Journal of Medical Virology 32263-270 (1992)

Enteric Non-A, Non-B Hepatitis: Epidemics, Animal Transmission, and Hepatitis E Virus Detection by the Polymerase Chain Reaction Shahid Jameel, Hemlata Durgapal, C.M. Habibullah, Mohammed Sultan Khuroo, and Subrat Kumar Panda Department of Pathology, All India Institute of Medical Sciences (H.D., S.K.P.), Virology Group, International Center for Genetic Engineering and Biotechnology (ICGEB) (S.J.), New Delhi, Department of Gastroenterology, Osmania General Hospital (C.M.H.), Hyderabad, and Department of Gastroenterology and Medicine, Sher-I-Kashmir Institute of Medical Sciences (M.S.K.), Srinagar, India We studied epidemics of viral hepatitis occurring at three different places in India. One was a combined epidemic due to hepatitis E virus (HEV) and hepatitis A virus (HAV) infections. In this epidemic, HAV affected children below 10 years of age, whereas HEV infected the young adult population. HEV was transmitted t o rhesus monkeys (Macaca mulata) and confirmed by the polymerase chain reaction (PCR) on bile from the animals. Fecal material from acutely infected patients in one of the epidemics was also found positive for HEV RNA by PCR. This may help in confirming the nature of future epidemics. The bile and liver from experimental animals can be used as a source of material for further virological and molecular biological studies of HEV.

ease, similar to hepatitis A, a high mortality rate of 10-20% has been observed in pregnant women, particularly in the third trimester of pregnancy [Khuroo et al., 19831. Recently, the genome of HEV was cloned and sequenced [Tam et al., 19911from the bile and liver ofa cynomolgusmonkey infected with fecal isolate from a Burmese patient with enteric non-A, non-B hepatitis. However, the inability to di,agnose conclusively and rapidly HEV infections is still a major hurdle. We describe the epidemiology of three separate epidemics of viral hepatitis in India, the development of a rhesus monkey model for the disease, and confirmatory detection of HEV in the bile of infected monkeys and the stools of acutely infected humans using the polymerase chain reaction (PCR).

MATERIALS AND METHODS Epidemics and Patients Clinical samples were collected from three epidemics: KEY WORDS: HEV, PCR, epidemic, hepatitis, Jammu city (February 19881, Hyderabad city and adbile, transmission joining Ranga Reddy district, South India (August 1990), and Aligarh, North India (March 1991). For collection of clinical samples, the epidemic sites were visINTRODUCTION ited with a team of local physicians. After consent, the Enteric hepatitis constitutes the major form of viral clinical history was taken. Blood (5-10 ml) was colhepatitis in adult populations of the developing world lected under aseptic conditions by cubital venipuncture and such epidemics are frequent in India and other using disposable syringes and needles. The serum was parts of South East Asia [Khuroo, 1980; Kane et al., separated and stored at -70°C. Fecal samples were col1984; Balayan et al., 1983; Molinie et al., 1988; Panda lected from patients with illness of less than 1 week. et al., 1989; Viswanathan, 1957; Myint et al., 1985; Sterile plastic jars (200 ml) were provided and patients Tandon et al., 19821. The agent, presumably similar to were asked to bring stools the next day which were then Caliciviruses has been observed frequently in the fecal stored at -70°C. Transportation of sera and stools from samples collected during the acute phase of the illness the epidemic site to the local coordinating centers and [Balayan et al., 1983; Bradley et al., 1988; Panda et al., then to the investigating laboratories was carried out 1989; Arankalle et al., 19881. This agent, termed hepa- in dry ice. titis E virus (HEV) has been transmitted successfully to several New and Old World primates, e.g., tamarins [Kane et al., 19841, cynomolgus monkeys [Bradley Accepted for publication January 17, 1992. et al., 1987; Krawczynski and Bradley, 19891, rhesus Address reprint requests to Subrat K. Panda, Department of monkeys [Panda et al., 1989; Gupta et al., 19901, and Pathology, All India Institute of Medical Sciences, New Delhi-110 chimpanzees. Although it causes a self-limiting dis- 029, India. 0 1992 Wilev-Liss, Inc.

