Diagnosis of Kyasanur forest disease by nested RT-PCR, real-time RT-PCR and IgM capture ELISA.

Kyasanur forest disease (KFD) is a zoonotic viral disease caused by infection by a Flavivirus, a member of the family Flaviviridae. KFD is a public health concern in the Karnataka State in southern India. Available conventional diagnostic tests such as virus isolation and serological tests, such as haemagglutination inhibition and complement fixation tests are time consuming. This study reports the development of a nested RT-PCR [nRT-PCR] and a TaqMan-based real-time RT-PCR and IgM antibodies capture ELISA [MAC-ELISA] for rapid and accurate diagnosis of suspected KFD cases. The nRT-PCR and the TaqMan-based real-time RT-PCR assays were developed using gene sequences of the NS-5/non-coding region. Both the assays detected KFD viral RNA in acute phase human serum samples and can provide early diagnosis of infection. Real-time RT-PCR was found to be more sensitive than nRT-PCR, which could detect 38 copies of KFDV RNA. MAC-ELISA was developed for the detection of recent infections. Although real-time RT-PCR and nRT-PCR require expensive reagents, expensive equipment and trained personnel, the developed MAC-ELISA can be used easily in the affected areas. These tests add to the existing diagnosis arsenal against haemorrhagic viruses that are prevalent in India. These assays will also help to extend our knowledge of the pathology of KFD virus and its associated clinical features, by measuring the viral titre during infection and at the time of seroconversion. Information, which is not available currently because of the lack of appropriate diagnostic methods. In addition, early laboratory diagnosis of KFDV infection will help in the application of appropriate control measures and management of KFD cases.

Evolution, dispersal and replacement of American genotype dengue type 2 viruses in India (1956-2005): selection pressure and molecular clock analyses.

This study reports the phylogeny, selection pressure, genotype replacement and molecular clock analyses of many previously unstudied dengue type 2 virus (DENV-2) strains, isolated in India over a time span of almost 50 years (1956-2005). Analysis of complete envelope (E) gene sequences of 37 strains of DENV-2 from India, together with globally representative strains, revealed that the American genotype, which circulated predominantly in India during the pre-1971 period, was then replaced by the Cosmopolitan genotype. Two previously unreported amino acid residues, one in the American (402I) and one in the Cosmopolitan (126K) genotypes, known to be involved functionally in the cellular tropism of the virus, were shown to be under positive selection pressure. The rate of nucleotide substitution estimated for DENV-2 was 6.5x10(-4) substitutions per site year(-1), which is comparable with earlier estimates. The time to the most recent common ancestor of the pre-1971 Indian strains and the American genotype was estimated to be between 73 and 100 years (1905-1932), which correlates with the historical record of traffic between India and South America and suggests transportation of the virus from the Americas. Post-1971 Indian isolates formed a separate subclade within the Cosmopolitan genotype. The estimated time to the most recent common ancestor of the Indian Cosmopolitan strains was about 47 years, with further estimates indicating the migration of DENV-2 from India to countries across the Indian ocean between 1955 and 1966. Overall, the present study increases our understanding of the events leading to the establishment and dispersal of the two genotypes in India.

Recent ancestry of Kyasanur Forest disease virus.

Kyasanur Forest disease virus (KFDV) is enzootic to India and maintained in ticks, mammals, and birds. It causes severe febrile illness in humans and was first recognized in 1957 associated with a high number of deaths among monkeys in Kyasanur Forest. Genetic analysis of 48 viruses isolated in India during 1957-2006 showed low diversity (1.2%). Bayesian coalescence analysis of these sequences and those of KFDVs from Saudi Arabia and the People's Republic of China estimated that KFDVs have evolved at a mean rate of approximately 6.4 x 10(-4) substitutions/site/year, which is similar to rates estimated for mosquito-borne flaviviruses. KFDVs were estimated to have shared a common ancestor in approximately 1942, fifteen years before identification of the disease in India. These data are consistent with the view that KFD represented a newly emerged disease when first recognized. Recent common ancestry of KFDVs from India and Saudi Arabia, despite their large geographic separation, indicates long-range movement of virus, possibly by birds.

Genetic characterization of Bagaza virus (BAGV) isolated in India and evidence of anti-BAGV antibodies in sera collected from encephalitis patients.

During investigations into the outbreak of encephalitis in 1996 in the Kerala state in India, an arbovirus was isolated from a Culex tritaeniorhynchus mosquito pool. It was characterized as a Japanese encephalitis and West Nile virus cross-reactive arbovirus by complement fixation test. A plaque reduction-neutralization test was performed using hyperimmune sera raised against the plaque-purified arbovirus isolate. The sera did not show reactivity with Japanese encephalitis virus and were weakly reactive with West Nile virus. Complete open reading frame sequence analysis characterized the arbovirus as Bagaza virus (BAGV), with 94.80 % nucleotide identity with African BAGV strain DakAr B209. Sera collected from the encephalitic patients during the acute phase of illness showed 15 % (8/53) positivity for anti-BAGV neutralizing antibodies. This is the first report of the isolation of BAGV from India. The presence of anti-BAGV neutralizing antibodies suggests that the human population has been exposed to BAGV.

Chikungunya outbreaks caused by African genotype, India.

Chikungunya fever is reported in India after 32 years. Immunoglobulin M antibodies and virus isolation confirmed the cause. Phylogenic analysis based on partial sequences of NS4 and E1 genes showed that all earlier isolates (1963-1973) were Asian genotype, whereas the current and Yawat (2000) isolates were African genotype.