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Homeodomain only protein (Hopx) is a regulator of cell differentiation and function, and it has also emerged as a crucial marker of specific developmental and differentiation potentials. Hopx expression and functions have been identified in some stem cells, tumors, and in certain immune cells. However, expression of Hopx in immune cells remains insufficiently characterized. Here we report a comprehensive pattern of Hopx expression in multiple types of immune cells under steady state conditions. By utilizing single-cell RNA sequencing (scRNA-seq) and flow cytometric analysis, we characterize a constitutive expression of Hopx in specific subsets of CD4+ and CD8+ T cells and B cells, as well as natural killer (NK), NKT, and myeloid cells. In contrast, Hopx expression is not present in conventional dendritic cells and eosinophils. The utility of identifying expression of Hopx in immune cells may prove vital in delineating specific roles of Hopx under multiple immune conditions.

BackgroundCell proliferation is a fundamental event during development, disease, and regeneration. Effectively tracking and quantifying proliferating cells and their derivatives is critical for addressing many research questions. Cell cycle expression such as for Ki67, proliferating cell nuclear antigen (PCNA), or aurora kinase B (Aurkb), or measurement of 5-bromo-2′-deoxyuridine (BrdU) or 3H-thymidine incorporation have been widely used to assess and quantify cell proliferation. These are powerful tools for detecting actively proliferating cells, but they do not identify cell populations derived from proliferating progenitors over time.AimsWe developed a new mouse tool for lineage tracing of proliferating cells by targeting the Aurkb allele.ResultsIn quiescent cells or cells arrested at G1/S, little or no Aurkb mRNA is detectable. In cycling cells, Aurkb transcripts are detectable at G2 and become undetectable by telophase. These findings suggest that Aurkb transcription is restricted to proliferating cells and is tightly coupled to cell proliferation. Accordingly, we generated an AurkbER Cre/+ mouse by targeting a tamoxifen inducible Cre cassette into the start codon of Aurkb. We find that the AurkbER Cre/+ mouse faithfully labels proliferating cells in developing embryos and regenerative adult tissues such as intestine but does not label quiescent cells such as post-mitotic neurons.ConclusionThe AurkbER Cre/+ mouse faithfully labels proliferating cells and their derivatives in developing embryos and regenerative adult tissues. This new mouse tool provides a novel genetic tracing capability for studying tissue proliferation and regeneration.

Editorial summary The heart is one of the least regenerative organs in the human body; adult cardiac myocytes divide at extremely low frequency. Therefore, meaningful induction of cardiac regeneration requires in-depth understanding of myocyte cell-cycle control. Recent insights into how myocytes can be coaxed into duplicating in vivo might inform emerging therapeutics.

Semaphorin ligands and cognate receptors are important in patterning the vasculature. Here, Aghajanian et al.report an unexpected role for ErbB2 in endothelial cells where it partners with Nrp1 to form a novel semaphoring holoreceptor required for embryonic vascular patterning.

Summary: Formation of the coronary vasculature is a complex and precisely coordinated morphogenetic process that begins with the formation of epicardium. The epicardium gives rise to many components of the coronary vasculature, including fibroblasts, smooth muscle cells, and endothelium. Hippo signaling components have been implicated in cardiac development and regeneration. However, a role of Hippo signaling in the epicardium has not been explored. Employing a combination of genetic and pharmacological approaches, we demonstrate that inhibition of Hippo signaling mediators Yap and Taz leads to impaired epicardial epithelial-to-mesenchymal transition (EMT) and a reduction in epicardial cell proliferation and differentiation into coronary endothelial cells. We provide evidence that Yap and Taz control epicardial cell behavior, in part by regulating Tbx18 and Wt1 expression. Our findings show a role for Hippo signaling in epicardial cell proliferation, EMT, and cell fate specification during cardiac organogenesis. : Singh et al. show that Hippo signaling components are expressed in proepicardial and epicardial cells and are required for coronary vasculature development. Yap and Taz regulate epicardial cell proliferation, EMT, and cell fate specification, in part by regulating Tbx18 and Wt1 expression. Keywords: epicardium, proepicardium, Hippo signaling, Yap, Taz, epithelial to mesenchymal transition (EMT)

In the adult dentate gyrus (DG) and in the proliferative zone lining the lateral ventricle (LV-PZ), radial glia-like (RGL) cells are neural stem cells (NSCs) that generate granule neurons. A number of molecular markers including glial fibrillary acidic protein (GFAP), Sox2 and nestin, can identify quiescent NSCs in these two niches. However, to date, there is no marker that distinguishes NSC origin of DG versus LV-PZ. Hopx, an atypical homeodomain only protein, is expressed by adult stem cell populations including those in the intestine and hair follicle. Here, we show that Hopx is specifically expressed in RGL cells in the adult DG, and these cells give rise to granule neurons. Assessed by non-stereological quantitation, Hopx-null NSCs exhibit enhanced neurogenesis evident by an increased number of BrdU-positive cells and doublecortin (DCX)-positive neuroblasts. In contrast, Sox2-positive, quiescent NSCs are reduced in the DG of Hopx-null animals and Notch signaling is reduced, as evidenced by reduced expression of Notch targets Hes1 and Hey2, and a reduction of the number of cells expressing the cleaved, activated form of the Notch1 receptor, the Notch intracellular domain (NICD) in Hopx-null DG. Surprisingly, Hopx is not expressed in RGL cells of the adult LV-PZ, and Hopx-expressing cells do not give rise to interneurons of the olfactory bulb (OB). These findings establish that Hopx expression distinguishes NSCs of the DG from those of the LV-PZ, and suggest that Hopx potentially regulates hippocampal neurogenesis by modulating Notch signaling.

Prior efforts to identify regulators of hematopoietic stem cell physiology have relied mainly on candidate gene approaches with genetically modified mice. Here we used a genome-wide association study (GWAS) strategy with the hybrid mouse diversity panel to identify the genetic determinants of hematopoietic stem/progenitor cell (HSPC) frequency. Among 108 strains, we observed ∼120- to 300-fold variation in three HSPC populations. A GWAS analysis identified several loci that were significantly associated with HSPC frequency, including a locus on chromosome 5 harboring the homeodomain-only protein gene (Hopx). Hopx previously had been implicated in cardiac development but was not known to influence HSPC biology. Analysis of the HSPC pool in Hopx−/− mice demonstrated significantly reduced cell frequencies and impaired engraftment in competitive repopulation assays, thus providing functional validation of this positional candidate gene. These results demonstrate the power of GWAS in mice to identify genetic determinants of the hematopoietic system.

