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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.

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.

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. Ancestral avian influenza A viruses are used to identify adaptive changes in viral polymerase and nucleoproteins that enable efficient replication and transmission in pigs.

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., Graphical abstract Image 1, Highlights • A bat haemosporidian (Hepatocystis) was detected in short nose fruit bats (Cynopterus brachyotis) in Singapore. • Infection was not associated with bat age, sex, sample location, body condition or monsoon season. • Infection was detected in only one bat species, indicating this Hepatocystis species may be host specific.

Work toward completion of the Annonaceae treatment for the Flora of Thailand revealed 18 species previously unrecorded for the country, six of them in the genus Fissistigma. In addition, several species previously placed in synonymy are re-instated, for which we propose three new combinations in the genera Mitrella, Monoon, and Sphaerocoryne.

BackgroundHuman infections with avian influenza viruses (AIV) represent a persistent public health threat. The principal risk factor governing human infection with AIV is from direct contact with infected poultry and is primarily observed in Asia and Egypt where live-bird markets are common.Areas of agreementChanging patterns of virus transmission and a lack of obvious disease manifestations in avian species hampers early detection and efficient control of potentially zoonotic AIV.Areas of controversyDespite extensive studies on biological and environmental risk factors, the exact conditions required for cross-species transmission from avian species to humans remain largely unknown.Growing pointsThe development of a universal (‘across-subtype’) influenza vaccine and effective antiviral therapeutics are a priority.Areas timely for developing researchSustained virus surveillance and collection of ecological and physiological parameters from birds in different environments is required to better understand influenza virus ecology and identify risk factors for human infection.

Bats are the reservoir for numerous human pathogens including coronaviruses. The factors leading to the emergence and sustained transmission of coronaviruses in humans are poorly understood. An outstanding question is how coronaviruses can accomplish a host switch with a likely mismatch between the surface protein spike of a bat virus and the human cellular receptor at the time of zoonotic virus transmission. To 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 cells and analyzed viral genetic changes. Here we observed extensive deletions within the spike and 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 do 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. Upon passaging this isolate in human cells, spike expression was restored due to acquisition of nucleotide insertions amongst virus subpopulations. Spike-independent infection of coronaviruses provides an alternative mechanism for viral maintenance in bats that does not rely on the compatibility of viral surface proteins and cellular entry receptors.

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.

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.

To understand the genetic diversity and patterns of circulation of rhinoviruses (RV) and enteroviruses (EV) in Singapore, we retrospectively screened 2950 nasal swab samples collected from adults presenting to primary care services with signs of febrile illness in Singapore during 2007‐2013 using sequencing and phylogenetic methods. Through sequencing and phylogenetic analysis, our results show the year‐round circulation of the three rhinovirus species, A, B, and C. A diverse set of RV/EV serotypes were detected in Singapore with a predominance of RV‐A in all years, whereas serotypes EV‐C A21 and EV‐D68 were only sporadically detected. This study highlights the previously unrecognized diversity and burden in the adult population in Singapore.

Avian influenza A(H9N2) virus isolated from a poultry worker in Pakistan in 2015 was closely related to viruses detected in poultry farms. Observed mutations in the hemagglutinin related to receptor-binding affinity and antigenicity could affect cross-reactivity with prepandemic H9N2 vaccine strains.

The emergence of a pandemic influenza virus may be better anticipated if we better understand the evolutionary steps taken by avian influenza viruses as they adapt to mammals. We used ancestral sequence reconstruction to resurrect viruses representing initial adaptive stages of the European avian-like H1N1 virus as it transitioned from avian to swine hosts. We demonstrate that efficient transmissibility in pigs was gained through stepwise adaptation after 1983. These time-dependent adaptations resulted in changes in hemagglutinin receptor binding specificity and increased viral polymerase activity. An NP-R351K mutation under strong positive selection increased the transmissibility of a reconstructed virus. The stepwise-adaptation of a wholly avian influenza virus to a mammalian host suggests a window where targeted intervention may have highest impact. Successful intervention will, however, require strategic coordination of surveillance and risk assessment activities to identify these adapting viruses and guide pandemic preparedness resources.

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.

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., Upper respiratory tract infection, Virus etiology, Singapore, Adults, Viral infection.

