We have developed a highly sensitive and specific qRT-PCR for detection and quantitation of dengue viruses. The assay will be a useful tool for differential diagnosis of dengue fever in a situation where a number of other clinically indistinguishable infectious diseases like malaria, Chikungunya, rickettsia and leptospira occur. The ability of the assay to detect DENV-2 in inoculated mosquitoes makes it a potential tool for detecting DENV in field-caught mosquitoes.

BackgroundDescriptions of dengue immunopathogenesis have largely relied on data from South-east Asia and America, while India is poorly represented. This study characterizes dengue cases from Pune, Western India, with respect to clinical profile and pro-inflammatory cytokines.Methodology/Principal FindingsIn 2005, 372 clinically suspected dengue cases were tested by MAC-ELISA and RT-PCR for dengue virus (DENV) aetiology. The clinical profile was recorded at the hospital. Circulating levels of IFN-γ, TNF-α, IL-6, and IL-8 were assessed by ELISA and secondary infections were defined by IgM to IgG ratio. Statistical analysis was carried out using the SPSS 11.0 version.Of the 372 individuals, 221 were confirmed to be dengue cases. Three serotypes, DENV-1, 2 and 3 were co-circulating and one case of dual infection was identified. Of 221 cases, 159 presented with Dengue fever (DF) and 62 with Dengue hemorrhagic fever (DHF) of which six had severe DHF and one died of shock. There was a strong association of rash, abdominal pain and conjunctival congestion with DHF. Levels of IFN-γ were higher in DF whereas IL-6 and IL-8 were higher in DHF cases (p<0.05). The mean levels of the three cytokines were higher in secondary compared to primary infections. Levels of IFN-γ and IL-8 were higher in early samples collected 2–5 days after onset than late samples collected 6–15 days after onset. IFN-γ showed significant decreasing time trend (p = 0.005) and IL-8 levels showed increasing trend towards significance in DHF cases (interaction p = 0.059). There was a significant association of IL-8 levels with thrombocytopenia and both IFN-γ and IL-8 were positively associated with alanine transaminase levels.Conclusions/SignificanceRash, abdominal pain and conjunctival congestion could be prognostic symptoms for DHF. High levels of IL-6 and IL-8 were shown to associate with DHF. The time trend of IFN-γ and IL-8 levels had greater significance than absolute values in DHF pathogenesis.

The envelope glycoprotein of the human immunodeficiency virus (HIV) utilizes CD4 as a receptor and CCR5 and/or CXCR4 as coreceptor to gain entry into the cell. The CCR5-tropic viruses, observed early in infection, could be important in transmission and the CXCR4-tropic viruses, observed late, may play an important role in disease progression. Viruses from 40 HIV-positive, asymptomatic or symptomatic individuals in India were isolated. Of 40 isolates 39 used CCR5. Thirty-three isolates were subtype C, 3 isolates were subtype A, and 4 isolates were HIV-2. Only 1 HIV-2 isolate, from a symptomatic individual, was dualtropic. Therefore, a majority of isolates from India belonged to subtype C and all the isolates utilized CCR5 exclusively irrespective of HIV disease status.

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Most strains of human immunodeficiency virus type 1 (HIV-1) which have only been carried in vitro in peripheral blood mononuclear cells (primary isolates) can be neutralized by antibodies, but their sensitivity to neutralization varies considerably. To study the parameters that contribute to the differential neutralization sensitivity of primary HIV-1 isolates, we developed a neutralization assay with a panel of genetically engineered cell lines (GHOST cells) that express CD4, one of eight chemokine receptors which function as HIV-1 coreceptors, and a Tat-dependent green fluorescent protein reporter cassette which permits the evaluation and quantitation of HIV-1 infection by flow cytometry. All 21 primary isolates from several clades could grow in the various GHOST cell lines, and their use of one or more coreceptors could easily be defined by flow cytometric analysis. Ten of these primary isolates, three that were CXCR4 (X4)-tropic, three that were CCR5 (R5)-tropic, and four that were dual- or polytropic were chosen for study of their sensitivity to neutralization by human monoclonal and polyclonal antibodies. Viruses from the X4-tropic category of viruses were first tested since they have generally been considered to be particularly neutralization sensitive. It was found that the X4-tropic virus group contained both neutralization-sensitive and neutralization-resistant viruses. Similar results were obtained with R5-tropic viruses and with dual- or polytropic viruses. Within each category of viruses, neutralization sensitivity and resistance could be observed. Therefore, sensitivity to neutralization appears to be the consequence of factors that influence the antibody-virus interaction and its sequelae rather than coreceptor usage. Neutralization of various viruses by the V3-specific monoclonal antibody, 447-52D, was shown to be dependent not only on the presence of the relevant epitope but also on its presentation. An epitope within the envelope of a particular virus is not sufficient to render a virus sensitive to neutralization by an antibody that recognizes that epitope. Moreover, conformation-dependent factors may overcome the need for absolute fidelity in the match between an antibody and its core epitope, permitting sufficient affinity between the viral envelope protein and the antibody to neutralize the virus. The studies indicate that the neutralization sensitivity of HIV-1 primary isolates is a consequence of the complex interaction between virus, antibody, and target cell.

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 . 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 6.5¾10 , 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 , 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.

Sera from human immunodeficiency virus-1-infected participants in the Multicenter AIDS Cohort Study were tested to assess the association between serum neutralizing antibodies (NAbs) and disease progression. Each of 14 pairs, retrospectively matched for age, sex, race, and CD4+ lymphocyte numbers early in the study, consisted of a rapid progressor (RP) who developed AIDS and a long-term nonprogressor (LTNP) who remained asymptomatic. Serum samples were drawn early, when all participants were asymptomatic, and late, when the RPs had developed clinical AIDS. The LTNPs and RPs had similar levels of NAbs against primary isolates at the early time point, indicating that NAb levels are not predictive of disease progression; at the late time point, the LTNPs had significantly higher titers because of an increase in the level of serum NAbs in the LTNPs and/or a decrease in the NAbs in the RPs. The patterns of neutralizing activity over time suggest that changes in effective NAbs against different viruses do not occur in parallel.

Dengue, a Flavivirus and chikungunya, an Alphavirus, transmitted by Aedes mosquitoes, are a cause of great concern to public health in India. Every year, thousands of individuals are affected and contribute to the burden of health care. Dengue outbreaks have continued since the 1950s but severity of disease has increased in the last two decades. Chikungunya outbreaks started in the 1960s and dwindled to sporadic cases until a resurgence in 2006. Based on the data of National Vector Borne Disease Control Programme (NVBDCP), the number of cases reported in 2013 was about 74 454 for dengue with 167 deaths and 18 639 for chikungunya. The number of cases reported is increasing, probably because of the availability of IgM detection kits produced and distributed by National Institute of Virology through NVBDCP and better reporting. In the absence of well-structured epidemiological studies, this review attempts to summarize reports on dengue and chikungunya outbreaks from various regions of India. For dengue, young adults are the major group affected; the severity of disease in India is still lower than that reported elsewhere in South-East Asia; and paediatric cases of dengue haemorrhagic fever have a high mortality. For chikungunya, all age groups are affected but severe manifestations are more often seen in children. Persisting arthralgia, neurological syndromes and non-neurological manifestations are recorded. Changes in the genotype and mutations in the genome have been detected for both dengue and chikungunya viruses. The review ends with a short summary of the most recent vector-control studies.