Summary: Loss of Pax3, a developmentally regulated transcription factor expressed in premigratory neural crest, results in severe developmental defects and embryonic lethality. Although Pax3 mutations produce profound phenotypes, the intrinsic transcriptional activation exhibited by Pax3 is surprisingly modest. We postulated the existence of transcriptional coactivators that function with Pax3 to mediate developmental functions. A high-throughput screen identified the Hippo effector proteins Taz and Yap65 as Pax3 coactivators. Synergistic coactivation of target genes by Pax3-Taz/Yap65 requires DNA binding by Pax3, is Tead independent, and is regulated by Hippo kinases Mst1 and Lats2. In vivo, Pax3 and Yap65 colocalize in the nucleus of neural crest progenitors in the dorsal neural tube. Neural crest deletion of Taz and Yap65 results in embryo-lethal neural crest defects and decreased expression of the Pax3 target gene, Mitf. These results suggest that Pax3 activity is regulated by the Hippo pathway and that Pax factors are Hippo effectors. : Hippo signaling is a conserved kinase cascade that mediates transcription through a Yap65/Taz-Tead complex and governs organ size. Manderfield et al. have identified Yap65 and Taz as coactivators of Pax factors and established neural crest as a novel site of Hippo signaling. The Pax3-Yap65/Taz complex is regulated by upstream Hippo kinases and is Tead independent. Deletion of Yap65 and Taz in neural crest generated differentiation defects in specific neural crest derivatives.

The recent development of targeted murine reporter alleles as proxies for intestinal stem cell activity has led to significant advances in our understanding of somatic stem cell hierarchies and dynamics. Analysis of these reporters has led to a model in which an indispensable reserve stem cell at the top of the hierarchy (marked by Bmi1 and Hopx reporters) gives rise to active intestinal stem cells (marked by an Lgr5 reporter). Despite these advances, controversy exists regarding the specificity and fidelity with which these alleles distinguish intestinal stem cell populations. Here, we undertake a comprehensive comparison of widely used proxy reporters including both CreERT2 and EGFP cassettes targeted to the Lgr5, Bmi1, and Hopx loci. Single-cell transcriptional profiling of these populations and their progeny reveals that reserve and active intestinal stem cells are molecularly and functionally distinct, supporting a two-stem-cell model for intestinal self-renewal.

Neurofibromatosis type 1 (NF1) is a common autosomal-dominant disorder associated with attention deficits and learning disabilities. The primary known function of neurofibromin, encoded by the NF1 gene, is to downregulate Ras activity. We show that nf1-deficient zebrafish exhibit learning and memory deficits and that acute pharmacological inhibition of downstream targets of Ras (MAPK and PI3K) restores memory consolidation and recall but not learning. Conversely, acute pharmacological enhancement of cAMP signaling restores learning but not memory. Our data provide compelling evidence that neurofibromin regulates learning and memory by distinct molecular pathways in vertebrates and that deficits produced by genetic loss of function are reversible. These findings support the investigation of cAMP signaling enhancers as a companion therapy to Ras inhibition in the treatment of cognitive dysfunction in NF1.

The death of cardiac myocytes resulting from myocardial infarction is a major cause of heart failure worldwide. Effective therapies for regenerating lost cardiac myocytes are lacking. Recently, the epicardium has been implicated as a source of inflammatory cytokines, growth factors and progenitor cells that modulate the response to myocardial injury. During embryonic development, epicardially-derived cells have the potential to differentiate into multiple cardiac lineages, including fibroblasts, vascular smooth muscle and potentially other cell types. In the healthy adult heart, epicardial cells are thought to be generally quiescent. However, injury of the adult heart results in reactivation of a developmental gene program in the epicardium, which leads to increased epicardial cell proliferation and differentiation of epicardium-derived cells (EPDCs) into various cardiac lineages. Recent work suggests that epicardial reactivation after injury is accompanied by, and contributes to, a robust inflammatory response. In this review, we describe the current status of research related to epicardial biology in cardiac development and regeneration, highlighting important recent discoveries and ongoing controversies.

SUMMARY Neurofibromatosis type 1 (NF1) is a common, dominantly inherited genetic disorder that results from mutations in the neurofibromin 1 (NF1) gene. Affected individuals demonstrate abnormalities in neural-crest-derived tissues that include hyperpigmented skin lesions and benign peripheral nerve sheath tumors. NF1 patients also have a predisposition to malignancies including juvenile myelomonocytic leukemia (JMML), optic glioma, glioblastoma, schwannoma and malignant peripheral nerve sheath tumors (MPNSTs). In an effort to better define the molecular and cellular determinants of NF1 disease pathogenesis in vivo, we employed targeted mutagenesis strategies to generate zebrafish harboring stable germline mutations in nf1a and nf1b, orthologues of NF1. Animals homozygous for loss-of-function alleles of nf1a or nf1b alone are phenotypically normal and viable. Homozygous loss of both alleles in combination generates larval phenotypes that resemble aspects of the human disease and results in larval lethality between 7 and 10 days post fertilization. nf1-null larvae demonstrate significant central and peripheral nervous system defects. These include aberrant proliferation and differentiation of oligodendrocyte progenitor cells (OPCs), dysmorphic myelin sheaths and hyperplasia of Schwann cells. Loss of nf1 contributes to tumorigenesis as demonstrated by an accelerated onset and increased penetrance of high-grade gliomas and MPNSTs in adult nf1a+/−; nf1b−/−; p53e7/e7 animals. nf1-null larvae also demonstrate significant motor and learning defects. Importantly, we identify and quantitatively analyze a novel melanophore phenotype in nf1-null larvae, providing the first animal model of the pathognomonic pigmentation lesions of NF1. Together, these findings support a role for nf1a and nf1b as potent tumor suppressor genes that also function in the development of both central and peripheral glial cells as well as melanophores in zebrafish.

