California sea lion (Zalophus californianus) lymph-node explant reveals involvement and possible transcriptional regulation of SLAM and nectin-4 during phocine distemper virus infection.

Phocine distemper virus (PDV) is a significant cause of mortality for phocid seals; however, the susceptibility of otariids to this virus is poorly understood. The authors used a lymph-node explant culture system from California sea lions (Zalophus californianus, CSL) to investigate: (1) the role of signaling lymphocyte activation molecule (SLAM) and nectin-4 in PDV infection and their cellular expression patterns, (2) if PDV induces transcriptional regulation of cell-entry receptors, and (3) the involvement of apoptosis in PDV infection. PDV replicated in the lymph-node explants with peak replication 3 days post-infection (dpi), but the replication was not sustained 4 to 5 dpi. The PDV+ cells co-localized SLAM and nectin-4. These cells expressed IBA1, indicating a histiocytic lineage. Comparison of receptor expression between infected and mock-infected lymph nodes suggested transcriptional downregulation of both receptors during the initial stage of infection and upregulation during the late stage of infection, but the values lack of statistical significance. Cleaved caspase-3+ cells were slightly increased in the infected lymph nodes compared with the mock-infected lymph node from 1 to 4 dpi, but without statistical significance, and a few apoptotic cells co-expressed PDV. The results suggest that lymph-node explants might be an important model to study PDV pathogenesis. CSLs have the potential to be infected with PDV, as they express both cell-entry receptors in histiocytes. The lack of statistical significance in the PDV replication, transcriptional regulation of viral receptors, and changes in apoptosis suggest that although CSL might be infected by PDV, they might be less susceptible than phocid species.

Ranking the risk of animal-to-human spillover for newly discovered viruses.

The death toll and economic loss resulting from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic are stark reminders that we are vulnerable to zoonotic viral threats. Strategies are needed to identify and characterize animal viruses that pose the greatest risk of spillover and spread in humans and inform public health interventions. Using expert opinion and scientific evidence, we identified host, viral, and environmental risk factors contributing to zoonotic virus spillover and spread in humans. We then developed a risk ranking framework and interactive web tool, SpillOver, that estimates a risk score for wildlife-origin viruses, creating a comparative risk assessment of viruses with uncharacterized zoonotic spillover potential alongside those already known to be zoonotic. Using data from testing 509,721 samples from 74,635 animals as part of a virus discovery project and public records of virus detections around the world, we ranked the spillover potential of 887 wildlife viruses. Validating the risk assessment, the top 12 were known zoonotic viruses, including SARS-CoV-2. Several newly detected wildlife viruses ranked higher than known zoonotic viruses. Using a scientifically informed process, we capitalized on the recent wealth of virus discovery data to systematically identify and prioritize targets for investigation. The publicly accessible SpillOver platform can be used by policy makers and health scientists to inform research and public health interventions for prevention and rapid control of disease outbreaks. SpillOver is a living, interactive database that can be refined over time to continue to improve the quality and public availability of information on viral threats to human health.

Classification of new morbillivirus and jeilongvirus sequences from bats sampled in Brazil and Malaysia.

As part of a broad One Health surveillance effort to detect novel viruses in wildlife and people, we report several paramyxovirus sequences sampled primarily from bats during 2013 and 2014 in Brazil and Malaysia, including seven from which we recovered full-length genomes. Of these, six represent the first full-length paramyxovirid genomes sequenced from the Americas, including two that are the first full-length bat morbillivirus genome sequences published to date. Our findings add to the vast number of viral sequences in public repositories, which have been increasing considerably in recent years due to the rising accessibility of metagenomics. Taxonomic classification of these sequences in the absence of phenotypic data has been a significant challenge, particularly in the subfamily Orthoparamyxovirinae, where the rate of discovery of novel sequences has been substantial. Using pairwise amino acid sequence classification (PAASC), we propose that five of these sequences belong to members of the genus Jeilongvirus and two belong to members of the genus Morbillivirus. We also highlight inconsistencies in the classification of Tupaia virus and Mòjiang virus using the same demarcation criteria and suggest reclassification of these viruses into new genera. Importantly, this study underscores the critical importance of sequence length in PAASC analysis as well as the importance of biological characteristics such as genome organization in the taxonomic classification of viral sequences.

