Virome characterization of game animals in China reveals a spectrum of emerging pathogens.

Game animals are wildlife species traded and consumed as food and are potential reservoirs for SARS-CoV and SARS-CoV-2. We performed a meta-transcriptomic analysis of 1,941 game animals, representing 18 species and five mammalian orders, sampled across China. From this, we identified 102 mammalian-infecting viruses, with 65 described for the first time. Twenty-one viruses were considered as potentially high risk to humans and domestic animals. Civets (Paguma larvata) carried the highest number of potentially high-risk viruses. We inferred the transmission of bat-associated coronavirus from bats to civets, as well as cross-species jumps of coronaviruses from bats to hedgehogs, from birds to porcupines, and from dogs to raccoon dogs. Of note, we identified avian Influenza A virus H9N2 in civets and Asian badgers, with the latter displaying respiratory symptoms, as well as cases of likely human-to-wildlife virus transmission. These data highlight the importance of game animals as potential drivers of disease emergence.

A selective sweep in the Spike gene has driven SARS-CoV-2 human adaptation.

The coronavirus disease 2019 (COVID-19) pandemic underscores the need to better understand animal-to-human transmission of coronaviruses and adaptive evolution within new hosts. We scanned more than 182,000 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes for selective sweep signatures and found a distinct footprint of positive selection located around a non-synonymous change (A1114G; T372A) within the spike protein receptor-binding domain (RBD), predicted to remove glycosylation and increase binding to human ACE2 (hACE2), the cellular receptor. This change is present in all human SARS-CoV-2 sequences but not in closely related viruses from bats and pangolins. As predicted, T372A RBD bound hACE2 with higher affinity in experimental binding assays. We engineered the reversion mutant (A372T) and found that A372 (wild-type [WT]-SARS-CoV-2) enhanced replication in human lung cells relative to its putative ancestral variant (T372), an effect that was 20 times greater than the well-known D614G mutation. Our findings suggest that this mutation likely contributed to SARS-CoV-2 emergence from animal reservoirs or enabled sustained human-to-human transmission.

A Genomic Perspective on the Origin and Emergence of SARS-CoV-2.

The ongoing pandemic of a new human coronavirus, SARS-CoV-2, has generated enormous global concern. We and others in China were involved in the initial genome sequencing of the virus. Herein, we describe what genomic data reveal about the emergence SARS-CoV-2 and discuss the gaps in our understanding of its origins.

The Egyptian Rousette Genome Reveals Unexpected Features of Bat Antiviral Immunity.

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans.

Reservoir host immune responses to emerging zoonotic viruses.

Zoonotic viruses, such as HIV, Ebola virus, coronaviruses, influenza A viruses, hantaviruses, or henipaviruses, can result in profound pathology in humans. In contrast, populations of the reservoir hosts of zoonotic pathogens often appear to tolerate these infections with little evidence of disease. Why are viruses more dangerous in one species than another? Immunological studies investigating quantitative and qualitative differences in the host-virus equilibrium in animal reservoirs will be key to answering this question, informing new approaches for treating and preventing zoonotic diseases. Integrating an understanding of host immune responses with epidemiological, ecological, and evolutionary insights into viral emergence will shed light on mechanisms that minimize fitness costs associated with viral infection, facilitate transmission to other hosts, and underlie the association of specific reservoir hosts with multiple emerging viruses. Reservoir host studies provide a rich opportunity for elucidating fundamental immunological processes and their underlying genetic basis, in the context of distinct physiological and metabolic constraints that contribute to host resistance and disease tolerance.

Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus.

The 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us of how little is known about biosafety level 4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics. VIDEO ABSTRACT.

Bat coronaviruses related to SARS-CoV-2 and infectious for human cells.