0 1992 WILEY-LISS, LNC.

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Screening of Sera The sera were first tested for bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) using commercially available test kits (Boehringer Mannheim, GmbH). The enzyme estimations were carried out using kinetic assays. Only those patients with the ALT and AST values 2.5 times over normal were included in the study for further screening. Samples were screened further for serum markers of hepatitis A and B. Tests were carried out to detect IgM antibody against hepatitis A virus (IgM anti-HAV), hepatitis B surface antigen (HBsAg), and IgM antibody against hepatitis B core antigen (IgM anti-HBc). Those positive for HBsAg were screened further for the hepatitis B “e” antigen (HBeAg) and antibody (anti-HBe). All these tests were carried out using micro-enzymelinked immunosorbent assay (ELISA) (Organon Teknika, The Netherlands) as per the manufacturer’s guidelines. Those cases positive for IgM antibody against either anti-HBc or anti-HAV were considered a s suffering from HBV or HAV infections, respectively. Those negative for both were considered as non-A, non-B hepatitis infections. Screening of Fecal Samples Fecal samples assigned serologically to the non-A, non-B group were screened by immunoelectron microscopy (IEM). Briefly, a 20% fecal suspension was prepared in phosphate-buffered saline (PBS) by vortexing vigorously with sterile 5 mm glass beads. The fecal suspension was centrifuged at 10,000 rpm in a Beckman JA20 rotor for 30 min a t 4°C. The clarified supernatant (100 pl) was incubated with 1:lO to 1:200 dilutions of autologous acute phase serum a t 37°C for 1 h r followed by 4°C overnight. The incubated mixture was then centrifuged at 25,OOOg in a Beckman SW50.1 rotor for 1 h r at 4°C. The pellet was resuspended in 50 pl of distilled water containing 0.1% bovine serum albumin. The resulting suspension was put onto 400 mesh carbon formavar coated copper grids (BioRad, USA) and stained with 3% phosphotungstic acid, pH 6.8. Grids were viewed in a Phillips CM 10 electron microscope a t 46,000 to 92,000 screen magnification. At least 100 fields were seen before regarding a sample as negative. Transmission to Rhesus Monkeys Sixteen adult rhesus monkeys (Macaca mulata) were used. Of these, 5 were bred in the Primate Research Facility a t AIIMS and 11were captured from the wild. All monkeys were between 6-8 k g in weight. Pre-inoculation physical examination and chest x-rays were carried out and the animals certified healthy by the veterinarian. Ethical clearance from the institutional committee for primate research was obtained. Baseline liver function test values were determined by performing three pre-inoculation bleedings. All animals were kept at the Primate Research Facility, AIIMS.

Jameel et al. For injections, fecal samples were made into 20% suspensions in 0.1 M PBS, pH 7.2, centrifuged a t 20,000 rpm in a Beckman JA20 rotor for 30 min at 4”C, and the supernatant filtered through a 0.22 pm filter (Millipore, USA). Of this suspension, 10 ml was administered intravenously to each animal. Five animals (M1878, M1879, M1880, M1882, and M1884) received a pooled fecal suspension from five acute hepatitis cases from the Jammu epidemic. The animals were bled weekly and fecal samples collected from day 10-day 29 post-inoculation. Bile was collected on day 23 and in two of the monkeys (M1879 and M1884) a partial hepatic resection was also carried out on day 23. Fecal material from one of the three monkeys (M1884) collected on days 19, 20, and 23 were pooled and used to inoculate three naive monkeys (M1877, M1883, and M1886). All three were followed up as above for bleeding and stool collection. Bile was collected from two of the animals (M1877 and M1886) on day 19 post-inoculation. Eight animals (M745, M1109, M1111, M1214, M1308, M1442, M1757, and M1937) received IEM-positive and pooled samples from the Hyderabad epidemic. The animals were followed up as described earlier for bleedings and stool collection. Bile was collected from four animals (M745, M1109, M1442, and M1757) on day 17 prior to elevation in transaminases, and on day 23 during elevated transaminase levels in the remaining four animals (M1111, M1214, M1308, and M1937). Two of the animals (M1308 and M1937) which were transmitted once with the Hyderabad epidemic samples were reinoculated with the same sample 6 months after attaining normal transaminase levels to check the possibility of immune protection. For collection of bile a n upper abdominal incision was made under general anesthesia induced by ketamine (1 mg/kg body weight). Needle biopsy of the liver was performed with a Menghini aspiration liver biopsy needle (1mm diameter).

