Recent studies have reported and provided nucleotide sequence data from divergent isolates of hepatitis E virus (HEV), including isolates from North America ...
Journal of General Virology (1999), 80, 169–177. Printed in Great Britain ...................................................................................................................................................................................................................................................................................
A divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis Youchun Wang,1, 2 Roger Ling,1 James C. Erker,3 Huayuan Zhang,2 Hemin Li,2 Suresh Desai,3 Isa K. Mushahwar3 and Tim J. Harrison1 1 Department of Medicine, Royal Free and University College Medical School of University College London, Royal Free Campus, Rowland Hill Street, London NW3 2PF, UK 2 Department of Hepatitis, National Institute for the Control of Pharmaceutical and Biological Products, Temple of Heaven, Beijing 100050, Peoples’ Republic of China 3 Experimental Biology Research, Abbott Laboratories, 1401 Sheridan Road, North Chicago, IL 60064, USA
Recent studies have reported and provided nucleotide sequence data from divergent isolates of hepatitis E virus (HEV), including isolates from North America and Africa. Sera were investigated from 29 Chinese patients with a diagnosis of acute hepatitis and who were negative for hepatitis viruses A–E by serology (HEV was excluded by testing for IgG antibody only). To determine whether some patients were infected with HEV but had yet to seroconvert to antibody positivity, RT–PCR was carried out with primers designed within conserved sequences of the HEV open reading frame (ORF) 1 and ORF2 regions. Fifteen patients were found to harbour sequences related to HEV. Analysis of the HEV products revealed that nucleotide sequences from nine of the sera closely matched Burmese-like HEV sequences (more than 92 % nucleotide identity across ORF1 and 88 % in ORF2). The remaining six HEV isolates were similar to each other but divergent from all other known HEV sequences (74 to 83 % nucleotide identity in ORF1 or ORF2). Phylogenetic analysis suggests that the six divergent isolates represent a fourth genotype of HEV, distinct from the previously described Burmese, Mexican and United States variants (genotypes 1, 2 and 3). This novel variant, referred to here as the Chinese genotype (genotype 4), may be responsible for a significant proportion of cases of acute hepatitis in China, as seen by the fact that 40 % of the HEVinfected patients in this study were genotype 4 positive.
Introduction The description at the end of the 1980s of hepatitis C virus (HCV, Choo et al., 1989) and hepatitis E virus (HEV ; Reyes et al., 1990) largely solved the enigma of non-A, non-B hepatitis (NANBH). While HEV resembles hepatitis A virus (HAV) in its route of transmission and in causing acute, resolving hepatitis, the epidemiology is quite different. HEV is generally found in developing countries. Major epidemics and sporadic infections, typically associated with faecal contamination of drinking water, have been described in various Asian and African countries and in Mexico. During epidemics, a percentage of Author for correspondence : Tim Harrison. Fax j44 171 830 2425. e-mail tjh!rfhsm.ac.uk The HEV ORF1 and ORF2 sequences reported here have been deposited with GenBank accession nos AF082081–AF082096.
0001-5735 # 1999 SGM
pregnant women develop unexplained fulminant hepatitis with a significant rate of mortality, reportedly as high as 20 %. Although chronic HEV infections have not been described, some acute episodes involve prolonged excretion of virus in the faeces (Nanda et al., 1995) and such infections may help to maintain a reservoir of virus between epidemics. It is also possible that the virus infects other hosts, as antibodies to HEV and HEV sequences have been detected in domestic livestock, such as sheep and pigs, and in rodents, such as rats (Balayan et al., 1990 ; Usmanov et al., 1994 ; Meng et al., 1997). The prototype nucleotide sequence of HEV was derived from a Burmese isolate (Tam et al., 1991). Other Asian isolates from Pakistan, India and China (Tsarev et al., 1992 ; Donati et al., 1997 ; Yin et al., 1993) resemble this prototype closely. HEV sequences described recently from Africa (Algeria and Chad ; van Cuyck-Gandre et al., 1997) (Morocco and Tunisia ; Chatterjee et al., 1997) and Central Asia (Tashkent, Uzbekistan BGJ
Y. Wang and others
