Use of nucleotide sequencing of the genomic cDNA fragments of the capsid/premembrane junction region for molecular epidemiology of dengue type 2 viruses.

The recent emergence of dengue hemorrhagic fever/dengue shock syndrome (DHF/ DSS) in India has been a source of concern. In the present study a quantitative comparison of 406 nucleotide long sequence from the capsid-premembrane junction region (C-PrM) of 9 dengue virus type 2 (DEN-2) isolates from Delhi with 10 DEN-2 isolates from diverse geographic areas provided sufficient information for estimating genetic relationships. The data indicated that the 1996 epidemic of DHF in Delhi was caused by genotype IV strains of DEN-2. This genotype, perhaps, displaced genotype V strains of DEN-2, which was circulating genotype in 1967. The period during which this displacement had occurred is not clear from the present study. Nonetheless, similar experience in four countries in Latin America and in Sri Lanka suggest that the introduction of new genotypes of DEN-2 displacing the circulating genotype may be associated with the appearance of DHF/DSS. More work is required to elucidate this hypothesis. Transitions at nucleotide positions 406 and 431 resulted in amino acid substitutions near (aa position 104, methionine --> valine) and at the hinge region (aa position 112, valine --> alanine) of C-PrM, respectively in all/most genotypes of group III and IV DEN-2 viruses analysed. Most of these virus strains have been isolated from DHF/DSS outbreaks. Significance of this observation is discussed. The data presented in this study suggest the utility of C-PrM sequence analysis for molecular epidemiology of dengue viruses.

Highly conserved nucleotide sequence and its deduced amino acids of the 5'-noncoding region and the capsid protein of a Bangkok isolate dengue-3 virus.

The dengue-3 virus genome encodes an uninterrupted open reading frame (ORF) flanked by 5' and 3' non-coding regions. The order of proteins encoded in dengue-3 virus ORF, as with other flaviviruses, is: Cap 5'-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'. The nucleotide sequence of the 5'-noncoding region and the capsid protein of dengue-3virus (a Bangkok isolate: CH53489 isolated by USAMC-AFRIMS in 1973) has been analyzed in both forward and reverse directions. The PCR-based cycle sequencing technique by the enzymatic method of Sanger et al (1977) using a sequencing primer 5'-end labeled with gamma32P-ATP is the method of our choice for sequencing analysis. One cDNA template was prepared by RT-PCR technique starting from the 5'-end nucleotide 1-465 of the dengue-3 genome. In our cycle sequencing experiments, the substitution of 7-deaza-dG was used for dG in DNA eliminated much of the secondary structures that produced gel artifacts. The final sequence result of this cDNA template was established from its sequence data determined on both strands in opposite directions. Alignment between the newly established nucleotide sequence as well as its deduced amino acid sequence of the Bangkok dengue-3 virus and the published sequence data of the dengue-3 prototype (H87) was manipulated by the PC-DOS-GIBIO-DNASIS TM 06-00 (Hitachi Software). According to the deduced amino acid sequence of the Bangkok dengue-3 virus, its C protein was found to be highly positively charged because of large numbers of lysine and arginine. The homology of the nucleotide sequence between the two dengue-3 virus revealed 97%. The deduced amino acid sequences from the nucleotides 95-465 of the two viruses showed the same indicating highly conserved capsid proteins. Multiple alignment of the nucleotide sequences as well as the deduced amino acid sequences among the Bangkok dengue-3 virus and other dengue 3 viruses also confirmed the highly conserved 5'-noncoding regions and the capsid proteins.

Comparative studies on immunoreactivity of truncated recombinant proteins of foot and mouth disease virus (FMDV) produced in E.coli and insect cells.

For effective FMD control programme, India needs large quantities of cheaper diagnostics in addition to vaccine. Diagnostic reagents produced through conventional methods may not be able to meet such requirements. Alternatively, rDNA technology using suitable heterologous systems that permit production of recombinant antigens to the most native form may be exploited. Studies conducted in our laboratory have led us to select carboxy terminal part of VP1 for expression and evaluation. The protein, which was purified from E.coli under denaturing conditions, was renatured and its reactivity was compared with the protein expressed in insect cells through recombinant baculovirus. The expressed protein in the insect cell whole lysate reacted more efficiently with antibodies raised against whole virus than the purified and renatured protein produced in E.coli. But for its lower reactivity, protein produced from E.coli was found to be suitable in type detection. In addition, the size of the protein is small (16 kD) and production and purification of it from E.coli may be cost effective. Hence, it may be exploited for FMDV typing.

