Hepatitis E as a Zoonosis.

Hepatitis E viruses in the family of Hepeviridae have been classified into 2 genus, 5 species, and 13 genotypes, involving different animal hosts of different habitats. Among all these genotypes, four (genotypes 3, 4, 7, and C1) of them are confirmed zoonotic causing sporadic human diseases, two (genotypes 5 and 8) were likely zoonotic showing experimental animal infections, and the other seven were not zoonotic or unconfirmed. These zoonotic HEV carrying hosts include pig, boar, deer, rabbit, camel, and rat. Taxonomically, all the zoonotic HEVs belong to the genus Orthohepevirus, which include genotypes 3, 4, 5, 7, 8 HEV in the species A and genotype C1 HEV in the species C. In the chapter, information of zoonotic HEV such as swine HEV (genotype 3 and 4), wild boar HEV (genotypes 3-6), rabbit HEV (genotype 3), camel HEV (genotype 7 and 8), and rat HEV (HEV-C1) was provided in detail. At the same time, their prevalence characteristics, transmission route, phylogenetic relationship, and detection technology were discussed. Other animal hosts of HEVs were introduced briefly in the chapter. All these information help peer researchers have basic understanding of zoonotic HEV and adopt reasonable strategy of surveillance and prevention.

Research on a rat model of genotype IV swine hepatitis E virus.

BACKGROUND: Hepatitis E virus (HEV) is an important zoonotic pathogen, Genotypes 3 and 4 are the main zoonotic genotype. Due to the lack of mature and effective culture cell lines, researches on genotype IV swine HEV (SHEV-4) infection and pathogenic mechanism have been carried out in pigs, gerbils and non-human primate models. OBJECTIVES: The aim of this study was to establish a rat infection model by intra-peritoneal infection with SHEV-4, which provided a new research idea and scientific basis for further revealing the mechanism of HEV infection and preventing HEV infection. METHODS: SHEV-4 virus was administered intra-peritoneally to 6- to 8-week-old mice to observe the serological changes and virus release. RESULTS: According to the results of the rat serum HEV IgG, ALT and AST levels, swine HEV, minus-strand HEV RNA can infect Sprague-Dawley rats across species, and there are no obvious clinical symptoms after infection. HEV RNA was detected in most tissues and organs after infection, but the viral load was low. The liver had pathological changes of chronic hepatitis. CONCLUSIONS: We found that the rat model of porcine HEV infection is a small animal model suitable for the study of HEV infection.

Deep Insight Into Long Non-coding RNA and mRNA Transcriptome Profiling in HepG2 Cells Expressing Genotype IV Swine Hepatitis E Virus ORF3.

Swine hepatitis E (swine HE) is a new type of zoonotic infectious disease caused by the swine hepatitis E virus (swine HEV). Open reading frame 3 (ORF3) is an important virulent protein of swine HEV, but its function still is mainly unclear. In this study, we generated adenoviruses ADV4-ORF3 and ADV4 negative control (ADV4-NC), which successfully mediated overexpression of enhanced green fluorescent protein (EGFP)-ORF3 and EGFP, respectively, in HepG2 cells. High-throughput sequencing was used to screen for differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs). The cis-target genes of lncRNAs were predicted, functional enrichment (Gene Ontology [GO] and Kyoto Encyclopedia of Genes and Genomes [KEGG]) was performed, and 12 lncRNAs with statistically significant different expressions (p ≤ 0.05 and q ≤ 1) were selected for further quantitative real-time reverse transcription (qRT-PCR) validation. In HepG2 cells, we identified 62 significantly differentially expressed genes (DEGs) (6,564 transcripts) and 319 lncRNAs (124 known lncRNAs and 195 novel lncRNAs) that were affected by ORF3, which were involved in systemic lupus erythematosus, Staphylococcus aureus infection, signaling pathways pluripotency regulation of stem cells, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, and platinum drug resistance pathways. Cis-target gene prediction identified 45 lncRNAs corresponding to candidate mRNAs, among which eight were validated by qRT-PCR: LINC02476 (two transcripts), RAP2C-AS1, AC016526, AL139099, and ZNF337-AS1 (3 transcripts). Our results revealed that the lncRNA profile in host cells affected by ORF3, swine HEV ORF3, might affect the pentose and glucuronate interconversions and mediate the formation of obstructive jaundice by influencing bile secretion, which will help to determine the function of ORF3 and the infection mechanism and treatment of swine HE.