High pathogenicity avian influenza (HPAI) virus H5N1 first emerged in Bangladesh in 2007. Despite the use of vaccines in chickens since 2012 to control HPAI, HPAI H5Nx viruses have continued to infect poultry, and wild birds, resulting in notable mass mortalities in house crows (Corvus splendens). The first HPAI H5Nx viruses in Bangladesh belonged to clade 2.2.2, followed by clade 2.3.4.2 and 2.3.2.1 viruses in 2011. After the implementation of chicken vaccination in 2012, these viruses were mostly replaced by clade 2.3.2.1a viruses and more recently clade 2.3.4.4b and h viruses. In this study, we reconstruct the phylogenetic history of HPAI H5Nx viruses in Bangladesh to evaluate the role of major host species in the maintenance and evolution of HPAI H5Nx virus in Bangladesh and reveal the role of heavily impacted crows in virus epidemiology. Epizootic waves caused by HPAI H5N1 and H5N6 viruses amongst house crows occurred annually in winter. Bayesian phylodynamic analysis of clade 2.3.2.1a revealed frequent bidirectional viral transitions between domestic ducks, chickens, and house crows that was markedly skewed towards ducks; domestic ducks might be the source, or reservoir, of HPAI H5Nx in Bangladesh, as the number of viral transitions from ducks to chickens and house crows was by far more numerous than the other transitions. Our results suggest viral circulation in domestic birds despite vaccination, with crow epizootics acting as a sentinel. The vaccination strategy needs to be updated to use more effective vaccinations, assess vaccine efficacy, and extension of vaccination to domestic ducks, the key reservoir.

Avian influenza virus (AIV) is of major concern to livestock, wildlife, and human health. In many countries in the world, including Bangladesh, AIV is endemic in poultry, requiring improving biosecurity. In Bangladesh, we investigated how variation in biosecurity practices in commercial chicken farms affected their AIV infection status to help guide AIV mitigation strategies. We collected pooled fecal swabs from 225 farms and tested the samples for the AIV matrix gene followed by H5, H7, and H9 subtyping using rRT-PCR. We found that 39.6% of chicken farms were AIV positive, with 13% and 14% being positive for subtypes H5 and H9, respectively. Using a generalized linear mixed effects model, we identified as many as 12 significant AIV risk factors. Two major factors promoting AIV risk that cannot be easily addressed in the short term were farm size and the proximity of the farm to a live bird market. However, the other ten significant determinants of AIV risk can be more readily addressed, of which the most important ones were limiting access by visitors (reducing predicted AIV risk from 42 to 6%), isolation and treatment of sick birds (42 to 7%), prohibiting access of vehicles to poultry sheds (38 to 8%), improving hand hygiene (from 42 to 9%), not sharing farm workers across farms (37 to 8%), and limiting access by wild birds to poultry sheds (37 to 8%). Our findings can be applied to developing practical and cost-effective measures that significantly decrease the prevalence of AIV in chicken farms. Notably, in settings with limited resources, such as Bangladesh, these measures can help governments strengthen biosecurity practices in their poultry industry to limit and possibly prevent the spread of AIV.

Abstract Novel coronavirus species of public health and veterinary importance have emerged in the first two decades of the twenty-first century, with bats identified as natural hosts for progenitors of many coronaviruses. Targeted wildlife surveillance is needed to identify the factors involved in viral perpetuation within natural host populations, and drivers of interspecies transmission. We monitored a natural colony of Egyptian rousette bats at monthly intervals across two years to identify circulating coronaviruses, and to investigate shedding dynamics and viral maintenance within the colony. Three distinct lineages were detected, with different seasonal temporal excretion dynamics. For two lineages, the highest periods of coronavirus shedding were at the start of the year, when large numbers of bats were found in the colony. Highest peaks for a third lineage were observed towards the middle of the year. Among individual bat-level factors (age, sex, reproductive status, and forearm mass index), only reproductive status showed significant effects on excretion probability, with reproductive adults having lower rates of detection, though factors were highly interdependent. Analysis of recaptured bats suggests that viral clearance may occur within one month. These findings may be implemented in the development of risk reduction strategies for potential zoonotic coronavirus transmission.

Abstract Background Bats are increasingly being recognized as important hosts for viruses, some of which are zoonotic and carry the potential for spillover within human and livestock populations. Biosurveillance studies focused on assessing the risk of pathogen transmission, however, have largely focused on the virological component and have not always considered the ecological implications of different species as viral hosts. The movements of known viral hosts are an important component for disease risk assessments as they can potentially identify regions of higher risk of contact and spillover. As such, this study aimed to synthesize data from both virological and ecological fields to provide a more holistic assessment of the risk of pathogen transmission from bats to people. Results Using radiotelemetry, we tracked the small-scale movements of Rousettus aegyptiacus, a species of bat known to host Marburg virus and other viruses with zoonotic potential, in a rural settlement in Limpopo Province, South Africa. The tracked bats exhibited seasonal variations in their movement patterns including variable usage of residential areas which could translate to contact between bats and humans and may facilitate spillover. We identified a trend for increased usage of residential areas during the winter months with July specifically experiencing the highest levels of bat activity within residential areas. July has previously been identified as a key period for increased spillover risk for viruses associated with R. aegyptiacus from this colony and paired with the increased activity levels, illustrates the risk for spillover to human populations. Conclusion This study emphasizes the importance of incorporating ecological data such as movement patterns with virological data to provide a better understanding of the risk of pathogen spillover and transmission.

High Pathogenicity Avian Influenza (HPAI) H5N1 outbreaks continue to wreak havoc on the global poultry industry and threaten the health of wild bird populations, with sporadic spillover in humans and other mammals, resulting in widespread calls to vaccinate poultry. Bangladesh has been vaccinating poultry since 2012, presenting a prime opportunity to study the effects of vaccination on HPAI H5N1circulation in both poultry and wild birds. We investigated the efficacy of vaccinating commercial poultry against HPAI H5N1 along with climatic and socio-economic factors considered potential drivers of HPAI H5N1 outbreak risk in Bangladesh. Using a multivariate modeling approach, we estimated that the rate of outbreaks was 18 times higher before compared to after vaccination, with winter months having a three times higher chance of outbreaks than summer months. Variables resulting in small but significant increases in outbreak rate were relatively low ambient temperatures for the time of year, literacy rate, chicken and duck density, crop density, and presence of highways; this may be attributable to low temperatures supporting viral survival outside the host, higher literacy driving reporting rate, density of the host reservoir, and spread of the virus through increased connectivity. Despite the substantial impact of vaccination on outbreaks, we note that HPAI H5N1 is still enzootic in Bangladesh; vaccinated poultry flocks have high rates of H5N1 prevalence, and spillover to wild birds has increased. Vaccination in Bangladesh thus bears the risk of supporting “silent spread,” where the vaccine only provides protection against disease and not also infection. Our findings underscore that poultry vaccination can be part of holistic HPAI mitigation strategies when accompanied by monitoring to avoid silent spread.