Antigenic structure of foot and mouth disease virus type A22 (Indian isolates).

Variations in foot and mouth disease virus are due to amino acid substitutions in the VP1, which is a major immunogen. Analysis of this hypervariable region is essential to know the antigenic structure of the serotype and is necessary to select a suitable vaccine strain. FMDV type A22 is one of the four prevailing virus types for which the vaccine is used regularly. To understand the antigenic structure of this type, carboxy- terminal region of VP1 from two field isolates and vaccine virus were sequenced and analysed. The results indicate that, Indian A22 has distinct antigenic structure.

Molecular biological characterization of enterovirus variant isolated from patients with aseptic meningitis

In Korea, there was a big outbreak of aseptic meningitis in 1993. Six clinical isolates of enterovirus were obtained from patients with aseptic meningitis and were identified as echovirus type 9 by serotyping with a pool of neutralizing antisera. For molecular characterization of the isolates, the nucleotide sequences of 5'-noncoding region (NCR), VP4, VP2, VP1, 2A and 2C regions of the isolates were compared with the corresponding regions of echovirus type 9 Hill and Barty strains. Unlike Hill strain, Barty strain contained a C-terminal extension to the capsid protein VP1 with an RGD (argnine-glycine-aspartic acid) motif. To determine whether similar structural features were present in our isolates, their nucleotide sequences including the VP1 region were analyzed. All isolates exhibited the VP1 extension with the RGD motif. We concluded the Korean isolates in the year of 1993 as the echovirus type 9 Barty strain although the isolates showed 15-20% nucleotide sequence differences in the several genomic regions.

Early events in virus-plant interactions

1. Plant viruses can only enter their host through a wounded plant cell. Once in the cytoplasm, the virion must be disassembled, and for certain viruses with a "+" RNA genome, cotranslational disassembly of virus particles has been described. 2. Subsequent to viral protein synthesis which requires the host translational machinery, the "+" RNA genome is replicated in the cytoplasm. Viral genome amplification requires at least one viral-coded non-structural protein in conjunction with one or more host factors. 3. Early events in virus infection can be studied in systems that hinder these events. This is the case of natural hosts that are resitant to viruses: mutant viruses which overcome such resistance have been described. It is also the case of genetically engineered plants that are protected from virus infection. Both types of systems should help in determining the mode of interaction involved, and possibly also the host factor(s) involved in the various steps of virus infection

Molecular cloning of coat protein gene of cucumber mosaic virus, CMV-U strain.

RNA isolation from purified cucumber mosaic virus, CMV-U strain and cDNA synthesis was carried out. The coat protein gene (RNA4) region was amplified selectively by polymerase chain reaction (PCR) with CMV RNA4-specific primers. Double-stranded cDNA was cloned in PRT103 vector at Xho1/Kpn1 site and about 1kb insert obtained. The insert was partly sequenced which showed 50% sequence homology with CMV-Q, C and WL strains.

Expression of the VP3-VP1 sequence of foot-and-mouth disease virus in Escherichia coli

1. cDNA recombinants containing the VP3 and VP1 sequences of foot-and-mouth disease virus were isolated and the VP3-VP1 sequence was reconstructed. 2. The reconstructed VP3-VP1 sequence was subcloned into expression vector pEX31b and a fusion protein of about 62,000 Da was expressed. 3. When injected into mice, the fusion protein was able to elicit the production of antibodies that recognized viral VP1 and VP3. 4. Antibodies present in sera from mice immunized with VP3-VP1 protein did not neutralize the foot-and-mouth disease virus in vitro

Nucleotide sequences of the VP1 capsid proteins of wild poliovirus types 1 and 3 from epidemic areas of Brazil

The nucleotide sequences encoding the capsid protein VP1 were determined for the wild polioviruses of serotypes 1 and 3 endemic to the northeastern region of Brazil. Compared with the corresponding Sabin vaccine strain sequences, the wild isolates differed at 20%(type 1) and 22%(type 3) of their nucleotide positions, and in 7%(type 1) and 11%(type 3) of their amino acid residues. The highest degree of amino acid heterogeneity occurred within the amino-terminal residues of the VP1 proteins. Intratypic amino acid differences also occurred in VP1 surface residues that form parts of antigenic sites for neutralizing antibodies