Hepatitis E Virus Isolates Research Articles

Rat Hepatitis E Virus Isolates Cluster among Urban Norway Rats (Rattus norvegicus) across a Roadway.

Hepatitis E virus (HEV) is a globally distributed pathogen that causes acute hepatitis in people. Recent human cases of HEV arising after contact with urban rats (Rattus spp.) have raised concerns regarding whether rats may be a source of HEV infection. We investigated whether urban Norway rats (Rattus norvegicus) could be a source of HEV in an underserved urban neighborhood of Vancouver, Canada. We found that 15% of rats tested positive for rat HEV, and that HEV status was associated with increasing rat body length and family relationships. Rat HEV isolates were clustered according to their location on either the east or west side of a busy roadway bisecting this neighborhood, suggesting that this street is a barrier to HEV spread. Widespread distribution of HEV among rats in this neighborhood poses potential human health risks, emphasizing the need to reduce close contact of people with rats and their excreta.

Endemic maintenance of human-related hepatitis E virus strains in synurbic wild boars, Barcelona Metropolitan Area, Spain

Hepatitis E virus (HEV), shared by humans, domestic animals, and wildlife, is an emerging global public health threat. Because wild boars are a major reservoir of HEV, the new zoonotic interfaces resulting from wild boar population increase and synurbization significantly contribute to increasing the risk of zoonotic transmission of HEV.This study characterizes HEV strains of synurbic wild boars and assesses their relationship with sympatric human and domestic swine HEV strains. We analyzed the faeces of 312 synurbic wild boars collected from 2016 to 2021 in the Barcelona Metropolitan Area (BMA), where there is a high density of wild boars, and found 7 HEV-positive samples among those collected between 2019 and 2020. The molecular analysis of these isolates, along with 6 additional wild boar HEV isolates from a previous study, allowed us to establish a close phylogenetic relationship between these HEV strains and human HEV isolates from sympatric blood donors and domestic pigs from Catalonia. HEV-positive wild boar samples belonged to piglet, juvenile and yearling individuals, but not adults, indicating the endemic maintenance of HEV in the wild boar population of the BMA by naïve young individuals. All wild boar HEV isolates in this study classified within HEV genotype 3.The results show, for the first time, a close molecular similarity between the HEV strains endemically maintained by the synurbic wild boars in the BMA and citizens from the same area and period. The data could also indicate that HEV infection presents a seasonal and interannual variability in wild boars of BMA. Further investigation is required to unveil the HEV transmission routes between synurbic wild boars and sympatric citizens. These findings can serve in other synurbic wildlife-human interfaces throughout the world.

A Broad-specificity Neutralizing Nanobody against Hepatitis E Virus Capsid Protein.

Hepatitis E virus (HEV) is a worldwide zoonotic and public health concern. The study of HEV biology is helpful for designing viral vaccines and drugs. Nanobodies have recently been considered appealing materials for viral biological research. In this study, a Bactrian camel was immunized with capsid proteins from different genotypes (1, 3, 4, and avian) of HEV. Then, a phage library (6.3 × 108 individual clones) was constructed using peripheral blood lymphocytes from the immunized camel, and 12 nanobodies against the truncated capsid protein of genotype 3 HEV (g3-p239) were screened. g3-p239-Nb55 can cross-react with different genotypes of HEV and block Kernow-C1/P6 HEV from infecting HepG2/C3A cells. To our knowledge, the epitope recognized by g3-p239-Nb55 was determined to be a novel conformational epitope located on the surface of viral particles and highly conserved among different mammalian HEV isolates. Next, to increase the affinity and half-life of the nanobody, it was displayed on the surface of ferritin, which can self-assemble into a 24-subunit nanocage, namely, fenobody-55. The affinities of fenobody-55 to g3-p239 were ∼20 times greater than those of g3-p239-Nb55. In addition, the half-life of fenobody-55 was nine times greater than that of g3-p239-Nb55. G3-p239-Nb55 and fenobody-55 can block p239 attachment and Kernow-C1/P6 infection of HepG2/C3A cells. Fenobody-55 can completely neutralize HEV infection in rabbits when it is preincubated with nonenveloped HEV particles. Our study reported a case in which a nanobody neutralized HEV infection by preincubation, identified a (to our knowledge) novel and conserved conformational epitope of HEV, and provided new material for researching HEV biology.

