Browsing by Author "Dassanayake, R.S."

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Absence of Wolbachia endobacteria in Sri Lankan isolates of the nematode parasite of animals Setaria digitata
(Elsevier, 2015) Voronin, D.; Abeykoon, A.M.; Gunawardene, Y.I.N.S.; Dassanayake, R.S.
Setaria digitata is an animal filarial parasite with natural hosts of cattle and buffaloes that causes mild disease conditions. Infection of non-permissive hosts such as goats, sheep and horses, by this nematode can cause cerebrospinal nematodiasis that leads to lumbar paralysis and the eventual death of the animals and inflicts considerable economic losses on livestock farmers. Wolbachia are obligate mutualistic endosymbionts for some filarial nematodes and are currently being targeted for the control of diseases caused by these parasites. However, little is known about the occurrence of this endosymbiont in the Setariidae family. In this work, worms collected from infected cattle in Sri Lanka were morphologically identified as S. digitata and tested for the presence of Wolbachia by PCR screening using the WSP- and Wolbachia-specific 16S rRNA and multilocus sequence typing primers that were designed to amplify the gatB, coxA, hcpA, ftsZ and fbpA sequences of Wolbachia. The presence of endobacteria in S. digitata was also examined by whole-mount immunofluorescence staining of the parasites and transmission electron microscopic studies. These analyses did not produce evidence of presence of Wolbachia or any other endosymbiotic bacteria in S. digitata, whereas such evidence was found in Brugia malayi, which was used as a positive control in this study. Copyright © 2014 Elsevier B.V. All rights reserved
Advances in Aedes mosquito vector control strategies using CRISPR/Cas9
(Springer, 2021) Wickramasinghe, P.D.S.U.; Silva, G.N.; Gunawardene, Y.I.N.S.; Dassanayake, R.S.
Advancements in genetic engineering have resulted in the development of mosquitoes with impaired vector competence, thereby limiting acquisition and transmission of pathogens. The main dengue (DENV) vector, Aedes aegypti, is an invasive species that have spread unwittingly across the world as a result of human trade and travel. The Ae. aegypti mosquito species has spread across tropical and subtropical regions, with higher presence in urban regions where rapid breeding patterns have shown in artificial containers. Identification of and treating an adequate number of mosquito breeding sites as a control measure have been done for the past couple of years, and yet improvement is far from the expectations, even with well-funded and well-organized initiatives. In order to stop the pathogen transmission, genetically modified mosquitoes (GMM) needs to be created and released. Despite many Aedes-related achievements, GMM creation has been challenging. The spread of particular genetic elements that impair vector competence, trigger deleterious recessive mutations, or skew a population's sex ratio can be used to prevent the spread of vector disease, or eradicate invasive organisms in a species-specific and eco-friendly manner. In recent years, genome editing strategies have evolved to make use of a variety of nucleases, ranging from sequence-specific zinc finger nucleases to modular TALENs (transcription activator-like effector nucleases) and most recently, RNA-guided nucleases adapted from bacterial adaptive immune systems, dubbed CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR associated systems). By combining these methods, a new era in gene editing had emerged. Generally, both of these gene editing technologies utilize sequence-specific nucleases to generate double-stranded DNA breaks (or nicks) in the target sequence, resulting in desired DNA modifications using endogenous DNA repair mechanisms. Since cells with DNA lesions are unable to divide further, the nuclease-generated strand breaks must be rapidly repaired by the cell to maintain the viability. CRISPR/Cas has been widely accepted for use in a variety of organisms, including insect species, with only minor optimization steps needed thus far. CRISPR/Cas9 technology transformed the process of engineering nucleases capable of cleaving complex genomic sequences. A complementary guide RNA (gRNA) directs the Cas9 endonuclease's operation to the specific DNA target site, enabling the editing of virtually any DNA sequence without complex protein engineering and selection procedures. Apart from genome editing, the specificity and flexibility of the CRISPR/Cas9 method enables unprecedented rapid development of genetically modified organisms with mutation systems for disease vector insect control. The stability and expression of the gene construct generated by CRISPR/Cas9 or any other method must be addressed before GMM are released, in order to make sure that pathogen transmission and formulation are interrupted robustly and completely. Spreading foreign antipathogen genes through gene drive strategies among wild mosquito populations strengthens the case for a more streamlined approach. Major fields that must be adequately assessed include risk evaluation and management, conducting studies to ensure human and environmental protection, developing effective control strategies built on comprehensive gene-driving systems, and adequately addressing the ethical, legal, and social consequences of GMM release. Although GMM is theoretically feasible as a disease control method, field releases should be made only when strong scientific evidence of human and environmental protection and effectiveness are presented, and public acceptance is addressed appropriately. This chapter discusses the diverse technological advances in generating Ae. aegypti mosquitoes which are resistant to dengue virus (DENV) and other diseases, as well as the biosafety and risk assessment of these procedures. Additionally, the chapter outlines a convincing path forward for developing successful genetic-based DENV control strategies based on CRISPR/Cas9, which could be expanded to control other arboviruses while maintaining biosafety.
