Evolution and ecology of Jeilongvirus among wild rodents and shrews in Singapore.

BACKGROUND: Jeilongvirus was proposed as a new genus within the Paramyxoviridae in 2018. The advancement in metagenomic approaches has encouraged multiple reports of Jeilongvirus detection following the initial species discovery, enriching species diversity and host range within the genus. However, Jeilongvirus remains understudied in Singapore, where interfaces between humans and small mammals are plentiful. METHODS: Here, we utilized metagenomic sequencing for the exploration of viral diversity in small mammal tissues. Upon discovery of Jeilongvirus, molecular screening and full genome sequencing was conducted, with the data used to conduct statistical modelling and phylogenetic analysis. RESULTS: We report the presence of Jeilongvirus in four species of Singapore wild small mammals, detected in their spleen and kidney. We show that full genomes of three Singapore Jeilongvirus encode for eight ORFs including the small hydrophobic and transmembrane proteins. All generated genomes cluster phylogenetically within the small mammal subclade, but share low genetic similarity with representative Jeilongvirus species. Statistical modelling showed no spatial or temporal patterns and differences among species, life history traits and habitat types. CONCLUSIONS: This study serves as a basis for understanding dynamics between Jeilongvirus and small mammal hosts in Singapore by displaying the virus generalist nature. In addition, the initial detection can help to invoke improved routine surveillance and detection of circulating pathogens in synanthropic hosts.

Advancing pathogen genomics in resource-limited settings.

Genomic sequencing has emerged as a powerful tool to enhance early pathogen detection and characterization with implications for public health and clinical decision making. Although widely available in developed countries, the application of pathogen genomics among low-resource, high-disease burden settings remains at an early stage. In these contexts, tailored approaches for integrating pathogen genomics within infectious disease control programs will be essential to optimize cost efficiency and public health impact. We propose a framework for embedding pathogen genomics within national surveillance plans across a spectrum of surveillance and laboratory capacities. We adopt a public health approach to genomics and examine its application to high-priority diseases relevant in resource-limited settings. For each grouping, we assess the value proposition for genomics to inform public health and clinical decision-making, alongside its contribution toward research and development of novel diagnostics, therapeutics, and vaccines.

Betacoronaviruses SARS-CoV-2 and HCoV-OC43 infections in IGROV-1 cell line require aryl hydrocarbon receptor.

The emergence of novel betacoronaviruses has posed significant financial and human health burdens, necessitating the development of appropriate tools to combat future outbreaks. In this study, we have characterized a human cell line, IGROV-1, as a robust tool to detect, propagate, and titrate betacoronaviruses SARS-CoV-2 and HCoV-OC43. IGROV-1 cells can be used for serological assays, antiviral drug testing, and isolating SARS-CoV-2 variants from patient samples. Using time-course transcriptomics, we confirmed that IGROV-1 cells exhibit a robust innate immune response upon SARS-CoV-2 infection, recapitulating the response previously observed in primary human nasal epithelial cells. We performed genome-wide CRISPR knockout genetic screens in IGROV-1 cells and identified Aryl hydrocarbon receptor (AHR) as a critical host dependency factor for both SARS-CoV-2 and HCoV-OC43. Using DiMNF, a small molecule inhibitor of AHR, we observed that the drug selectively inhibits HCoV-OC43 infection but not SARS-CoV-2. Transcriptomic analysis in primary normal human bronchial epithelial cells revealed that DiMNF blocks HCoV-OC43 infection via basal activation of innate immune responses. Our findings highlight the potential of IGROV-1 cells as a valuable diagnostic and research tool to combat betacoronavirus diseases.

Host, season, habitat and climatic factors as drivers of Asian rodent tick (Ixodes granulatus) (Acari: Ixodidae) occurrence and abundance in Southeast Asia.

