Hepatitis E, which is enterically transmitted, is the most common cause of acute ... Phylogenetic analysis of several isolates of hepatitis E virus (HEV) from Asia ...
Am. J. Trop. Med. Hyg., 62(2), 2000, pp. 187–189 Copyright 䉷 2000 by The American Society of Tropical Medicine and Hygiene
SHORT REPORT: PHYLOGENETICALLY DISTINCT HEPATITIS E VIRUSES IN PAKISTAN ´ , H. Y. ZHANG, S. A. TSAREV, R. L. WARREN, J. D. CAUDILL, N. J. SNELLINGS, L. BE ´ GOT, H. VAN CUYCK-GANDRE B. L. INNIS, AND C. F. LONGER Department of Virus Diseases, and Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, District of Columbia; Unite´ de Virologie, Centre de Recherches du Service de Sante´ des Arme´es, Grenoble, France
Abstract. Hepatitis E, which is enterically transmitted, is the most common cause of acute hepatitis in much of Asia. Phylogenetic analysis of several isolates of hepatitis E virus (HEV) from Asia suggests that transmission of this virus is geographically restricted. In Sarghoda, Pakistan, HEV Sar-55 was isolated from a 1987 outbreak. It belongs to the Central-Asian cluster of the Asian sub-genotype. We now report the complete sequence of a second Pakistan HEV from a 1988 outbreak in Abbottabad. The Abbottabad nucleotide sequence was compared with 15 other complete HEV sequences using statistical methods of phylogenetic analysis. The analysis showed that Abbottabad HEV belongs to the South Asia cluster of the Asian sub-genotype. The sequence differences of the 2 Pakistan isolates recovered only one year apart suggest that HEV of 2 distinct origins circulate in Pakistan. TABLE 1 Chronologic list of complete hepatitis E virus genome sequences analyzed
Hepatitis E is a waterborne disease transmitted to humans by the fecal-oral route and is caused by hepatitis E virus (HEV). Seroepidemiologic studies suggest that HEV is widely transmitted and that most adults in south Asia have been infected.1 This virus has an RNA genome of approximately 7.5 kb. To date, 15 isolates, defined as HEV contained in a specific patient specimen, have been fully sequenced; together, they comprise 5 genotypes: genotype I (Asia-Africa), genotype II (United States), genotype III (Mexico), genotype IV (Beijing, China), and genotype V (European).2–4 Genotype I is the most complex, having 2 sub-genotypes: African and Asian. The Asian sub-genotype, now represented by 11 isolates, consists of 2 genetic clusters: Central Asian and South Asia. The Central Asian cluster includes isolates from China (Xinjiang) and Pakistan (Sar-55); partially sequenced isolates from Kirgizia and Uzbekistan also belong to this cluster. The South Asian cluster includes isolates from Burma, India, and Nepal.2,5 Hepatitis E is very common in Pakistan.6–8 Nevertheless, only a single isolate from Sargodha has been genomically and phenotypically characterized.9 In this study, we report the full sequence of a second Pakistan HEV isolate from Abbottabad (Abb-2B), which is phylogenetically distinct from Sar-55, and speculate on their origins. Fecal samples containing HEV from archived specimens collected during an outbreak investigation of hepatitis E in Abbottabad were provided by J. Ticehurst (Walter Reed Army Institute of Research). We determined the consensus sequence of HEV isolate Abb-2B (7,143 nucleotides) from cDNA using methods previously reported.10,11 We performed phylogenetic analysis of Abb-2B and all other full-length HEV genomes (n ⫽ 15) in the GenBank database (Table 1) using PHYLIP and PAUP programs in GCG release 10.0 (University of Wisconsin, Madison, WI) as previously described.2 Both methods of phylogenetic analysis placed the Abb2B isolate in the South Asian cluster of genotype I, whereas Sar-55, the other Pakistan isolate, was placed in the Central Asia cluster of genotype I (Figure 1). The certainty of this difference is greater than 95%, as determined by 1,000 replicate bootstrap analysis.12 The analyses suggest that Abb-2B (1988) is the earliest phylogenetic representative of a cluster of HEV that has
Strain*
1982-Burma 1987-Mexico 1987-Pakistan (Sargodha) (Sar-55) 1987-China-B (Xinjiang) (strain HeBei) 1987-China-D (Xinjiang) 1987-China-C (Xinjiang) (strain K52-87) 1988-Pakistan (Abbottabad) (Abb-2B) 1989-Myanmar (Burma) 1990-India (Hyderabad) 1992-Nepal (Kathmandu) (strain TK-15/92) 1992-Fulminant (India?) 1993-India (Madras) 1995-USA-H (US-1, from human) 1996-USA-H (US-2, from human) 1996-USA-S (from swine)
GenBank accession no.
