Transient transmission of Chikungunya virus in Singapore exemplifies successful mitigation of severe epidemics in a vulnerable population.

OBJECTIVES: Singapore experienced two major outbreaks of chikungunya in 2008-09 and 2013-14. Despite repeated virus introductions, fresh local outbreaks have not emerged after 2014. The present study reviews the success of chikungunya control in Singapore, despite repeated introduction of virus strains, presence of competent vectors and an immunologically naïve population. METHODS: Chikungunya virus (CHIKV) sequences (421 envelope 1 genes and 56 polyproteins) were analysed to distinguish the indigenous virus groups from 2008 to 2020. Vector surveillance data was used to incriminate the vector/s associated with local outbreaks. The population exposure to CHIKV was determined by assessing the seroprevalence status in three cohorts of sera collected in 2009 (n=2,008), 2013 (n=2,000) and 2017 (n=3,615). RESULTS: Four distinct groups of CHIKV of East, Central and South African genotype have mainly circulated since 2008, transmitted primarily by Aedes albopictus. The age weighted CHIKV IgG prevalence rates were low (1-5%) and showed a non-significant increase from 2009 to 2013, but a significant decrease in 2017. In contrast, the prevalence of CHIKV neutralising antibodies in the population increased significantly from 2009 to 2013, with no significant change in 2017, but the levels remained below 2%. CONCLUSIONS: The evidence suggested that surveillance and vector control strategies implemented were robust to avert severe epidemics, despite repeated introduction of virus strains, presence of competent vectors and an immunologically naïve population.

Evolution, heterogeneity and global dispersal of cosmopolitan genotype of Dengue virus type 2.

Dengue virus type 2 (DENV-2) contributes substantially to the dengue burden and dengue-related mortality in the tropics and sub-tropics. DENV-2 includes six genotypes, among which cosmopolitan genotype is the most widespread. The present study investigated the evolution, intra-genotype heterogeneity and dispersal of cosmopolitan genotype to understand unique genetic characteristics that have shaped the molecular epidemiology and distribution of cosmopolitan lineages. The spatial analysis demonstrated a wide geo-distribution of cosmopolitan genotype through an extensive inter-continental network, anchored in Southeast Asia and Indian sub-continent. Intra-genotype analyses using 3367 envelope gene sequences revealed six distinct lineages within the cosmopolitan genotype, namely the Indian sub-continent lineage and five other lineages. Indian sub-continent lineage was the most diverged among six lineages and has almost reached the nucleotide divergence threshold of 6% within E gene to qualify as a separate genotype. Genome wide amino acid signatures and selection pressure analyses further suggested differences in evolutionary characteristics between the Indian sub-continent lineage and other lineages. The present study narrates a comprehensive genomic analysis of cosmopolitan genotype and presents notable genetic characteristics that occurred during its evolution and global expansion. Whether those characteristics conferred a fitness advantage to cosmopolitan genotype in different geographies warrant further investigations.

Gravitrap deployment for adult Aedes aegypti surveillance and its impact on dengue cases.

House Index, Container Index, and Breteau Index are the most commonly used indices for dengue vector surveillance. However, these larval indices are a poor proxy for measuring the adult population-which is responsible for disease transmission. Information on the adult distribution and density are important for assessing transmission risk as well as for developing effective control strategies. This study introduces a new entomological index, Gravitrap aegypti index (GAI), which estimates the adult female Aedes aegypti population in the community and presents its association with dengue cases. Gravitraps were deployed across 34 treatment sites in Singapore from September 2013 to September 2016. The GAI, derived from the Gravitrap surveillance data, was analysed to investigate the spatio-temporal patterns of the Ae. aegypti population in Singapore. The index was further categorised into low, moderate, and high-risk groups and its association with dengue cases were examined. A Before-After Control Impact analysis was performed to evaluate the epidemiology impact of Gravitrap system on dengue transmission. The Ae. aegypti population exhibits a seasonal pattern, and spatial heterogeneity in Ae. aegypti abundance was observed among treatment sites. The Ae. aegypti population was also found to be unevenly distributed among floors of an apartment block, with low floors (floors 1-4) having a higher abundance of mosquitoes trapped than mid (floors 5-8) and high (floors ≥9) floors. Areas with high GAI were shown to have higher dengue case count. Gravitrap has also demonstrated to be a good dengue control tool. The contribution of cases by treatment sites to the national numbers was lower after Gravitraps deployment. The GAI, which is of better relevance to dengue transmission risk, could be recommended as an indicator for decision making in vector control efforts, and to monitor the spatio-temporal variability of the adult Aedes population in the country. In addition, findings from this study indicate that Gravitraps can be used as a dengue control tool to reduce dengue transmission.

SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes.

BACKGROUND: The monitoring of vectors is one of the key surveillance measures to assess the risk of arbovirus transmission and the success of control strategies in endemic regions. The recent re-emergence of Zika virus (ZIKV) in the tropics, including Singapore, emphasizes the need to develop cost-effective, rapid and accurate assays to monitor the virus spread by mosquitoes. As ZIKV infections largely remain asymptomatic, early detection of ZIKV in the field-caught mosquitoes enables timely implementation of appropriate mosquito control measures. RESULTS: We developed a rapid, sensitive and specific real-time reverse transcription polymerase chain reaction (rRT-PCR) assay for the detection of ZIKV in field-caught mosquitoes. The primers and PCR cycling conditions were optimized to minimize non-specific amplification due to cross-reactivity with the genomic material of Aedes aegypti, Aedes albopictus, Culex quinquefasciatus, Culex tritaeniorhynchus, Culex sitiens and Anopheles sinensis, as well as accompanying microbiota. The performance of the assay was further evaluated with a panel of flaviviruses and alphaviruses as well as in field-caught Ae. aegypti mosquitoes confirmed to be positive for ZIKV. As compared to a probe-based assay, the newly developed assay demonstrated 100% specificity and comparable detection sensitivity for ZIKV in mosquitoes. CONCLUSIONS: Being a SYBR Green-based method, the newly-developed assay is cost-effective and easy to adapt, thus is applicable to large-scale vector surveillance activities in endemic countries, including those with limited resources and expertise. The amplicon size (119 bp) also allows sequencing to confirm the virus type. The primers flank relatively conserved regions of ZIKV genome, so that, the assay is able to detect genetically diverse ZIKV strains. Our findings, therefore, testify the potential use of the newly-developed assay in vector surveillance programmes for ZIKV in endemic regions.

Feeding host range of Aedes albopictus (Diptera: Culicidae) demonstrates its opportunistic host-seeking behavior in rural Singapore.

Aedes albopictus (Skuse) is a competent vector of arboviruses of public health importance, including dengue virus (DENV) and chikungunya virus viruses. Ae. albopictus is the primary vector of chikungunya virus in Singapore. However, despite being ubiquitous, it plays a secondary role in DENV transmission. The vectorial capacity of Ae. albopictus for DENV in field settings appears to be weak because dengue primarily occurs in Aedes aegypti (L.)-dominated, urban settings of the country. As host-seeking behavior is one of the determinants of vectorial capacity, we screened 6,762 female Ae. albopictus from rural, semiurban, and urban locations in Singapore for avian and nonavian bloodmeals using two polymerase chain reaction-sequencing assays developed in-house. The majority (83.2%, n = 79) of bloodmeals from rural and semiurban areas were from humans. However, Ae. albopictus was also found to feed on shrews, swine, dogs, cats, turtles, and multiple hosts in rural settings. In urban areas, all positive bloodmeals were from humans. There were no avian bloodmeals. Our findings testify that Ae. albopictus is highly anthropophagic even in rural settings, but become opportunistic in extremely low human abundance. This opportunistic feeding behavior warrants further investigations into the vectorial capacity of Ae. albopictus to assess its role in arbovirus transmission in endemic habitats.