Detection of poliovirus, hepatitis A virus and rotavirus from sewage and water samples.

A modified adsorption-elution technique for concentration of enteric viruses from sewage and water samples was developed. The viruses in water were concentrated by negatively charged membrane filtration, eluted with 2.9% tryptose phosphate broth containing 6% glycine pH 9.0, and reconcentrated using centrifugation by a speedVac concentrator. The presence of poliovirus, hepatitis A virus (HAV) RNA, and rotavirus antigen was determined by cell culture isolation, nested polymerase chain reaction (nested PCR), and enzyme-linked immunosorbent assay (ELISA), respectively. A total of 100 sewage and water samples were collected from various sources in congested communities in Bangkok, concentrated and examined for those enteric viruses. Of 20 surface water samples from canals which located near sewage drains, 15% were positive for HAV RNA by nested PCR. Of 48 domestic sewage samples from man-holes of underground sewers, 8% were positive for rotavirus antigen by ELISA. Even though the samples were concentrated 256-2,000 fold, poliovirus was not found by isolation in cell culture.

Neurological manifestations of dengue infection.

BACKGROUND: Severe forms of dengue, the most important arboviral infection of man, are associated with haemorrhagic disease and a generalised vascular leak syndrome. The importance of dengue as a cause of neurological disease is uncertain. METHODS: During 1995, all patients with suspected CNS infections admitted to a referral hospital in southern Vietnam were investigated by culture, PCR, and antibody measurement in serum and CSF for dengue and other viruses. FINDINGS: Of 378 patients, 16 (4.2%) were infected with dengue viruses, compared with four (1.4%) of 286 hospital controls (odds ratio [95% CI] 3.1 [1.7-5.8]). Five additional dengue positive patients with CNS abnormalities were studied subsequently. No other cause of CNS infection was identified. Seven infections were primary dengue, 13 secondary, and one was not classified. Ten patients had dengue viruses isolated or detected by PCR, and three had dengue antibody in the CSF. 12 of the 21 had no characteristic features of dengue on admission. The most frequent neurological manifestations were reduced consciousness and convulsions. Nine patients had encephalitis. No patient died, but six had neurological sequelae at discharge. Phylogenetic analysis of the four DEN-2 strains isolated mapped them with a DEN-2 strain isolated from a patient with dengue haemorrhagic fever, and with other strains previously isolated in southern Vietnam. INTERPRETATION: In dengue endemic areas patients with encephalitis and encephalopathy should be investigated for this infection, whether or not they have other features of the disease.

Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity.

Viremia titers in serial plasma samples from 168 children with acute dengue virus infection who were enrolled in a prospective study at 2 hospitals in Thailand were examined to determine the role of virus load in the pathogenesis of dengue hemorrhagic fever (DHF). The infecting virus serotype was identified for 165 patients (DEN-1, 46 patients; DEN-2, 47 patients; DEN-3, 47 patients, DEN-4, 25 patients). Patients with DEN-2 infections experienced more severe disease than those infected with other serotypes. Eighty-one percent of patients experienced a secondary dengue virus infection that was associated with more severe disease. Viremia titers were determined for 41 DEN-1 and 46 DEN-2 patients. Higher peak titers were associated with increased disease severity for the 31 patients with a peak titer identified (mean titer of 107.6 for those with dengue fever vs. 108.5 for patients with DHF, P=.01). Increased dengue disease severity correlated with high viremia titer, secondary dengue virus infection, and DEN-2 virus type.

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.

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.

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.

Acute jaundice in Vientiane, Lao People's Democratic Republic.

