Using spatial genetics to quantify mosquito dispersal for control programs.

BACKGROUND: Hundreds of millions of people get a mosquito-borne disease every year and nearly one million die. Transmission of these infections is primarily tackled through the control of mosquito vectors. The accurate quantification of mosquito dispersal is critical for the design and optimization of vector control programs, yet the measurement of dispersal using traditional mark-release-recapture (MRR) methods is logistically challenging and often unrepresentative of an insect's true behavior. Using Aedes aegypti (a major arboviral vector) as a model and two study sites in Singapore, we show how mosquito dispersal can be characterized by the spatial analyses of genetic relatedness among individuals sampled over a short time span without interruption of their natural behaviors. RESULTS: Using simple oviposition traps, we captured adult female Ae. aegypti across high-rise apartment blocks and genotyped them using genome-wide SNP markers. We developed a methodology that produces a dispersal kernel for distance which results from one generation of successful breeding (effective dispersal), using the distance separating full siblings and 2nd- and 3rd-degree relatives (close kin). The estimated dispersal distance kernel was exponential (Laplacian), with a mean dispersal distance (and dispersal kernel spread σ) of 45.2 m (95% CI 39.7-51.3 m), and 10% probability of a dispersal > 100 m (95% CI 92-117 m). Our genetically derived estimates matched the parametrized dispersal kernels from previous MRR experiments. If few close kin are captured, a conventional genetic isolation-by-distance analysis can be used, as it can produce σ estimates congruent with the close-kin method if effective population density is accurately estimated. Genetic patch size, estimated by spatial autocorrelation analysis, reflects the spatial extent of the dispersal kernel "tail" that influences, for example, the critical radii of release zones and the speed of Wolbachia spread in mosquito replacement programs. CONCLUSIONS: We demonstrate that spatial genetics can provide a robust characterization of mosquito dispersal. With the decreasing cost of next-generation sequencing, the production of spatial genetic data is increasingly accessible. Given the challenges of conventional MRR methods, and the importance of quantified dispersal in operational vector control decisions, we recommend genetic-based dispersal characterization as the more desirable means of parameterization.

Characterization of Fowlpox virus in chickens and bird-biting mosquitoes: a molecular approach to investigating Avipoxvirus transmission.

Avian pox is a highly contagious avian disease, yet relatively little is known about the epidemiology and transmission of Avipoxviruses. Using a molecular approach, we report evidence for a potential link between birds and field-caught mosquitoes in the transmission of Fowlpox virus (FWPV) in Singapore. Comparison of fpv167 (P4b), fpv126 (VLTF-1), fpv175-176 (A11R-A12L) and fpv140 (H3L) gene sequences revealed close relatedness between FWPV strains obtained from cutaneous lesions of a chicken and four pools of Culex pseudovishnui, Culex spp. (vishnui group) and Coquellitidea crassipes caught in the vicinity of the study site. Chicken-derived viruses characterized during two separate infections two years later were also identical to those detected in the first event, suggesting repeated transmission of closely related FWPV strains in the locality. Since the study location is home to resident and migratory birds, we postulated that wild birds could be the source of FWPV and that bird-biting mosquitoes could act as bridging mechanical vectors. Therefore, we determined whether the FWPV-positive mosquito pools (n=4) were positive for avian DNA using a polymerase chain reaction-sequencing assay. Our findings confirmed the presence of avian host DNA in all mosquito pools, suggesting a role for Cx. pseudovishnui, Culex spp. (vishnui group) and Cq. crassipes mosquitoes in FWPV transmission. Our study exemplifies the utilization of molecular tools to understand transmission networks of pathogens affecting avian populations, which has important implications for the design of effective control measures to minimize disease burden and economic loss.

Construction sites as an important driver of dengue transmission: implications for disease control.