Wild birds are considered the natural reservoir of influenza A viruses (IAVs) making them critical for IAV surveillance efforts. While sea ducks have played a role in novel IAV emergence events that threatened food security and public health, very few surveillance samples have been collected from sea duck hosts. From 2014&ndash, 2018, we conducted surveillance focused in the Mississippi flyway, USA at locations where sea duck harvest has been relatively successful compared to our other sampling locations. Our surveillance yielded 1662 samples from sea ducks, from which we recovered 77 IAV isolates. Our analyses identified persistence of sea duck specific IAV lineages across multiple years. We also recovered sea duck origin IAVs containing an H4 gene highly divergent from the majority of North American H4-HA with clade node age of over 65 years. Identification of IAVs with long branch lengths is indicative of substantial genomic change consistent with persistence without detection by surveillance efforts. Sea ducks play a role in the movement and long-term persistence of IAVs and are likely harboring more undetected IAV diversity. Sea ducks should be a point of emphasis for future North American wild bird IAV surveillance efforts.

During the SARS epidemic in 2003/2004, a number of deletions were observed in ORF8 of SARS-CoV, and eventually deletion variants became predominant, leading to the hypothesis that ORF8 was an evolutionary hot spot for adaptation of SARS-CoV to humans. However, due to the successful control of the SARS epidemic, the importance of these deletions for the epidemiological fitness of SARS-CoV in humans could not be established. The emergence of multiple SARS-CoV-2 strains with ORF8 deletions, combined with evidence of a robust immune response to ORF8, suggests that the lack of ORF8 may assist with host immune evasion. In addition to providing a key insight into the evolutionary behavior of SARS-CoV-2 as the virus adapts to its new human hosts, the emergence of ORF8 deletion variants may also impact vaccination strategies., To date, limited genetic changes in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome have been described. Here, we report a 382-nucleotide (nt) deletion in SARS-CoV-2 that truncates open reading frame 7b (ORF7b) and ORF8, removing the ORF8 transcription regulatory sequence (TRS) and eliminating ORF8 transcription. The earliest 382-nt deletion variant was detected in Singapore on 29 January 2020, with the deletion viruses circulating in the country and accounting for 23.6% (45/191) of SARS-CoV-2 samples screened in this study. SARS-CoV-2 with the same deletion has since been detected in Taiwan, and other ORF7b/8 deletions of various lengths, ranging from 62 nt to 345 nt, have been observed in other geographic locations, including Australia, Bangladesh, and Spain. Mutations or deletions in ORF8 of SARS-CoV have been associated with reduced replicative fitness and virus attenuation. In contrast, the SARS-CoV-2 382-nt deletion viruses showed significantly higher replicative fitness in vitro than the wild type, while no difference was observed in patient viral load, indicating that the deletion variant viruses retained their replicative fitness. A robust antibody response to ORF8 has been observed in SARS-CoV-2 infection, suggesting that the emergence of ORF8 deletions may be due to immune-driven selection and that further deletion variants may emerge during the sustained transmission of SARS-CoV-2 in humans.

Summary Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with a 382-nucleotide deletion (∆382) in the open reading frame 8 (ORF8) region of the genome have been detected in Singapore and other countries. We investigated the effect of this deletion on the clinical features of infection. Methods We retrospectively identified patients who had been screened for the ∆382 variant and recruited to the PROTECT study—a prospective observational cohort study conducted at seven public hospitals in Singapore. We collected clinical, laboratory, and radiological data from patients' electronic medical records and serial blood and respiratory samples taken during hospitalisation and after discharge. Individuals infected with the ∆382 variant were compared with those infected with wild-type SARS-CoV-2. Exact logistic regression was used to examine the association between the infection groups and the development of hypoxia requiring supplemental oxygen (an indicator of severe COVID-19, the primary endpoint). Follow-up for the study's primary endpoint is completed. Findings Between Jan 22 and March 21, 2020, 278 patients with PCR-confirmed SARS-CoV-2 infection were screened for the ∆382 deletion and 131 were enrolled onto the study, of whom 92 (70%) were infected with the wild-type virus, ten (8%) had a mix of wild-type and ∆382-variant viruses, and 29 (22%) had only the ∆382 variant. Development of hypoxia requiring supplemental oxygen was less frequent in the ∆382 variant group (0 [0%] of 29 patients) than in the wild-type only group (26 [28%] of 92; absolute difference 28% [95% CI 14–28]). After adjusting for age and presence of comorbidities, infection with the ∆382 variant only was associated with lower odds of developing hypoxia requiring supplemental oxygen (adjusted odds ratio 0·07 [95% CI 0·00–0·48]) compared with infection with wild-type virus only. Interpretation The ∆382 variant of SARS-CoV-2 seems to be associated with a milder infection. The observed clinical effects of deletions in ORF8 could have implications for the development of treatments and vaccines. Funding National Medical Research Council Singapore.