PHYLOGENETIC ANALYSIS OF TWELVE BULGARIAN SEQUENCES BASED ON PARTIAL OPEN READING FRAME 2 GENOME FRAGMENT OF HEPATITIS E VIRUS

Background: Hepatitis E virus (HEV) causes both acute and chronic liver inflammation. HEV is transmitted through the fecal-oral mechanism and infects both animals and humans. The virus belongs to the Hepeviridae family and its genome is a single stranded RNA molecule. Thanks to molecular sequencing methods different genotypes and subgenotypes have been established. The aim of the present study was to identify and characterize Bulgarian HEV sequences by applying Sanger sequencing technique for a genome fragment in Open Reading Frame 2 (ORF2 region). Material and methods: Twelve retrospective samples from patients with serologically confirmed HEV infection (anti-HEV IgM and IgG positive) were sequenced by Sanger sequencing. Sequencing data were analysed by BioEdit, MEGA11 and NCBI Genbank software tools. Results: The results revealed that all isolates assign to species Paslahepevirus balayani. Phylogenetic analysis showed that HEV isolates were characterized with considerable genetic diversity. The sequences were sub-clustered into the following subgenotypes: HEV-3e, 3m, 3f and 3c. Conclusion: We successfully applied the Sanger method for hepatitis E virus RNA sequencing. The established heterogeneity of subgenotypes requires further study in order to determine the circulation of all possible subgenotypes of HEV in the country.

Molecular identification and genotyping of hepatitis E virus from Southern Punjab, Pakistan.

Hepatitis E is a global health concern. Hepatitis E virus (HEV) infection is endemic in Pakistan. HEV has four genotypes: HEV-1 through HEV-4. The genotypes HEV-1 and HEV-2 are associated with infection in humans, especially in countries with poor sanitation. The genotypes HEV-3 and HEV-4 are zoonotic and human infection takes place by consuming undercooked meat or being in contact with animals. The present study was designed to ascertain the presence of HEV in the Southern Punjab region of Pakistan. First, blood samples (n = 50) were collected from patients suspected of infection with the hepatitis E virus from the Multan District. The serum was separated and the samples were initially screened using an HEV IgM-ELISA. Second, the ELISA-positive samples were subjected to PCR and were genetically characterized. For PCR, the RNA extraction and complementary DNA synthesis were done using commercial kits. The HEV ORF2 (Open Reading Frame-2, capsid protein) was amplified using nested PCR targeting a 348bp segment. The PCR amplicons were sequenced and an evolutionary tree was constructed using MEGA X software. A protein model was built employing the SWISS Model after protein translation using ExPASy online tool. The positivity rate of anti-HEV antibodies in serum samples was found as56% (28/50). All Pakistani HEV showed homology with genotype 1 and shared common evolutionary origin and ancestry with HEV isolates of genotype 1 of London (MH504163), France (MN401238), and Japan (LC314158). Sequence analysis of motif regions assessment and protein structure revealed that the sequences had a similarity with the reference sequence. These data suggest that genotype 1 of HEV is circulating in Pakistan. This finding could be used for the diagnosis and control of HEV in the specific geographic region focusing on its prevalent genotype.

The hepatitis E virus ORF1 hypervariable region confers partial cyclophilin dependency.