BACKGROUND: In 2013 and 2014, Singapore experienced its worst dengue outbreak known-to-date. Mosquito breeding in construction sites stood out as a probable risk factor due to its association with major dengue clusters in both years. We, therefore, investigated the contribution of construction sites to dengue transmission in Singapore, highlighting three case studies of large construction site-associated dengue clusters recorded during 2013-16. METHODS: The study included two components; a statistical analysis of cluster records from 2013 to 2016, and case studies of three biggest construction site-associated clusters. We explored the odds of construction site-associated clusters growing into major clusters and determined whether clusters seeded in construction sites demonstrated a higher tendency to expand into major clusters. DENV strains obtained from dengue patients residing in three major clusters were genotyped to determine whether the same strains expanded into the surroundings of construction sites. RESULTS: Despite less than 5% of total recorded clusters being construction site-associated, the odds of such clusters expanding into major clusters were 17.4 (2013), 9.2 (2014), 3.3 (2015) and 4.3 (2016) times higher than non-construction site clusters. Aedes premise index and average larvae count per habitat were also higher in construction sites than residential premises during the study period. The majority of cases in clusters associated with construction sites were residents living in the surroundings. Virus genotype data from three case study sites revealed a transmission link between the construction sites and the surrounding residential areas. CONCLUSIONS: Significantly high case burden and the probability of cluster expansion due to virus spill-over into surrounding areas suggested that construction sites play an important role as a driver of sustained dengue transmission. Our results emphasise that the management of construction-site associated dengue clusters should not be limited to the implicated construction sites, but be extended to the surrounding premises to prevent further transmission.

Microbial survey of ready-to-eat salad ingredients sold at retail reveals the occurrence and the persistence of Listeria monocytogenes Sequence Types 2 and 87 in pre-packed smoked salmon.

BACKGROUND: As the preparation of salads involves extensive handling and the use of uncooked ingredients, they are particularly vulnerable to microbial contamination. This study aimed to determine the microbial safety and quality of pre-packed salads and salad bar ingredients sold in Singapore, so as to identify public health risks that could arise from consuming salads and to determine areas for improvement in the management of food safety. RESULTS: The most frequently encountered organism in pre-packed salad samples was B. cereus, particularly in pasta salads (33.3%, 10/30). The most commonly detected organism in salad bar ingredients was L. monocytogenes, in particular seafood ingredients (44.1%, 15/34), largely due to contaminated smoked salmon. Further investigation showed that 21.6% (37/171) of the pre-packed smoked salmon sold in supermarkets contained L. monocytogenes. Significantly higher prevalence of L. monocytogenes and higher Standard Plate Count were detected in smoked salmon at salad bars compared to pre-packed smoked salmon in supermarkets, which suggested multiplication of the organism as the products move down the supply chain. Further molecular analysis revealed that L. monocytogenes Sequence Type (ST) 2 and ST87 were present in a particular brand of pre-packed salmon products over a 4-year period, implying a potential persistent contamination problem at the manufacturing level. CONCLUSIONS: Our findings highlighted a need to improve manufacturing and retail hygiene processes as well as to educate vulnerable populations to avoid consuming food prone to L. monocytogenes contamination.

Epidemic resurgence of dengue fever in Singapore in 2013-2014: A virological and entomological perspective.

BACKGROUND: Dengue resurged in Singapore during 2013-14, causing an outbreak with unprecedented number of cases in the country. In the present study, we summarise the epidemiological, virological and entomological findings gathered through the dengue surveillance programme and highlight the drivers of the epidemic. We also describe how the surveillance system facilitated the preparedness to moderate epidemic transmission of dengue in the country. METHODS: The case surveillance was based on a mandatory notification system that requires all medical practitioners to report clinically-suspected and laboratory-confirmed cases within 24 hours. The circulating Dengue virus (DENV) populations were monitored through an island wide virus surveillance programme aimed at determining the serotypes and genotypes of circulating virus strains. Entomological surveillance included adult Aedes surveillance as well as premise checks for larval breeding. RESULTS: A switch in the dominant serotype from DENV-2 to DENV-1 in March 2013 signalled a potential spike in cases, and the alert was corroborated by an increase in average Aedes house index. The alert triggered preparedness and early response to moderate the impending outbreak. The two-year outbreak led to 22,170 cases in 2013 and 18,338 in 2014, corresponding to an incidence rate of 410.6 and 335.0 per 100,000 population, respectively. DENV-1 was the dominant serotype in 2013 (61.7 %, n = 5,071) and 2014 (79.2 %, n = 5,226), contributed largely by a newly-introduced DENV-1 genotype III strain. The percentage of houses with Ae. aegypti breeding increased significantly (p < 0.001) from 2012 (annual average of 0.07 %) to 2013 (annual average of 0.14 %), followed by a drop in 2014 (annual average of 0.10 %). Aedes breeding data further showed a wide spread distribution of Ae. aegypti in the country that corresponded with the dengue case distribution pattern in 2013 and 2014. The adult Aedes data from 34 gravitrap sentinel sites revealed that approximately 1/3 of the monitored sites remained at high risk of DENV transmission in 2013. CONCLUSIONS: The culmination of the latest epidemic is likely to be due to a number of demographic, social, virological, entomological, immunological, climatic and ecological factors that contribute to DENV transmission. A multi-pronged approach backed by the epidemiological, virological and entomological understanding paved way to moderate the case burden through an integrated vector management approach.