An ARV1 homologue from a filarial nematode is functional in yeast
(London School Of Hygiene And Tropical Medicine, 2019) Herath, H.M.L.P.B.; Gunawardene, Y.I.N.S.; Pathiranage, M.; Wickramasinghe, P.D.S.U.; Wickramatunga, P.G.T.S.; Dassanayake, R.S.
The transmembrane protein, ARV1, plays a key role in intracellular sterol homeostasis by controlling sterol distribution and cellular uptake. To date, only the ARV1s from yeast and humans have been characterized to some extent. In this study, the ARV1 of an animal filarial parasite, Setaria digitata (SdARV1), was characterized; its cDNA was 761 bp and encoded a protein of 217 amino acids, with a predicted molecular weight of 25 kDa, containing a highly conserved ARV1 homology domain and three transmembrane domains in the bioinformatic analyses. Information required to cluster members belonging to a particular taxon has been revealed in phylogenetic analyses of ARV1 sequences derived from different organisms. Reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that SdARV1 was expressed in different developmental stages - microfilariae and adult male and female worms. Experiments carried out with a single copy of the SdARV1 under the control of the PMA-1 promoter in a temperature-sensitive Saccharomyces cerevisiae mutant strain indicated full complementation of the mutant phenotype, with growth at a non-permissive temperature (37°C). Microscopic observations of cellular morphology with Gram staining revealed alteration of the shape from shrunken to oval, in mutant and complemented strains, respectively. Assessment of free sterol levels extracted from mutant yeast and complemented strains indicated that the level of sterol was significantly higher in the former compared to the latter, which had sterol levels similar to those of the wild type. Thus, the results of the current study suggest that SdARV1 is ubiquitously expressed in different developmental stages of S. digitata, and that it is a true functional homologue of mammalian and yeast ARV1s, which have crucial phylogenetic information that follows classical evolutionary trends. Finally, this is the first study to report the biological function of nematode ARV1.
Assessment of developmental and reproductive fitness of dengue-resistant transgenic Aedes aegypti and Improvement of fitness using antibiotics
(Hindawi Pub. Co., 2021) Ramyasoma, H.P.B.K.D.; Gunawardene, Y.I.N.S.; Hapugoda, M.; Dassanayake, R.S.
BACKGROUND: Genetic modification offers opportunities to introduce artificially created molecular defence mechanisms to vector mosquitoes to counter diseases causing pathogens such as the dengue virus, malaria parasite, and Zika virus. RNA interference is such a molecular defence mechanism that could be used for this purpose to block the transmission of pathogens among human and animal populations. In our previous study, we engineered a dengue-resistant transgenic Ae. aegypti using RNAi to turn off the expression of dengue virus serotype genomes to reduce virus transmission, requiring assessment of the fitness of this mosquito with respect to its wild counterpart in the laboratory and semifield conditions. METHOD: Developmental and reproductive fitness parameters of TM and WM have assessed under the Arthropod Containment Level 2 conditions, and the antibiotic treatment assays were conducted using co-trimoxazole, amoxicillin, and doxycycline to assess the developmental and reproductive fitness parameters. RESULTS: A significant reduction of developmental and reproductive fitness parameters was observed in transgenic mosquito compared to wild mosquitoes. However, it was seen in laboratory-scale studies that the fitness of this mosquito has improved significantly in the presence of antibiotics such as co-trimoxazole, amoxicillin, and doxycycline in their feed. CONCLUSION: Our data indicate that the transgenic mosquito produced had a reduction of the fitness parameters and it may lead to a subsequent reduction of transgenic vector density over the generations in field applications. However, antibiotics of co-trimoxazole, amoxicillin, and doxycycline have shown the improvement of fitness parameters indicating the usefulness in field release of transgenic mosquitoes.
Bioinformatics and DNA micro-arrays in post genomic analysis
(Author, 2009) Dassanayake, R.S.; Gunawardene, Y.I.N.S.
No abstract available
Clinical utility of PCR and real time PCR assays for Cytomegalovirus, hepatitis B and hepatitis C infections.
(Sri Lanka Association for the Advancement of Science, 2008) Dassanayake, R.S.; de Silva, P.; Weerasena, J.; Gunawardene, Y.I.N.S.; Manamperi, A.
Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, Reactivation of cytomegalovirus (CMV), Hepatitis B (HBV) and C (HCV) viruses from the status of latency is seen in immunocompromised individuals and such reactivation is often associated with morbidity and mortality in such individuals. The prevalence of these viral infections in a selected population of patients referred to the Molecular Diagnostic Laboratory at the Durdan's Hospital, Colombo, during the period from August 2007 to May 2008 were studied using qualitative PCR assays. All specimens from patients with suspected clinical diagnoses of either CMV or HBV or HCV infections were analyzed. Of 176 samples analyzed for CMV 78 were positive (37 males, 29 females) and majority of them are patients from a nephrology unit. Out of 40 and 10 samples analyzed from males and females, respectively, 22 and 4 were positive for HBV. Twenty six samples were analyzed for HCV and only 6 were fond to be infected with viruses and all of them were from males. Although PCR detection of these viral DNA/RNA is a sensitive method to detect infection, it lacks specificity for the detection of active viral disease and for monitoring the efficacy of antiviral therapy. Therefore, Real-time PCR (RT-PCR) assays for the detection and quantification of CMV-DNA, HBV-DNA and HCV-RNA were developed using SYBRgreen1 chemistry. The assays developed are capable of detecting viral particles in blood samples and quantifying viral DNA accurately over a broad range of input target copies (102 - 108copies/ml) and therefore, can be used to predict the reactivation of viruses by comparing with published kinetic criteria in clinical guidelines. Post PCR analyses of Real-time PCR products by agarose gel electrophoresis revealed bands having the same intensity for a wide range of target copies (103 -108copies/ml). In contrast, RT-PCR elicited higher cycle threshold for the descending order of concentration of target copies. Therefore, based on these results, it is evident that the intensity of conventional PCR bands should not be used for the assessment of viral reactivation or for monitoring therapeutic intervention and for this purpose RT-PCR is the method of choice
Could quantitative real-time polymerase chain reaction assay serve as an alternative test method to evaluate human epidermal growth factor receptor 2 status of gastric carcinoma in the South Asian setting?
(Indian Society of Gastroenterology/Springer India, 2019) Kannangara, D.K.S.; Lokuhetty, M.D.S.; Subasinghe, D.; Gunawardene, Y.I.N.S.; Dassanayake, R.S.
BACKGROUND:Human epidermal growth factor receptor 2 (HER2) protein overexpression and/or HER2 gene amplification are/is linked to a dismal outcome of gastric carcinoma (GCa). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are key methods to identify patients for HER2 targeted therapy. Drawbacks of both the methods warrant novel tests. Hence, we evaluated the value of quantitative real-time polymerase chain reaction (qPCR) as an alternative test method, relative to IHC to detect HER2 status of GCa and to find relationship between these results with demographic/clinicopathological data.METHOD:Twenty GCa patients with known IHC HER2 scores were evaluated. qPCR was performed for the HER2 gene and amyloid precursor protein (reference gene) in formalin-fixed paraffin-embedded GCa tissue. Cycle threshold values (Ct) were analyzed using the Pfaffl method to detect HER2 gene amplification.RESULTS:HER2 positivity rates by IHC and qPCR were 20% and 35%, respectively. The sensitivity and specificity of qPCR were 67% and 76%, respectively, relative to IHC. qPCR results were reproducible. The diagnostic consistency between IHC and qPCR (κ = 0.146) was slightly agreeable (0.01 < k < 0.20), with a 65% concordance. Based on McNemar's test, there was no significant difference between the results of the two tests. IHC HER2 protein expression had relationship with the tumor (TNM) stage and Lauren histological type (p < 0.05). Positive HER2 gene expression by qPCR showed relationship with depth of invasion, lymph node involvement, and degree of differentiation (p < 0.05).CONCLUSION:Cost-effective qPCR could serve as an alternative test method for detection of HER2 status of GCa. Both HER2 overexpression by IHC and gene amplification by qPCR are associated with adverse clinicopathological features
Determination of appropriate positioning of the ovitraps for dengue mosquito surveillance
(Faculty of Graduate Studies, University of Kelaniya, 2015) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.
Three months ovitrap survey was conducted to assess the suitable position in placing the ovitraps for dengue vector mosquito surveillance and this study was initiated due to loss of valuable data from our previous studies as a result of physical damage of the ground kept ovitarps. Thirty four households in the Ragama Medical Officer of Health area in Gampaha District were selected to conduct the ovitrap survey during the period of May to July, 2015 to select the most appropriate positioning of the ovitrap. The conventional black plastic ovitraps (3.2x 2.7 cm) were used in this purpose to collect aquatic stages of Aedes mosquitoes while placing plywood paddle (4 x 0.5 cm) over the upper rim of each coded ovitrap. A total of 136 ovitraps were used in the study site providing four ovitraps (2 each indoor & outdoor) for each house while one of the ovitraps of indoor and outdoor being hung and other being kept on the ground. In positioning ovitraps, the outdoor ones were kept 3m away from the house while leaving indoor ovitraps in the living room in close proximity to racks/hanging clothes or partially shaded places. Following collection of samples at each week, ovitraps were washed thoroughly, refilled with new water and a new paddle, and corresponding data were recorded and analyzed. These analyses revealed that number of larvae and the number of Aedes mosquito eggs present in the two different ovitrap positions (Ground kept vs Hung) were not significantly different; in spite of significant difference (P=0.001) between the outside and inside placements. Further, significantly higher values were observed for both number of mosquito eggs and larvae present in each ovitrap kept outside (60 and 13 respectively) than those placed inside (32 and 3 respectively). Furthermore, slightly higher values were observed for hung ovitraps (49 and 9 respectively) than ones kept on the ground (43 and 7 respectively). Finally, ovitrap placed above the ground level was selected in continuing the routine ovitrap survey, as there was considerable reduction of mechanical damage to the latter thus facilitating continuous data collection.