Analysis of serum samples from patients with acute jaundice by means of enzyme-linked immunosorbent assay and polymerase chain reaction testing provided the first profile of this condition in Vientiane, Lao PDR, in 1995 and 1996. In a case-control, hospital-based study, evidence of acute infections due to hepatitis A and B viruses was found in 14% and 10% of cases, respectively. Hepatitis E virus, however, did not appear to contribute to clinically recognized acute jaundice. Similarly, antibody to hepatitis C virus was recognized in almost equal proportions of cases (8%) and controls (6%), thus representing probable background infections. The detection of hepatitis G virus marks the first report of this virus in Lao PDR. The large proportion (21%) of new leptospiral infections in cases without acute hepatitis A or B was notable. This finding suggests significant regional underreporting of leptospirosis as a cause of acute jaundice. The limited laboratory diagnostic capabilities for confirming a differential diagnosis of leptospirosis contribute to the lack of attention paid to this important health problem.

High prevalence of hepatitis G viremia among kidney transplant patients in Thailand.

Patients receiving kidney transplants (KT) are at high risk for blood borne viral infections. To determine the prevalence of a recently discovered hepatitis G virus (HGV) in this patient group, reverse transcription-polymerase chain reaction (RT-PCR) employing primers derived from the NS5 region of the viral genome was utilized. HGV RNA was detected in 40 of 94 KT patients (43%), as compared to 3 of 69 healthy subjects (4.3%). Cocirculation of HGV and hepatitis C virus (HCV) RNA was detected in 12 patients (13%). Comparison of patients with and without HGV revealed that the former had received hemodialysis before transplantation for a significantly longer duration than the latter (28 vs. 17 months, respectively; P < 0.05). The amount of blood transfused and mean levels of liver enzymes, including alkaline phosphatase, alanine transaminase, and aspartate transaminase, were the same in both groups. Sequence analysis of 275-base pair DNA clones obtained from 2 patients revealed approximately 92% sequence homology to the published HGV and GB virus C sequences. These results suggested that HGV infection among Thai KT patients was high and the role of HGV in causing liver disease remains to be determined.

Rapid diagnosis of dengue viremia by reverse transcriptase-polymerase chain reaction using 3'-noncoding region universal primers.

A reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed as a rapid diagnostic test of dengue viremia. To detect dengue viruses in serum or plasma specimens, a pair of universal primers was designed for use in the RT-PCR. Using these primers, the 3'-noncoding region of dengue virus types 1, 2, 3, and 4 could be amplified, but not those of other flaviviruses, such as West Nile virus, Japanese encephalitis virus, and yellow fever virus, or the alphavirus Sindbis virus. The sensitivity of the RT-PCR assay was similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture. Combining a silica method for RNA isolation and RT-PCR dengue virus could be detected in a 6-hr assay. In a preliminary study using this method, we detected dengue virus in 38 of 39 plasma specimens from which dengue virus had been isolated by mosquito inoculation. We then applied this method for detecting dengue viremia to 117 plasma samples from 62 children with acute febrile illnesses in a dengue-endemic area. We detected dengue viremia in 19 of 20 samples obtained on the day of presentation, which had been confirmed as acute dengue infection by mosquito inoculation and antibody responses. The overall sensitivity of this method was 91.4% (32 of 35; 95% confidence interval [CI] = 82.2-100%). The results from testing plasma samples from febrile nondengue patients showed a specificity of 95.4% (42 of 44; 95% CI = 89.3-100%).

Hemorrhagic fever in Cambodia is caused by dengue viruses: evidence for transmission of all four serotypes.

Hemorrhagic fever (HF) has been widespread in Cambodia and thought to be due to dengue virus although laboratory confirmation has been lacking. Between 1980 and 1995, 49,420 cases of HF and 3,032 deaths were reported. Cases increased during this period; large epidemics of HF occurred every two to three years. In 1995 there were 10,208 cases of HF with 424 deaths. Over a two day period in August 1995, 40 consecutive cases were investigated at the National Pediatric Hospital in Phnom Penh, Cambodia. All 40 cases were confirmed as dengue by virus identification and/or serology. Mean age was 6.5 years. Of 39 patients with complete medical records, the diagnoses were: dengue fever (n = 3), dengue hemorrhagic fever (DHF) grade 2 (n = 21), DHF grade 3 (n = 10), and DHF grade 4 (n = 5). The serologic response was secondary in 95%. Dengue virus was identified in 13 of 40 cases. All four dengue serotypes were identified. The high frequency of secondary infections, the low mean age of admission, and identification of all four dengue serotypes support the national statistics to show that DHF is highly endemic in Cambodia.