Synthetic Peptides Containing Three Neutralizing Epitopes of Genotype 4 Swine Hepatitis E Virus ORF2 induced Protection against Swine HEV Infection in Rabbit.

Genotype 4 hepatitis E virus (HEV) is a zoonotic pathogen transmitted to humans through food and water. Previously, three genotype 4 swine HEV ORF2 peptides (407EPTV410, 410VKLYTS415, and 458PSRPF462) were identified as epitopes of virus-neutralizing monoclonal antibodies that partially blocked rabbit infection with swine HEV. Here, individual and tandem fused peptides were synthesized, conjugated to keyhole limpet hemocyanin (KLH), then evaluated for immunoprotection of rabbits against swine HEV infection. Forty New Zealand White rabbits were randomly assigned to eight groups; groups 1 thru 5 received three immunizations with EPTV-KLH, VKLYTS-KLH, PSRPF-KLH, EPTVKLYTS-KLH, or EPTVKLYTSPSRPF-KLH, respectively; group 6 received truncated swine HEV ORF2 protein (sp239), and group 7 received phosphate-buffered saline. After an intravenous swine HEV challenge, all group 7 rabbits exhibited viremia and fecal virus shedding by 2-4 weeks post challenge (wpc), seroconversion by 4-9 wpc, elevated alanine aminotransferase (ALT) at 2 wpc, and severe liver lymphocytic venous periphlebitis. Only 1-2 rabbits/group in groups 1-4 exhibited delayed viremia, fecal shedding, seroconversion, increased ALT levels, and slight liver lymphocytic venous periphlebitis; groups 5-6 showed no pathogenic effects. Collectively, these results demonstrate that immunization with a polypeptide containing three genotype 4 HEV ORF2 neutralizing epitopes completely protected rabbits against swine HEV infection.

Swine hepatitis E virus: Cross-species infection, pork safety and chronic infection.

Swine hepatitis E virus (swine HEV) belongs to the species Orthohepevirus A within the genus Orthohepevirus in the family Hepeviridae. Four different genotypes of swine HEV within the species Orthohepevirus A have been identified so far from domesticated and wild swine population: genotypes 3 (HEV-3) and 4 (HEV-4) swine HEVs are zoonotic and infect humans, whereas HEV-5 and HEV-6 are only identified from swine. As a zoonotic agent, swine HEV is an emerging public health concern in many industrialized countries. Pigs are natural reservoir for HEV, consumption of raw or undercooked pork is an important route of foodborne HEV transmission. Occupational risks such as direct contact with infected pigs also increase the risk of HEV transmission in humans. Cross-species infection of HEV-3 and HEV-4 have been documented under experimental and natural conditions. Both swine HEV-3 and swine HEV-4 infect non-human primates, the surrogates of man. Swine HEV, predominantly HEV-3, can establish chronic infection in immunocompromised patients especially in solid organ transplant recipients. The zoonotic HEV-3, and to lesser extent HEV-4, have also been shown to cause neurological diseases and kidney injury. In this review, we focus on the epidemiology of swine HEV, host and viral determinants influencing cross-species HEV infection, zoonotic infection and its associated pork safety concern, as well as swine HEV-associated chronic infection and neurological diseases.

Experimental infection of rabbit with swine-derived hepatitis E virus genotype 4.

Hepatitis E virus (HEV) is a zoonotic virus that is capable of causing cross-species infection. Rabbits can be experimentally infected with human- and swine-derived HEV-4 in the species Orthohepevirus A, and avian-derived HEV-3 in the species Orthohepevirus B suggesting rabbits can serve as an animal model for zoonotic HEV infection study. However, these studies show that the infectivity of swine HEV isolates in rabbits is not consistent. In this study, the animal study was conducted by the experimental infection of rabbit with a swine-derived HEV-4 isolated in China (designated CHN-SD-sHEV) for 28 weeks post-inoculation (wpi) in compassion to that infected with a rabbit-derived HEV-3 (designated HEV-SX-rHEV). Two rabbits were euthanized every 2 wpi for pathological examinations. The results showed that rabbits infected with CHN-SD-sHEV had the viremia and virus fecal shedding from 1 wpi to 22 wpi and seroconverted from 10 to 28 wpi. Meanwhile, elevated ALT levels were detected at 2 wpi. Moreover, virus replication was confirmed by the detection of both positive- and negative-strand HEV RNAs in the livers and spleens. Diarrhea and hepatocellular lesions were also observed in some animals. In contrast, rabbits experimentally infected with CHN-SX-rHEV exhibited earlier seroconversion, viremia and virus fecal shedding and hepatocellular lesions. Taken together, our data demonstrate that in comparison to the previously reported cases, the swine-derived HEV-4 isolated in China could cross-species infect rabbit accompanied with prolonged virus fecal shedding and liver lesions.

