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.

Simian homologues of human herpesviruses and implications for novel viral introduction to free-living mountain gorillas.

The endangered mountain gorilla (Gorilla beringei beringei) in Rwanda, Uganda, and the Democratic Republic of Congo is frequently in contact with humans through tourism, research activities, and illegal entry of people into protected gorilla habitat. Herpesviruses, which are ubiquitous in primates, have the potential to be shared in any setting where humans and gorillas share habitat. Based on serological findings and clinical observations of orofacial ulcerated lesions resembling herpetic lesions, an alpha-herpesvirus resembling human herpes simplex virus type 1 (HSV-1) has long been suspected to be present in human-habituated mountain gorillas in the wild. While the etiology of orofacial lesions in the wild has not been confirmed, HSV-1 has been suspected in captively-housed mountain gorillas and confirmed in a co-housed confiscated Grauer's gorilla (Gorilla beringei graueri). To better characterize herpesviruses infecting mountain gorillas and to determine the presence/absence of HSV-1 in the free-living population, we conducted a population-wide survey to test for the presence of orally shed herpesviruses. DNA was extracted from discarded chewed plants collected from 294 individuals from 26 groups, and samples were screened by polymerase chain reaction using pan-herpesvirus and HSV-1-specific assays. We found no evidence that human herpesviruses had infected free-ranging mountain gorillas. However, we found gorilla-specific homologs to human herpesviruses, including cytomegaloviruses (GbbCMV-1 and 2), a lymphocryptovirus (GbbLCV-1), and a new rhadinovirus (GbbRHV-1) with similar characteristics (i.e., timing of primary infection, shedding in multiple age groups, and potential modes of transmission) to their human counterparts, human cytomegalovirus, Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, respectively.

Sylvatic Transmission of Chikungunya Virus among Nonhuman Primates in Myanmar.

Nonhuman primates living in proximity to humans increase risks for sylvatic arbovirus transmission. We collected serum samples from nonhuman primates in Hlawga National Park near Yangon, Myanmar, and detected antibodies against chikungunya (33%) and Japanese encephalitis (4%) viruses. Buffer zones between primate and human communities might reduce cross-species arbovirus transmission.

Possibility for reverse zoonotic transmission of SARS-CoV-2 to free-ranging wildlife: A case study of bats.

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 (ß-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 ß-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 ß-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.

Early detection of wildlife morbidity and mortality through an event-based surveillance system.

The ability to rapidly detect and respond to wildlife morbidity and mortality events is critical for reducing threats to wildlife populations. Surveillance systems that use pre-diagnostic clinical data can contribute to the early detection of wildlife morbidities caused by a multitude of threats, including disease and anthropogenic disturbances. Here, we demonstrate proof of concept for use of a wildlife disease surveillance system, the 'Wildlife Morbidity and Mortality Event Alert System', that integrates pre-diagnostic clinical data in near real-time from a network of wildlife rehabilitation organizations, for early and enhanced detection of unusual wildlife morbidity and mortality events. The system classifies clinical pre-diagnostic data into relevant clinical classifications based on a natural language processing algorithm, generating alerts when more than the expected number of cases is recorded across the rehabilitation network. We demonstrated the effectiveness and efficiency of the system in alerting to events associated with both common and emerging diseases. Tapping into this readily available unconventional general surveillance data stream offers added value to existing wildlife disease surveillance programmes through a relatively efficient, low-cost strategy for the early detection of threats.

TRANSTHORACIC ECHOCARDIOGRAPHIC EVALUATION AND SERUM CARDIAC TROPONIN VALUES IN ANESTHETIZED HEALTHY FEMALE SOUTHERN SEA OTTERS (ENHYDRA LUTRIS NEREIS).

Information about antemortem cardiac evaluation in sea otters (Enhydra lutris) is limited, despite well-established clinical care and rehabilitation procedures and a reported elevated risk of cardiac disease for this species. Serum cardiac troponin I (cTnI) concentration and echocardiographic assessment are two ways of screening for and diagnosing cardiac disease. However, no baseline data or reference intervals for either evaluation are published for sea otters. The objectives of this prospective study were to establish serum cTnI concentrations and echocardiographic technique and quantitative measurements in anesthetized healthy female southern sea otters (Enhydra lutris nereis) (n=15). Serum cTnI values were assessed by a high-sensitivity assay. Serum cTnI concentration ranged from <0.006 to 0.038 ng/ml. A complete echocardiogram, including two-dimensional and M-mode modalities, was performed. Echocardiographic measurements for left atrial size, aorta size, left ventricular structure, and left ventricular function were reported. The median left atrial size to aorta ratio was 1.22 (range 0.80-1.59) in short-axis and 1.70 (range 1.39-2.15) in long-axis. The median left ventricular internal dimension was 3.53 cm (range 2.87-4.92 cm) when assessed in two dimensions and 3.58 cm (range 2.80-4.48 cm) by M-mode. Serum concentrations of cTnI and transthoracic echocardiography may represent valuable tools for the antemortem diagnosis of cardiac disease in sea otters.