Herpes simplex virus type-2, cytomegalovirus and Epstein-Barr virus infection in acute non A to E hepatitis Thai patients.

A significant number of acute non A to E hepatitis cases are reported in Thailand every year, and the etiologies of these cases are unknown. Members of the herpesviridae family have been reported to cause either a self limited or fatal hepatitis in a small proportion of patients in other parts of the world. To determine whether herpesviruses may play a role in acute non A to E hepatitis, sera from 32 acute hepatitis patients without markers for acute hepatitis A to E virus infection were examined for IgM to herpesvirus type 2 (HSV-2), cytomegalovirus (CMV), and Epstein-Barr virus (EBV) using commercially available assays. IgM to HSV-2 was detected in four sera, IgM to CMV was detected in one serum, and IgM to EBV was detected in one serum. All of the acute non A to E hepatitis patients recovered and none had underlying conditions associated with impaired immunity. These results suggest that herpesviruses should be considered in the differential diagnosis for Thai patients with hepatitis.

Etiology of acute non-A, B, C hepatitis in Thai patients: preliminary study.

To better characterize the etiology of acute non-A, B, C hepatitis, 24 sera from 50 acute hepatitis without acute markers for hepatitis A, B, and C were examined for acute markers for the hepatitis E virus (HEV), cytomegalovirus (CMV), herpes simplex virus type 2 (HSV-2), and Epstein-Barr virus. Immunoglobulin M (IgM) specific for HEV, HSV-2, and CMV was detected using ELISA and total Ig specific to EBV was determined by standard indirect immunofluorescence. IgM to CMV was not observed in sera from any of the patients; whereas, IgM to HEV was detected in sera from 2 patients and IgM to HSV-2 was detected in 5 of 24 acute hepatitis patients. In addition, high titer of antibody was found in 2 of the patients. This results indicate that HSV-2 and HEV circulate in Thailand and are responsible for a small proportion of non-A, B, C hepatitis in Thailand.

The replicating intermediates of herpes simplex virus type 1 DNA are relatively short.

Herpes simplex virus type 1 (HSV-1) replication is thought to occur via a rolling circle type of mechanism, generating large DNA concatemers from which unit length genomes are subsequently cleaved. In this report, we have employed field inversion gel electrophoresis (FIGE), Southern blot hybridization, and endonuclease digestion, to identify and characterize these DNAs. Two species of HSV-1 DNA: (1) genome-length and (2) DNA that remained at the electrophoresis origin (referred to as well-associated DNA) were detected. To ascertain that the latter was large in size and not virion DNA trapped at the origin with high molecular weight cellular DNA, the infected cell DNA was digested with a restriction enzyme that does not cut the viral DNA. In order to do this HSV-1 strain 1702, lacking any XbaI sites in its genome, was utilized. After digestion of samples with XbaI, and FIGE, cellular DNA was seen to migrate into the gel; however, the viral DNA remained in the sample wells. Pulse labeling experiments showed that this large DNA was processed to 150 kb genome lengths. Endonuclease digestion of the well-associated DNA revealed that it contained a greater ratio of joint to terminal fragments than virion DNA-a characteristic of long concatemers. Quantitation of the terminal fragments revealed mainly L termini. Surprisingly, the ratio of joint to terminal fragments was 2.5 suggesting that the lengths of concatemers were short (in the order of 1-2 genome lengths) and that the well association was due to conformation rather than concatemeric length. Because one of these genome lengths is present as the replication intermediate, the growing tail must be less than genome length. Thus genome lengths must be processed from the replication intermediate soon after they are completed.

