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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.

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.

Bats are responsible for the zoonotic transmission of several major viral diseases, including those leading to the 2003 SARS outbreak and likely the ongoing COVID-19 pandemic. While comparative genomics studies have revealed characteristic adaptations of the bat innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the viral tolerance in bats. Here we report the establishment of genome-wide RNA interference (RNAi) and CRISPR libraries for the screening of the model megabat, Pteropus alecto. We used the complementary RNAi and CRISPR libraries to interrogate P. alecto cells for infection with two different viruses: mumps virus and influenza A virus, respectively. Independent screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells and human cells. The MTHFD1 inhibitor, carolacton, potently blocked replication of several RNA viruses, including SARS-CoV-2. We also discovered that bats have lower expression levels of MTHFD1 than humans. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad-spectrum antiviral therapy.

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.

Bats are reservoir hosts of many zoonotic viruses with pandemic potential. We utilized single-cell transcriptome sequencing (scRNA-seq) to analyze the immune response in bat lungs upon in vivo infection with a double-stranded RNA virus, Pteropine orthoreovirus PRV3M. Bat neutrophils were distinguished by high basal IDO1 expression. NK cells and T cells were the most abundant immune cells in lung tissue. Three distinct CD8

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.

Background Singapore was previously endemic for JEV but the threat of JE remains. There is continued JEV transmission in animal hosts despite the banning of pig farming in 1992. Surveillance of mosquitoes, ardeid birds and swine gives public health officials useful warning signs for the monitoring of JEV. In this study, we aim to develop a JE Risk Map based on the data gathered by spatial and quantitative survey of the JE-associated vectors and animal hosts. Methods For over a year across the island, the distribution and population of herons were visually surveyed and the distribution of wild boars was captured with wildlife camera traps. Mosquito population data was collected from 16 sites with ecological factors suitable for JE transmission. The animal and vector data were then spatially analysed using open-sourced GIS software. Results Four JE high risk clusters were interpolated from the combined risk factors of data points. A JEV positive Culex tritaeniorynchus pool was subsequently detected in the largest of these JE high risk clusters.

Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Y&uacute, nnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Y&uacute, nnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term.

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&ndash, 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.

Bats are important reservoirs and vectors in the transmission of emerging infectious diseases. Many highly pathogenic viruses such as SARS-CoV and rabies-related lyssaviruses have crossed species barriers to infect humans and other animals. In this study we monitored the major roost sites of bats in Singapore, and performed surveillance for zoonotic pathogens in these bats. Screening of guano samples collected during the survey uncovered a bat coronavirus ({\textless}span class="jp-italic"{\textgreater}Betacoronavirus{\textless}/span{\textgreater}) in {\textless}span class="jp-italic"{\textgreater}Cynopterus brachyotis{\textless}/span{\textgreater}, commonly known as the lesser dog-faced fruit bat. Using a capture-enrichment sequencing platform, the full-length genome of the bat CoV was sequenced and found to be closely related to the bat coronavirus HKU9 species found in Leschenault's rousette discovered in the Guangdong and Yunnan provinces.

Filoviruses, especially Ebola virus (EBOV) and Marburg virus (MARV), are notoriously pathogenic and capable of causing severe haemorrhagic fever diseases in humans with high lethality1,2. The risk of future outbreaks is exacerbated by the discovery of other bat-borne filoviruses of wide genetic diversity globally3–5. Here we report the characterization of a phylogenetically distinct bat filovirus, named Měngla virus (MLAV). The coding-complete genome of MLAV shares 32–54% nucleotide sequence identity with known filoviruses. Phylogenetic analysis places this new virus between EBOV and MARV, suggesting the need for a new genus taxon. Importantly, despite the low amino acid sequence identity (22–39%) of the glycoprotein with other filoviruses, MLAV is capable of using the Niemann–Pick C1 (NPC1) as entry receptor. MLAV is also replication-competent with chimeric MLAV mini-genomes containing EBOV or MARV leader and trailer sequences, indicating that these viruses are evolutionally and functionally closely related. Finally, MLAV glycoprotein-typed pseudo-types transduced cell lines derived from humans, monkeys, dogs, hamsters and bats, implying a broad species cell tropism with a high risk of interspecies spillover transmission. Měngla virus (MLAV) is a phylogenetically distinct bat filovirus, whose genome shares 32–54% nucleotide sequence identity with known filoviruses. MLAV glycoprotein-typed pseudo-types can transduce cell lines derived from humans, monkeys, dogs, hamsters and bats.

In the version of this Letter originally published, in the ‘Phylogenetic analysis’ section of the Methods, the authors mistakenly stated that the GenBank accession number for the Ravn virus genome sequence was FJ750958. The correct accession number is DQ447649 for Ravn virus, Kenya, 1987. Accordingly, the label ‘RAVN2007’ in Fig. 1b should have been ‘RAVV1987’. This mistake does not change any conclusions in this study. This statement and figure have now been amended in all versions of the Letter, and the Supplementary Information file has been updated accordingly.

