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.

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.

VKORC1 mutations in rodent populations of a tropical city-state as an indicator of anticoagulant rodenticide resistance.

Anticoagulant rodenticides are commonly used in rodent control because they are economical and have great deployment versatility. However, rodents with Single Nucleotide Polymorphism (SNP) mutations within the Vkorc1 gene are resistant to the effects of anticoagulant rodenticide use and this influences the effectiveness of control strategies that rely on such rodenticides. This study examined the prevalence of rat SNP mutations in Singapore to inform the effectiveness of anticoagulant rodenticide use. A total of 130 rat tail samples, comprising 83 Rattus norvegicus (63.8%) and 47 Rattus rattus complex (36.2%) were conveniently sampled from November 2016 to December 2019 from urban settings and sequenced at exon 3 of Vkorc1. Sequencing analysis revealed 4 synonymous and 1 non-synonymous mutations in Rattus rattus complex samples. A novel synonymous mutation of L108L was identified and not previously reported in other studies. Non-synonymous SNPs were not detected in the notable codons of 120, 128 and 139 in R. norvegicus, where these regions are internationally recognised to be associated with resistance from prior studies. Our findings suggest that the prevalence of anticoagulant rodenticide resistance in Singapore is low. Continued monitoring of rodenticide resistance is important for informing rodent control strategies aimed at reducing rodent-borne disease transmission.

Association of rodents with man-made infrastructures and food waste in Urban Singapore.

BACKGROUND: Rodent population control is an important measure in reducing the risk of rodent-borne disease transmission. In this study, we examined rodent activity in the sanitary waste network around the household waste-collection bin chamber of an urban residential apartment block. METHODS: We utilised infra-red camera traps to determine the pattern of rodent activity in a rodent-infested bin chamber and its associated sanitary waste network. Multivariable logistic regression was performed to assess the risk factors that were independently associated with rodent activity in the bin chambers. RESULT: The camera trap surveillance showed that the rodents were active in the bin chamber and sanitary network both in the day and at night. In the cross-sectional study, rodent activity in the bin chambers was independently associated with broken floor traps [Adjusted odds ratio (AOR): 36.7, CI: 21.3-66.3], calendar month [Log-likelihood ratio test (LRT) p = 0.002] and Town Council [LRT p = 0.004] variables. In restricted analysis, rodent activity in bin chambers was independently associated with defects in the wastewater pipe under the chamber [AOR: 12.3, CI: 4.3-51.7]. CONCLUSION: Our study suggests that urban municipal management councils should prioritize rodent control resources in areas according to the factors that increase the risk of rodent infestation.

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.

Non-Invasive Dengue Diagnostics-The Use of Saliva and Urine for Different Stages of the Illness.

Dengue diagnosis is largely dependent on clinical symptoms and routinely confirmed with laboratory detection of dengue virus in patient serum samples collected via phlebotomy. This presents a challenge to patients not amenable to venipuncture. Non-invasive methods of dengue diagnosis have the potential to enhance the current dengue detection algorithm. In this study, samples from dengue infected patients were collected between January 2012 until September 2012 and September 2013 until December 2013 in two different setups. Panel A samples (blood, urine, and saliva) were collected daily when the 39 patients were hospitalised and during their follow-up visits while Panel B samples (saliva) were collected from 23 patients during the acute stage of dengue. Using DENV PCR on Panel A, from day 2 to day 4 post fever onset, serum showed the best overall positivity followed by saliva and urine (100%/82.1%/67.9%). From day 5 until day 10 post fever onset, serum and urine had similar positivity (67.4%/61.2%), followed by saliva (51.3%). Beyond day 10 post fever onset, DENV was undetectable in sera, but urine and saliva showed 56.8% and 28.6% positivity, respectively. DENV in urine was detectable up until 32 days post fever. Panel B results showed overall sensitivity of 32.4%/36% (RNA/NS1) for DENV detection in saliva. Our results suggest that the urine-based detection method is useful especially for late dengue detection, where DENV is undetected in sera but still detectable in urine. This provides a potential tool for the physician to pick up new cases in an area where there is ongoing dengue transmission and subsequently prompt for intensified vector control activities.

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.

Detection of Japanese Encephalitis Virus in Culex Mosquitoes in Singapore.

Mosquito-borne flaviviruses are emerging pathogens of an increasing global public health concern because of their rapid increase in geographical range and the impact of climate change. Japanese encephalitis virus (JEV) and West Nile virus (WNV) are of concern because of the risk of reemergence and introduction by migratory birds. In Singapore, human WNV infection has never been reported and human JEV infection is rare. Four sentinel vector surveillance sites were established in Singapore to understand the potential risk posed by these viruses. Surveillance was carried out from August 2011 to December 2012 at Pulau Ubin, from March 2011 to March 2013 at an Avian Sanctuary (AS), from December 2010 from October 2012 at Murai Farmway, and from December 2010 to December 2013 at a nature reserve. The present study revealed active JEV transmission in Singapore through the detection of JEV genotype II in Culex tritaeniorhynchus collected from an Avian Sanctuary. Culex flavivirus (CxFV), similar to the Quang Binh virus isolated from Cx. tritaeniorhynchus in Vietnam and CxFV-LSFlaviV-A20-09 virus isolated in China, was also detected in Culex spp. (vishnui subgroup). No WNV was detected. This study demonstrates the important role that surveillance plays in public health and strongly suggests the circulation of JEV among wildlife in Singapore, despite the absence of reported human cases. A One Health approach involving surveillance, the collaboration between public health and wildlife managers, and control of mosquito populations remains the key measures in risk mitigation of JEV transmission in the enzootic cycle between birds and mosquitoes.

