IgM-capture ELISA of serum samples collected from Filipino dengue patients.

Viral antigens for 4 dengue serotypes were produced in C6/36 Aedes albopictus cells. These were used as assay antigens for IgM-capture ELISA to detect IgM antibodies in sera of dengue patients from 3 hospitals in Metro Manila, Philippines. A total of 378 serum samples came from National Children's Hospital (NCH), San Lazaro Hospital (SLH), and St Luke's Medical Center (SLMC), from January to November 1995. Three hundred and four (304) out of 378 serum samples, or 80.42% showed positive IgM ELISA titer against at least one of the 4 assay antigens. Dengue type 4 (D4) antigen detected antibodies in 61.90% (234/378) of these serum samples, whereas type 1 (D1), type 3 (D3), and type 2 (D2) had detection rates of 60.05% (227/378), 50.79% (192/378) and 49.47% (187/378) respectively. Although the results show that both D1 and D4 are the most effective antigens in identifying dengue infections for this batch of samples, the use of a cocktail of antigens is still recommended. The results of this study are the basis for the IgM-capture ELISA protocol presently applied for the laboratory confirmation of dengue cases in the Philippines.

Molecular evolution, distribution and genetic relationship among the dengue 2 viruses isolated from different clinical severity.

Twenty-two strains of dengue 2 virus, isolated in China, Latin America, New Guinea and Thailand were subjected to phylogenetic analysis. The UPGMA analysis was carried out on each gene region of dengue virus and demonstrated that outcome from most of the gene regions showed similar results except those from NS4B and YUTR with very short nucleotide length. Among ten regions examined, the results from E gene documented the geographical differences of the virus strains most clearly and all the American strains (Mara 4, IQT1797 and S1) were distantly related to the Asian isolates. As for the 16 Thai strains isolated in 1993, they were clustered into three groups and a strain from a DSS patient formed a distinct branch compared to the other two groups. This finding from phylogenetic analysis is consistent with earlier conclusion and support the severity related subtyping of dengue 2 virus based on amino acid changes.

Infection of dengue 2 virus strains isolated from patients exhibiting different disease severities to human peripheral blood leukocytes and production of cytokines in the infected culture supernatant.

Three strains of type 2 dengue virus (DV-2), which had been isolated from patients exhibiting different disease severity, were inoculated to primary culture of human peripheral blood leukocytes from 3 healthy donors. The percentage of dengue antigen positive cells was highest for the strain isolated from a case of dengue shock syndrome, followed by the strain isolated from a case of dengue hemorrhagic fever, and lowest for the strain isolated from a mild case of dengue fever (DF). Generally, similar trend was observed for the amount of some cytokines released into infected culture supernatant, such as interleukin 6, and tumor necrosis factor-alpha. However, such a trend was not observed for interleukin 1 beta.

The first report on human cases serologically diagnosed as Japanese encephalitis in Indonesia.

Although Japanese encephalitis (JE) virus was isolated from mosquitos in 1974, human JE cases have never been reported in Indonesia in spite of the prevalence of anti-JE antibodies among human and pig populations as well as abundant JE vector mosquitos. In this report, we describe serological diagnosis of JE cases in Bali. Indonesia. using IgM-capture ELISA both on serum and cerebrospinal fluid (CSF) of the patients. In the first series of our investigation (Series 1), we examined serum specimens from 12 patients with clinical diagnosis of viral encephalitis, meningitis or dengue hemorrhagic fever (DHF), and found 2 possible JE cases. In the next series (Series 2), we examined both serum and CSF from encephalitis patients and gave laboratory diagnosis of JE. One of them was suspected to have concomitant or recent infection with dengue virus, probably type 3. These results strongly indicated that JE has been prevalent in Bali, Indonesia.

Identification of a flavivirus isolated from mosquitos in Chiang Mai Thailand.

A virus isolate, ThCAr105/92, from a pool of mosquitos, Culex tritaeniorhynchus, collected in Chiang Mai, Thailand in 1992, appeared to be a member of the genus Flavivirus of the family Flaviviridae, based on the reverse transcription polymerase chain reaction (RT-PCR) using flavivirus cross-reacting primer pairs, electron microscopic examination, and serological tests. However, RT-PCR using Japanese encephalitis (JE) virus-specific primers showed that the isolate was different from JE virus. Sucrose density gradient sedimentation of the virus replicated in C6/36 cells indicated that the virus is relatively unstable in the infected culture fluids at 37 degrees C. Antibody prepared against this virus and a virus seed for the isolate were tested by cross neutralization against a panel of flaviviruses and the results showed that the new isolate was a distinct subtype of Tembusu virus.

Dengue virus infection among children with undifferentiated fever in Karachi.