Highly pathogenic avian influenza virus subtype H5N1 endangers poultry, wildlife, and human health and is enzootic in large parts of Asia, with live bird markets (LBMs) as putative hotspots for their maintenance, amplification, and spread. To mitigate the extent of these and other avian influenza viruses (AIV) of concern, we aimed to increase our quantitative understanding of the factors determining the presence of avian influenza virus in LBM stalls. Between 2016 and 2017, we collected fecal or offal samples from 1008 stalls in 113 LBMs across the Dhaka and Rajshahi districts in Bangladesh. For each stall, samples were pooled and tested for the AIV matrix gene, followed by H5 and H9 subtyping using rRT-PCR. We detected Influenza A viral RNA in 49% of the stalls. Of the AIV positive samples, 52% and 24% were determined to be H5 and H9 viruses, respectively, which are both subtypes of considerable health concern. We used generalized linear mixed effect modelling to study AIV presence in individual stalls within LBMs as a function of 13 out of the 20 risk factors identified by FAO. We found that small and feasible improvements in cleaning and disinfection frequency, installing running water in stalls, and not mixing different breeds of chicken in the same cages had large impacts on the presence of AIV in stalls (Odds ratios 0.03–0.05). Next, cleaning vehicles used in poultry transport, not selling waterfowl with chickens in the same stall, buying stock directly from commercial farms, separating sick birds from healthy ones, and avoiding access by wild birds like house crows, also had major effects on lowering the risk of stalls having AIV (Odds ratios 0.16–0.33). These findings can be directly used in developing practical and affordable measures to reduce the prevalence of AIV in LBMs. Also, in settings with limited resources like Bangladesh, such mitigation may significantly contribute to reducing AIV circulation amongst poultry and spillover to wildlife and humans.

Live bird markets (LBMs) are critical for poultry trade in many developing countries that are regarded as hotspots for the prevalence and contamination of avian influenza viruses (AIV). Therefore, we conducted weekly longitudinal environmental surveillance in LBMs to determine annual cyclic patterns of AIV subtypes, environmental risk zones, and the role of climatic factors on the AIV presence and persistence in the environment of LBM in Bangladesh. From January 2018 to March 2020, we collected weekly fecal and offal swab samples from each LBM and tested using rRT-PCR for the M gene and subtyped for H5, H7, and H9. We used Generalized Estimating Equations (GEE) approaches to account for repeated observations over time to correlate the AIV prevalence and potential risk factors and the negative binomial and Poisson model to investigate the role of climatic factors on environmental contamination of AIV at the LBM. Over the study period, 37.8% of samples tested AIV positive, 18.8% for A/H5, and A/H9 was, for 15.4%. We found the circulation of H5, H9, and co-circulation of H5 and H9 in the environmental surfaces year-round. The Generalized Estimating Equations (GEE) model reveals a distinct seasonal pattern in transmitting AIV and H5. Specifically, certain summer months exhibited a substantial reduction of risk up to 70–90% and 93–94% for AIV and H5 contamination, respectively. The slaughtering zone showed a significantly higher risk of contamination with H5, with a three-fold increase in risk compared to bird-holding zones. From the negative binomial model, we found that climatic factors like temperature and relative humidity were also significantly associated with weekly AIV circulation. An increase in temperature and relative humidity decreases the risk of AIV circulation. Our study underscores the significance of longitudinal environmental surveillance for identifying potential risk zones to detect H5 and H9 virus co-circulation and seasonal transmission, as well as the imperative for immediate interventions to reduce AIV at LBMs in Bangladesh. We recommend adopting a One Health approach to integrated AIV surveillance across animal, human, and environmental interfaces in order to prevent the epidemic and pandemic of AIV.

The pathogens that cause most emerging infectious diseases in humans originate in animals, particularly wildlife, and then spill over into humans. The accelerating frequency with which humans and domestic animals encounter wildlife because of activities such as land-use change, animal husbandry, and markets and trade in live wildlife has created growing opportunities for pathogen spillover. The risk of pathogen spillover and early disease spread among domestic animals and humans, however, can be reduced by stopping the clearing and degradation of tropical and subtropical forests, improving health and economic security of communities living in emerging infectious disease hotspots, enhancing biosecurity in animal husbandry, shutting down or strictly regulating wildlife markets and trade, and expanding pathogen surveillance. We summarize expert opinions on how to implement these goals to prevent outbreaks, epidemics, and pandemics.

Spillovers of Nipah virus (NiV) from Pteropus bats to humans occurs frequently in Bangladesh, but the risk for spillover into other animals is poorly understood. We detected NiV antibodies in cattle, dogs, and cats from 6 sites where spillover human NiV infection cases occurred during 2013–2015.

Knowledge of the dynamics and genetic diversity of Nipah virus circulating in bats and at the human-animal interface is limited by current sampling efforts, which produce few detections of viral RNA. We report a series of investigations at Pteropus medius bat roosts identified near the locations of human Nipah cases in Bangladesh during 2012–2019. Pooled bat urine was collected from 23 roosts; 7 roosts (30%) had >1 sample in which Nipah RNA was detected from the first visit. In subsequent visits to these 7 roosts, RNA was detected in bat urine up to 52 days after the presumed exposure of the human case-patient, although the probability of detection declined rapidly with time. These results suggest that rapidly deployed investigations of Nipah virus shedding from bat roosts near human cases could increase the success of viral sequencing compared with background surveillance and could enhance understanding of Nipah virus ecology and evolution.

Avian influenza viruses (AIV) have been frequently detected in live bird markets (LBMs) around the world, primarily in urban areas, and have the ability to spillover to other species, including humans. Despite frequent detection of AIV in urban LBMs, the contamination of AIV on environmental surfaces in rural and peri-urban LBMs in Bangladesh is poorly documented. Therefore, we conducted this study to determine the prevalence of AIV subtypes within a subset of peri-urban and rural LBMs in Bangladesh and to further identify associated risk factors. Between 2017 and 2018, we collected faecal and offal samples from 200 stalls in 63 LBMs across four sub-districts. We tested the samples for the AIV matrix gene (M-gene) followed by H5, H7, and H9 subtypes using real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). We performed a descriptive analysis of market cleanliness and sanitation practices in order to further elucidate the relationship between LBM biosecurity and AIV subtypes by species, sample types, and landscape. Subsequently, we conducted a univariate analysis and a generalized linear mixed model (GLMM) to determine the risk factors associated with AIV contamination at individual stalls within LBMs. Our findings indicate that practices related to hygiene and the circulation of AIV significantly differed between rural and peri-urban live bird markets. 42.5% (95% CI: 35.56–49.67) of stalls were positive for AIV. A/H5, A/H9, and A HA/Untyped were detected in 10.5% (95% CI: 6.62–15.60), 9% (95% CI: 5.42–13.85), and 24.0% (95% CI: 18.26–30.53) of stalls respectively, with no detection of A/H7. Significantly higher levels of AIV were found in the Sonali chicken strain compared to the exotic broiler, and in offal samples compared to fecal samples. In the GLMM analysis, we identified several significant risk factors associated with AIV contamination in LBMs at the stall level. These include: landscape (AOR: 3.02; 95% CI: 1.18–7.72), the number of chicken breeds present (AOR: 2.4; 95% CI: 1.01–5.67), source of birds (AOR: 2.35; 95% CI: 1.0–5.53), separation of sick birds (AOR: 3.04; 95% CI: 1.34–6.92), disposal of waste/dead birds (AOR: 3.16; 95% CI: 1.41–7.05), cleaning agent (AOR: 5.99; 95% CI: 2.26–15.82), access of dogs (AOR: 2.52; 95% CI: 1.12–5.7), wild birds observed on site (AOR: 2.31; 95% CI: 1.01–5.3). The study further revealed a substantial prevalence of AIV with H5 and H9 subtypes in peri-urban and rural LBMs. The inadequate biosecurity measures at poultry stalls in Bangladesh increase the risk of AIV transmission from poultry to humans. To prevent the spread of AIV to humans and wild birds, we suggest implementing regular surveillance at live bird markets and enhancing biosecurity practices in peri-urban and rural areas in Bangladesh.