Longitudinal analysis of pinnipeds in the northwest Atlantic provides insights on endemic circulation of phocine distemper virus.

Phocine distemper virus (PDV) is a morbillivirus that circulates within pinnipeds in the North Atlantic. PDV has caused two known unusual mortality events (UMEs) in western Europe (1988, 2002), and two UMEs in the northwest Atlantic (2006, 2018). Infrequent cross-species transmission and waning immunity are believed to contribute to periodic outbreaks with high mortality in western Europe. The viral ecology of PDV in the northwest Atlantic is less well defined and outbreaks have exhibited lower mortality than those in western Europe. This study sought to understand the molecular and ecological processes underlying PDV infection in eastern North America. We provide phylogenetic evidence that PDV was introduced into northwest Atlantic pinnipeds by a single lineage and is now endemic in local populations. Serological and viral screening of pinniped surveillance samples from 2006 onward suggest there is continued circulation of PDV outside of UMEs among multiple species with and without clinical signs. We report six full genome sequences and nine partial sequences derived from harbour and grey seals in the northwest Atlantic from 2011 through 2018, including a possible regional variant. Work presented here provides a framework towards greater understanding of how recovering populations and shifting species may impact disease transmission.

Serological Evidence for Henipa-like and Filo-like Viruses in Trinidad Bats.

Bat-borne zoonotic pathogens belonging to the family Paramxyoviridae, including Nipah and Hendra viruses, and the family Filoviridae, including Ebola and Marburg viruses, can cause severe disease and high mortality rates on spillover into human populations. Surveillance efforts for henipaviruses and filoviruses have been largely restricted to the Old World; however, recent studies suggest a potentially broader distribution for henipaviruses and filoviruses than previously recognized. In the current study, we screened for henipaviruses and filoviruses in New World bats collected across 4 locations in Trinidad near the coast of Venezuela. Bat tissue samples were screened using previously established reverse-transcription polymerase chain reaction assays. Serum were screened using a multiplex immunoassay to detect antibodies reactive with the envelope glycoprotein of viruses in the genus Henipavirus and the family Filoviridae. Serum samples were also screened by means of enzyme-linked immunosorbent assay for antibodies reactive with Nipah G and F glycoproteins. Of 84 serum samples, 28 were reactive with ≥1 henipavirus glycoprotein by ≥1 serological method, and 6 serum samples were reactive against ≥1 filovirus glycoproteins. These data provide evidence of potential circulation of viruses related to the henipaviruses and filoviruses in New World bats.

CARNIVORE PROTOPARVOVIRUS 1 (PARVOVIRUSES) AT THE DOMESTIC-WILD CARNIVORE INTERFACE IN INDIA.

Carnivore protoparvovirus 1 (CP1, earlier called Feline panleukopenia virus) variants such as canine parvovirus (CPV) and feline parvovirus (FPV) are significant, emerging, multihost pathogens of domestic and wild carnivores. The diversity of CP1 variants was studied between 2008 and 2014 in Wayanad, India, where flagship wildlife species such as tigers (Panthera tigris) and leopards (Panthera pardus) coexist alongside domestic carnivores, including dogs (Canis lupus familiaris) and cats (Felis catus). Using polymerase chain reaction, FPV and CPV sequences were obtained from the heart blood of a necropsied leopard individual for the first time in the world and from rectal swabs of three sympatric and clinically ill domestic dogs. CP1 amplicons were also detected in a tiger. Cross-species transmission possibilities were identified, as the closest relatives to the leopard FPV sequence were found in domestic cats from a neighboring state.

NONINVASIVE SAMPLING FOR DETECTION OF ELEPHANT ENDOTHELIOTROPIC HERPESVIRUS AND GENOMIC DNA IN ASIAN (ELEPHAS MAXIMUS) AND AFRICAN (LOXODONTA AFRICANA) ELEPHANTS.