and Osh, Kirgizia ; Chatterjee et al., 1997) are distinct from previous isolates but nonetheless cluster phylogenetically with the Asian prototype. All of these Burmese-like isolates share more than 90 % nucleotide identity, with the exception of sequences within the hypervariable region of open reading frame (ORF) 1. In contrast, the single isolate from Mexico (Huang et al., 1992) is clearly divergent from the Burmese-like isolates, sharing approximately 75 % nucleotide identity. Despite this divergence, immunoassays developed to detect immunodominant epitopes encoded by ORF2 and ORF3 generally cross-react with known isolates. This divergence is reflected in the design of assays for anti-HEV antibodies, which employ peptides and recombinant antigens from both the Burmese and Mexican prototypes (Yarbough et al., 1991 ; Dawson et al., 1992). HEV isolates significantly divergent from the Burmese and Mexican prototypes have been identified in the USA. While almost all clinical cases of hepatitis E in the USA, and other developed countries, are in travellers returning from endemic areas, anti-HEV in normal blood donors is not invariably associated with travel (Dawson et al., 1992). A case of acute hepatitis E in a non-traveller from Minnesota was reported recently, demonstrating the presence of a significantly divergent variant, HEV-US1, in the USA (Kwo et al., 1997 ; Schlauder et al., 1998). HEV-US1 is approximately as similar to the Mexican and Burmese-like isolates as the Mexican isolate is to the Burmese-like isolates, i.e. approximately 75 % nucleotide identity. This HEV-US1 isolate shows a significant degree of nucleotide identity to HEV sequences recently isolated from herds of swine in the USA, approximately 92 % (Meng et al., 1997). Hepatitis E is endemic in China and epidemics have also been reported. For example, around 120 000 individuals were infected during a prolonged outbreak of enterically transmitted NANBH in Xinjiang between July 1986 and April 1988 (Zhuang et al., 1991). Cloning and sequencing of several virus isolates confirmed that the cause was HEV (Aye et al., 1992 ; Bi et al., 1993 ; Yin et al., 1993). In 1991, around 1800 sera from sporadic cases of acute hepatitis from 11 Chinese cities (not including Xinjiang) were assayed for anti-HEV IgG, with 8n6 % testing positive (Zhu et al., 1996). In areas where immunization against HAV has been introduced, as many as 20 % of cases of sporadic acute hepatitis may be positive for anti-HEV IgG (H. Zhang, unpublished observations). Despite rapid identification and characterization of viruses associated with hepatitis, cases of acute hepatitis of presumed virus aetiology continue to be described worldwide and have been designated non-A–E hepatitis. While commercial and research-based assays are readily available to identify HEV infections, their success in identifying divergent variants of the virus is unclear. To investigate the possible virus aetiology of acute non-A–E hepatitis in China, we have determined the prevalence of HEV in anti-HEV IgG-negative sera. RT–PCR was used to detect sequences within conserved regions of HEV BHA
ORFs 1 and 2. In addition to the identification of HEV infections attributable to Burmese-like isolates, several members of a novel genotype of HEV were identified.
Methods
Serum samples. Sera from 29 patients from China with a clinical diagnosis of acute (sporadic) hepatitis were collected from Beijing (sample code prefix B), Liaoning province (prefix S) and Henian province (prefix H). All sera were negative for anti-HAV IgM, hepatitis B virus surface antigen (HBsAg) and anti-HCV, as determined by commercial assays licensed by the Chinese Ministry of Public Health, for HBV DNA and HCV RNA, and for anti-HEV IgG using an in-house peptide assay accredited by the Chinese National Reference Laboratory, and were diagnosed provisionally as carrying non-A–E hepatitis.
RT–PCR. RNA was extracted from 140 µl serum with a QIAmp viral RNA kit (Qiagen), yielding 50 µl purified RNA. RT–PCR was carried out in a 50 µl reaction with 15 µl purified RNA, 25 pmol each primer, 4n5 U AMV reverse transcriptase and 5 U Taq polymerase. Thirty-five cycles of 94 mC for 1 min, 50 mC for 45 s and 72 mC for 1 min, with a final extension of 72 mC for 10 min, were used for amplification. Second-round reactions were carried out in a volume of 50 µl with 5 µl first-round product, 25 pmol each primer and 5 U Taq polymerase. Parameters were the same as the first round except that only 30 cycles of amplification were carried out. Consensus oligonucleotide primers for HEV ORF1 and ORF2 were designed within regions of sequence conserved between the full-length sequences from Asia, Mexico and the USA (J. C. Erker, S. M. Desai, G. G. Schlauder, G. J. Dawson & I. K. Mushahwar, unpublished results). The ORF1 primers are positioned within the methyltransferase region at nucleotides 56–79 and 473–451 of the Burmese isolate (GenBank accession no. M73218), amplifying a product of 418 bp : ConsORF1-s1, 5h CTGGCATYACTACTGCYATTGAGC and ConsORF1-a1, 5h CCATCRARRCAGTAAGTGCGGTC. The ORF2 primers, at positions 6298–6321 and 6494–6470 of the Burmese isolate, produce a product of 197 bp : ConsORF2-s1, 5h GACAGAATTRATTTCGTCGGCTGG and ConsORF2-a1, 5h CTTGTTCRTGYTGGTTRTCATAATC. Secondround internal primers amplify 287 and 145 bp products for ORF1 and ORF2, respectively : ConsORF1-s2, 5h CTGCCYTKGCGAATGCTGTGG ; ConsORF1-a2, 5h GGCAGWRTACCARCGCTGAACATC ; ConsORF2-s2, 5h GTYGTCTCRGCCAATGGCGAGC ; and ConsORF2-a2, 5h GTTCRTGYTGGTTRTCATAATCCTG.
