The unique riverine ecology of hepatitis E virus transmission in South-East Asia

TRANSACTIONSOFTHEROYALSOCIETYOFTROPICALMEDICINEANDHYGIENE(1999)93,255-260

The unique riverine ecology of hepatitis E virus transmission in South-East Asia

Andrew L. Corwin’, Ngc _ lven T. K. Tien2. Khanthonn Bounlu3. Tarot Winarno4, Maid: ~~_ ~ ~~~~-- l~-.- yP.Putri’,KantiLaras’, Bia P. Larasati’, Nono Sukri’, Timothy’Endy’, H. A. Sulaiman6 and Kenneth C. Hyams’ I US NavalMedical Research Unit No. 2, Jakarta, Indonesia; 2Pasteur Institute, Ho ChiMinh City, Viet Nam; ‘Center-for National Laborato y and Epidemiology, Ministry of Health, Lao PDR; 4Provincial Health Service, Sintang District, West Kalimantan, Indonesia; ‘Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; 6Medical Faculty, University of Indonesia, Jakarta, Indonesia; 7Naval Medical Research Institute, Bethesda, Ma yland, USA

Abstract The ecology of hepatitis E virus (HEV) transmission in South-East Asia was assessed from a review of 6 published and 3 unpublished NAMRU-2 reports of hepatitis outbreak investigations, cross-sectional prevalence studies, and hospital-based case-control studies. Findings from Indonesia and Viet Nam show epidemic foci centred in jungle, riverine environments. In contrast, few cases of acute, clinical hepatitis from cities in Indonesia, Viet Nam and Laos could be attributed to HEV. When communities in Indonesia were grouped into areas of low (<40%), medium (40-60%), and high (>60%) prevalence of anti-HEV antibodies, uses of river water for drinking and cooking, personal washing, and human excreta disposal were all significantly associated with high prevalence of infection. Conversely, boiling of river drinking water was negatively associated with higher prevalence (P < 0.01). The protective value ofboiling river water was also shown in sporadic HEV transmission in Indonesia and in epidemic and sporadic spread in Viet Nam. Evidence from Indonesia indicated that the decreased dilution of HEV in river water due to unusually dry weather contributed to risk of epidemic HEV transmission. But river flooding conditions and contamination added to the risk of HEV infection in Viet Nam. These findings attest to a unique combination of ecological and environmental conditions predisposing to epidemic HEV spread in South-East Asia.

Keywords: hepatitis E virus, hepatitis E virus antibodies, prevalence, ecology, epidemiology, South-East Asia, Indonesia, Viet Nam, Laos

Introduction Hepatitis E virus (HEV) transmission occurs predo-

minantly in the developing world. First identified during the 1956 outbreak in India, involving over 29 000 cases, epidemic and sporadic HEV infections have since been recognized in many developing regions except for South America, although antibody to the virus was recently detectedfromBrazil (VISWANATHAN,~~~~~;BALAYAN, ~~~O;BRADLEY, 1992; PANG etal., 1995; PARANA etal., 1997). In China, some 120 000 cases of HEV were associated with an epidemic in Xinjiang during 1986- 88 (AYE et al, 1992). In South-East Asia, HEV in epidemic form has been reported from Myamnar, Viet NamandIndonesia (BALAYAN,~~~O;BRADLEY,~~~~; CORWIN et al., 1996a; MAST et al., 1996).

HEV infections reuorted from develoued countries in contrast are generally acquired from tiavel-related ex- posures. Only l-2% of US blood donors have been found to be HEV-seroreactive, and few infections have been documented in Europe. In the USA and Singapore, acute, clinically recognized hepatitis E has been attrib- utedtoforeigntravel (BALAYAN,~~~O;BRADLEY,~~~~; MAST et al., 19%‘).