Development of a quantitative PCR assay to evaluate HER2 status of Gastric carcinoma in a cohort of Sri Lankan patients
(Sri Lanka Medical Association, 2016) Kannangara, D.K.S.; Subasinghe, D.; Lokuhetti, M.D.S.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.
INTRODUCTION AND OBJECTIVES: Human epidermal growth factor receptor2(HER2) protein overexpression and/or HER2gene amplification is linked to dismal outcome of Gastric carcinoma(GCa). Immunohistochemistry(IHC) and fluorescence in situ hybridization(FISH) are key-methods to identify patients for HER2 targeted therapy. Drawbacks of both methods warrant novel tests. The study aimed to determine whether quantitative Polymerase Chain Reaction (qPCR) could serve as a supplementary-method to evaluate HER2 status of GCa in a cohort of Sri Lankan patients and investigate correlation between HER2 assessed by different methods and clinic-pathological features. METHOD: Twenty GCa-patients with known IHC-HER2 scores were evaluated. qPCR was performed for HER2gene and Ameloid precursor protein (reference gene) in Formalin fixed paraffin embedded GCa tissue. Threshold values(Ct) were analyzed using Pfaffl-method to detect HER2gene amplification. RESULTS: HER2positivity by IHC(protein) and qPCR(gene) were 20% and 35% respectively. Sensitivity and specificity of qPCR was 67% and 76% respectively and results were reproducible. HER2protein positivity was correlated with Tumour TNM-stage and Lauren-histological types(P<0.05). Positive expression of HER2gene was correlated with depth of tumour invasion, differentiation and Lymph node-status(P<0.05). Diagnostic consistency between IHC and qPCR(κ=0.146) was slightly agreeable(0.01
Development of modified mismatch PCR-RFLP to screen mutations in codon 12 and 13 of K- ras gene of colorectal (CRC) patients in Sri Lanka
(Sri lanka Medical Association, 2015) Dhilhani, M.F.F.; de Zoysa, M.I.M.; Chandrasekharan, N.V.; Gunawardene, Y.I.N.S.; Lokuhetti, M.D.S.; Dassanayake, R.S.
INTRODUCTION AND OBJECTIVES: Mutations in K-ras codon 12, 13 of exon 2 are known to affect prognosis and impart resistance to anti EGFR monoclonal antibody therapy in CRC. Although several diagnostic tools have been developed for K-ras mutation testing, these procedures are too expensive or time consuming. Oufaim was to develop an effective, reliable and inexpensive method for the detection of K-ras mutations in codons 12 and 13 of exon 2 in CRC patients in Sri Lanka, and to relate the mutational status to liver metastasis, METHOD: The mismatch PCR-RFLP was developed and used to screen mutations in codon 12 and 13 for DMA isolated from paraffinized tumour tissue of 30 CRC patients followed up for 5 year after surgery to detect liver metastasis. Cross-tabulations were generated between K-ras mutations and the metastatic status. The Chi Square test was used to indicate statistical significance of the association. RESULTS: Analysis of banding pattern obtained from restriction digestion of PCR amplified region containing codon 12 and/or 13 of KRAS gene of 14(46.6%) CRC patients revealed the presence of mutations. Of the 30 patients, 13(43.3%) had developed liver metastases. There was a significant association between the presence of a K-ros mutation and the occurrence of liver metastasis (X2=4.693, p=0.003). CONCLUSION: This mismatch PCR-RFLP protocol is a suitable method to screen codon 12 and 13 mutation of K-ros gene to predict liver metastasis. Presence of these mutations is associated with the occurrence of liver metastasis during the first 5 years after surgery.
Development of single-strand conformational polymorphism to screen for mutations in hotspot regions of beta-globin gene of beta-thalassemia patients of Sri Lanka
(SEAMEO Regional Tropical Medicine and Public Health Project, 2013) Dassanayake, R.S.; Mahadevan, K.; Gunawardene, Y.I.N.S.