Sequence analysis. Second-round PCR products were excised from agarose gels, purified and ligated into pCR2.1 (Invitrogen) or Pinpoint Xa1 T (Promega) vectors. One to three clones were sequenced manually with the k40 or reverse sequencing primers and Sequenase version 2.0 (Amersham) or automatically with an ABI model 373 DNA sequencer and the ABI sequencing ready reaction kit (Perkin-Elmer) according to the manufacturer’s instructions. Sequences used for the phylogenetic analysis were of isolates from Mexico (Mex, GenBank accession no. M74506), Burma (Bur1, M73218 ; Bur2, D10330), Pakistan (P1, M80581), China (C1, D11092 ; C2, L25547 ; C3, M94177 ; C4, D11093 ; C5, L08816 ; C6, L25595), India (I1, X98292 ; I2, X99441), Morocco (Mor12, AF010423 ; Mor23, AF010424), Tashkent, Uzbekistan (Tash, AF010426), Osh, Kirgizia (Osh, AF010425) and the USA (US1, AF060668 ; US2, AF060669). The HEV ORF1 sequences reported here are : B1, AF082081 ; B6, AF082085 ; B7, AF082086 ; S13, AF082092 ; and S15, AF082093. The HEV ORF2 sequences reported are : B2, AF082082 ; B3, AF082083 ; B4, AF082084 ; H3, AF082087 ; H8, AF082088 ; S1, AF082089 ; S5, AF082095 ; S9, AF082096 ; S10, AF082090 ; S11, AF082091 ; and S15, AF082094.
Chinese variant of HEV
Phylogenetic analysis. Nucleotide sequences were aligned using PILEUP (GCG version 9.0, Genetics Computer Group, Madison, WI, USA). These alignments were analysed with the DNADIST program of PHYLIP (version 3.5c ; Felsenstein, 1993) to calculate evolutionary distances between sequences. Unrooted phylogenetic trees were generated using FITCH and RETREE with the midpoint rooting option. The final output was visualized using the program TreeView (Page, 1996). The robustness of the phylogenetic trees was assessed by bootstrap analysis of the sequence alignments with the programs SEQBOOT, DNADIST and NEIGHBOUR from the PHYLIP package, using 1000 resamplings of the data. The consensus tree was generated with CONSENSE. Bootstrap values greater than 70 % provide significant evidence for phylogenetic grouping.
Results and Discussion Sequencing revealed that products amplified from the sera of 15 of the 29 patients were HEV-related (Table 1). Serum from only one individual, S15, allowed amplification of both the ORF1 and ORF2 regions. The ORF1 degenerate primer set amplified 43 % of these HEV sequences successfully. In contrast, the ORF2 set was much more efficient, amplifying products from 90 % of the positive individuals tested. While both primer sets amplify Burmese-like and US isolates successfully (J. C. Erker, S. M. Desai, G. G. Schlauder, G. J. Dawson & I. K. Mushahwar, unpublished results), further characterization of these primers may be necessary to improve their sensitivity on discrepant samples. Perhaps alternative amplification conditions such as ‘ touchdown ’ PCR may be required. It is also likely that, as additional novel HEV sequences are amplified and extended, the primers may require modification. The HEV sequences amplified from nine patients shared a high degree of identity with HEV isolates described previously. The 242 nucleotide ORF1 sequences of B1, B2, B6, B7 and S13 were most identical to each other, with 96 to 99 % identity. These sequences were approximately 92 to 95 % identical to isolates from Burma, China, Pakistan, Africa and India, with the exception of 98 % identity to the Indian isolate referred to here as I1 (Donati et al., 1997). Approximately 80 % identity was shared with the Mexican isolates and 74 % with the isolates from the USA. The 98 nucleotide ORF2 sequences from S1, S10, S11 and H8 shared 97 to 99 % identity with each other and 88 to 99 % identity with other isolates from Asia and Africa. These sequences were approximately 86 % identical to the Mexican isolate and 80 % identical to the US isolates. HEV ORF2 sequences from the six remaining positive patients (B3, B4, S5, S9, S15 and H3) shared 90 to 96 % identity with each other, but only approximately 79 to 83 % identity with HEV sequences from Asia, Africa, Mexico and the USA and the four ORF2 sequences reported above. This level of identity is similar to that seen between the Burmese-like and Mexican (79 to 86 %), the Burmese-like and US (73 to 85 %) and the Mexican and US (83 %) isolates, suggesting that these six isolates may form a distinct group. Of these six novel HEV samples, only S15 yielded an ORF1 product. This product was 76 to 80 % identical to the Asian, African, Mexican and US
Table 1. HEV ORF1 and ORF2 RT–PCR results for 29 Chinese isolates The presence of HEV ORF1 and ORF2 in each isolate was determined by RT–PCR. HEV genotypes determined by sequence analysis are indicated. , Not tested ; , not applicable.
Isolate B1† B2† B3† B4 B5 B6† B7† H3† H4 H5 H6 H8 S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13 S14 S15 S16 S17
HEV ORF1
HEV ORF2
HEV genotype
j j k k k j j j\k* k k k k k k k k k k j\k* k k k k j k j k k
j j k j k k j\k* j j k k k j k k k j j j k k k j k k
1c 1c 4b 4b 1c 1c 4a 1c 1c 4b 4c 1c 1c 1c 4a
* Positive by ethidium bromide staining but sequence analysis showed no relationship to HEV. † Follow-up serum samples were obtained and tested for anti-HEV IgG.
isolates, as well as to the five ORF1 isolates reported above. The Burmese-like, Mexican and US isolates possess similar identity (75 to 80 %) between groups, supporting the suggestion that S15 is a member of a distinct group. There is a high degree of conservation between the different isolates of HEV. However, analysis of the nucleotide alignments of the ORF1 and ORF2 regions demonstrates significant genetic differences between geographically distributed isolates (Fig. 1 a, b). The consensus sequence shown is generally derived from the Burmese-like isolates. Of interest is the finding that the majority of the nucleotide substitutions within ORF1 and ORF2 do not result in amino acid substitutions. Within ORF1, of 85 amino acid positions 17 BHB
Y. Wang and others (a)
(b)
Fig. 1. Nucleotide sequence alignment of HEV ORF1 (a) and ORF2 (b). Sequences of amplification primers have been removed. The consensus sequence is shown at the bottom (Cons). Only those nucleotides which differ from the consensus are shown ; hyphens indicate agreement with the consensus. Nucleotide changes resulting in amino acid substitutions are shaded. The potential methyltransferase domains within ORF1 are underlined and labelled (Koonin et al., 1992).