HEV is a small, single-stranded RNA virus similar in structure to the caliciviruses. HEV spread is generally water-borne, particularly in epidemics. Unlike hepatitis A virus (HAV), there is little evidence to suggest person- to-person transmission. Because HEV is principally spread by human and possibly animal faecal contamina- tion of water resources, transmission is associated with poor water-related hygiene and sanitary conditions (VIS- WANATHAN,~~~~~;KANE etaZ., 1984; B~y~N,1990; BRADLEY, 1992; MAST&ALTER, 1993).Alsoin con- trast to HAV, HEV is characterized by (i) a longer incubation period ofup to 9 weeks, (ii) prolonged clinical course of illness, and (iii) poor protective value of serum immune globulin. Particularly notable and unique rela- tive to HAV is the high case-fatality rate (CFR) in pregnant women (1 O-24%) associated with H&V ir&ec- tions KI-IUROO. ~~~O;DECOCK etal.. 1987: BALAYAN. 1990;‘ REYES 8( BA&JDY, 1991; 'BRAD&Y, 1942,

Corresponding author: CDR Andrew Lee Corwin, US NAM- RU-2, Box 3 Unit 8132, APO AP96520-8132; phone +62 21 421 4457 to 4463, fax +62 21424 4507, e-mail corwin@smtp.namru:!.go.id

CENTERS FOR DISEASE CONTROLAND PREVENTION, 1993;MAsT&AL~~~,1993).

This review, mostly of the literature as well as a few unpublished research findings from studies conducted or supported by the US Naval Medical Research Unit No. 2 (NAMRU-2) in Jakarta, Indonesia, is intended to show that in South-East Asia the epidemic form of HEV transmission may be a function of a unique, predomi- nantly rural, riverine ecology. The data also indicate that extremes of rainfall conditions, both flooding and drought, combined with a background of specific river usage practices, contribute to the risk of HEV transmis- sion.

Methods and Analysis Examules of both enidemic and sooradic HEV trans-

mission ‘were identifieh from our inbestigations of rural and urban settings in South-East Asia. First, recognized foci of epidemic HEV transmission in jungle, riverine areas were investigated in Indonesian Borneo (West Kalimantan)and Eastern Java (Bondowoso), and in the An Giang Province of the Mekong River Delta region of Viet Nam which borders Cambodia (CORWIN et al., 1995,1996a, 1997; unpublished data; HAU et al., 1999). Second, the importance of HEV in acute, sporadic hepatitis was examined in a multi-hospital-based study of HEV that included the cities of Takarta. Indonesia (unpublished data), Hanoi and Ho C& Ming, Viet Nam (CORWIN et al., 1996b) and Vientiane, Lao PDR (BO~NLU et al., 1998) Data from studies conducted in Indonesia, Viet Nam, and Laos were included in the analysis.

Epidemiological approach Three approaches were used to investigate HEV

transmission in 8 reported studies: (i) outbreak investi- gations; (ii) cross-sectional prevalence studies; and (iii) hospital-based, case-control studies. Data pertaining to demographics, the environment, medical history, and risk-related behaviours had been obtained bv use of a standardized questionnaire administered by trained in- terviewers. Except for hosnital-based studies. and 1 HEV outbreak investigation described from Vie; Nam, the household served as the principal sampling unit.

Outbreak investigation. In Indonesia, an investigation of a 1991 HEV outbreak was carried out among com-

256

munities on the Inggar River of Borneo, providing a retrospective epidemiological evaluation (CORWIN et al., 1995). Another outbreak of hepatitis, that affected rural villages along the Balut River in eastern Java, was investigated in 1998, and subsequently attributed to HEV (unpublished data). From Viet Nam, an HEV outbreak investigation was conducted in 1994 among communities along a 60-km stretch of the Hau River, a tributary of the Mekong River (CORWIN et al., 1996a).

Cross-sectional seroprevalence studies. Communities in Indonesian Borneo (contiguous to the 1991 outbreak area described above) located along a 150~km stretch of the Kapuas River (into which the Inggar River flows) were surveyed in 1995. Targeted for study purposes was the affected population from a 1987 HEV outbreak where HEV was first recognized in Indonesia. For com- parison a population further downstream of the Kapuas River that was more urban was studied (CORWIN et al., 1997).

In the Mekong Delta river region of Viet Nam, the serourevalence of anti-HEV antibodies was studied in

I

communities surveyed from the area adjacent to the foci of epidemic HEV transmission in 1994 (HAU et aZ., 1999).

