Molecular epidemiology of measles in India, 2005-2010.

Measles is a childhood disease that causes great morbidity and mortality in India and worldwide. Because measles surveillance in India is in its infancy, there is a paucity of countrywide data on circulating Measles virus genotypes. This study was conducted in 21 of 28 States and 2 of 7 Union Territories of India by MeaslesNetIndia, a national network of 27 centers and sentinel practitioners. MeaslesNetIndia investigated 52 measles outbreaks in geographically representative areas from 2005 through June 2010. All outbreaks were serologically confirmed by detection of antimeasles virus immunoglobulin M (IgM) antibodies in serum or oral fluid samples. Molecular studies, using World Health Organization (WHO)-recommended protocols obtained 203 N-gene, 40 H-gene, and 4 M-gene sequences during this period. Measles genotypes D4, D7, and D8 were found to be circulating in various parts of India during the study period. Further phylogenetic analysis revealed 4 lineages of Indian D8 genotypes: D8a, D8b, D8c, and D8d. This study generated a large, countrywide sequence database that can form the baseline for future molecular studies on measles virus transmission pathways in India. This study has created support and capabilities for countrywide measles molecular surveillance that must be carried forward.

Chandipura virus growth kinetics in vertebrate cell lines, insect cell lines & embryonated eggs.

BACKGROUND & OBJECTIVES: Since not much information on Chandipura virus is available, an attempt was made to study the growth kinetics of the virus in certain vertebrate, invertebrate cell lines and embryonated chicken eggs. METHODS: Comparative study of Chandipura virus (CHPV) growth kinetics in three vertebrate cell lines [Vero E6, Rhabdo myosarcoma (RD), Porcine stable kidney (PS) cell lines], two insect cell lines [Aedes aegypti (AA) and Phlebotomus papatasi (PP-9) cell lines] and embryonated pathogen free chicken eggs was conducted, by tissue culture infective dose 50 per cent (TCID(50)) and indirect immunofluorescence assay (IFA). RESULTS: All the cell lines and embryonated egg supported the growth of CHPV and yielded high virus titre. The vertebrate cell lines showed distinct cytopathic effect (CPE) within 4-6 h post infection (PI), while no CPE was observed in insect cell lines. PP-9 cell line was the most sensitive system to CHPV as viral antigen could be detected at 1 h PI by IFA. INTERPRETATION & CONCLUSIONS: Our results demonstrated that all the systems were susceptible to CHPV and achieved high yield of virus. However, the PP-9 cell line had an edge over the others due to its high sensitivity to the virus which might be useful for detection and isolation of the virus during epidemics.

Ganjam virus.

Ganjam virus (GANV), a member of genus Nairovirus of family Bunyavirdae is of considerable veterinary importance in India. Though, predominantly tick borne, GANV was also isolated from mosquitoes, man and sheep. Neutralizing and complement fixing antibodies to GANV have been detected in animal and human sera collected from different parts of the country. Thirty three strains of GANV have been isolated from India, mainly from Haemaphysalis ticks. The virus replicated in certain vertebrate and mosquito cell lines and found pathogenic to laboratory animals. One natural infection and five laboratory-acquired infections in men were also reported. GANV is antigenically related to Nairobi sheep disease virus (NSDV) of Africa, which is highly pathogenic for sheep and goats causing 70-90 per cent mortality among the susceptible population. Recent molecular studies have demonstrated that GANV is an Asian variant of NSDV and both these viruses are related to the dreaded Crimean Congo haemorrhagic fever (CCHF) group viruses. The versatility of the virus to replicate in different arthropod species, its ability to infect sheep, goat and man makes it an important zoonotic agent.

Detection of chandipura virus from sand flies in the genus Sergentomyia (Diptera: Phlebotomidae) at Karimnagar District, Andhra Pradesh, India.

A total of 191 adult sand flies belonging to the genus Sergentomyia were collected from seven villages in Karimnagar and Warangal districts of Andhra Pradesh State, India, after an outbreak of encephalitis due to Chandipura virus (CHPV). Fifteen pools, each containing two specimens, were tested by reverse transcriptase-polymerase chain reaction and sequencing. One pool of Sergentomyia from Kolanur village in Karimnagar District was positive for CHPV.

A new cell line from the embryonic tissues of Culex tritaeniorhynchus and its susceptibility to certain flaviviruses.

