Hepatitis E virus in Nepal: similarities with the Burmese and Indian variants.

Hepatitis E has been the predominant type of acute hepatitis in Nepal both in adults and children, in sporadic and epidemic forms. We examined six hepatitis E virus (HEV) isolates obtained during an 8-year period, from 1987 to 1995, in the Kathmandu valley of Nepal. Analysis of portions of the putative helicase, polymerase and capsid genes demonstrated close genetic relatedness among themselves (> 96.4% identity) and with the Burmese (> 95.5%) and Indian (> 95.3%) isolates, and less so with the African (> 94.4%) and the Chinese (> 91%) isolates within the Asian genotype. Phylogenetic analysis placed the Nepali isolates in the Burma-India evolutionary branch and showed that the oldest isolate, TK78/87 was more similar to the Burmese isolates whereas the most recent isolates were closer to the Indian ones. Assuming no frameshifts, the Nepali isolates showed high amino acid conservation, but also unique changes when compared to other HEV isolates. Amino acid residue 614 of the capsid protein was identified as a possible marker to distinguish the Burma-Nepal-India from the China-Central Asian Republics subgenotype, and the Mexico genotype.

Isolation of Japanese encephalitis virus from clinical specimens using a continuous mosquito cell line.

During the 1983 Japanese encephalitis (JE) epidemic in northern Thailand, we systematically attempted to isolate JE virus (JEV) from clinical specimens collected from 49 consecutive JE patients at 1 provincial hospital. Fresh acute plasma and cerebrospinal fluid (CSF) samples and postmortem brain samples were immediately inoculated onto cultured monolayers of Aedes pseudoscutellaris (LSTM-AP-61) cells which had been shipped to the epidemic site. None of 49 plasma samples yielded virus. None of 30 fresh CSF samples from nonfatal cases yielded virus, but 5 of 15 (33%) CSF samples from fatal cases did. Inoculation of fresh brain specimens obtained at autopsy yielded virus in every case attempted (7 of 7), whereas postmortem needle biopsy specimens of brain yielded virus in only 1 of 4 cases. Isolates were most frequently successful using thalamic tissue (6 of 7 cases), but isolates were also commonly obtained from frontal cortex (4/7), occipital cortex (4/7), cerebellum (4/7), medulla (4/7) and pons (2/7).

Sensitivity and specificity of a universal primer set for the rapid diagnosis of dengue virus infections by polymerase chain reaction and nucleic acid hybridization.

A set of sense and anti-sense oligomeric DNA primers, degenerate in the third "wobble" base position of codons so as to match all known dengue virus sequences, was evaluated as universal primers in a polymerase chain reaction (PCR) assay for the rapid diagnosis of dengue virus infections. Virus-specific complementary DNA (cDNA) was prepared by reverse transcription (RT) of total RNA extracted from serum. Amplified cDNA was identified by nucleic acid hybridization with four serotype-specific, oligomeric DNA probes. Using sera from patients admitted with dengue fever, RT/PCR followed by nucleic acid hybridization using radiolabeled probes was 68% sensitive (50/74; 95% confidence interval [CI] = 57-78%) and 100% specific. Chemiluminescent detection of hybridized products was 62% sensitive (26/42; 95% CI = 46-75%). Using specimens from which a virus isolate had been obtained, RT/PCR followed by nucleic acid hybridization with radiolabeled probes was 80% sensitive (40/50; 95% CI = 69-91%) and 100% specific. The results suggest that RT/PCR using degenerate primers is a sensitive and specific method for the detection of dengue viruses in clinical specimens.

An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate.

The diagnostic sensitivity and specificity of detection of anti-dengue IgM by antibody capture enzyme-linked immunosorbent assay (ELISA) was investigated in dengue infections in a variety of clinical settings. Sera from uninfected controls were uniformly negative. Serial specimens from experimental and natural infections showed that viremia and fever terminated as anti-dengue IgM became detectable. Anti-dengue IgM appeared in most cases by the 3rd afebrile day of illness and declined to undetectable levels after 30-60 days. Assay sensitivity was 78% in admission sera (924/1,183; 95% CI = 75-81%) and 97% in paired sera (1,030/1,062; 95% CI = 96-98%) thus exceeding or matching the performance of the hemagglutination-inhibition assay. Measurement of the anti-dengue IgM to anti-Japanese encephalitis IgM ratio correctly identified all sera from 112 patients with strictly defined Japanese encephalitis and 98% (307/312; 95% CI = 96-99%) of sera from patients whose dengue infections were confirmed by virus isolation. Dengue infections could be classified as primary or secondary by determining the ratio of units of dengue IgM to IgG antibody. We propose that measurement of dengue and Japanese encephalitis IgM and IgG antibodies upon admission and discharge from hospital care should replace the hemagglutination inhibition assay as the standard dengue serologic technique in regions where these 2 viruses co-circulate.