Beta-thalassemia is prevalent in Sri Lanka and imposes a heavy economic and social burden in the country due to the patients' life-long need for regular blood transfusion and treatment with iron chelation therapy. Thus, there is a need to develop a rapid, reliable and effective population-based presymptomatic and prenatal screening method for beta-thalassemia. Single strand conformational polymorphism (SSCP) technique was developed as an adjunct for the previously developed allele-specific PCR (ASP) technique to screen the presence of mutationsin beta-globin gene. A hotspot region of beta-globin gene containing 98% of known beta-thalassemia mutations was amplified from 24 clinically diagnosed beta-thalassemia patients and two normal individuals. Two overlapping amplicons of 238 bp and 268 bp were subjected to SSCP analysis. The SSCP banding patterns of these two fragments from beta-thalassemia patients were different from the corresponding regions of normal individuals. Sequence analysis of these regions revealed the presence of 4 mutations in the form of deletion and substitution that have not been reported previously from Sri Lanka. Therefore, the SSCP protocol developed in this study together with ASP should provide an appropriate screening approach for presymptomatic and parental diagnosis of beta-thalassemia in the Sri Lankan population.
Development of siRNA mediated RNA interference and functional analysis of novel parasitic nematode-specific protein of Setaria digitata
(Academic Press, 2018) Somarathne, M.B.C.L.; Gunawardene, Y.I.N.S.; Chandrasekharan, N.V.; Dassanayake, R.S.
Despite the differences of the host, parasitic nematodes may share commonalities in their parasitizing genes. Setaria digitata novel protein (SDNP) is such an entity which is parasitic nematode-specific and having sequence similarities with those of W. bancrofti, B. malayi, Loa loa and Onchocerca volvulus. Post-transcriptional gene silencing by siRNA mediated RNA interference (RNAi) is a widely used technique in functional genomics. Though the technique has been used in several free-living, plant and animal parasitic nematodes, it has not yet been tried out for the filarial worm S. digitata. In this study, we developed an effective siRNA delivery method by microinjection and utilized the siRNAi tool to knockdown SDNP to study the phenotypic and cellular changes associated with the interference. qPCR analysis revealed, a significant reduction of SDNP transcript levels following siRNA microinjection into S. digitata adult worms. Similarly, immunohistochemical staining indicated a reduction of SDNP protein expression. Furthermore, worms treated with siRNA showed a significant reduction of microfilariae release together with embryonic lethality by arresting an early developmental stage compared to non-treated worms. A distinct motility reduction was also observed in treated worms compared to non-treated counterparts. This is the first report of the amenability of S. digitata to the siRNA induced RNAi. The presence of inter-domain linkers of muscle-specific twitchin kinase and calcium-dependent protein kinase isoform CDPK1 together with what our results revealed suggest that SDNP is most likely a protein involved in muscle movement and growth and development of the nematode. Hence SDNP has the characteristics of a potential drug target.
Engineering miR-shRNA based molecule to interfere replication of dengue virus in transgenic Aedes aegypti mosquitoes: Bioinformatics approach
(Moleclar Medicine Unit, Faculty of Medicine, University of Kelaniya, Sri Lanka, 2015) Ramyasoma, H.P.B.K.D.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.; Kajan, M.; Abeyewickreme, W.
BACKGROUND: The genus Flavivirus of the family Flaviviridae includes several vector-borne viruses to which the four serotypes of dengue viruses (DENV-1,-2,-3 and 4) belong to and DENV viruses have a messenger like positive polarity, single-stranded RNA genome approximately 11kb in length which encodes three structural proteins (C-prM-E) and seven Non-Structural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). RNA interference (RNAi) and its properties as a tool has heralded a new era in functional genomics and short double stranded RNAs mediated by RNAi has become a powerful tool for post transcriptional gene silencing. Therefore, this study took the advantage the latter biological phenomenon and designed a multiple miR-shRNA (multi-mir-shRNA) molecule using bioinformatic approach to be effective to block the replication of all dengue serotypes of Sri Lanka. METHODS: Genome sequences of DENV strains belonged to serotypes 1 and 3 isolated from Sri Lanka deposited in GenBank were analyzed for potential sequences for the best siRNA target sites and identified two such sites from DENV 1 and DENV 3 from non structural protein coding sequence of NS5 and structural protein coding sequence of prM consensus regions, respectively. Two more siRNA targets reported from previous study chosen from upstream and downstream of non coding region effective for silencing all DENV serotypes were also used in designing mir-shRNA sequences. The stem region of miR1175 pre-miRNA sequence (miRBase ref: MI0013470) of Aedes aegypi was then replaced with each selected siRNA targets to generate the DENV effective miR-shRNAs and generated miR-shRNAs connected together by placing restriction endonuclease sites between each other to obtain multi-mir-shRNA containing four loop and stem structures. The transgenic gene cassette containing Ae. aegypti carboxypeptidase A promoter, multi-miR-shRNAs and poly adenylation signal of Simian Virus 40 Major Capsid VP1 was then constructed. Expression of this effecter molecule can be achieved by the induction of Ae. aegypti carboxypeptidase A (AeCPA) promoter following blood meal which then ensures activation of RNAi at the time of virus enter into the midgut of mosquito. RESULTS: Folding patterns of the transcript of the designed multi-mir-shRNA cluster were analyzed using online bioinfomatic tool, mfold and the secondary structure of this transcript shown to have optimum endogenous miRNA cleavge/processing with the lowest -ΔG indicating the ability of this design to exert RNAi in mosquito Ae. aegypti. CONCLUSION: Designing multi-miR-shRNA in bioinformatic means an effective way to construct the effector molecule that could exert the maximum RNAi against DENV. However, the effect of design will have to be demonstrated first by transforming to Ae. aegypti mosquitoes and then by evaluating the inhibition of DENV replication in mosquito.