BHC
Chinese variant of HEV
Table 2. Phylogenetic distances within and between genotypes for ORF1 and ORF2 The range given represents the minimum to maximum genetic differences within and between groups of sequences derived from the genotypes and regions shown. , Not determined, as only a single sequence has been reported. Phylogenetic distance (substitutions per position) Region sequenced
China (genotype 4)*
ORF1 (242 nucleotides) China* (n l 1) Burma† (n l 19) Mexico (n l 1) USA (n l 2) ORF2 (98 nucleotides) China* (n l 6) 0n0103–0n1350 Burma† (n l 15) Mexico (n l 1) USA (n l 2)
Burma (genotype 1)†
Mexico (genotype 2)
USA (genotype 3)
0n2768–0n3142 0n0–0n0972
0n2867 0n2341–0n2663
0n3058–0n3204 0n3034–0n4055 0n2857–0n3158 0n087
0n2189–0n3407 0n0–0n1331
0n1919–0n2608 0n1904–0n2346
0n2508–0n3438 0n2308–0n3565 0n3044–0n3194 0n0633
* Refers to isolates B3, B4, H3, S5, S9 and S15. † Refers to all Burmese-like HEV sequences (Bur1, Bur2, C1, C2, C3, C4, C5, C6, P1, I1, I2, Chad, Mor12, Mor23, Tash, Osh, B2, B6, B7, H8, S1, S10 and S11).
show variability, three of which are located within putative methyltransferase domains. There are seven amino acid substitutions that occur only in a single isolate. S15 is the most divergent, with 10 amino acid substitutions, only one of which is unique. Within ORF2, there are six amino acid positions out of 32 where substitutions occur, with no more than two substitutions in a given isolate. These amino acid changes, as well as the many nucleotide changes, indicate the presence of distinct groups of HEV, definable genotypes and potential subtypes. Phylogenetic analysis of these sequences reveals at least four unique genetic groups of HEV. Although the number of data-points analysed here is minimal, Schlauder et al. (1998) have shown that analysis even of small regions of the HEV genome yields evolutionary distances and relationships similar to those produced from the full-length genome. A summary of the evolutionary distances calculated for the ORF1 region is shown in Table 2. Isolates B2, B6, B7, S5 and S13 group strongly with the Burmese-like sequences, with distances of 0n0472 to 0n0972 substitutions per position to members of this group. Distances of S15 to the other HEV isolates are all greater than 0n2768 substitutions. Similar distances are seen between Burmese-like isolates and the Mexican or US isolates (0n2341 to 0n4055 substitutions) and between the Mexican isolate and the US isolates (0n2857 to 0n3158 substitutions). These data suggest that S15 is as related to the variants reported previously from Asia, Mexico and the USA as these variants are to each other. Analysis of the ORF1 distances suggests that isolates within a single group would have genetic
Fig. 2. Histogram representing the distribution of genetic distances calculated for the ORF1 region. Bars below the histogram indicate genetic distances characteristic of within-genotype (a), between-genotype (b), within-subtype (c) and between-subtype (d) differences.
distances of less than 0n0972 nucleotide substitutions per position across this region, while the distances between groups would be greater than 0n2341 substitutions. This is demonstrated by the histogram shown in Fig. 2. The genetic distances clearly group into two main sets, separating distances within a genotype (less than 0n15) and those between genotypes (greater than 0n235). Distances of less than 0n15 may be further divided into two groups, those less than 0n035, representing distances within potential subtypes, and those greater than 0n045, showing distances between subtypes. The evolutionary distances of B3, B4, H3, S5, S9 and S15 BHD
Y. Wang and others
(a)
(b)
Fig. 3. Unrooted phylogenetic trees produced from (a) 242 nucleotides from ORF1 and (b) 98 nucleotides from ORF2. Genotypes are indicated in boxes, subtypes in circles. Branch lengths are proportional to the genetic distance ; scale bars indicate 0n1 nucleotide substitutions per position. BOOTSTRAP values for the various branches are shown as percentages of trees obtained from 1000 resamplings of the data.