Global shifts in mammalian population trends reveal key predictors of virus spillover risk.

Emerging infectious diseases in humans are frequently caused by pathogens originating from animal hosts, and zoonotic disease outbreaks present a major challenge to global health. To investigate drivers of virus spillover, we evaluated the number of viruses mammalian species have shared with humans. We discovered that the number of zoonotic viruses detected in mammalian species scales positively with global species abundance, suggesting that virus transmission risk has been highest from animal species that have increased in abundance and even expanded their range by adapting to human-dominated landscapes. Domesticated species, primates and bats were identified as having more zoonotic viruses than other species. Among threatened wildlife species, those with population reductions owing to exploitation and loss of habitat shared more viruses with humans. Exploitation of wildlife through hunting and trade facilitates close contact between wildlife and humans, and our findings provide further evidence that exploitation, as well as anthropogenic activities that have caused losses in wildlife habitat quality, have increased opportunities for animal-human interactions and facilitated zoonotic disease transmission. Our study provides new evidence for assessing spillover risk from mammalian species and highlights convergent processes whereby the causes of wildlife population declines have facilitated the transmission of animal viruses to humans.

Suspected Exposure to Filoviruses Among People Contacting Wildlife in Southwestern Uganda.

Background: Human and filovirus host interactions remain poorly understood in areas where Ebola hemorrhagic fever outbreaks are likely to occur. In the Bwindi region of Uganda, a hot spot of mammalian biodiversity in Africa, human livelihoods are intimately connected with wildlife, creating potential for exposure to filoviruses. Methods: We tested samples from 331 febrile patients presenting to healthcare facilities near Bwindi Impenetrable Forest, Uganda, by polymerase chain reaction (PCR) analysis and Western blot, using recombinant glycoprotein antigens for Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus. Behavioral data on contact with wildlife were collected to examine risk factors for filovirus seropositivity. Results: All patients were negative for active filovirus infection, by PCR analysis. However, patients were seroreactive to SUDV (4.7%), EBOV (5.3%), and BDBV (8.9%), indicating previous exposure. Touching duikers was the most significant risk factor associated with EBOV seropositivity, while hunting primates and touching and/or eating cane rats were significant risk factors for SUDV seropositivity. Conclusions: People in southwestern Uganda have suspected previous exposure to filoviruses, particularly those with a history of wildlife contact. Circulation of filoviruses in wild animals and subsequent spillover into humans could be more common than previously reported.

ATYPICAL CHLAMYDIACEAE IN WILD POPULATIONS OF HAWKS ( BUTEO SPP.) IN CALIFORNIA.

Chlamydiaceae bacteria infect many vertebrate hosts, and previous reports based on polymerase chain reaction (PCR) assays and serologic assays that are prone to cross-reaction among chlamydial organisms have been used to describe the prevalence of either DNA fragments or antibodies to Chlamydia spp. in wild raptorial populations. This study reports the PCR-based prevalence of Chlamydiaceae DNA that does not 100% match any avian or mammalian Chlamydiaceae in wild populations of hawks in California Buteo species. In this study, multimucosal swab samples ( n = 291) for quantitative PCR (qPCR) and plasma ( n = 78) for serology were collected from wild hawks. All available plasma samples were negative for antibodies using a C. psittaci-specific elementary body agglutination test (EBA; n = 78). For IgY antibodies all 51 available samples were negative using the indirect immunofluorescent assay. The overall prevalence of Chlamydiaceae DNA detection in wild Buteo species sampled was 1.37% (4/291) via qPCR-based analysis. Two fledgling Swainson's hawks ( Buteo swainsoni) and two juvenile red-tailed hawks ( Buteo jamaicensis) were positive by qPCR-based assay for an atypical chlamydial sequence that did not 100% match any known C. psittaci genotype. Positive swab samples from these four birds were sequenced based on the ompA gene and compared by high-resolution melt analysis with all known avian and mammalian Chlamydiaceae. The amplicon sequence did not 100% match any known avian chlamydial sequence; however, it was most similar (98.6%) to C. psittaci M56, a genotype that is typically found in muskrats and hares. Culture and full genome sequence analysis of Chlamydia spp. isolated from diseased hawks will be necessary to classify this organism and to better understand its epizootiology and potential health impact on wild Buteo populations in California.