Hepatitis E virus (HEV) is an emerging pathogen responsible for more than 20 million cases of acute hepatitis globally per annum. Healthy individuals typically have a self-limiting infection, but mortality rates in some populations such as pregnant women can reach 30 %. A detailed understanding of the virus lifecycle is lacking, mainly due to limitations in experimental systems. In this regard, the cyclophilins are an important family of proteins that have peptidyl-prolyl isomerase activity and play roles in the replication of a number of positive-sense RNA viruses, including hepatotropic viruses such as hepatitis C virus (HCV). Cyclophilins A and B (CypA/B) are the two most abundant Cyps in hepatocytes and are therefore potential targets for pan-viral therapeutics. Here, we investigated the importance of CypA and CypB for HEV genome replication using sub-genomic replicons. Using a combination of pharmacological inhibition by cyclosporine A (CsA), and silencing by small hairpin RNA we find that CypA and CypB are not essential for HEV replication. However, we find that silencing of CypB reduces replication of some HEV isolates in some cells. Furthermore, sensitivity to Cyp silencing appears to be partly conferred by the sequence within the hypervariable region of the viral polyprotein. These data suggest HEV is atypical in its requirements for cyclophilin for viral genome replication and that this phenomenon could be genotype- and sequence-specific.

Comparative analysis of the open reading frames protein genes of genotype 4 Hepatitis E virus in swine and wild boar

The goal of this study was to determine the molecular diversity of the open reading frames (ORFs) ORF1, ORF2, ORF3 protein genes from full-length genomes of genotype 4 hepatitis E virus (HEV) from pigs and wild boars at protein and gene level. Statistical technique Shannon entropy was used for mutational analysis of ORF1–ORF3 protein genes to identify amino acid substitutions in the HEV 4 sequences isolated from pigs and wild boars that were most susceptible to mutations. Gene selective pressure for genes was estimated using Tajima’s neutrality test. The ORF regions of 11 swine and 11 wild boar genotype 4 HEV isolates with complete genomes from the GenBank database were analyzed comparatively. The total number of polymorphic sites was determined. Nonsynonymous (amino acid changing) and synonymous (amino acid preserving) substitutions were identified in ORF1, ORF2, ORF3 in swine and wild boar HEV 4 isolates. No evidence of recombination was found for ORFs in 11 swine HEV 4 isolates, ORF2, ORF3 in 8 wild boar HEV 4 isolates. However, a recombination fragment with a length of 430 nucleotides was detected in the ORF1 gene of 3 wild boar HEV 4 isolates. Positive D Tajima factors were determined for ORF1, ORF2, ORF3 genes of swine HEV 4 and ORF1, ORF2 genes of wild boar HEV 4. While a negative value of D Tajima’s factor was determined for ORF3 gene of wild boar HEV 4. Molecular characteristics showing principal distinctions between the open-reading frames of swine and wild boar genotype 4 hepatitis E virus were obtained. Wild boar ORF1 is characterized by lower nucleotide diversity π value (0.144) and higher number of segregated sites S value (1,688) comparing with higher π value (0.159) and lower S value (1,602) of swine ORF1. Positive values of D Tajima’s factor for ORF1, ORF2 ORF3 genes of swine HEV 4 and ORF1, ORF2 genes of wild boar HEV 4 show on positive selection of these genes. Negative value of D Tajima’s factor for ORF3 gene of wild boar HEV 4 indicates onto purifying selection decreasing variability in ORF3 gene of wild boar HEV 4. The largest number of amino acid variation sites (19.2%) was found for wild boar HEV 4 ORF3 followed by swine HEV 4 ORF3 (15.7%) comparing with other swine and wild boars HEV 4 ORFs

Prevalence of hepatitis E virus and its association with adverse pregnancy outcomes in pregnant women in China.

