Primary structure of open reading frame 2 and 3 of the hepatitis E virus isolated from Morocco

The nucleotide sequence from position 5,014 to 7,186 of the hepatitis E virus (HEV) genome was determined using a set of 10 polymerase chain reaction (PCR) fragments amplified directly from a pool of fecal specimens obtained from patients with well-documented epidemic HEV infection in Morocco. This sequence contains the 3′-terminal region of open reading frame 1 (ORF1), full length ORF2 and ORF3, and a portion of the 3′-noncoding region. The HEV Morocco nucleotide sequence was compared with the corresponding sequences of 13 HEV strains. A region of ORF2 that overlaps with ORF3 was found to be the most conserved region of ORF2, whereas a protein segment encoded by this region was found to be the most variable. Theoretical RNA secondary structure analysis predicted that this region may be folded into a strong secondary structure that may constrain nucleotide sequence variability. In addition, the nucleotide sequence comparison revealed that the HEV Morocco sequence is most homologous to the sequences of the HEV Asian strains compared with the HEV Mexico, swine, and US strains. Phylogenetic analysis performed on the entire ORF2 and ORF3 sequences and on a small fragment of ORF2 allowed classification of the HEV Morocco strain together with a few other known African strains as a separate subtype within the Asian–African genotype. J. Med. Virol. 57:126–133, 1999. Published 1999 Wiley-Liss, Inc.     

Molecular cloning and sequencing of the Mexico isolate of hepatitis E virus (HEV).

Hepatitis E virus (HEV) is the major causative agent of hepatitis E or what was formerly known as enterically transmitted non-A, non-B hepatitis. The disease has a worldwide distribution but occurs principally in developing countries in any of three forms: large epidemics, smaller outbreaks, or sporadic infections. Genetic variation of different HEV strains was previously noted and it will be important to determine the extent to which this variation may pose problems in the diagnosis and treatment of HEV infection. To analyze differences at the genetic level between HEV(Mexico; M) and the previously characterized HEV(Burma; B) and HEV(Pakistan; P) isolates, overlapping cDNAs were cloned from samples obtained from an infected human and an experimentally inoculated cynomolgus macaque. These cDNA clones, representing the nearly complete (7185-bp) genome of HEV(M), confirmed an expression strategy for the virus that involves the use of 3 forward open reading frames (ORFs). The HEV(M) strain has an overall 76 and 77% nucleic acid identity with the HEV(B) strain and HEV(P) strain, respectively; however, the degree of sequence variation was not uniform throughout the viral genome. A hypervariable region was identified in ORF1 that exhibited a 58 and 54% nucleic acid sequence and 13% amino acid similarity with the Burma strain and the Pakistan strain, respectively. A large number of the nucleotide differences occurred at the third codon position, with the deduced amino acid sequences similarity of 83, 93, and 87% between HEV(M) and HEV(B) isolates in ORF1, ORF2, and ORF3, respectively, and with 84, 93, and 87% amino acid identities between HEV(M) and HEV(P) isolates in ORF1, ORF2, and ORF3, respectively. The nucleotide sequences derived from the highly conserved regions of HEV genome will be useful in developing polymerase chain reaction-based tests to confirm the viral infection. Knowledge of the extent of the sequence variation encountered with HEV will not only aid in the future development of diagnostic and vaccine reagents but also further our understanding of how HEV strain variation might impact the pathological outcome of infection.     

Geographical distribution of hepatitis E virus genotypes

Hepatitis E virus (HEV) is the major agent of acute hepatitis in developing countries where the infection occurs sporadically or in large waterborne epidemics. HEV, classified in the Caliciviridae, is not culturable. The detection of HEV RNA by RT-PCR in serum and stool samples is reliable during the 7 to 15 days following the onset of the disease. Restriction endonuclease analysis, cloning and sequencing of PCR products allow a phylogenetic analysis of HEV isolates. Although they belong to a single serotype, strains recovered from different geographical regions display a significant genetic heterogeneity. Sequencing data from ORF1 and ORF2 regions has led to the characterization of 3 distinct genotypes: genotype I gathering the Asian and African subgenotypes; genotype II gathering swine and human US strains; genotype III limited to the Mexico prototype. Novel variants are currently described from Africa (Nigeria), China and Europe (Greece and Italy). Each genotype appears to be related to a well defined geographical area. Nevertheless, a genetic variability is observed within endemic regions such as Asia or Africa. Nigerian endemic isolates especially could represent an intermediate stage in the evolutionary process towards genetic diversity. The animal reservoir, proved by the detection of HEV sequences by PCR among pigs in Nepal and in the USA, could help to resolve unanswered questions about the origin of HEV genotypes, their spread and evolution.     

