Ru-Tong Huang, Xiao-Yu Li, Xiao-Bing Xia, Xi-Tong Yuan, Min-Xia Liu, De-Rong Li, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing 100850, China
Dr. Ru-Tong Huang, male, born on 1942-11-11 in Zhejiang Province, gra duated from Shanghai Fudan University as a postgraduate in 1966, now associate professor of microbiology, majoring aetiology of medical virology, having 60 pape rs published.
Supported by the Foundation of General Logistics Department of C hinese PLA, No.9608037
Correspondence to: Dr. Ru-Tong Huang, Institute of Micr obiology and Epidemiology, Academy of Military Medical Sciences, Beijing 1008 50, China
Telephone: +86-10-66931535, Fax. +86-10-68213044
Received: 1999-01-20

Subject headings: hepatitis E virus; antibodies; viral/analy sis; nucleotide sequences; sequence analysis

Huang RT, Li XY, Xia XB, Yuan XT, Liu MX, Li DR. Antibody detection and sequence analysis of sporadic HEV in Xiamen region. World J Gastroentero, 1999;5(3):270-272

INTRODUCTION
Hepatitis E virus (HEV) is transmitted through a fecal-oral route^［1］. H EV induces acute hepatitis and is responsible for a significant portion of the fulminant hepatitis in epidemic and sporadic cases, especially in the mixed infection patients and women in their third trimester of pregnancy^［1］. It has been reported that HEV infection is more prevalent in underdeveloped and develo ping countries in Asia, Africa, and Central America, but is rare in developed co untries^［1］. In China, a large outbreak occurred between 1986 and 1988 in Xinjiang, and sporadic spread was often found in other regions.
      HEV is a non enveloped virus, approximately 27nm-34nm in diameter and has a positive-sense, single-stranded RNA genome of approximately 7.2k b. The viral genome consists of three discontinuous open reading frames (ORFs). Since the molecular cloning and sequencing of HEV were described^［2］, s everal genomic analyses of HEV strains obtained from different geographic areas have been reported^［3］. The existing variations on the gene structure of HEV strains from some regions of China was reported by us^［4］. In this st udy, after the collection of the serum samples of patients with acute hepatitis in Xiamen, anti-HEV antibody and HEV RNA in serum were detected, further HEV RN A was cloned and sequenced. The results are described and discussed.

PATIENTS AND METHODS
Patients
From September 1996 to March 1997, 81 serum of patients (71 male and 10 female, aged 13-69 years) with acute hepatitis at clinic and admitted to the Infections Disease Hospital in Xiamen were collected. These serum samples were provided by professor LIAO Mian-Chu and were stored at -25℃ before test. The sera sho wed elevated ALT levels.The serum of patients with acute hepatitis was tested fo r detection of serum IgM anti-HAV, HBsAg, IgM anti-HBc, anti-HCV and anti-HE V antibodies. According to above detections, all samples suspected of hepatitis E were taken to our laboratory and were further studied. One case (sample No.3, Zho u, male aged 52 years) used in the determination of sequence was diagnosed as fu lminant hepatitis. He had complained of tiredness, anorexia, urine-yellow, jaun dice in skin and sclera and spider nevus, with ALT 20420 nmol·s^-1/L and SB-205.2μmol/L, and virus markers of anti-HAV IgM and anti HEV IgM positive.

Detection of anti-HEV antibody
Anti-HEV IgG and IgM antibodies were further detected by ELISA with recombinant antigens (Institute of Virology, Chinese Academy of Preventive Medicine) accord ing to the manufacturer′s instructions.

Cloning and sequencing of HEV RNA
Two sets of pair primers were synthesized at Institute of Microbiology of Chines e Science Academy according to the Burmese HEV sequence^［3］. The sequence of each oligonucleotide primer was: outer primers (F1) 5′ GCT ATT ATG GAG GAG TGT GG 3′ and (R1) 5′CAG GGC CCC AAT TCT TCT 3′, inner primers (F2) 5′G CG TGG ATC TTG CAG GCC 3′ and (R2) 5′TTC AAC TTC AAG CCA CAG CC 3′. HEV RNA was extracted from-200μL serum by proteinase K (10g/L) guanidine thioc yanate buffer (4.2M guanidine thiocyanate and 5g/L N-lauroyl sar cosine and 0.025mol/L Tris-HCl, pH 8.0)phenol/choroform^［5］ . All viral RNA were used to be transcribed and nested-PCR^［4］. The am plified PCR products were ligated into pGEM-T vector (Promega products) accordi ng to the manufacturer′s instructions. The ligation mixture was transformed to E. coli strain JM 109. The positive clones were picked up and cultured in LB medium. The positive recombinant clones were identified and sequenced. The nucl eotide sequence (location 4522-4761) of X-S1 isolate of HEV was compared with those of other known HEV strains. Percentage of similarity and divergence were calculated as described previously^［4］.

RESULTS
Detection of anti-HEV and HEV RNA in serum samples of patients with acute hepatitis
Twelve of 81 serum samples of acute hepatitis in Xiamen were positive for anti- HEV IgG, of them, 11 were also positive for anti-HEV IgM. Eight of 12 serum sam ples of positive anti-HEV IgG were used to detect HEV RNA, 2 samples were found positive for HEV RNA. The results are shown in Table 1.