Hospital-based, case-control studies of acute hepatitis. A standardized case-control design and data collection instrument were used to evaluate the HEV infection in acute, clinically recognized hepatitis, from 4 urban centres (unpublished data; CORWIN et aZ., 1996b; un- published data; BOUNLU et aZ., 1998) in 3 South-East Asia countries: Indonesia, Viet Nam, and Laos. The duration of each study was 12 consecutive months: the first study began in 1993 and the last in 1997. Cases (175-200) were selected on the basis of clinical criteria for suspected acute hepatitis, and controls were selected to match the demographic characteristics of cases.

Serological tests Laboratory testing was coordinated by the US NAM-

RU-2, Jakarta, Indonesia. Serawere tested by commercial enzyme immunosorbent assay for IgG antibody to HEV (Abbott Laboratories, Abbott Park, IL) and IgGantibody to HAV (Abbott Laboratories). Sera from acute hepatitis studiesalsoweretestedforIgMantibodytoHAVandIgM antibody to hepatitis B core antigen (HBcAg) (Abbott Laboratories). Sera from patients with signs and symp- toms of acute hepatitis that were positive for IgG antibody to HEV were further tested for IgM antibody to HEV (Genelabs Diagnostics PTE, Singapore).

HEV transmission had not been\ecoanized in eoidemic form was found to areas. Background

be lower than in-outbreak-affected (community-acquired) HEV infec-

ANDREW L. CORWIN ETAL.

Specimens found positive for IgG antibody to HEV were also tested by polymerase chain reaction (PCR) at the Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand (TAM et al., 199 1). The oligonucleotide primers used in HEV PCR were HEV I’4653 (5’-TTT-TCA-GGT-GGC-TGC-C- 3’) and HEV P4874 (5’-GGG-CCC-CAA-TTC-TTC- T-3’) for RT-PCR; and HEV I’4704 (5’-ATA-CCG- TCA-GAG-CCC-AGG-3’) and HEV I?4845 (5’-GGC- GAA-GCG-CAC-GAC-ATC-3’) for nested PCR. Re- liability evaluation using positive and negative controls conducted at AFRIMS indicated high test sensitivity and specificity (unpublished data). Additionally, IgM anti- body to HEV was detected from sera using Western blotting as previously described (HE et aZ., 1993).

HEV studies in South-East Asia Indonesia

A 199 1 HEV outbreak in Sintang, West Kalimantan, affected over 2500 neonle. An attack rate of 90 cases/ 1000 population and case-fatality rate (CFR) of 14% among pregnant women were estimated (CORWIN et al., 1995). Notable was the relatively high seropreva- lence of IgG antibody to HEV 2 years following this outbreak. Overall, anti-HEV prevalence was 59% among 445 study subjects. There was no significant difference (P > 0.05) in HEVprevalence between cases (72%) with a history of acute jaundice and subjects without jaundice (61%). Similarly, the prevalence of anti-HEV 7 years after the 1987 epidemic was 50% in an area adjacent to the 1991 outbreak (CORWIN et al.. 1997) (Fig. 1). Finally, ‘first time’ epidemic HEV transmission h In- donesia outside of Borneo was recognized March/April 1998. A preliminary attack rate of 13% was estimated, ranging by community from 10% to 19% (unpublished data). However. 415 (43%) of the 962 studv subiects surveyed were positive for’IgG antibody to*HEV, of which 49% had serological evidence of a recent infection based on RT-PCR testing.

In the area of the 1987 West Kalimantan outbreak, continuinn (snoradic) HEV transmission was found bv comparini I&V infection in the population living (aged 27 years: prevalence 53%) during the epidemic with young children born after the event (aged <7 years: prevalence 16%; P < 0.000 1). However, the prevalence of anti-HEV antibodies in neiahbourine areas where

n Locations of hepatitis E outbreak

Fig. 1. Geographical distribution of recognized hepatitis E outbreaks, South-East Asia, 199 l-94.

HEPATITISEVIRUSINSOUTH-EASTASIA 257

tions in non-outbreak-affected communities surveyed for comparative purposes in West Kalimantan were identified in 23% of the population (CORWIN et al., 1997).