Hepatitis E virus (HEV) infection has become a global concern, especially in pregnant women. However, the association between HEV prevalence and age, gravidity and parity of pregnant women remains unclear. Pregnant women (n=19,762) were enrolled for HEV prevalence and associated adverse pregnancy outcomes investigation in Qujing City, Yunnan Province of China from May 2019 to December 2020. The seroprevalence of HEV was 11.6% (2,297/19,762; 95% CI:11.2%-12.1%). About 11.4% (2,247/19,762; 95% CI:10.9%-11.8%) were positive for anti-HEV IgG antibody, 0.1% (22/19,762; 95% CI:0.1%-0.2%) were positive for anti-HEV IgM antibody, and 0.1% (28/19,762; 95% CI:0.1%-0.2%) were positive for both anti-HEV IgM and IgG antibodies. Sixty-one out of 2,297 anti-HEV-antibodies-positive pregnant women were positive for HEV RNA. Phylogenetic analysis revealed that all HEV isolates from pregnant women belong to genotype 4. Age, gravidity and parity are associated with increased prevalence of HEV. Pregnant women positive for HEV-IgG antibody bear a higher risk for an adverse pregnancy history and liver injury with elevated alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels than anti-HEV-negative pregnant women. Furthermore, seropositive pregnant women suffered a higher adverse maternal outcomes risk (crude odds ratio [cOR]=1.29; 95% CI: 1.16-1.43; adjusted odds ratio [aOR]=1.40, 95% CI: 1.25-1.55 for anti-HEV-IgG-positive pregnant women and cOR=1.38, 95% CI: 1.02-1.86; aOR=1.43, 95% CI: 1.05-1.95 for anti-HEV-IgM-positive pregnant women) and fetal outcomes risk (cOR=1.80, 95% CI: 1.61-2.01; aOR=1.77, 95% CI: 1.57-1.99) than anti-HEV-negative pregnant women. Adverse pregnancy outcomes of HEV infection are aggravated by age, gravidity and parity. In this study, we demonstrated high prevalence of HEV in pregnancy women in China, and HEV infection can cause various adverse maternal and neonatal outcomes.

Search for the putative RNA thermometers in the genome of Hepatitis E virus

Currently, some temperature-sensitive elements in bacteria are known. Structurally and functionally different RNA thermometers control a variety of cellular processes in bacteria including virulence. Up-to-date experimental confirmation of RNA thermometers functioning in viruses was obtained only for West Nile virus. But other, unknown yet, types of RNA thermometers may exist in nature. The goal of this study was the determination of conservative stem-loop structures in the swine, wild boars’ hepatitis E virus (HEV) genome which may act as RNA thermometers. The search for putative RNA thermometers in the swine HEV which is a common pathogen in the pig population worldwide was executed. Bioinformatics analysis was used to predict the secondary structure of the linear RNA fragments and to determine the melting temperature of the potential hairpins in the HEV genome. 108 swine, wild boars genotype 3 and genotype 4 HEV isolates with complete genomes from the GenBank database were analyzed for the availability of stem-loop structures. Conservative hairpin with the putative thermoregulating function was found in genotype 3 HEV isolates from pig and wild boar for 64 HEV isolates from 108 analyzed ones. The stem of the hairpin with a length of 37 nt contains two AUG start codons of translation initiation and the melting temperature of the hairpin is equal to 38–42 °С for ionic strength of 0.165 M Na+. These hairpins contain a metastable element (one or two bulges) in the stem. Conservative secondary stem-loop structures with putative thermoregulating function for genomic RNA of 64 HEV isolates with complete genome were found by bioinformatics analysis. These hairpins contain a metastable element (one or two bulges) in the stem like an RNA thermometer of West Nile virus and satisfy the necessary and sufficient conditions of RNA thermometer formation. Determined stem-loop structures are proposed as putative thermoregulator elements because they are highly conservative uncanonical structures that are present in the genomes of 64 HEV isolates from 108 analyzed ones

Quantification of hepatitis E virus in raw pork livers using droplet digital RT-PCR

Hepatitis E virus (HEV) is the causative agent of hepatitis E. Some of the rise in hepatitis E infection in China may be linked to undercooked pork. In this study, we established a reverse transcription droplet digital PCR (RT-ddPCR) method to detect HEV in raw pork livers. The detection limit of the assay for HEV RNA was as low as 1.81 copies/μL. The suggested approach was validated on 14 samples, demonstrating greater sensitivity, specificity, and anti-interference performance features than RT-qPCR. Furthermore, we amplified the partial ORF2 gene by nested RT-PCR and sequenced for the HEV RNA positive samples. The prevalence of HEV in all collected samples was 2.24% (14/626), and the viral load was between 8.0 copies/μL and 8975 copies/μL. Specifically, the virus was detected in 10.62% (12/113) of the samples collected from the bio-safety disposal centers for dead livestock and poultry, in 0.67% (2/300) of the samples collected from the slaughterhouses, and none of the samples collected from the retail markets was HEV RNA positive. The subsequent phylogenetic analysis revealed that all HEV isolates belonged to the subtype 4d, which is one of the most common subtypes in northern China.