Prevalence of hepatitis E virus (HEV) infection in various pig farms from Shaanxi Province, China: First detection of HEV RNA in pig semen.

The zoonotic transmission of hepatitis E virus (HEV) is mainly mediated by HEV genotypes 3 and 4, with domestic pigs serving as an important reservoir for both genotypes. In China, genotype 4 HEV is the primary prevalent genotype on pig farms. In this study, the prevalence of HEV infection in pig herds of Shaanxi Province was investigated. Serological testing detected anti-HEV antibody-positive pigs in five selected cities, with 13 of 17 farms harbouring at least one positive pig (76.47%). Within positive farms, the proportion of positive pigs ranged from 1.6% to 37.5%. Genetic detection analyses of faecal samples revealed that pigs in four cities and on nine of 17 farms were positive for sequences homologous to a partial ORF2-coding region of HEV (306 bp), as were 6 of 53 bile and 1 of 26 semen samples. Meanwhile, DNA coding for partial HEV ORF1 (1,080 bp) and a longer gene segment coding for partial ORF2 (1,594 bp) were successfully amplified from RNA isolated from pig semen from one HEV-positive pig. Sequence comparisons of partial ORF2 gene sequences showed that HEV isolates from Shaanxi Province shared the highest identity (81.4%-96.1%) with genotype 4 HEV. Phylogenetic tree analysis grouped these isolates into three subgenotypes (4d, 4h and 4i), with subgenotype 4i the predominant subgenotype. In addition, the HEV isolate from pig semen belonged to subgenotype 4i HEV based on phylogenetic trees constructed both using partial ORF1 and ORF2 gene sequences. In conclusion, HEV infection is endemic on pig farms of Shaanxi Province, China, and 4i is the predominant HEV subgenotype. More important, this is the first study demonstrating detection of HEV RNA in pig semen, suggesting that artificial insemination can transmit HEV in pigs.

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

Hepatitis 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 that 625DFCP628, 458PSRPF462, and 407EPTV410 peptides 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.IMPORTANCE Genotype 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 studied in vitro and in 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.

Construction of a recombinant baculovirus expressing swine hepatitis E Virus ORF2 and preliminary research on its immune effect.

In the swine hepatitis E virus (HEV), open reading frame 2 (ORF2) is rich in antigenic determinants and neutralizing epitopes that could induce immune protection. We chose the Bac-to-Bac® Baculovirus Expression System to express fragments containing the critical neutralizing antigenic sites within the HEV ORF2 protein of pigs to obtain a recombinant baculovirus. The fragment of swine HEV ORF2 region (1198-1881bp) was cloned into vector pFastBacTM. A recombinant baculovirus, rBacmid-ORF2, was obtained after transposition and transfection. The molecular mass of the recombinant protein was 26 kDa. Mice were immunized by the intraperitoneal and oral routes with cell lysates of recombinant baculovirus rBacmid-ORF2. Serum and feces of the mice were collected separately at 0, 14, 28, and 42 d after immunization and the antibody levels of IgG and secretory IgA against swine HEV were determined using an enzyme-linked immunosorbent assay. The results suggested that rBacmid-ORF2 induced antibodies of the humoral and mucosal immune responses in mice and that the oral route was significantly superior to the intraperitoneal route. This is the first study to demonstrate that that recombinant baculovirus swine HEV ORF2 could induce humoral and mucosal immune responses in mice.

Hepatitis E as a Zoonosis.