Occurrence and Antimicrobial Resistance Traits of Escherichia coli from Wild Birds and Rodents in Singapore.

Antimicrobial resistance (AMR) in Escherichia coli (E. coli) poses a public health concern worldwide. Wild birds and rodents, due to their mobility, are potential vehicles for transmission of AMR bacteria to humans. Ninety-six wild birds' faecal samples and 135 rodents' droppings samples were collected and analysed in 2017. Forty-six E. coli isolates from wild birds and rodents were subjected to AMR phenotypic and genotypic characterisation. The proportion of E. coli isolates resistant to at least one of the antimicrobials tested from wild birds (80.8%) was significantly higher than that of isolates from rodents (40.0%). The proportion of E. coli isolates resistant to each antimicrobial class for wild birds was 3.8% to 73.1% and that for rodents was 5.0% to 35.0%. Six out of 26 E. coli isolates from wild birds (23.1%) and two out of 20 (10.0%) isolates from rodents were multi-drug resistant (MDR) strains. These MDR E. coli isolates were detected with various antimicrobial resistance genes such as blaTEM-1B and qnrS1 and could be considered as part of the environmental resistome. Findings in this study suggested that wild birds and rodents could play a role in disseminating antimicrobial resistant E. coli, and this underscores the necessity of environment management and close monitoring on AMR bacteria in wild birds and rodents to prevent spreading of resistant organisms to other wildlife animals and humans.

Serological evidence of continued Japanese encephalitis virus transmission in Singapore nearly three decades after end of pig farming.

BACKGROUND: Singapore used to report an annual average of 14 cases of Japanese encephalitis, but ever since the abolishment of pig farms in the early 1990s, the local incidence rate for Japanese encephalitis virus (JEV) infections has reduced drastically. Studies done in the early 2000s demonstrated the presence of JEV-specific antibodies in animals such as wild boars, dogs, chickens and goats on the offshore island and peripheral parts of the Singapore, indicative of prior JEV exposure. A JEV wildlife and sentinel chicken surveillance system was initiated in 2010 through to 2017 to study the animal host seroprofiles. RESULTS: A total of 12/371 (3.23%) of resident bird samples, 24/254 (9.45%) of migratory bird samples and 10/66 (15.16%) of wild boar samples were positive for the presence of JEV antibodies. Seroconversions in sentinel chickens were observed at two time points. Through this study, two sites with active transmission of JEV amongst avian or porcine hosts were identified. CONCLUSIONS: JEV transmission in animal hosts has continued despite the phasing out of pig farming nearly thirty years ago; however, the public health risk of transmission remains low. Environmental management for mosquito population remains key to keeping this risk low.

Salmonella in Retail Food and Wild Birds in Singapore-Prevalence, Antimicrobial Resistance, and Sequence Types.

Non-typhoidal salmonellosis is a leading cause of foodborne zoonosis. To better understand the epidemiology of human salmonellosis, this study aimed to determine the prevalence, antimicrobial resistance and sequence types of Salmonella in retail food and wild birds (proximity to humans) in Singapore. We analyzed 21,428 cooked and ready-to-eat food and 1,510 residual faecal samples of wild birds collected during 2010-2015. Thirty-two Salmonella isolates from food and wild birds were subjected to disc diffusion and multi-locus sequence typing (MLST). Salmonella was isolated from 0.08% (17/21,428) of food and 0.99% (15/1510) of wild birds. None of the isolates from wild birds (n = 15) exhibited phenotypic resistance, while the isolates from food (47.1%, 8/17) showed a high prevalence of phenotypic resistance to, at least, one antimicrobial. These findings suggested that the avian Salmonella isolates had been subjected to less antimicrobial selection pressure than those from food samples. MLST revealed specific sequence types found in both food and wild birds. The study can guide future studies with whole-genome analysis on a larger number of isolates from various sectors for public health measures.

WIP1 phosphatase is a negative regulator of NF-kappaB signalling.

Post-translational modifications of NF-kappaB through phosphorylations enhance its transactivation potential. Much is known about the kinases that phosphorylate NF-kappaB, but little is known about the phosphatases that dephosphorylate it. By using a genome-scale siRNA screen, we identified the WIP1 phosphatase as a negative regulator of NF-kappaB signalling. WIP1-mediated regulation of NF-kappaB occurs in both a p38-dependent and independent manner. Overexpression of WIP1 resulted in decreased NF-kappaB activation in a dose-dependent manner, whereas WIP1 knockdown resulted in increased NF-kappaB function. We show that WIP1 is a direct phosphatase of Ser 536 of the p65 subunit of NF-kappaB. Phosphorylation of Ser 536 is known to be essential for the transactivation function of p65, as it is required for recruitment of the transcriptional co-activator p300. WIP1-mediated regulation of p65 regulated binding of NF-kappaB to p300 and hence chromatin remodelling. Consistent with our results, mice lacking WIP1 showed enhanced inflammation. These results provide the first genetic proof that a phosphatase directly regulates NF-kappaB signalling in vivo.