To date, the SARS-CoV-2 genome has been considered genetically more stable than SARS-CoV or MERS-CoV. Here we report a 382-nt deletion covering almost the entire open reading frame 8 (ORF8) of SARS-CoV-2 obtained from eight hospitalized patients in Singapore. The deletion also removes the ORF8 transcription-regulatory sequence (TRS), which in turn enhances the downstream transcription of the N gene. We also found that viruses with the deletion have been circulating for at least four weeks. During the SARS-CoV outbreak in 2003, a number of genetic variants were observed in the human population [1], and similar variation has since been observed across SARS-related CoVs in humans and bats. Overwhelmingly these viruses had mutations or deletions in ORF8, that have been associated with reduced replicative fitness of the virus [2]. This is also consistent with the observation that towards the end of the outbreak sequences obtained from human SARS cases possessed an ORF8 deletion that may be associated with host adaptation [1]. We therefore hypothesise that the major deletion revealed in this study may lead to an attenuated phenotype of SARS-CoV-2.

Bartonella species are arthropod-borne bacterial pathogens that infect numerous mammalian species. Small mammals play an important role as natural reservoirs of many Bartonella species, maintaining the greatest diversity of Bartonella described to date. Although Bartonella research has been conducted in Southeast Asia, no studies have been undertaken on small mammals in Singapore. Here, we report the detection and description of Bartonella in small mammals in Singapore during the period of November 2011 to May 2014. BartonellaDNA was detected in 20.8% (22/106) of small mammal spleens with a PCR amplifying the beta subunit of bacterial RNA polymerase (rpoB) gene. Commensal species Rattus norvegicus and Rattus tanezumi had the highest prevalence, 75% (3/4) and 34,5% (10/29), followed by Suncus murinus 30% (6/20), Tupaia glis 16,7% (1/6) and Mus castaneus 13.3% (2/15). Phylogenetic analysis of 18 rpoB gene sequences revealed five Bartonella genotypes circulating in the small mammals of Singapore. Bayesian tip-significance testing demonstrated strong structuring in the geographical signal, indicating that distribution of Bartonella species is correlated to the distribution of their hosts. Major deforestation and fragmentation in Singapore favour synanthropic species that traverse habitats and increase the possibility of spillover to incidental hosts.

Bats are unique mammals that are reservoirs of high levels of virus diversity. Although several of these viruses are zoonotic, the majority are not. Astroviruses, transmitted fecal-orally, are commonly detected in a wide diversity of bat species, are prevalent at high rates and are not thought to directly infect humans. These features make astroviruses useful in examining virus evolutionary history, epidemiology in the host, and temporal shedding trends. Our study screened for the presence of astroviruses in bats in Singapore, reconstructed the phylogenetic relations of the polymerase genes and tested for population characteristics associated with infection. Of the seven species screened, astroviruses were detected in Rhinolophus lepidus and Eonycteris spelaea. The R. lepidus sequences grouped with other Rhinolophus astrovirus sequences from China and Laos, while the Eoncyteris sequences formed a distinct clade with astroviruses from Rousettus spp. in Laos and Pteropus giganteus in Bangladesh, but not with other E. spelaea sequences. Longitudinal collections of Eonycteris feces demonstrated variable shedding. Juvenile status of bats was a risk factor for astroviruses. This study highlights the diversity of astroviruses in nectivorous and insectivorous bats in Singapore and provides a predictive framework for understanding astrovirus infection in these bats. It also suggests that in addition to host phylogenetic relatedness, host ecology, such as roosting behavior, may drive co-infections, virus maintenance and spillover.

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.

An H1N1 subtype influenza A virus with all eight gene segments derived from wild birds (including mallards), ducks and chickens, caused severe disease outbreaks in swine populations in Europe beginning in 1979 and successfully adapted to form the European avian‐like swine (EA‐swine) influenza lineage. Genes of the EA‐swine lineage that are clearly segregated from its closest avian relatives continue to circulate in swine populations globally and represent a unique opportunity to study the adaptive process of an avian‐to‐mammalian cross‐species transmission. Here, we used a relaxed molecular clock model to test whether the EA‐swine virus originated through the introduction of a single avian ancestor as an entire genome, followed by an analysis of host‐specific selection pressures among different gene segments. Our data indicated independent introduction of gene segments via transmission of avian viruses into swine followed by reassortment events that occurred at least 1–4 years prior to the EA‐swine outbreak. All EA‐swine gene segments exhibit greater selection pressure than avian viruses, reflecting both adaptive pressures and relaxed selective constraints that are associated with host switching. Notably, we identified key amino acid mutations in the viral surface proteins (H1 and N1) that play a role in adaptation to new hosts. Following the establishment of EA‐swine lineage, we observed an increased frequency of intrasubtype reassortment of segments compared to the earlier strains that has been associated with adaptive amino acid replacements, disease severity and vaccine escape. Taken together, our study provides key insights into the adaptive changes in viral genomes following the transmission of avian influenza viruses to swine and the early establishment of the EA‐swine lineage.