Hepatitis E (HE) virus infection is not limited to spread from human to human but also occurs between animals and more importantly as zoonotic spread from animals to humans. Genotyping of strains from hepatitis E virus-infected patients has revealed that these infections are not all caused by genotypes 1 or 2 but often by genotypes 3 or 4. Therefore, it is important to understand the striking difference between the spread of genotypes 1 and 2 in countries with poor sanitary standards and the spread of genotypes 3 and 4 in countries with good sanitary standards. The number of animal species known to be infected with HEV is expanding rapidly. The finding of HEV in new host species always raises the question regarding the zoonotic potential of these newfound strains. However, as new strains are found, the complexity increases.Certain genotypes are known to have the ability of zoonotic spread from certain animal species and these animals may even constitute an infection reservoir. Some animal species may contribute to zoonotic infections albeit on a smaller scale, while others are believed to be of minor or no importance at all. This chapter reviews possible sources of zoonotic hepatitis E virus infection.

Molecular detection of hepatitis E virus in feces and slurry from swine farms, Rio Grande do Sul, Southern Brazil / Detecção molecular do vírus da hepatite E em fezes e chorume de propriedades suinícolas, Rio Grande do Sul, Sul do Brasil

Hepatitis E virus (HEV) is highly disseminated among swine herds worldwide. HEV is also a threat to public health, since particularly genotypes 3 and 4 may cause acute hepatitis in human beings. No previous studies were done on the occurrence of HEV in environmental samples in Rio Grande do Sul, Brazil. In the present study, reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to detect the presence of HEV in swine feces and in effluents from slurry lagoons in farms located in the municipality of Teutônia, inside the area of swine husbandry in the state. Pooled fecal samples from the floor of pig barns from 9 wean-to-finish farms and liquid manure samples were collected from the slurry lagoons from 8 of these farms. From the pooled fecal samples, 8/9 were positive for the HEV ORF1 gene by RT-PCR; all the slurry lagoon samples were positive for HEV RNA (100%). The identity of the HEV ORF1 amplicons was confirmed by sequencing belonging to HEV genotype 3, which was previously shown to be circulating in South America.

O vírus da hepatite E (HEV) é altamente disseminado entre rebanhos suínos no mundo todo. O HEV é também uma ameaça à saúde pública, já que os genótipos 3 e 4 podem causar hepatite aguda em seres humanos. Não há estudos anteriores sobre a ocorrência de HEV em amostras ambientais no Rio Grande do Sul. No presente estudo, empregou-se transcrição reversa e reação em cadeia da polimerase (RT-PCR) para detectar a presença de HEV em fezes de suínos e efluentes de lagoas de chorume em fazendas localizadas no município de Teutônia, representativo da região de maior produção de suínos no estado. Pools de amostras fecais foram coletadas a partir do chão de galpões de suínos provenientes de 9 propriedades de terminação; outra amostra de esterco líquido foi coletada das lagoas de chorume de 8 dessas fazendas. A partir das amostras fecais reunidas, 8/9 foram positivas para o gene ORF1 de HEV por PCR convencional; todas as amostras de lagoas de chorume foram positivas para RNA de HEV (100%). A identificação dos produtos de amplificação de HEV ORF1 foi confirmada por sequenciamento pertencente ao HEV genótipo 3, o qual foi previamente detectado na América do Sul.

Genetic characterization and serological prevalence of swine hepatitis E virus in Shandong province, China.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is classified into four major genotypes (1 to 4) and swine is the main natural reservoir for genotypes 3 and 4. In this study, a total of 106 bile samples from a slaughterhouse in the Shandong province of China were tested for the partial ORF2 gene of HEV by RT-nPCR to determine the virus genotypes, and two indirect ELISA were developed for the detection of swine HEV specific IgM and IgG antibodies in 980 serum samples from 24 farms, in order to investigate the seroprevalence. Thirty-two out of 106 (30.2%) bile samples were positive for HEV and a high degree of partial ORF2 sequence similarity (86.8-100%) was observed among 20 samples. The viral sequences belonged to genotype 4, subtypes 4a and 4d. One complete genome sequence of a subtype 4d HEV was further determined and characterized. The seroprevalence of HEV IgG and IgM antibodies was 100% (24/24) and 41.7% (10/24) for herds, and 66.4% (651/980) and 1.6% (16/980) for the individual pigs, respectively. These results suggested a high prevalence of genotype 4 of swine HEV infection both in swine farms and at the slaughterhouse in Shandong province, which further raise public-health concerns for zoonosis and pork safety.

