Yansheng Geng

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.

Serological Prevalence of Hepatitis E Virus in Domestic Animals and Diversity of Genotype 4 Hepatitis E Virus in China

Pigs have been confirmed to be reservoirs of some genotypes of hepatitis E virus (HEV), and other nonhuman species are also likely infected with the virus. To assess the prevalence of HEV infection in domestic animals in China, 3579 serum samples, including 1967 swine, 700 goat, and 912 cattle sera, were collected from 26 provinces across the country and tested for HEV antibodies and antigen using enzyme immunoassays. The results showed that 82.2% of the swine samples, but only 10.4% and 28.2% of cattle and goat sera, were anti-HEV positive respectively. The prevalence of anti-HEV antibody in animals varied from province to province, ranging from 10.9% to 100% in pigs, 0% to 48% in goats, and 0% to 92.9% in cattle. About 1.9% of pigs, 1.6% of goats, and 0.8% of cattle tested in the study were positive for HEV antigen. Some samples, including all HEV antigen-positive samples, were tested for HEV-specific RNA using reverse transcription polymerase chain reaction. Fifteen swine samples, but none from the goats or cattle, were found to be HEV RNA positive. Sequence and phylogenetic analyses classified all the swine HEV isolates into HEV genotype 4, which was further divided into four subgroups. This study demonstrated that HEV infection is widespread in domestic animals, particularly pigs, in China. The HEV genotype infecting pigs in China was genotype 4. However, the isolates displayed considerable genetic diversity.

Virus host protein interaction network analysis reveals that the HEV ORF3 protein may interrupt the blood coagulation process

Hepatitis E virus (HEV) is endemic worldwide and a major cause of acute liver disease in developing countries. However, the molecular mechanisms of liver pathology and clinical disease are not well understood for HEV infection. Open reading frame 3 (ORF3) of HEV encodes a small phosphoprotein, which is assumed to be involved in liver pathology and clinical disease. In this study, the interactions between the HEV ORF3 protein and human proteins were investigated using a stringent, high-throughput yeast two-hybrid (Y2H) analysis. Thirty two proteins were shown to interact with genotype 1 ORF3, 28 of which have not been reported previously. These novel interactions were evaluated by coimmunoprecipitation of protein complexes from transfected cells. We found also that the ORF3 proteins of genotype 4 and rabbit HEV interacted with all of the human proteins identified by the genotype 1 ORF3 protein. However, the putative ORF3 protein derived from avian HEV did not interact with the majority of these human proteins. The identified proteins were used to infer an overall interaction map linking the ORF3 protein with components of the host cellular networks. Analysis of this interaction map, based on functional annotation with the Gene Ontology features and KEGG pathways, revealed an enrichment of host proteins involved in complement coagulation, cellular iron ion homeostasis and oxidative stress. Additional canonical pathway analysis highlighted the enriched biological pathways relevant to blood coagulation and hemostasis. Consideration of the clinical manifestations of hepatitis E reported previously and the results of biological analysis from this study suggests that the ORF3 protein is likely to lead to an imbalance of coagulation and fibrinolysis by interacting with host proteins and triggering the corresponding pathological processes. These results suggest critical approaches to further study of the pathogenesis of the HEV ORF3 protein.

Evaluation of an antigen‐capture EIA for the diagnosis of hepatitis E virus infection

An enzyme immunoassay (EIA) has been developed for hepatitis E virus (HEV) antigen (HEV‐Ag) detection and marketed in China. This study aimed to evaluate the sensitivity of the assay and assess the value of HEV‐Ag detection in the diagnosis of HEV infection in comparison with HEV RNA detection. Using serial dilutions of a genotype 4 HEV strain, significant correlation was found between the EIA (S/CO) and HEV RNA (IU/mL) concentration in the range 103.5 to 100.5 IU/mL HEV RNA, the Pearson correlation coefficient r approached 0.97. The EIA detection limit was 54.6 IU/mL, compared to 24 IU/mL for HEV RNA using real‐time RT‐PCR. In clinical samples from hepatitis E patients, the HEV‐Ag and HEV RNA positivity rates were 55.6% (65/117) and 60.7% (71/117) in sera and 76.7% (56/73) and 84.9% (62/73) in stools, and the concordance of these two markers was 77.8% in sera and 80.8% in stools. In serum samples, the HEV‐Ag positivity rate and the concordance between HEV‐Ag and HEV RNA were inversely proportional to the presence of anti‐HEV antibody. The presence of anti‐HEV IgG could reduce the S/CO of the HEV‐Ag EIA. These results reveal a significant correlation between the detection of HEV‐Ag and HEV RNA. The sensitivity of the HEV‐Ag EIA was lower than real‐time RT‐PCR but could be higher than conventional nested RT‐PCR. Therefore, the detection of HEV‐Ag in serum and faeces is valuable for the diagnosis and prognosis of HEV infection in developing regions where real‐time RT‐PCR is not available.

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.

Comparison of hepatitis E virus genotypes from rabbits and pigs in the same geographic area: no evidence of natural cross-species transmission between the two animals.

Domesticated pigs have been shown to be a reservoir of genotypes 3 and 4 hepatitis E virus (HEV). Farmed rabbits were recently recognized as the host of a novel virus, rabbit HEV. In order to determine whether HEV is transmitted naturally between rabbits and pigs, a survey on HEV infections was conducted in rabbits and pigs aged 2-4 months from rabbit and pig farms located near to each other in nine villages in three counties of Hebei Province, China. The overall anti-HEV antibody positivity rates in serum samples of swine and rabbits were 61.7% (58/94) and 23.2% (67/289), and the positive rates for HEV RNA were 23.4% (22/94) and 10% (29/289), respectively. In addition, 37 of 125 swine fecal samples (29.6%) were HEV RNA positive. The nucleotide sequences of a 304 bp region within HEV ORF2 have identity ranging from 84.5% to 100% among the rabbit isolates and from 82.3% to 100% among the swine isolates. In contrast, the nucleotide identity between the two species groups was only 72-76.6%. Consequently, the two groups were clearly separated in the phylogenetic tree that showed all of the rabbit isolates are closely related to the rabbit HEV reported recently and the swine isolates belong to genotype 4, including subgenotypes 4a, 4c and 4d. The results showed that HEV is highly prevalent in farmed rabbits and pigs in these areas. However, genotype 4 HEV and rabbit HEV are circulating separately in pigs and rabbits in the same area. In conclusion, there was no evidence of cross-species transmission of HEV between pigs and rabbits. The frequency of HEV transmission events between these two animal species is likely low in commercial farms.

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.

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.