A Systematic Meta-analysis of Immune Signatures in Patients With Acute Chikungunya Virus Infection.

BACKGROUND: Individuals infected with chikungunya virus (CHIKV) normally exhibit a variety of clinical manifestations during the acute phase of infection. However, studies in different patient cohorts have revealed that disease manifestations vary in frequency. METHODS: Disease profiles between patients with acute CHIKV-infection and febrile patients without CHIKV were compared and examined to determine whether any clinical presentations were associated with the clinical outcome of CHIKV infection. Circulatory immune mediators profiles were then characterized and compared with data from 14 independent patient cohort studies. The particular immune mediator signature that defines acute CHIKV infection was determined. RESULTS: Our findings revealed a specific pattern of clinical presentations of joint-specific arthralgia from this CHIKV cohort. More importantly, we identified an immune mediator signature dominated by proinflammatory cytokines, which include interferon α and γ and interleukin 2, 2R, 6, 7, 12, 15, 17, and 18, across different patient cohorts of CHIKV load associated with arthralgia. CONCLUSIONS: To our knowledge, this is the first study that associated levels of CHIKV load with arthralgia as an indicator of acute CHIKV infection. Importantly, our findings also revealed specific immune mediator signatures that can be used to better define CHIKV infection.

Molecular epidemiology of dengue in Malaysia: 2015-2021.

Dengue has been one of the major public health problems in Malaysia for decades. Over 600,000 dengue cases and 1,200 associated fatalities have been reported in Malaysia from 2015 to 2021, which was 100% increase from the cumulative total of dengue cases reported during the preceding 07-year period from 2008 to 2014. However, studies that describe the molecular epidemiology of dengue in Malaysia in recent years are limited. In the present study, we describe the genetic composition and dispersal patterns of Dengue virus (DENV) by using 4,004 complete envelope gene sequences of all four serotypes (DENV-1 = 1,567, DENV-2 = 1,417, DENV-3 = 762 and DENV-4 = 258) collected across Malaysia from 2015 to 2021. The findings revealed that DENV populations in Malaysia were highly diverse, and the overall heterogeneity was maintained through repetitive turnover of genotypes. Phylogeography analyses suggested that DENV dispersal occurred through an extensive network, mainly among countries in South and East Asia and Malaysian states, as well as among different states, especially within Peninsular Malaysia. The results further suggested Selangor and Johor as major hubs of DENV emergence and spread in Malaysia.

Case report: Zika surveillance complemented with wastewater and mosquito testing.

BACKGROUND: In June 2023, a local cluster of 15 Zika cases was reported in a neighbourhood in Northeastern Singapore. The last significant local transmission of Zika virus (ZIKV) with more than 450 cases was in 2016-2017. To monitor the situation and mitigate further transmission, case, entomological and wastewater-based surveillance were carried out. METHODS: Primary healthcare practitioners and the community were alerted to encourage timely case identification. Surveillance was enhanced through testing of Aedes mosquitoes collected from the National Gravitrap surveillance system, and wastewater samples were collected from a network of autosamplers deployed at manholes across the country. FINDINGS: ZIKV RNA was detected in mosquito pools (3/43; 7%) and individual mosquitoes (3/82; 3.7%) captured, and in wastewater samples (13/503) collected from the vicinity of the cluster of cases. Respective samples collected from other sites across the country were negative. The peak detection of ZIKV RNA in mosquitoes and wastewater coincided temporally with the peak in the number of cases in the area (15-25 May 2023). INTERPRETATION: The restriction of ZIKV signals from wastewater and mosquitoes within the neighbourhood suggested limited ZIKV transmission. The subsequent waning of signals suggested effectiveness of control measures. We demonstrate the utility of wastewater-based surveillance of ZIKV, which complements existing case- and entomological-based surveillance. The non-intrusive approach is particularly useful to monitor diseases such as Zika, which generally causes silent or mild infections, but may cause severe outcomes such as congenital Zika syndrome. FUNDING: This study was funded by Singapore's Ministry of Finance and the National Environment Agency, Singapore.

