Interlaboratory Validation of a Detection Method for Hepatitis E Virus RNA in Pig Liver

Background: In the last years, the number of notified hepatitis E cases in humans has continuously increased in Europe. Foodborne infection with the zoonotic hepatitis E virus (HEV) genotype 3 is considered the major cause of this disease. Undercooked liver and raw sausages containing the liver of pigs and wild boar are at high risk of containing HEV. However, so far, no standardized method for the detection of HEV-RNA in pig liver is available. Methods: An international collaborative study on method reproducibility involving 11 laboratories was performed for an HEV-RNA detection method, which consists of steps of sample homogenization, RNA extraction and real-time RT-PCR detection, including a process control. Naturally contaminated pork liver samples containing two different amounts of HEV and a HEV-negative pork liver sample were tested by all laboratories using the method. Results: Valid results were retrieved from 10 laboratories. A specificity of 100% and a sensitivity of 79% were calculated for the method. False negative results were only retrieved from the sample containing very low HEV amounts near the detection limit. Conclusions: The results show that the method is highly specific, sufficiently sensitive and robust for use in different laboratories. The method can, therefore, be applied to routine food control as well as in monitoring studies.

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Hepatitis E Virus RNA in Commercial Porcine Livers in The Netherlands

Journal of Food Protection ◽

10.4315/0362-028x-70.12.2889 ◽

2007 ◽

Vol 70 (12) ◽

pp. 2889-2895 ◽

Cited By ~ 144

Author(s):

MARTIJN BOUWKNEGT ◽

FROUKJE LODDER-VERSCHOOR ◽

WIM H. M. VAN DER POEL ◽

SASKIA A. RUTJES ◽

ANA MARIA DE RODA HUSMAN

Keyword(s):

The Netherlands ◽

Hepatitis E Virus ◽

Blot Hybridization ◽

Rna Extraction ◽

Southern Blot Hybridization ◽

Hepatitis E ◽

Extraction Methods ◽

High Dose ◽

Genotype 3 ◽

Potential Health

Human hepatitis E virus (HEV) infections by genotype 3 strains in industrialized countries are hypothesized to be caused by pigs. To examine this hypothesis, the potential health risks of transmission routes should be examined. Possible foodborne transmission was studied by quantifying the presence and infectivity of HEV in commercial porcine livers in The Netherlands. A comparison of four tissue disruption and seven RNA extraction methods revealed that mechanical disruption followed by silica-based RNA extraction gave the highest RNA yields and was therefore employed on commercial porcine livers. Four (6.5%) of 62 porcine livers were HEV RNA positive by reverse transcriptase PCR and Southern blot hybridization. Each positive liver was estimated to contain ∼65 PCR-detectable units per g. Sequences were obtained for three of four positive livers and classified as HEV genotype 3. Ninety-three percent similarity to Dutch human HEV sequences and 97% similarity to Dutch swine HEV sequences were observed. To determine whether positive livers contained infectious HEV particles, extracts from livers with known HEV RNA sequences were inoculated intravenously in pigs. Two control pigs were included: one was inoculated with a high dose known to result in infection (104 PCR-detectable units of HEV RNA), and the other was inoculated with a lower concentration of virus that equaled the concentration of PCR-detectable units in commercial livers (∼20 PCR-detectable units). Infection was observed in the high-dose control, but not in other pigs, suggesting a dose-dependent response in pigs. Hence, the implications of HEV RNA in commercial porcine livers in The Netherlands are unknown. However, HEV RNA is present in commercial porcine livers, and sufficient heating of porcine livers before consumption as precautionary measure is recommended.

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Detection and characterization of infectious Hepatitis E virus from commercial pig livers sold in local grocery stores in the USA

Journal of General Virology ◽

10.1099/vir.0.82613-0 ◽

2007 ◽

Vol 88 (3) ◽

pp. 912-917 ◽

Cited By ~ 248

Author(s):

A. R. Feagins ◽

T. Opriessnig ◽

D. K. Guenette ◽

P. G. Halbur ◽

X.-J. Meng

Keyword(s):

Hepatitis E Virus ◽

Phylogenetic Analyses ◽

Hepatitis E ◽

Infectious Hepatitis ◽

Health Concern ◽

Grocery Stores ◽

Genotype 3 ◽

Pig Liver ◽

Virus Shedding ◽

The Usa

Hepatitis E virus (HEV) is a zoonotic pathogen of which pigs are reservoirs. To determine the presence of HEV RNA in commercial pig livers sold in local grocery stores in the USA, 127 packages of commercial pig liver were purchased and tested by a universal RT-PCR assay capable of detecting all four known HEV genotypes. Among the 127 livers tested, 14 were positive for HEV RNA. Sequence and phylogenetic analyses revealed that the 14 isolates all belonged to genotype 3. An animal study was subsequently conducted in pigs to determine whether the PCR-positive pig livers still contained infectious virus. The results showed that pigs inoculated with two of the three PCR-positive pig-liver homogenates became infected, as evidenced by the detection of faecal virus shedding, viraemia and seroconversion. The data demonstrated that commercial pig livers sold in grocery stores are contaminated by HEV and that the contaminating virus remains infectious, thus raising a public-health concern for food-borne HEV infection.

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Complete Genome Sequences of Two Swiss Hepatitis E Virus Isolates from Human Stool and Raw Pork Sausage

Genome Announcements ◽

10.1128/genomea.00888-17 ◽

2017 ◽

Vol 5 (35) ◽

Cited By ~ 11

Author(s):

Jakub Kubacki ◽

Cornel Fraefel ◽

Marco Jermini ◽

Petra Giannini ◽

Gladys Martinetti ◽

...

Keyword(s):

Hepatitis E Virus ◽

Hepatitis E ◽

Genotype 3 ◽

Genome Sequences ◽

Virus Genotype ◽

Pig Liver ◽

Pork Sausage ◽

Full Length Genome ◽

Virus Isolates ◽

Human Stool

ABSTRACT We present here the full-length genome sequences of two hepatitis E virus genotype 3 (HEV-3) isolates from a human stool sample from a patient with acute hepatitis and a raw sausage containing pig liver. Sequence analysis implies that Swiss HEV isolates may form a novel subgroup of HEV-3 viruses.

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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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