Detection of viruses using discarded plants from wild mountain gorillas and golden monkeys.

Infectious diseases pose one of the most significant threats to the survival of great apes in the wild. The critically endangered mountain gorilla (Gorilla beringei beringei) is at high risk for contracting human pathogens because approximately 60% of the population is habituated to humans to support a thriving ecotourism program. Disease surveillance for human and non-human primate pathogens is important for population health and management of protected primate species. Here, we evaluate discarded plants from mountain gorillas and sympatric golden monkeys (Cercopithecus mitis kandti), as a novel biological sample to detect viruses that are shed orally. Discarded plant samples were tested for the presence of mammalian-specific genetic material and two ubiquitous DNA and RNA primate viruses, herpesviruses, and simian foamy virus. We collected discarded plant samples from 383 wild human-habituated mountain gorillas and from 18 habituated golden monkeys. Mammalian-specific genetic material was recovered from all plant species and portions of plant bitten or chewed by gorillas and golden monkeys. Gorilla herpesviral DNA was most consistently recovered from plants in which leafy portions were eaten by gorillas. Simian foamy virus nucleic acid was recovered from plants discarded by golden monkeys, indicating that it is also possible to detect RNA viruses from bitten or chewed plants. Our findings show that discarded plants are a useful non-invasive sampling method for detection of viruses that are shed orally in mountain gorillas, sympatric golden monkeys, and potentially other species. This method of collecting specimens from discarded plants is a new non-invasive sampling protocol that can be combined with collection of feces and urine to evaluate the most common routes of viral shedding in wild primates. Am. J. Primatol. 78:1222-1234, 2016. © 2016 Wiley Periodicals, Inc.

Assessing ecological correlates of marine bird declines to inform marine conservation.

Identifying drivers of ecosystem change in large marine ecosystems is central for their effective management and conservation. This is a sizable challenge, particularly in ecosystems transcending international borders, where monitoring and conservation of long-range migratory species and their habitats are logistically and financially problematic. Here, using tools borrowed from epidemiology, we elucidated common drivers underlying species declines within a marine ecosystem, much in the way epidemiological analyses evaluate risk factors for negative health outcomes to better inform decisions. Thus, we identified ecological traits and dietary specializations associated with species declines in a community of marine predators that could be reflective of ecosystem change. To do so, we integrated count data from winter surveys collected in long-term marine bird monitoring programs conducted throughout the Salish Sea--a transboundary large marine ecosystem in North America's Pacific Northwest. We found that decadal declines in winter counts were most prevalent among pursuit divers such as alcids (Alcidae) and grebes (Podicipedidae) that have specialized diets based on forage fish, and that wide-ranging species without local breeding colonies were more prone to these declines. Although a combination of factors is most likely driving declines of diving forage fish specialists, we propose that changes in the availability of low-trophic prey may be forcing wintering range shifts of diving birds in the Salish Sea. Such a synthesis of long-term trends in a marine predator community not only provides unique insights into the types of species that are at risk of extirpation and why, but may also inform proactive conservation measures to counteract threats--information that is paramount for species-specific and ecosystem-wide conservation.

PSOROPTES INFESTATION AND TREATMENT IN AN ISOLATED POPULATION OF BIGHORN SHEEP (OVIS CANADENSIS).

The authors captured bighorn sheep (Ovis canadensis) comprising a small population in the San Bernardino Mountains of California and evaluated the degree of infestation by mites of the genus Psoroptes for each individual. The animals were treated with two novel methods: amitraz-impregnated collars and cyfluthrin-impregnated ear tags and recaptured the following year to evaluate the effect of treatment. The authors compared data on degree of infestation for animals recaptured in the posttreatment year, detected no significant interyear differences in infestation severity scores among animals treated with amitraz or cyfluthrin, and could not detect any differences between treatment types. However, a significant (P<0.10) decreased pattern in severity scores from the beginning to the end of treatments was detected, suggesting a cumulative therapeutic value in repeated annual treatments across the 3-yr period. Additionally, the authors detected a lower median mite severity score between 2000 and a later capture in 2006. These positive outcomes may be the result of previous treatments during 2000-2002, but environmental covariates not accounted for could have been contributing factors. Avermectin drugs with longer release profiles may be a more effective treatment option in this and other small bighorn sheep populations that are compromised with mite infestations.