Survey of hepatitis E virus in pork products and pig stools in Nakhon Pathom Province, Thailand.

BackgroundHepatitis E virus (HEV) is an important public health threat resulting in more than 3 million symptomatic cases and 70,000 deaths annually. HEV is classified into at least eight genotypes, and five are associated with human infection. Genotypes 1 and 2 primarily affect humans, whereas genotypes 3 and 4 circulate in both humans and swine and are considered zoonotic viruses. Previous studies in Central Thailand have reported human HEV isolates with high similarity to swine strains and high seroprevalence in pigs, suggesting the potential for pig‐to‐human transmission.ObjectivesThis study aimed to detect and analyse HEV in pork products and pig stools collected from local markets and pig farms in Nakhon Pathom Province in Central Thailand.MethodsA total of 177 pig stool and 214 pork product samples were detected for HEV by using RT–PCR amplification. Next, nucleotide sequencing and phylogenetic analysis were performed.ResultsWe found one sample of pork products (1/214, 0.5%), which was a pig liver sample (1/51, 2.0%), and 49 HEV‐positive samples in pig stools (49/177, 27.7%). Phylogenetic analysis showed that all these HEV sequences belonged to genotype 3, with a high correlation between our samples and HEV from humans and swine was previously reported in Thailand.ConclusionsThis study suggested that the consumption of poorly sanitized or uncooked animal meat or food and frequent exposure to pig stools may be risk factors for HEV infections in humans.

HuH-7-Lunet BLR Cells Propagate Rat Hepatitis E Virus (HEV) in a Cell Culture System Optimized for HEV.

The family Hepeviridae comprises the species Orthohepevirus A–D (HEV-A to -D). HEV-C genotype 1 (HEV-C1, rat HEV) is able to infect humans. This study investigated whether an optimized HEV-A cell culture system is able to propagate the cell culture-derived rat HEV, and if de novo isolation of the virus from rat liver is possible. We tested the liver carcinoma cell lines PLC/PRF/5, HuH-7, and HuH-7-Lunet BLR for their susceptibility to HEV-C1 strains. Cells were infected with the cell culture-derived HEV-C1 strain R63 and rat liver-derived strain R68. Cells were maintained in MEMM medium, which was refreshed every 3–4 days. The viral load of HEV-C1 was determined by RT-qPCR in the supernatant and expressed as genome copies per mL (c/mL). Rat HEV replication was most efficient in the newly introduced HuH-7-Lunet BLR cell line. Even if the rat HEV isolate had been pre-adapted to PLC/PRF/5 by multiple passages, replication in HuH-7-Lunet BLR was still at least equally effective. Only HuH-7-Lunet BLR cells were susceptible to the isolation of HEV-C1 from the liver homogenate. These results suggest HuH-7-Lunet BLR as the most permissive cell line for rat HEV. Our HEV-C1 cell culture system may be useful for basic research, the animal-free generation of large amounts of the virus as well as for the testing of antiviral compounds and drugs.

Avian Hepatitis E Virus ORF2 Protein Interacts with Rap1b to Induce Cytoskeleton Rearrangement That Facilitates Virus Internalization.