The Asian rodent tick (Ixodes granulatus) occurs throughout much of Asia, it frequently bites humans, and zoonotic pathogens, such as Borrelia burgdorferi (sensu lato) and Rickettsia honei, have been detected within it. Unfortunately, the ecology of I. granulatus remains poorly known, including drivers of its abundance and the interaction ecology with its sylvatic hosts. To elucidate the ecology of this medically important species, the habitat preferences of I. granulatus were assessed in Singapore and Malaysia. Ixodes granulatus showed strong associations with old forest habitats, though across different age classes of old forest there was limited variation in abundance. Ixodes granulatus was absent from other habitats including young forest, scrubland, and parks/gardens. Within its sylvatic rodent hosts, a range of factors were found to be statistically significant predictors of I. granulatus load and/or infestation risk, including sex and body condition index. Male rodents were significantly more likely to be infested and to have higher loads than females, similarly, animals with a lower body condition index were significantly more likely to be infested. Proactive public health efforts targeted at preventing bites by this tick should carefully consider its ecology to minimise ecological overlap between humans and I. granulatus.

The genomic landscape of swine influenza A viruses in Southeast Asia.

Swine are a primary source for the emergence of pandemic influenza A viruses. The intensification of swine production, along with global trade, has amplified the transmission and zoonotic risk of swine influenza A virus (swIAV). Effective surveillance is essential to uncover emerging virus strains; however gaps remain in our understanding of the swIAV genomic landscape in Southeast Asia. More than 4,000 nasal swabs were collected from pigs in Cambodia, yielding 72 IAV-positive samples by RT-qPCR and 45 genomic sequences. We unmasked the cocirculation of multiple lineages of genetically diverse swIAV of pandemic concern. Genomic analyses revealed a novel European avian-like H1N2 swIAV reassortant variant with North American triple reassortant internal genes, that emerged approximately seven years before its first detection in pigs in 2021. Using phylogeographic reconstruction, we identified south central China as the dominant source of swine viruses disseminated to other regions in China and Southeast Asia. We also identified nine distinct swIAV lineages in Cambodia, which diverged from their closest ancestors between two and 15 B.P., indicating significant undetected diversity in the region, including reverse zoonoses of human H1N1/2009 pandemic and H3N2 viruses. A similar period of cryptic circulation of swIAVs occurred in the decades before the H1N1/2009 pandemic. The hidden diversity of swIAV observed here further emphasizes the complex underlying evolutionary processes present in this region, reinforcing the importance of genomic surveillance at the human-swine interface for early warning of disease emergence to avoid future pandemics.

Cencurut virus: A novel Orthonairovirus from Asian house shrews (Suncus murinus) in Singapore.

Orthonairovirus is a genus of viruses in the family Nairoviridae, order Bunyavirales, with a segmented circular RNA genome. They typically infect birds and mammals and are primarily transmitted by ectoparasites such as ticks. Four of nine Orthonairovirus genogroups can infect humans, with Crimean-Congo hemorrhagic fever virus infections displaying case fatality rates up to 40%. Here, we discover and describe a novel Orthonairovirus as Cencurut virus (CENV). CENV was detected in 34 of 37 Asian house shrews (Suncus murinus) sampled in Singapore and in a nymphal Amblyomma helvolum tick collected from an infected shrew. Pairwise comparison of CENV S, M, and L segments had 95.0 to 100% nucleotide and 97.5 to 100% amino acid homology within CENV genomes, suggesting a diverse viral population. Phylogenetic analysis of the individual gene segments showed that CENV is related to Erve, Lamgora, Lamusara, and Thiafora viruses, with only 49.0 to 58.2% nucleotide and 41.7 to 61.1% amino acid homology, which has previously been detected in other shrew species from France, Gabon, and Senegal respectively. The high detection frequency suggests that CENV is endemic among S. murinus populations in Singapore. The discovery of CENV, from a virus family with known zoonotic potential, underlines the importance of surveillance of synanthropic small mammals that are widely distributed across Southeast Asia.

Determinants of influenza and pneumococcal vaccine uptake among preschool children in Singapore.

