National Institute for Communicable Diseases and Department of Medical Virology, University of Witwatersrand, Private Bag X4, Sandringham 2131, South Africa
Correspondence
Hazel Maila
hazelm{at}nicd.ac.za
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ABSTRACT |
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MAIN TEXT |
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HEV is prevalent in Africa and epidemics have been identified in Algeria, the Ivory Coast, Sudan, Somalia (Bradley, 1992) and Djibouti (Coursaget et al., 1996
), with smaller outbreaks reported in Morocco (Benjelloun et al., 1997
), Ethiopia (Tsega et al., 1991
), Chad (van Cuyck-Gandre et al., 1996
) and Kenya (Mast et al., 1994
), while sporadic cases of HEV have been identified in Egypt (Tsarev et al., 1999
), Nigeria (Buisson et al., 2000
) and Tunisia (Coursaget et al., 1996
).
The first report of hepatitis E in southern Africa was the published account of a typical waterborne outbreak with 273 cases and at least four deaths in Maun, northern Botswana, in 1985 (Byskov et al., 1989). The disease appeared to have affected 12 % of the population and pregnant women were most severely affected. An earlier outbreak in southern Africa, which occurred in Namibia's Kavango region in 1983 and affected people living in settlements lacking potable water and waste disposal facilities, was only recently characterized and identified by molecular methods as epidemic HEV (Isaacson et al., 2000
). The disease was usually mild, except in pregnant women who made up six of the seven fatal infections reported in this outbreak. HEV was detected by RT-PCR in faeces from nine of 16 patients tested and their phylogeny was later established from a consensus sequence of 296 bp derived from the extreme 3' region of open reading frame 2 (ORF2) (He et al., 2000
). Other indications of hepatitis E infection in southern Africa include reports from the small kingdom of Swaziland, where van Rensburg et al. (1995)
examined Mozambican refugees, and South Africa (Grabow et al., 1996
; Tucker et al., 1996
).
In this paper, we have confirmed for the first time, by amplification and sequencing of isolates, that an outbreak of hepatitis E also occurred in the Kavango region of Namibia in 1995. In addition, we have shown by phylogenetic analysis that this outbreak was not caused by the same strain as the 1983 outbreak in the same area.
A non-A, non-B hepatitis outbreak involving more than 600 people occurred in 1995/6 in the Kavango region of northern Namibia, centred on the town of Rundu (Surveillance Bulletin, 1996). The source of the outbreak was suspected to be the water supply, which was compromised by drought and disturbances in reticulation following work on the pipes approximately 6 months earlier. Patients included both males and females within the age range between 5 days and 80 years (median 25 years), a total bilirubin range of 17724 µmol l-1, an alanine aminotransferase range of 266780 IU l-1 and an aspartate transaminase range of 2510 540 IU l-1. Patients presented with symptoms of viral hepatitis (jaundice and hypochondrial pain). There were at least three fulminant cases, including one pregnant woman.
Patients' sera were tested for HEV IgG using the Abbott HEV EIA kit after onset of illness. Mean optical density per cut-off ratio for the four sequenced specimens 8039, 7948, 7801 and 7798 were, respectively, 2·56, 6·36, 1·79 and 1·79 (positive cut-off 0·15). HEV antibodies were detected in sera specimens of approximately 75 % of icteric patients. The incidence was 13 % in patients aged <10 years and 81 % in those aged 50 years. No antibodies were detected in any of the children under the age of 5 years (Surveillance Bulletin, 1996
).
Clinical materials, including human stool and serum samples, obtained from patients were stored at -70 °C and used for this study. Approval to undertake the study was obtained from the Ministry of Health and Social Services of the Republic of Namibia and consent was obtained from the patients to use their specimens for HEV studies.
Stool samples were clarified by vigorous shaking in a 20 % suspension of chloroform containing 0·5 % gelatin-buffered saline and then centrifuged for 30 min at 6000 r.p.m. at 4 °C (model J-21B; Beckman Instruments). The supernatant was filtered (0·45 µm Millex-HA millipore filters; Millipore) and stored at -20 °C.