A cluster of acute hepatitis E infection in United Nations Bangladeshi peacekeepers in Haiti.

In the fall of 1995, within a month of deployment to Haiti for peacekeeping duty, four Bangladeshi soldiers developed acute icteric hepatitis in rapid succession. Hepatitis E virus (HEV) was found to be the etiology by demonstrating HEV genomic sequences in serum samples by the polymerase chain reaction (PCR) and serologically by the detection of elevated IgM titers to HEV. No case had serologic evidence of acute hepatitis A or C infection. The soldiers had probably acquired their infection while living in a cantonment area outside Dhaka, Bangladesh for one month prior to deployment. Cloning and sequencing of amplified PCR products demonstrated a single strain suggestive of a common source of infection. Furthermore, high genomic identity with Asian strains of HEV and dissimilarity with the Mexican strain was demonstrated, verifying that the strain had indeed been imported. Human waste management from the Bangladesh camp in Haiti was strictly controlled and no secondary cases were observed. A convenience sample of 105 (12%) soldiers from the Bangladesh battalion (850 men) revealed anicteric or asymptomatic HEV infection in seven (7%) of 105. This report contains the first demonstration of acute hepatitis E in natives of Bangladesh and demonstrates the power of the PCR in the rapid diagnosis and epidemiologic analysis of HEV infection. More importantly, this cluster demonstrates the importation of an important infectious disease by multinational peacekeepers to a potentially susceptible host country.

Short report: hepatitis E infection in the Brazilian Amazon.

This is the first report of serologic evidence of hepatitis E infection in Brazil. During a community-based survey of healthy individuals, six of 97 gold miners in the Amazon region of Mato Grosso had antibody to the virus. The mining camps have poor sanitation with a great potential for fecal-oral transmission of disease. Since levels of hepatitis E antibodies may quickly wane, studies to directly measure the incidence of seroconversion are planned to determine the intensity of transmission in this area.

Fatal outcome in Japanese encephalitis.

Forty-nine consecutive patients with laboratory-confirmed acute Japanese encephalitis were studied to identify risk factors present at hospital admission which were associated with a fatal outcome. Sixteen patients (33%) died. The following constellation of findings correlated with a fatal outcome: infectious virus in cerebrospinal fluid (CSF), low levels of Japanese encephalitis virus-specific IgG and IgM in both CSF and serum, and a severely depressed sensorium. Age, sex, days ill before admission, distance from home to the hospital, past medical history, CSF protein content, and CSF leukocyte count were not significant risk factors. Among patients hospitalized for acute Japanese encephalitis, a vigorous virus-specific immunoglobulin response, both systemically and locally within the central nervous system, is a good marker for survival, and may be an inherently important factor in recovery from illness.

Production of lethal infection that resembles fatal human disease by intranasal inoculation of macaques with Japanese encephalitis virus.

Twelve rhesus macaques (Macaca mulatta) challenged intranasally with a wild-type Japanese encephalitis virus (JEV) developed clinical signs 11-14 days later. Tissues from the cerebral cortex, cerebellum, brainstem, thalamus, meninges, and all levels of the spinal cord were stained for JEV antigen with hyperimmune mouse ascitic fluid and streptavidin-alkaline phosphatase; immunofluorescent staining was also done on frozen sections. Viral antigen was found in all cell layers of the cerebellum, the gray matter of the thalamus and brainstem, and the ventral horn of all levels of the spinal cord. Staining was limited to neurons and their processes. Histopathologic changes were limited to the nervous system and characterized by nonsuppurative meningoencephalitis. These results were comparable with those of previous studies done with human autopsy tissues. Intranasal inoculation of rhesus monkeys with JEV was effective in producing clinical disease comparable with natural disease in humans and may serve as a model to evaluate protective efficacy of candidate JEV vaccines.

Preparation of an attenuated dengue 4 (341750 Carib) virus vaccine. II. Safety and immunogenicity in humans.