Assessing the Risk of Dengue Virus Local Transmission: Study on Vector Competence of Italian Aedes albopictus.

The frequency of locally transmitted dengue virus (DENV) infections has increased in Europe in recent years, facilitated by the invasive mosquito species Aedes albopictus, which is well established in a large area of Europe. In Italy, the first indigenous dengue outbreak was reported in August 2020 with 11 locally acquired cases in the Veneto region (northeast Italy), caused by a DENV-1 viral strain closely related to a previously described strain circulating in Singapore and China. In this study, we evaluated the vector competence of two Italian populations of Ae. albopictus compared to an Ae. aegypti lab colony. We performed experimental infections using a DENV-1 strain that is phylogenetically close to the strain responsible for the 2020 Italian autochthonous outbreak. Our results showed that local Ae. albopictus is susceptible to infection and is able to transmit the virus, confirming the relevant risk of possible outbreaks starting from an imported case.

Evolution and heterogeneity of multiple serotypes of Dengue virus in Pakistan, 2006-2011.

BACKGROUND: Even though dengue has been recognized as one of the major public health threats in Pakistan, the understanding of its molecular epidemiology is still limited. The genotypic diversity of Dengue virus (DENV) serotypes involved in dengue outbreaks since 2005 in Pakistan is not well studied. Here, we investigated the origin, diversity, genetic relationships and geographic distribution of DENV to understand virus evolution during the recent expansion of dengue in Pakistan. METHODS: The study included 200 sera obtained from dengue-suspected patients from 2006 to 2011. DENV infection was confirmed in 94 (47%) sera by a polymerase chain reaction assay. These included 36 (38.3%) DENV-2, 57 DENV-3 (60.6%) and 1 DENV-4 (1.1%) cases. Sequences of 13 whole genomes (6 DENV-2, 6 DENV-3 and 1 DENV-4) and 49 envelope genes (26 DENV-2, 22 DENV-3 and 1 DENV-4) were analysed to determine the origin, phylogeny, diversity and selection pressure during virus evolution. RESULTS: DENV-2, DENV-3 and DENV-4 in Pakistan from 2006 to 2011 shared 98.5-99.6% nucleotide and 99.3-99.9% amino acid similarity with those circulated in the Indian subcontinent during the last decade. Nevertheless, Pakistan DENV-2 and DENV-3 strains formed distinct clades characterized by amino acid signatures of NS2A-I116T + NS5-K861R and NS3-K590R + NS5-S895L respectively. Each clade consisted of a heterogenous virus population that circulated in Southern (2006-2009) and Northern Pakistan (2011). CONCLUSIONS: DENV-2, DENV-3 and DENV-4 that circulated during 2006-2011 are likely to have first introduced via the southern route of Pakistan. Both DENV-2 and DENV-3 have undergone in-situ evolution to generate heterogenous populations, possibly driven by sustained local DENV transmission during 2006-2011 periods. While both DENV-2 and DENV-3 continued to circulate in Southern Pakistan until 2009, DENV-2 has spread in a Northern direction to establish in Punjab Province, which experienced a massive dengue outbreak in 2011.

Singapore's 5 decades of dengue prevention and control-Implications for global dengue control.