Among the 127 households surveyed after the 199 1 outbreak in West Kalimantan, 112 (88%) had a mini- mum of 1 anti-HEV reactor. However, only 63% of households had > 1 positive family member. There was no anoarent increased risk of HEV infection associated w&*&creased family size (CORwIN et al., 1995). Finally, findings from a survey conducted in 1994 from West Kalimantan region of the 1987 outbreak provided a more complete picture of transmission resulting from both epidemic and sporadic HEV transmission: 94% of 178 households in the outbreak area had > 1 family member positive for IgG antibody to HAV compared with 52% for IgG antibody to HEV (P < 0.0001) (CORWIN et aZ., 1997).

In urban Jakarta, prevalences of background HEV infections (positive for IgG antibody to HEV) among cases and control subjects (non-jaundiced, hospital inpatients) were very low, and differed little: 5%.and 2%. resnectivelv (urmublished data). No data are avail- able for-IgM an&odi to HEV (Tadle).

Indo-china (Viet Nam and Laos) Another study of epidemic HEV transmission in

South-East Asia involved an outbreak during 1994 in An Giang Province, Viet Nam (Fig. 1). IgG antibody to HEV was recognized in 76% of cases compared with 36% of matched community and geographical controls (P < 0.001). As in Indonesia, there was no evidence of familial clustering of seropositive individuals in Viet Nam (CORWIN et al., 1996a).

Data presented in Table show low prevalence of anti- HEV antibodies in acute, clinically recognized hepatitis from urban settings. In Hanoi and Vientiane, 2% of 375 and 4% of 52 acute episodes of suspected hepatitis, respectively, were attributed to recent HEV infections (CORWIN~~~Z., 1996b;BOUNLUetaL, 1998).Noserum sample from hospitalized case subjects in Ho Chi Minh or Jakarta was positive for IgM antibody to HEV (unpublished data). Background IgG antibody to HEV, reflecting previous infections, varied little between acute jaundiced case and control populations at each study location in Indo-china. Notable was the high proportion of controls positive for IgG antibody to HEV. similar to cases, from Hanoi and Vientiane: 14% ofco&rolsvs 12% of&es and 17% ofcontrolsvs 16% of cases, respectively (CORWIN et al., 1996b; BOIJNLU et al., 1998).

Role of river ecology Indonesia

Water use related to hygiene and sanitation was clearly associated with the risk of HEV infection. In the 1991 West Kalimantan outbreak area, HEV prevalence in- creased with the usage of river water for drinking and

Table. Study of acute clinical hepatitis E in South- East Asia

Locality IgG

anti-HEV IgG IgM

anti-HEV” anti-HEV

Vientiane

Jakarta

Hanoi

Ho Chi Minh City

16% 16% (n $16)

(n = ;21) 14%

(n 0 187) (n = 6)

‘In the absence of IgM anti-HAV and IgG anti-HBc. bNot available.

cooking (P < O.OOl), bathing (P < O.OOOl), and excre- ment disposal (P< 0.001; Fig. 2). Conversely, the prevalence of anti-HEV antibodies decreased as the practice of boiling drinking water increased (P = 0.02; Fig. 2) (CORWIN et al, 1995).

Adding to the risk of epidemic and sporadic HEV transmission in the 1987 outbreak affected and compari- son study areas of West Kalimantan were the practices of bathing, human waste disposal, drinking, and launder- ing. Usage of river water for bathing and human waste disposal was significantly associated with positivity for IgG anti-HEV antibodies in both outbreak and compari- son areas. In the comparison area, drinking and washing with river water were found to be strongly associated (P<O.OOOl) with prevalence of IgG anti-HEV anti- bodies (Fig. 3) (CORWIN, et aZ., 1997).

There was no evidence from the 1991 outbreak in West Kalimantan that the community-specific preva- lence of HEV declined downstream along the same river, as the current becomes swifter and waters deeper. How- ever, the prevalence of clinical signs and symptoms compatible with acute hepatitis during the actual out- break significantly decreased with community proximity further downriver: ranging from a community high of 35%toalowof<l%(P<O~OOl;Fig.4)(CORWINetal., 1995).

Viet Nam The protective value of boiling river water for drinking

purposes was suggested during the 1994 HEV outbreak in An Giang Province: the proportion of subjects positive for IgG antibody to HEV who boiled water for drinking was lower (40%) compared with those who did not (55%) (P < 0.05) (CORWIN etal., 1995a). Inanadjacent non-outbreak area, the practice of boiling river water for drinking was again significantly (P < 0.01) associated with the absence of IgG antibody to HEV.