Hand, foot and mouth disease (HFMD) is a viral infectious disease caused by human Enterovirus A, particularly Enterovirus 71 (EV71) and Coxsackievirus 16 (CA16) serotypes, with EV71 infection associated with severe neurological complications and mortality. Lots of attention has been placed on elucidating viral epitopes, which is useful for EV71 viral research. In this study, a murine monoclonal antibody (mAb 4) specific for EV71 was generated and mapped to target the N-terminal region of VP1 capsid protein, spanning amino acid residues 12-19 (IGDSVSRA). mAb 4 can cross-react with all the 11 representative EV71 subgenotypes (A, B1-5, C1-5), but not with the representative strain of CA16 as demonstrated by immunofluorescence assay (IFA). BLAST analyses of this epitope against all Enterovirus entries in Genbank also demonstrated that this epitope is unique in EV71, but not other Enterovirus such as CA16 It may be useful for structural study of VP1 morphogenesis during infection and also applications for identification of EV71 infection.

Background Human Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no efficient universal antibody test available to detect EV71 infections. Methodology/Principal Finding In the present study, an epitope-blocking ELISA was developed to detect specific antibodies to human EV71 viruses in human or animal sera. The assay relies on a novel monoclonal antibody (Mab 1C6) that specifically binds to capsid proteins in whole EV71 viruses without any cross reaction to any EV71 capsid protein expressed alone. The sensitivity and specificity of the epitope-blocking ELISA for EV71 was evaluated and compared to microneutralization using immunized animal sera to multiple virus genotypes of EV71 and coxsackieviruses. Further, 200 serum sample from human individuals who were potentially infected with EV71 viruses were tested in both the blocking ELISA and microneutralization. Results indicated that antibodies to EV71 were readily detected in immunized animals or human sera by the epitope blocking ELISA whereas specimens with antibodies to other enteroviruses yielded negative results. This assay is not only simpler to perform but also shows higher sensitivity and specificity as compared to microneutralization. Conclusion The epitope-blocking ELISA based on a unique Mab 1C6 provided highly sensitive and 100% specific detection of antibodies to human EV71 viruses in human sera.

BACKGROUND: Enterovirus 71 (EV71) is the main causative agent of Hand, Foot and Mouth disease (HFMD) and is associated with severe neurologic complications and mortalities. At present, there is no vaccine or therapeutic available for treatment. METHODOLOGY/PRINCIPAL FINDING: In this study, we generated two mAbs, denoted as mAb 51 and 53, both targeting the same linear epitope on VP1 capsid protein, spanning amino acids 215-219. In comparison, mAb 51 belonging to isotype IgM possesses neutralizing activity in vitro, whereas, mAb 53 belonging to isotype IgG1 does not have any neutralizing ability, even towards its homologous strain. When mAb 51 at 10 µg/g of body weight was administered to the 2-week-old AG129 mice one day prior to lethal challenge, 100% in vivo passive protection was observed. In contrast, the isotype control group mice, injected with an irrelevant IgM antibody before the challenge, developed limb paralysis as early as day 6 post-infection. Histological examination demonstrated that mAb 51 was able to protect against pathologic changes such as neuropil vacuolation and neuronal loss in the spinal cord, which were typical in unprotected EV-71 infected mice. BLAST analyses of that epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 (CA16). CONCLUSION: We have defined a linear epitope within the VP1 protein and demonstrated its neutralizing ability to be isotype dependent. The neutralizing property and highly conserved sequence potentiated the application of mAb 51 and 53 for protection against EV71 infection and diagnosis respectively.

Over the last decade, frequent epidemic outbreaks of hand, foot and mouth disease have been observed in the Asia-Pacific region. Hand, foot and mouth disease is caused by different viruses from the enterovirus family, mainly coxsackievirus A16 and enterovirus 71 (EV71) from the human enterovirus A family. Severe disease and neurological complications are associated more often with EV71 infection, and can lead occasionally to fatal brain stem encephalitis in young children. The rapid progression and high mortality of severe hand, foot and mouth disease makes the direct detection of antigens early in infection essential. The best method for virus detection is the use of specific monoclonal antibodies. The generation and characterization of a monoclonal antibody specific for the 3D polymerase of human enterovirus A and the development of a virus detection dot blot assay are described. A recombinant 3CD protein from EV71 C4 strain was used as an immunogen to generate monoclonal antibodies (MAbs). Screening of hybridoma cells led to the isolation of monoclonal antibody 4B12 of the immunoglobulin IgG1 isotype. MAb 4B12 recognizes the linear epitope DFEQALFS close to the active site of the 3D polymerase, corresponding to amino acid positions 53-60 of 3D and 1784-1791 of enterovirus 71 polyprotein. The presence of 3D polymerase and its precursor 3CD proteinase in purified virus particles was confirmed. MAb 4B12 was used successfully to detect all enterovirus 71 subgenotypes in a denaturing dot blot assay with a sensitivity of 10 pg of 3D protein and 10(4) tissue culture infective dose of virus particles.