Genetic variation of Japanese encephalitis virus in Taiwan.

A 280-nucleotide sequence from the capsid-premembrane (C/preM) gene region of 44 Japanese encephalitis (JE) virus strains isolated in Taiwan from mosquitoes from 1983 to 1994, and 3 strains, (Ling [1965], Chang [1965], and HV1 [1958]) isolated from human brain were analyzed by direct sequencing of reverse transcription-polymerase chain reaction (RT-PCR) amplified products and compared with the corresponding sequences of reference strains. The overall sequence homology of the 47 isolates was > or = 93.3%. Taking 12% nucleotide divergence as a cut-off value, all isolates fell into genotype 3, which included strains from Japan, China, the Philippines, Sri Lanka, India, and Nepal. High nucleotide homology was observed among isolates from different regions of Taiwan and different time periods; on the other hand, high variation existed among isolates from the same region and time period. Phylogenetic analysis showed that the 47 Taiwan isolates fell into three clusters. Twenty-five isolates formed cluster 1, 18 isolates cluster 2, and four isolates cluster 3. Isolates in cluster 1 showed greater (< or = 2.9%) intragroup divergence compared to those in cluster 2 (< or = 1.1%) or cluster 3 (< or = 0.7%). The majority of isolates from northern (73.3%) and central (60%) Taiwan belonged to cluster 1, whereas most isolates (66.7%) from southern Taiwan belonged to cluster 2. Comparison with other Asian JE virus strains showed that isolates of cluster 1 were more specific to Taiwan than isolates of cluster 2 and cluster 3.

Complete nucleotide sequence of the genome of Japanese encephalitis virus ling strain: the presence of a 25-nucleotide deletion in the 3'-nontranslated region.

The complete sequence of the genome of the Japanese encephalitis virus (JEV) Ling strain isolated from the brain of a patient in Taiwan in 1965 was cloned by using the reverse transcription-polymerase chain reaction method. Seven overlapping cDNA clones that span the entire virus genome were isolated and sequenced to determine the complete nucleotide sequence, which is 10,951 nucleotides in length. As reported for three other JEV strains (Beijing-1, SA-14, and JaOArS982), the Ling strain contains 95 nucleotides in the 5' nontranslated region (NTR), followed by a single open reading frame of 10,296 nucleotides. However, the length of the 3' NTR of JEV Ling is 560 nucleotides, 25 nucleotides shorter than that of other JEV strains sequenced to date. Comparison of nucleotide and amino acid sequences among these four JEV strains showed that nucleotide (amino acid) sequence divergence in the translated region varied from 1.25% to 3.27% (0.49-1.63%). The nucleotide (amino acid) divergences between the Ling and Beijing-1 strains were 1.25% (0.87%) and between the SA-14 and JaOArS982 strains were 1.42% (0.49%). These values are lower than those found between the Ling and SA-14 [2.44% (1.02%)] or the Ling and JaOArS982 strains [2.84% (0.93%)], as well as those between Beijing-1 and SA-14 [3.14% (1.60%)] or Beijing-1 and JaOArS982 [3.27% (1.63%)] strains. Sequence comparisons of subregions of the genomes i.e., structural genes, nonstructural genes, or individual genes, showed divergence similar to that obtained by comparing the entire sequence. It is likely that the JEV sequence divergence between two human isolates or between two mosquito isolates is lower than that between a human isolate and a mosquito isolate.

Processing of Japanese encephalitis virus non-structural proteins: NS2B-NS3 complex and heterologous proteases.

Processing of Japanese encephalitis (JE) virus non-structural (NS) proteins expressed by recombinant vaccinia viruses was analysed to characterize the responsible viral protease. Analysis of the processing of polyprotein NS2A-2B-3' containing the N-terminal 322 amino acids of NS3 revealed products consistent with cleavages at the predicted intergenic junctions as well as at one or possibly two sites within NS2A. Cleavage at the alternate site(s) containing the cleavage sequence motif within NS2A could possibly explain the production of the NS1' protein in JE virus-infected cells. Polyprotein NS2A-d2B-3' containing a large deletion within NS2B was cleavage-defective, despite the presence of the proposed NS3 protease domain. Cleavage of NS2A-d2B-3' was restored if NS2B or NS2A-2B was supplied in trans, providing evidence that NS2B is strictly required for NS3 proteolytic activity. NS2B- or NS3-specific sera raised against the bacterial TrpE fusion protein co-precipitated NS2B and NS3 or NS3' from the lysate of JE virus or recombinant virus-infected cells. Thus both protease components are associated as a complex, presumably representing the active JE virus protease. JE virus and the analogous dengue 4 (DEN-4) protease components were employed to examine the activity of heterologous proteases. The defective cleavage of JE virus NS2A-d2B-3' was complemented by heterologous DEN-4 NS2B, whereas the defective cleavage of DEN-4 NS2A-d2B-3' was not corrected by heterologous JE virus NS2B. This suggests that the heterologous JE virus NS2B-DEN-4 NS3 protease is not active, despite the considerable sequence conservation of NS2B and NS3 between the two viruses. The cleavage activity was restored by replacement of the C-terminal 80 amino acids of JE virus NS2B with the corresponding DEN-4 sequence, consistent with the notion that the C-terminal region contains amino acid residues for interaction with DEN-4 NS3.