ABSTRACTAvian hepatitis E virus (HEV) causes liver diseases and multiple extrahepatic disorders in chickens. However, the mechanisms involved in avian HEV entry remain elusive. Herein, we identified the RAS-related protein 1b (Rap1b) as a potential HEV-ORF2 protein interacting candidate. Experimental infection of chickens and cells with an avian HEV isolate from China (CaHEV) led to upregulated expression and activation of Rap1b both in vivo and in vitro. By using CaHEV capsid as mimic of virion to treat cell in vitro, it appears that the interaction between the viral capsid and Rap1b promoted cell membrane recruitment of the downstream effector Rap1-interacting molecule (RIAM). In turn, RIAM further enhanced Talin-1 membrane recruitment and retention, which led to the activation of integrin α5/β1, as well as integrin-associated membrane protein kinases, including focal adhesion kinase (FAK). Meanwhile, FAK activation triggered activation of downstream signaling molecules, such as Ras-related C3 botulinum toxin substrate 1 RAC1 cell division cycle 42 (CDC42), p21-activated kinase 1 (PAK1), and LIM domain kinase 1 (LIMK1). Finally, F-actin rearrangement induced by Cofilin led to the formation of lamellipodia, filopodia, and stress fibers, contributes to plasma membrane remodeling, and might enhance CaHEV virion internalization. In conclusion, our data suggested that Rap1b activation was triggered during CaHEV infection and appeared to require interaction between CaHEV-ORF2 and Rap1b, thereby further inducing membrane recruitment of Talin-1. Membrane-bound Talin-1 then activates key Integrin-FAK-Cofilin cascades involved in modulation of actin kinetics, and finally leads to F-actin rearrangement and membrane remodeling to potentially facilitate internalization of CaHEV virions into permissive cells.IMPORTANCE Rap1b is a multifunctional protein that is responsible for cell adhesion, growth, and differentiation. The inactive form of Rap1b is phosphorylated and distributed in the cytoplasm, while active Rap1b is prenylated and loaded with GTP to the cell membrane. In this study, the activation of Rap1b was induced during the early stage of avian HEV infection under the regulation of PKA and SmgGDS. Continuously activated Rap1b recruited its effector RIAM to the membrane, thereby inducing the membrane recruitment of Talin-1 that led to the activation of membrane α5/β1 integrins. The triggering of the signaling pathway-associated Integrin α5/β1-FAK-CDC42&RAC1-PAK1-LIMK1-Cofilin culminated in F-actin polymerization and membrane remodeling that might promote avian HEV virion internalization. These findings suggested a novel mechanism that is potentially utilized by avian HEV to invade susceptible cells.

Effect of Hepatitis E Virus RNA Universal Blood Donor Screening, Catalonia, Spain, 2017‒2020.

Hepatitis E virus (HEV) is the major cause of acute viral hepatitis in several countries in Europe. HEV is acquired mainly by consumption of contaminated pork but can also be transmitted through blood transfusion. HEV infection is usually self-limited but can become persistent in immunocompromised persons. During the first 30 months of HEV RNA universal screening of blood donations in Catalonia, Spain, we identified 151 HEV RNA–positive donations (1/4,341 blood donations). Most infected donors reported consumption of pates and sausages, and 58% were negative for HEV IgM and IgG. All HEV isolates belonged to genotype 3. All infected donors spontaneously resolved the infection, and no neurologic symptoms and reinfections were observed after 1 year of follow-up. Since the implementation of HEV RNA universal screening, no new cases of transfusion-transmitted HEV infection were reported. Our data indicate HEV screening of blood donations provides safer blood for all recipients, especially for immunosuppressed persons.

Characterization of rabbit hepatitis E virus isolated from a feral rabbit

Rabbit hepatitis E virus (HEV) has been detected among rabbits and recently isolated from immunocompromised patients, suggesting zoonotic transmission. In this study, HEV infection among feral rabbits (Oryctolagus cuniculus) was assessed by detection of anti-HEV antibodies and HEV RNA. The prevalence of anti-HEV antibodies in sera was of 33 % (20/60) and HEV RNA was detected from only one of fecal swabs (1.7 %, 1/58). Furthermore, one naïve rabbit was intravenously inoculated with the suspension of the HEV-positive fecal specimen, exhibiting persistent HEV shedding in feces, intermittent viremia, seroconversion to anti-HEV IgM and IgG, and high alanine aminotransferase (ALT) values, indicating persistent HEV infection. The isolate JP-59 had a length of 7,282 bp excluding a poly (A) tail and possessed the characteristic 93 bp-insertion in ORF1. Phylogenetic analysis indicated that JP-59 formed a cluster with other rabbit HEV isolates from rabbits and human origin. The JP-59 shared the nucleotide sequence identities less than 87 % with other rabbit HEVs, suggesting that a novel rabbit HEV strain was circulating in Japan.

Trends in hepatitis E virus infection: Analyses of the long-term screening of blood donors in Hokkaido, Japan, 2005-2019.