Sera were collected from a total of 122 children, comprising 117 cases with undifferentiated fever and 5 cases with dengue hemorrhagic fever (DHF), during June to September 1994 in Karachi, Pakistan. Sera were tested by the IgM-capture ELISA using dengue type 1 (D1), dengue type 2 (D2), West Nile (WN), and Japanese encephalitis (JE) viral antigens. Among 92 single sera from undifferentiated fever cases, IgM antibodies were detected in 5 cases by D1, 8 cases by D2, and 5 cases by WN antigens, respectively. Corresponding number of positives among 25 paired sera from undifferentiated fever cases were 3 by D1, 6 by D2, and 1 by WN antigen. Four out of 5 DHF cases possessed anti-D1 as well as anti-D2 IgM antibodies. Only a single DHF case was positive for anti-WN IgM antibodies. Anti-JE IgM antibodies were not detected in any of the tested serum specimens. Clinical manifestations of undifferentiated fever patients were generally non specific, but the percentage of children with anemia, hepatomegaly and splenomegaly was higher in patients possessing anti-dengue IgM antibodies than those without. Among the groups with anti-dengue IgM antibodies, those possessing only anti-D2 but not anti-D1 IgM antibodies showed higher percentages with cough, edema, and splenomegaly. The results indicated that up to 26% of the undifferentiated fever cases were caused by dengue virus infection in Karachi, Pakistan.

The effect of nonionic detergent on dengue and Japanese encephalitis virus antigens in antigen detection ELISA and IgM-capture ELISA.

In order to simplify dengue and Japanese encephalitis (JE) IgM-ELISA, we have been trying to produce antigens as infected C6/36 cell culture fluid. In this study, we examined the effect of nonionic detergents, which were used to inactivate viral infectivity, on dengue and JE antigen titers as well as the results in an IgM-capture ELISA. In the antigen detection ELISA, antigen titers were not significantly reduced after treatment with nonionic detergents (Nonidet P-40 or Triton X-100, at 0.01 to 0.1% final concentration). In contrast, in the IgM-capture ELISA, the color development was significantly reduced when the antigens were pretreated with nonionic detergents. The results suggest that certain epitopes which react with anti-viral IgM antibodies, but not IgG antibodies, have been destroyed by treatment with nonionic detergents. The results indicate that we cannot use nonionic detergents to inactivate the infectivity of assay antigens.

Comparison of sequences of E/NS1 gene junction of dengue type 3 virus following culture subpassage in C6/36 cells to study the possible occurrence of mutations.

The aim of this study was to determine whether mutations could occur in the dengue virus genome following three subpassages of the virus in a mosquito cell line. This was done because sources of virus isolates used for sequencing studies are usually maintained in cell lines rather than in patients' sera. Therefore it must be assured that no mutation occurred during the passaging. For this purpose, sequencing was carried out using the polymerase chain reaction (PCR) products of the envelope/non-structural protein 1 junction region (280 nucleotides) of dengue type 3 virus. Sequence data were compared between the virus from a patient's serum against the virus subpassaged three times in the C6/36 cell line. We found that the sequence data of the virus from serum was identical to the virus that was subpassaged three times in C6/36 cell line.

Cross-sectional serosurvey for Japanese encephalitis specific antibody from animal sera in Malaysia 1993.

Serum specimens were collected from 6 species of animals living in 9 states of Malaysia including Sabah, North Borneo in 1993. Antibodies against Japanese encephalitis (JE) virus in these sera were detected by means of hemagglutination-inhibition (HI) and neutralization (NT) tests. By HI test, 702 of 2,152 (32.6%) sera showed positive results. Higher positive rates were obtained by the NT test, in which 1,787 of 1,927 (92.7%) sera had antibodies against JE virus. All serum specimens with positive HI were confirmed as positive by the NT. Swine sera showed especially higher rates of antibody positive and higher antibody titers compared with other animals. These results suggest that JE infections are widely distributed among many animals of Malaysia, and pig is the most susceptible amplifier host for JE virus.

The use of polymerase chain reaction (PCR) as a diagnostic tool for dengue virus.

This study describes the use of polymerase chain reaction as a diagnostic tool for detecting and typing of dengue virus. PCR was compared against virus isolation. First RT-PCR was done using dengue consensus primers after which positive samples were subjected to RT-PCR using type-specific primers. This study shows that the local strains of the dengue virus could be detected using the chosen primers. Furthermore, RT-PCR was found to be more sensitive than virus isolation in identifying the dengue positive samples.

Genotype determination of three dengue type 2 virus strains from Myanmar by sequencing E/NSI gene junction.

Genotype of three dengue-2 virus strains from Myanmar was determined as genotype II by sequencing 240 nucleotide long fragment across the E/NS1 gene junction by the primer extension dideoxy chain termination method, applying direct sequencing of the PCR product. These strains were isolated from a dengue shock syndrome (DSS) patient and two patients with dengue hemorrhagic fever (DHF) grade 1, in Yangon (Rangoon), Myanmar (Burma), in 1987. Sequence homology of all three strains were highest (96%) to New Guinea C strain (genotype II), lesser homology (93%) to Jamaican 1409 strain (genotype III), and the least homology (91%) to PR 159/S1 strain (genotype I). Two DHF strains revealed only 2 nucleotide and 3 nucleotide differences compared with DSS strain, all at the 3rd position of the codons which resulted in silent mutations.