M73218 M74506 M80581 M94177 D11093 L25595 AF185822 D10330 AF076239 AF051830 X98292 X99441 AF060668 AF060669 AF082843
* Year and place of isolation, strain designation if assigned.
been very successful, spreading across South Asia and causing disease in at least 4 countries for more than a decade (1982–1992). In contrast, Sar-55 (1987) is a phylogenetically late derivative from the Central Asian cluster of HEV; during the same period (1986–1988), ancestral isolates from this cluster caused the largest outbreak of hepatitis E ever recorded, in the Xinjiang Autonomous Region of China.13 These 2 Pakistani isolates were recovered 18 months apart in cities only 300 km from each other, but because they are so distinct they must have had separate origins. There are many examples of travelers importing HEV.14–18 Dissemination of imported HEV is then determined by social and environmental factors. Since hepatitis E is endemic in Pakistan, these factors presumably favor dissemination of imported HEV. The geography of South Asia provides no barrier to movement of HEV from east to west; therefore, it is not surprising to find a South Asian HEV similar to Abb-2B in Pakistan. On the other hand, harsh geography makes travel from Central Asia south to Pakistan less frequent. We speculate that Sar-55 was an unusual introduction of HEV into Pakistan from China to the north, whereas Abb-2B represents HEV endemic throughout Pakistan. Sequence analysis of HEV isolates from old and new sporadic cases of hepatitis
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´ AND OTHERS VAN CUYCK-GANDRE
FIGURE 1. Bootstrap 50% majority rule consensus phylogenetic trees of hepatitis E virus (HEV) isolates based on complete genomes. The trees were generated from the PileUp alignment of 16 HEV sequences including the sequence reported in this study. Branch lengths are proportional to the evolutionary distance between sequences. The numbers represent bootstrap values (expressed as percentage of all trees) obtained from 1,000 replicates. Roman numerals identify genotypes (⬎15% non-identity), Arabic numbers identify sub-genotypes (⬎7.5% non-identity), and letters identify less different but distinct phylogenetic clusters within sub-genotypes. Since no full genome examples from Africa are available, this sub-genotype is absent from these trees. A, neighbor-joining analysis; the scale is percent of genetic distances. B, maximum parsimony analysis; the scale is number of evolutionary changes.
HEPATITIS E VIRUS IN PAKISTAN
E can address our speculation. Phylogenetic analysis promises to be a powerful epidemiologic tool. 7. Acknowledgments: We thank the Department of Preventive Medicine and Biometrics, Uniformed Services University of the Health Sciences (Bethesda, MD) and the Pakistan-United Laboratory for Sero-Epidemiology (Rawalpindi, Pakistan) for performing the outbreak investigation that yielded the Abbottabad specimens. We also thank J. Ticehurst who conceived of this work, and the National Cancer Institute for computer time and staff support at the Frederick Biomedical Supercomputing Center, Frederick Cancer Research and Development Center (Frederick, MD). H. van Cuyck-Gandre´ was a National Research Council Associate when she did this work. Financial support: This work was supported by the U.S. Army Medical Research and Materiel Command. Authors’ addresses: H. van Cuyck-Gandre´ , Unite´ de Virologie, Centre de Recherches du service de Sante´ des Arme´ es, 24, Ave des Maquis du Gre´ sivaudan, BP 87, 38702 La Tronche Cedex, France. H. Y. Zhang, S. A. Tsarev, J. D. Caudill, N. J. Snellings, B. L. Innis, and C. F. Longer, Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, DC 20307-5100. R. L. Warren, Department of Bacterial Diseases, Walter Reed Army Institute of Research, Washington, DC 20307-5100. L. Be´ got, Unite´ de Virologie, Centre de Recherches du Service de Sante´ des Arme´ es, Grenoble, France. REFERENCES
1. Balayan MS, 1997. Type E hepatitis: state of the art. Int J Infect Dis 2: 113–120. 2. Tsarev SA, Binn LN, Gomatos PJ, Arthur RR, Monier MK, van Cuyck-Gandre H, Longer CF, Innis BL, 1999. Phylogenetic analysis of hepatitis E virus isolates from Egypt. J Med Virol 57: 68–74. 3. Wang Y, Ling R, Erker JC, Zhang H, Li H, Desai S, Mushahwar IK, Harrison TJA, 1999. Divergent genotype of hepatitis E virus in Chinese patients with acute hepatitis. J Gen Virol 80: 169–177. 4. Schlauder GG, Desai SM, Zanetti AR, Tassopoulos NC, Mushahwar IK, 1999. Novel hepatitis E virus (HEV) isolates from Europe: evidence for additional genotypes of HEV. J Med Virol 57: 243–251. 5. Gouvea V, Snellings N, Popek MJ, Longer CF, Innis BL 1998. Hepatitis E virus: complete genome sequence and phylogenetic analysis of a Nepali isolate. Virus Res 57: 21–26. 6. Iqbal M, Ahmed A, Qamar A, Dixon K, Duncan JF, Islam NU,
8.