Polymerase Chain Reaction Extraction ofRNA. Bile (25 p1) was diluted to 250 p1 into a solution containing 10 mM Tris, pH 8.0,5 mM ethyl diaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate (SDS), 10 pg tRNA, and 25 pg Proteinase K. After vigorous mixing, the mixture was kept a t 55°C for 30 min. An equal volume of phenol-chloroform (1:l) was added and the mixture kept at 65°C for another 15 min. The aqueous layer was then extracted with a n equal volume of chloroform. Two hundred microliters of the aqueous layer was precipitated with 85 pl of 7.5 M ammonium acetate and 750 p1 of ethanol a t -70°C for 1-2 hr. The RNA pellet was rinsed twice with 70% ethanol, dried, resuspended in 25 p1 RNase-free water, and stored at -70°C. For isolation of RNA from stools, the guanidinium isothiocyanate-acid phenol method [Chomczynski and Sacchi, 19871 was used. Briefly, a toothpick tip full of stool was taken and resuspended with vigorous vortexing into 250 pl of solution D (4 M guanidinium isothio-

PCR for HEV

cyanate, 0.75 M sodium citrate, 0.5% Sarkosyl, and 0.1 M 2-mercaptoethanol). After acid phenol treatment, the RNA in the aqueous phase was precipitated sequentially with isopropanol and ethanol. The pellets were washed with 70% ethanol, resuspended in 50 pl RNasefree water, and stored a t -70°C. Primers. The selection of oligonucleotide primers for PCR amplification was based on the partial nucleotide sequence of HEV available in literature [Reyes et al., 1990; Genebank accession # M324001. Two of the primer sequences (3043, 3044) and a PCR product a s positive control were kindly provided by Dr. R. Purcell (National Institutes of Health, USA). The nucleotide sequences of the primers, with the HEV-specific sequences underlined, were 3043

5’-CGGGATCCACACACATCTGAGCTACATTCGTGAGCT-3’

3044

: 5’-CGGAATTCAAAGGCATCCATGGTGTTTGAGAA-

TGAC-3’

HEV-1: 5’-GGAATTCGACTCCACCCAGAATAACTT-3’ HEV-3 : 5‘-GGAATTCACAGCCGGCGATCAGGACAG-3’ DET-3 : 5’-ACTCCTCCATAATAGCACACTCTAGACCCAGAG-3‘.

The amplification primer pairs 3043/3044 and HEV1IHEV-3 were selected to amplify DNA fragments of 591 bp and 343 bp, respectively. Further, HEV-I/ HEV-3 are internal to 304313044, thus enabling nested PCR to be performed. DET-3 is a n internal oligonucleotide used for confirmation of PCR amplification specificity by Southern hybridization. Oligonucleotide syntheses were carried out on a n automated DNA synthesizer (Model 380B; Applied Biosystem, USA) using phosphoramidite chemistry. The crude oligonucleotides were precipitated with ammonium acetate and ethanol prior to use as primers in the PCR reaction. PCR amplifications. Prior to PCR amplification, the RNA was reverse transcribed into (-)cDNA. Five to ten microliters of the extracted RNA was heated to 95°C for 5 min and rapidly chilled on ice. In a final volume of 20 p1, this was made up to 50 mM Tris-HC1, pH 8.3,75 mM KC1,3 mM MgCl,, 10 mM dithiothreitol (DTT), 500 pM each dNTP, 1 nmol random hexameric primers (Sigma, USA), 20 units RNasin (Promega, USA), and 200 units M-MuLv reverse transcriptase (BRL, USA). After a 90 min incubation at 37”C, the reaction was heated to 95°C for 5 min and stored frozen at -20°C. For PCR amplification, 80 pl of a PCR reaction mix was added to 20 pl of the cDNA mix such that the final 100 pl volume contained 10 mM Tris-HC1, pH 8.3, 50 mM KCl, 2.5 mM MgCl,, 0.01% gelatin (1X PCR buffer), 200 pM each dNTP, 25 pmol of 3043 and 3044 primers and 1 unit of Taq Polymerase (Cetus-Perkin Elmer, USA or Stratagene, USA). After overlaying with 90 pl of mineral oil (Sigma), the amplifications were carried out in a thermocycler (Perkin Elmer or Coy, USA) with the following cycling program: 94°C denaturation for 1 min, 50°C annealing for 1 min, and