Detection and characterisation of swine hepatitis E virus in New Zealand

The objectives of the present study were to establish the presence of hepatitis E virus (HEV) in New Zealand pigs, first by testing for HEV antibody in pig herds throughout New Zealand to measure the herd prevalence, then by attempting to amplify HEV genomic sequences by PCR. Antibody was measured by two independently designed ELISA serology tests. HEV RNA fragments were amplified by RT-PCR of nucleic acid extracted from faeces of 10-12-week-old piglets using primers targeting ORF1, ORF2, and ORF2/3. PCR products were subject to phylogenetic analysis. Antibody to HEV was found throughout New Zealand pig herds as well as in the different age groups within the herds. Twenty herds from 22 tested were positive for HEV antibody (91% herd prevalence). Phylogenetic analysis of the amplified sequences placed this New Zealand strain of HEV closest to the human European strain It-1 (AF 110390) and U.S. swine strain (AF 082843) with 88% and 83% similarity respectively in ORF1. It was concluded that HEV is widely distributed in the New Zealand pig population. Phylogenetic analysis shows that this is a new HEV strain, grouping most closely with the United States/European cluster, which includes HEV strains of both human and swine origin.     

Clinical and epidemiological implications of swine hepatitis E virus infection

In nonendemic areas, most patients with acute hepatitis E were infected through traveling to endemic areas. However, some patients did not have a history of foreign travel before infection. Furthermore, high seroprevalence rates of antibody to hepatitis E virus (anti-HEV) were found in the general adult population in some countries without any recorded outbreak of hepatitis E. The significance of anti-HEV assay in these subjects remains obscure. To study if swine might be a source of HEV infection, HEV was tested in sera of 235 pigs in Taiwan, and from 5 patients with acute HEV infection who either denied or did not provide any foreign travel history. Three (1.3%) pigs had detectable swine HEV RNA. The swine and human HEV strains from Taiwan formed a monophyletic group, distinct from three previously reported groups: the United States human and swine HEV strains, the Mexico strain, and the largest group composed of the Asian and the African strains. The identity of nucleotide sequences was 84-95% between swine and human HEV strains in Taiwan, and 72-79% between Taiwan strains and those from different areas. The predicted amino acid sequence of a Taiwan swine HEV strain within the peptide 3-2 used in commercial anti-HEV assay showed a high identity (91-94%) with those of other human and swine HEV strains. Swine may be a reservoir of HEV and subclinical swine HEV infection may occur. Cross-reactivity of current anti-HEV assay may account for the high prevalence rate of anti-HEV in the general population in nonendemic areas.     

Genetic heterogeneity of hepatitis E virus in Darfur, Sudan, and neighboring Chad

The within-outbreak diversity of hepatitis E virus (HEV) was studied during the outbreak of hepatitis E that occurred in Sudan in 2004. Specimens were collected from internally displaced persons living in a Sudanese refugee camp and two camps implanted in Chad. A comparison of the sequences in the ORF2 region of 23 Sudanese isolates and five HEV samples from the two Chadian camps displayed a high similarity (>99.7%) to strains belonging to Genotype 1. But four isolates collected in one of the Chadian camps were close to Genotype 2. Circulation of divergent strains argues for possible multiple sources of infection.     