Partial nucleotide sequence of Japanese encephalitis virus ling strain genome and comparison of the encoded structural proteins and nonstructural protein NS1 among Japanese encephalitis virus strains.

Approximately 4000 nucleotides of the 5'-terminal portion of Japanese encephalitis virus (JEV) Ling strain genome were cloned and sequenced. This nucleotide sequence and its encoded C-PrM-E-NS1 polyprotein sequences were also compared with the corresponding sequences of other JE virus strains. Results demonstrated that the nucleotide sequence homology varied from 97.1 to 99.3% and the amino acid homology 98.6 to 98.9%. Based on homology, the Ling strain was closer to the Beijing-1 strain than to the SA14 and JaOArS982 strains. However, only on comparison of the E sequence, which neutralization, hemagglutination-inhibition and complement fixation antigenic determinants are located, between Ling and other JEV strains demonstrated that nucleotide sequence homology varied from 97.1% to 99.3% and amino acid homology from 98.6% to 99.2%. The Ling strain JEV is more closely related to the Beijing-1 strain than to the Nakayama NIH, SA14 and JaOArS982 strains in that order. Based on this analysis, the Taiwanese JEV strain appears to be more closely related to the Chinese strain than to the Japanese strain. Also, JEV strains isolated in humans are more closely related to each other than to JEV strains of mosquito isolates.

Processing of dengue type 4 and other flavivirus nonstructural proteins.

Dengue type 4 (DEN4) and other flaviviruses employ host and viral proteases for polyprotein processing. Most proteolytic cleavages in the DEN4 nonstructural protein (NS) region are mediated by the viral NS2B-NS3 protease. The N-terminal third of NS3, containing sequences homologous to serine protease active sites, is the protease domain. To determine required sequences in NS2B, deletions were introduced into DEN4 NS2B-30% NS3 cDNA and the expressed polyproteins assayed for self-cleavage. A 40 amino acid segment within NS2B was essential. Sequence analysis of NS2B predicts that this segment constitutes a hydrophilic domain surrounded by hydrophobic regions. Hydrophobicity profiles of other flavivirus NS2Bs show similar patterns. Cleavage of DEN4 NS1-NS2A requires an octapeptide sequence at the NS1 C terminus and downstream NS2A. Comparison of the analogous octapeptide sequences among flaviviruses indicates a consensus cleavage sequence of (P8)/Met/Leu-Val-Xaa-Ser-Xaa-Val-Ala(P1), where Xaa are non-conserved amino acids. The effects on cleavage of amino acid substitutions in this consensus sequence were analyzed. Most substitutions of the conserved residues interfered with cleavage, whereas substitutions of non-conserved residues had little or no effect. These findings indicate that the responsible enzyme recognizes well-defined sequences at the cleavage site.

Increased immunogenicity and protective efficacy in outbred and inbred mice by strategic carboxyl-terminal truncation of Japanese encephalitis virus envelope glycoprotein.

We constructed recombinant vaccinia viruses expressing the full-length envelope (E) glycoprotein of Japanese encephalitis virus (JEV) or a strategically truncated E glycoprotein, approximately 80% of the N-terminal sequence, and compared their antigenic structure and protective immunity in mice. The truncation site in the JEV E glycoprotein sequence corresponds to the position that had been shown to increase the immunogenicity of dengue type 4 or type 2 virus E glycoprotein. Analysis of the JEV E glycoprotein in recombinant virus-infected cells showed that C-terminally truncated E retains an antigenic structure similar to that of the full-length E glycoprotein. The full-length JEV E glycoprotein was detected predominantly intracellularly, while a small fraction (< 2%) was present on the cell surface. On the other hand, the truncated 80% E glycoprotein exhibited an alteration in the intracellular transport pathway resulting in increased accumulation (10-25%) on the cell surface and secretion (6-10%) into the medium. The C-terminally truncated E glycoprotein induced a greater antibody response and a higher level of protective immunity than did the full-length E glycoprotein in outbred CD-1 mice as well as in two strains of inbred mice that differ in their resistance to intraperitoneal (ip) JEV infection. In the case of outbred CD-1 and inbred C57/Bl mice, which possess a dominant autosomal genetic locus that controls resistance to a high dose of ip infection of JEV or the capacity to acquire resistance to intracerebral JEV infection, truncated E glycoprotein induced a higher titer of JEV neutralizing antibodies.