Hepatitis E virus egress depends on the exosomal pathway, with secretory exosomes derived from multivesicular bodies

Our previous studies indicated that hepatitis E virus (HEV) forms membrane-associated particles in the cytoplasm, most likely by budding into intracellular vesicles, and requires the multivesicular body (MVB) pathway to release virus particles, and the released HEV particles with a lipid membrane retain the trans-Golgi network protein 2 on their surface. To examine whether HEV utilizes the exosomal pathway to release the virus particles, we analysed whether the virion release from PLC/PRF/5 cells infected with genotype 3 HEV (strain JE03-1760F) is affected by treatment with bafilomycin A1 or GW4869, or by the introduction of a small interfering RNA (siRNA) against Rab27A or Hrs. The extracellular HEV RNA titre was increased by treatment with bafilomycin A1, but was decreased by treatment with GW4869. The relative levels of virus particles released from cells depleted of Rab27A or Hrs were decreased to 16.1 and 11.5 %, respectively, of that released from cells transfected with negative control siRNA. Electron microscopic observations revealed the presence of membrane-associated virus-like particles with a diameter of approximately 50 nm within the MVB, which possessed internal vesicles in infected cells. Immunoelectron microscopy showed positive immunogold staining for the HEV ORF2 protein on the intraluminal vesicles within the MVB. Additionally, immunofluorescence analysis indicated the triple co-localization of the ORF2, ORF3 and CD63 proteins in the cytoplasm, as specific loculated signals, supporting the presence of membrane-associated HEV particles within the MVB. These findings indicate that membrane-associated HEV particles are released together with internal vesicles through MVBs by the cellular exosomal pathway.

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Tumour susceptibility gene 101 and the vacuolar protein sorting pathway are required for the release of hepatitis E virions

Journal of General Virology ◽

10.1099/vir.0.035378-0 ◽

2011 ◽

Vol 92 (12) ◽

pp. 2838-2848 ◽

Cited By ~ 56

Author(s):

Shigeo Nagashima ◽

Masaharu Takahashi ◽

Suljid Jirintai ◽

Toshinori Tanaka ◽

Tsutomu Nishizawa ◽

...

Keyword(s):

Susceptibility Gene ◽

Multivesicular Body ◽

Hepatitis E ◽

Negative Control ◽

Dominant Negative ◽

Wild Type ◽

Orf3 Protein ◽

Virus Particles ◽

Virion Release ◽

Vacuolar Protein

We have previously demonstrated that an intact PSAP motif in the ORF3 protein is required for the formation and release of membrane-associated hepatitis E virus (HEV) particles with ORF3 proteins on their surface. In this study, we investigated the direct interaction between the ORF3 protein and tumour susceptibility gene 101 (Tsg101), a cellular factor involved in the budding of viruses containing the P(T/S)AP late-domain, in PLC/PRF/5 cells expressing the wild-type or PSAP-mutated ORF3 protein and Tsg101 by co-immunoprecipitation. Tsg101 bound to wild-type ORF3 protein, but not to the PSAP-inactive ORF3 protein. To examine whether HEV utilizes the multivesicular body (MVB) pathway to release the virus particles, we analysed the efficiency of virion release from cells upon introduction of small interfering RNA (siRNA) against Tsg101 or dominant-negative (DN) mutants of Vps4 (Vps4A and Vps4B). The relative levels of virus particles released from cells depleted of Tsg101 decreased to 6.4 % of those transfected with negative control siRNA. Similarly, virion egress was significantly reduced by the overexpression of DN forms (Vps4AEQ or Vps4BEQ). The relative levels of virus particles released from cells expressing Vps4AEQ and Vps4BEQ were 19.2 and 15.6 %, respectively, while the overexpression of wild-type Vps4A and Vps4B did not alter the levels of virus release. These results indicate that the ORF3 protein interacts with Tsg101 through the PSAP motifs in infected cells, and that Tsg101 and the enzymic activities of Vps4A and Vps4B are involved in HEV release, thus suggesting that HEV requires the MVB pathway for egress of virus particles.

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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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