This paper summarises the lessons learnt in dengue epidemiology, risk factors, and prevention in Singapore over the last half a century, during which Singapore evolved from a city of 1.9 million people to a highly urban globalised city-state with a population of 5.6 million. Set in a tropical climate, urbanisation among green foliage has created ideal conditions for the proliferation of Aedes aegypti and Aedes albopictus, the mosquito vectors that transmit dengue. A vector control programme, largely for malaria, was initiated as early as 1921, but it was only in 1966 that the Vector Control Unit (VCU) was established to additionally tackle dengue haemorrhagic fever (DHF) that was first documented in the 1960s. Centred on source reduction and public education, and based on research into the bionomics and ecology of the vectors, the programme successfully reduced the Aedes House Index (HI) from 48% in 1966 to <5% in the 1970s. Further enhancement of the programme, including through legislation, suppressed the Aedes HI to around 1% from the 1990s. The current programme is characterised by 4 key features: (i) proactive inter-epidemic surveillance and control that is stepped up during outbreaks; (ii) risk-based prevention and intervention strategies based on advanced data analytics; (iii) coordinated inter-sectoral cooperation between the public, private, and people sectors; and (iv) evidence-based adoption of new tools and strategies. Dengue seroprevalence and force of infection (FOI) among residents have substantially and continuously declined over the 5 decades. This is consistent with the observation that dengue incidence has been delayed to adulthood, with severity highest among the elderly. Paradoxically, the number of reported dengue cases and outbreaks has increased since the 1990s with record-breaking epidemics. We propose that Singapore's increased vulnerability to outbreaks is due to low levels of immunity in the population, constant introduction of new viral variants, expanding urban centres, and increasing human density. The growing magnitude of reported outbreaks could also be attributed to improved diagnostics and surveillance, which at least partially explains the discord between rising trend in cases and the continuous reduction in dengue seroprevalence. Changing global and local landscapes, including climate change, increasing urbanisation and global physical connectivity are expected to make dengue control even more challenging. The adoption of new vector surveillance and control tools, such as the Gravitrap and Wolbachia technology, is important to impede the growing threat of dengue and other Aedes-borne diseases.

Lineage Replacement Associated with Fitness Gain in Mammalian Cells and Aedes aegypti: A Catalyst for Dengue Virus Type 2 Transmission.

Shifting of virus serotypes and clade replacement events are known to drive dengue epidemics. However, only a few studies have attempted to elucidate the virus attributes that contribute to such epidemics. In 2007, Singapore experienced a dengue outbreak affecting more than 8000 individuals. The outbreak ensued with the shuffling of dominant clades (from clade I to clade II) of Dengue virus 2 (DENV-2) cosmopolitan genotype, at a time when the Aedes premise index was significantly low. Therefore, we hypothesized that clade II had higher epidemic potential and fitness than clade I. To test this hypothesis, we tested the replication and apoptotic qualities of clade I and II isolates in mammalian cells and their ability to infect and disseminate in a field strain of Ae. Aegypti. Our findings indicated that clade II replicated more efficiently in mammalian cells than clade I and possessed higher transmission potential in local vectors. This could collectively improve the epidemic potential of clade II, which dominated during the outbreak in 2007. The findings exemplify complex interactions between the emergence, adaptation and transmission potential of DENV, and testify the epidemiological importance of a deeper understanding of virus and vector dynamics in endemic regions.

Isolation and Genetic Characterization of Japanese Encephalitis Virus Two Decades after Its Elimination in Singapore.

Japanese encephalitis virus (JEV) is an important arbovirus in Asia that can cause serious neurological disease. JEV is transmitted by mosquitoes in an enzootic cycle involving porcine and avian reservoirs, in which humans are accidental, dead-end hosts. JEV is currently not endemic in Singapore, after pig farming was abolished in 1992; the last known human case was reported in 2005. However, due to its location along the East-Asian Australasian Flyway (EAAF), Singapore is vulnerable to JEV re-introduction from the endemic regions. Serological and genetic evidence in the last decade suggests JEV's presence in the local fauna. In the present study, we report the genetic characterization and the first isolation of JEV from 3214 mosquito pools consisting of 41,843 Culex mosquitoes, which were trapped from April 2014 to May 2021. The findings demonstrated the presence of genotype I of JEV (n = 10), in contrast to the previous reports of the presence of genotype II of JEV in Singapore. The genetic analyses also suggested that JEV has entered Singapore on several occasions and has potentially established an enzootic cycle in the local fauna. These observations have important implications in the risk assessment and the control of Japanese encephalitis in non-endemic countries, such as Singapore, that are at risk for JEV transmission.