To determine safety and immunogenicity, a single 0.5 ml dose of a monovalent live-attenuated dengue (DEN) 4 (341750 Carib) vaccine was given sc to 3 groups of flavivirus nonimmune volunteers in increasing concentrations. Two recipients received 10(3) plaque forming units (PFU)/dose (1:100 dilution of stock vaccine). One remained asymptomatic, but became viremic between days 12 and 15, experienced a mild elevation of temperature (37.4 degrees C), and developed DEN-4 specific antibody. Neither recipient of the 10(4) PFU became infected. Eight volunteers then received undiluted vaccine (10(5) PFU). Viremia and antibody (neutralizing, hemagglutination inhibition, and IgM) developed in 5 of the 8 (63%). These 5 volunteers also developed a scarcely noticeable macular, blanching rash and minimal temperature elevations (37.3, 38.1, 37, 37.9, and 37.9 degrees C). Clinically insignificant decreases in total white blood cell, lymphocyte, and polymorphonuclear cell counts and an elevation in mononuclear cell counts occurred in association with viremia. This vaccine is safe, reasonably immunogenic, and suitable for further evaluation.

An intranasal challenge model for testing Japanese encephalitis vaccines in rhesus monkeys.

Placebo-controlled field efficacy trials of new Japanese encephalitis (JE) vaccines may be impractical. Therefore, an animal model to evaluate efficacy of candidate JE vaccines is sought. Previous work has shown that exposure of monkeys to JE virus (JEV) via the intranasal route results in encephalitis. Here we report the further development of this model and the availability of titered virus stocks to assess the protective efficacy of JE vaccines. To determine the effective dose of our JE challenge virus, dilutions of a stock JEV (KE-93 isolate) were inoculated into four groups of three rhesus monkeys. A dose-dependent response was observed and the 50% effective dose (ED50) was determined to be 6.0 x 10(7) plaque forming units (pfu). Among animals that developed encephalitis, clinical signs occurred 9-14 days postinoculation. Infection with JEV was confirmed by detection of JEV in nervous tissues and IgM to JEV in the cerebrospinal fluid. Viremia with JEV was also detected intermittently throughout infection. Validation of the model was performed using a known effective JE vaccine and saline control. One ED90 of virus (2.0 x 10(9) pfu) was used as a challenge dose. Four of four animals that received saline control developed encephalitis while one of four monkeys administered the JE vaccine did so. This study demonstrates that the virus strain, route of inoculation, dose, and the outcome measure (encephalitis) are suitable for assessment of protective efficacy of candidate JE vaccines.

Safety, immunogenicity, and protective efficacy of NYVAC-JEV and ALVAC-JEV recombinant Japanese encephalitis vaccines in rhesus monkeys.

Two poxvirus-vectored vaccines for Japanese encephalitis (JE), NYVAC-JEV and ALVAC-JEV, were evaluated in rhesus monkeys for safety, immunogenicity, and protective efficacy. The vaccines were given to four monkeys each on study days 0 and 28 along with saline placebo on day 7. For controls, the licensed BIKEN JE vaccine and a saline placebo were given to other groups of four monkeys on days 0, 7, and 28. No systemic effects were observed. All injection site reactions were mild. All vaccines elicited appreciable JE-specific neutralizing antibody responses. However, a more rapid increase and higher peak level of antibody were seen in the BIKEN group as compared with the NYVAC-JEV and ALVAC-JEV groups. The peak neutralizing antibody level in the NYVAC-JEV group was higher than that of the ALVAC-JEV group. Antibody persisted in all four BIKEN recipients through 273 days of follow-up, whereas, the antibody level decreased to the threshold of detection in two NYVAC-JEV and all four ALVAC-JEV recipients by day 120. On day 273, all monkeys were given a booster dose. A rapid increase in neutralizing antibody was seen in all vaccine recipients by seven days. Two months after the booster dose, all monkeys were challenged intranasally with one 90% effective dose of JE virus. Four recipients of saline, three of ALVAC-JEV, one of NYVAC-JEV, and one of BIKEN experienced encephalitis. This study suggests that the NYVAC-JEV and ALVAC-JEV vaccines are safe and immunogenic in monkeys and that the NYVAC-JEV and BIKEN vaccines are effective in protecting monkeys from encephalitis.

Japanese encephalitis vaccine (inactivated, BIKEN) in U.S. soldiers: immunogenicity and safety of vaccine administered in two dosing regimens.

