Reemergence of dengue virus type-3 (subtype-III) in India: implications for increased incidence of DHF & DSS.

BACKGROUND: Dengue virus infection has recently taken endemic proportion in India implicating all the four known dengue serotypes. There was a major dengue outbreak in northern India including Delhi in October- December, 2003 and again in 2004. We have carried out a detailed investigation of the 2004 outbreak by Serosurveillance, RT-PCR, nested PCR, virus isolation and genotyping. We also report the molecular epidemiological investigation of these outbreaks. RESULTS: The serological investigation of 162 suspected serum samples using an in-house dengue dipstick ELISA revealed 11%-IgM, 51%-IgG and 38%-both IgM and IgG antibody positivity. The RT-PCR analysis revealed presence of dengue RNA in 17 samples. Further subtyping and genotyping by nested PCR and nucleotide sequencing of C-prM gene junction revealed the association of subtype III of dengue virus type 3 in the outbreak. CONCLUSION: The sudden shifting and dominance of the dengue virus serotype-3 (subtype III) replacing the earlier circulating serotype-2 (subtype IV) is a point of major concern and may be attributed to increased incidence of DHF and DSS in India.

Emergence of dengue virus type-3 in northern India.

During the last few decades dengue has reemerged in several parts of Southeast Asia, including India. A major outbreak of dengue infection occurred in northern India during October to December 2003. To determine the etiology, we carried out serological, virological and molecular investigations of this outbreak. A total of 76 dengue suspected patient blood samples were collected from Gwalior, Madhya Pradesh and Delhi, India. Serological investigations carried out using an in-house Dipstick ELISA protocol revealed the presence of anti-dengue antibodies in 53 patients. Twelve of them (22%) had a positive IgM response, indicative of primary infection, and 22 of them (42%) revealed only IgG antibodies, indicative of secondary infection. RT-PCR analysis employing dengue group specific amplimer revealed the presence of dengue specific RNA in four acute phase samples. These four RT-PCR positive samples were further processed for virus isolation in C6/36 cells and suckling mice, yielding four dengue virus isolates. The Nested PCR analysis employing serotype specific amplimer revealed the presence of dengue-3 specific 389 bp amplicon. This study confirmed the reemergence of dengue virus type-3 in a dominant form in India after a gap of nine years. Earlier, dengue virus type-2 was implicated as the etiology of a major dengue epidemic in Delhi in 1996 and Gwalior in 2001. The implication of dengue type-3 as etiology of a DHF epidemic in neighboring Sri Lanka and Bangladesh recently confirms the reemergence of dengue type-3 as the dominant form on the Indian subcontinent.

Production and characterization of monoclonal antibody specific to recombinant dengue multi-epitope protein.

Monoclonal antibodies against novel dengue recombinant protein were produced following immunization of Balb/c mice with recombinant dengue multi-epitope protein (r-DMEP) expressed in Escherichia coli vector and purified in a single-step chromatography system. Antigenicity of r-DMEP was evaluated by dot enzyme immunoassay. Mice were immunized intraperitoneally with five doses each of 100 microg of this novel antigen at 1-week intervals and a final intravenous booster dose prior to the fusion. Hybridomas resulted from fusion of myeloma cells and splenocytes using PEG-1500 as an additive. Selection of the hybrids was done using HAT medium, and the hybrids thus selected were finally screened qualitatively and quantitatively by dot and plate immunoassays, respectively. Five antibody secretory hybrid clones exhibited specific reactivity against r-DMEP by dot-ELISA, whereas a lone clone was found to be cross-reactive with Japanese encephalitis virus (JEV). Monoclonal antibodies (MAbs) specific to r-DME protein recognized the envelope and non-structural epitopes by Western blot analysis. These MAbs were further checked for their diagnostic efficacy using dengue suspected clinical samples and found overall sensitivity and specificity for DRDE dipstick ELISA. MAb-based dipstick ELISA results were 85%, 75% and 85%, 90%, respectively.

Rapid detection and differentiation of dengue virus serotypes by a real-time reverse transcription-loop-mediated isothermal amplification assay.

The development and validation of a one-step, real-time, and quantitative dengue virus serotype-specific reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay targeting the 3' noncoding region for the rapid detection and differentiation of dengue virus serotypes are reported. The RT-LAMP assay is very simple and rapid, wherein the amplification can be obtained in 30 min under isothermal conditions at 63 degrees C by employing a set of four serotype-specific primer mixtures through real-time monitoring in an inexpensive turbidimeter. The evaluation of the RT-LAMP assay for use for clinical diagnosis with a limited number of patient serum samples, confirmed to be infected with each serotype, revealed a higher sensitivity by picking up 100% samples as positive, whereas 87% and 81% of the samples were positive by reverse transcription-PCR and virus isolation, respectively. The sensitivity and specificity of the RT-LAMP assay for the detection of viral RNA in patient serum samples with reference to virus isolation were 100% and 93%, respectively. The optimal assay conditions with zero background and no cross-reaction with other closely related members of the Flavivirus family (Japanese encephalitis, West Nile, and St. Louis encephalitis viruses) as well as within the four serotypes of dengue virus were established. None of the serum samples from healthy individuals screened in this study showed any cross-reaction with the four dengue virus serotype-specific RT-LAMP assay primers. These findings demonstrate that RT-LAMP assay has the potential clinical application for detection and differentiation of dengue virus serotypes, especially in developing countries.

Emergence and continued circulation of dengue-2 (genotype IV) virus strains in northern India.

Dengue (DEN) is an acute mosquito borne viral disease of mankind. Off late it has become an important public health concern in Southeast Asia. Although, all the four known dengue virus serotypes (DEN-1 to 4) are reported from time to time, in the recent past, DEN-2 has emerged as the predominant type, being the causative agent of several outbreaks of dengue fever (DF) and dengue haemorrhagic fever (DHF) in India. To elucidate the true molecular epidemiology of these viruses, we have sequenced C-prM gene junction (454 nucleotides) of 11 DEN-2 viruses directly from patient serum. The C-prM gene junction was amplified initially by reverse transcription-polymerase chain reaction followed by automated DNA sequencing. These sequences provide unique information with regard to molecular epidemiology when compared to other DEN-2 sequences from diverse geographic origins. The sequence analysis revealed that most of the mutations in this region remained silent, except a few at the carboxy-terminal of the capsid. Reported phylogenetic analysis classifies DEN-2 viruses into five distinct genotypes. The Gwalior DEN-2 viruses, included in the present study were classified into genotype-IV, and were found to be most closely related to Delhi 1996 DEN-2 viruses and FJ 10/11 strains prevalent in the Fujian state of China. However, two earlier Indian isolates of DEN-2 were classified into genotype-V. The present study indicates that genotype V of DEN-2 has been replaced by genotype IV during the past decade, which continues to circulate silently in north India, and have the potential to reemerge and cause major epidemics of DF and DHF.