ORF2 to each other range from 0n0103 to 0n1350 nucleotide substitutions per position (Table 2). This is similar to the genetic distances seen between Burmese-like isolates, which have distances of 0n0 to 0n1331 substitutions per position, and to that between the US isolates, 0n0633 substitutions. Isolates S1, S10, S11 and H8 group with the Burmese-like isolates, well within the distances stated above. The isolates B3, B4, H3, S5, S9 and S15 show distances of 0n1919 to 0n3438 substitutions per position to the Burmese-like, the Mexican and the US isolates. This is similar to the distances between the Burmeselike, the Mexican and the US groups (0n1904 to 0n3565 substitutions). These genetic distances suggest that, within a given HEV genotype, evolutionary distances across this ORF2 BHE
region will be less than 0n1350 nucleotide substitutions per position, while those between genotypes will be greater than 0n1904 substitutions. The presence of four genetically distinct groups or genotypes of HEV is further demonstrated by the unrooted phylogenetic trees produced from the ORF1 and the ORF2 alignments (Fig. 3 a, b). In each instance, the B3, B4, H3, S5, S9 and S15 sequences group on a distinct major branch of the tree. There are three other major branches, one representing the US isolates (US1 and US2), another representing the Mexican isolate (M1) and the last containing the Burmese-like isolates from Asia and Africa. This branching is supported by strong bootstrap values for ORF1, with all of the major branches having values over 71n3 %. Many of the bootstrap values for the ORF2 phylogenetic analysis are also significant. Interestingly, while the branching within the B3, B4, H3, S5, S9 and S15 group of sequences is strongly supported, the node at the base of this major branch is only supported in 49n8 % of the trees. This may be attributable to the many common nucleotide substitutions between the Mexican sequence and the US isolates or these six Chinese sequences (Fig. 1 b), causing the Mexican sequence to group with B3, B4, H3, S5, S9 and S15 in 45 % of the trees and with the US isolate in 5 % of the trees. While the results obtained from the ORF2 region weakly mirror those of the ORF1 region, as well as those of other investigators (Schlauder et al., 1998 ; Chatterjee et al., 1997 ; van Cuyck-Gandre et al., 1997), these results may indicate that this region of the HEV genome is nearing the minimum length and variability required for accurate typing. The division of HEV isolates into four distinct genotypes is demonstrated clearly by sequence and phylogenetic analyses. There is also an indication of the existence of subtypes, particularly within the ORF1 region (Fig. 2). Within the Burmese-like group, there are many nucleotide substitutions that consistently occur in combination within ORF1, particularly between positions 119 and 179 in Fig. 1 (a), suggesting as many as five separate subgroups. These five subgroups are illustrated by the five minor branches on the phylogenetic tree (Fig. 3 a), with each grouping having bootstrap values greater than 77n6 %. The genetic distances within each of these five subgroups are less than 0n0338 substitutions per nucleotide (Table 3). Distances between subgroups are greater than 0n0428 substitutions, except for the single distance of 0n0341 substitutions between an Indian isolate (I1) and the Tashkent isolate (Tash). Based on these genetic distances, it is possible that the two isolates from the USA may represent two subtypes, as the ORF1 distance between isolates is 0n087 substitutions. While the overall grouping between the Burmese-like ORF2 sequences on the phylogenetic tree is similar to that for ORF1, genetic distances and bootstrap values do not clearly support the separation into five distinct subtypes. In particular, there are no Central Asian isolates for which sequence data are available across the region of analysis. The Bur1, Bur2 and I2 isolates form a closely related subgroup
Chinese variant of HEV
Table 3. Phylogenetic distances for ORF1 and ORF2 within and between potential subtypes of HEV The range given represents the minimum to maximum genetic differences within and between groups of sequences derived from the potential subtypes shown. , Not determined, as only a single sequence has been reported. Phylogenetic distance (substitutions per position) Region sequenced
Subtype 1a
ORF1 (242 nucleotides) 1a (n l 3) 0n0125–0n0338 1b (n l 7) 1c (n l 5)
Subtype 1b
Subtype 1c
Subtype 1d
Subtype 1e
0n0428–0n0730 0n0–0n0294
0n0553–0n0868 0n0510–0n0822 0n0042–0n0167
0n0561–0n0742 0n0695–0n0923 0n0680–0n0969
0n0695–0n0785 0n0561–0n0740 0n0428–0n0650 (0n0341)* 0n0695–0n0785 0n0126
1d (n l 2) 1e (n l 2)
0n0042
ORF2 (98 nucleotides) 1a (n l 3) 1b (n l 7) 1c (n l 4) 1d (n l 1) 4a (n l 2) 4b (n l 3) 4c (n l 1)
1a 0n0–0n0208
4a 0n0313
1b 0n0417–0n0866 0n0–0n0426
1c 0n0417–0n0748 0n0206–0n0528 0n0–0n0103
4b 0n1103–0n1350 0n0103–0n0209
4c 0n0973–0n1096 0n0980–0n1096
1d 0n1088–0n1331 0n0836–0n0872 0n0868–0n0973
* Genetic distance between the I1 and Tash isolates.
