In-silico Designing and Testing of Primers for Sanger Genome Sequencing of Dengue Virus Types of Asian Origin.

Rarity in reporting whole genome sequence of Dengue virus from dengue endemic countries leaves lacunae in understanding regional pattern of virus mutation and ultimately leading to non-understanding of transmission pattern and clinical outcomes emerging at regional levels. Due to inter-serotype genomic similarity and intra-serotype genomic diversity, appropriate designing of primer pairs appears as an exhaustive exercise. Present paper reports new Dengue virus type-specific primer which may help in characterizing virus specific to Asian origin. Genomes of dengue virus serotypes of Asian region were searched and using advanced bioinformatics tools, serotype specific primers were designed and tested for their targeted amplification efficiency. 19 primers sets for DENV-1, 18 primer sets for DENV-2, 17 for DENV-3 and 18 for DENV-4 were designed. In-silico and experimental testing of the designed primers were performed on virus isolated from both clinical isolates and passaged cultures. While all 17 and 18 primer sets of DENV-3 and DENV-2 respectively yielded good quality sequencing results; in case of DENV-4, 16 out of 18 primer sets and in DENV-1, 16 out of 19 primer sets yielded good results. Average sequencing read length was 382 bases and around 82% nucleotide bases were Phred quality QV20 bases (representing an accuracy of circa one miscall every 100 bases) or higher. Results also highlighted importance of use of primer development algorithm and identified genomic regions which are conservative, yet specific for developing primers to achieve efficiency and specificity during experiments.

Age of initial cohort of dengue patients could explain the origin of disease outbreak in a setting: a case control study in Rajasthan, India.

Dengue fever (DF) and dengue hemorrhagic fever (DHF) is a public health problem with 390 million cases reported in world annually. In Rajasthan, DF with DHF is being reported for about two decades. For undertaking interventions into disease transmission, locating origin of transmission is very important. Present paper reports retrospective analysis of the hospital reported cases of dengue during the year 2013-2014 undertaken in Barmer, Rajasthan. To address task of investigating outbreak, detailed analysis of the data on serological test results (Mac-ELISA assay of NS1, IgG and IgM) performed by local hospital, Balotra was made. The domestic breeding containers were examined for the presence of larvae and adult forms of Aedes aegypti by visiting individual households as well as common places of human aggregation like schools and hospitals. The analysis showed that first dengue cases started from the lot of school going children and then followed by adults and finally during peak period of infection only children around 1-2 years got infected. The subsequent entomological investigations during the outbreak showed school as principal source of mosquito breeding. Present investigations highlight that schools (March to April) play the role of primary sites of disease transmission and should be preferred for undertaking vector control operations to prevent dengue transmission from getting aggravated.

Rare occurrence of natural transovarial transmission of dengue virus and elimination of infected foci as a possible intervention method.

Transovarial transmission of dengue virus has been studied in 33 districts of Rajasthan, India. Small proportion (1.09%) of breeding containers positive for the virus and their elimination has been demonstrated as a possible intervention method of disease control. Dengue virus was isolated from individual mosquitoes employing Indirect Fluorescence Antibody Test and Reverse Transcriptase Polymerase Chain Reaction. Out of 1,30,525 containers examined only 1432(1.09%) showed transovarially transmitted virus activity. Elimination of larvae from all the 1432 virus positive containers resulted in substantial control over prospective transmission of dengue. The study highlights rarity of transovarial transmission under natural conditions and sensitizes whether elimination of vertically infected foci could be used as a new intervention method.

Multiple dengue virus types harbored by individual mosquitoes.

The existing knowledge on pathogenesis and aetiology of DHF establishes that Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS) are caused by two subsequent infections of two different serotypes of dengue affecting a common human population with a time gap. Present studies have been undertaken on 212 laboratory reared infected individual mosquitoes from larvae collected from 31 dengue endemic towns of Rajasthan, India. Type specific DEN viruses were detected from individual mosquitoes employing RT-PCR. In 78.7% of 212 infected individual mosquitoes studied, vertically transmitted multiple DENV types were observed. We report for the first time that single mosquitoes contain multiple dengue virus types.

Activation of p16 gene silenced by DNA methylation in cancer cells by phosphoramidate derivatives of 2'-deoxyzebularine.

We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2'-deoxyzebularine 5'-triphosphate (dZTP). Because 2'-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine 5'-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent, and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.