Epidemiological study of hepatitis E virus infection among students and workers in Hebei Province of China.

AIMS: Hepatitis E caused by the hepatitis E virus (HEV) is prevalent worldwide. In China, considerable shifts in the epidemiology of hepatitis E have been observed over the last two decades, with ongoing changes in the prevalence of HEV. METHODS: This study, in conjunction with the health examinations for students and workers, aims to estimate the seroprevalence and assess the risk factors of HEV infection in general population in Hebei province, China. Epidemiological information was collected using a specific questionnaire and blood samples were collected from each participant during the process of health examination. Anti-HEV IgG and IgM in sera were tested using the Wantai ELISA assay kits. Logistic regression modelling was used to identify associated risk factors. RESULTS: The average positive rate of anti-HEV IgG in students (6-25 years) was 3.4%. One (0.2%) student was anti-HEV IgM positive, while also testing positive for IgG. The HEV seroprevalence was not related to students' gender, school, or family residence. In occupational populations, the overall seropositivity rate was 13.3% for anti-HEV IgG and 0.67% for IgM. HEV seropositivity increased significantly with age, ranging from 3.8% to 18.6% in age groups, and differed significantly among four occupation groups: farmers (17.6%), food supply workers (18.0%), other non-farm workers (14.7%) and healthcare workers (5.9%) (p = 0.002). Multivariable logistic analysis confirmed the significant correlations of seroprevalence with age and occupation. CONCLUSIONS: The study found a low seroprevalence of HEV in children and young adults in Hebei Province, China. Advanced age correlates with higher seroprevalence in occupational populations, indicating an accumulation of HEV infection over time. Seroprevalence varied significantly among different occupation groups, suggesting the important role of occupational exposure for HEV infection.

Detection of Hepatitis E Virus in Rabbits and Rabbit Meat from Slaughterhouses in Hebei Province of China.

Background: Hepatitis E virus (HEV) is a zoonotic pathogen. HEV has been found to be widely prevalent in rabbits. Its isolates are classified into HEV-3, rabbit subgenotype (HEV-3ra). The routes of human infection with HEV-3ra remain unclear; however, foodborne transmission is possible when asymptomatically infected animals enter the food chain. The prevalence of HEV infection in slaughtered rabbits and the presence of HEV in rabbit meat were evaluated in this study. Materials and Methods: In three slaughterhouses in Hebei province, China, samples of rabbit blood were collected during the slaughter process, and muscle, liver, and cavity juice were collected from the rabbit carcasses. Anti-HEV antibody in serum samples was detected using enzyme-linked immunosorbent assay. HEV RNA was tested in all samples by reverse transcription nested PCR (RT-nested PCR). The final amplicons of RT-nested PCR were sequenced and phylogenetically analyzed. Results: Of the 459 serum samples, 50 [10.9%, 95% confidence interval (CI): 8.1-13.7] were positive for anti-HEV antibody, and 17 (3.7%, 95% CI: 2.0-5.4) were positive for HEV RNA. HEV RNA was detected in 7 of 60 liver samples (11.7%, 95% CI: 3.3-20) and 2 cavity juice samples from semi-eviscerated carcasses, but was not detected in any muscle sample from either the eviscerated or semi-eviscerated carcasses. All the detected HEV strains belonged to HEV-3ra and related most closely with the rabbit HEV sequence previously reported in China. Conclusion: A portion of rabbits were in the viremia period of HEV infection at the slaughter age, resulting in the possibility of HEV carriage by rabbit carcass, particularly semi-eviscerated carcass containing liver. These findings suggest a potential risk of HEV transmission from raw rabbit products entering the food chain, whereas the presence of HEV appeared to be lower in the eviscerated carcass than in the semi-eviscerated carcass.

Hepatitis E Virus.

Since the sequence of hepatitis E virus (HEV) was determined from a patient with enterically transmitted non-A, non-B hepatitis in 1989, similar sequences have been isolated from many different animals, including pigs, wild boars, deer, rabbits, bats, rats, chicken, and trout. All of these sequences have the same genomic organization, which contains open reading frames (ORFs) 1, 2, and 3, although their genomic sequences are variable. Some have proposed that they be classified as new family, Hepeviridae, which would be further divided into different genera and species according to their sequence variability. The size of these virus particles generally ranged from 27 to 34 nm. However, HEV virions produced in cell culture differ in structure from the viruses found in feces. Those from cell culture have a lipid envelope and either lack or have a little ORF3, whereas the viruses isolated from feces lack a lipid envelope but have ORF3 on their surfaces. Surprisingly, most of the secreted ORF2 proteins from both these sources are not associated with HEV RNA.