Elephant endotheliotropic herpesvirus (EEHV) hemorrhagic disease (EEHV-HD) threatens Asian elephant (Elephas maximus) population sustainability in North America. Clusters of cases have also been reported in African elephants (Loxodonta africana). Risk to range country elephant populations is unknown. Currently, EEHV detection depends upon sampling elephants trained for invasive blood and trunk wash collection. To evaluate noninvasive sample collection options, paired invasively collected (blood, trunk wash and oral swabs), and noninvasively collected (chewed plant and fecal) samples were compared over 6 wk from 9 Asian elephants and 12 African elephants. EEHV shedding was detected simultaneously in a paired trunk wash and fecal sample from one African elephant. Elephant γ herpesvirus-1 shedding was identified in six chewed plant samples collected from four Asian elephants. Noninvasively collected samples can be used to detect elephant herpesvirus shedding. Longer sampling periods are needed to evaluate the clinical usefulness of noninvasive sampling for EEHV detection.

First evidence of a new simian adenovirus clustering with Human mastadenovirus F viruses.

BACKGROUND: Adenoviruses play an important role as human pathogens, though most infections are believed to be asymptomatic. The over 100 human adenovirus types are classified into seven species (A-G), some of which include simian adenoviruses. Recent findings have highlighted that simian adenoviruses have a zoonotic potential and that some human adenoviruses are likely the result of relatively recent spillover events. METHODS: In order to evaluate the risks associated with primates hunted and sold as bushmeat, multiple samples from 24 freshly killed monkeys were collected in the Republic of the Congo and tested for adenovirus DNA by PCRs targeting the conserved DNA polymerase and hexon genes. RESULTS: The DNA of a novel simian adenovirus was detected in a moustached monkey (Cercopithecus cephus) by the DNA polymerase PCR, but not by the hexon PCR. The 275 nucleotide amplicon was most closely related to members of the Human mastadenovirus F species (93% HAdV-40 and 89% HAdV-41 amino acid identity), rather than to other known simian adenoviruses. CONCLUSIONS: The phylogenetic clustering with Human mastadenovirus F sequences suggests a common ancestor, more recent than the last common ancestor of humans and moustached monkeys. The findings increase concerns about the zoonotic potential of simian adenoviruses and highlight the need for more research and surveillance on the issue.

Evolutionary Dynamics and Global Diversity of Influenza A Virus.

UNLABELLED: The increasing number of zoonotic infections caused by influenza A virus (IAV) subtypes of avian origin (e.g., H5N1 and H7N9) in recent years underscores the need to better understand the factors driving IAV evolution and diversity. To evaluate the current feasibility of global analyses to contribute to this aim, we evaluated information in the public domain to explore IAV evolutionary dynamics, including nucleotide substitution rates and selection pressures, using 14 IAV subtypes in 32 different countries over a 12-year period (2000 to 2011). Using geospatial information from 39,785 IAV strains, we examined associations between subtype diversity and socioeconomic, biodiversity, and agricultural indices. Our analyses showed that nucleotide substitution rates for 11 of the 14 evaluated subtypes tended to be higher in Asian countries, particularly in East Asia, than in Canada and the United States. Similarly, at a regional level, subtypes H5N1, H5N2, and H6N2 exhibited significantly higher substitution rates in East Asia than in North America. In contrast, the selection pressures (measured as ratios of nonsynonymous to synonymous evolutionary changes [dN/dS ratios]) acting on individual subtypes showed little geographic variation. We found that the strongest predictors for the detected subtype diversity at the country level were reporting effort (i.e., total number of strains reported) and health care spending (an indicator of economic development). Our analyses also identified major global gaps in IAV reporting (including a lack of sequences submitted from large portions of Africa and South America and a lack of geolocation information) and in broad subtype testing which, until addressed, will continue to hinder efforts to track the evolution and diversity of IAV around the world. IMPORTANCE: In recent years, an increasing number of influenza A virus (IAV) subtypes, including H5N1, H7N9, and H10N8, have been detected in humans. High fatality rates have led to an increased urgency to better understand where and how novel pathogenic influenza virus strains emerge. Our findings showed that mutational rates of 11 commonly encountered subtypes were higher in East Asian countries than in North America, suggesting that there may be a greater risk for the emergence of novel pathogenic strains in East Asia. In assessing the potential drivers of IAV subtype diversity, our analyses confirmed that reporting effort and health care spending were the best predictors of the observed subtype diversity at the country level. These findings underscore the need to increase sampling and reporting efforts for all subtypes in many undersampled countries throughout the world.