The safety and immunogenicity of Japanese encephalitis (JE) vaccine (Nakayama strain, monovalent / BIKEN) was studied in 538 U.S. soldiers in 1990. Three doses of vaccine from three consecutively manufactured lots were given on days 0, 7, and either 14 or 30. Serum for antibody determination was drawn at months 0, 2, and 6. Japanese encephalitis plaque reduction neutralization tests were performed by three laboratories on each specimen. Five hundred twenty-eight (98%) participants completed the immunization series. All recipients without antibody before immunization developed neutralizing antibody against JE virus. There were no differences in geometric mean titer among the three test lots at months 2 and 6. Soldiers who received the third dose on day 30 had higher titers at both time points. Antibody to yellow fever had no significant effect on immune response to vaccine. Conclusions drawn from analysis of serologic data from the three labs were nearly identical. Symptoms were generally limited to mild local effects and were reduced in frequency with each subsequent does in the series (21% to 11%; P < 0.0001). Generalized symptoms were rare (e.g., fever = 5%) with no reported cases of anaphylaxis.

Phase 1 studies of Walter Reed Army Institute of Research candidate attenuated dengue vaccines: selection of safe and immunogenic monovalent vaccines.

We describe the results of initial safety testing of 10 live-attenuated dengue virus (DENV) vaccine candidates modified by serial passage in primary dog kidney (PDK) cells at the Walter Reed Army Institute of Research. The Phase 1 studies, conducted in 65 volunteers, were designed to select an attenuated vaccine candidate for each DENV serotype. No recipient of the DENV candidate vaccines sustained serious injury or required treatment. Three vaccine candidates were associated with transient idiosyncratic reactions in one volunteer each, resulting in their withdrawal from further clinical development. Increasing PDK cell passage of DENV-1, DENV-2, and DENV-3 candidate vaccines increased attenuation for volunteers, yet also decreased infectivity and immunogenicity. This effect was less clear for DENV-4 candidate vaccines following 15 and 20 PDK cell passages. Only one passage level each of the tested DENV-2, -3, and -4 vaccine candidates was judged acceptably reactogenic and suitable for expanded clinical study. Subsequent studies with more recipients will further establish safety and immunogenicity of the four selected vaccine candidates: DENV-1 45AZ5 PDK 20, DENV-2 S16803 PDK 50, DENV-3 CH53489 PDK 20, and DENV-4 341750 PDK 20.

Genotyping of hepatitis E virus in clinical specimens by restriction endonuclease analysis.

The genomic variability of hepatitis E virus (HEV) was examined by restriction endonuclease analysis (REA) of four genomic cDNA copies comprising a 499 bp segment of the putative polymerase gene, a 264 bp segment of the helicase gene, and two, 680 bp and 448 bp, segments of the capsid gene. Analysis of the deduced restriction sites of all 27 HEV sequences currently available in the GenBank, and digestion of reverse-transcribed and nested PCR amplified segments obtained from six Nepali isolates were used to devise and test a REA genotyping assay. The assay allowed easy discrimination between the Mexico and Asian genotypes, and the classification of the Asian genotypes into three, or perhaps four subgenotypes. In addition, endonucleases identifiers of individual isolate or clusters of isolates were found. This assay permits rapid identification of a large number of HEV isolates directly from clinical specimens for studies on the molecular epidemiology and evolution of HEV.

Virus isolations from mosquitoes collected during the 1982 Japanese encephalitis epidemic in northern Thailand.

From 16 June to 15 August, 1982 CDC light traps were used to collect mosquitoes in the province of Kamphaengphet, N. Thailand. 353,042 mosquitoes comprising 59 species were collected and identified, and 345,173 were placed in pools for attempted virus isolation by inoculation of C6/36 Aedes albopictus mosquito cell cultures. Viruses were isolated from 63 mosquito pools. These comprised 56 flaviviruses, identified as 35 isolates of Japanese encephalitis (JE) virus strains, 18 strains of Tembusu (TEM) virus and three untyped flaviviruses (FLA); three alphaviruses, identified as the first isolates of Getah (GET) virus to have been made in Thailand; and four viruses which are still unidentified. Most virus isolates were from Culex tritaeniorhynchus mosquitoes collected in carbon dioxide baited light traps. JE virus was isolated only over a ten-day period and the last isolate was obtained one week before the peak of admission of human encephalitis cases at Kamphaengphet Provincial Hospital. Rapid screening of isolates grown on Ae. pseudoscutellaris (LSTM-AP-61) mosquito cells by indirect immunofluorescence using flavivirus group-specific and JE-specific monoclonal antibodies showed a high degree of correlation with plaque reduction neutralization tests. An antigen capture enzyme immunoassay (EIA) test successfully identified about 50% of the JE virus positive pools, but the method saved considerable processing time.