Cross-species transmission can often lead to deleterious effects in incidental hosts. Parvoviruses have a wide host range and primarily infect members of the order Carnivora. Here we describe juvenile common palm civet cats (Paradoxurus musangus) that were brought to the Singapore zoo and fell ill while quarantined. The tissues of two individual civets that died tested PCR-positive for parvovirus infection. Phylogenetic analysis revealed this parvovirus strain falls in a basal position to a clade of CPV that have infected dogs in China and Uruguay, suggesting cross-species transmission from domestic to wild animals. Our analysis further identified these viruses as genotype CPV-2a that is enzootic in carnivores. The ubiquity of virus infection in multiple tissues suggests this virus is pathogenic to civet cats. Here we document the cross-species transmission from domestic dogs and cats to wild civet populations, highlighting the vulnerability of wildlife to infectious agents in companion animals.

Background A number of serious human adenovirus (HAdV) outbreaks have been recently reported: HAdV-B7 (Israel, Singapore, and USA), HAdV-B7d (USA and China), HAdV-D8, -D54, and -C2 (Japan), HAdV-B14p1 (USA, Europe, and China), and HAdV-B55 (China, Singapore, and France). Methods To understand the epidemiology of HAdV infections in Singapore, we studied 533 HAdV-positive clinical samples collected from 396 pediatric and 137 adult patients in Singapore from 2012 to 2018. Genome sequencing and phylogenetic analyses were performed to identify HAdV genotypes, clonal clusters, and recombinant or novel HAdVs. Results The most prevalent genotypes identified were HAdV-B3 (35.6%), HAdV-B7 (15.4%), and HAdV-E4 (15.2%). We detected 4 new HAdV-C strains and detected incursions with HAdV-B7 (odds ratio [OR], 14.6; 95% confidence interval [CI], 4.1–52.0) and HAdV-E4 (OR, 13.6; 95% CI, 3.9–46.7) among pediatric patients over time. In addition, immunocompromised patients (adjusted OR [aOR], 11.4; 95% CI, 3.8–34.8) and patients infected with HAdV-C2 (aOR, 8.5; 95% CI, 1.5–48.0), HAdV-B7 (aOR, 3.7; 95% CI, 1.2–10.9), or HAdV-E4 (aOR, 3.2; 95% CI, 1.1–8.9) were at increased risk for severe disease. Conclusions Singapore would benefit from more frequent studies of clinical HAdV genotypes to identify patients at risk for severe disease and help guide the use of new antiviral therapies, such as brincidofovir, and potential administration of HAdV 4 and 7 vaccine., Human adenovirus (HAdV) outbreaks are often inexplicable with sparse clinical epidemiological data. We sought to describe clinical HAdV genotypes and identify risk factors associated with severe disease among HAdV-positive patients seen at 2 hospitals in Singapore.

Routine surveillance and surveillance in response to influenza virus outbreaks in avian species in Vietnam in 2009–2013 resulted in the isolation of numerous H5N1 influenza viruses of clades 1.1.2, 2.3.2.1a, 2.3.2.1b, 2.3.2.1c, and 2.3.4.1. Consistent with other studies, we found that viruses of clade 2.3.2.1c were dominant in Vietnam in 2013 and circulated in the northern, central, and southern parts of the country. Phylogenetic analysis revealed reassortment among viruses of clades 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c; in contrast, no reassortment was detected between clade 2.3.2.1 viruses and viruses of clades 1.1.2 or 2.3.4.1, respectively. Deep-sequencing of 42 of the 53 isolated H5N1 viruses revealed viral subpopulations encoding variants that may affect virulence, host range, or sensitivity to antiviral compounds; viruses isolates containing these subpopulations may have a higher potential to transmit and adapt to mammals. Among the viruses sequenced, relatively high numbers of non-synonymous nucleotide polymorphisms were detected in three viruses isolated from barn swallows, possibly suggesting influenza virus adaption to this host.