Young children are at increased risk of severe illness from influenza and pneumococcal infections. The World Health Organization (WHO) recommends vaccination with influenza and pneumococcal conjugate vaccine (PCV). However, in Singapore, vaccine uptake remains suboptimal relative to other routine childhood immunisations. Limited information exists regarding determinants of influenza and pneumococcal vaccine uptake in children. We estimated vaccine uptake and investigated factors associated with influenza and pneumococcal vaccination status by age group using data from a cohort study on acute respiratory infections in children attending preschools in Singapore. We recruited children aged two to six years at 24 participating preschools from June 2017 to July 2018. We determined the proportion of children immunised with influenza vaccine and PCV, and investigated socio-demographic factors associated with vaccine uptake using logistic regression models. Among 505 children, 77.5% were of Chinese ethnicity, and 53.1% were male. History of influenza vaccination was 27.5% of which 11.7% had been vaccinated within the past 12 months. In multivariable analyses, factors associated with influenza vaccine uptake were 'children living in landed property' (aOR = 2.25, 95% CI [1.07-4.67]) and 'history of hospitalisation due to cough' (aOR = 1.85, 95% CI [1.00-3.36]). Nearly three-quarters of participants (70.7% 95%CI: [66.6-74.5]) reported prior PCV vaccination. PCV uptake was higher for younger children. 'Higher parental education' (OR = 2.83, 95% CI [1.51,5.32]), 'household income' (OR = 1.26, 95% CI [1.08,1.48]) and 'smokers in household' (OR = 0.48, 95% CI [0.31,0.74]) were significantly associated with PCV uptake in univariable analyses. Only 'smokers in household' remained significantly associated with PCV uptake (aOR = 0.55, 95% CI [0.33,0.91]) in the adjusted model. Our results indicate that episodes of severe respiratory illness are a cue to influenza vaccination suggesting that doctors are more likely to recommend influenza vaccines to high-risk children. For PCV, our findings suggest overall greater awareness and education on the benefit of PCV vaccination is required.

Spike-Independent Infection of Human Coronavirus 229E in Bat Cells.

Bats are the reservoir for numerous human pathogens, including coronaviruses. Despite many coronaviruses having descended from bat ancestors, little is known about virus-host interactions and broader evolutionary history involving bats. Studies have largely focused on the zoonotic potential of coronaviruses with few infection experiments conducted in bat cells. To determine genetic changes derived from replication in bat cells and possibly identify potential novel evolutionary pathways for zoonotic virus emergence, we serially passaged six human 229E isolates in a newly established Rhinolophus lepidus (horseshoe bat) kidney cell line. Here, we observed extensive deletions within the spike and open reading frame 4 (ORF4) genes of five 229E viruses after passaging in bat cells. As a result, spike protein expression and infectivity of human cells was lost in 5 of 6 viruses, but the capability to infect bat cells was maintained. Only viruses that expressed the spike protein could be neutralized by 229E spike-specific antibodies in human cells, whereas there was no neutralizing effect on viruses that did not express the spike protein inoculated on bat cells. However, one isolate acquired an early stop codon, abrogating spike expression but maintaining infection in bat cells. After passaging this isolate in human cells, spike expression was restored due to acquisition of nucleotide insertions among virus subpopulations. Spike-independent infection of human coronavirus 229E may provide an alternative mechanism for viral maintenance in bats that does not rely on the compatibility of viral surface proteins and known cellular entry receptors. IMPORTANCE Many viruses, including coronaviruses, originated from bats. Yet, we know little about how these viruses switch between hosts and enter human populations. Coronaviruses have succeeded in establishing in humans at least five times, including endemic coronaviruses and the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In an approach to identify requirements for host switches, we established a bat cell line and adapted human coronavirus 229E viruses by serial passage. The resulting viruses lost their spike protein but maintained the ability to infect bat cells, but not human cells. Maintenance of 229E viruses in bat cells appears to be independent of a canonical spike receptor match, which in turn might facilitate cross-species transmission in bats.

Genomic epidemiology of SARS-CoV-2 in Cambodia, January 2020 to February 2021.