The virus in a 1 ml sample of clarified stool or sera was concentrated by centrifugation at 23 000 r.p.m. for 1 h at 4 °C (Jouan MR22i centrifuge; The Scientific Group). The pellet was resuspended in 140 µl PBS and viral RNA extracted using the QIAamp Viral RNA isolation kit (Qiagen) according to the manufacturer's protocol.
First-round PCR and reverse transcription were carried out in a 50 µl reaction mixture with 10 µl purified RNA, 400 nM of each primer, 200 µM of each dNTP, 0·625 U Super-therm DNA polymerase and 1x PCR reaction buffer (JMR Holdings), 1·5 mM MgCl2, 6 U AMV reverse transcriptase and 10 U RNasin (Roche Diagnostics). Viral RNA was reverse transcribed at 43 °C for 45 min and the cDNA amplified by 35 cycles of PCR (94 °C for 45 s, 48 °C for 45 s and 72 °C for 1 min) and a final extension step at 72 °C for 7 min. Second-round reactions were carried out in a volume of 100 µl with 10 µl first-round product and the same PCR reaction conditions.
The outer primers (sense 5'-CGCAACCTCACCCCTGGTAACAC-3', nt 925947; anti-sense 5'-CAGAAAGAAGGAGGGCACAAGC-3', nt 19912012) amplified a 1088 bp target region while the inner primers (sense 5'-CTCAGCAGGATAAGGGTATTGC-3', nt 12561277; anti-sense 5'-CTATAACTCCCGAGTTTTACC-3', nt 19631983) produced a 728 bp amplicon. All numbering is from the start of ORF2.
Sequencing of PCR products was performed using the ABI Sequencing Ready Reaction kit (Perkin Elmer) as specified by the manufacturer. The sequencing reaction was purified from excess dye terminators using the Centri-Sep Spin-Columns purification kit (Princeton Separations). Automated sequencing was performed using the ABI PRISM 377 DNA Sequencer (Perkin Elmer).
The nucleotide and amino acid alignments, consensus sequences, distance matrices and phylogenetic analysis were obtained using DNAMAN software (version 4.0, Lynnon BioSoft). Using 100 bootstrap replicates, a bootstrap value >70 % defined a stable phylogenetic grouping (Muerhoff et al., 1997). Trees were created using TREEVIEW (Win32, version 1.6.1). Nucleotide sequences were translated using DNASIS (Hitachi software, version 2.5).
Four Namibian sequences isolated from four different patients' stool specimens (HEV IgG positive) were compared with GenBank sequence data from 20 other HEV isolates over a 451 bp region of ORF2 (nt 13071757). The bootstrapped tree, rooted on the Chinese isolate T1 (Fig. 1a), grouped the isolates into the four conventional genotypes (nomenclature according to Wang et al., 2000
and Widdowson et al., 2003
). Because our sequence data only overlapped previously published sequence data from two other African outbreaks (1997/98 Nigeria and 1983 Namibia) over 180 bp, a second tree was drawn over this shorter region (Fig. 1b
) to include these strains. The partitioning of the common species was similar in both trees with the Afro-Asian isolates clustering in genotype I, the Mexican isolate and the newly defined 1995 Namibian isolates in genotype II, the United States isolates in genotype III and the Chinese isolate T1 in genotype IV. These four genetic clusters are consistent with previous studies. The four 1995 Nambian sequences shared a nucleotide identity of between 98·4 and 99·8 % and were 85·886·3 % similar to the Mexican isolate over the 451 bp region but were only 77·679·6 % similar to other African isolates. The 1995 Namibian sequences shared a similar nucleotide identity (98·3100 %) over the 180 bp region and were 88·991·1 % similar to the 1997/98 Nigerian isolates and 85·686·1 % similar to the Mexican isolate, all in genotype II, but only 77·878·9 % similar to the 1983 Namibian isolate in genotype I. Genotype I partitions into two subgenotypes with different African isolates clustering together and away from the Asian isolates. The 1995 Namibian sequences, however, grouped together as a separate clade of genotype II and were most similar to the 1997/98 Nigerian isolates. The 1997/98 Nigerian isolates were previously shown to cluster together with the Mexican isolate (Buisson et al., 2000
) and it is therefore likely that the same HEV strain circulating in Africa at this time caused both the 1995 and the 1997/98 outbreaks.