Weather factors Indonesia

In West Kalimantan 91% of study households re- ported unusually dry weather leading up to and during the 199 1 HEV outbreak. Trend analvsis of weather data indicated subnormal rainfall in the months leading up to the outbreak period in September 1991. In the months just before the outbreak, only 19 cm of rain were recorded in August 1991, compared with an August meanvalueof209cmforalltheyears 1985-93 (CORWIN et aZ., 1995).

Viet Nam In Viet Nam, climatic conditions were also linked to

epidemic HEV transmission in the 1994 outbreak. How- ever, unusually heavy rainfall occurred during the nor- mally dry months leading up to the outbreak. In May 1994 (early in the outbreak), 341 cm of rain fell in the affected area, compared with a monthly (May) mean of 94 cm recorded for the preceding 4 years (1990-93). This was the second highest rate of monthly rainfall throughout the entire 1990-94 period. Additionally, significant flooding of the Mekong River was reported earlier in the year and during the outbreak period. Flooding in the months preceding the 1994 outbreak, as reflected by river depth (in meters), exceeded the highest monthly measures recorded for any of the previous 4 years (CORWIN et al., 1996a). Anecdotal reports from local health officials also suggest increased annual occurrence of community-acquired infections during the rainy season.

Discussion Populations affected by HEV outbreaks in Indonesia

and Viet Nam have been located on major riverine systems in areas best characterized as rural, with a poorly developed sanitary infrastructure (COR~IN et al., 1995; CORWIN et al., 1996a). That the 2 distantly separated

258 ANDREW L. CORWIN ETAL.

Drinking and Cooking Bathing

Area Prevalence for IgG anti-HEV Area Prevalence for IgG anti-HEV

Excrement Disposal

Area Prevalence for IgG anti-HEV Area Prevalence for IgG anti-HEV

Low: < 40% Medium: 40 - 60% High: > 60%

Fig. 2. Percentage of people in the 199 1 Kalimantan hepatitis E outbreak area using river water for various purposes, and prevalence of IgG antibodies against hepatitis E.

Case ChUOl

Bathing

(3% Control

Human Waste Disposal

Case Control

Drinking

Case Control CCL% Control

Washing Boiled Water

Fig. 3. Percentage of people with IgG antibodies to hepatitis E virus and various risk factors in the 1987 outbreak-affected and comparison study areas of West Kalimantan.

outbreaks described in this review shared (i) the same riverine-type settings, and (ii) water-associated risks against a background of universal river usage, attests to the importance of this ecology in HEV spread in South- East Asia. Other outbreaks fi-om within and outside the region, as in Myanmar and Pakistan, have been attrib- uted to faecally contaminated water supplies resulting from temporary sanitary system breakdowns (KANE et al., 1984).

Epidemic HEV transmission appears relatively con- fined, occurring in limited areas with definable geogra- phical borders. Also, uniquely separate episodes of

epidemic HEV spread (repeated outbreaks), recognized from the same district (Sintang, West Kalimantan) and 4 years apart, affected 2 contiguous although geographi- cally distinct populations (CORWIN et al., 1995; CORWIN et al., 1997). This suggests maintenance of over time HEV transmission in an animal and/or human reservoir.

Study observations suggest that river water is a primary source of HEV infection in South-East Asia. In addition to river usage for fish farming and transportation, populations in outbreak-affected areas of Indonesia and Viet Nam share a dependence on river water for most personal hygiene and sanitary needs. In West Kaliman-

HEPATITISEVIRUSINSOUTH-EASTASIA 259

A

High > 60%

Fig. 4. (A) Area-specific prevalence of hepatitis E virus, Sintang District, West Kalimantan, September 1993. (B) Percentage of population with signs and symptoms compatible with acute hepatitis in West Kalimantan August/September 199 1.

tan, Indonesia, outhouses over the water on extended docks are used by individuals, the family unit, and the community at large; at the same time bathing, washing of clothes, and drinking of water take place. The frequency of usage of rivers and tributaries for drinking and cooking, bathing and human excreta disposal was related to an increased prevalence of anti-HEV antibodies. Also, the practice of regularly boiling river water for drinking and cooking purposes was associated with decreased area-specific anti-HEV antibody prevalence (CORWIN er al., 1995; CORWIN et aZ., 1997). The absence of HEV as a cause of acute, clinical hepatitis from study cities in South-East Asia is probably an attribute of improved sanitary systems that characterize more urbanized areas.