Engineering RNA interference-based dengue virus resistance in the mosquito vector aedes aegypti: The current status and future directions
(Springer, 2021) Denipitiyage, S.D.; Gunawardene, Y.I.N.S.; Federico, Z.; Dassanayake, R.S.
Dengue is an acute, febrile disease caused by the dengue viruses (DENV) comprising four serotypes and transmitted by the mosquito vector Ae. aegypti. DENV are single-stranded, positive-sense RNA viruses of the family Flaviviridae. Dengue is declared as a current significant challenge in the Southeast Asia, imposing growing burden on infected populations. To date, dengue control has mostly relied on vector control strategies which have largely become ineffective. There is, therefore, an urgent need for novel vector control strategies. Development of genetically modified mosquito vectors to manipulate disease-vectoring populations has gathered increased interest in recent time. RNAi-mediated viral resistance contributes to the suppression of viruses, including DENV in the mosquito vector Ae. aegypti. With recent advances in the field of molecular biology, we and other scientists are continuing to engineer genes that confer virus resistance to reduce transmission rates of DENV and introducing these genes into the mosquito genome. Even though scientists successfully generated mosquito refractory to DENV2–4, no mosquito refractory to all four serotypes has been developed to date. This limitation can be overcome by systematic analysis of the molecular mechanisms of RNAi in the mosquito vector Ae. aegypti. An enhanced understanding of RNAi function in the mosquito vector Ae. aegypti will facilitate the application of RNAi to control the transmission of the dengue disease in the future. Here, based on current understanding of the RNAi, we discuss the mechanisms of RNAi in the mosquito vector Ae. aegypti. We also provide guidelines for optimal design of RNAi experiments in Ae. aegypti with the possible risks associated with them along with proposed solutions.
Evaluating novel effective primers to amplify heterozygous alleles of second, third and fourth exons of HLA-A;-B;-C;-DRB1 and-DQB1 loci using sequencing-based typing.
(National Science Foundation, 2019) Perera, P.C.D.; Upamali, B.D.N.; Gunawardene, Y.I.N.S.; Dassanayake, R.S.
ABSTRACT: Human leukocyte antigen (HLA) typing is one of the most crucial steps that determines the success of an organ transplant. However, HLA typing is a challenging task due to the diversity of HLA alleles, which is caused by high polymorphism of the region and high number of guanine and cytosine bases that limits the degree of amplification. Low resolution serology typing that is currently employed in Sri Lanka may fail to identify subtle differences in certain alleles, which may affect the long-term survival of the organ recipient. Therefore, a low cost, high-resolution DNA-based typing method for the HLA loci of Sri Lankans was developed based on polymerase chain reaction (PCR) amplification followed by Sanger sequencing, which is considered to be the gold standard for HLA typing. With minimised PCR bias and equal chances of amplifying all the alleles curated so far, a novel set of primers were designed to amplify the second and third exons of alleles in group specific PCR. To increase the resolution of alleles further, the fourth exon was also amplified using novel primers designed in this study and primers reported in the literature. Touchdown PCR and hot-start PCR were used to optimise PCR conditions so that non-specific amplifications are minimal. SBTengine® (version 3.12.0.2724) software was used in assigning the sequence chromatogram to the allele sequence. Seventeen new primers were designed in this study to ensure the amplification and identification of both alleles in heterozygous individuals that were previously unable to be identified using primers reported in the literature. © 2019, National Science Foundation. All rights reserved.
Evaluation of the effects of Aedes vector indices and climatic factors on dengueincidence in Gampaha District, Sri Lanka
(Hindawi Publishing Corporation, 2019) Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Udayanga, N.W.B.A.L.; Abeyewickreme, W.