The first case of coronavirus disease 2019 (COVID-19) in Cambodia was confirmed on 27 January 2020 in a traveller from Wuhan. Cambodia subsequently implemented strict travel restrictions, and although intermittent cases were reported during the first year of the COVID-19 pandemic, no apparent widespread community transmission was detected. Investigating the routes of severe acute respiratory coronavirus 2 (SARS-CoV-2) introduction into the country was critical for evaluating the implementation of public health interventions and assessing the effectiveness of social control measures. Genomic sequencing technologies have enabled rapid detection and monitoring of emerging variants of SARS-CoV-2. Here, we detected 478 confirmed COVID-19 cases in Cambodia between 27 January 2020 and 14 February 2021, 81.3 per cent in imported cases. Among them, fifty-four SARS-CoV-2 genomes were sequenced and analysed along with representative global lineages. Despite the low number of confirmed cases, we found a high diversity of Cambodian viruses that belonged to at least seventeen distinct PANGO lineages. Phylogenetic inference of SARS-CoV-2 revealed that the genetic diversity of Cambodian viruses resulted from multiple independent introductions from diverse regions, predominantly, Eastern Asia, Europe, and Southeast Asia. Most cases were quickly isolated, limiting community spread, although there was an A.23.1 variant cluster in Phnom Penh in November 2020 that resulted in a small-scale local transmission. The overall low incidence of COVID-19 infections suggests that Cambodia's early containment strategies, including travel restrictions, aggressive testing and strict quarantine measures, were effective in preventing large community outbreaks of COVID-19.

Co-circulation of alpha- and beta-coronaviruses in Pteropus vampyrus flying foxes from Indonesia.

Bats are important reservoirs for alpha- and beta-coronaviruses. Coronaviruses (CoV) have been detected in pteropodid bats from several Southeast Asian countries, but little is known about coronaviruses in the Indonesian archipelago in proportion to its mammalian biodiversity. In this study, we screened pooled faecal samples from the Indonesian colonies of Pteropus vampyrus with unbiased next-generation sequencing. Bat CoVs related to Rousettus leschenaultii CoV HKU9 and Eidolon helvum CoV were detected. The 121 faecal samples were further screened using a conventional hemi-nested pan-coronavirus PCR assay. Three positive samples were successfully sequenced, and phylogenetic reconstruction revealed the presence of alpha- and beta-coronaviruses. CoVs belonging to the subgenera Nobecovirus, Decacovirus and Pedacovirus were detected in a single P. vampyrus roost. This study expands current knowledge of coronavirus diversity in Indonesian flying foxes, highlighting the need for longitudinal surveillance of colonies as continuing urbanization and deforestation heighten the risk of spillover events.

TMEM41B and VMP1 modulate cellular lipid and energy metabolism for facilitating dengue virus infection.

Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are two ER-associated lipid scramblases that play a role in autophagosome formation and cellular lipid metabolism. TMEM41B is also a recently validated host factor required by flaviviruses and coronaviruses. However, the exact underlying mechanism of TMEM41B in promoting viral infections remains an open question. Here, we validated that both TMEM41B and VMP1 are essential host dependency factors for all four serotypes of dengue virus (DENV) and human coronavirus OC43 (HCoV-OC43), but not chikungunya virus (CHIKV). While HCoV-OC43 failed to replicate entirely in both TMEM41B- and VMP1-deficient cells, we detected diminished levels of DENV infections in these cell lines, which were accompanied by upregulation of the innate immune dsRNA sensors, RIG-I and MDA5. Nonetheless, this upregulation did not correspondingly induce the downstream effector TBK1 activation and Interferon-beta expression. Despite low levels of DENV replication, classical DENV replication organelles were undetectable in the infected TMEM41B-deficient cells, suggesting that the upregulation of the dsRNA sensors is likely a consequence of aberrant viral replication rather than a causal factor for reduced DENV infection. Intriguingly, we uncovered that the inhibitory effect of TMEM41B deficiency on DENV replication, but not HCoV-OC43, can be partially reversed using exogenous fatty acid supplements. In contrast, VMP1 deficiency cannot be rescued using the metabolite treatment. In line with the observed phenotypes, we found that both TMEM41B- and VMP1-deficient cells harbor higher levels of compromised mitochondria, especially in VMP1 deficiency which results in severe dysregulations of mitochondrial beta-oxidation. Using a metabolomic profiling approach, we revealed distinctive global dysregulations of the cellular metabolome, particularly lipidome, in TMEM41B- and VMP1-deficient cells. Our findings highlight a central role for TMEM41B and VMP1 in modulating multiple cellular pathways, including lipid mobilization, mitochondrial beta-oxidation, and global metabolic regulations, to facilitate the replication of flaviviruses and coronaviruses.