Epidemiology of Hepatitis E.

Hepatitis E virus (HEV) is globally prevalent with relatively high percentages of anti-HEV immunoglobulin G-positive individuals in the populations of developing and developed countries. There are two distinct epidemiological patterns of hepatitis E. In areas with high disease endemicity, primarily developing countries in Asia and Africa, this disease is caused mainly by genotypes HEV-1 or HEV-2; both genotypes transmit predominantly through contaminated water and occur as either outbreaks or sporadic cases of acute hepatitis. The acute hepatitis has the highest attack rate in young adults and is particularly severe among pregnant women. In developed countries, sporadic cases of locally acquired HEV-3 or HEV-4 infection are observed. The reservoir of HEV-3 and HEV-4 is believed to be animals, such as pigs, with zoonotic transmission to humans. The affected persons are often elderly, and persistent infection has been well documented among immunosuppressed persons. A subunit vaccine has been shown to be effective in preventing clinical disease and has been licensed in China.

Transmission of Hepatitis E Virus.

Transmission of hepatitis E virus (HEV) occurs predominantly by the fecal-oral route. Large epidemics of hepatitis E in the developing countries of Asia and Africa are waterborne and spread through contaminated drinking water. The reservoir of HEV in developed countries is believed to be in animals with zoonotic transmission to humans, possibly through direct contact or the consumption of undercooked contaminated meat. And HEV transmission through blood transfusion, organ transplantation, and vertical transmission has been reported.

Stability and transmissibility of SARS-CoV-2 in the environment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing the ongoing global coronavirus disease 2019 (COVID-19) pandemic, is believed to be transmitted primarily through respiratory droplets and aerosols. However, reports are increasing regarding the contamination of environmental surfaces, shared objects, and cold-chain foods with SARS-CoV-2 RNA and the possibility of environmental fomite transmission of the virus raises much concern and debate. This study summarizes the current knowledge regarding potential mechanisms of environmental transmission of SARS-CoV-2, including the prevalence of surface contamination in various settings, the viability and stability of the virus on surfaces or fomites, as well as environmental factors affecting virus viability and survival such as temperature and relative humidity. Instances of fomite transmission, including cold-chain food transmission, and the importance of fomite transmission in epidemics, are discussed. The knowledge gaps regarding fomite transmission of SARS-CoV-2 are also briefly analyzed.

Detection of Hepatitis E Virus in Raw Pork and Pig Viscera As Food in Hebei Province of China.

Hepatitis E virus (HEV) is an important human pathogen with pigs serving as the main natural animal reservoir. In China, pork is the most popular meat, while pig viscera are also widely consumed. The aim of this study was to evaluate the prevalence of HEV among pigs at slaughter, and assess the presence of HEV in raw pork and pig viscera as food. Samples of pig blood, raw pork, liver, kidney, and blood curd were collected from slaughterhouse or (and) retail market. Anti-HEV antibody in serum samples was detected using enzyme-linked immunosorbent assay based on an ORF2 antigen sandwich kit. HEV RNA was tested by reverse transcription nested polymerase chain reaction (RT-nested PCR) and the viral load was further assessed using quantitative real-time PCR. The final amplicons of RT-nested PCR were sequenced and undergone phylogenetic analysis. Prevalence of antibodies to HEV was 90.4% (104/115) in pigs at slaughtered level, and one serum sample was HEV RNA positive (0.9%, 1/115). HEV RNA was detected in liver, kidney, and blood samples with positivity of 6.1% (7/114), 3.1% (4/129), and 1.2% (2/170) respectively with viral loads ranged 102.4-104.4 (2.4Log-4.4Log) genome equivalents per gram, but not in pork. The HEV RNA prevalence in both liver and kidney were statistically higher than in pork. Phylogenetic analysis showed that all obtained sequences belonged to HEV genotype 4, which were divided into subtypes 4a, 4b, 4d, and 4i, highly identical to the known human and swine HEV sequences in China. The results indicate that raw pig viscera are more likely to harbor HEV than pork, suggesting a higher transmission risk related to consuming pig organs.