A new cell line from the embryonic tissue of Culex tritaeniorhynchus mosquito was established. Morphological studies carried out at the 45th passage level (P-45) showed four different cell types viz. epithelial-like cells, fibroblast-like cells, giant cells and vacuolated cells. Karyological studies indicated diploid (2n = 6) chromosomes in majority of cells irrespective of passage level. A twelve-fold increase of cell number was observed in 10 days at P-49. The cells could be preserved in liquid nitrogen for more than 40 months. Isoenzyme profile analysis with four enzymes clearly indicated that this cell line was derived from C. tritaeniorhynchus. This cell line was susceptible to Japanese encephalitis (JEV) and West Nile viruses (WNV) but not to Dengue 1-4 (DEN 1-4) viruses. Protein of 38 K was detected in the membrane fraction of the cells from this and the C6/36 cell line, which was found to bind DEN 1-4 viruses. These data suggest that DEN viruses bind to this membrane protein and probably enter into the cells but do not continue further in the replication process.

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.

Antigen distribution pattern of West Nile virus in Culex tritaeniorhynchus, Culex vishnui and Culex pseudovishnui mosquitoes.

Distribution of West Nile (WN) virus antigen in different tissues of mosquitoes was studied in three species viz., Culex tritaeniorhynchus, C. vishnui and C. pseudovishnui. Overall per cent positivity was higher in the intra thoracically inoculated as compared to the orally infected mosquitoes, suggesting the existence of a midgut barrier. In a small number of mosquitoes salivary glands were found negative even though fluorescence was seen in the respective head squashes, suggesting salivary gland barrier in these mosquitoes. There was no difference in the per cent salivary gland and salivary gland area positivity between these three species. Presence of virus antigen in the ovaries of these three species on the 3rd post infection day suggests the possibility of transovarial transmission of virus even in the first gonotrophic cycle, which is of epidemiological importance.

Development of an inactivated candidate vaccine against Chandipura virus (Rhabdoviridae: Vesiculovirus).

A Vero cell based vaccine candidate against Chandipura (CHP) virus (Rhabdoviridae: Vesiculovirus), was developed and evaluated for immunogenicity in mice. Virus was purified by ultracentrifugation on 30% glycerol cushion followed by differential centrifugation on 10-60% sucrose gradient and inactivated with ß-propio lactone at a concentration of 1:3500. The inactivated product was blended with aluminium phosphate (3%) and immunized 4-week-old Swiss albino mice. Neutralizing antibodies in the range of 1:10 to 160 and 1:80 to 1:320 was detected with 85% and 100% sero-conversion after 2nd and 3rd dose, respectively. All the immunized mice with antibody titer above 1:20 survived live virus challenge. The vaccine candidate has potential to be an efficient vaccine against CHP virus.

West Nile virus isolates from India: evidence for a distinct genetic lineage.

The complete genomic sequence of one human isolate of West Nile virus (WNV) and the partial genomic sequences of 14 other strains from India isolated in the period 1955-1982 from different hosts and geographical areas were determined. Phylogenetic analyses based on complete and partial genomic sequences (921 nt of the C-prM-E region) revealed that WNV could be classified into five distinct groups that differed from each other by 20-25% at the complete genome level and by 20-26% using partial sequences. Of the Indian isolates, 13 formed a distinct genetic lineage, lineage 5, whereas two isolates, one from a human patient (1967) and another from a bat (1968), were related closely to lineage 1 strains. The complete genomic sequence of the Indian isolate, 804994, showed 20-22% genetic divergence from the previously proposed lineage 1 and 2 strains and 24-25% divergence from isolates of the newly proposed lineages 3 (Rabensburg isolate 97-103 of 1997) and 4 (Russian isolate LEIV-Krnd88-190 of 1998). Similarly, the partial genomic sequences of the Indian isolates showed 21-26% divergence from lineage 1 and 2 strains and from the Rabensburg (97-103) and Russian (LEIV-Krnd88-190) isolates. Cross-neutralization using strain-specific polyclonal antibodies against lineage 1 strain Eg-101 and representative Indian strains suggests substantial antigenic variation. This study documents circulation of WNV strains typical to India for 27 years and the introduction of lineage 1 strains during 1967-1968. These results indicate strongly that WNV should be classified into five genetic lineages, with Indian viruses constituting the distinct genetic lineage 5.