265

72°C extension for 2 min. A total of 25 cycles were carried out followed by a final extension a t 72°C for 7 min. For nested PCR, 10 pl amplified material from the first set was removed and reamplified a s above with primers HEV-1 and HEV-3. Following amplification, 10 p1 of the reaction was analyzed on a 2%agarose gel (BioRad), followed by ethidium bromide staining. For Southern blotting, DNA from the stained gel was transferred by capillary action to a nylon membrane (Zetaprobe; BioRad) in a buffer containing 10 mM NaOH, 1mM EDTA. Following overnight transfer, the membrane was rinsed briefly in blotting buffer and the DNA denatured with 0.4 M NaOH for 10 min. The membrane was neutralized by rinsing briefly in 2 x SSC, air dried, and then baked a t 80°C under vacuum for 60 min. Prehybridization was done for 3 4 hr a t 50°C in 25-50 ml of the following solution: 5 x SSC, 20 mM sodium phosphate, pH 7,7% SDS, 10 x Denhardt’s mix, and 100 pglml of freshly denatured salmon sperm DNA. Hybridization was done at 50°C overnight with 25 ml of fresh prehybridization solution containing about lo7 cpm of end-labeled DET-3 oligonucleotide. The membrane was then washed as follows: a) twice for 30 min each a t 50°C in 3 x SSC, 10 x Denhardt’s mix, 5% SDS, and 25 mM sodium phosphate, pH 7.5; b) once for 30 rnin at 50°C in 2 X SSC, 2% SDS. Autoradiography was carried out at room temperature for 2 hr. False positive PCR results were avoided by strict application of the published control measures [Kwok and Higuchi, 19891.

RESULTS Epidemics Samples from three epidemics were used in this study. The Jammu epidemic occurred in Jammu city in January-February 1988. Of the 176,800 people exposed to infection, 518 cases of icteric viral hepatitis were recorded over a period of 10 weeks. The disease was water-borne due to fecal contamination of the water supplies. The age distribution was 31.8 5 13.8 years. Thirty pregnant women were affected. Two deaths were recorded, both occurring in pregnant women. Thirtyfive sera were tested for viral markers of which 2 were positive for IgM anti-HAV, 2 positive for HBsAg (but negative for IgM anti-HBc), and the rest negative for all markers of HAV and HBV. The Hyderabad epidemic was centered around a small taluk town, Ibrahimpatnam, located 38 km from the main city. It had 5,000 houses and a population of 18,000. The cause of epidemic was due to contamination of drinking water with sewage. The town obtains its water supply from one overhead tank which draws water from three tube wells. One of the tube wells was adjacent to a liquid sewer disposal site and drainage was by open drains leading to a large pond. A total of 281 samples were collected, 80% of which were obtained from the clinic of a local medical practitioner and the rest from a local government health clinic.

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TABLE I. Etiological Distribution Total no. of cases tested Hyderabad 245 September-October 1990 Aligarh 96 March 1991 Jammu 35 February 1988 Total 376

of Acute Viral Hepatitis During Three Epidemics

HAV

HBV

(%)

(yo)

57 (23)

-

2 (5.7) 59 (15.7)

HAV and HBV

29 (11.8) 19 (19)

-

-

-

48 (12.7)

01 (0.26)

Non-A, non-B

(W

01 (.4)

158 (64.48) 77 (89.2) 33 (94) 268 (71.3)

TABLE 11. Age Distribution of Hepatitis A and Hepatitis E During Two Epidemics of Viral Hepatitis* No. of cases Non-A, non-B HAV HBV Age group ALG HYD ALG HYD ALG HYD ALG HYD 1-5 6-10 11-15 16-20 2 1-25 26-30 31-35 36-40 41-45 46-50 56-60 >60

1 10 20 15 9 2 -

3

1 2

99 21 18 22 17 27 11 5 3 6 7

4 2 2 6 6 4 2 1

53 11 12 14 11 22 9 4 3 4 7

-

2

-

-

-

~~

~

*ALG, Aligarh epidemic; HYD, Hyderabad epidemic.