High seroprevalence of hepatitis E virus in rabbit slaughterhouse workers.

Hepatitis E virus (HEV) was first detected in rabbits in the year 2009. Rabbit HEV is now known to be widely prevalent in rabbits and tentatively assigned into genotype 3 (HEV-3) as subgenotype-3ra (HEV-3ra). However, its role in human infection remains undetermined. This study was conducted to investigate the prevalence of HEV infection among rabbit slaughterhouse workers and to identify whether the workers exposed to rabbits are at a higher risk of HEV infection. Seventy-five workers at rabbit slaughterhouses and a control group of 421 general adults in the same area in Hebei province, China, were serologically examined for anti-HEV antibodies. HEV seroprevalences between the slaughterhouse workers and the general adults were compared. Age-adjusted prevalence of anti-HEV immunoglobulin G (IgG) in the rabbit slaughterhouse workers and control group was 46.1% and 10.8% respectively. The slaughterhouse workers had significantly higher seroprevalence and an approximately 6.9-fold increased risk for being seropositive for anti-HEV IgG as compared to the general population (odds ratio, 6.9; 95% CI: 4.3, 10.9). In slaughterhouse workers, anti-HEV IgG positive rate was positively associated with working years; in general adults, this rate was positively associated with age. The prevalence of anti-HEV immunoglobulin M (IgM) among exposed workers (6.7%) was significantly higher than that of control groups (1.2%). In conclusion, the seroprevalence of HEV is significantly higher in slaughterhouse workers than in general adults indicating that occupational exposure to rabbits is a potential risk factor for HEV infection.

Hepatitis E virus was not detected in feces and milk of cows in Hebei province of China: No evidence for HEV prevalence in cows.

Hepatitis E virus (HEV) is an important human pathogen, with pigs and other species serving as natural animal reservoirs. Recently, the report of detection of genotype 4 HEV in dairy milk with high positive rate in Yunnan province of China has attracted extensive attention. To assess the zoonotic risk of cows as HEV reservoir and transmission of HEV through dairy milk, 467 fecal samples of cows, 276 fresh milk samples, and 140 retail milk samples were collected across Hebei Province, China, from March 2017 to May 2018, and detected for HEV RNA. Fecal samples of rabbit or pig were also collected for HEV detection from farms of mixed farming with cows or farms neighboring cow farms. HEV RNA was not detected in any cow feces or in any milk samples, but 9.3% feces of pigs and 18.9% feces of rabbits were positive for HEV RNA. In addition, all of the dairy milk samples undergone HEV antigen and anti-HEV antibody detections, but none was positive. Phylogenetic analysis showed that all of the HEV isolates from pigs belonged to genotype 4 and those from rabbits were genotype 3-rabbit HEV. The results indicate that, currently in Hebei province of China, HEV is not apparently prevalent in cows and hence there is no zoonotic transmission risk through dairy milk towards humans, albeit the genotype 4 and 3 (rabbit) HEV are prevalent in pigs and rabbits respectively.

Valuable antibody detection method for classifying hepatitis E virus genotypes.

Nucleotide-based methods are conventionally used to classify the hepatitis E virus (HEV) genotypes. A serological enzyme immunoassay (EIA) using open reading frame 3 (ORF3) C-terminal peptides was developed to conveniently and accurately classify and evaluate the genotypes of HEV. The sera of mice immunized with HEV genotype 1, 3, and 4 reacted highly specifically to the peptides of the corresponding genotypes. Most (84.2%) clinical sera infected with HEV genotype 4 were positive for anti-HEV antibodies when tested with the ORF3 peptides of genotype 4, but were negative for genotypes 1 and 3. Monkey and clinical serial sera infected with HEV reacted strongly to the homologous genotype ORF3 peptides. The indirect EIAs were more sensitive, with stronger reactivity, than commercial anti-HEV immunoglobulin G assays when serial sera from monkeys infected with HEV genotype 1 or 4 were tested. All our results indicate that the serological typing EIA assays described in this study are more effective and convenient for the classification of HEV genotypes than molecular approaches, and can be used to screen large numbers of serum samples and differentiate genotypes for the diagnosis of HEV infections.