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By successfully amplifying, sequencing and analysing four HEV isolates from a non-A, non-B hepatitis outbreak in Rundu, Namibia, in 1995, we are able to confirm that the agent was indeed HEV, as suspected from the results of the serological assays at the time. Furthermore we are able to report the presence of a second unique HEV strain in southern Africa, indicating that HEV genotypes may be more widely distributed than previously thought.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Bradley, D. W. (1992). Hepatitis E: epidemiology, aetiology and molecular biology. Rev Med Virol 2, 1928.
Buisson, Y., Grandadam, M., Nicand, E. & 7 other authors (2000). Identification of a novel hepatitis E virus in Nigeria. J Gen Virol 81, 903909.
Byskov, J., Wouters, J. S. M., Sathekge, T. J. & Swanepoel, R. (1989). An outbreak of suspected water-borne epidemic non-A non-B hepatitis in northern Botswana with a high prevalence of hepatitis B carriers and hepatitis delta markers among patients. Trans R Soc Trop Med Hyg 83, 110116.[Medline]
Coursaget, R., Buisson, Y., Enogat, N. & 8 other authors (1996). Hepatitis E virus infections in France and Africa. In Enterically-Transmitted Hepatitis Viruses, pp. 201212. Edited by Y. Buisson, P. Coursaget & M. Kane. France: La Simarre, Joue-les-Tours.
Grabow, W. O. K., Taylor, M. B. & Webber, L. M. (1996). Hepatitis E virus in South Africa. S Afr J Sci 92, 178180.
He, J., Binn, L. N., Tsarev, S. A., Hayes, C. G., Frean, J. A., Isaacson, M. & Innis, B. L. (2000). Molecular characterization of a hepatitis E virus isolate from Namibia. J Biomed Sci 7, 334338.[CrossRef][Medline]
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. J Med Virol 55, 300304.[CrossRef][Medline]
Irshad, M. (1999). Hepatitis E virus: an update on its molecular, clinical and epidemiological characteristics. Intervirology 42, 252262.[CrossRef][Medline]
Isaacson, M., Frean, J., He, J., Seriwatana, J. & Innis, B. L. (2000). An outbreak of hepatitis E in Northern Namibia, 1983. Am J Trop Med Hyg 62, 619625.
Krawczynski, K., Aggarwal, R. & Kamili, S. (2000). Hepatitis E. Infect Dis Clin North Am 14, 669687.[Medline]
Mast, E., Polish, L., Favorov, M. & 8 other authors (1994). Hepatitis E among refugees in Kenya: apparent person to person transmission, evidence for age-dependent disease expression and new serological assays. In Viral Hepatitis and Liver Disease, 1994, pp. 375378. Edited by K. Nishioka, H. Suzuki, S. Mishiro & T. Oda. Tokyo: Springer.
Muerhoff, A. S., Smith, D. B., Leary, T. P., Erker, J. C., Desai, S. M. & Mushahwar, I. K. (1997). Identification of GB virus C variants by phylogenetic analysis of 5'-untranslated and coding region sequences. J Virol 71, 65016508.[Abstract]
Purcell, R. H. (1996). Hepatitis E virus. In Fields Virology, 3rd edn, vol. 2, pp. 28312843. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. Philadelphia: LippincottRaven.
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 virus isolated from a patient with acute hepatitis reported in the United States. J Gen Virol 79, 447456.[Abstract]
Schlauder, G. G., Frider, B., Sookoian, S., Castano, G. C. & Mushahwar, I. K. (2000). Identification of 2 novel isolates of hepatitis E virus in Argentina. J Infec Dis 182, 294297.[CrossRef][Medline]
Surveillance Bulletin, January (1996). Hepatitis, NIV. South African Virus Laboratories, p. 11.