A Look inside the Replication Dynamics of SARS-CoV-2 in Blyth's Horseshoe Bat (Rhinolophus lepidus) Kidney Cells.

Bats are considered the natural reservoir of numerous emerging viruses such as severe acute respiratory syndrome coronaviruses (SARS-CoVs). There is a need for immortalized bat cell lines to culture and investigate the pathogenicity, replication kinetics, and evolution of emerging coronaviruses. We illustrate the susceptibility and permissiveness of a spontaneously immortalized kidney cell line (Rhileki) from Blyth's horseshoe bat (R. lepidus) to SARS-CoV-2 virus, including clinical isolates, suggesting a possible virus-host relationship. We were able to observe limited SARS-CoV-2 replication in Rhileki cells compared with simian VeroE6 cells. Slower viral replication in Rhileki cells was indicated by higher ct values (RT-PCR) at later time points of the viral culture and smaller foci (foci forming assay) compared with those of VeroE6 cells. With this study we demonstrate that SARS-CoV-2 replication is not restricted to R. sinicus and could include more Rhinolophus species. The establishment of a continuous Rhinolophus lepidus kidney cell line allows further characterization of SARS-CoV-2 replication in Rhinolophus bat cells, as well as isolation attempts of other bat-borne viruses. IMPORTANCE The current COVID-19 pandemic demonstrates the significance of bats as reservoirs for severe viral diseases. However, as bats are difficult to establish as animal models, bat cell lines can be an important proxy for the investigation of bat-virus interactions and the isolation of bat-borne viruses. This study demonstrates the susceptibility and permissiveness of a continuous kidney bat cell line to SARS-CoV-2. This does not implicate the bat species Rhinolophus lepidus, where these cells originate from, as a potential reservoir, but emphasizes the usefulness of this cell line for further characterization of SARS-CoV-2. This can lead to a better understanding of emerging viruses that could cause significant disease in humans and domestic animals.

Serological exposure in Bactrian and dromedary camels in Kazakhstan to a MERS or MERS-like coronavirus.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a camel-borne zoonotic virus endemic across Eastern Africa and the Middle East, with evidence of circulation in Bangladesh and Mongolia. To determine if MERS-CoV was present in Kazakhstan, in 2017-2018, we collected swabs and sera from Bactrian camels (n = 3124) and dromedary (n = 5083). The total seropositivity was 0.54% in Bactrian camels and 0.24% in dromedaries; however, we did not detect MERS-CoV RNA in swab samples. There was no difference in the probability of infection between species or sex, but younger camels had a higher probability of being seropositive, suggesting a recent introduction of the virus to Kazakhstan. The infection of both camel species indicates that they both may play a role as natural reservoirs. These results reinforce the need for continual surveillance, especially at the camel-human interface to understand the risk of zoonotic exposure.

Correction: Filovirus-reactive antibodies in humans and bats in Northeast India imply zoonotic spillover.

[This corrects the article DOI: 10.1371/journal.pntd.0007733.].

Ancestral sequence reconstruction pinpoints adaptations that enable avian influenza virus transmission in pigs.

