Hepatitis E virus (HEV) is a positive-sense single-stranded RNA virus that belongs to the genus Hepevirus in the family Hepeviridae.1–3 The HEV is the causative agent of acute or fulminant hepatitis E, primarily transmitted by the fecal–oral route.4 The relatively high mortality rate in HEV-infected pregnant women, up to 28%, is unique among hepatitis viruses.1,5 Hepatitis E is a zoonotic disease, with swine, wild boars, and wild deer serving as the reservoir for human infections.6–8 Four genotypes of HEV (G1–G4) have been detected in humans and G3 and G4 HEV are responsible for sporadic and autochthonous infections in both humans and other animal species worldwide.9–12
The HEV is a public health concern in many Asian and African countries where sanitation conditions are insufficient.13,14 Large waterborne outbreaks with high attack rates among young adults have been described in regions characterized by poor sanitary conditions in countries such as China, India, Somalia, and Uganda.15 However, there have been no reports of HEV infection in the Philippines. No information about hepatitis E patients or HEV infection in animals has been reported, and no sequence data have been deposited from this country. There is also no report of the HEV-pollution status of the environmental sewage water. With the hypothesis that environmental water samples may reflect the prevalence of HEV circulation, we examined river water samples to investigate HEV in the environment in one of the most densely populated cities in the world, Manila City, a metropolitan area in the Philippines with over 10 million residents.
A total of 12 water samples were collected from rivers that run through Manila City. Six sampling sites were selected (Figure 1). Sampling sites 1 to 3 were in the Pasig River, sites 4 and 5 were in the Paranaque River, and site 6 was in the Las Pinas River. These rivers receive the wastewater from the residents nearby. Water samples were drawn at all locations during both the dry season (December 23, 2012) and the wet season (July 23, 2013), and were named D1 to D6 and W1 to W6, respectively. The water samples were kept at 4°C during transport.
The concentration and purification of these water samples was carried out as described previously16; briefly, 500 mL of water was collected from each sampling site, and centrifuged at 3,000 rpm for 30 min at 4°C. Then, 2.5 mM MgCl2 was added to the supernatant to a final concentration of 0.05 mM. The pH value was adjusted to 3.5. The solution was filtered through a 0.45-μm mixed cellulose ester membrane filter (Merck Millipore, Tokyo, Japan) by a positive-pressure pump. Absorbents on the filter were then eluted with 10 mL of 3% beef extract solution by ultrasonication, three times. The solution was centrifuged at 12,000 rpm for 30 min, and the supernatant was stored at −80°C until RNA extraction.
The RNA was extracted using the MagNA Pure LC Total Nucleic Acid isolation kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's recommendations. Reverse transcription was performed with a high-capacity cDNA reverse transcription kit (ABI Applied Biosystems, Carlsbad, CA) at 25°C for 10 min, 37°C for 120 min, and followed by 85°C for 5 min in a 20-μL reaction mixture containing 1 μL reverse transcriptase, 2 μL of the random primer, 1 μL RNase inhibitor, 2 μL RT buffer, 0.8 μL 10-mM deoxynucleoside triphosphates, 8 μL RNA, and 5.2 μL distilled water. A nested reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to amplify a portion of the ORF2 genome, based on the method described previously.17
By RT-PCR, three samples (W4, W5, and W6) of the 12 water samples were positive for HEV RNA. Excluding the primer sequences, the length of the nested RT-PCR products was 338 nucleotides corresponding to nt 5959-6296 in the ORF2 of the Myanmar strain (D10330). The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and cloned into TA cloning vector pCR2.1 (Invitrogen, Carlsbad, CA). Each of 20 clones was sequenced. The clones with the same nucleotide sequence were counted as one strain. Finally, 21 HEV strains were obtained (GenBank accession nos. KF546257–KF546277), of which five strains were isolated from W4, 10 strains from W5, and six strains from W6. Phylogenetic analysis indicated that all 21 strains were G3 HEV. With the exception of strain W5-13, the other 20 strains' sequences belonged to sub-genotype 3a,18 separated into four clusters (cluster 1 to 4) with nucleotides sequence identities of 89.6–99.7% (Figure 2). In cluster 1, the sequences of three strains isolated from W6 were close to that of HEV strain EF530663 (isolated from a patient in Hungary) with nucleotide sequence identities of 92.3% to 92.6%. The nucleotide sequences of all nine of the strains in cluster 2 detected from W5 were close to that of a Japan swine HEV strain (AB094215) with identities of 91.1–92.6%. Cluster 3 contained six strains three from W4 and three from W6. Their sequences were close to that of AB671098, isolated from a Japanese donor, with nucleotide sequence identities of 93.5–94.4%. Cluster 4 comprised two strains from W4, with sequences close to the Japan strain AB 807429 (identities of 91.7–92.0%). The strain W5-13 does not belong to any known sub-genotype and shares identities of 84.0–84.3%, 90.2–91.7%, 85.5–88.2%, and 83.7–84.0%, with the Philippines HEV strains in clusters 1 to 4, respectively. The strain W5-13 thus constitutes a new sub-genotype of G3 HEV.
A basic local alignment tool (BLAST) analysis showed that the nucleotide sequence identities between these HEV strains detected in the Philippines and other HEV strains that have been published in GenBank were lower than 94.4%, indicating that area-specific HEV strains are circulating in the Philippines. All 21 of the HEV strains we detected in the river water were collected during the wet season, suggesting that the wet season presents a higher risk of individuals in the area contracting HEV infections.