Findings from Indonesia show a probable dose- response effect associated with clinical (HEV) disease. Further upstream, communities in the outbreak area had attack rates considerably higher, possibly because of greater concentration of virus from excreta and other human and/or animal waste products in shallow, slow- moving river water. Reduced dilution added to the opportunity of exposure (CORWIN et al., 1995). In outbreaks reported from Somalia, severe morbidity and high case-fatality rates were also attributed to HEV inoculum size: greater exposure resulting from poor dilution in river and well water (BILE et al., 1994). Negligible familial clustering (compared with anti- HAV antibodies) of HEV infection indicates that per- son-to-person contact contributes little to the mechan- istic spread of HEV, irrespective of epidemic or sporadic acquired infections (CORWIN et al., 1995; CORWIN et al., 1997). A similar finding was reported from a comparative study of intra-familial transmission versus waterborne spread during a bimodal, waterborne HEV outbreak in India (AGGARWAL & NAIK, 1994). In urban Rangoon (Myanmar), however, there was no apparent systems’ breakdown affecting water sanitation, although the possibility was acknowledged (MYINT et al., 1985).

Outbreaks in other geographical areas indicate a

primary role for river water in HEV transmission. Villages in Somalia that depended on river water during the 1988 HEV outbreak had higher attack rates than those relying on pond or well water (BILE et d., 1994). Epidemic conditions in Nepal were also attributed to significant rains (KANE et aZ., 1984; SHRESTHA, 1991; CiAYSON et al., i995).

The nooulation-based nrevalence of anti-HEV anti- bodies in non-outbreak arias adjacent to recognized foci of HEV transmission, in both Indonesia and Viet Nam, was found to be significantly lower. Nevertheless, spora- dic (ongoing) HEV infections in such areas were evident, indicating that unique conditions of decreased dilution of virus or increased contamination are necessary for an epidemic (CORWIN et al., 1997).

In Viet Nam, recognized clinical disease associated with the 1994 HEV outbreak was principally a male (15- 40 years) phenomenon (CORm et al., 1996a). Simi- larly, the highest attack rates of epidemic HEV spread in Indonesia were among the same adult population (COR- WIN et al., 1995; CORWIN et cd., 1997). This suggests a possible age-related occupational water exposure, and possible gender bias in health care utilization patterns. Similar findings relative to adults aged 14-40 years have been reported from the New Delhi (India) and other epidemic experiences (MELNICK, 1957; VISWANA- THAN, 1957b; FAVOROV et al., 1992).

In conclusion, climatic extremes affecting river ecol- ogy: heavy rainfall linked to flooding in Viet Nam, or subnormal rainfall in Indonesia, have probably contrib- uted to the favourable conditions that influenced epi- demic or cyclic HEV spread. Anecdotal information from the Mekong Delta Region also indicates a dramatic annual rise in cases during the flooding of the river system (CORWTN et al., 1995; CORWIN et al., 1997). In Nepal, major peaks in epidemic HEV occurrence coincided with monsoon rains (KANE et aZ., 1984). Excessive run-off associated with heavy rains and flooding probably adds to the mixing of contaminated matter with water supplies as

ANDREW L. CORWIN ETAL.

infected human and/or animal waste materials are washed into rivers and other water sources.

In order for HEV epidemics to occur, unique combi- nations of water-related factors appear necessary.

References Aggarwal, R. & Naik, S. R. (1994). Hepatitis E: intra familial

transmission versus waterborne spread. Journal of Hepatology, 21,718-723.

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. (1990). HEV infection: historical perspectives, global epidemiology, and clinical features. In: Viral Hepatitis and Liver Diseases, Proceedings of the 1990 International Sympo- sium on Viral Hepatitis and Liver Disease. Hollinger, F. B., Lemon. S. M. & Mareolis. H. (editors). Baltimore: Williams & Wilkins, pp. 498-551. ’ ’ ’

Bile, K., Isse, A., Mohammad, O., Allebeck, I’., Nilsson, L., Norder, H., Mushawar, I. K. & Magnuis, L. 0. (1994). Contrasting role of river and wells as sources of drinking water on attack and fatality rates in a hepatitis E epidemic in Somalia. American Journal of Tropical Medicine and Hygiene, 54.466-474.