Coronaviruses are positive-stranded RNA viruses that infect a variety of hosts, resulting in a range of symptoms from gastrointestinal illness to respiratory distress. Bats are reservoirs for a high diversity of coronaviruses, and focused surveillance detected several strains genetically similar to MERS-coronavirus, SARS-coronavirus, and the human coronaviruses 229E and NL63. The bat fauna of central Asia, which link China to eastern Europe, are relatively less studied than other regions of the world. Kazakhstan is the world’s ninth largest country; however, little is understood about the prevalence and diversity of bat-borne viruses. In this study, bat guano was collected from bat caves in three different sites of southern Kazakhstan that tested positive for coronaviruses. Our phylogenetic reconstruction indicates these are novel bat coronaviruses that belong to the genus Alphacoronavirus. In addition, two distinct lineages of Kazakhstan bat coronaviruses were detected. Both lineages are closely related to bat coronaviruses from China, France, Spain, and South Africa, suggesting that co-circulation of coronaviruses is common in multiple bat species with overlapping geographical distributions. Our study highlights the need for collaborative efforts in understudied countries to increase integrated surveillance capabilities toward better monitoring and detection of infectious diseases.

Avian influenza virus (AIV) surveillance in Antarctica during 2013 revealed the prevalence of evolutionarily distinct influenza viruses of the H11N2 subtype in Adélie penguins. Here we present results from the continued surveillance of AIV on the Antarctic Peninsula during 2014 and 2015. In addition to the continued detection of H11 subtype viruses in a snowy sheathbill during 2014, we isolated a novel H5N5 subtype virus from a chinstrap penguin during 2015. Gene sequencing and phylogenetic analysis revealed that the H11 virus detected in 2014 had a >99.1% nucleotide similarity to the H11N2 viruses isolated in 2013, suggesting the continued prevalence of this virus in Antarctica over multiple years. However, phylogenetic analysis of the H5N5 virus showed that the genome segments were recently introduced to the continent, except for the NP gene, which was similar to that in the endemic H11N2 viruses. Our analysis indicates geographically diverse origins for the H5N5 virus genes, with the majority of its genome segments derived from North American lineage viruses but the neuraminidase gene derived from a Eurasian lineage virus. In summary, we show the persistence of AIV lineages in Antarctica over multiple years, the recent introduction of gene segments from diverse regions, and reassortment between different AIV lineages in Antarctica, which together significantly increase our understanding of AIV ecology in this fragile and pristine environment. IMPORTANCE Analysis of avian influenza viruses (AIVs) detected in Antarctica reveals both the relatively recent introduction of an H5N5 AIV, predominantly of North American-like origin, and the persistence of an evolutionarily divergent H11 AIV. These data demonstrate that the flow of viruses from North America may be more common than initially thought and that, once introduced, these AIVs have the potential to be maintained within Antarctica. The future introduction of AIVs from North America into the Antarctic Peninsula is of particular concern given that highly pathogenic H5Nx viruses have recently been circulating among wild birds in parts of Canada and the Unites States following the movement of these viruses from Eurasia via migratory birds. The introduction of a highly pathogenic influenza virus in penguin colonies within Antarctica might have devastating consequences.

Summary Coronaviruses are a diverse group of viruses that infect mammals and birds. Bats are reservoirs for several different coronaviruses in the Alphacoronavirus and Betacoronavirus genera. They also appear to be the natural reservoir for the ancestral viruses that generated the severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus outbreaks. Here, we detected coronavirus sequences in next‐generation sequence data created from Eonycteris spelaea faeces and urine. We also screened by PCR urine samples, faecal samples and rectal swabs collected from six species of bats in Singapore between 2011 and 2014, all of which were negative. The phylogenetic analysis indicates this novel strain is most closely related to lineage D Betacoronaviruses detected in a diverse range of bat species. This is the second time that coronaviruses have been detected in cave nectar bats, but the first coronavirus sequence data generated from this species. Bat species from which this group of coronaviruses has been detected are widely distributed across SE Asia, South Asia and Southern China. They overlap geographically, often share roosting sites and have been witnessed to forage on the same plant. The addition of sequence data from this group of viruses will allow us to better understand coronavirus evolution and host specificity.

Here we summarize an April 2016, 7-patient cluster of human adenovirus (HAdV) infections in a cardiothoracic surgery intensive care unit. We show that the patients were infected with a single HAdV21b type. Rapid HAdV typing diagnostics and effective antiviral interventions are needed for immunocompromised patients suffering from HAdV infections.

