Medicine
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This repository contains the published and unpublished research of the Faculty of Medicine by the staff members of the faculty
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Item Multiple dengue virus serotypes resistant transgenic Aedes aegypti fitness evaluated under laboratory conditions.(Landes Bioscience, 2020) Ramyasoma, H.P.B.K.D.; Dassanayake, R.S.; Hapugoda, M.; Capurro, M.L.; Silva Gunawardene, Y.I.N.ABSTRACT:Dengue viruses (DENV) are the wildest transmitted arbovirus members of the family Flaviviridae, genus Flavivirus. Dengue viruses are composed of four serotypes, DENV1, 2, 3, and 4, and these viruses can cause dengue fever and dengue haemorrhagic fever or dengue shock syndrome, when infecting humans. RNA interference (RNAi) is a self-defence mechanism, which can be used to prevent invasions of RNA viruses to the host. Genetically engineering a host with an RNAi molecule that targets a single virus serotype may develop escape mutants, and can cause unusual dominance over other serotypes. Therefore, the simultaneous targeting of multiple serotypes is necessary to block DENV transmission. Here, we report the development of transgenic Aedes aegypti based on a bioinformatically designed multiple miRshRNA (microRNA-based shRNA) DNA sequence under the control of a blood-meal induced promoter, Carboxypeptidase A, to induce RNAi for DENV in Aedes aegypti, and demonstrate the expression of a synthetic multiple shRNA polycistronic cluster having RNA interference sequences to target DENV genomes. The transgenic mosquitoes have lower rates of infection, dissemination, and transmission for DENV2 and DENV4 compared to wild mosquitoes, with a significant reduction of dengue copy number and antigen levels in the midgut. These levels of DENV were low enough to make transgenic mosquitoes stop the DENV transmission from infected host to a susceptible host and refractory to DENV2 and DENV4 infection. Such multiple resistance in Ae. aegypti has not been documented previously. Laboratory fitness measurement of transgenic Ae. aegypti showed results comparable to other reported transgenic mosquitoes. KEYWORDS: RNA interference; aedes aegypti; dengue disease; multiple miRshRNA; piggyBacItem Detection of Dengue Viral Migration to Sri Lanka(19th Conference on Postgraduate Research, International Postgraduate Research Conference 2018, Faculty of Graduate Studies,University of Kelaniya, Sri Lanka, 2018) Withanage, G.P.; Hapuarachchi, H.C.; Gunawardene, Y.I.N.S.; Hapugoda, M.D.Dengue is one of the most important mosquito-borne viral infectionsin Sri Lanka.The causative agent is Dengue Viruses (DENV) and the primary vector of the virus is Aedesaegypti(Linnaeus) while Ae. albopictus (Skuse) is the subsidiary vector. The current research was focused on the detection of DENV serotypes and genotypes circulating in mosquitoes during the dengue epidemic in June and July, 2017 in the EriyawetiyaGramaNiladhari division, where one of the dengue high-risk area in Kelaniya Medical Officer of Health (MOH) area in the District of Gampaha, Sri Lanka. Aedesmosquitoes were collected following WHO guidelinesandthe field-caught mosquitoes were transported to the laboratory for species identification and subsequent analysis. Head and thorax of each mosquito was removed and mosquito samples were pooled separately. Total RNA was extracted from mosquito samples and semi-nested Polymerase Chain Reaction (PCR) was performed to identify DENV serotypes present in the mosquito samples. The results of the PCR indicated the presence of DENV2 in both Ae. aegypti (1/5) and Ae. albopictus (1/27) mosquitoes. Then complete Envelope (E) gene was amplified with DENV2 specific primers for genotyping of the virus which is required to identify the molecular evolution of the DENV2. Prior to sequencing the PCR products were purified and sequencing results were analyzed usingLaserGene software. The generated sequences were aligned with retrieved DENV2 sequences available at NCBI database and the phylogenetic trees were developed using MEGA7 software with General Time Reversible (GTR) substitution model with gamma distributed rates. The robustness of clades was determined by using bootstrap analysis of 500 replicates. The result of the phylogenetic analysis illustrates that the E gene sequences of DENV2 obtained from two DENVpositive mosquito poolsbelong to DENV2 Cosmopolitan Clade Ib, which has been the dominant strain in South-East Asia, specially Singapore, Indonesia, Malaysia, and China since August, 2015.The evidence suggests recent introduction of this DENV strain into Sri LankaItem Silent transmission as a risk factor affecting transmission of dengue fever.(Faculty of Veterinary Medicine and Animal Science, University of Peradeniya, 2007) Hapangama, H.A.D.C.; Hapugoda, M.D.; Gunawardene, Y.I.N.S.; Premaratna, R.; Dayanath, M.Y.D.; Abeyewickreme, W.The global incidence of dengue fever has increased by more than four-folds over the last 30 years, making it the most threatening mosquito-borne viral disease at present. Objective of this study is to determine the role of silent transmission •on' incidence of dengue. A total of 40 households, living within a 300 m radius of seven selected ‘confirmed dengue cases at different dengue high risk localities in Gampaha District were recruited for this study. A minimum of three inhabitants were tested for anti-dengue antibodies using a commercial kit to determine the prevalence of silent transmission on dengue infection in each households. Entomological surveillance was carried out in all seven localities. Out of 40 households, 26 (65%) houses were positive for dengue viral infection. Total of 148 inhabitants (68 Males); mean age; 35.9 years were enrolled. Of the 148, 41 (27.7%) had evidence of exposure to dengue virus [positive for IgM: 28/41(68.4%), IgM'& IgG: 7/41(17%) and IgG: 6/41(14.6%)]. Out of 28 primary infections, 20(71.4%) were asymptomatic. Of