Spatiotemporal patterns and risk factors for lead exposure in endangered California condors during 15 years of reintroduction.

Large-scale poisoning events are common to scavenging bird species that forage communally, many of which are in decline. To reduce the threat of poisoning and compensate for other persistent threats, management, including supplemental feeding, is ongoing for many reintroduced and endangered vulture populations. Through a longitudinal study of lead exposure in California condors (Gymnogyps californianus), we illustrate the conservation challenges inherent in reintroduction of an endangered species to the wild when pervasive threats have not been eliminated. We evaluated population-wide patterns in blood lead levels from 1997 to 2011 and assessed a broad range of putative demographic, behavioral, and environmental risk factors for elevated lead exposure among reintroduced California condors in California (United States). We also assessed the effectiveness of lead ammunition regulations within the condor's range in California by comparing condor blood lead levels before and after implementation of the regulations. Lead exposure was a pervasive threat to California condors despite recent regulations limiting lead ammunition use. In addition, condor lead levels significantly increased as age and independence from intensive management increased, including increasing time spent away from managed release sites, and decreasing reliance on food provisions. Greater independence among an increasing number of reintroduced condors has therefore elevated the population's risk of lead exposure and limited the effectiveness of lead reduction efforts to date. Our findings highlight the challenges of restoring endangered vulture populations as they mature and become less reliant on management actions necessary to compensate for persistent threats.

Enteric pathogens and antimicrobial resistance in turkey vultures (Cathartes aura) feeding at the wildlife-livestock interface.

Free-flying turkey vultures (Cathartes aura) were sampled in California to investigate the fecal shedding prevalence and antimicrobial susceptibility of Salmonella enterica, Campylobacter spp., and Escherichia coli. Nine different serotypes of Salmonella enterica were detected in cloacal swabs from turkey vultures, and 6% of vultures were shedding Campylobacter spp.. Turkey vultures sampled at a location with range sheep were more likely to shed tetracycline-resistant E. coli, suggesting that proximity to livestock facilities could facilitate acquisition of drug-resistant bacteria in avian scavengers. These findings illustrate the importance of assessing drug-resistant pathogen transfer at the livestock-wildlife interface.

Targeting surveillance for zoonotic virus discovery.

We analyzed a database of mammal-virus associations to ask whether surveillance targeting diseased animals is the best strategy to identify potentially zoonotic pathogens. Although a mixed healthy and diseased animal surveillance strategy is generally best, surveillance of apparently healthy animals would likely maximize zoonotic virus discovery potential for bats and rodents.

Novel strains of Campylobacter cause diarrheal outbreak in Rhesus macaques (Macaca mulatta) of Kathmandu Valley.

Campylobacter spp. is often underreported and underrated bacteria that present real health risks to both humans and animals, including non-human primates. It is a commensal microorganism of gastrointestinal tract known to cause gastroenteritis in humans. Commonly found in many wild animals including non-human primates (monkeys- Rhesus macaques) these pathogens are known to be a common cause of diarrhea in humans in many parts of developing and under developed countries. Rhesus macaques from the two holy sites in Kathmandu (Pashupati and Swoyambhu) were included in this cross-sectional study. Diarrheal samples of monkeys were analyzed to detect and characterize the pathogen using 16S rRNA-based PCR screening, followed by DNA sequencing and phylogenetic analysis. Out of a total 67 collected diarrheal samples, Campylobacter spp. were detected in the majority of the samples (n = 64; 96%). DNA sequences of the amplified PCR products were successfully obtained from 13 samples. Phylogenetic analysis identified Candidatus Campylobacter infans (n = 10, Kimura-2 parameter (K2P) pairwise distance values of 0.002287). Remaining three sequences might potentially belong to a novel Campylobacter species/sub-species- closely relating to known species of C. helviticus (K2P pairwise distance of 0.0267). Both Candidatus Campylobacter infans and C. helvitucus are known to infect humans and animals. Additionally, we also detected the bacteria in water and soil samples from the sites. Campylobacter spp. caused the 2018 diarrhea outbreak in Rhesus macaques in the Kathmandu valley. Campylobacter might be one of the important contributing pathogens in diarrheal outbreaks-both in humans and animals (monkeys) in Nepal. Due to close interactions of these animals with humans and other animals, One Health approach might be the most effective way to prevent and mitigate the threat posed by this pathogen.

Highly pathogenic avian influenza A (H5N1) in marine mammals and seabirds in Peru.