IntroductionAntimicrobial resistance (AMR) is a growing global health threat for humans and animals. Environmental contamination of antimicrobials from human and domestic animal feces has been linked to AMR in wildlife populations, including rhesus macaques. This study aimed to describe the eco-epidemiology of AMR within Salmonella and Staphylococcus species isolated from rhesus macaques.MethodsWe followed macaque groups for 4 h per day (2 days) to observe the direct and indirect contact rate and type between macaques and people and livestock. We collected 399 freshly defecated, non-invasive fecal samples from macaques at seven sites in Bangladesh in January–June 2017. Bacterial isolation and identification were conducted using culture, biochemical characteristics, and polymerase chain reaction (PCR). An antimicrobial susceptibility test (AST) for 12 antimicrobials for each organism was conducted using the Kirby–Bauer disc diffusion method.ResultsThe overall prevalence of Salmonella spp. and Staphylococcus spp. in rhesus macaques was 5% (n = 18; 95% CI: 3–7%) and 16% (n = 64; 95% CI: 13–20%), respectively. All the isolated Salmonella spp. and most of the Staphylococcus spp. (95%; 61/64; 95% CI: 86.9–99%) were resistant to at least one antimicrobial. The odds of a fecal sample having antimicrobial-resistant Salmonella spp (OR = 6.6; CI: 0.9–45.8, P = 0.05) and Staphylococcus spp. (OR = 5.6; CI: 1.2–26, P = 0.02) were significantly higher in samples collected at peri-urban sites than those collected at rural and urban sites. Salmonella spp. were most frequently resistant to tetracycline (89%), azithromycin (83%), sulfamethoxazole-trimethoprim (50%), and nalidixic acid (44%). Staphylococcus spp. were found to be highly resistant to ampicillin (93%), methicillin (31%), clindamycin (26%), and rifampicin (18%). Both bacterial species produced colonies with multidrug resistance to up to seven antimicrobials. Direct and indirect contact rates (within 20 m for at least 15 min) and resource sharing between macaques and people were higher in urban sites, while macaque-livestock contact rates were higher in rural sites.DiscussionThe study shows that resistant microorganisms are circulating in rhesus macaque, and direct and indirect contact with humans and livestock might expand the resistant organisms.

Abstract Bat-borne viruses in the Henipavirus genus have been associated with zoonotic diseases of high morbidity and mortality in Asia and Australia. In Africa, the Egyptian rousette bat species (Rousettus aegyptiacus) is an important viral host in which Henipavirus-related viral sequences have previously been identified. We expanded these findings by assessing the viral dynamics in a southern African bat population. A longitudinal study of henipavirus diversity and excretion dynamics identified 18 putative viral species circulating in a local population, three with differing seasonal dynamics, and the winter and spring periods posing a higher risk of virus spillover and transmission. The annual peaks in virus excretion are most likely driven by subadults and may be linked to the waning of maternal immunity and recolonization of the roost in early spring. These results provide insightful information into the bat-host relationship that can be extrapolated to other populations across Africa and be communicated to at-risk communities as a part of evidence-based public health education and prevention measures against pathogen spillover threats.

Dysregulation of the imprinted H19/IGF2 locus can lead to Silver-Russell syndrome (SRS) in humans. However, the mechanism of how abnormal H19/IGF2 expression contributes to various SRS phenotypes remains unclear, largely due to incomplete understanding of the developmental functions of these two genes. We previously generated a mouse model with humanized H19/IGF2 imprinting control region (hIC1) on the paternal allele that exhibited H19/Igf2 dysregulation together with SRS-like growth restriction and perinatal lethality. Here, we dissect the role of H19 and Igf2 in cardiac and placental development utilizing multiple mouse models with varying levels of H19 and Igf2. We report severe cardiac defects such as ventricular septal defects and thinned myocardium, placental anomalies including thrombosis and vascular malformations, together with growth restriction in mouse embryos that correlated with the extent of H19/Igf2 dysregulation. Transcriptomic analysis using cardiac endothelial cells of these mouse models shows that H19/Igf2 dysregulation disrupts pathways related to extracellular matrix and proliferation of endothelial cells. Our work links the heart and placenta through regulation by H19 and Igf2, demonstrating that accurate dosage of both H19 and Igf2 is critical for normal embryonic development, especially related to the cardiac-placental axis.

Abstract Background Traditional, instructor led, in-person training of CPR skills has become more challenging due to COVID-19 pandemic. We compared the learning outcomes of standard in-person CPR training (ST) with alternative methods of training such as hybrid or online-only training (AT) on CPR performance, quality, and knowledge among laypersons with no previous CPR training. Methods We searched PubMed and Google Scholar for relevant articles from January 1995 to May 2020. Covidence was used to review articles by two independent researchers. Effective Public Health Practice Project (EPHPP) Quality Assessment Tool was used to assess quality of the manuscripts. Results Of the 978 articles screened, twenty met the final inclusion criteria. All included studies had an experimental design and moderate to strong global quality rating. The trainees in ST group performed better on calling 911, time to initiate chest compressions, hand placement and chest compression depth. Trainees in AT group performed better in assessing scene safety, calling for help, response time including initiating first rescue breathing, adequate ventilation volume, compression rates, shorter hands-off time, confidence, willingness to perform CPR, ability to follow CPR algorithm, and equivalent or better knowledge retention than standard teaching methodology. Conclusion AT methods of CPR training provide an effective alternative to the standard in-person CPR for large scale public training.