the 7 secondary infections, 1(14.28%) was asymptomatic. Of the 6 previous exposures to dengue 4(66.67%) were asymptomatic. Of the 7 localities investigated, 1 had >50%, 4 had >25% and 2 had <25% clustering of cases. Ae. albopictus found in all seven localities and Ae. aegypti found only in two localities. This study suggests presence of silent transmission of dengue virus with a trend towards clustering around cases and also this shows that the presence of vectors increases the incidence of dengue.Item Detection of dengue viruses in vector mosquitoes collected from localities with reported dengue cases in the Gampaha District, 2008-2009(Sri Lanka Association for the Advancement of Science, 2010) Hapugoda, M.D.; Abeyewickreme, W.; Peris, I.; Warakagoda, S.; Perera, V.; Weerasinghe, I.Dengue is an important mosquito-borne flaviviral infection in Sri Lanka. Aedes aegypti is considered as the predominant vector of dengue and Ae. albopictus is considered as a subsidiary vector of dengue in Sri Lanka. Detection of dengue virus in these two vector species is important for control activities. Objective of this study was to examine transmission of dengue viruses by Ae. aegypti and Ae. albopictus mosquitoes in the field. Hundred dengue patients confirmed by both clinically and serologically were selected. Mosquito surveillance was conducted by visiting patient's premise within one week of notification of a case. A buffer zone covering 500 m radius in each confirmed patient's premise was surveyed. Larvae and adults were collected using standard larval surveillance techniques and a back-pack aspirator respectively. Adult and larvae were pooled (1-50 larvae/adult per pool). Single step single tube R -PCR assay followed by Semi-Nested-PCR agarose gel electrophoresis was performed. Ethical permission for this study was obtained from the Ethical Review Committee of the Faculty of Medicine, University of Kelaniya. Informed written consent was obtained from each patient for conducting mosquito surveillance at his/her premise. From 100 confirmed dengue case reported stations, 10% (10/100) and 60% (60/100) pools of Ae. aegypti and Ae. albopictus larvae were collected respectively. Adult mosquito collection showed 8% (8/100) and 30% (30/100) of Ae. aegypti andAe.albopictus respectively. These results showed high density of Ae. albopictus mosquitoes. Dengue 3 viral infection was detected in 1 and 7 pools of Ae. aegypti and Ae. albopictus respectively. Transmission of dengue serotype 3 virus by Ae. albopictus and high density of this species in selected areas were observed. Although Ae. aegypti is still the main vector initiating outbreaks and transmitting the disease in city centers, the possibility of Ae. albopictus playing a major role in this region should not be ignored.Theseadultmosquitoes may acquire dengue viruses through horizontal or vertical transmission. Alargenumber of mosquito pools needed to test for confirmation.Acknowledgements: Financial assistance from the WHO/SEARO (WHO/SEARO SN1167)and technical assistance from the IAEA (IAEA TC SRL TC 5/042) are gratefullyacknowledged.Item Knowledge, attitudes and practices (KAP) on dengue control in Gampaha district.(Sri Lanka Association for the Advancement of Science, 2008) Abeyewickreme, W.; Hapangama, H.A.D.C.; Gunawardene, Y.I.N.S.; Hapugoda, M.D.; Gunawardena, N.K.; Wickremasinghe, A.R.Dengue/Dengue Hemorrhagic Fever (DHF) has become a major public health problem in many parts of the tropics. In Sri Lanka, it is endemic in some parts of the country with outbreaks of dengue/ DHF. The present study was done to assess 1) knowledge regarding dengue among the general population in the district of Gampaha, 2) whether simple preventive measures are being practiced in the community. A cross-sectional survey was conducted in selected 2000 households in the Gampaha District from June - August, 2007 using a pre-tested structured questionnaire to assess the level of knowledge, attitudes and practices regarding dengue. The majority of the respondents interviewed were females (65.2%). More than 90% have had secondary education (90.2%). The main source of water supply were pipe borne (43.4%) and well water (40.6%). 64.3% households stored water for washing, drinking and cleaning purposes. More than 95% of the respondents had heard about dengue fever and its transmission. 91.3% cited that their main source of information on dengue was from television/radio. 34.3% had either received advice or participated in a training programme on dengue prevention and 7.8% had received support materials. The main preventive measure used to reduce the mosquito nuisance was personal protection with repellents (73.6%). Most of the respondents (56.8%) felt that no action was taken by the government to control mosquitoes. When respondents views were taken, the suggestions made by them to improve dengue control included, fogging (31.8%), educating people (30.5%), treating water (24.3%) and cleaning the environment (19.5%). When questions were directed at possible methods for community participation for dengue control, the majority were in favour of removing solid waste (84.15%), eliminating stagnant water collections (40.7%), removing larvae (8.75%) and covering all the water containers using lids (8.1%). The community had good understanding on dengue and the main source of information was from the electronic media. However it was found that good knowledge itself does not necessarily lead to good practices. The respondents' attitudes were found to be good and most of them were supportive of control measures. Mass media is an important means of conveying health messages to the public, thus research and development of educational strategies designed to improve behaviour and practices of effective control measures through mass media among the community are recommended. Financial support by World Health Organization TDR/WHO Multi Country Study on "Eco-Bio-Social Research on Dengue in Asia" is acknowledged.