Detection of Serum Antibodies to Hepatitis E Virus Based on HEV Genotype 3 ORF2 Capsid Protein Expressed in Nicotiana benthamiana

Hepatitis E virus (HEV) is an important human pathogen. In addition to humans, HEV has also been identified in pig, chicken, mongoose, deer, rat, rabbit and fish. There are four recognized and two putative genotypes of mammalian HEV. Genotypes 1 and 2 are restricted to humans, while genotypes 3 and 4 are zoonotic. The recently identified rabbit HEV is a distant member of genotype 3. Here, we first expressed and purified the recombinant capsid protein of rabbit HEV and showed that the capsid protein of rabbit HEV cross-reacted with antibodies raised against avian, rat, swine and human HEV. Conversely, we showed that antibodies against rabbit HEV cross-reacted with capsid proteins derived from chicken, rat, swine and human HEV. Since pigs are the natural host of genotype 3 HEV, we then determined if rabbit HEV infects pigs. Twenty pigs were divided into five groups of four each and intravenously inoculated with PBS, US rabbit HEV, Chinese rabbit HEV, US rat HEV and swine HEV, respectively. Results showed that only half of the pigs inoculated with rabbit HEV had low levels of viraemia and faecal virus shedding, indicative of active but not robust HEV infection. Infection of pigs by rabbit HEV was further verified by transmission of the virus recovered from pig faeces to naïve rabbits. Pigs inoculated with rat HEV showed no evidence of infection. Preliminary results suggest that rabbit HEV is antigenically related to other HEV strains and infects pigs and that rat HEV failed to infect pigs.

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Characterization of Three Novel Linear Neutralizing B-Cell Epitopes in the Capsid Protein of Swine Hepatitis E Virus

Journal of Virology ◽

10.1128/jvi.00251-18 ◽

2018 ◽

Vol 92 (13) ◽

pp. e00251-18 ◽

Cited By ~ 4

Author(s):

Yiyang Chen ◽

Baoyuan Liu ◽

Yani Sun ◽

Huixia Li ◽

Taofeng Du ◽

...

Keyword(s):

B Cell ◽

Capsid Protein ◽

Hepatitis E Virus ◽

Hepatitis E ◽

Disease Diagnosis ◽

Antigenic Structure ◽

Human Populations ◽

Genotype 3 ◽

Genotype 4 ◽

B Cell Epitopes

ABSTRACTHepatitis E virus (HEV) causes liver disease in humans and is thought to be a zoonotic infection, with domestic animals, including swine and rabbits, being a reservoir. One of the proteins encoded by the virus is the capsid protein. This is likely the major immune-dominant protein and a target for vaccination. Four monoclonal antibodies (MAbs), three novel, 1E4, 2C7, and 2G9, and one previously characterized, 1B5, were evaluated for binding to the capsid protein from genotype 4 swine HEV. The results indicated that625DFCP628,458PSRPF462, and407EPTV410peptides on the capsid protein comprised minimal amino acid sequence motifs recognized by 1E4, 2C7, and 2G9, respectively. The data suggested that 2C7 and 2G9 epitopes were partially exposed on the surface of the capsid protein. Truncated genotype 4 swine HEV capsid protein (sp239, amino acids 368 to 606) can exist in multimeric forms. Preincubation of swine HEV with 2C7, 2G9, or 1B5 before addition to HepG2 cells partially blocked sp239 cell binding and inhibited swine HEV infection. The study indicated that 2C7, 2G9, and 1B5 partially blocked swine HEV infection of rabbits better than 1E4 or normal mouse IgG. The cross-reactivity of antibodies suggested that capsid epitopes recognized by 2C7 and 2G9 are common to HEV strains infecting most host species. Collectively, MAbs 2C7, 2G9, and 1B5 were shown to recognize three novel linear neutralizing B-cell epitopes of genotype 4 HEV capsid protein. These results enhance understanding of HEV capsid protein structure to guide vaccine and antiviral design.IMPORTANCEGenotype 3 and 4 HEVs are zoonotic viruses. Here, genotype 4 HEV was studied due to its prevalence in human populations and pig herds in China. To improve HEV disease diagnosis and prevention, a better understanding of the antigenic structure and neutralizing epitopes of HEV capsid protein are needed. In this study, the locations of three novel linear B-cell recognition epitopes within genotype 4 swine HEV capsid protein were characterized. Moreover, the neutralizing abilities of three MAbs specific for this protein, 2C7, 2G9, and 1B5, were studiedin vitroandin vivo. Collectively, these findings reveal structural details of genotype 4 HEV capsid protein and should facilitate development of applications for the design of vaccines and antiviral drugs for broader prevention, detection, and treatment of HEV infection of diverse human and animal hosts.