Storage stability of dengue IgM and IgG antibodies in whole blood and serum dried on filter paper strips detected by ELISA.

Blood specimens from 133 patients clinically diagnosed as dengue virus infection by physicians in Nakhon Phanom Hospital, Thailand, were examined to detect anti-dengue IgM and IgG antibodies by antibody capture ELISA. The blood specimens were divided into 3 types of storage; (1) frozen serum aliquots, (2) whole blood dried on filter paper strips, and (3) sera dried on filter paper strips. These specimens were stored for the periods of 1, 3, 4, and 5 months, at -20 degrees C in the case of frozen serum aliquots, or at room temperature in the case of specimens dried on filter paper strips, before examined in paralleled by the ELISA. Anti-dengue IgG antibodies were stable for at least 5 months of storage as dried whole blood or serum on filter paper strips. So were the anti-dengue IgM antibodies in the dried whole blood from secondary dengue cases. Anti-dengue IgM antibodies from primary dengue cases declined slowly in whole blood and more rapidly in serum, both dried on filter paper strips. In the serum dried on filter paper strips, even anti-dengue IgM antibodies from secondary cases decreased significantly on storage. We suggest that diagnosis on dengue infections by IgM-capture ELISA should be performed within 1 month after the test specimens are collected as whole blood, not as serum, when the filter paper method is used for sample collection.

Comparative nucleotide and deduced amino acid sequence of the envelope glycoprotein gene among three dengue virus type 2 strains isolated from patients with different disease severities in Maha Sarakham, northeast Thailand.

The nucleotide (nt) sequence of the envelope glycoprotein (E) gene of dengue virus type 2 was determined by the primer-extension dideoxy chain-termination method for 3 dengue virus type 2 (D2) strains which had been isolated from patients with dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS), in Maha Sarakham, Northeast Thailand, in 1986-1987. Their nt sequences were essentially the same except for a single silent nt replacement in each DHF and DSS strain compared with DF strain. Therefore, these 3 strains possessed identical deduced amino acid (AA) sequences in their E protein. The result indicated that the primary structure of the E protein of D2 virus is not related to the clinical severity of the infected patients. Eleven nt replacements which resulted in 4 amino acid replacements were found to be unique to these 3 Northeast Thai strains. Sequence similarity showed that the 3 Northeast Thai strains were closest to the DSS isolate (H) followed by the DHF isolate (D) identified in Bangkok in 1980.

Partial sequence analysis of type 1 dengue virus coding for the nonstructural hydrophilic protein NS-3

A complementary DNA copy of DEN-1 RNA was synthesized using reverse transcriptase and a random primer. The double-stranded DNA copy was cloned at the Sma I site of the pUC13 vector and was used to transform Escherichia coli JM83. Among the transformants selected for characterization by nucleotide sequence analysis, we report here one that codes for a region of nonstructural hydrophilic protein, NS-3. Computer analysis of this sequence (967 bp) showed abouth 87% conservation at the amino acid level and 79% at the nucleotide level when compared with dengue serotypess 2,3 and 4 and Japanese encephalitis virus. This suggest an important function which is common to all four serotypes. Comparision of the cloned region with sequences of the above strains also suggest conservation of hydrophobic regions

Development of the second generation Japanese encephalitis (JE) vaccine.

Recent analysis of Japanese encephalitis (JE) virus genome RNA, especially its nucleotide sequence, revealed the localization of virion envelope glycoprotein (E) gene on the virus genome. Since the E protein is the major protective antigen related with the virus neutralization, attempts have been made to produce the second generation JE vaccine by expressing the E protein using recombinant DNA technologies. These studies will eventually lead to control JE by mass-vaccination in several Asian countries where JE is one of the major public health problems.

Flavivirus infections in Chiang Mai area, Thailand, in 1982.

Infection by JE virus still constitutes major cause of encephalitis in Chiang Mai Area, although some cases of possible dengue encephalopathy were observed. In spite of many apparent encephalitis cases, infection of vector mosquitoes by JE virus was not demonstrated. Virus isolation from hospitalized patients showed that the principal type of dengue virus circulating in Chiang Mai in 1982 was type 1 virus. Seroepidemiological survey on healthy humans indicated that the northern part of Chiang Mai Province in the region of the Maekong Valley has not yet been invaded so much by dengue viruses, compared with the Chiang Mai Valley, where dengue infection apparently became more prevalent than 12 years ago. The survey also indicated that the spread of JE virus in the study area was not uniform. Survey on vertebrates showed that anti-JE antibodies were highly prevalent among swine, horses, mules, sheep, and dogs. On the other hand, antibody prevalence was low in monkeys, ducks, and sparrows, and was negative among chickens and lizards. IgM-ELISA appeared to help differential diagnosis on JE from dengue even when the HI test did not give positive results.