Development of an efficient wastewater testing protocol for high-throughput country-wide SARS-CoV-2 monitoring.

Wastewater-based surveillance has been widely used as a non-intrusive tool to monitor population-level transmission of COVID-19. Although various approaches are available to concentrate viruses from wastewater samples, scalable methods remain limited. Here, we sought to identify and evaluate SARS-CoV-2 virus concentration protocols for high-throughput wastewater testing. A total of twelve protocols for polyethylene glycol (PEG) precipitation and four protocols for ultrafiltration-based approaches were evaluated across two phases. The first phase entailed an initial evaluation using a small sample set, while the second phase further evaluated five protocols using wastewater samples of varying SARS-CoV-2 concentrations. Permutations in the pre-concentration, virus concentration and RNA extraction steps were evaluated. Among PEG-based methods, SARS-CoV-2 virus recovery was optimal with 1) the removal of debris prior to processing, 2) 2 h to 24 h incubation with 8% PEG at 4 °C, 3) 4000 xg or 14,000 xg centrifugation, and 4) a column-based RNA extraction method, yielding virus recovery of 42.4-52.5%. Similarly, the optimal protocol for ultrafiltration included 1) the removal of debris prior to processing, 2) ultrafiltration, and 3) a column-based RNA extraction method, yielding a recovery of 38.2%. This study also revealed that SARS-CoV-2 RNA recovery for samples with higher virus concentration were less sensitive to changes in the PEG method, but permutations in the PEG protocol could significantly impact virus yields when wastewater samples with lower SARS-CoV-2 RNA were used. Although both PEG precipitation and ultrafiltration methods resulted in similar SARS-CoV-2 RNA recoveries, the former method is more cost-effective while the latter method provided operational efficiency as it required a shorter turn-around-time (PEG precipitation, 9-23 h; Ultrafiltration, 5 h). The decision on which method to adopt will thus depend on the use-case for wastewater testing, and the need for cost-effectiveness, sensitivity, operational feasibility and scalability.

Minimizing errors in RT-PCR detection and quantification of SARS-CoV-2 RNA for wastewater surveillance.

Wastewater surveillance for pathogens using reverse transcription-polymerase chain reaction (RT-PCR) is an effective and resource-efficient tool for gathering community-level public health information, including the incidence of coronavirus disease-19 (COVID-19). Surveillance of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) in wastewater can potentially provide an early warning signal of COVID-19 infections in a community. The capacity of the world's environmental microbiology and virology laboratories for SARS-CoV-2 RNA characterization in wastewater is increasing rapidly. However, there are no standardized protocols or harmonized quality assurance and quality control (QA/QC) procedures for SARS-CoV-2 wastewater surveillance. This paper is a technical review of factors that can cause false-positive and false-negative errors in the surveillance of SARS-CoV-2 RNA in wastewater, culminating in recommended strategies that can be implemented to identify and mitigate some of these errors. Recommendations include stringent QA/QC measures, representative sampling approaches, effective virus concentration and efficient RNA extraction, PCR inhibition assessment, inclusion of sample processing controls, and considerations for RT-PCR assay selection and data interpretation. Clear data interpretation guidelines (e.g., determination of positive and negative samples) are critical, particularly when the incidence of SARS-CoV-2 in wastewater is low. Corrective and confirmatory actions must be in place for inconclusive results or results diverging from current trends (e.g., initial onset or reemergence of COVID-19 in a community). It is also prudent to perform interlaboratory comparisons to ensure results' reliability and interpretability for prospective and retrospective analyses. The strategies that are recommended in this review aim to improve SARS-CoV-2 characterization and detection for wastewater surveillance applications. A silver lining of the COVID-19 pandemic is that the efficacy of wastewater surveillance continues to be demonstrated during this global crisis. In the future, wastewater should also play an important role in the surveillance of a range of other communicable diseases.

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.

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.

Non-intrusive wastewater surveillance for monitoring of a residential building for COVID-19 cases.

