Lakhmir Singh

Coat protein gene diversity among Chrysanthemum virus B isolates from India

Summary.The complete coat protein (CP) sequences from 29 Indian isolates of Chrysanthemum virus B (CVB) were determined and analysed in relation to other previously characterized carlaviruses. The CP genes of the Indian CVB isolates were highly heterogeneous, sharing nucleotide sequence identities of 74–98%. Based on phylogenetic analyses, the isolates formed three groups potentially representing either two or three major CVB strain groupings. Recombination analysis revealed at least one definite recombination event involving the exchange of sequences between members of different groups. To our knowledge this is the first reported evidence of homologous recombination in carlaviruses.

Genomic sequence analysis of four new chrysanthemum virus B isolates: evidence of RNA recombination

Chrysanthemums worldwide suffer from a high incidence of infection with chrysanthemum virus B (CVB), a member of the genus Carlavirus, family Betaflexiviridae. Three major lineages or strains of this virus have been found in India, but none have been characterized beyond the genetic variation they display in their coat protein genes. Here, we describe the analysis of four near-complete genome sequences (from the three lineages) representing the genetic diversity of these strains. Ranging in size from 8815 to 8855 nucleotides (excluding the polyA tail), these four isolates have a genome organization very similar to that of the recently reported Japanese isolate of CVB, with which they share between 70 and 73% genome-wide sequence identity. We present further evidence that recombination may feature quite prominently in the evolution of CVB.

Immunodiagnostics for Cherry virus A and Cherry necrotic rusty mottle virus

Cherry necrotic rusty mottle virus (CNRMV) and Cherry virus A (CVA) are important graft transmitted viruses in the family Betaflexiviridae, infecting cherry. It is believed that CVA causes severe symptoms and disease in combination with other stone fruit viruses in both cherry and non-cherry hosts while CNRMV infected tree show reduced growth, significant yield loss and early death. Detection of these viruses is crucial for their sanitation, indexing and certificate programmes. In this study, a polyclonal antibody was produced against recombinant coat protein (CP) of CVA and CNRMV expressed in Escherichia coli for the development serological based diagnostics. The coat protein gene of CVA and CNRMV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using specifically designed primer pair with restriction sites at both ends and further cloned and expressed in bacterial expression vector (pET32a). CP coding region of both the viruses was expressed separately and successfully as a fusion protein. The fusion proteins were purified and directly used for rabbit immunizations. Antibodies were purified, conjugated with alkaline phosphatase and used in DAS-ELISA. A total of 74 and 43 samples were checked for CVA and CNRMV, respectively. In this analysis, 40/74 samples were found positive for CVA and 20/43 tested samples were positive for CNRMV by DAS-ELISA, further confirmed by RT-PCR. Antibodies raised against recombinant CNRMV CP also detected the virus consistently in western blot analysis with high sensitivity and specificity. IC-RT-PCR was also developed for the detection of CVA using the produced antibody. To the best of our knowledge, this is the first report of DAS-ELISA and IC-RT-PCR based diagnostics for CVA, and first report of DAS-ELISA based diagnostics for CNRMV from India.

Intermolecular Interactions of Chrysanthemum virus B Coat Protein: Implications for Capsid Assembly

Chrysanthemum virus B coat protein constitutes the viral capsid which, besides other functions, encapsulates and protects the viral nucleic acid. We have demonstrated homotypic interaction of the coat protein subunits, essentially important for dimer formation, which is the first step during capsid assembly in vivo and in vitro. Interaction capacity of full length and truncated protein has been investigated and important regions have been identified through protein-protein interaction in yeast and by co-immunoprecipitation assays. Complete coat protein was found to interact strongly with similar subunits. Constructs with 102 amino acids from the N-terminal and 64 amino acids from C-terminal were found to be inconsequential for dimer formation as they did not show any interaction with similar subunits or with full length protein when analysed for β-galactosidase or histidine prototrophy. Results suggest that the region of 98–184 amino acids from the middle plays an important role in the process, probably without the involvement of N- and C- terminals.