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Advances in Hepatitis E Virus Biology and Pathogenesis

Viruses ◽

10.3390/v13020267 ◽

2021 ◽

Vol 13 (2) ◽

pp. 267

Author(s):

Shaoli Lin ◽

Yan-Jin Zhang

Keyword(s):

Pregnant Women ◽

Hepatitis E Virus ◽

Case Fatality ◽

Hepatitis E ◽

Host Cells ◽

High Rate ◽

Rapid Progression ◽

Genotype 1 ◽

Genotype 3 ◽

Zoonotic Infections

Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV is a positive-sense single-stranded RNA virus. Human HEV strains mainly belong to four major genotypes in the genus Orthohepevirus A, family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. HEV infection with genotype 1 and 2 mainly presents as acute and self-limiting hepatitis in young adults. However, HEV infection of pregnant women with genotype 1 strains can be exacerbated to fulminant hepatitis, resulting in a high rate of case fatality. As pregnant women maintain the balance of maternal-fetal tolerance and effective immunity against invading pathogens, HEV infection with genotype 1 might dysregulate the balance and cause the adverse outcome. Furthermore, HEV infection with genotype 3 can be chronic in immunocompromised patients, with rapid progression, which has been a challenge since it was reported years ago. The virus has a complex interaction with the host cells in downregulating antiviral factors and recruiting elements to generate a conducive environment of replication. The virus-cell interactions at an early stage might determine the consequence of the infection. In this review, advances in HEV virology, viral life cycle, viral interference with the immune response, and the pathogenesis in pregnant women are discussed, and perspectives on these aspects are presented.

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Infection dynamics and persistence of hepatitis E virus on pig farms – a review

Porcine Health Management ◽

10.1186/s40813-021-00189-z ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

M. Meester ◽

T. J. Tobias ◽

M. Bouwknegt ◽

N. E. Kusters ◽

J. A. Stegeman ◽

...

Keyword(s):

Risk Factors ◽

Hepatitis E Virus ◽

Risk Mitigation ◽

Transmission Rate ◽

Hepatitis E ◽

Main Body ◽

Genotype 3 ◽

Infection Dynamics ◽

Pig Farms ◽

Slaughter Pigs

Abstract Background Hepatitis E virus (HEV) genotype 3 and 4 is a zoonosis that causes hepatitis in humans. Humans can become infected by consumption of pork or contact with pigs. Pigs are the main reservoir of the virus worldwide and the virus is present on most pig farms. Main body Though HEV is present on most farms, the proportion of infected pigs at slaughter and thus the level of exposure to consumers differs between farms and countries. Understanding the cause of that difference is necessary to install effective measures to lower HEV in pigs at slaughter. Here, HEV studies are reviewed that include infection dynamics of HEV in pigs and on farms, risk factors for HEV farm prevalence, and that describe mechanisms and sources that could generate persistence on farms. Most pigs become infected after maternal immunity has waned, at the end of the nursing or beginning of the fattening phase. Risk factors increasing the likelihood of a high farm prevalence or proportion of actively infected slaughter pigs comprise of factors such as farm demographics, internal and external biosecurity and immunomodulating coinfections. On-farm persistence of HEV is plausible, because of a high transmission rate and a constant influx of susceptible pigs. Environmental sources of HEV that enhance persistence are contaminated manure storages, water and fomites. Conclusion As HEV is persistently present on most pig farms, current risk mitigation should focus on lowering transmission within farms, especially between farm compartments. Yet, one should be aware of the paradox of increasing the proportion of actively infected pigs at slaughter by reducing transmission insufficiently. Vaccination of pigs may aid HEV control in the future.

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Hepatitis E Virus Occurrence in Pigs Slaughtered in Italy

Animals ◽

10.3390/ani11020277 ◽

2021 ◽

Vol 11 (2) ◽

pp. 277

Author(s):

Eleonora Chelli ◽

Elisabetta Suffredini ◽

Paola De Santis ◽

Dario De Medici ◽

Santina Di Bella ◽

...