After experiencing several cases of transfusion-transmitted hepatitis E (TT-HE) in Hokkaido, Northern Japan, hepatitis E virus (HEV) screening in blood donors, using a nucleic acid amplification test (NAT), was introduced in 2005. The frequency of HEV RNA-positive donations (2005-2019) was investigated, and the HEV RNA-positive specimens were phylogenetically analyzed. In August 2014, the 20-pooled NAT (20P-NAT) was replaced with an individual-NAT (ID-NAT) system. Until 2019, the frequency of HEV RNA-positive donors was 0.011% (289/2,638,685) with 20P-NAT and 0.043% (597/1,379,750) with ID-NAT, and no TT-HE cases were observed in Hokkaido. The prevalence among male, but not female donors, increased significantly between 2015 and 2019. Eighty-nine percent of HEV isolates from donors were genotype 3 and the remainder were genotype 4, and many clusters existed in each genotype. ALT levels at the time of donation were significantly higher in donors with genotype 4. Four subgenotypes, namely 3a (37%), 3b (41%), 3e (6%), and 4c (10%), comprised 94% of the total. During this period, the most identified subgenotype, 3a, transitioned to 3b. Majority of the HEV strains within the same clusters were detected in the same geographical region around the same period. Many of the human HEV isolates were shown to coexist closely with animal HEV isolates phylogenetically. In Hokkaido, multiple divergent HEV strains have been circulating, and small outbreaks of hepatitis E have occurred in the last 15 years. The results suggested that HEV NAT can contribute significantly in ensuring safety during blood transfusions.

Sequence to structure analysis of the ORF4 protein from Hepatitis E virus.

Hepatitis E virus (HEV) is the main cause of acute hepatitis worldwide. HEV accounts for up to 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (G1) HEV. The contributing factors in adverse cases during pregnancy in women due to HEV infection is still debated. The mechanism underlying the pathogenesis of viral infection is attributed to different genomic component of HEV, i.e., open reading frames (ORFs): ORF1, ORF2, ORF3 and ORF4. Recently, ORF4 has been discovered in enhancing the replication of GI isolates of HEV through regulation of an IRES-like RNA element. However, its characterization through computational methodologies remains unexplored. In this novel study, we provide comprehensive overview of ORF4 protein's genetic and molecular characteristics through analyzing its sequence and different structural levels. A total of three different datasets (Human, Rat and Ferret) of ORF4 genomes were built and comparatively analyzed. Several non-synonymous mutations in conjunction with higher entropy values were observed in rat and ferret datasets, however, limited variation was observed in human ORF4 genomes. Higher transition to tranversion ratio was observed in the ORF4 genomes. Studies have reported the association of intrinsic disordered proteins (IDP) with drug discovery due to its role in several signaling and regulatory processes through protein-protein interactions (PPIs). As PPIs are potent drug target sources, thus the ORF4 protein was explored by analyzing its polypeptide structure in order to shed light on its intrinsic disorder. Pressures that lead towards preponderance of disordered-promoting amino acid residues shaped the evolution of ORF4. The intrinsic disorder propensity analysis revealed ORF4 protein (Human) as a highly disordered protein (IDP). Predominance of coils and lack of secondary structure further substantiated our findings suggesting its involvement in binding to ligand molecules. Thus, ORF4 contributes to cellular signaling processes through protein-protein interactions, as IDPs are targets for regulation to accelerate the process of drug designing strategies against HEV infections.

Clinical Outcomes and Prevalence of Hepatitis E Virus (HEV) Among Non-A-C Hepatitis Patients in Egypt.