烟台沿海地区人源和猪源戊型肝炎病毒基因型别的研究

目的调查烟台市沿海地区人源与猪源戊型肝炎病毒（HEV）基因型别的相关性。方法应用逆转录一巢式聚合酶联反应（RT—nPcR）方法对当地急性散发戊型肝炎患者、正常人群中抗HEV-IgM阳性者和当地养猪场的猪进行HEVRNA检测,并对HEVRNA阳性标本进行克隆测序和序列分析。结果16例急性散发戊型肝炎患者中有7例粪便标本HEVRNA阳性;51份IgM阳性正常人群血清标本中有1份HEVRNA阳性;34份猪胆汁标本中有1份HEVRNA阳性。序列分析发现该地区HEV人株与猪株在ORF2部分区域的核苷酸序列同源性为87％～98．1％。7株患者的戊肝病毒基因型和1株猪的戊肝病毒基因型均为Ⅳ型,基因序列同源性在87％～98．1％之间;其中有6例患者和猪的基因序列同源性在93．9％～98．1％之间,为Ⅳ型a亚型;1例患者和猪的基因序列同源性为87％,为Ⅳ型d亚型。正常人群的1例戊肝病毒基因型为Ⅰ型d亚型。该地区人与猪HEV的ORF2的部分基因片段与HEVⅠ～Ⅳ型的代表株进行比较,核苷酸序列同源性分别是82．5％～100％,81．7％～92．9％,81．4％～93．9％,84．9％～100％。结论该地区人群中流行的HEV存在2个基因型3个亚型,主要以基因Ⅳa型为主,与猪群中流行的HEV基因Ⅳa型同源性较高;HEVI型在人群中散在存在。     

Development and validation of an improved RT-PCR assay with nested universal primers for detection of hepatitis E virus strains with significant sequence divergence

Recent studies revealed that hepatitis E virus (HEV) genomes are more variable than previously thought and well-conserved regions suitable for designing universal primers are limited. In this study, based on alignment of 70 full-length HEV sequences of genotypes 1-4, a part of the ORF2/ORF3 overlapping region was found to be the best target region for PCR amplification of various HEV strains. Using the newly designed primers, an RT-PCR method (ORF2/3-137 PCR) that amplifies a 137-nucleotide (nt) sequence within the ORF2/ORF3 overlapping region and is capable of amplifying all known HEV sequences was developed. When compared with the previous RT-PCR method (ORF2-457 PCR) that amplifies a 457 nt ORF2 sequence, ORF2/3-137 PCR was two to three times more sensitive than ORF2-457 PCR upon testing serial dilutions of three HEV RNA-positive serum samples. The ORF2/3-137 PCR assay could detect viraemia in five patients with acute or fulminant hepatitis E 3-14 days longer than ORF2-457 PCR after disease onset. All 41 ORF2-457 PCR-positive serum samples of various genotypes tested positive for HEV RNA by the ORF2/3-137 PCR assay. Since the amplicons of ORF2/3-137 PCR contain variable sequences, a phylogenetic tree of the ORF2/3-137 products could clearly distinguish the different HEV genotypes.     

Genetic variability of hepatitis E virus within and between three epidemics in India

Hepatitis E virus (HEV) is an important cause of epidemic and sporadic acute viral hepatitis in many developing countries, including India. We evaluated the genetic variability within two regions (a 476-nt long ORF1 segment and a 304-nt long ORF2 segment) from specimens collected during three outbreaks in the cities of Karnal (1987), Yamunanagar (1989), and Meerut (1996), India, and from one patient, residing in Lucknow, India, who had a case of sporadic hepatitis (1996). Within an outbreak, sequences in the ORF1 and ORF2 regions were 99.3-100.0% identical. However, when strains were compared between outbreaks, identity in the ORF1 and ORF2 region was 97.1-99.2 and 96.4-100.0%, respectively. A comparison of these sequences to previously published Indian ORF1 and ORF2 sequences revealed even lower similarities, 95.2-98.5 and 95.1-98.7%, respectively. One patient in the Meerut outbreak had genomic sequences that differed substantially from the other patients affected during this outbreak and probably reflected a sporadic infection. The sporadic hepatitis E strain from Lucknow clustered with a previously described HEV strain from a patient with fulminant hepatic failure (FHF). Our data suggest that the ORF1 and ORF2 segments can be used to study the molecular epidemiology of HEV infection and indicate that much remains to be determined about the genetic variability of Indian HEV strains.     