Keyword(s):

Hepatitis E Virus ◽

Phylogenetic Analyses ◽

Hepatitis E ◽

Rt Pcr ◽

High Seroprevalence ◽

Genotype 3 ◽

Sporadic Cases ◽

Wild Boar Meat ◽

Small Clusters ◽

Frequent Exposure

In Europe, foodborne transmission has been clearly associated to sporadic cases and small clusters of hepatitis E in humans linked to the consumption of contaminated pig liver sausages, raw venison, or undercooked wild boar meat. In Europe, zoonotic HEV-genotype 3 strains are widespread in pig farms but little information is available on the prevalence of HEV positive pigs at slaughterhouse. In the present study, the prevalence of HEV-RNA positive pigs was assessed on 585 animals from 4 abattoirs located across Italy. Twenty-one pigs (3.6%) tested positive for HEV in either feces or liver by real-time RT-PCR. In these 21 pigs, eight diaphragm muscles resulted positive for HEV-RNA. Among animals collected in one abattoir, 4 out of 91 plasma tested positive for HEV-RNA. ELISA tests for the detection of total antibodies against HEV showed a high seroprevalence (76.8%), confirming the frequent exposure of pigs to the virus. The phylogenetic analyses conducted on sequences of both ORF1 and ORF2 fragments, shows the circulation of HEV-3c and of a novel unclassified subtype. This study provides information on HEV occurrence in pigs at the slaughterhouse, confirming that muscles are rarely contaminated by HEV-RNA compared to liver, which is the most frequently positive for HEV.

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Hepatitis E Virus in Croatia in the “One-Health” Context

Pathogens ◽

10.3390/pathogens10060699 ◽

2021 ◽

Vol 10 (6) ◽

pp. 699

Author(s):

Anna Mrzljak ◽

Lorena Jemersic ◽

Vladimir Savic ◽

Ivan Balen ◽

Maja Ilic ◽

...

Keyword(s):

Hepatitis E Virus ◽

Current Knowledge ◽

Hepatitis E ◽

Human Case ◽

Interspecies Transmission ◽

Genotype 3 ◽

Chronic Patients ◽

Indirect Contact ◽

The One ◽

Foodborne Infection

Hepatitis E virus (HEV) is the most common cause of viral hepatitis globally. The first human case of autochthonous HEV infection in Croatia was reported in 2012, with the undefined zoonotic transmission of HEV genotype 3. This narrative review comprehensively addresses the current knowledge on the HEV epidemiology in humans and animals in Croatia. Published studies showed the presence of HEV antibodies in different population groups, such as chronic patients, healthcare professionals, voluntary blood donors and professionally exposed and pregnant women. The highest seroprevalence in humans was found in patients on hemodialysis in a study conducted in 2018 (27.9%). Apart from humans, different studies have confirmed the infection in pigs, wild boars and a mouse, indicating the interspecies transmission of HEV due to direct or indirect contact or as a foodborne infection. Continued periodical surveys in humans and animals are needed to identify the possible changes in the epidemiology of HEV infections.

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Efficient Production of Chimeric Hepatitis B Virus-Like Particles Bearing an Epitope of Hepatitis E Virus Capsid by Transient Expression in Nicotiana benthamiana

Life ◽

10.3390/life11010064 ◽

2021 ◽

Vol 11 (1) ◽

pp. 64

Author(s):

Gergana Zahmanova ◽

Milena Mazalovska ◽

Katerina Takova ◽

Valentina Toneva ◽

Ivan Minkov ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Nicotiana Benthamiana ◽

Hepatitis E Virus ◽

Chimeric Protein ◽

Hepatitis E ◽

Core Antigen ◽

Efficient Production ◽

Virus Like Particles ◽

B Virus

The core antigen of hepatitis B virus (HBcAg) is capable of self-assembly into virus-like particles (VLPs) when expressed in a number of heterologous systems. Such VLPs are potential carriers of foreign antigenic sequences for vaccine design. In this study, we evaluated the production of chimeric HBcAg VLPs presenting a foreign epitope on their surface, the 551–607 amino acids (aa) immunological epitope of the ORF2 capsid protein of hepatitis E virus. A chimeric construct was made by the insertion of 56 aa into the immunodominant loop of the HBcAg. The sequences encoding the chimera were inserted into the pEAQ-HT vector and infiltrated into Nicotiana benthamiana leaves. The plant-expressed chimeric HBcHEV ORF2 551–607 protein was recognized by an anti-HBcAg mAb and anti-HEV IgG positive swine serum. Electron microscopy showed that plant-produced chimeric protein spontaneously assembled into “knobbly” ~34 nm diameter VLPs. This study shows that HBcAg is a promising carrier platform for the neutralizing epitopes of hepatitis E virus (HEV) and the chimeric HBcAg/HEV VLPs could be a candidate for a bivalent vaccine.

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