Isolation of bovine viral diarrhea virus 1, a pestivirus from autopsied lamb specimen from Tamil Nadu, India.

An epizootic of febrile illness among the Madras red breed of sheep had occurred in 1994 in Verrapuram, Chennai, India. The epizootic was suspected as Rift Valley fever (RVF)-like sickness based on clinical features. However, its etiological agent could neither be isolated nor implicated conclusively. During the post-epizootic period a male lamb died of similar clinical features and the spleen was immediately collected. Inoculation of spleen suspension in infant mouse brain yielded a virus that was serially passaged in infant mice and rhabdomyosarcoma (RD) cells. Electron microscopic observations revealed virus particles resembling flaviviruses. RT-PCR performed on extracted total RNA from infected cells and mouse brains with flavivirus-specific or RVF-specific primers gave negative results. However, an amplicon of 280 bp was obtained with pestivirus-specific primers from the 5'-UTR. Further, a nested PCR yielded a product of 157 bp. Nucleotide sequencing of the 157 bp product showed 100% homology to BVDV-1. Western blot analysis with a flavivirus envelope protein-specific MAb revealed three proteins of 33 K, 45 K and 55 K. Further studies suggested that the 33 K and 55 K proteins were glycosylated. This is the first report of isolation of BVDV-1 from a lamb in India.

A new continuous cell line from larval hemocytes of Spodoptera litura (F.).

A new cell line has been established from larval hemocytes of the moth, S. litura (tobacco cut worm). It took 147 days to form a monolayer and one year for the first 17 passages. At present, the culture is at 86th passage level and is designated NIV-SU-1095. Three cell types could be distinguished, viz. plasmatocytes (53%), prohemocytes (36%) and granular hemocytes (11%). The chromosome number was very high, 74% metaphase cells showed more than 100 chromosomes. The cells could be cryopreserved. The cells were susceptible to the baculoviruses, Autographa californica nuclear polyhedrosis virus and S. litura nuclear polyhedrosis virus (SLNPV). Plaques could be observed on 7th post infection day with SLNPV. Six cloned cell lines have been developed of which clone II-1F was more sensitive to both the baculoviruses compared to the original cell line.