The systematic position of two enigmatic Annonaceae species from China, Desmos saccopetaloides (W. T. Wang) P. T. Li and Desmos yunnanensis (Hu) P. T. Li, has been controversial, with both species having been transferred between several different genera within subfamilies Annonoideae and Malmeoideae. Phylogenetic analyses of eight chloroplast regions (matK, ndhF, ndhF-rpl32, psbA-trnH, rbcL, rpl32-trnL, trnL-F, and ycf1; ca. 9.2kb, 66 taxa) unambiguously placed D. saccopetaloides in a subclade of tribe Miliuseae, nested among the genera Monoon, Neo-uvaria, Phaeanthus, Sageraea, and Stelechocarpus. This relationship was also supported by endosperm rumination patterns in the seed; other morphological characters furthermore indicated that D. saccopetaloides has closer affinities with Monoon, Neo-uvaria, and Phaeanthus rather than either Sageraea or Stelechocarpus. Desmos saccopetaloides is distinguished from these genera by its leaf-opposed inflorescences, sepaloid outer petals, saccate inner petals with basal glandular tissue, moniliform monocarps with uniseriate seeds, and rectangular disulculate pollen with two cryptoapertures. On the basis of the combined molecular phylogenetic and morphological data, we propose a new genus, Wangia, to accommodate D. saccopetaloides. The molecular phylogenetic analyses furthermore indicated that D. yunnanensis belongs to the genus Dasymaschalon: examination of the type collections revealed that it is conspecific with Dasymaschalon obtusipetalum, although the combination Dasymaschalon yunnanense has nomenclatural priority.

Bats are unique mammals, exhibit distinctive life history traits and have unique immunological approaches to suppression of viral diseases upon infection. High-throughput next-generation sequencing has been used in characterizing the virome of different bat species. The cave nectar bat, Eonycteris spelaea, has a broad geographical range across Southeast Asia, India and southern China, however, little is known about their involvement in virus transmission. Here we investigate the diversity and abundance of viral communities from a colony of Eonycteris spelaea residing in Singapore. Our results detected 47 and 22 different virus families from bat fecal and urine samples, respectively. Among these, we identify a large number of virus families including Adenoviridae, Flaviviridae, Reoviridae, Papillomaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, and Polyomaviridae. In most cases, viral sequences from Eonycteris spelaea are genetically related to a group of bat viruses from other bat genera (e.g., Eidolon, Miniopterus, Rhinolophus and Rousettus). The results of this study improve our knowledge of the host range, spread and evolution of several important viral pathogens. More significantly, our findings provide a baseline to study the temporal patterns of virus shedding and how they correlate with bat phenological trends.

Surveillance of H9N2 is currently focused on areas central to the commercial poultry industry. This study determined the prevalence of H9N2 virus in commercial and backyard poultry flocks in Punjab Province, Pakistan. Oral and tracheal swabs were collected from commercial and backyard poultry from January 2015 through June 2016. Antisera against H5, H7, H9, and Newcastle disease viruses were used for virus identification. Molecular confirmation was made by reverse transcription PCR. Avian influenza virus subtypes H5 and H7 were not detected. The H9N2 virus was isolated in 5.7% of 905 tested flocks (5-10 birds/flock). Prevalence in commercial and backyard poultry was 6.7% of 687 flocks and 2.7% of 218 flocks, respectively. Hemagglutinin and neuraminidase-gene-based phylogenetic analysis of commercial and backyard poultry isolates showed 100% homology. Within sublineage B2 of Pakistan, identity among most recent isolates (2015) was 100%, compared to 75%-99% identity with previously isolated viruses (2010-12), indicating continued virus evolution. Most of the previously reported and currently studied viruses were isolated near the Pakistan-India border. Phylogenetic analysis showed that Pakistani and Indian isolates were closely related, indicating that avian influenza virus transmission may occur across this border.Prevalencia y filogenia del virus de la influenza H9N2 en avicultura de traspatio y comercial en Pakistán. La vigilancia del virus de influenza H9N2 se centra actualmente en áreas centrales de la industria avícola comercial. Este estudio determinó la prevalencia del virus de influenza H9N2 en parvadas comerciales y de traspatio en la provincia de Punjab, Pakistán. Se recolectaron hisopos orales y traqueales de aves comerciales y de traspatio desde enero del 2015 hasta junio del 2016. Se usaron antisueros contra los virus de la enfermedad H5, H7, H9 y contra Newcastle para la identificación del virus. La confirmación molecular se realizó mediante transcripción reversa y PCR. No se detectaron los subtipos H5 y H7 del virus de la influenza aviar. El virus H9N2 se aisló en 5.7% de 905 parvadas analizadas (5-10 aves/parvada). La prevalencia en aves comerciales y domésticas fue del 6.7% de 687 bandadas y de 2.7% de 218 bandadas, respectivamente. El análisis filogenético basado en el gene de la hemaglutinina y de la neuraminidasa de los aislamientos de aves comerciales y de traspatio mostró 100% de identidad genética. Dentro del sublinaje B2 de Pakistán, la identidad entre los aislados más recientes (2015) fue del 100%, en comparación con el 75% al 99% de identidad con los virus aislados previamente (años 2010 al 2012), lo que indica la continua evolución del virus. La mayoría de los virus previamente reportados y estudiados actualmente se aislaron cerca de la frontera entre Pakistán e India. El análisis filogenético mostró que los aislamientos de Pakistán e India estaban estrechamente relacionados, lo que indica que la transmisión del virus de la influenza aviar puede ocurrir a través de esta frontera.