Wastewater-based surveillance for SARS-CoV-2 has been used for the early warning of transmission or objective trending of the population-level disease prevalence. Here, we describe a new use-case of conducting targeted wastewater surveillance to complement clinical testing for case identification in a small community at risk of COVID-19 transmission. On 2 July 2020, a cluster of COVID-19 cases in two unrelated households residing on different floors in the same stack of an apartment building was reported in Singapore. After cases were conveyed to healthcare facilities and six healthy household contacts were quarantined in their respective apartments, wastewater surveillance was implemented for the entire residential block. SARS-CoV-2 was subsequently detected in wastewaters in an increasing frequency and concentration, despite the absence of confirmed COVID-19 cases, suggesting the presence of fresh case/s in the building. Phone interviews of six residents in quarantine revealed that no one was symptomatic (fever/respiratory illness). However, when nasopharyngeal swabs from six quarantined residents were tested by PCR tests, one was positive for SARS-CoV-2. The positive case reported episodes of diarrhea and the case's stool sample was also positive for SARS-CoV-2, explaining the SARS-CoV-2 spikes observed in wastewaters. After the case was conveyed to a healthcare facility, wastewaters continued to yield positive signals for five days, though with a decreasing intensity. This was attributed to the return of recovered cases, who had continued to shed the virus. Our findings demonstrate the utility of wastewater surveillance as a non-intrusive tool to monitor high-risk COVID-19 premises, which is able to trigger individual tests for case detection, highlighting a new use-case for wastewater testing.

Molecular Analysis of the Bloodmeals of Culex spp. Mosquitoes at Natural Habitats in Singapore to Investigate the Potential Risk of Japanese Encephalitis Virus and West Nile Virus Transmission.

Japanese encephalitis virus (JEV) and West Nile virus (WNV) are arboviruses primarily transmitted by Culex spp. mosquitoes. Birds are the primary hosts for JEV and WNV. Recent WNV outbreaks in Europe and United States and their association with migratory birds highlight the importance of understanding the feeding host preference of potential vectors for outbreak preparedness, especially in nonendemic settings. Singapore is nonendemic to JEV and WNV, but is a stopover site for migratory birds of the East Asian-Australasian Flyway. Therefore, we elucidated the feeding host range of Culex spp. mosquitoes captured in four natural (bird) habitats in Singapore from January 2011 to December 2012. We characterized feeding host DNA in field-caught mosquitoes using a PCR sequencing-based assay targeting the mitochondrial gene regions. Of 22,648 mosquitoes captured, 21,287 belonged to the Culex vishnui subgroup. The host DNA analysis showed that mosquitoes from the Cx. vishnui subgroup are opportunistic biters, feeding on a range of birds and mammals. Cx. vishnui subgroup, Culex sitiens and Culex bitaeniorhynchus, was primarily ornithophagic, although they fed opportunistically on mammals, including humans. Culex gelidus and Culex quinquefasciatus, in contrast, fed mainly on mammals. The presence of ornitho- and anthropophilic mosquito vectors and susceptible avian and mammalian hosts poses a risk spill-over transmission of JEV and WNV among humans, should these viruses be introduced through migratory birds and establish persistent transmission in resident birds and animal hosts in Singapore.

Environmental Contamination of SARS-CoV-2 in a Non-Healthcare Setting.

Fomite-mediated transmission has been identified as a possible route for the spread of COVID-19 disease caused by SARS-CoV-2. In healthcare settings, environmental contamination by SARS-CoV-2 has been found in patients' rooms and toilets. Here, we investigated environmental presence of SARS-CoV-2 in non-healthcare settings and assessed the efficacy of cleaning and disinfection in removing virus contamination. A total of 428 environmental swabs and six air samples was taken from accommodation rooms, toilets and elevators that have been used by COVID-19 cases. By using a reverse transcription polymerase chain reaction assay, we detected two SARS-CoV-2 RNA positive samples in a room where a COVID-19 patient stayed prior to diagnosis. The present study highlights the risk of fomite-mediated transmission in non-healthcare settings and the importance of surface disinfection in spaces occupied by cases. Of note, neither air-borne transmission nor surface contamination of elevators, which were transiently exposed to infected individuals, was evident among samples analyzed.