The Aligarh epidemic followed as one of the city’s open drains became clogged due to lack of cleaning. The water supply was intermittent, very superficial, and crossed the sewer line at many places. That the disease was water-borne was further substantiated by the observation that the maximum number of cases originated from two hostels on the campus of the local university, and that both of these hostels received their water supply from the same overhead storage tank. We were able to collect samples only from the patients coming to two of the university’s hospitals. The distribution pattern of various agents responsible for the disease is described in Table I. In the Hyderabad epidemic there were 29 (11%) patients positive for IgM anti-HBc indicating HBV infection. Whereas this is higher than normal and indicative of the high rate of transmission in these areas, the high incidence of HBV infection (19%) in Aligarh reflects a bias due to collection of samples from patients admitted to hospitals. However, the Jammu epidemic was solely of non-A, non-B type with only two cases of HAV. The Hyderabad epidemic was a mixed epidemic with non-A, non-B (64.5%)as the major component and HAV (23%) a s the associated component, affecting mostly children. In the Aligarh epidemic, no HAV cases were seen, possibly due to the predominance of young adults in the study population. The age-related incidence of HAV and non-A, non-B infections for the Hyderabad

and Aligarh epidemics is presented in Table 11. Almost all cases of HAV-related hepatitis occurred in children below 10 years of age, whereas non-A, non-B hepatitis affected both adults and children, with a prepoderance in the young adult population. The incidence of HAV and non-A, non-B together clearly demonstrates the difference between the two in a n endemic area. There was no significant difference between the transaminase values, disease symptoms, and outcome among the HAV and non-A, non-B groups. There were four pregnant women in the non-A, non-B group and no complications were observed in them. However, in Jammu epidemic 30 pregnant women were affected with two deaths. Fecal samples collected from the non-A, non-B group were screened by IEM. Virus-like particles (VLPs) were observed in 5 of the 18 stools examined. The acutephase sera from patients cross-agglutinated VLPs from each other.

Transmission to Rhesus Monkeys Three (M1878, M1879, and M1884) of the five animals that received fecal samples from the Jammu epidemic exhibited transaminase elevations between day 6 and day 50 post-inoculation (Fig. 1). Liver biopsies taken during enzyme elevation showed mild, nonspecific necroinflammatory changes. Electron microscopy revealed dilation of endoplasmic reticulum and degen-

267

PCR for HEV

ALT/AST 150 -

M-1878 B

-

0

10

20

30

40

60

70 0

60

160

160

M-1879 B,L

100

100

M-1880

60

60

,

C 10

20

30

40

60

60

70

C

0

10

20

30

40

60

60

70

B,L 150i

M-1884 M-1882

B

100 -

I

-

50

u

n

v -

0

10

20

30

40

60

60

70

0‘

0

10

20

30

40

60

DAYS POST INOCULATION Fig. 1. The ALT (.) and AST (+) profiles of rhesus monkeys infected with fecal suspension from the epidemic at Jammu city. Arrows indicate the days on which bile fluid (B) and liver biopsies (L) were taken.

60

70

Jameel et al.

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from the Hyderabad epidemic, four animals (M1111, M1214, M1308, and M1937) developed elevated transaminase levels. In the other four animals (M745, M1109, M1442, and M17571, bile collection and partial hepatectomy were carried out on day 17 post-inoculation which was before any detectable rise in transaminases. Though post-operative transaminase increase took place, i t was not possible to ascertain whether it was due to disease or operative procedures. The two animals (M1308 and M1937) which were inoculated with a n infectious stool 6 months post-convalescence did not develop any increase in transaminase levels up to 2 months post-inoculation.