BackgroundHepatitis E virus (HEV) is an emerging infectious agent that causes acute hepatitis in developing and developed countries. Diagnosis of HEV infection has not been routinely done in Egyptian hospitals, and clinicians do not prescribe ribavirin (RBV) for acute hepatitis cases of unknown etiology (AHUE). We aimed to screen patients with AHUE for the presence of HEV markers and to determine the complications associated with HEV infection.Patients and MethodsHEV markers (anti-HEV IgM, anti-HEV IgG, and HEV RNA) were assessed in patients with AHUE (n=300) admitted to Assiut University Hospitals. RT-qPCR was used to detect the viral load and sequencing analysis was carried out to determine the genotype of the detected viruses. Phylogenetic tree was constructed to evaluate the genetic relatedness between the isolates. Laboratory parameters and the outcomes of infection were determined.ResultsAcute HEV infection (AHE) was detected in 30 out of 300 (10%) of AHUE patients. Anti-HEV IgM, HEV RNA, and anti-HEV IgG were reported in 83%, 50%, and 43% of the samples, respectively. HEV RNA load ranged from 5×102 IU/mL to 1.1×104 IU/mL. Sequencing of the isolated viruses revealed that five viruses belong to HEV-1 and one isolate belongs to HEV-3 with high homology to the virus recently isolated from the cow and goat milk in the Egyptian villages. Although previous reports showed that attenuated HEV isolates were circulating in Egypt, four out of 30 patients (13%) developed coagulopathy and hepatic encephalopathy and died due to fulminant hepatic failure (FHF) within 3–6 weeks of hospitalization. Age, malignancy, and a history of pre-existing liver diseases were a risky factor for FHF development.ConclusionAHE is common in Upper Egypt. Older patients with malignancy and/or a history of liver diseases are risky. HEV diagnosis and treatment become pivotal in Egyptian hospitals to reduce the fatality rate and they should start urgently and promptly.

Phylogenetic analysis of two new complete genomes of the hepatitis E virus (HEV) genotype 3 from Thailand

Hepatitis E virus (HEV) is a causative agent of acute viral hepatitis globally. Evolutionary phylogeny classifies the HEV into eight genotypes that correlate with the viral transmission. Only four genotypes have been proven to be responsible for transmission in humans. However, there has been no report on the genomics and genotyping of HEV in Thailand during the past ten years. Here, we identified the genotype distributions of the Thai isolates of HEV and we sequenced two HEV genomes. We screened for 18 Thai isolates of HEV from Siriraj Hospital in Bangkok, from 2014–2016. The HEV genomes were sequenced from the serum and feces of a patient. The results showed that all Thai isolates of HEV were identified as genotype 3 (HEV-3). The ORF2 and genome phylogenies suggested two subgenotypes, called 3.1 and 3.2. The Thai isolates of HEV were frequently found in the subgenotype 3.1. The genome sequences of the two Thai isolates of HEV from the serum and fecal samples of the same patient showed 91% nucleotide similarity with the HEV genotype 3. Comparisons between the HEV genome and the ORF2 phylogenies illustrated that the ORF2 tree can be used to identify HEV genotypes, but it has less phylogenetic power for the HEV evolution. The two new genome sequences of HEV-3 from Thailand could contribute valuable information to the HEV genome study. (226 words)

Characterization of a Novel Rat Hepatitis E Virus Isolated from an Asian Musk Shrew (Suncus murinus).

The Asian musk shrew (shrew) is a new reservoir of a rat hepatitis E virus (HEV) that has been classified into genotype HEV-C1 in the species Orthohepevirus C. However, there is no information regarding classification of the new rat HEV based on the entire genome sequences, and it remains unclear whether rat HEV transmits from shrews to humans. We herein inoculated nude rats (Long-Evans rnu/rnu) with a serum sample from a shrew trapped in China, which was positive for rat HEV RNA, to isolate and characterize the rat HEV distributed in shrews. A rat HEV strain, S1129, was recovered from feces of the infected nude rat, indicating that rat HEV was capable of replicating in rats. S1129 adapted and grew well in PLC/PRF/5 cells, and the recovered virus (S1129c1) infected Wistar rats. The entire genomes of S1129 and S1129c1 contain four open reading frames and share 78.3–81.8% of the nucleotide sequence identities with known rat HEV isolates, demonstrating that rat HEVs are genetically diverse. We proposed that genotype HEV-C1 be further classified into subtypes HEV-C1a to HEV-C1d and that the S1129 strain circulating in the shrew belonged to the new subtype HEV-C1d. Further studies should focus on whether the S1129 strain infects humans.