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°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.Dengue virus is a mosquito-borne flavivirus and the most widely prevalent arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America (4). There are four distinct serotypes, DEN-1, DEN-2, DEN-3, and DEN-4, which produce a spectrum of illness ranging from inapparent infection to moderate febrile illness and severe and fatal hemorrhagic disease. In recent years, dengue fever (DF) and its more serious forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), have emerged as a major public health problems, with expanded geographic distributions and increased epidemic activities (20).Dengue virus infection induces a life-long protective immunity to the homologous serotype but confers only partial and transient protection against subsequent infection by the other three serotypes. Therefore, multiple and sequential infections with the four dengue virus serotypes would be expected for people living in a region where dengue is hyperendemic due to the lack of cross-protective neutralizing antibodies. Seroepidemiological studies have shown that secondary infection is a major risk factor for DHF and DSS through antibody-dependent enhancement (6, 16). Therefore, rapid detection and differentiation between primary and secondary dengue virus infections and determination of the dengue virus serotypes of past and current ...

The standardization and validation of a one-step, single-tube, accelerated, quantitative reverse transcription (RT) loop-mediated isothermal amplification (RT-LAMP) assay targeting the E1 gene for the rapid and real-time detection of Chikungunya virus (CHIKV) are reported. A linear relationship between the amount of template and time of positivity value over a range of 2 ؋ 10 8 to 2 ؋ 10 2 copies was obtained. The feasibility of CHIKV RT-LAMP for clinical diagnosis was validated with patient serum samples from an ongoing epidemic in Southern India. Optimal assay conditions with zero background were established for the detection of low levels of CHIKV in acute-phase patient serum samples. The comparative evaluation of the RT-LAMP assay with acute-phase patient serum samples demonstrated exceptionally higher sensitivity by correctly identifying 21 additional positive borderline cases that were missed by conventional RT-PCR (P < 0.0001) with a detection limit of 20 copies. The quantification of virus load in patient serum samples was also determined from the standard curve based on their time of positivity and was found to be in the range of 2 ؋ 10 8 to 2 ؋ 10 1 copies. In addition, the field applicability of the RT-LAMP assay was also demonstrated by standardizing SYBR Green I-based RT-LAMP wherein the amplification was carried out in a water bath at 63°C for 60 min, which was followed by monitoring gene amplification with the naked eye through color changes. These findings demonstrated that the RT-LAMP assay is a valuable tool for rapid, real-time detection as well as quantification of CHIKV in acute-phase serum samples without requiring any sophisticated equipment and has potential usefulness for clinical diagnosis and surveillance of CHIKV in developing countries.

Background: Dengue virus type 1 (DENV-1) have been mostly circulating silently with dominant serotypes DENV-2 and DENV-3 in India. However recent times have marked an increase in DENV-1 circulation in yearly outbreaks. Many studies have not been carried out on this virus type, leaving a lacunae pertaining to the circulating genotypes, since its earliest report in India. In the present study, we sequenced CprM gene junction of 13 DENV-1 isolated from Delhi and Gwalior (North India) between 2001-2007 and one 1956 Vellore isolate as reference. For comparison, we retrieved 11 other Indian and 70 global reference sequences from NCBI database, making sure that Indian and global isolates from all decades are available for comparative analysis.

An outbreak of viral encephalitis occurred in Gorakhpur, India, from July through November 2005. The etiologic agent was confirmed to be Japanese encephalitis virus by analyzing 326 acute-phase clinical specimens for virus-specific antibodies and viral RNA and by virus isolation. Phylogenetic analysis showed that these isolates belonged to genogroup 3.

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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.

Chikungunya fever is an important arboviral infection prevalent throughout Africa and Southeast Asia. Recently, in 2006, it has reemerged in many parts of India, affecting more than a million persons. A detail serological, virological, and molecular investigation of this unprecedented outbreak was carried out by collecting and studying 540 samples from all the affected regions of India during this epidemic. An in-depth investigation revealed the presence of anti-Chikungunya antibodies in 68% of the samples and genomic RNA in 49% of them. In addition 32 Chikungunya viruses were isolated from 45 representative polymerase chain reaction-positive samples. The nucleotide sequences of partial E1 gene of 25 representative Chikungunya viruses were deciphered. The sequence analysis indicated that all the isolates of this epidemic belonged to the new Indian Ocean island clade of East Central South (ECS) African genotype. This study conclusively proved the genotype shift from Asian to ECS African as the major factor in the reemergence of Chikungunya in an unprecedented outbreak in India after a gap of 32 years.

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