A thymidine kinase-negative HSV-1 strain establishes a persistent infection in SCID mice that features uncontrolled peripheral replication but only marginal nervous system involvement.

A detailed knowledge of the pathogenesis of infections caused by thymidine-kinase (TK)-deficient herpes simplex virus type 1 (HSV-1) strains is important because such mutants can arise during treatment of HSV infections with acyclovir--especially in immunocompromised patients--and also because TK-negative mutants may become useful for the therapy of intracranial tumors. In this work, we studied the pathogenesis of a genetically engineered TK-negative HSV-1 strain dlsptk, in SCID mice (mice with severe combined immunodeficiency) after corneal infection. We found that dlsptk established a persistent infection that kills SCID mice within 80.2 +/- 21.3 days. The cause of death seemed to be related to uncontrolled viral replication in the superficial and deep facial tissues of the animals. Viremia probably did not occur, as judged by the inability to detect infectious virus and viral gene expression in various internal organs. However, the virus did reach the nervous system, most probably by axonal transport from the primary site of the infection. Virus-specific DNA reached low but detectable levels in the trigeminal ganglia and the brainstems by 7 days p.i. and remained at low levels for up to 50 days p.i. as determined by spot blot analysis. By in situ hybridization and immunostaining we determined that, in some of the neurons of the trigeminal ganglia infected by the virus, viral latency was established. However, our results suggested that in other infected neurons viral replication occurred and virus spread to surrounding nonneuronal cells and to the central nervous system. This work provides a new model in which the pathogenesis of infections caused by TK-deficient HSV strains in immunocompromised hosts can be effectively studied and which may also help to identify the potential side effects of the therapy of intracranial tumors with TK-negative HSV strains.

Herpes simplex virus type 1 mutant strain in1814 establishes a unique, slowly progressing infection in SCID mice.

Ocular infection of immunocompetent (BALB/c) mice with wild-type herpes simplex virus type 1 (HSV-1) 17+ may lead to acute fatal encephalitis; however, in surviving animals, a latent (nonproductive) infection of the nervous system is established. In contrast, 17+ infection invariably kills mice with severe combined immunodeficiency (SCID mice) within 2 weeks. Ocular infection of immunocompetent mice with a mutant HSV-1 strain, in1814, which does not produce a functional alpha-transinducing protein, results in no detectable viral replication in the nervous system during the time corresponding to the acute phase of infection, no mortality, and the establishment of latency. In SCID mice, however, the in1814 virus establishes a unique, slowly progressing infection. In studying the courses of in1814 infection in SCID and BALB/c mice, we found that although intact B- and/or T-lymphocytic functions were required for the control of viral replication in the nervous system, some of the infected neurons of SCID mice seemed to be able to restrict in1814 replication and harbor the virus in a latent state.

Analysis of immediate-early transcripts of equine cytomegalovirus.

Equine cytomegalovirus (ECMV) contains a linear, double-stranded DNA genome composed of a 146-kbp unique region flanked by a pair of 18-kbp direct repeat (DR) sequences at the termini. Cycloheximide, actinomycin D, and phosphonoacetic acid were applied to infected cell cultures to divide viral transcription into immediate-early (IE), early, and late phases. Eight IE transcripts were identified and mapped to two regions (I and II) of the viral genome. Two of these IE RNAs (13.0 and 5.5 kb in size) were transcribed from region I, which is located within the DR regions; these IE genes are diploid. The other IE transcripts (17.0, 9.0, 7.2, 6.8, 4.5, and 4.2 kb) originated from region II. IE region II is adjacent to region I and spans both unique and DR sequences at the left terminus of the genome. Region II IE transcripts are spliced and transcribed in the opposite direction from region I IE transcripts. IE transcripts from region I were present throughout the replication cycle, whereas those from region II were more abundant during the IE stage than at the early and late stages of infection. These studies demonstrate that ECMV differs from other herpesviruses in the organization and unusually large transcription units of its IE genes.