The establishment of reference sequence for SARS-CoV-2 and variation analysis

Starting around December 2019, an epidemic of pneumonia, which was named COVID-19 by the World Health Organization, broke out in Wuhan, China, and is spreading throughout the world. A new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the Coronavirus Study Group of the International Committee on Taxonomy of Viruses was soon found to be the cause. At present, the sensitivity of clinical nucleic acid detection is limited, and it is still unclear whether it is related to genetic variation. In this study, we retrieved 95 full-length genomic sequences of SARAS-CoV-2 strains from the National Center for Biotechnology Information and GISAID databases, established the reference sequence by conducting multiple sequence alignment and phylogenetic analyses, and analyzed sequence variations along the SARS-CoV-2 genome. The homology among all viral strains was generally high, among them, 99.99% (99.91%-100%) at the nucleotide level and 99.99% (99.79%-100%) at the amino acid level. Although overall variation in open-reading frame (ORF) regions is low, 13 variation sites in 1a, 1b, S, 3a, M, 8, and N regions were identified, among which positions nt28144 in ORF 8 and nt8782 in ORF 1a showed mutation rate of 30.53% (29/95) and 29.47% (28/95), respectively. These findings suggested that there may be selective mutations in SARS-COV-2, and it is necessary to avoid certain regions when designing primers and probes. Establishment of the reference sequence for SARS-CoV-2 could benefit not only biological study of this virus but also diagnosis, clinical monitoring and intervention of SARS-CoV-2 infection in the future.     

A novel genotype of hepatitis E virus prevalent among farmed rabbits in China

In total, 335 serum samples were collected from rabbits from two farms in Gansu province, China, and tested for anti‐hepatitis E virus (HEV) antibody using EIA and for HEV RNA using nested RT‐ PCR with ORF2 primers. The overall prevalence of anti‐HEV antibody and HEV RNA was 57.0% (191/335) and 7.5% (25/335), respectively. The positivity rate of HEV RNA in the anti‐HEV antibody negative group (7.6% (11/144)) did not differ significantly from that in the positive group (7.3% (14/191)). The concordance between HEV RNA and anti‐HEV antibody was 43.3% with no significant correlation (P < 0.05). All 25 amplicons from the ORF2 region were cloned and sequenced. On the basis of nucleotide sequence comparison, they had 84–99% identity to each other and 73–77%, 70–76%, 75–82%, 71–77%, and 53–65% with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively. Samples that were positive with the ORF2 primers were amplified using ORF1 region primers; 17 were positive and shared 71–78%, 73–76%, 74–82%, 72–78%, and 39–58% identity with the corresponding regions of genotypes 1, 2, 3, 4, and avian HEV, respectively, at the nucleotide level. Two representative full‐length sequences were determined. These two sequences shared 85% identity with each other and had 74%, 73%, 78–79%, 74–75%, and 46–47% identity to full‐length genotypes 1, 2, 3, 4, and avian HEV, respectively. Thus, the sequences isolated from the rabbits represent a novel genotype of HEV. This study provides novel information about HEV genotypes infecting rabbits as well as evidence of a new mammalian genotype of HEV. J. Med. Virol. 81:1371–1379, 2009. © 2009 Wiley‐Liss, Inc.     

Partial sequence comparison of eight new Chinese strains of hepatitis E virus suggests the genome sequence is relatively stable

Partial genomic sequences representing 420 nucleotides of a nonstructional region, 480 nucleotides of the putative RNA polymerase region, and 540 nucleotides of the structural region of epidemic-associated Chinese strains of hepatitis E virus (HEV) were obtained by direct sequencing of PCR-amplified DNA. Comparison with previously published HEV sequences showed a clear relatedness of all Chinese strains to each other and to a Pakistani strain (Sar-55). All eight Chinese strains examined had very similar sequences (98.5-99.8% homology) in the regions examined and were much closer to the Pakistani strain (Sar-55) (97.9-98.4% homology) than to the Burmese strain (92.5-93.3% homology). Sequence comparisons of the three genomic regions in the Chinese strains indicated that the RNA polymerase region was much more conserved than the other nonstructural region or the structural region. HEV isolates from three remote geographic regions of China had sequences closely related to each other.     

Detection of hepatitis E virus in patients sera in southern Spain

The aim of the present study was to detect acute Hepatitis E virus (HEV) infection in patients with abnormal alanine transaminase (ALT) in which other viral hepatitis infections had been excluded in southern Spain, an area adjacent to regions where this disease is endemic. Of 336 sera tested 30 (8.92%) were positive for IgM antibodies against HEV (anti-HEV IgM) and 7 (2.08%) were negative in a repeated assay. Immunoblot analysis (IBA) was applied to the 37 positive sera in the first assay; its results were positivity for 26 (7.73%), ambiguous for 5 and negative for 6 sera. Amplification of ORF1 and ORF2 of HEV by means of nested RT-PCR was carried out with the 37 sera that were either positive or ambiguous by ELISA; a positive result was obtained only with one serum for the ORF2 protein. IgM antibodies against the HEV ORF2 protein could be a useful marker in the diagnosis of acute infection and a substitute for the determination of viral RNA in serum; this is of both diagnostic and epidemiological importance as it would allow the patients transmitting the infection to be recognized by means of a simple determination of antibodies. The sequence of the ORF2 fragment of HEV occurring in samples taken from both humans and animals amplified in this study has considerable homology with the sequences of HEV strains/isolates of European origin. These results demonstrate that an autochthonous HEV circulates in Spain.     