Constant monitoring of Aedes vector indices such as Aedes mosquito abundance and ovitrap data is important for the control of dengue epidemics. Therefore, the current study attempted to evaluate the effect of larval and climatic factors on the incidence of dengue outbreaks in the Gampaha district. Based on the distribution of previously reported dengue cases, 34 households in Narangodapaluwa PHI area, Ragama, Sri Lanka, were selected randomly, and entomological surveillance was done fortnightly using adult mosquito catches and larval surveillance techniques for a period of two years. Further, weekly ovitrap surveillance was conducted for one year, by maintaining four ovitraps in a single house, two indoors and two outdoors at ground and at a height of 1.5-2 m. Based on the findings, larval indices, namely, Breteau index (BI), House index (HI), and Container index (CI), were calculated, along with the Ovitrap index (OI). The study area was positive for Ae. albopictus with an adult capturing range of 1~15/34 households. BI initially remained < 3%, which subsequently decreased up to 0. No significant difference in OI was found between the ovitraps placed at ground level and at a height of 1.5-2m (p>0.05), 95% level of confidence. The OI varied from 56.9% to 94.7% during the study period of 12 months, indicating two peaks at the monsoons. Statistics of one-way ANOVA revealed a significant difference in the monthly OI during the study period (p≤0.001) with two peaks representing the monsoonal rainfall patterns. Pearson's correlation analysis revealed that the association between dengue cases and larval indices (BI, CI, HI, and OI) and meteorological parameters was not significant (p<0.05). Migration of mosquitoes and patients could be considered as possible factors affecting the absence of a significant relationship.
Evaluation of the Pyrethroid Resistance based on Voltage-Gated Sodium Channel (VGSC) Mutations in Aedes aegypti populations of Colombo, Gampaha and Kandy Districts in Sri Lanka
(International Postgraduate Research Conference 2019, Faculty of Graduate Studies, University of Kelaniya, Sri Lanka, 2019) Ranathunge, T.; Udayanga, L.; Sarasija, S.; Karunathilaka, S.; Nawarathne, S.; Rathnarajah, H.; Dulficar, F.F.; Shafi, F.N.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.
Many countries focus on chemical based vector control strategies to restrict the disease transmissions, where pyrethroid insecticides are widely used as the first line of defense against Ae. aegypti. However, the constant use of insecticides have proven to induce insecticide resistance in mosquitoes. The knockdown resistance (kdr) occurs due to mutations in the Voltage Sensitive Sodium Channel (VSSC) or mutations in the Voltage-Gated Sodium Channel (VGSC), coded by the VSSC gene. Only three kdr mutations namely, the V1016G, S989P, and F1534C have been confirmed as commonly occurring amino acid substitutions among mosquito populations in Southeast Asia. Therefore, to extend this observation, current study was conducted to evaluate the prevalence of V1016G and F1534C mutations among Ae. aegypti mosquito populations in three different geographical regions of Sri Lanka. Immature (both pupae and larvae) stages of Ae. aegypti mosquitoes were collected from Colombo, Gampaha and Kandy districts from March to December 2018 and samples were transported to the Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya. A total of 855 Ae. aegypti larvae were collected from all districts and polymerase chain reaction (PCR) assay for molecular genotyping of mutations was performed for collected all Ae. aegypti larvae (III instar), to identify the prevalence of kdr mutations in the three Ae. aegypti populations. The frequencies of the resistant and susceptible kdr alleles were determined by using the Hardy–Weinberg Equilibrium for each of the point mutation. The Ae. aegypti populations from Colombo, Gampha and Kandy districts showed 40.07% (123/307), 39.58% (114/288) and 19.58% (47/240) of V1016G and F1534C mutations, respectively. The wild type (RR) genotype remained predominant within all the three districts, whereas the homogenous (SS) mutation genotype occurred only in minority. Further, the F1534C was predominant in Ae. aegypti populations of all districts. Among the kdr mutation population, heterogeneous genotyping (RS) for both V1016G and F1534C was prominent, while SS genotyping for V1016G mutation was not observed in the Kandy district. The findings clearly denote that long-term insecticide applications and multiple use of pyrethroids has led to the progression of insecticide resistance among local Ae. aegypti populations. Therefore, evaluation of the prevalence levels of these kdr mutations highlights the necessity for shifting towards novel vector control strategies
Evolutionary selective trends of insect/mosquito antimicrobial defensin peptides containing cysteine-stabilized alpha/beta motifs
(Elsevier, 2007) Dassanayake, R.S.; Gunawardene, Y.I.N.S.; Tobe, S.S.