and the single African isolate (Chad) groups independently. However, the two subgroups encompassing the Burmese-like isolates from China, India and Pakistan have genetic distances which overlap. Distances less than 0n0426 substitutions are seen within each of these potential subgroups, but distances greater than 0n0206 substitutions per position are seen between these subgroups. This is most likely the result of the short length and high degree of conservation of the ORF2 region analysed. Analysis of the phylogenetic tree and genetic distances of the B3, B4, S5, S15 and H3 ORF2 sequences suggests the presence of subtypes within this genotype (Table 3). Isolates S15 and H3 group together, with a distance of 0n0313 substitutions per position. Isolates B3, B4 and S5 also group closely, with distances less than 0n0209 substitutions. S9 forms a unique branch, with distances greater than 0n0973 substitutions to the other members of this genotype, distances that are similar to those between the other potential subtypes. These data, as well as those from the Burmese-like ORF2 analysis, again suggest that the US isolates may form two subtypes, as the genetic distance between the US ORF2 sequences is 0n0633 substitutions. Following the recent expansion of the number of HEV variants for which sequence data are available, a consistent nomenclature needs to be established. Sequence and phylogenetic analyses support the establishment of four main
genotypes of HEV, which group in accordance with four potential HEV strains. These include the Burmese (genotype 1), Mexican (genotype 2) and US genotypes (genotype 3) and the Chinese genotype described here (genotype 4). Genotype 1 represents all isolates with identity to the original HEV isolate from Burma. This includes isolates from Burma, India, Pakistan, China, Central Asia and Africa. A tentative designation of subtypes can also be established. Separation of genotype 1 into at least three subtypes is supported by phylogenetic analysis of the available full-length sequences (J. C. Erker, S. M. Desai, G. G. Schlauder, G. J. Dawson & I. K. Mushahwar, unpublished results), full-length ORF2\3 analysis (Schlauder et al., 1998) and restriction endonuclease digestion of PCR products (Gouvea et al., 1998). These groups include subtype 1a (Bur1, Bur2 and I2), subtype 1b (C1, C2, C3, C4, C5, C6 and P1) and subtype 1c (I1). The ORF1 and ORF2 analyses presented here increase the number of isolates belonging to subtype 1c (B1, B2, B6, B7, S1, S10, S11, S13 and H8) as well as suggesting two additional subtypes, subtype 1d (Tash and Osh) and subtype 1e (Mor12, Mor23 and Chad). The establishment of subtypes 1d and 1e is further supported by the analysis of additional sequences from these isolates. Analysis of the hypervariable region of ORF1 and the ORF2\3 overlap has demonstrated that these isolates are divergent from, yet most closely related to, the Asian isolates (Chatterjee et al., 1997 ; van CuyckGandre et al., 1997). Subtypes of genotype 2 cannot be BHF
Y. Wang and others
determined, as only a single Mexican isolate has been reported. Two human isolates have been identified for genotype 3, each possibly representing a distinct subtype, subtypes 3a (US1) and 3b (US2). The HEV sequences identified in swine in the USA (Meng et al., 1997) may represent a third subtype, 3c (data not shown). Genotype 4 may be further divided into three groups, subtypes 4a (S15 and H3), 4b (B3, B4 and S5) and 4c (S9). As sequences of the novel isolates from Africa and Central Asia and the Chinese isolates described here are extended, and as additional isolates are identified, the divisions between genotypes and potential subtypes may become more clearly defined. Partial sequences of two HEV isolates from Guangzhou, China have been reported (Huang et al., 1995). Analysis of 210 nucleotide sequences within the RNA-dependent RNA polymerase region of ORF1 suggests that these isolates (G9 and G20) are distinct from the Burmese-like and Mexican strains, with less than 80 % identity. These sequences are also distinct from the HEV-US1 and HEV-US2 isolates (Schlauder et al., 1998). The relationship of G9 and G20 to isolates of the novel Chinese variant could not be determined, because we have not analysed the analogous region from any of the isolates described here. Follow-up serum samples were obtained from six of the patients who were positive for HEV by RT–PCR, and tested for antibodies using the Genelabs (Singapore) anti-HEV assay. Sera from B1, B2, B3, B6 and B7 were collected 1 month after acute infection and H3 was collected 3 months after acute infection. Patients B2, B6 and B7, who were infected with Burmese-like isolates, were anti-HEV IgG positive. The sampleto-cut-off ratios (S\CO) were 1n1, 3n3 and 3n8, respectively. Patient B1 was antibody negative using this test, with an S\CO ratio of 0n05. Anti-HEV IgG could not be detected in the follow-up sera from the two patients infected with the novel Chinese variant, B3 and H3. The samples had S\CO ratios of 0n4 and 0n11, respectively. Since Burmese- and Mexican-based assays have been shown to detect antibodies to Burmese-like and Mexican HEV readily, as well as IgG to US isolates (Bradley, 1995 ; Schlauder et al., 1998), it is interesting that antibodies were not detected. Further investigation is required to determine whether antibody titres were below the levels detectable with a peptide assay, which may be less sensitive than assays based on recombinant antigens (Mast et al., 1998), or whether the Chinese variant represents a distinct serotype of HEV. There are many aspects related to the reporting and analysis of HEV sequences that require consistency. Foremost among these, GenBank citations should be as complete as possible for all isolates. The geographical origins of several sequences within GenBank are unclear, or are too broad to allow accurate conclusions about the regional distribution of genotypes or subtypes to be reached. Incomplete citation or isolate designation may also interfere with the analysis of the six full-length sequences from China. It seems that the isolates BHG