Highly pathogenic avian influenza (HPAI) A/H5N1 viruses (lineage 2.3.4.4b) are rapidly invading the Americas, threatening wildlife, poultry, and potentially evolving into the next global pandemic. In November 2022 HPAI arrived in Peru, triggering massive pelican and sea lion die-offs. We report genomic characterization of HPAI/H5N1 in five species of marine mammals and seabirds (dolphins, sea lions, sanderlings, pelicans and cormorants). Peruvian viruses belong to lineage 2.3.4.4b, but they are 4:4 reassortants where 4 genomic segments (PA, HA, NA and MP) position within the Eurasian lineage that initially entered North America from Eurasia, while the other 4 genomic segments (PB2, PB1, NP and NS) position within the American lineage (clade C) that circulated in North America. These viruses are rapidly accruing mutations, including mutations of concern, that warrant further examination and highlight an urgent need for active local surveillance to manage outbreaks and limit spillover into other species, including humans.

Correction: Detection of novel coronaviruses in bats in Myanmar.

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

Ecological characterization of 175 low-pathogenicity avian influenza viruses isolated from wild birds in Mongolia, 2009-2013 and 2016-2018.

BACKGROUND: Since 2005, highly pathogenic avian influenza A H5N1 viruses have spread from Asia worldwide, infecting poultry, humans and wild birds. Subsequently, global interest in avian influenza (AI) surveillance increased. OBJECTIVES: Mongolia presents an opportunity to study viruses in wild birds because the country has very low densities of domestic poultry and supports large concentrations of migratory water birds. METHODS: We conducted AI surveillance in Mongolia over two time periods, 2009-2013 and 2016-2018, utilizing environmental fecal sampling. Fresh fecal samples were collected from water bird congregation sites. Hemagglutinin (HA) and neuraminidase (NA) subtypes of positive samples were identified through viral isolation or molecular assays, with pathogenicity determined by HA subtype or sequencing the HA cleavage site. RESULTS: A total of 10,222 samples were collected. Of these, 7,025 fecal samples were collected from 2009 to 2013, and 3,197 fecal samples were collected from 2016 to 2018. Testing revealed 175 (1.7%) positive samples for low-pathogenicity influenza A, including 118 samples from 2009 to 2013 (1.7%) and 57 samples from 2016 to 2018 (1.8%). HA and NA subtyping of all positives identified 11 subtypes of HA and nine subtypes of NA in 29 different combinations. Within periods, viruses were detected more frequently during the fall season than in the early summer. CONCLUSION: Mongolia's critical wild bird habitat is positioned as a crossroad of multiple migratory flyways. Our work demonstrates the feasibility of using an affordable environmental fecal sampling approach for AI surveillance and contributes to understanding the prevalence and ecology of low-pathogenicity avian influenza viruses in this important location, where birds from multiple flyways mix.

Rapid genomic surveillance of SARS-CoV-2 in a dense urban community of Kathmandu Valley using sewage samples.

Understanding disease burden and transmission dynamics in resource-limited, low-income countries like Nepal are often challenging due to inadequate surveillance systems. These issues are exacerbated by limited access to diagnostic and research facilities throughout the country. Nepal has one of the highest COVID-19 case rates (915 cases per 100,000 people) in South Asia, with densely-populated Kathmandu experiencing the highest number of cases. Swiftly identifying case clusters (hotspots) and introducing effective intervention programs is crucial to mounting an effective containment strategy. The rapid identification of circulating SARS-CoV-2 variants can also provide important information on viral evolution and epidemiology. Genomic-based environmental surveillance can help in the early detection of outbreaks before clinical cases are recognized and identify viral micro-diversity that can be used for designing real-time risk-based interventions. This research aimed to develop a genomic-based environmental surveillance system by detecting and characterizing SARS-CoV-2 in sewage samples of Kathmandu using portable next-generation DNA sequencing devices. Out of 22 sites in the Kathmandu Valley from June to August 2020, sewage samples from 16 (80%) sites had detectable SARS-CoV-2. A heatmap was created to visualize the presence of SARS-CoV-2 infection in the community based on viral load intensity and corresponding geospatial data. Further, 47 mutations were observed in the SARS-CoV-2 genome. Some detected mutations (n = 9, 22%) were novel at the time of data analysis and yet to be reported in the global database, with one indicating a frameshift deletion in the spike gene. SNP analysis revealed possibility of assessing circulating major/minor variant diversity on environmental samples based on key mutations. Our study demonstrated the feasibility of rapidly obtaining vital information on community transmission and disease dynamics of SARS-CoV-2 using genomic-based environmental surveillance.