Evaluation of viruses and their association with ocular lesions in pinnipeds in rehabilitation.

OBJECTIVE: To assess whether corneal lesions in stranded pinnipeds were associated with viral infections, and to identify the potential pathogen(s) associated with the lesions. ANIMALS STUDIED: Twenty-nine California sea lions (Zalophus californianus), 18 northern elephant seals (Mirounga angustirostris), and 34 Pacific harbor seals (Phoca vitulina richardsii). PROCEDURE: DNA and RNA were extracted from ocular swabs, corneal tissue, and aqueous humor and screened for herpesvirus, adenovirus, poxvirus, and calicivirus families by PCR. RESULTS: The results indicated a high overall prevalence of viruses, with adenoviruses and herpesviruses detected in all three host species. Three novel adenoviruses (PhAdV-1, PhAdV-2, OtAdV-2) and two novel herpesviruses (PhHV-6, OtHV-4) were detected. There were no statistical differences in the prevalence of viral infection or coinfection among groups of individuals with or without corneal lesions, nor were lesion type, onset, or presence of concurrent disease significantly associated with a viral infection. CONCLUSIONS: The results suggested that viral presence in ocular tissues was common, not significantly associated with ocular disease and thus should not preclude release of an otherwise healthy animal. We could not confirm a correlation of virus presence with lesion due to the high percentage of virus-positive, clinically normal animals. This implied that seals and sea lions can have ocular tissues infected with several viruses without having readily evident associated lesions. This difficulty in correlating viral presence, particularly herpesviruses, with ocular lesions was also a common finding in studies with terrestrial species and highlighted the difficulty of confirming a virus as a primary pathogen in ocular lesions.

Capacity building efforts and perceptions for wildlife surveillance to detect zoonotic pathogens: comparing stakeholder perspectives.

BACKGROUND: The capacity to conduct zoonotic pathogen surveillance in wildlife is critical for the recognition and identification of emerging health threats. The PREDICT project, a component of United States Agency for International Development's Emerging Pandemic Threats program, has introduced capacity building efforts to increase zoonotic pathogen surveillance in wildlife in global 'hot spot' regions where zoonotic disease emergence is likely to occur. Understanding priorities, challenges, and opportunities from the perspectives of the stakeholders is a key component of any successful capacity building program. METHODS: A survey was administered to wildlife officials and to PREDICT-implementing in-country project scientists in 16 participating countries in order to identify similarities and differences in perspectives between the groups regarding capacity needs for zoonotic pathogen surveillance in wildlife. RESULTS: Both stakeholder groups identified some human-animal interfaces (i.e. areas of high contact between wildlife and humans with the potential risk for disease transmission), such as hunting and markets, as important for ongoing targeting of wildlife surveillance. Similarly, findings regarding challenges across stakeholder groups showed some agreement in that a lack of sustainable funding across regions was the greatest challenge for conducting wildlife surveillance for zoonotic pathogens (wildlife officials: 96% and project scientists: 81%). However, the opportunity for improving zoonotic pathogen surveillance capacity identified most frequently by wildlife officials as important was increasing communication or coordination among agencies, sectors, or regions (100% of wildlife officials), whereas the most frequent opportunities identified as important by project scientists were increasing human capacity, increasing laboratory capacity, and the growing interest or awareness regarding wildlife disease or surveillance programs (all identified by 69% of project scientists). CONCLUSIONS: A One Health approach to capacity building applied at local and global scales will have the greatest impact on improving zoonotic pathogen surveillance in wildlife. This approach will involve increasing communication and cooperation across ministries and sectors so that experts and stakeholders work together to identify and mitigate surveillance gaps. Over time, this transdisciplinary approach to capacity building will help overcome existing challenges and promote efficient targeting of high risk interfaces for zoonotic pathogen transmission.

Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions.

Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity-the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples.