9.
10.
11.
12. 13. 14.
15.
16.
17.
18.
189
Rauf A, Bryan JP, Malik IA, Legters LJ, 1989. An outbreak of enterically transmitted non-A, non-B hepatitis in Pakistan. Am J Trop Med Hyg 40: 438–443. Malik IA, Qureshi MS, Luqman M, Qamar MA, Ahmed A, Legters LJ, Ahmad M, Akhtar MA, 1998. Epidemics of nonA, non-B hepatitis in Pakistan. Trop Doct 18: 99–101. Ticehurst J, Popkin TJ, Bryan JP, Innis BL, Duncan JF, Ahmed A, Iqbal M, Malik KI, Kaoikian AZ, Legters LJ, Purcell RH, 1992. Association of hepatitis E virus with outbreak of hepatitis in Pakistan: serologic responses and pattern of virus excretion. J Med Virol 36: 84–92. Tsarev SA, Emerson SU, Reyes GR, Tsareva TS, Legters LJ, Malik IA, Iqbal M, Purcell RH 1992. Characterization of a prototype strain of hepatitis E virus. Proc Natl Acad Sci USA 89: 559–563. van Cuyck-Gandre´ H, Zhang HY, Tsarev SA, Clements NJ, Cohen SJ, Caudill JD, Coursaget P, Buisson Y, Warren RL, Longer CF, 1995. Partial sequence of HEV isolates from North Africa and Pakistan: comparison with known sequences. Buisson Y, Coursaget P, Kane M, eds. Enterically-Transmitted Hepatitis Viruses. Tours, France: La Simarre, 301–310. van Cuyck-Gandre´ H, Zhang HY, Tsarev SA, Clements NJ, Cohen SJ, Caudill JD, Buisson Y, Coursaget P, Warren RL, Longer CF, 1997. Characterization of hepatitis E virus (HEV) from Algeria and Chad by partial genome sequence. J Med Virol 53: 340–347. Hillis DM, Bull JJ, 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol 42: 182–192. Huang RT, Li DR, Wei J, Huang XR, Yuan XT, Tian X, 1992. Isolation and identification of hepatitis E virus in Xinjiang, China. J Gen Virol 73: 1143–1148. Drabick JJ, Gambel JM, Gouvea VS, Caudill JD, Sun W, Hoke Jr CH, Innis BL, 1997. A cluster of acute hepatitis E infection in United Nations Bangladeshi peacekeepers in Haiti. Am J Trop Med Hyg 57: 449–454. Shidrawi RG, Skidmore SJ, Coleman JC, Dayton R, MurrayLyon IM, 1994. Hepatitis E: an important cause of imported non-A, non-B hepatitis among migrant workers in Qatar. J Med Virol 43: 412–414. Skaug K, Hagen IJ, von der Lippe B, 1994. Three cases of acute hepatitis E virus infection imported into Norway. Scand J Infect Dis 26: 137–139. Zaaijer HL, Kok M, Lelie PN, Timmerman RJ, Chau K, van der Pal HJ, 1993. Hepatitis E in The Netherlands: imported and endemic. Lancet 341: 826. Skidmore SJ, Yarbough PO, Gabor KA, Tam AW, Reyes GR, Flower AJ, 1991. Imported hepatitis E in UK. Lancet 337: 1541.