Abstract Mitigating the risk of acquiring coronaviruses including SARS-CoV-2 requires awareness of the survival of virus on high-touch environmental surfaces (HITES) and skin, and frequent use of targeted microbicides with demonstrated efficacy. The data on stability of infectious SARS-CoV-2 on surfaces and in suspension have been put into perspective, as these inform the need for hygiene. We evaluated the efficacies of formulated microbicidal actives against alpha- and beta-coronaviruses, including SARS-CoV-2. The coronaviruses SARS-CoV, SARS-CoV-2, human coronavirus 229E, murine hepatitis virus-1, or MERS-CoV were deposited on prototypic HITES or spiked into liquid matrices along with organic soil loads. Alcohol-, quaternary ammonium compound-, hydrochloric acid-, organic acid-, p-chloro-m-xylenol-, and sodium hypochlorite-based microbicidal formulations were evaluated per ASTM International and EN standard methodologies. All evaluated formulated microbicides inactivated SARS-CoV-2 and other coronaviruses in suspension or on prototypic HITES. Virucidal efficacies (≥ 3 to ≥ 6 log10 reduction) were displayed within 30 s to 5 min. The virucidal efficacy of a variety of commercially available formulated microbicides against SARS-CoV-2 and other coronaviruses was confirmed. These microbicides should be useful for targeted surface and hand hygiene and disinfection of liquids, as part of infection prevention and control for SARS-CoV-2 and emerging mutational variants, and other emerging enveloped viruses.

Abstract Background Improved understanding of the foraging ecology of bats in the face of ongoing habitat loss and modification worldwide is essential to their conservation and maintaining the substantial ecosystem services they provide. It is also fundamental to assessing potential transmission risks of zoonotic pathogens in human-wildlife interfaces. We evaluated the influence of environmental and behavioral variables on the foraging patterns of Pteropus lylei (a reservoir of Nipah virus) in a heterogeneous landscape in Cambodia. Methods We employed an approach based on animal-movement modeling, which comprised a path-segmentation method (hidden Markov model) to identify individual foraging-behavior sequences in GPS data generated by eight P. lylei. We characterized foraging localities, foraging activity, and probability of returning to a given foraging locality over consecutive nights. Generalized linear mixed models were also applied to assess the influence of several variables including proxies for energetic costs and quality of foraging areas. Results Bats performed few foraging bouts (area-restricted searches) during a given night, mainly in residential areas, and the duration of these decreased during the night. The probability of a bat revisiting a given foraging area within 48 h varied according to the duration previously spent there, its distance to the roost site, and the corresponding habitat type. We interpret these fine-scale patterns in relation to global habitat quality (including food-resource quality and predictability), habitat-familiarity and experience of each individual. Conclusions Our study provides evidence that heterogeneous human-made environments may promote complex patterns of foraging-behavior and short-term re-visitation in fruit bat species that occur in such landscapes. This highlights the need for similarly detailed studies to understand the processes that maintain biodiversity in these environments and assess the potential for pathogen transmission in human-wildlife interfaces.

Bats are a likely reservoir of zoonotic coronaviruses (CoVs). Here, analyzing bat CoV sequences in China, the authors find that alpha-CoVs have switched hosts more frequently than betaCoVs, identify a bat family and genus that are highly involved in host-switching, and define hotspots of CoV evolutionary diversity.

Abstract Background Many ecologically important plants are pollinated or have their seeds dispersed by fruit bats, including the widely distributed African straw-colored fruit bats (Eidolon helvum). Their ability to fly long distances makes them essential for connecting plant populations across fragmented landscapes. While bats have been implicated as a reservoir of infectious diseases, their role in disease transmission to humans is not well understood. In this pilot study, we tracked E. helvum to shed light on their movement patterns in Tanzania and possible contact with other species. Methods Tracking devices were deployed on 25 bats captured in the Morogoro Municipal and Kilombero District area near the Udzungwa Mountains of Tanzania. Nightly flight patterns, areas corresponding to foraging bouts and feeding roosts, and new day roosts were determined from bat movement data and characterized according to their proximity to urban built-up and protected areas. Sites for additional environmental surveillance using camera traps were identified via tracking data to determine species coming in contact with fruits discarded by bats. Results Tracking data revealed variability between individual bat movements and a fidelity to foraging areas. Bats were tracked from one to six nights, with a mean cumulative nightly flight distance of 26.14 km (min: 0.33, max: 97.57) based on data from high-resolution GPS tags. While the majority of their foraging locations were in or near urban areas, bats also foraged in protected areas, of which the Udzungwa Mountains National Park was the most frequented. Camera traps in fruit orchards frequented by tracked bats showed the presence of multiple species of wildlife, with vervet monkeys (Chlorocebus pygerythrus) observed as directly handling and eating fruit discarded by bats. Conclusions Because we observed multiple interactions of animals with fruits discarded by bats, specifically with vervet monkeys, the possibility of disease spillover risk exists via this indirect pathway. With flight distances of up to 97 km, however, the role of E. helvum in the seed dispersal of plants across both protected and urban built-up areas in Tanzania may be even more important, especially by helping connect increasingly fragmented landscapes during this Anthropocene epoch.

The 1998 Nipah virus outbreak in Malaysia resulted in major financial losses to the multi-million-dollar swine industry. While productivity and biosecurity of pig farms have improved since, biosecurity in some farms remains substandard with farmers struggling to adapt to current national pig farming policies. Farm viability and animal health depends on farmers' role as decision-makers in managing disease threats and other aspects of farm management. This study aimed to describe the mental model of farmers in making decisions about disease prevention and control measures during the 1998 Nipah virus outbreak, and in 2019, 20 years after the last reported Nipah case. Using a qualitative approach, in-depth, semi-structured interviews with 12 pig farmers (mostly small-scale or medium-scale farms) were conducted in three states in Malaysia. Data were analyzed via inductive content analysis. Thirty-six unique dimensions in the mental model were identified, representing six interrelated themes corresponding to participants' decision making related to disease prevention and control: drivers of action to prevent and control disease; perception of practice options; individual determinants of familiar practices; external social factors; external economic factors; and additional external factors. Key drivers of disease control and prevention responses during the Nipah outbreak included heightened perception of risk, emotions, perceived economic loss, and subjective norms whereas key drivers in 2019 included perception of risk, perceived effectiveness, perceived benefits, and other dimensions such as perception of the future, perceived economic cost, barriers, and loss. An unfavorable future outlook, perceived economic factors, and socio-political and personal factors currently hinders farm improvement and adoption of Pig Farming Areas (PFAs) and Modern Pig Farming (MPF) systems. Private sector service providers and veterinarians are highly influential in advocating for good biosecurity, herd health, and animal health intervention practices. Insights gained can inform the development of strategic policies and interventions.