Gut bacterial communities in Atlantic bottlenose dolphins (Tursiops truncatus) throughout a disease-driven (Morbillivirus) unusual mortality event.

Gut microbiomes are important determinants of animal health. In sentinel marine mammals where animal and ocean health are connected, microbiome impacts can scale to ecosystem-level importance. Mass mortality events affect cetacean populations worldwide, yet little is known about the contributory role of their gut bacterial communities to disease susceptibility and progression. Here, we characterized bacterial communities from fecal samples of common bottlenose dolphins, Tursiops truncatus, across an unusual mortality event (UME) caused by dolphin Morbillivirus (DMV). 16S rRNA gene sequence analysis revealed similar diversity and structure of bacterial communities in individuals stranding before, during, and after the 2013-2015 Mid-Atlantic Bottlenose Dolphin UME and these trends held in a subset of dolphins tested by PCR for DMV infection. Fine-scale shifts related to the UME were not common (10 of 968 bacterial taxa) though potential biomarkers for health monitoring were identified within the complex bacterial communities. Accordingly, acute DMV infection was not associated with a distinct gut bacterial community signature in T. truncatus. However, temporal stratification of DMV-positive dolphins did reveal changes in bacterial community composition between early and late outbreak periods, suggesting that gut community disruptions may be amplified by the indirect effects of accumulating health burdens associated with chronic morbidity.

Pinniped electroencephalography: Methodology and findings in California sea lions (Zalophus californianus).

This study was designed to identify abnormalities in the electroencephalograms (EEGs) recorded from stranded California sea lions (Zalophus californianus) with suspected domoic acid (DA) toxicosis. Recordings from animals presenting for non-neurological issues were also obtained to better understand the normal EEG (background activity and transient events) in this species, as, to date, studies have focused on examining natural sleep in pinnipeds. Most animals were sedated for electrode placement and EEG acquisition with some receiving antiepileptic medications or isoflurane during the procedure. A total of 103 recordings were read and scored from 0 (normal) to 3 (severely abnormal). Epileptiform discharges, consisting of spikes, sharp waves, slow waves, and/or spike waves, were present in all EEGs with scores of 1, 2, or 3. The distribution of these events over the scalp varied. While often generalized, others were lateralized over one hemisphere, bifrontal, bioccipital, and/or bitemporal, while some discharges were multifocal. Findings were different between sea lions and occasionally changed within the EEG on a given sea lion. No clinical seizures were observed during the recording but a few sea lions had findings consistent with electroencephalographic seizures. When available, supporting diagnostic results obtained from magnetic resonance imaging (MRI) and/or necropsy/histopathology were described, as well as the status of those sea lions that recovered and were released with satellite tags.

Correction: Detection of novel coronaviruses in bats in Myanmar.

[This corrects the article DOI: 10.1371/journal.pone.0230802.].

Antibiotic resistance genes of public health importance in livestock and humans in an informal urban community in Nepal.

Efforts to mitigate the increasing emergence of antimicrobial resistance (AMR) will benefit from a One Health perspective, as over half of animal antimicrobials are also considered medically important in humans, and AMR can be maintained in the environment. This is especially pertinent to low- and middle-income countries and in community settings, where an estimated 80% of all antibiotics are used. This study features AMR genes found among humans, animals, and water at an urban informal settlement in Nepal with intensifying livestock production. We sampled humans, chickens, ducks, swine, and water clustered by household, as well as rodents and shrews near dwellings, concurrently in time in July 2017 in southeastern Kathmandu along the Manohara river. Real-time qualitative PCR was performed to screen for 88 genes. Our results characterize the animal-human-environmental interfaces related to the occurrence of specific resistance genes (blaSHV-1 (SHV(238G240E) strain), QnrS, ermC, tetA, tetB, aacC2, aadA1) associated with antibiotics of global health importance that comprise several drug classes, including aminoglycosides, beta-lactams, tetracyclines, macrolides, and fluoroquinolones. By characterizing risk factors across AMR genes of public health importance, this research highlights potential transmission pathways for further investigation and provides prioritization of community-based prevention and intervention efforts for disrupting AMR transmission of critically important antibiotics used in both humans and animals in Nepal.