Influenza A H10N7 virus with a hemagglutinin gene of North American origin was detected in Australian chickens and poultry abattoir workers in New South Wales, Australia, in 2010 and in chickens in Queensland, Australia, on a mixed chicken and domestic duck farm in 2012. We investigated their genomic origins by sequencing full and partial genomes of H10 viruses isolated from wild aquatic birds and poultry in Australia and analyzed them with all available avian influenza virus sequences from Oceania and representative viruses from North America and Eurasia. Our analysis showed that the H10N7 viruses isolated from poultry were similar to those that have been circulating since 2009 in Australian aquatic birds and that their initial transmission into Australia occurred during 2007 and 2008. The H10 viruses that appear to have developed endemicity in Australian wild aquatic birds were derived from several viruses circulating in waterfowl along various flyways. Their hemagglutinin gene was derived from aquatic birds in the western states of the United States, whereas the neuraminidase was closely related to that from viruses previously detected in waterfowl in Japan. The remaining genes were derived from Eurasian avian influenza virus lineages. Our analysis of virological data spanning 40 years in Oceania indicates that the long-term evolutionary dynamics of avian influenza viruses in Australia may be determined by climatic changes. The introduction and long-term persistence of avian influenza virus lineages were observed during periods with increased rainfall, whereas bottlenecks and extinction were observed during phases of widespread decreases in rainfall. These results extend our understanding of factors affecting the dynamics of avian influenza and provide important considerations for surveillance and disease control strategies.

Since 2013, there have been several alarming influenza-related events; the spread of highly pathogenic avian influenza H5 viruses into North America, the detection of H10N8 and H5N6 zoonotic infections, the ongoing H7N9 infections in China and the continued zoonosis of H5N1 viruses in parts of Asia and the Middle East. The risk of a new influenza pandemic increases with the repeated interspecies transmission events that facilitate reassortment between animal influenza strains; thus, it is of utmost importance to understand the factors involved that promote or become a barrier to cross-species transmission of Influenza A viruses (IAVs). Here, we provide an overview of the ecology and evolutionary adaptations of IAVs, with a focus on a review of the molecular factors that enable interspecies transmission of the various virus gene segments.

Background Undifferentiated acute febrile illness (AFI) is a common presentation among adults in primary care settings in Singapore but large gaps exist in the understanding of the characteristics of these patients. We studied clinical and epidemiological characteristics of AFI patients and factors associated with delayed recovery from AFI. Methods We performed a secondary data analysis using data from the Early DENgue infection and outcome (EDEN) study on 2046 adult patients presenting at 5 Singapore polyclinics between December 2007 and February 2013 with a history of fever (≥38 °C) for less than 72 h. We used an accelerated failure time model to investigate factors associated with delayed recovery from AFI. Results The mean age of patients was 36.6 years, 65 % were male, 51 % were of Chinese ethnicity, and 75 % lived in public housing. Median illness duration was 5 days (interquartile range, 3–7). In multivariable analysis, the unemployed and white collar workers had longer illness duration compared with blue collar workers (time ratio (TR), 1.10; 95 % confidence interval (CI), 1.03–1.17 and TR, 1.08; 95 % CI, 1.02–1.15, respectively). Patients with more symptoms at initial consultation had slower recovery (TR, 1.03 per additional symptom; 95 % CI, 1.02–1.03). Other clinical factors were also associated with longer duration of illness, including use of analgesics (TR, 1.21; 95 % CI, 1.15–1.28); use of cough medicines (TR, 1.14; 95 % CI, 1.08–1.20); use of antibiotics (TR, 1.14; 95 % CI, 1.07–1.21); and hospitalization (TR, 1.59; 95 % CI, 1.39–1.82). Compared to patients with normal WBC count at first consultation, those with low WBC count had slower recovery (TR, 1.14; 95 % CI, 1.07–1.21), while the reverse was observed among patients with high WBC count (TR, 0.94; 95 % CI, 0.88–1.00). Conclusions Differences in illness duration among different types of employment may reflect differences in their underlying general health status. Early identification of factors delaying recovery could help triage management in a primary care setting. In-depth characterization of fever etiology in Singapore will improve surveillance and control activities.