Cloning and sequencing of HEV
The amplified PCR positive products obtained from X-S1 and X-S6 samples were purified and ligated into pGEM-T vector, then transformed to E.coli JM 109. One of two white specks obtained from X-S1 had a band of about 239bp in gel electrophoresis. Two white specks of the X-S6 were positive and a band of 239bp was found after digested by EcoRⅠ and Hind-Ⅲ. One positi ve clone of X-S1 was sequenced. HEV sequence of the X-S1 isolate could be reco gnized, this HEV X-S1 strain was neither lost nor inserted in length 239bp compared with Burma strain of HEV. Aberrance occurred in 48 bases, 5 of them took place at the first codon position, 4 at the second position and 39 at the third codon position. The nucleotide sequence of X-S1 isolate is shown in Figure 1.

Table 1 Results of anti-HEV antibody and HEV RNA detection in seru m of patients from Xiamen

^aA＞0.30+x- of negative samples: positive.^bNot done.

Figure1(PDF) Comparison of the nucleotide sequences amon g four strains of HEV.

Comparison of the nucleotide sequence of HEV
The sequence of HEV X-S1 strain was compared with the Chinese (G-20), Burmese (B-121) and Mexican strains of HEV. The similarity of nucleotide sequences was 85.4%, 79.2% and 76.4% respectively. The divergence of nucleotide sequences w as 14.2%, 19.9% and 22.3% respectively.

DISCUSSION
This paper reports the results of serological survey on hepatitis E virus infection in Xiamen population. The serum samples from 81 patients with acute hepatitis were tested for anti-HEV IgG and IgM antibodies with HEV recombination antigen by ELISA. Twelve (14.8%) of 81 patients with acute hepatitis had antibody to HEV, 11 were positive for IgM anti-HEV and 2 positive for HEV RNA. The results show that there is hepatitis E virus infection in Xiamen again, but there has been no documented report of detection of HEV RNA in anti-HEV positive patients from this area.
      The mixed infection of HEV and HBV were described previously^［4］. This paper reports the co-infection of HEV and HAV. This case is positive for both ant ibodies of HAV and HEV. The patient presented as severe hepatitis in the clinica l characteristics, with very high ALT (20420nmol·s^-1/L). This shows t hat both the HEV and HAV are transmitted primarily through a fecal-oral route.In this study, the partial nucleotide sequence of Xiamen X-S1 isolate of sporad ic HEV was described and compared. It is shown that Xiamen (X-S1) strain and Gu angzhou (G-20) strain^［4］are most identical to each other (85.4%), wit h a lower range of identities to the Burmese strain^［2］and Mexican strai n^［3］ (80.1%-77.3%). The nucleotide sequences of the X-S1 strain and t he G-20 strain may belong to a novel and unique branch. Similar results have been reported by other investigators^［6,7］. Recently the HEV-US-1 strain w as discovered by George G. Schlauder^［8］, it is significantly divergent f rom other human HEV isolates, which may be the fourth genotype. The discovery of these HEV variants may be important in understanding the worldwide distribution of HEV infection.

REFERENCES
1    Gust ID, Purcell RH. Report of a workshop: waterborne non-A, non-B hepatitis.J Infect Dis,1987;156:630-635
2    Tam AW, Smith MM, Guerra ME, Huang CC, Bradley DW, Fry KE，Reyes GR. Hepatitis E virus (HEV): molecular cloning 
      and sequencing of the full-length viral genome. Virology, 1991;185:120-131
3    Huang CC, Nguyen D, Fernandez J, Yun KY, Fry KE, Bradley DW，Tam AW, Reyes GR. Molecular cloning and sequencing 
      of the Mexico isolate of hepatitis E virus. Virology, 1992;191:550-558
4   Huang RT, Nakazono N, Ishii K, Kawamata O, Kawaguchi R, Tsukada Y. Existing variations on the gene structure of 
      hepatitis E virus strains from some regions of China.J Med Virol, 1995;47:303-308
5    Tsarev SA, Emerson SU, Reyes GR, Tsareva TS, Legters LJ, Malik IA, Iqbal M, Purcell RH. Characterization of a prototype 
      strain of hepatitis E virus. Proc Natl Acad Sci USA,1992;89:559-563
6    Wei SJ, Walsh P, Tong YB, Dong HQ, Cai XL. Nucleic acid sequence analysis of the sporadic hepatitis E virus strains in
      Guangzhou.Chin J Microbiol Immunol, 1998;18:92-95
7    Wu JC, Sheen IJ, Chiang TY, Sheng WY, Wang YJ, Chan CY, Lee SD. The impact of traveling to endemic areas on the 
      spread of hepatitis E virus infection: epidemiological and molecular analyses.Hepatology,1998;27:1415-1420
8    Schlauder GG, Dawson GJ, Erker JC, Kwo PY, Kningge F, Smalley L, Rosenblatt JE, Desai SM, Mushahwar IK. The 
      sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in 
      the United States. J Gen Virol, 1998;79:447-456 

Reviews	Add
more>>