Understanding the evolutionary adaptations that enable avian influenza viruses to transmit in mammalian hosts could allow better detection of zoonotic viruses with pandemic potential. We applied ancestral sequence reconstruction to gain viruses representing different adaptive stages of the European avian-like (EA) H1N1 swine influenza virus as it transitioned from avian to swine hosts since 1979. Ancestral viruses representing the avian-like precursor virus and EA swine influenza viruses from 1979-1983, 1984-1987 and 1988-1992 were reconstructed and characterized. Glycan-binding analyses showed stepwise changes in the haemagglutinin receptor-binding specificity of the EA swine influenza viruses-that is, from recognition of both α2,3- and α2,6-linked sialosides to recognition of α2,6-linked sialosides only; however, efficient transmission in piglets was enabled by adaptive changes in the viral polymerase protein and nucleoprotein, which have been fixed since 1983. PB1-Q621R and NP-R351K increased viral replication and transmission in piglets when introduced into the 1979-1983 ancestral virus that lacked efficient transmissibility. The stepwise adaptation of an avian influenza virus to a mammalian host suggests that there may be opportunities to intervene and prevent interspecies jumps through strategic coordination of surveillance and risk assessment activities.

H5Nx Viruses Emerged during the Suppression of H5N1 Virus Populations in Poultry.

Highly pathogenic avian influenza (HPAI) H5 viruses have posed a substantial pandemic threat through repeated human infection since their emergence in China in 1996. Nationwide control measures, including vaccination of poultry, were implemented in 2005, leading to a sharp reduction in H5N1 virus outbreaks. In 2008, novel non-N1 subtype (H5Nx) viruses emerged, gradually replacing the dominant H5N1 subtype and causing global outbreaks. The cause of this major shift in the ecology of HPAI H5 viruses remains unknown. Here, we show that major H5N1 virus lineages underwent population bottlenecks in 2006, followed by a recovery in virus populations between 2007 and 2009. Our analyses indicate that control measures, not competition from H5Nx viruses, were responsible for the H5N1 decline, with an H5N1 lineage capable of infecting poultry and wild birds experiencing a less severe population bottleneck due to circulation in unaffected wild birds. We show that H5Nx viruses emerged during the successful suppression of H5N1 virus populations in poultry, providing an opportunity for antigenically distinct H5Nx viruses to propagate. Avian influenza vaccination programs would benefit from universal vaccines targeting a wider diversity of influenza viruses to prevent the emergence of novel subtypes. IMPORTANCE A major shift in the ecology of highly pathogenic avian influenza (HPAI) H5 viruses occurred from 2008 to 2014, when viruses with non-N1 neuraminidase genes (termed H5Nx viruses) emerged and caused global H5 virus outbreaks. Here, we demonstrate that nationwide control measures, including vaccination in China, successfully suppressed H5N1 populations in poultry, providing an opportunity for antigenically distinct H5Nx viruses to emerge. In particular, we show that the widespread use of H5N1 vaccines likely conferred a fitness advantage to H5Nx viruses due to the antigenic mismatch of the neuraminidase genes. These results indicate that avian influenza vaccination programs would benefit from universal vaccines that target a wider diversity of influenza viruses to prevent potential emergence of novel subtypes.

Genetic Characterization of Highly Pathogenic Avian Influenza A(H5N8) Virus in Pakistani Live Bird Markets Reveals Rapid Diversification of Clade 2.3.4.4b Viruses.

The highly pathogenic (HPAI) avian influenza A(H5N1) viruses have undergone reassortment with multiple non-N1-subtype neuraminidase genes since 2008, leading to the emergence of H5Nx viruses. H5Nx viruses established themselves quickly in birds and disseminated from China to Africa, the Middle East, Europe and North America. Multiple genetic clades have successively evolved through frequent mutations and reassortment, posing a continuous threat to domestic poultry and causing substantial economic losses. Live bird markets are recognized as major sources of avian-to-human infection and for the emergence of zoonotic influenza. In Pakistan, the A(H5N1) virus was first reported in domestic birds in 2007; however, avian influenza surveillance is limited and there is a lack of knowledge on the evolution and transmission of the A(H5) virus in the country. We collected oropharyngeal swabs from domestic poultry and environmental samples from six different live bird markets during 2018-2019. We detected and sequenced HPAI A(H5N8) viruses from two chickens, one quail and one environmental sample in two markets. Temporal phylogenetics indicated that all novel HPAI A(H5N8) viruses belonged to clade 2.3.4.4b, with all eight genes of Pakistan A(H5N8) viruses most closely related to 2017 Saudi Arabia A(H5N8) viruses, which were likely introduced via cross-border transmission from neighboring regions approximately three months prior to virus detection into domestic poultry. Our data further revealed that clade 2.3.4.4b viruses underwent rapid lineage expansion in 2017 and acquired significant amino acid mutations, including mutations associated with increased haemagglutinin affinity to human α-2,6 receptors, prior to the first human A(H5N8) infection in Russian poultry workers in 2020. These results highlight the need for systematic avian influenza surveillance in live bird markets in Pakistan to monitor for potential A(H5Nx) variants that may arise from poultry populations.