Cross-species transmission can often lead to deleterious effects in incidental hosts. Parvoviruses have a wide host range and primarily infect members of the order Carnivora. Here we describe juvenile common palm civet cats (Paradoxurus musangus) that were brought to the Singapore zoo and fell ill while quarantined. The tissues of two individual civets that died tested PCR-positive for parvovirus infection. Phylogenetic analysis revealed this parvovirus strain falls in a basal position to a clade of CPV that have infected dogs in China and Uruguay, suggesting cross-species transmission from domestic to wild animals. Our analysis further identified these viruses as genotype CPV-2a that is enzootic in carnivores. The ubiquity of virus infection in multiple tissues suggests this virus is pathogenic to civet cats. Here we document the cross-species transmission from domestic dogs and cats to wild civet populations, highlighting the vulnerability of wildlife to infectious agents in companion animals.

Dasymaschalon and the closely related genera Desmos, Friesodielsia and Monanthotaxis together comprise ca. 170 species of trees, shrubs and woody climbers distributed in tropical Africa and tropical Asia. These genera form the desmoid clade, which, because of the presence of diverse flower and fruit syndromes including different types of pollination chambers and moniliform monocarps, offers an opportunity to investigate potentially ecologically significant shifts in flower and fruit characters. Despite its morphological diversity, however, generic delimitation within the desmoid clade is problematic and the intergeneric relationships of the constituent genera are only poorly understood. Bayesian, maximum likelihood and maximum parsimony analyses of plastid DNA sequence data ( matK, psbA-trnH, ndhF, rbcL, trnL-F; ca. 5.4 kb; 52 taxa) were used to clarify phylogenetic relationships within the desmoid clade. The evolution and taxonomic utility of selected fruit and flower characters was investigated with likelihood and parsimony ancestral character reconstructions. The results indicate problems in the current delimitations of Dasymaschalon and Friesodielsia. Friesodielsia as currently circumscribed is polyphyletic, with African Friesodielsia species allied to the African genus Monanthotaxis, and only distantly related to Asian representatives. The majority of Dasymaschalon species form a strongly supported clade, but three species are more closely related to Asian species of Friesodielsia. Ancestral character reconstructions indicate that seed number and monocarp shape are of limited value in generic circumscriptions, and that the three-petalled corolla characteristic of Dasymaschalon evolved independently twice within the desmoid clade. Disruptions to homeotic gene expression or strong selective pressure for a partial enclosure of the mature stamens and carpels by the corolla are hypothesised to underlie the parallel evolution of pollination chambers formed by outer petal homologues subsequent to inner petal loss

Aim African–Asian disjunctions are common in palaeotropical taxa, and are typically explained by reference to three competing hypotheses: (1) ‘rafting’ on the Indian tectonic plate, enabling Africa-to-Asia dispersal; (2) migration via Eocene boreotropical forests; and (3) transoceanic long-distance dispersal. These hypotheses are tested using Uvaria (Annonaceae), which is distributed in tropical regions of Africa, Asia and Australasia. Recent phylogenetic reconstructions of the genus show a clear correlation with geographical provenance, indicating a probable origin in Africa and subsequent dispersal to Asia and then Australasia. Ancestral areas and migration routes are inferred and compared with estimates of divergence times in order to distinguish between the prevailing dispersal hypotheses. Location Palaeotropics. Methods Divergence times in Uvaria are estimated by analysing the sequences of four DNA regions (matK, psbA–trnH spacer, rbcL and trnL–F) from 59 Uvaria species and 77 outgroup species, using a Bayesian uncorrelated lognormal (UCLD) relaxed molecular clock. The ancestral area of Uvaria and subsequent dispersal routes are inferred using statistical dispersal–vicariance analysis (s-diva). Results Uvaria is estimated to have originated in continental Africa 31.6 Ma [95% highest posterior density (HPD): 38.4–25.1 Ma] between the Middle Eocene and Late Oligocene. Two main migration events during the Miocene are identified: dispersal into Madagascar around 17.0 Ma (95% HPD: 22.3–12.3 Ma); and dispersal into Asia between 21.4 Ma (95% HPD: 26.7–16.7 Ma) and 16.1 Ma (95% HPD: 20.1–12.1 Ma). Main conclusions Uvaria fruits are widely reported to be consumed by primates, and are therefore unlikely candidates for successful long-distance transoceanic dispersal. The other biogeographical hypotheses, involving rafting on the Indian tectonic plate, and dispersal via the European boreotropical forests associated with the Eocene thermal maximum, can be discounted due to incongruence with the divergence time estimates. An alternative scenario is suggested, involving dispersal across Arabia and central Asia via the tropical forests that developed during the late Middle Miocene thermal maximum (17–15 Ma), associated with the ‘out-of-Africa’ dispersal of primates. The probable route and mechanism of overland dispersal between Africa and Asia for tropical plant groups during the Miocene climatic optimum are clarified based on the Uvaria data.