Systematic identification of hepatitis E virus ORF2 interactome reveals that TMEM134 engages in ORF2-mediated NF-κB pathway.

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E. Open reading frame 2 (ORF2) encodes the capsid protein of HEV, which is the main structural protein and may participate, together with the host factors, in viral entry and egress. However, it is still not clear which host proteins are involved in the interaction with ORF2 and what the functions of these ORF2-interacting proteins are. In this study, we have applied a split-ubiquitin yeast two-hybrid screening approach in combination with the pull-down and coimmunoprecipitation assays, identified and validated multiple interacting partners of ORF2 of genotype 1 and 4, which have diverse biological functions. Among these novel candidates that have not been previously reported, we have found that 20 of them are located in endoplasmic reticulum. TMEM134, which interacts and co-localizes with ORF2 in the endoplasmic reticulum, negatively regulates ORF2-mediated inhibition of the NF-κB signaling pathway. Our study for the first time has systematically mapped the ORF2 interactome in two genotypes of HEV, providing a new insight of understanding the virus-host interaction during the pathogenesis of HEV, and may offer potential therapeutic targets to intervene the HEV life cycle.

Hepatitis E Virus.

Since the sequence of hepatitis E virus (HEV) was determined from a patient with enterically transmitted non-A, non-B hepatitis in 1989, similar sequences have been isolated from many different animals, including pigs, wild boars, deer, rabbits, bats, rats, chicken, and trout. All of these sequences have the same genomic organization, which contains open reading frames (ORFs) 1, 2, and 3, although their genomic sequences are variable. Some have proposed that they be classified as new family, Hepeviridae, which would be further divided into different genera and species according to their sequence variability. The size of these virus particles generally ranged from 27 to 34 nm. However, HEV virions produced in cell culture differ in structure from the viruses found in feces. Those from cell culture have a lipid envelope and a little ORF3 on their surfaces, whereas the viruses isolated from feces lack lipid envelope and ORF3. Surprisingly, most of the secreted ORF2 protein from both these sources is not associated with HEV RNA.

Epidemiology of Hepatitis E.

Hepatitis E virus (HEV) is globally prevalent with relatively high percentages of anti-HEV immunoglobulin G-positive individuals in the populations of developing and developed countries. There are two distinct epidemiologic patterns of hepatitis E. In areas with high disease endemicity, primarily developing countries in Asia and Africa, this disease is caused mainly by genotype 1 or 2 HEV, both of which transmit predominantly through contaminated water and occur as either outbreaks or as sporadic cases of acute hepatitis. The acute hepatitis caused by either of these two genotypes has the highest attack rate in young adults, and the disease is particularly severe among pregnant women. In developed countries, sporadic cases of locally acquired genotype 3 or 4 HEV infection are observed. The reservoir of genotype 3 and 4 HEV is believed to be animals, such as pigs, with zoonotic transmission to humans. The affected persons are often elderly, and persistent infection has been well documented among immunosuppressed persons. A subunit vaccine has been shown to be effective in preventing clinical disease and has been licensed in China.

Transmission of Hepatitis E Virus.

Transmission of hepatitis E virus (HEV) occurs predominantly by the fecal-oral route. Large epidemics of hepatitis E in the developing countries of Asia and Africa are waterborne and spread through contaminated drinking water. The reservoir of HEV in developed countries is believed to be in animals with zoonotic transmission to humans, possibly through direct contact or the consumption of undercooked contaminated meat. HEV transmission through blood and vertical transmission have also been reported.

Detection and assessment of infectivity of hepatitis E virus in urine.

BACKGROUND & AIMS: Hepatitis E virus (HEV) is known to be excreted in the stool but there has been no report of its presence in urine. This study investigated the presence of HEV RNA and antigen (HEV-Ag) in urine and its possible transmission. METHODS: Serum and urine samples from patients with chronic or acute HEV infection and HEV infected monkeys were tested for viral and biochemical markers. Liver and kidney biopsies from the infected monkeys were analyzed by histopathology and immunohistochemistry. The infectivity of HEV from urine was assessed by inoculation into monkeys. RESULTS: HEV RNA and HEV-Ag were detected persistently in the urine of a patient with chronic HEV infection. Subsequently, HEV RNA was detected in the urine of three of the eight (37.5%) acute patients, all of whom had detectable HEV-Ag in their urine. HEV RNA and HEV-Ag were also detectable in the urine of HEV infected monkeys. The ratio of HEV-Ag to RNA in the urine of the infected monkeys was significantly higher than in their sera and feces. The parameters of routine urinalysis remained within the normal ranges in the hepatitis E patients and infected monkeys, however, pathological changes and HEV-Ag were observed in the kidneys of the infected monkeys. Furthermore, one of two monkeys became infected with HEV after inoculation with urine from another infected monkey. CONCLUSIONS: HEV infection may result in kidney injury and the urine may pose a risk of transmission. HEV-Ag detection in urine may be valuable for diagnosis of ongoing HEV infection.