reported here as C2 and C6, as well as C3 and C5, may be identical except for sequence modifications at the 5h or 3h termini. If this is the case, genetic distances of 0n0 000 nucleotide substitutions per position are purely artefactual. Additionally, future analysis of HEV isolates should include as long a sequence as possible, either from a single region of the genome or from multiple shorter regions. Currently, there is a high degree of variability in the regions of the HEV genome targeted for analysis. We suggest that the ORF1 consensus primers presented here may be used effectively in future studies. The current primers work sufficiently well to amplify known and unknown samples under standard amplification conditions. This region has been shown to be sufficient in length and variability to provide genotype and subtype results similar to full-length genome analysis. The rapid expansion of the data set of HEV sequences, with recent reports of distinct Burmese-like isolates from Africa and Central Asia, novel isolates from the USA, HEV sequences in US swine and the novel Chinese variant reported here, demonstrates that HEV is a worldwide problem. While studies are needed to determine the prevalence of these new isolates in human populations, clinicians need to be aware of the potential for underestimation of acute hepatitis E infection. Following the submission of this manuscript, Hsieh et al. (1998) reported the isolation of a unique variant of HEV from four Taiwanese patients. These 92 nucleotide ORF1 sequences were more than 96 % identical to each other but only 71 to 79 % identical to the Burmese-like and Mexican isolates. These Taiwanese isolates are also 76n1 to 80n4 % identical to the HEVUS sequences. However, the ORF1 sequence of the genotype 4 isolate reported here (S15) is 87 to 89n3 % identical to these Taiwanese isolates, suggesting that these five isolates may be members of the same genetic group. Hsieh et al. (1998) demonstrated that the Taiwanese isolates represent a new strain of HEV, based on the presence or absence of endonuclease restriction sites. Nucleotide alignment and phylogenetic analysis of this 92 nucleotide region demonstrate the presence of the four genetic groups presented above and the potential for subtypes, as well as the close relationship of the Taiwanese and S15 sequences (data not shown). As the number of HEV isolates continues to increase, the need to establish guidelines for forming genetic groups and consistent nomenclature becomes urgent. The data provided here, although based on limited sequence information, allow the formation of consistent genetic groups using robust phylogenetic analysis, the ability to amplify multiple regions of the genome with consensus primers, and an easily interpretable nomenclature. Youchun Wang is the recipient of a Research Development Award in Tropical Medicine from the Wellcome Trust. We are grateful to the following for supplying serum samples : Professor Zhao Guizhen (Chinese Medical University, Shen Yang, Liaoning Province), Dr Li Rongcheng and Ms Gongjian (Health and Anti-epidemic Centre of Guangxi, Nanning) and Ms Liu Xin (Henian Institute of Hepatology, Henian).
Chinese variant of HEV Critical review of the manuscript by Dr A. Scott Muerhoff is highly appreciated by the authors.
Kwo, P. Y., Schlauder, G. G., Carpenter, H. A., Murphy, P. J., Rosenblatt, J. E., Dawson, G. J., Mast, E. E., Krawczynski, K. & Balan, V. (1997). Acute hepatitis E by a new isolate acquired in the United States.
Mayo Clinic Proceedings 72, 1133–1136.
References Aye, T. T., Uchida, T., Ma, X. Z., Iida, F., Shikata, T., Zhuang, H. & Win, K. M. (1992). Complete nucleotide sequence of a hepatitis E virus
isolated from the Xinjiang epidemic (1986–1988) of China. Nucleic Acids Research 20, 3512. Balayan, M. S., Usmanov, R. K., Zamyatina, N. A., Djumalieva, D. I. & Karas, F. R. (1990). Experimental hepatitis E infection in domestic pigs.
Journal of Medical Virology 32, 58–59. Bi, S. L., Purdy, M. A., McCaustland, K. A., Margolis, H. S. & Bradley, D. W. (1993). The sequence of hepatitis E virus isolated directly from a
single source during an outbreak in China. Virus Research 28, 233–247. Bradley, D. W. (1995). Hepatitis E virus : a brief review of the biology, molecular virology and immunology of a novel virus. Journal of Hepatology 22 (Suppl. 1), 140–145. Chatterjee, R., Tsarev, S., Pillot, J., Coursaget, P., Emerson, S. U. & Purcell, R. H. (1997). African strains of hepatitis E virus that are distinct
from Asian strains. Journal of Medical Virology 53, 139–144. Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. & Houghton, M. (1989). Isolation of a cDNA clone derived from a blood-
Mast, E. E., Alter, M. J. & Holland, P. V. (1998). Evaluation of assays for antibody to hepatitis E virus by a serum panel. Hepatology 27, 857–861. Meng, X. J., Purcell, R. H., Halbur, P. G., Lehman, J. R., Webb, D. M., Tsareva, T. S., Haynes, J. S., Thacker, B. J. & Emerson, S. U. (1997). A
novel virus in swine is closely related to the human hepatitis E virus. Proceedings of the National Academy of Sciences, USA 94, 9860–9865. Nanda, S. K., Ansari, I. H., Acharya, S. K., Jameel, S. & Panda, S. K. (1995). Protracted viremia during acute sporadic hepatitis E virus
infection. Gastroenterology 108, 225–230. Page, R. D. (1996). TreeView : an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357–358. Reyes, G. R., Purdy, M. A., Kim, J. P., Luk, K. C., Young, L. M., Fry, K. E. & Bradley, D. W. (1990). Isolation of a cDNA from the virus
responsible for enterically transmitted non-A, non-B hepatitis. Science 247, 1335–1339. Schlauder, G. G., Dawson, G. J., Erker, J. C., Kwo, P. Y., Knigge, M. F., Smalley, D. L., Rosenblatt, J. E., Desai, S. M. & Mushahwar, I. K. (1998). The sequence and phylogenetic analysis of a novel hepatitis E
borne non-A, non-B viral hepatitis genome. Science 244, 359–362.
virus isolated from a patient with acute hepatitis reported in the United States. Journal of General Virology 79, 447–456.