Equine herpesvirus 1 sequence near the left terminus codes for two open reading frames.

We have previously reported the sequence of the equine herpesvirus one genomic termini that are homologous to the genomic termini of other herpesviruses. In this paper, we present the nucleotide sequence adjacent to the left terminus sequence (map units 0.0087 to 0.0237). This sequence codes for two open reading frames (ORF) which are homologous to ORF2 and ORF3 of the varicella-zoster virus genome and are located at colinear positions. The L region sequence presented here also contains a segment that is involved in the generation of the genome of EHV-1 DI particles through recombination with sequences mapping within the internal portion of the inverted repeat sequences of the short region.

Identification of the site of recombination in the generation of the genome of DI particles of equine herpesvirus type 1.

Defective interfering particles (DIPs) are generated by serial, undiluted propagation of equine herpesvirus type 1 (EHV-1). DIP-rich preparations of EHV-1 mediate oncogenic transformation and persistent infection in permissive hamster embryo fibroblasts. The defective genomes consist of reiterations of sequences from the left terminus (0.00 to 0.04 map units) of the long (L) region covalently linked to sequences from the inverted repeats (0.78 to 0.79, 0.83 to 0.87, 0.91 to 0.95, and 0.99 to 1.00 map units) of the short (S) region of the standard genome. We have identified and determined the nucleotide sequences of these segments of the standard genome as well as the component of the defective DNA that contains the site at which these two viral sequences recombined. Comparison of these sequences revealed that there is an 8-nucleotide sequence that is common to both the left terminus sequences and the inverted repeat sequences. These 8-nucleotide identical sequences are located at 3.25 kbp from the left terminus and at 9 kbp downstream of the L-S junction. The recombination between the left terminus and the inverted repeat sequences occurred at the site of homology and resulted in the generation of a novel open reading frame. The last 97 amino acids of an open reading frame of 469 amino acids encoded by sequences within the inverted repeats were replaced by a sequence of 68 amino acids encoded by a 204-bp sequence mapping at 0.023 map units. It will be of interest to determine whether this altered open reading frame, generated by recombination of sequences separated by more than 110,000 bp in the standard genome, plays a role in the varied outcomes of infection mediated by EHV-1 DIPs.

Viral transcripts in cells infected with defective interfering particles of equine herpesvirus type 1.

Equine herpesvirus type 1 (EHV-1) preparations enriched in defective interfering particles (DIPs) have previously been demonstrated to mediate the coestablishment of persistent infection and oncogenic transformation in primary hamster embryo fibroblasts. In this study, it was demonstrated that infection of a rabbit kidney (RK) cell line with EHV-1 DIP-enriched preparations also results in the establishment of persistent infection. Viral transcription was characterized in RK cells infected with DIP-enriched stocks and compared to viral transcription in RK cells infected with standard (STD) EHV-1. During the first 8 hr of infection with the DIP-enriched EHV-1 preparation, viral DNA sequences which are conserved in the DIP genome were predominantly expressed. Thus, these transcripts originate from DNA sequences that contain the components of the defective genome which originates from DNA sequences mapping at 0.00-0.04 of the Long region terminus and within two portions of the Short region inverted repeats (IR), 0.78-0.79 and 0.83-0.865 of the internal IRs and 0.99-1.00 and 0.915-0.95 of the terminal IRs. The overwhelming majority of viral transcripts that were synthesized in the DIP-enriched infections appeared to correspond to transcripts expressed in STD infection as assessed by Northern hybridization analysis but the synthesis of transcripts originating from sequences not conserved in the defective genome was significantly delayed. However, some high molecular weight RNA species that were synthesized in STD infections were not detected in DIP-enriched infections. Studies utilizing metabolic inhibitors indicated that viral transcription in DIP-enriched infections, like that of STD cytocidal infection, is regulated in an immediate early, early and late manner.