Persistent hepatitis e virus genotype 4 infection in a child with acute lymphoblastic leukemia.

INTRODUCTION: In general, the hepatitis E virus (HEV) causes acute, self-limiting hepatitis. Prolonged and chronic infections caused by HEV genotype 3 have been found in some immunosuppressed patients in developed countries. CASE PRESENTATION: Here we report a Chinese boy with acute lymphoblastic leukemia, who developed hepatitis E during a period of intensive chemotherapy. Twenty months after the initial infection, HEV viremia was reappeared in the patient, with detectable anti-HEV IgM and IgG and modestly elevated serum transaminases. Sequence analysis of the viral RNAs revealed the reactivation of the HEV genotype 4d strain, indicating viral persistence in the patient. CONCLUSIONS: To our knowledge, this is the first chronic case confirmed by the prolonged presence of HEV RNA in china. It is also the first reported persistent hepatitis E infection caused by HEV genotype 4.

Hepatitis E virus open reading frame 3 protein interacts with porcine liver-specific plasminogen and α2-antiplasmin.

Hepatitis E virus (HEV) is a hepatotropic virus that causes acute and, occasionally, chronic viral hepatitis. At least four recognized genotypes of mammalian HEV have been identified. Genotypes 3 and 4 are zoonotic in humans and pigs are the major host. The 7.2 kb genome of HEV contains three open reading frames: ORF1, ORF2, and ORF3. ORF1 encodes a nonstructural protein and the ORF2 protein is the main capsid protein but the precise functions of ORF3 protein remain obscure. To explore the role of ORF3 in the porcine host, the genotype 4 ORF3 protein was used for yeast two-hybrid screening to find cellular binding partners encoded by a pig liver cDNA library, two porcine liver specific proteins, plasminogen and α2-antiplasmin were identified. Their interactions with the ORF3 protein were validated by chemiluminescent Co-Immunoprecipitation assay and by Western blotting in Co-IP and His pull-down assay. The biological significance of these interactions and their possible role in the HEV infection are discussed.

Genotype analysis of hepatitis E virus from sporadic hepatitis E cases in northern China.

Hepatitis E is an important public health problem in many countries. However, there is no definite conclusion about the zoonotic reservoir, transmission patterns and risk factors of hepatitis E in the human population. The aim of this study was to analyze the epidemiological and viral genotype characteristics of hepatitis E cases in northern China. Surveillance was conducted in two hospitals in Liaoning and Hebei province from July 2010 to June 2012. Out of a total of 116 diagnosed patients, 88 (75.9%) were male and 28 (24.1%) were female and most (73%) were in the age group 40-70 years. In both hospitals, cases were diagnosed more frequently in March than in other months. HEV RNA was amplified from 41 patients and characterized by nucleotide sequencing and phylogenetic analysis. Most of the isolates (37 strains, 90.3%) were genotype 4, including subgenotypes 4a, 4b, 4d, 4h, 4i and a new subgenotype. One subgenotype 3a strain was isolated from Baoding, Hebei province. Three genotype 1b strains were found from patients in Jinzhou, Liaoning province. Most of the genotype 4 strains and the genotype 3 strains were phylogenetically related to known swine isolates. In conclusion, the finding that HEV infects mostly middle-aged and elderly men and that the incidence spiked in March may reflect the zoonotic transmission characteristics of HEV infection. Pigs, but not rabbits, were the important reservoirs in this area, because genotype 4 HEV was found to be responsible for the majority hepatitis E cases. However, genotype 1 is still present in northern China. Also, the first isolation of genotype 3 HEV in this area indicates that alternative routes of HEV transmission might exist.

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.