The results of this study beg the question, what is the source of HEV detected in the Manila City rivers? Because no epidemiological information about HEV in the Philippines is currently available, for human patients, animal outbreaks, or genetic sequences, it is difficult to speculate about the sources of HEV. However, because the HEV is primarily transmitted by the fecal–oral route, HEV might be present in rivers containing human or animal stool. In this study, all of the HEV strains were detected from sampling sits 4–6, located in the Paranaque River and the Las Pinas River. None of the water samples from the Pasig River (sampling sites 1–3) were found to be HEV RNA positive. The Paranaque River and the Las Pinas River are considerably smaller than the Pasig River, and flow through a residential area having high population density. The degree of wastewater pollution is higher for sampling sites 4–6 than for sampling sites 1–3. All of the HEV detected in the river water samples belonged to G3. Genotype 3 HEV can be isolated not only from infected humans but is known to be zoonotic and has also been isolated from domestic swine and wild boars, wild deer, mongoose, and rabbits.6,7,9,11,19,20 The rivers were probably contaminated with HEV by human or animal excrement, or both.
In conclusion, we have detected and here reported HEV in the Philippines for the first time, and showed that G3 HEV in particular is circulating in the rivers of Manila City. To fully elucidate and address the HEV infection situation in the Philippines, it will be necessary to collect and analyze hepatitis patients' information and investigate the prevalence of HEV infection in swine and wild animals in these areas.
We thank Hiroyuki Katayama (University of Tokyo) for helpful discussions.
Meng XJ, Anderson DA, Arankalle VA, Emerson SU, Harrison TJ, Jameel S, Okamoto H, 2012. Hepeviridae. King AM, Carstens MJ, Lefkowitz EJ, eds. Virus Taxonomy. Ninth Report of the ICTV. London: Elsevier/Academic Press, 1021–1028.
Balayan MS, Andjaparidze AG, Savinskaya SS, Ketiladze ES, Braginsky DM, Savinov AP, Poleschuk VF, 1983. Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal-oral route. Intervirology 20: 23–31.
Tei S, Kitajima N, Takahashi K, Mishiro S, 2003. Zoonotic transmission of hepatitis E virus from deer to human beings. Lancet 362: 371–373.
Li TC, Chijiwa K, Sera N, Ishibashi T, Etoh Y, Shinohara Y, Kurata Y, Ishida M, Sakamoto S, Takeda N, Miyamura T, 2005. Hepatitis E virus transmission from wild boar meat. Emerg Infect Dis 11: 1958–1960.
Colson P, Borentain P, Queyriaux B, Kaba M, Moal V, Gallian P, Heyries L, Raoult D, Gerolami R, 2010. Pig liver sausage as a source of hepatitis E virus transmission to humans. J Infect Dis 202: 825–834.
Zhao C, Ma Z, Harrison TJ, Feng R, Zhang C, Qiao Z, Fan J, Ma H, Li M, Song A, Wang Y, 2009. A novel genotype of hepatitis E virus prevalent among farmed rabbits in China. J Med Virol 81: 1371–1379.
Yamamoto H, Suzuki J, Matsuda A, Ishida T, Ami Y, Suzaki Y, Adachi I, Wakita T, Takeda N, Li TC, 2012. Hepatitis E virus outbreak in monkey facility, Japan. Emerg Infect Dis 18: 2032–2034.
Nakamura M, Takahashi K, Taira K, Taira M, Ohno A, Sakugawa H, Arai M, Mishiro S, 2006. Hepatitis E virus infection in wild mongooses of Okinawa, Japan: demonstration of anti-HEV antibodies and a full-genome nucleotide sequence. Hepatol Res 34: 137–140.
Arankalle VA, Tsarev SA, Chadha MS, Alling DW, Emerson SU, Banerjee K, Purcell RH, 1995. Age-specific prevalence of antibodies to hepatitis A and E viruses in Pune, India, 1982 and 1992. J Infect Dis 171: 447–450.
Tao Z, Song Y, Wang H, Zhang Y, Yoshida H, Ji S, Xu A, Song L, Liu Y, Cui N, Ji F, Li Y, Chen P, Xu W, 2012. Intercity spread of echovirus 6 in Shandong Province, China: application of environmental surveillance in tracing circulating enteroviruses. Appl Environ Microbiol 78: 6946–6953.
Li TC, Ochiai S, Ishiko H, Wakita T, Miyamura T, Takeda N, 2012. A retrospective study on imported hepatitis E in Japan. Travel Med Infect Dis 10: 80–85.
Lu L, Li C, Hagedorn CH, 2006. Phylogenetic analysis of global hepatitis E virus sequences: genetic diversity, subtypes and zoonosis. Rev Med Virol 16: 5–36.
Meng XJ, Purcell RH, Halbur PG, Lehman JR, Webb DM, Tsareva TS, Haynes JS, Thacker BJ, Emerson SU, 1997. A novel virus in swine is closely related to the human hepatitis E virus. Proc Natl Acad Sci USA 94: 9860–9865.
Li TC, Saito M, Ogura G, Ishibashi O, Miyamura T, Takeda N, 2006. Serologic evidence for hepatitis E virus infection in mongoose. Am J Trop Med Hyg 74: 932–936.