Japanese encephalitis virus in Bangkok: factors influencing vector infections in three suburban communities.

An unexpected outbreak of Japanese encephalitis (JE) in Bangkok in 1985 led us to investigate the vector ecology of urban JE from January 1986 to June 1987 at three suburban sites that displayed a wide range of factors imputed to influence JE transmission. Culex tritaeniorhynchus Giles and Cx. gelidus Theobald, suspected vectors, comprised 71-96% of all mosquitoes collected by CO2-baited CDC traps at the three sites. Mean of mosquito abundance per two trap-nights per month ranged from 28 to 5,728 mosquitoes at the sites of lowest and highest abundance, respectively. Cx. tritaeniorhynchus yielded more JE isolates (n = 16) than Cx. gelidus (n = 7), but the minimum infection rates of the two species (number of JE isolates per 1,000 mosquitoes tested; MIR, 0.17 and 0.47, respectively) were comparable and covaried with vector abundance. Moreover, the proportion of sentinel pigs that had JE antibodies generally increased proportionately with vector abundance at the sites. Vector abundance was high in monsoon (May-October), moderate in transition (March-April and November-December), and low in dry (January-February) seasons. Mosquitoes collected in monsoon seasons yielded 96% of the JE isolates, whereas 4 and 0% of the isolates were obtained from transition and dry season collections, respectively. More pigs seroconverted in monsoon and transition seasons than in dry seasons. Indices of JE transmission activity (vector abundance, pig seroconversions, and MIRs) increased proportionately with rainfall. Despite higher indices at the site of greatest vector abundance than elsewhere, the risk of human infection appeared greatest at the site with moderate vector abundance because of its greatest human population density.

Anti-rabies virus IgM in serum and cerebrospinal fluid from rabid dogs.

An anti-rabies IgM antibody capture radio immunoassay was used to test serum and cerebrospinal fluid from 37 dogs held in quarantine for suspicion of rabies. Rabies was confirmed in dogs that died by mouse inoculation and subsequent examination of mouse brains by fluorescent antibody technique to detect rabies antigen. The mean counts per minute (CPM) of iodinated anti-rabies gamma globulin coupled IgM rabies antibody in CSF and serum from rabid dogs were significantly higher than in CSF and serum from non-rabid dogs. Mean CPM from rabid dogs was greater in CSF than in sera, in contrast with non-rabid dogs, from which mean cpm was higher in sera than CSF, suggesting that antibody may have been synthesized in the CSF. To evaluate this test further, a dog was infected by rabies virus, and serial serum and CSF specimens were collected until the time of death. IgM anti-rabies antibody developed in the CSF and serum 29 days following infection, and rose just before the dog died of rabies on day 34. The rabies MAC RIA is potentially useful as a diagnostic method in quarantined dogs with rabies-like illness. Perhaps more importantly, it may be applied to better understand the immunopathogenicity of rabies.

A longitudinal study of Japanese encephalitis in suburban Bangkok, Thailand.

A one-year study of Japanese encephalitis (JE) in a small focus of transmission was conducted in suburban Bangkok in 1985. Monthly data were collected on weather, vector density, sentinel pig and chick JE antibody seroconversions, and epidemiology as related to human JE cases. The primary vector species were found to be Culex gelidus and Culex tritaeniorhynchus; from which one isolate each was obtained in March and June, respectively. Pig JE antibody seroconversion peaked in April (the hottest month), with secondary peaks following in July and December. Chick seroconversions were found only in June and July. Human cases (7) in the primary focus occurred from May-July, and started 2 months following the finding of the first JEV isolate in mosquitoes and 1 month following mass JEV seroconversion in pigs. Overall, the attack rate in the focus (0.83/10(5] was greater than 4 times that of the rest of Bangkok (0.19/10(5]. Attack rates were highest in 0-9 and 10-19 year-old groups, respectively. Indications are that JEV is transmitted to humans in Bangkok at least 10 out of 12 months per year, but that cases are concentrated in the May to July period.

Blood value changes in flavivirus-inoculated Macaca fascicularis monkeys.

Blood values were analysed in eighteen cynomolgus monkeys on pre-and post-neurovirulence testing of dengue-2 and yellow fever vaccine viruses, dengue-2 parental and Japanese encephalitis viruses. Certain changes between blood chemistry, hematology and serology were observed and briefly discussed.