Host specificity of Hepatocystis infection in short-nosed fruit bats (Cynopterus brachyotis) in Singapore.

Haemosporidians infect a wide diversity of bat genera and species, yet little is known about their transmission cycles or epidemiology. Though several recent studies have focused on the genus Hepatocystis, an Old World parasite primarily infecting bats, monkeys, and squirrels, this group is still understudied with little known about its transmission and molecular ecology. These parasites lack an asexual erythrocytic stage, making them unique from the Plasmodium vertebrate life cycle. In this study, we detected a prevalence of 31% of Hepatocystis in short-nosed fruit bats (Cynopterus brachyotis) in Singapore. Phylogenetic reconstruction with a partial cytochrome b sequence revealed a monophyletic group of Hepatocystis from C. brachyotis in Malaysia, Singapore, and Thailand. There was no relationship with infection and bat age, sex, location, body condition or monsoon season. The absence of this parasite in the five other bat species sampled in Singapore indicates this Hepatocystis species may be host restricted.

Etiology of febrile respiratory infections in the general adult population in Singapore, 2007-2013.

Pathogens that cause upper respiratory infections are numerous and specific preventive and therapeutic strategies are scarce. In order to ascertain the etiological agents resulting in upper respiratory tract infections (URTI) in adults in Singapore, nasal swab samples were collected from 2057 patients presenting with fever at primary healthcare clinics in Singapore from December 2007 to February 2013. Samples were tested using the Luminex NxTAG Respiratory Pathogen Panel that includes 22 respiratory pathogen targets. Patient-reported symptoms and vital signs were recorded and full blood and differential counts taken. Pathogens were detected in the following order of frequency: influenza viruses, rhino-/enteroviruses, coronaviruses, parainfluenza viruses, pneumoviruses, adenovirus, bocavirus and C. pneumoniae. Fifteen virus species were detected as part of coinfections, in which rhinoviruses were the most commonly observed pathogen. Our results suggest that influenza viruses are the main etiological agents, but multiple other respiratory viruses contribute to the total burden of URTI in adults in Singapore.

Genetic diversity and expanded host range of astroviruses detected in small mammals in Singapore.

Astroviruses are a genetically diverse group of viruses that infect a wide range of hosts, including small mammals. Small mammals were trapped at 19 sites across Singapore from November 2011 to May 2014. Pooled oropharyngeal and rectal swabs (n = 518) and large intestine tissue (n = 107) were screened using a PCR to detect the presence of Astrovirus RNA-dependent RNA polymerase (RdRp) gene. Astroviruses were detected in 93 of 625 (14.9%) of samples tested, with eight of 11 species of rats, shrews, and squirrels testing positive. This is the first detection of astroviruses in seven species (Callosciurus notatus, Mus castaneus, Rattus tanezumi, Rattus tiomanicus, Sundamys annandalei, Suncus murinus and Tupaia glis). Phylogenetic analysis of 10 RdRp gene sequences revealed that astroviruses from Singapore small mammals fall in three distinct clades, one that is specific to the common treeshrew (Tupaia glis), and two comprised of multiple species. One of these includes viruses from the cave nectar bat (Eonycteris spelaea), two rodent species, and a squirrel, suggesting that virus spillover from bats to small mammals may have occurred. Our results show an increased host range for astroviruses and highlight their potential for intra- and inter-species transmission.