Analysis of the complete genome sequences of one swine and two human hepatitis E virus genotype 4 strains isolated in Beijing, China.

Full-length sequences were determined and analyzed for two human (MO and W3) and one swine (W2-5) hepatitis E virus (HEV) isolates from Beijing, China. The genomes of the three strains were composed of 7242, 7239, 7239 nucleotides, respectively, excluding the poly (A) tails, and were 84% identical to each other. All were classified into genotype 4. Sequence analysis shows that the 2 human isolates have up to 91-94% nucleotide identity in full length genome with swine strains isolated in China, while the swine isolate share 92% identity with the human strain T1 from Beijing. At the amino acid level, the three strains share 94%, 97% and 89-92% identity in the ORF1, ORF2 and ORF3, proteins respectively. The human strains MO and W3 have the highest identity, 97%, 98-99% and 96-98% in ORFs 1-3, respectively, to swine strains CHN-XJ-SW13 and CHN-XJ-SW33 from Xinjiang, China, while swine strain W2-5 has highest identity with the human strain HE-JA2, 96%, 99% and 91% in ORFs 1-3, respectively. Genotype specific amino acid substitutions were found at a single site in all three ORFs by sequences alignment, and genotype specific short sequences (5-10aa in length) were found in ORF1 and the C-terminus of ORF3. However, no difference was found at any amino acid position that discriminates between human and swine HEVs within genotype 4 for any of the three ORFs. These results indicated that the genotype 4 HEV strains from humans and pigs in China may evolve from the common ancestor.

Detection and Genetic Characterization of Isolates of Hepatitis E Virus from Pigs and Human in Chungnam Region of Korea

Swine hepatitis E virus (HEV) has been reported as a new zoonotic agent due to its close genomic resemblance to the human HEV. Recently this virus is indicated as one of the important pathogens in xenotransplantation that uses pig as a donor animal. We carried out to investigate the prevalence of HEV infections among the pigs and human population in Chungnam region using a nested RT-PCR for detection of a part of HEV ORF2 gene. The sequences of the amplified DNA were analyzed and the genetical divergency were characterized. A total of 18 HEV strains, comprising 16 strains from pig and 2 strains from human, were genetically isolated from the fecal and serum samples. Among the isolates, 5 strains (2.5%) were detected from 200 swine sera and 2 strains (2.0%) from 100 human sera. All of the 16 swine strains were isolated from the pigs at 3 month of age, but none of age groups revealed the positive for swine HEV RNA. In comparison of the nucleotide sequence between 16 swine HEV and 2 human HEV isolates, the range of identities was 91.5% to 100%. Two human HEV isolates shared 99.7% homology. In phylogenetic analysis, all of the isolates were classified into genotype III, and the 18 isolates were also closely related to the prototype of swine HEV and human HEV strains isolated in the United States and others recently identified from swine in Japan and Netherland.

Detection and Genetic Characterization of Isolates of Hepatitis E Virus from Pigs and Human in Chungnam Region of Korea

Swine hepatitis E virus (HEV) has been reported as a new zoonotic agent due to its close genomic resemblance to the human HEV. Recently this virus is indicated as one of the important pathogens in xenotransplantation that uses pig as a donor animal. We carried out to investigate the prevalence of HEV infections among the pigs and human population in Chungnam region using a nested RT-PCR for detection of a part of HEV ORF2 gene. The sequences of the amplified DNA were analyzed and the genetical divergency were characterized. A total of 18 HEV strains, comprising 16 strains from pig and 2 strains from human, were genetically isolated from the fecal and serum samples. Among the isolates, 5 strains (2.5%) were detected from 200 swine sera and 2 strains (2.0%) from 100 human sera. All of the 16 swine strains were isolated from the pigs at 3 month of age, but none of age groups revealed the positive for swine HEV RNA. In comparison of the nucleotide sequence between 16 swine HEV and 2 human HEV isolates, the range of identities was 91.5% to 100%. Two human HEV isolates shared 99.7% homology. In phylogenetic analysis, all of the isolates were classified into genotype III, and the 18 isolates were also closely related to the prototype of swine HEV and human HEV strains isolated in the United States and others recently identified from swine in Japan and Netherland.