Takahashi, M., Nishizawa, T., Yoshikawa, A., Sato, S., Isoda, N., Ido, K., Sugano, K. & Okamoto, H. (2002). Identification of two distinct genotypes of hepatitis E virus in a Japanese patient with acute hepatitis who had not travelled abroad. J Gen Virol 83, 19311940.
Tassopoulos, N. C., Krawczynski, K., Hatzakis, A., Katsoulidou, A., Delladetsima, I., Koutelou, M. G. & Trichopoulos, D. (1994). Case report: role of hepatitis E virus in the etiology of community-acquired non-A, non-B hepatitis in Greece. J Med Virol 42, 124128.[Medline]
Tsarev, S. A., Binn, L. N., Gomatos, P. J., Arthur, R. R., Monier, M. K., van Cuyck-Gandre, H., Longer, C. F. & Innis, B. L. (1999). Phylogenetic analysis of hepatitis E virus isolates from Egypt. J Med Virol 57, 6874.[CrossRef][Medline]
Tsega, E., Krawczynski, K., Hansson, B. G., Nordenfelt, E., Negusse, Y., Alemu, W. & Bahru, Y. (1991). Outbreak of acute hepatitis E virus infection among military personnel in Northern Ethiopia. J Med Virol 34, 232236.[Medline]
Tucker, T. J., Kirsch, R. E., Louw, S. J., Isaacs, S., Kannemeyer, J. & Robson, S. C. (1996). Hepatitis E in South Africa: evidence for sporadic spread and increased seroprevalence in rural areas. J Med Virol 50, 117119.[CrossRef][Medline]
van Cuyck-Gandre, H., Zhang, H. Y., Clements, N. J., Cohen, S. G., Caudill, J. D., Coursaget, P., Buisson, Y., Warren, R. L. & Longer, C. F. (1996). Partial sequence of HEV isolates from North Africa and comparison with known HEV sequences. In Enterically-Transmitted Hepatitis Viruses: Proceedings of the International Symposium on Enterically-Transmitted Hepatitis Viruses (1995: Paris, France), pp. 301310. Edited by Y. Buisson, P. Coursaget & M. Kane. France: La Simarre, Joue-les-Tours.
van Cuyck-Gandre, H., Zhang, H. Y., Tsarev, S. A., Warren, R. L., Caudill, J. D., Snellings, N. J., Begot, L., Innis, B. L. & Longer, F. (2000). Short report: phylogenetically distinct hepatitis E viruses in Pakistan. Am J Trop Med Hyg 62, 187189.
van Rensburg, E. J., Lemmer, H. R. & Joubert, J. J. (1995). Prevalence of viral infections in Mozambican refugees in Swaziland. East Afr Med J 72, 588590.[Medline]
Wang, Y., Zhang, H., Ling, R., Li, H. & Harrison, T. J. (2000). The complete sequence of hepatitis E virus genotype 4 reveals an alternative strategy for translation of open reading frames 2 and 3. J Gen Virol 81, 16751686.
Widdowson, M. A., Jaspers, W. J., van der Poel, W. H. M., Verschoor, F., de Roda Husman, A. M., Winter, H. L. J., Zaaijer, H. L. & Koopmans, M. (2003). Cluster of cases of acute hepatitis associated with hepatitis E virus infection acquired in The Netherlands. Clin Infect Dis 36, 2933.[Medline]
Worm, H. C., Schlauder, G. G., Wurzer, H. & Mushahwar, I. K. (2000). Identification of a novel variant of hepatitis E virus in Austria: sequence, phylogenetic and serological analysis. J Gen Virol 81, 28852890.
Zaaijer, H. L., Kok, M., Lelie, P. N., Timmerman, R. J., Chau, K. & van der Pal, H. J. H. (1993). Hepatitis E in The Netherlands: imported and endemic. Lancet 341, 826.[Medline]
Zanetti, A. R., Dawson, G. J. & The Study Group of Hepatitis E (1994). Hepatitis type E in Italy: a seroepidemiological survey. J Med Virol 42, 318320.[Medline]
Received 20 August 2003;
accepted 30 September 2003.