Despite molecular and serologic evidence of Nipah virus in bats from various locations, attempts to isolate live virus have been largely unsuccessful. We report isolation and full-genome characterization of 10 Nipah virus isolates from Pteropus medius bats sampled in Bangladesh during 2013 and 2014.

The COVID-19 pandemic highlights the substantial public health, economic, and societal consequences of virus spillover from a wildlife reservoir. Widespread human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also presents a new set of challenges when considering viral spillover from people to naïve wildlife and other animal populations. The establishment of new wildlife reservoirs for SARS-CoV-2 would further complicate public health control measures and could lead to wildlife health and conservation impacts. Given the likely bat origin of SARS-CoV-2 and related beta-coronaviruses (β-CoVs), free-ranging bats are a key group of concern for spillover from humans back to wildlife. Here, we review the diversity and natural host range of β-CoVs in bats and examine the risk of humans inadvertently infecting free-ranging bats with SARS-CoV-2. Our review of the global distribution and host range of β-CoV evolutionary lineages suggests that 40+ species of temperate-zone North American bats could be immunologically naïve and susceptible to infection by SARS-CoV-2. We highlight an urgent need to proactively connect the wellbeing of human and wildlife health during the current pandemic and to implement new tools to continue wildlife research while avoiding potentially severe health and conservation impacts of SARS-CoV-2 "spilling back" into free-ranging bat populations.

The ongoing coronavirus disease 2019 (COVID-19) pandemic has had devastating health and socio-economic impacts. Human activities, especially at the wildlife interphase, are at the core of forces driving the emergence of new viral agents. Global surveillance activities have identified bats as the natural hosts of diverse coronaviruses, with other domestic and wildlife animal species possibly acting as intermediate or spillover hosts. The African continent is confronted by several factors that challenge prevention and response to novel disease emergences, such as high species diversity, inadequate health systems, and drastic social and ecosystem changes. We reviewed published animal coronavirus surveillance studies conducted in Africa, specifically summarizing surveillance approaches, species numbers tested, and findings. Far more surveillance has been initiated among bat populations than other wildlife and domestic animals, with nearly 26,000 bat individuals tested. Though coronaviruses have been identified from approximately 7% of the total bats tested, surveillance among other animals identified coronaviruses in less than 1%. In addition to a large undescribed diversity, sequences related to four of the seven human coronaviruses have been reported from African bats. The review highlights research gaps and the disparity in surveillance efforts between different animal groups (particularly potential spillover hosts) and concludes with proposed strategies for improved future biosurveillance.

Abstract Background 99mTc-sestamibi single-photon emission computed tomography/computed tomography (SPECT/CT) has recently been explored for the characterization of indeterminate renal masses. As judged by increased intra-tumoral radiotracer uptake, we have previously reported the excellent diagnostic performance characteristics of this test for identifying benign/indolent oncocytomas and hybrid oncocytic/chromophobe tumors (HOCTs). In this study, we investigated potential molecular mechanisms underlying the discriminatory ability of 99mTc-sestamibi SPECT/CT for renal masses. Fifty renal masses imaged with 99mTc-sestamibi SPECT/CT prior to surgical resection were evaluated by immunohistochemistry for mitochondrial content and expression of the multi-drug resistance pump 1 (MDR1/P-gp). Immunohistochemical staining was scored semi-quantitatively, and results were compared across renal tumor histologies and correlated with 99mTc-sestamibi uptake. Results In total, 6/6 (100%) and 2/2 (100%) HOCTs demonstrated strong mitochondrial content staining combined with low MDR1 staining. Clear cell renal cell carcinoma showed an opposite pattern with the majority having low mitochondrial (14/26, 54%) and high MDR1 staining (18/26, 69%). Other tumor types were more variable in staining pattern, although the staining pattern reliably predicted 99mTc-sestamibi uptake in almost all tumors except chromophobe renal cell carcinoma. Conclusions Our findings confirm that renal tumors with high mitochondrial content and relatively low MDR pump expression activity accumulate 99mTc-sestamibi and allow for the accurate diagnosis of the benign/indolent tumor class that includes oncocytomas and HOCTs. For masses in which MDR activity outweighs the presence of mitochondria, the tumors appear cold on 99mTc-sestamibi SPECT/CT, allowing for high confidence in the diagnosis of renal cell carcinoma.

Preventing emergence of new zoonotic viruses depends on understanding determinants for human risk. Nipah virus (NiV) is a lethal zoonotic pathogen that has spilled over from bats into human populations, with limited person-to-person transmission. We examined ecologic and human behavioral drivers of geographic variation for risk of NiV infection in Bangladesh. We visited 60 villages during 2011–2013 where cases of infection with NiV were identified and 147 control villages. We compared case villages with control villages for most likely drivers for risk of infection, including number of bats, persons, and date palm sap trees, and human date palm sap consumption behavior. Case villages were similar to control villages in many ways, including number of bats, persons, and date palm sap trees, but had a higher proportion of households in which someone drank sap. Reducing human consumption of sap could reduce virus transmission and risk for emergence of a more highly transmissible NiV strain.

Dromedary camels are bred domestically and imported into Bangladesh. In 2015, of 55 camels tested for Middle East respiratory syndrome coronavirus in Dhaka, 17 (31%) were seropositive, including 1 bred locally. None were PCR positive. The potential for infected camels in urban markets could have public health implications and warrants further investigation.

Cell-type-specific 3D organization of the genome is unrecognizable during mitosis. It remains unclear how essential positional information is transmitted through cell division such that a daughter cell recapitulates the spatial genome organization of the parent. Lamina-associated domains (LADs) are regions of repressive heterochromatin positioned at the nuclear periphery that vary by cell type and contribute to cell-specific gene expression and identity. Here we show that histone 3 lysine 9 dimethylation (H3K9me2) is an evolutionarily conserved, specific mark of nuclear peripheral heterochromatin and that it is retained through mitosis. During mitosis, phosphorylation of histone 3 serine 10 temporarily shields the H3K9me2 mark allowing for dissociation of chromatin from the nuclear lamina. Using high-resolution 3D immuno-oligoFISH, we demonstrate that H3K9me2-enriched genomic regions, which are positioned at the nuclear lamina in interphase cells prior to mitosis, re-associate with the forming nuclear lamina before mitotic exit. The H3K9me2 modification of peripheral heterochromatin ensures that positional information is safeguarded through cell division such that individual LADs are re-established at the nuclear periphery in daughter nuclei. Thus, H3K9me2 acts as a 3D architectural mitotic guidepost. Our data establish a mechanism for epigenetic memory and inheritance of spatial organization of the genome.