Bounlu, K., Insisiengmay, S., Vanthanouvong, K., Saykham, Widjaya, S., Iinuma, K., Matsubayashi, K., Laras, K., Putri, M. P., Endy, T., Vaughn, D., Raengsakulrach, B., Hyams, K. C., Hayden, M., Scheffel, C. & Corwin, A. L. (1998). Acute jaundice in Vientiane, Lao People’s Democratic Republic. Journal of Clinical Infectious Diseases, 27, 7 17- 72 1.

Bradley, D. W. (1992). Hepatitis E: epidemiology, aetiology, and molecular biology. Reviews in Medical Virology, 2,19-28.

Centers for Disease Control and Prevention (1993). Hepatitis E among U.S. travelers, 198991992. Morbidity and Mortality Weekly Report, 42, l-4.

Clavson. E.. Mvint, K. S. A., Snitbhan. R.. Vauahn. D. W.. Innis,B. S., Ghan, L., Cheung, P. & Shresma, M. I’: (1995): Viremia, fecal shedding, and IgM and IgG responses in patients with hepatitis E. Journal of Infectious Diseases, 172, 927-933.

Cotwin, A., Jarot, K., Lubis, I., Nasution, K., Suparmawo, S., Sumardiati, A., Widodo, S., Nazir, S., Omdortf, G., Choi, Y., Tan, R., Sie, A., Wignall, S., Graham, R. & Hyams, K. (1995). Two years’ investigation of epidemic hepatitis E virus transmission in West Kalimantan (Borneo), Indonesia. Transactions of the Royal Society of Tropical Medicine and Hygiene, 89,262-265.

Corwin. A. L.. Khiem. H. B.. Clavson. E. T.. Sac. P. K.. Nhune. V. T.-T., Yen, V. T:, Cut; C. T. T.; Vaughn, D., M&en, fi Richie, T. L., Putri, M. P., He, J., Graham, R., Wignall, S. & Hvams, K. C. (1996a). A waterborne outbreak of henatitis E virus transmission in southwestern Vietnam. American Jour- nal of Tropical Medicine and Hygiene, 54, 559-562.

Corwin, A. L., Dai, T. C., DUC, D. D., Suu, P. I., Van, N. T., Ha, L. D., Janick, M., Kanti, L., Sie, A., Soderquist, R., Graham, R., Wignall, S. & Hyams, K. C. (1996b). Acuteviral hepatitis E in Hanoi, Viet Nam. Transactions of the Royal Society of Tropical Medicine and Hygiene, 90, 647-648.

Corwin, A., Putri, M. P., Winarno, J., Lubis, I., Suparmanto, S., Surnardiati, A., Laras, K., Tan, R., Master, J., Warner, G., Wignall, F. S., Graham, R. & Hyams, K. C. (1997). Epidemic and sporadic hepatitis E virus transmission in West Kaliman- tan (Borneo), Indonesia. American Journal of Tropical Medi- cine and Hygiene, 57,62-65.

DeCock, K. M., Bradley, D. W., Sandford, N. L., Govindar- ajan, S., Maynard, J. E. & Redeker, A. G. (1987). Epidemic non-A, non-B hepatitis in patients from Pakistan. Annals of InternalMedicine, 106, 227-230.

Favorov, M. O., Fields, H. A., Purdy, M. A., Yashina, T. L., Aleksandrov, A. G., Alter, M. J., Yarasheva, D. M., Bradley, D. W. & Margolis, H. S. (1992). Serologic identification of

hepatitis E virus infections in epidemic and endemic settings. Journal of Medical Virology, 36, 246-250.

Hau, C. H., Hien, T. T., Tien, N. T. K., Khiem, H. B., Sac, P. K., Nhung, V. T., Larasati, R. P., Laras, K., Putri, M. P., Doss, R., Hyams, K. C. & Corwin, A. L. (1999). Prevalence of enteric hepatitis A and E viruses in the Mekong river delta region of Viemam. American Journal of Tropical Medicine and Hvtiene. 60.277-280.