Dawson, G. J., Chau, K. H., Cabal, C. M., Yarbough, P. O., Reyes, G. R. & Mushahwar, I. K. (1992). Solid-phase enzyme-linked immunosorbent
Tam, A. W., Smith, M. M., Guerra, M. E., Huang, C. C., Bradley, D. W., Fry, K. E. & Reyes, G. R. (1991). Hepatitis E virus (HEV) – molecular
assay for hepatitis E virus IgG and IgM antibodies utilizing recombinant antigens and synthetic peptides. Journal of Virological Methods 38, 175–186. Donati, M. C., Fagan, E. A. & Harrison, T. J. (1997). Sequence analysis of full length HEV clones derived directly from human liver in fulminant hepatitis E. In Viral Hepatitis and Liver Disease, pp. 313–316. Edited by M. Rizzetto, R. H. Purcell, J. L. Gerin & G. Verme. Turin : Edizioni Minerva Medica. Felsenstein, J. (1993). PHYLIP inference package, version 3.5c. Department of Genetics, University of Washington, Seattle, WA, USA. Gouvea, V., Hoke, C. H., Jr & Innis, B. L. (1998). Genotyping of hepatitis E virus in clinical specimens by restriction endonuclease analysis. Journal of Virological Methods 70, 71–78. Hsieh, S. Y., Yang, P. Y., Ho, Y. P., Chu, C. M. & Liaw, Y. F. (1998).
Identification of a novel strain of hepatitis E virus responsible for sporadic acute hepatitis in Taiwan. Journal of Medical Virology 55, 300–304. Huang, C. C., Nguyen, D., Fernandez, J., Yun, K. Y., Fry, K. E., Bradley, D. W., Tam, A. W. & Reyes, G. R. (1992). Molecular cloning and
sequencing of the Mexico isolate of hepatitis E virus (HEV). Virology 191, 550–558. Huang, R. T., Nakazono, N., Ishii, K., Kawamata, O., Kawaguchi, R. & Tsukada, Y. (1995). Existing variations on the gene structure of hepatitis
E virus strains from some regions of China. Journal of Medical Virology 47, 303–308. Koonin, E. V., Gorbalenya, A. E., Purdy, M. A., Rozanov, M. N., Reyes, G. R. & Bradley, D. W. (1992). Computer-assisted assignment of
functional domains in the nonstructural polyprotein of hepatitis E virus – delineation of an additional group of positive-strand RNA plant and animal viruses. Proceedings of the National Academy of Sciences, USA 89, 8259–8263.
cloning and sequencing of the full-length viral genome. Virology 185, 120–131. Tsarev, S. A., Emerson, S. U., Reyes, G. R., Tsareva, T. S., Legters, L. J., Malik, I. A., Iqbal, M. & Purcell, R. H. (1992). Characterization of a
prototype strain of hepatitis E virus. Proceedings of the National Academy of Sciences, USA 89, 559–563. Usmanov, R. K., Balayan, M. S., Dvoinikova, O. V., Alymbayeva, D. B., Zamyatina, N. A., Kazachkov, Y. A. & Belov, V. I. (1994). Experimental
hepatitis E infection in lambs. Voprosy Virusologii 39, 165–168. van Cuyck-Gandre, H., Zhang, H. Y., Tsarev, S. A., Clements, N. J., Cohen, S. J., Caudill, J. D., Buisson, Y., Coursaget, P., Warren, R. L. & Longer, C. F. (1997). Characterization of hepatitis E virus (HEV) from
Algeria and Chad by partial genome sequence. Journal of Medical Virology 53, 340–347. Yarbough, P. O., Tam, A. W., Fry, K. E., Krawczynski, K., McCaustland, K. A., Bradley, D. W. & Reyes, G. R. (1991). Hepatitis E virus :
identification of type-common epitopes. Journal of Virology 65, 5790– 5797. Yin, S., Tsarev, S. A., Purcell, R. H. & Emerson, S. U. (1993). Partial sequence comparison of eight new Chinese strains of hepatitis E virus suggests the genome sequence is relatively stable. Journal of Medical Virology 41, 230–241. Zhu, X. J., Zhuang, H., Zhu, W. F., Ma, W. M., Xu, C., Xu, D. Z., Chen, Y. J., Wei, B. J., Lou, G. Q. & Ma, Y. X. (1996). Studies on etiology and
epidemiology of sporadic acute hepatitis from 11 cities. China Public Health 12, 58–60. Zhuang, H., Cao, X. Y., Liu, C. B. & Wang, G. M. (1991). Epidemiology of hepatitis E in China. Gastroenterologia Japonica 26 (Suppl. 3), 135–138. Received 5 June 1998 ; Accepted 27 August 1998
BHH