Bats are implicated as natural reservoirs for a wide range of zoonotic viruses including SARS and MERS coronaviruses, Ebola, Marburg, Nipah, Hendra, Rabies and other lyssaviruses. Accordingly, many One Health surveillance and viral discovery programs have focused on bats. In this report we present viral metagenomic data from bats collected in the Kingdom of Saudi Arabia [KSA]. Unbiased high throughput sequencing of fecal samples from 72 bat individuals comprising four species; lesser mouse-tailed bat (Rhinopoma hardwickii), Egyptian tomb bat (Taphozous perforatus), straw-colored fruit bat (Eidolon helvum), and Egyptian fruit bat (Rousettus aegyptiacus) revealed molecular evidence of a diverse set of viral families: Picornaviridae (hepatovirus, teschovirus, parechovirus), Reoviridae (rotavirus), Polyomaviridae (polyomavirus), Papillomaviridae (papillomavirus), Astroviridae (astrovirus), Caliciviridae (sapovirus), Coronaviridae (coronavirus), Adenoviridae (adenovirus), Paramyxoviridae (paramyxovirus), and unassigned mononegavirales (chuvirus). Additionally, we discovered a bastro-like virus (Middle East Hepe-Astrovirus), with a genomic organization similar to Hepeviridae. However, since it shared homology with Hepeviridae and Astroviridae at ORF1 and in ORF2, respectively, the newly discovered Hepe-Astrovirus may represent a phylogenetic bridge between Hepeviridae and Astroviridae.

Mojiang virus (MojV) is the first henipavirus identified in a rodent and known only by sequence data, whereas all other henipaviruses have been isolated from bats (Hendra virus, Nipah virus, Cedar virus) or discovered by sequence data from material of bat origin (Ghana virus). Ephrin-B2 and -B3 are entry receptors for Hendra and Nipah viruses, but Cedar virus can utilize human ephrin-B1, -B2, -A2 and -A5 and mouse ephrin-A1. However, the entry receptor for MojV remains unknown, and its species tropism is not well characterized. Here, we utilized recombinant full-length and soluble forms of the MojV fusion (F) and attachment (G) glycoproteins in membrane fusion and receptor tropism studies. MojV F and G were functionally competent and mediated cell–cell fusion in primate and rattine cells, albeit with low levels and slow fusion kinetics. Although a relative instability of the pre-fusion conformation of a soluble form of MojV F was observed, MojV F displayed significantly greater fusion activity when heterotypically paired with Ghana virus G. An exhaustive investigation of A- and B-class ephrins indicated that none serve as a primary receptor for MojV. The MojV cell fusion phenotype is therefore likely the result of receptor restriction rather than functional defects in recombinant MojV F and G glycoproteins.

[This corrects the article DOI: 10.1371/journal.pone.0194647.].

We report on a study conducted in Guangdong Province, China, to characterize behaviors and perceptions associated with transmission of pathogens with pandemic potential in highly exposed human populations at the animal-human interface. A risk factor/exposure survey was administered to individuals with high levels of exposure to wildlife. Serological testing was performed to evaluate prior infection with several wildlife viral pathogens. Follow up serology was performed on a subset of the cohort as well as close contacts of individuals. 1,312 individuals were enrolled in the study. Contact with a wide range of wildlife species was reported in both occupational and occasional contexts. The overall proportion of individuals seropositive to any of the tested wildlife pathogens was approximately 4.0%. However, persons employed as butchers demonstrated a seropositivity of 9.0% to at least one pathogen of interest. By contrast, individuals working as hunters had lower rates of seropositivity. Among the study population, a number of other behaviors showed correlation with seropositivity, including contact with particular wildlife species such as field rats. These results demonstrate the need to further explore zoonotic risks of particular activities regarding wildlife contact, and to better understand risks of persons working as butchers with wildlife species.

Macacine herpesvirus 1 (MaHV1; B virus) naturally infects macaques (Macaca spp.) and can cause fatal encephalitis in humans. In Peninsular Malaysia, wild macaques are abundant, and translocation is used to mitigate human–macaque conflict. Most adult macaques are infected with MaHV1, although the risk for transmission to persons who handle them during capture and translocation is unknown. We investigated MaHV1 shedding among 392 long-tailed macaques (M. fascicularis) after capture and translocation by the Department of Wildlife and National Parks in Peninsular Malaysia, during 2009–2011. For detection of MaHV1 DNA, PCR was performed on urogenital and oropharyngeal swab samples. Overall, 39% of macaques were shedding MaHV1 DNA; rates of DNA detection did not differ between sample types. This study demonstrates that MaHV1 was shed by a substantial proportion of macaques after capture and transport and suggests that persons handling macaques under these circumstances might be at risk for exposure to MaHV1.

Bats are recognized reservoirs for many emerging zoonotic viruses of public health importance. Identifying and cataloguing the viruses of bats is a logical approach to evaluate the range of potential zoonoses of bat origin. We characterized the fecal pathogen microbiome of both insectivorous and frugivorous bats, incorporating 281 individual bats comprising 20 common species, which were sampled in three locations of Yunnan province, by combining reverse transcription polymerase chain reaction (RT-PCR) assays and next-generation sequencing. Seven individual bats were paramyxovirus-positive by RT-PCR using degenerate primers, and these paramyxoviruses were mainly classified into three genera (Rubulavirus, Henipavirus and Jeilongvirus). Various additional novel pathogens were detected in the paramyxovirus-positive bats using Illumina sequencing. A total of 7066 assembled contigs (≥200 bp) were constructed, and 105 contigs matched eukaryotic viruses (of them 103 belong to 2 vertebrate virus families, 1 insect virus, and 1 mycovirus), 17 were parasites, and 4913 were homologous to prokaryotic microorganisms. Among the 103 vertebrate viral contigs, 79 displayed low identity (

The source of human infection with Middle East respiratory syndrome coronavirus remains unknown. Molecular investigation indicated that bats in Saudi Arabia are infected with several alphacoronaviruses and betacoronaviruses. Virus from 1 bat showed 100% nucleotide identity to virus from the human index case-patient. Bats might play a role in human infection.