He,Jz Tam, A. W., Yarbough, P. O., Reyes, G. R. & Carl, M. (1993). Expression and diagnostic utility of hepatitis E virus: putative stictural proteins expressed in insect cells. Journal of ClinicalMicrobiologv, 31, 2167-2173.

Kane, M. A., Bradley, D. W., Shrestha, S. M., Maynard, J. E., Cook, E. H., Mishra, R. P. & Joshi, D. D. (1984). Epidemic non-A, non-B hepatitis in Nepal: recovery of a possible etioloeic aeent and transmission studies in marmosets. Your- nal of yhe A;*terican Medical Association, 252, 3 140-3 145‘:

Khuroo, M. S. (1980). Study of an epidemic of non-A, non-B heoatitis: oossibilitv of another human henatitis virus distinct from post:transfuscon non-A, non-B type.~AmericanJournal of Medicine, 68, 8 18-824.

Mast, E. E. & Alter, M. J. (1993). Epidemiology of viral hepatitis: an overview. Seminars in Virology, 4, 273-283.

Mast, E. E., Purdy, M. A. & Krawczynski, K. (1996). Hepatitis E. Bailliere’s Clinics in Gasrroenterology, 10, 227-242.

Mast, E. E., Kuramoto. I. K.. Favorov. M. O., Schoenine, V. R.. Burkholder, B. T., Shapiro, C. N. & Holland, P. V. 71997): Prevalence of and risk factors for antibody to hepatitis E virus seroreactivity among blood donors in northern California. Journal of Infectious Diseases, 176,34-40.

Melnick, J. L. (1957). A water-borne urban epidemic of hepatitis. In: Hepatitis Frontiers, Hartman, F. W., LoGrippo, G. A., Matffer, J. G. & Barron, J. (editors). Boston: Little Brown. vv. 21 l-225. _-_

Myint, H. L. A., Myint, M. S., Tun, K., Thein, M. M. & Khin, M. T. (1985). A clinical and epidemiological study of an enidemic of non-A non-B henatitis in Raneoon. American Jburnal of Tropical Medicine an; Hygiene, 34, i 183 - 1189.

Pang, L., Alencar, F. E., Cerutti, C. Jr, Milhous, W. K., Andrade, A. L., Oliveira, R., Kanesa-Thasan, N., MaCarthy, P. 0. & Hoke, C. H. Jr (1995). Short report: hepatitis E infection in the Brazilian Amazon. American Journal of Tropical Medicine and Hygiene, 52,347-348.

Parana, R., Cotrim, H. P., Cartey-Boennec, M. L., Trepo, C. & Lvra. L. (1997). Prevalence ofheoatitisEvirusIeGantibodies in patients from a referral unit of liver disease: in Salvador, Bahia, Brazil. American Journal of Tropical Medicine and Hygiene, 57,60-6 1.

Reyes, G. R. & Baraudy, B. M. (199 1). Molecular biology of non-A non-B agents: hepatitis C and hepatitis E viruses. Advances in Virus Research, 40, 57 - 102.

Shrestha, S. M. (199 1). Enteric non-A, non-B hepatitis in Neval: clinical and eoidemioloaical observations. In: Viral Heiatitts C, D and E, Shitkata, y., Purcell, R. H. & Uchida, T. (editors). Amsterdam: Elsevier Science Publishers, PD. _- 265-275.

Tam, A. W., Smith, M. M., Guerra, M. E., Huang, E. E., Bradley, D. W., Fry, K. E. & Reyes, G. R. (1991). Hepatitis E virus (HEV): molecular cloning and sequencing of the full- length viral genome. Virology, 185, 120- 13 1.

Viswanathan, R. (1957a). Infectious hepatitis in Delhi (1955-56): epidemiology. IndianJournal ofMedical Research, 45,71-76.

Viswanathan, R. (1957b). Certain epidemiological features of infectious hepatitis during the Delhi epidemic, 1955-1956. In: Hepatitis Frontiers, Hartman, F. W., LoGrippo, G. A., yo;F;rhJ. G. & Barron, J. (editors). Boston: Little Brown, pp.

Received 6 October 1998; revised 4 December 1998; accepted for publication 1 February I999