广州地区散发性戊型肝炎病毒基因片段序列分析

目的对广州地区散发性戊型肝炎病毒（ＨＥＶ）作基因片段序列分析。方法用逆转录套式聚合酶链反应（ＲＴ－ｎｅｓｔｅｄＰＣＲ）检测了８例广州地区散发性戊肝患者粪便和血清中的ＨＥＶＲＮＡ，其中３例粪便阳性。对阳性ＰＣＲ产物进行基因克隆、核酸序列分析。结果广州地区Ｇ１、Ｇ２、Ｇ３株与缅甸株（Ｂ）、巴基斯坦株（Ｐ）、墨西哥株（Ｍ）、中国新疆株（Ｃｈ１．１）和广州血清株（Ｇ－９）的核苷酸和氨基酸的同源性均值分别为８０．６７％和８８．６０％（Ｂ）、８１．２５％和８９．２０％（Ｐ）、７７．４５％和８４．８１％（Ｍ）、８１．２５％和８９．２０％（Ｃ）、９７．８５％和９６．２０％（Ｇ）。结论广州ＨＥＶ株有一定程度的变异。     

Full-genome nucleotide sequence of a hepatitis E virus strain that may be indigenous to Japan

We identified hepatitis E virus (HEV) RNA in serum from a Japanese patient with acute hepatitis, who had never been abroad. The full-genome nucleotide sequence of the HEV isolate (JRA1) from this patient was composed of 7227 nucleotides excepting the poly(A) tail and had ORF1 coding for 1703 amino acids (aa), ORF2 coding for 660 aa, and ORF3 coding for 122 aa. This Japanese strain showed approximately 87% nucleotide similarity to human and swine strains reported from the United States, while it had only 73-76% similarity to Asian and Mexican strains. Here we report the characteristics of the HEV-JRA1 isolate, which might be the first example of an indigenous strain(s) of HEV in Japan.     

Molecular virology of hepatitis E virus

This review details the molecular virology of the hepatitis E virus (HEV). While replicons and in vitro infection systems have recently become available, a lot of information on HEV has been generated through comparisons with better-studied positive-strand RNA viruses and through subgenomic expression of viral open reading frames. These models are now being verified with replicon and infection systems. We provide here the current knowledge on the HEV genome and its constituent proteins - ORF1, ORF2 and ORF3. Based on the available information, we also modify the existing model of the HEV life cycle.     

Imported and autochthonous hepatitis E virus strains in Spain

Hepatitis E virus (HEV) causes hepatitis E, an acute liver disease displaying diverse epidemiological patterns that correlate with the genetic diversity of the virus. Only a few strains have been characterized to date from cases of hepatitis E in Spain. Using three sets of new, HEV‐specific primers, viral genome fragments were amplified from serum samples from 13 patients with acute hepatitis in different regions of Spain. Direct sequencing of these fragments and analysis of sequences lead to identify six genotype 1, six genotype 3, and one genotype 4 viral strains. Genotype 1 sequences were found in the clade with subtype 1a strains, and were amplified from travelers from India and Bangladesh, and from an African immigrant. Genotype 3 sequences were found in the clade with subtype 3f strains, were always amplified from patients who did not travel abroad recently, and were closely related to sequences from swine strains isolated in Spain. Patients infected by these strains lived in different regions and were mainly men aged above 50 years. The single genotype 4 sequence detected was amplified from a traveler returning from Vietnam. Hepatitis E is both an imported and an autochthonous disease in Spain, and closely related HEV genotype 3f strains are responsible for infections acquired locally in different regions of the country within a given time. Studies involving a significant number of human, swine, and environmental viral strains collected prospectively are, however, required in order to confirm a swine origin for autochthonous HEV genotype 3 human infections. J. Med. Virol. 81:1743–1749, 2009. © 2009 Wiley‐Liss, Inc.