolate of HEV genotype 1 showed the appearance of virus in the stools on day 4 postinoculation (5.65-6.85 log copies/mg) and in the serum on day 7 postinoculation (6.0-6.93 log copies/mL). Peak HEV RNA levels in the stool and serum coincided with peak alanine aminotransferase (ALT) levels observed on day 22 postinoculation in the two chimpanzees. At the time of detection of IgG anti-HEV in serum, viral RNA was no longer detectable in the stool or serum and ALT values had returned to normal levels in both chimpanzees, suggesting the efficacy of the immune response in terminating viral replication. Quantitative evaluation of HEV RNA in humans may allow determining the role of virus levels in the pathogenesis and transmission of HEV.
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Affiliation(s)
- X Li
- Division of Viral Hepatitis, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
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