A LT 1AST M-1886

0

m

20

30

40

60

60

PCR Amplification for HEV Detection

I

0

M-1883

m

10

SO

40

60

60

M-1877 0

0 0

m

20

so

40

60

60

DAYS POST I NOC U L AT I 0N Fig. 2. The ALT (.) and AST ( + ) profiles of the three rhesus monkeys inoculated with pooled (19,21,23rd days) acute phase fecal sample from primary inoculated monkey (M1884, Fig. 1).Arrows indicate the day on which bile fluid (B) was taken.

eration of mitochondria a s described by us previously [Panda et al., 19891. The presence of VLPs was confirmed in the feces of one animal (M1884) by IEM. A fecal suspension from this animal was further able to transmit infection to a fresh set of animals in which all three of the inoculated animals (M1877, M1883, and M1886) developed elevated transaminase levels (Fig. 2). Of the eight animals inoculated with fecal material

To evaluate the role of HEV in non-A, non-B-related disease, we utilized PCR amplification [Mullis et al., 19861 for the detection of HEV genomic sequences in clinical isolates. Stools from acutely infected individuals and bile fluid and liver biopsies from experimentally infected rhesus monkeys were tested for HEV. This was facilitated by the availability of a partial genomic sequence for a HEV isolate [Reyes et al., 1990; Genebank accession # M324001. Oligonucleotide primers and probes synthesized from the known sequence were used in this PCR detection scheme. Amplification with the two sets of primers in a nested PCR strategy gave rise to DNA fragments corresponding to 591 bp and 343 bp in positive samples [Figs. 3, 41. The HEVspecific nature of these amplified fragments was further substantiated by performing Southern hybridization with a n oligonucleotide probe positioned between the primer sequences in the HEV genomic sequence (Fig. 3B). Bile was taken from two infected monkeys (M1879 and M1884) belonging to the primary group and from one monkey (M1877) belonging to the secondary group inoculated with stools from the Jammu epidemic. Although all three were positive by the PCR method, amplification results are shown only for M1877 (Fig. 3, lane 6). Eight animals were inoculated with IEM-positive stools from the Hyderabad epidemic. Of these, four were positive (M745, M1109, M1757, and M1937) and the other four were negative (M1111, M1214, M1308, and M1442) for HEV sequences (Fig. 3). It is interesting to note t h a t three (M745, M1109, and M1757) of the four positive biles were isolated just prior to elevation in transaminases. Conversely, three of the four biles isolated during peak transaminase levels were found to be negative for HEV (M1111, M1214, and M1308). Stools from five of the Hyderabad epidemic patients serologically classified as non-A, non-B were tested by PCR amplification for HEV. Of these, two were positive (Fig. 4, lanes 5 and 6). One of these stools (lane 6) was also positive for VLPs by IEM and was present in the pool used to transmit infection to eight monkeys in the Hyderabad group. Partial liver resections were taken from two monkeys (M1879 and M1884) in the Jammu epidemic

PCR for HEV

269

Fig. 4. PCR amplification of HEV RNA from stools of acute hepatitis patients. Lanes 14%patients from Hyderabad epidemic. Rxn2+, reaction positive control for primers HEV-1I HEV-3; Rxnl+, reaction positive control for nested PCR. Arrowheads indicate positions of amplified products. Fig. 3. PCR amplification of HEV RNA from monkey bile analyzed on 2% agarose gels by (A) ethidium bromide staining, and (B) Southern hybridization with DET-3, a n internal oligonucleotide end-labeled with 32P.Lane 1: M1111; lane 2 M1214; lane 3: M1308; lane 4 M1937; lane 5 M1877; lane 6: M745; lane 7 M1109; lane 8: M1442; lane 9 M1757. Ext -, extraction negative control; +,reaction positive control; Neg, reaction negative control. Amplified products of 591 bp and 343 bp generated during nested PCR are indicated.

group. RNA isolated from these livers when PCR amplified for HEV was also found to be positive (data not shown). We have thus established the detection of HEV sequences by PCR amplification in stools, bile and liver tissue, and confirmed that the epidemics of Jammu and Hyderabad had a n HEV-related disease component.

