Molecular typing of dengue virus isolated from field caught mosquito specimens by reverse transcriptase polymerase chain reaction (rt-pcr)

Abstract

The Dengue viruses are borne by the Aedes mosquito. They cause dengue fever in most tropical areas of the world. The detection and successful typing of dengue virus (DENV) from field caught Aedes mosquito is important for virological surveillance and implementation of epidemiologic control measure. We have applied a rapid and sensitive, nested reverse transcription-polymerase chain reaction (RT-PCR) assay for the type-specific detection of dengue viruses in artificially infected and in field caught mosquitoes for first time in Bangladesh. Small scale bionomics study was done in few selected areas of Dhaka city to help find the recent dengue prevalent season, dengue vector breeding sites and the peak season of vector population. Findings of bionomics study have increased the chance of getting dengue infected mosquitoes from field. In bionomics study of Ae. aegypti higher number of mosquito population (85.15% eggs, larvae or pupae) were found in artificial container than in natural container (14.85%), indicating recent creation and availability of huge breeding ground for Aedes aegypti. Increased use of non biodegradable plastics, pot gardening, water logging and water storage habit of Dhaka city dwellers may have influenced higher mosquito densities by providing many more breeding sites. Larvae survey showed the Breteau Index of 20-40, well above the risk level for dengue virus transmission and the post monsoon period (August to October) was found to be the most affected period for larval growth. The Indoor resting adult populations start rising from August and the peak mosquito population and higher biting rate were found from September to November, which was consistent with the larvae survey. Field studies were conducted during November-December 2010, 2011 and from July to December 2012. Adult Aedes aegypti mosquitoes were caught from selected dengue prevalent areas of Dhaka city and tested for the presence of dengue virus by ELISA and RT-PCR method. The RT-PCR assay was sensitive enough to detect dengue virus in lab infected mosquito- thorax pool. Almost all ELISA positive mosquito specimens were found to be positive for dengue virus by RT-PCR (90% sensitivity compared to ELISA). Approximately 4.8%, (9 out of 188) mosquito pools (9-13 mosquitoes /pool) were found to be positive for dengue viruses. Of the nine RT-PCR positive Ae. aegypti pools six pools were (66.66%) positive for dengue virus 3 (DEN3), two (22.2%) were positive for DEN 2 virus and only one (11.11%) were positive for dengue 4 (DEN 4) viruses. No DEN1 virus positive pool could be recorded during this study. The predominant virus type responsible for the current dengue fever is DEN 3 and multiple virus serotypes also co-circulating in Dhaka city. Majority of the dengue virus-positive mosquitoes harbored DEN-3 virus, which had been the predominant dengue virus type responsible for the local dengue epidemic since 1964. Multiple serotypes were found in Dhanmondi R/A (serotype 3 and 4) and Mirpur (serotype 2 and 3). DEN-3 alone was detected in Rampura and DEN-2 in Dhaka University Campus and no dengue virus was detected in Shegunbagicha. Ae aegypti caught at the five indoor stations had MIR (minimum infection rate) ranging from 0.00-1.63 per 100 mosquitoes with an MIR of 0.72 for the combined stations. Dhanmondi residential area showed the highest (1.63) and Shegunbagicha showed the lowest (0.00). Dengue positive mosquito pools were found between end of the September to mid October in three dengue prevalent seasons 2010, 2011 and 2012. These findings give us impression that the predominant perhaps the most virulent serotype virus type responsible for the current dengue fever is DEN-3 and multiple virus serotypes are also co-circulating in Dhaka city. We found that the overall rate of dengue virus infection per 100 Ae. aegypti females during the study period was as high as 0.72, giving us an impression that a low grade infection and transmission is currently present in Dhaka city. Evidence of more than one viral serotypes should be taken as a warning that outbreaks of DHF might occur in the future, although a low grade infection and transmission is currently present in Dhaka city. RT-PCR based surveillance of dengue viral infection in Ae. aegypti mosquitoes could serve as an early warning monitoring system of dengue outbreak.