Rapid detection and characterization of Chikungunya virus by RT-PCR in febrile patients from Kerala, India.

There has been a resurgence and prevalence of fever with symptoms of Chikungunya (CHIK) and increased death toll in Kerala, the southern-most state of India. The objective of this study was to develop a rapid detection method to determine the presence of CHIK- virus in the serum samples collected from febrile patients in Kerala, India. Serum specimens were analyzed for CHIK viral RNA by RT-PCR using primers specific for nsP1 and E1 genes. Five out of twenty clinical samples were positive for CHIK virus. The partial sequences of the E1 and nsP1 genes of the strain, IndKL01 were highly similar to the Reunion strains and the recently isolated Indian strains. A novel substitution, A148V, was detected in the E1 gene of the isolate, IndKL02. The detection procedure used in this study was simple, sensitive and rapid (less than 4 hr). This result suggests that CHIK viruses similar to the Reunion strains, which had resulted in high morbidity and mortality rates, may have caused the recent Chikungunya outbreak in India. The effect of the variant, E1-A148V, in the virulence and the rate of transmission of the virus deserves further investigation.

Cloning, characterization, and expression of a new cry2Ab gene from Bacillus thuringiensis strain 14-1.

Bacillus thuringiensis is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of B. thuringiensis are promising candidates for management of resistance development in insects owing to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. The cry2Ab gene was found to lack a functional promoter and, hence, is cryptic in nature. The cry2Ab7 gene was cloned from a new indigenous B. thuringiensis strain, 14-1. Nucleotide sequencing of the cry2Ab gene cloned from B. thuringiensis strain 14-1 revealed an open reading frame of 1902 bp. The deduced amino acid sequence of Cry2Ab of B. thuringiensis strain 14-1 showed a variation in three amino acid residues in comparison to the holotype sequence, Cry2Ab1. Expression of the newly cloned cry2Ab gene was studied in an acrystalliferous strain of B. thuringiensis (4Q7) by fusing the cry2Ab gene downstream of cry2Aa promoter and orf1 + orf2 sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a spore-crystal mixture obtained from transformants of B. thuringiensis strain 4Q7 showed production of Cry2Ab protein of about 65 kDa. Alkali solubilized Cry2Ab7 protein showed toxicity against Helicoverpa armigera neonates.