DISCUSSION We describe the epidemiological profile of three epidemics of viral hepatitis in India. One of these was a mixed epidemic of HAV and HEV. Although both HAV and HEV are water-borne pathogens, the infections they cause have rarely been observed together in a single epidemic. This study provides comparative data from a single mixed epidemic. It was observed t h a t whereas the incidence of HAV was restricted to children under 10 years of age, HEV infections were widely distributed among all age groups, but with a preponder-

ance in the young adult population (Table 11). No difference was observed in the sex distribution, transaminase values, or outcome of disease between the HAV and HEV-related disease groups. The Hyderabad epidemic confounded our impression that HEV infections do not produce long-lasting immunity. Our reinoculation data support the presence of anti-HEV immunity in monkeys for at least 6 months post-convalescence. However, this challenge was carried out with the same stool specimen which was initially infectious in these animals. More data are needed on cross-challenge with infectious materials from the same as well as different epidemics to address conclusively the presence or absence of long-term immunity. Although recent observations involving serological cross-reactivity and limited sequence information do not support a high degree of variation, the neutralizing epitopes may contain hypervariable regions. This remains to be confirmed with the availability of complete genomic sequences of a number of isolates worldwide. By the use of a PCR amplification strategy we established t h a t two of the epidemics contain a n HEV-related disease component. Like others [Ray et al., 1991I we were able to detect HEV in stools from acutely infected humans. The presence of HEV in bile and liver

Jameel et al.

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tissue from experimentally infected animals was confirmed. The pattern of positivity in the bile of monkeys was interesting. It appears that HEV is maintained in bile prior to elevation of aminotransferases and falls once the enzyme levels increase. This is also the case for HEV in stools as assessed by IEM. It appears, then, that HEV after infecting liver cells is shed and accumulates in the bile before excretion. Since the presence of HEV is not uniform and is dependent on the time of collection, a PCR-based test for HEV RNA may not be a very reliable diagnostic test. In the absence of reliable serological markers, however, PCR has been helpful in establishing the etiology of disease and for identifying infectious clinical samples. The presence of HEV RNA in the bile of three out of four monkeys prior to transaminase elevations can also be considered a s a n indication of subclinical infection which may be prevalent in human population. Only detailed seroepidemiological studies will reveal the extent of such subclinical infections. We reported earlier t h a t rhesus monkeys can be used for HEV transmission [Panda et al., 19891. Here, we confirm those earlier observations by applying PCRbased detection to look specifically for HEV RNA in experimentally infected animals. We are currently using the bile and liver tissue of experimentally infected animals to clone and sequence the Indian strain of HEV.

ACKNOWLEDGMENTS This work received financial assistance under the Indo-US Vaccine Action Program at AIIMS and internal funding from ICGEB. One of us (S.J.)was a Biotechnology Career Fellow of the Rockefeller Foundation during the course of this work and acknowledges the material and scientific support rendered by Dr. A. Siddiqui as part of this fellowship program. REFERENCES Arankalle VA, Tiechurst J, Sreenivasan MA, Kapikian AZ, Popper H, Pavri KM, Purcell RH (1988): Aetiological association of a virus like particle with enterically transmitted non-A, non-B hepatitis. Lancet 1:550-554. Balayan MS, Andjaparidze AG, Savinskaya SS, Ketiladze ES, Braginsky EM, Savinow AP, Poleschuk VF (1983):Evidence for a virus in non-A, non-B hepatitis transmitted via the fecal oral route. Intervirology 120:23-31. Bradley DW, Andjaparidze A, Cook E H J r , McCaustland K, Balayan M, Stetler H, Velaque 0, Robertson B, Humphrey C, Kane M, Weisfuse I (1988): Aetiological agent of enterically transmitted non-A, non-B hepatitis. Journal of General Virology 69:731-738. Bradley DW, Krawczynski K, Cook EH Jr, McCaustland KA, Humphrey CD, Spelbring J E , Myint H, Maynard J E (1987): Enterically transmitted non-A, non-B hepatitis: serial passage of disease in cynomolgus macaques and tamarins and recovery of disease asso-

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Enteric non-A, non-B hepatitis: epidemics, animal transmission, and hepatitis E virus detection by the polymerase chain reaction.

We studied epidemics of viral hepatitis occurring at three different places in India. One was a combined epidemic due to hepatitis E virus (HEV) and h...
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