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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.

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.

Viral antigen production was examined in the culture fluid of Aedes albopictus clone C6/36 cell line incubated at 28 degrees C and 37 degrees C after infection with four strains of dengue type 4 (DEN-4) virus which were isolated from patients with different clinical severities. During the observation period from day 1 to day 18, the number of infected cells at each day for all four strains did not show any significant difference (P >0.05). Antigen production as determined by the hemagglutination (HA) test and sandwich enzyme linked immunosorbent assay (ELISA) was higher at 28 degrees C than at 37 degrees C for all four DEN-4 virus strains examined. The amount of viral antigen produced was highest for CT93-74 strain (dengue hemorrhagic fever syndrome (DHF) grade II) and was significantly different in comparison to other strains (P <0.001). This strain was followed by CT93-158 and CT93-129 strains (both of DHF grade I), and CT93-77 strain (dengue fever (DF)). The viral antigen production was apparently proportional to the clinical severity of the patient from whom the virus was isolated. These results show that CT93-74 strain could be used to produce DEN-4 virus antigen of sufficiently high titer in the C6/36 cell culture instead of classical extraction of this antigen from suckling mouse brains.

A 37-year-old Japanese male was admitted to Nagasaki University Hospital with abrupt onset of biphasic fever, general malaise and myalgia 9 days after coming back to Japan from Manila. He developed a rubella like erythematous rash 3 days after admission and purpuric eruption one week after admission. A biopsied specimen from the purpura revealed lymphocytic vasculitis with T cell dominance and without immunoglobulin or complement deposition around the blood vessels. RT-PCR analysis on peripheral blood mononuclear cells using dengue virus specific primers confirmed the diagnosis of type 3 dengue fever. PCR analysis using virus specific primers is a rapid and valuable method for making a correct diagnosis of dengue fever.

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.

In order to localize denaturation-resistant neutralizing epitope(s) in the C-terminal 180 amino acids of Japanese encephalitis (JE) virus E-protein, four recombinant clones encoding different or overlapping nucleotide sequences were constructed by PCR from a recombinant plasmid pS22. The amplified fragments were cloned into PCR 1000 vector, and then transferred into Escherichia coli expression vector pRIT2T. The inserted genes were expressed as fusion proteins with protein-A and examined for their antigenicity and immunogenicity by Western blotting and mouse immunization, respectively. Among the four recombinant fusion proteins, the highest neutralizing antibody titre was obtained by the one expressed by the recombinant clone pRIT2T-B3, which carried the coding sequence of amino acid number 373-399 of JE virus E protein. The results indicated that this short region of 27 amino acids sequence near the C-terminal of JE virus E protein possesses neutralizing epitope(s). These data should assist in the design of an efficient subunit vaccine against JE virus infection in future.

A chimeric human and simian immunodeficiency virus carrying the tat, rev, vpu, env, and nef genes of human immunodeficiency virus type 1 was generated. The chimeric virus, NM-3n, grew competently in peripheral blood mononuclear cells from cynomolgus monkeys like the parental SIVmac. Two cynomolgus monkeys and one rhesus monkey inoculated with NM-3n raised antibodies to SIVmac Gag and HIV-1 Env. The antibodies raised in the cynomolgus monkeys persisted for at least 1.7 years. The antibodies contained virus neutralizing activity not only to the original chimeric virus but also to the parental HIV-1. Infectious viruses were isolated from one of the cynomolgus monkeys 37 and 63 weeks after inoculation and from the rhesus monkey continuously from 6 weeks after infection onward. The recovered virus maintained its chimeric structure but included several clones with mutations in the env V3 region. When the recovered virus was inoculated to another rhesus monkey, no difference in the frequency of virus recovery was seen from the originally infected monkeys. These carrier monkeys have so far shown no sign of the disease.

We have recently reported the isolation of a human immunodeficiency virus type 1 (HIV-1), KB-1gp32 carrying a shorter size (32 kDa) of transmembrane glycoprotein (TMP) from TALL-1 cells persistently infected with KB-1gp41 virus strain (Shimizu et al., 1990a). Endoglycosidase treatments showed that the different size of the TMP between the two strains was due to a truncation of 9 kDa of polypeptide in the KB-1gp32 TMP coding region. Sequence analysis revealed the substitution of a CAG codon to a TAG stop codon just downstream of the putative membrane-spanning domain of the TMP of KB-1gp32. This resulted in a truncation of some 133 amino acids of the cytoplasmic domain of TMP. The data indicate that a premature stop codon in KB-1gp32 has been introduced during adaption of the parental virus to TALL-1 cells. We have constructed two chimeric clones between the env region of a clone pKB-1, derived from KB-1gp32, and an infectious molecular clone pNL-432. We have also constructed a site-directed mutant of pNL-432 carrying a premature stop codon at the same position as the env stop codon of pKB-1. Among the three clones carrying a premature stop codon in env, only one chimeric clone was infectious to TALL-1 but not MT-2 cells. This clone contained the entire tat, rev, vpu, and env genes of pKB-1. The pNL-432 mutant was not infectious. The results suggest that some sequences of pKB-1 might compensate for the truncation of the TMP during replication in TALL-1 cells.

This study was carried out to define the role of cattle as an amplifier of Rift Valley fever. Three areas of different density of cattle population were surveyed. Cattle do not seem to play a significant role as an amplifier of the virus in human beings.