Insect defensins containing cysteine-stabilized alpha/beta motifs (Cs-alpha/beta defensin) are cationic, inducible antibacterial peptides involved in humoral defence against pathogens. To examine trends in molecular evolution of these antimicrobial peptides, sequences similar to the well-characterized Cs-alpha/beta defensin peptide of Anopheles gambiae, using six cysteine residues as landmarks, were retrieved from genomic and protein databases. These sequences were derived from different orders of insects. Genes of insect Cs-alpha/beta defensin appear to constitute a multigene family in which the copy number varies between insect species. Phylogenetic analysis of these sequences revealed two main lineages, one group comprising mainly lepidopteran insects and a second, comprising Hemiptera, Coleoptera, Diptera and Hymenoptera insects. Moreover, the topology of the phylogram indicated dipteran Cs-alpha/beta defensins are diverse, suggesting diversity in immune mechanisms in this order of insects. Overall evolutionary analysis indicated marked diversification and expansion of mature defensin isoforms within the species of mosquitoes relative to non-mosquito defensins, implying the presence of finely tuned immune responses to counter pathogens. The observed higher synonymous substitution rate relative to the nonsynonymous rate in almost all the regions of Cs-alpha/beta defensin of mosquitoes suggests that these peptidesare predominately under purifying selection. The maximum-likelihood models of codon substitution indicated selective pressure at different amino acid sites in mosquito mature Cs-alpha/beta defensins is differ and are undergoing adaptive evolution in comparison to non-mosquito Cs-alpha/betadefensins, for which such selection was inconspicuous; this suggests the acquisition of selective advantage of the Cs-alpha/beta defensins in the former group. Finally, this study represents the most detailed report on the evolutionary strategies of Cs-alpha/beta defensins of mosquitoes in particular and insects in general, and indicates that insect Cs-alpha/beta defensins have evolved by duplication followed by divergence, to produce a diverse set of paralogues
Functional analysis of a novel parasitic nematode-specific protein of Setaria digitata larvae in Culex quinquefasciatus by siRNA mediated RNA interference
(BioMed Central, 2018) Somarathne, M.B.C.L.; Gunawardene, Y.I.N.S.; Chandrasekharan, N.V.; Ellepola, A.N.B.; Dassanayake, R.S.
BACKGROUND: Functional analysis of animal parasitic nematode genes is often quite challenging due to the unavailability of standardised in vitro culture conditions and lack of adequate tools to manipulate these genes. Therefore, this study was undertaken to investigate the suitability of Culex quinquefasciatus, as an in vivo culture platform for Setaria digitata larvae and RNA interference (RNAi), as a post-transcriptional gene silencing tool to study the roles of a vital gene that encodes a novel parasitic nematode-specific protein (SDNP). RESULTS: The red colour fluorescence detected following RNAi injection to the thorax of C. quinquefasciatus indicated the uptake of dsRNA by S. digitata larvae. The reduction of SDNP transcripts in siRNA treated larvae compared to non-treated larvae, as determined by qPCR, indicated that the siRNA pathway is operational in S. digitata larvae. The observation of motility reductions and deformities during the development indicated the association of SDNP in larvae locomotion and development processes, respectively. The irregularities in the migration of larvae in mosquitoes and elevated survival rates of mosquitoes compared to their untreated counterparts indicated reduced parasitism of S. digitata larvae in mosquitoes upon targeted downregulation of SDNP by siRNA treatment. CONCLUSION: SDNP plays vital roles in muscle contraction, locomotion, development processes, larval development and parasitism of S. digitata. Its ubiquitous presence in parasitic nematodes and its absence in their hosts provide a tantalising prospect of the possibility of targeting SDNP for future development of anthelmintic drugs. The susceptibility of the larval stages of S. digitata for RNAi in Culex quinquefasciatus was also demonstrated for the first time in this study.
Genetic improvements to the sterile insect technique (SIT) for the control of mosquito population
(Springer, 2021) Dilani, P.V.D.; Gunawardene, Y.I.N.S.; Dassanayake, R.S.
Mosquito-borne diseases are becoming a major health problem worldwide. At present, the principal method of controlling these diseases entirely depends on the mosquito vector control strategies. However, traditional control methods which are focussed on reducing mosquito populations through environmental management and the application of insecticides are largely ineffective. Hence, various control methods, including the release of sterile insect technique (SIT), have been proposed for the reduction of the mosquito population. As a species-specific control strategy, SIT offers considerable environmental benefits and a chemical-free option for insect control. However, the application of the SIT to mosquito control consistently suffered from lack of efficient sexing system, high fitness cost and operational difficulty in ionizing radiation, density-dependent nature of the target mosquito population and various other technical issues. The intervention of genetic engineering has led to several improvements in the operation or security of SIT programmes. The advent of mosquito transgenesis has paved the way for novel approaches in mosquito control. One possibility is a release of insects carrying dominant lethal (RIDL) strategy by engineering self-limiting gene, which offers solutions for many drawbacks of traditional SIT by providing genetic sterilization, genetic sexing, genetic containment and provision of genetic markers while maintaining its environmentally benign and species-specific utility. The success of this strategy often depends on how genetic modification affects the fitness of the mosquitoes. With several improvements and modifications allowing minimum fitness load, RIDL is now available for a wide range of mosquitoes such as Aedes aegypti, Aedes albopictus and Anopheles stephensi with field-testing possibilities. However, with solid epidemiological evidence and community support, widespread implementation of these strategies might reverse the current alarming global mosquito vector-borne diseases.