Surveillance for potentially zoonotic viruses in rodent and bat populations and behavioral risk in an agricultural settlement in Ghana.

BACKGROUND: In Ghana, the conversion of land to agriculture, especially across the vegetative belt has resulted in fragmented forest landscapes with increased interactions among humans, domestic animals, and wildlife. METHODS: We investigated viruses in bats and rodents, key reservoir hosts for zoonotic viral pathogens, in a small agricultural community in the vegetation belt of Ghana. We also administered questionnaires among the local community members to learn more about people's awareness and perceptions of zoonotic disease risks and the environmental factors and types of activities in which they engage that might influence pathogen transmission from wildlife. RESULTS: Our study detected the RNA from paramyxoviruses and coronaviruses in rodents and bats, including sequences from novel viruses with unknown zoonotic potential. Samples collected from Epomophorus gambianus bats were significantly more likely to be positive for coronavirus RNA during the rainy season, when higher numbers of young susceptible individuals are present in the population. Almost all community members who responded to the questionnaire reported contact with wildlife, especially bats, rodents, and non-human primates in and around their homes and in the agricultural fields. Over half of the respondents were not aware or did not perceive any zoonotic disease risks associated with close contact with animals, such as harvesting and processing animals for food. To address gaps in awareness and mitigation strategies for pathogen transmission risks, we organized community education campaigns using risk reduction and outreach tools focused around living safely with bats and rodents. CONCLUSIONS: These findings expand our knowledge of the viruses circulating in bats and rodents in Ghana and of the beliefs, perceptions, and practices that put community members at risk of zoonotic virus spillover through direct and indirect contact with bats and rodents. This study also highlights the importance of community engagement in research and interventions focused on mitigating risk and living safely with wildlife.

Coronavirus and Paramyxovirus Shedding by Bats in a Cave and Buildings in Ethiopia.

Bats are important hosts of zoonotic viruses with pandemic potential, including filoviruses, MERS-Coronavirus (CoV), SARS-CoV -1, and likely SARS-CoV-2. Viral infection and transmission among wildlife are dependent on a combination of factors that include host ecology and immunology, life history traits, roosting habitats, biogeography, and external stressors. Between 2016 and 2018, four species of insectivorous bats from a readily accessed roadside cave and buildings in Ethiopia were sampled and tested for viruses using consensus PCR assays for five viral families/genera. Previously identified and novel coronaviruses and paramyxoviruses were identified in 99 of the 589 sampled bats. Bats sampled from the cave site were more likely to test positive for a CoV than bats sampled from buildings; viral shedding was more common in the wet season; and rectal swabs were the most common sample type to test positive. A previously undescribed alphacoronavirus was detected in two bat species from different taxonomic families, sampling interfaces, geographic locations, and years. These findings expand knowledge of the range and diversity of coronaviruses and paramyxoviruses in insectivorous bats in Ethiopia and reinforce that an improved understanding of viral diversity and species-specific shedding dynamics is important for designing informed zoonotic disease surveillance and spillover risk reduction efforts.

Presence of Recombinant Bat Coronavirus GCCDC1 in Cambodian Bats.

Bats have been recognized as an exceptional viral reservoir, especially for coronaviruses. At least three bat zoonotic coronaviruses (SARS-CoV, MERS-CoV and SARS-CoV-2) have been shown to cause severe diseases in humans and it is expected more will emerge. One of the major features of CoVs is that they are all highly prone to recombination. An extreme example is the insertion of the P10 gene from reoviruses in the bat CoV GCCDC1, first discovered in Rousettus leschenaultii bats in China. Here, we report the detection of GCCDC1 in four different bat species (Eonycteris spelaea, Cynopterus sphinx, Rhinolophus shameli and Rousettus sp.) in Cambodia. This finding demonstrates a much broader geographic and bat species range for this virus and indicates common cross-species transmission. Interestingly, one of the bat samples showed a co-infection with an Alpha CoV most closely related to RsYN14, a virus recently discovered in the same genus (Rhinolophus) of bat in Yunnan, China, 2020. Taken together, our latest findings highlight the need to conduct active surveillance in bats to assess the risk of emerging CoVs, especially in Southeast Asia.