The animal reservoir of SARS-CoV-2 is unknown despite reports of SARS-CoV-2-related viruses in Asian Rhinolophus bats1-4, including the closest virus from R. affinis, RaTG13 (refs. 5,6), and pangolins7-9. SARS-CoV-2 has a mosaic genome, to which different progenitors contribute. The spike sequence determines the binding affinity and accessibility of its receptor-binding domain to the cellular angiotensin-converting enzyme 2 (ACE2) receptor and is responsible for host range10-12. SARS-CoV-2 progenitor bat viruses genetically close to SARS-CoV-2 and able to enter human cells through a human ACE2 (hACE2) pathway have not yet been identified, although they would be key in understanding the origin of the epidemic. Here we show that such viruses circulate in cave bats living in the limestone karstic terrain in northern Laos, in the Indochinese peninsula. We found that the receptor-binding domains of these viruses differ from that of SARS-CoV-2 by only one or two residues at the interface with ACE2, bind more efficiently to the hACE2 protein than that of the SARS-CoV-2 strain isolated in Wuhan from early human cases, and mediate hACE2-dependent entry and replication in human cells, which is inhibited by antibodies that neutralize SARS-CoV-2. None of these bat viruses contains a furin cleavage site in the spike protein. Our findings therefore indicate that bat-borne SARS-CoV-2-like viruses that are potentially infectious for humans circulate in Rhinolophus spp. in the Indochinese peninsula.

HIV reservoirs as obstacles and opportunities for an HIV cure.

The persistence of HIV reservoirs remains a formidable obstacle to achieving sustained virologic remission in HIV-infected individuals after antiretroviral therapy (ART) is discontinued, even if plasma viremia has been successfully suppressed for prolonged periods of time. Numerous approaches aimed at eradicating the virus, as well as maintaining its prolonged suppression in the absence of ART, have had little success. A better understanding of the pathophysiologic nature of HIV reservoirs and the impact of various interventions on their persistence is essential for the development of successful therapeutic strategies against HIV or the long-term control of infection. Here, we discuss the persistent HIV reservoir as a barrier to cure as well as the current therapeutic strategies aimed at eliminating or controlling the virus in the absence of ART.

Immunological features of bats: resistance and tolerance to emerging viruses.

Bats are among the most diverse mammalian species, representing over 20% of mammalian diversity. The past two decades have witnessed a disproportionate spillover of viruses from bats to humans compared with other mammalian hosts, attributed to the viral richness within bats, their phylogenetic likeness to humans, and increased human contact with wildlife. Unique evolutionary adaptations in bat genomes, particularly in antiviral protection and immune tolerance genes, enable bats to serve as reservoirs for pandemic-inducing viruses. Here, we discuss current limitations and advances made in understanding the role of bats as drivers of pandemic zoonoses. We also discuss novel technologies that have revealed spatial, dynamic, and physiological factors driving virus and host coevolution.

Lessons from the host defences of bats, a unique viral reservoir.

There have been several major outbreaks of emerging viral diseases, including Hendra, Nipah, Marburg and Ebola virus diseases, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS)-as well as the current pandemic of coronavirus disease 2019 (COVID-19). Notably, all of these outbreaks have been linked to suspected zoonotic transmission of bat-borne viruses. Bats-the only flying mammal-display several additional features that are unique among mammals, such as a long lifespan relative to body size, a low rate of tumorigenesis and an exceptional ability to host viruses without presenting clinical disease. Here we discuss the mechanisms that underpin the host defence system and immune tolerance of bats, and their ramifications for human health and disease. Recent studies suggest that 64 million years of adaptive evolution have shaped the host defence system of bats to balance defence and tolerance, which has resulted in a unique ability to act as an ideal reservoir host for viruses. Lessons from the effective host defence of bats would help us to better understand viral evolution and to better predict, prevent and control future viral spillovers. Studying the mechanisms of immune tolerance in bats could lead to new approaches to improving human health. We strongly believe that it is time to focus on bats in research for the benefit of both bats and humankind.

Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins.

The current outbreak of coronavirus disease-2019 (COVID-19) poses unprecedented challenges to global health1. The new coronavirus responsible for this outbreak-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-shares high sequence identity to SARS-CoV and a bat coronavirus, RaTG132. Although bats may be the reservoir host for a variety of coronaviruses3,4, it remains unknown whether SARS-CoV-2 has additional host species. Here we show that a coronavirus, which we name pangolin-CoV, isolated from a Malayan pangolin has 100%, 98.6%, 97.8% and 90.7% amino acid identity with SARS-CoV-2 in the E, M, N and S proteins, respectively. In particular, the receptor-binding domain of the S protein of pangolin-CoV is almost identical to that of SARS-CoV-2, with one difference in a noncritical amino acid. Our comparative genomic analysis suggests that SARS-CoV-2 may have originated in the recombination of a virus similar to pangolin-CoV with one similar to RaTG13. Pangolin-CoV was detected in 17 out of the 25 Malayan pangolins that we analysed. Infected pangolins showed clinical signs and histological changes, and circulating antibodies against pangolin-CoV reacted with the S protein of SARS-CoV-2. The isolation of a coronavirus from pangolins that is closely related to SARS-CoV-2 suggests that these animals have the potential to act as an intermediate host of SARS-CoV-2. This newly identified coronavirus from pangolins-the most-trafficked mammal in the illegal wildlife trade-could represent a future threat to public health if wildlife trade is not effectively controlled.

Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins.

The ongoing outbreak of viral pneumonia in China and across the world is associated with a new coronavirus, SARS-CoV-21. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection2. Although bats are probable reservoir hosts for SARS-CoV-2, the identity of any intermediate host that may have facilitated transfer to humans is unknown. Here we report the identification of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity in the receptor-binding domain to SARS-CoV-2. The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of new coronaviruses and should be removed from wet markets to prevent zoonotic transmission.

Structural basis of receptor recognition by SARS-CoV-2.

A novel severe acute respiratory syndrome (SARS)-like coronavirus (SARS-CoV-2) recently emerged and is rapidly spreading in humans, causing COVID-191,2. A key to tackling this pandemic is to understand the receptor recognition mechanism of the virus, which regulates its infectivity, pathogenesis and host range. SARS-CoV-2 and SARS-CoV recognize the same receptor-angiotensin-converting enzyme 2 (ACE2)-in humans3,4. Here we determined the crystal structure of the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 (engineered to facilitate crystallization) in complex with ACE2. In comparison with the SARS-CoV RBD, an ACE2-binding ridge in SARS-CoV-2 RBD has a more compact conformation; moreover, several residue changes in the SARS-CoV-2 RBD stabilize two virus-binding hotspots at the RBD-ACE2 interface. These structural features of SARS-CoV-2 RBD increase its ACE2-binding affinity. Additionally, we show that RaTG13, a bat coronavirus that is closely related to SARS-CoV-2, also uses human ACE2 as its receptor. The differences among SARS-CoV-2, SARS-CoV and RaTG13 in ACE2 recognition shed light on the potential animal-to-human transmission of SARS-CoV-2. This study provides guidance for intervention strategies that target receptor recognition by SARS-CoV-2.

AKT signaling modulates latent viral reservoir viability in HIV-1-infected blood-brain barrier pericytes.

Despite antiretroviral therapy (ART), chronic forms of HIV-associated neurocognitive disorders (HAND) affect an estimated 50% of individuals living with HIV, greatly impacting their quality of life. The prevailing theory of HAND progression posits that chronic inflammation arising from the activation of latent viral reservoirs leads to progressive damage in the central nervous system (CNS). Recent evidence indicates that blood-brain barrier (BBB) pericytes are capable of active HIV-1 infection; however, their latent infection has not been defined. Given their location and function, BBB pericytes are poised to be a key viral reservoir in the development of HAND. We present the first transcriptional analysis of uninfected, active, and latent human BBB pericytes, revealing distinct transcriptional phenotypes. In addition, we demonstrate that latent infection of BBB pericytes relies on AKT signaling for reservoir survival. These findings provide insight into the state of reservoir maintenance in the CNS during HIV-1 infection and provide novel targets for reservoir clearance.

Wildlife Immune Responses to Mycobacterium bovis and to Bacille of Calmette-Guerin.

Bovine tuberculosis (bTB) is a zoonotic bacterial disease presenting public health, veterinary, and economic threats around the globe. Although cattle producers rely on regular testing and management practices to minimize domestic herd exposure, wildlife species around the world continue to be the main reservoirs for disease. Wildlife reservoirs for bTB include the Eurasian badger (Meles meles) in Great Britain and Ireland, the brushtail possum (Trichosurus vulpecula) in New Zealand, wild boar (Sus scrofa) in Spain, as well as white-tailed deer (Odocoileus virginianus) in the United States and red deer (Cervus elaphus) in Spain. Although all reservoir species share the ability to infect cattle, they differ in transmission capability, disease pathogenesis, diagnostic detection, and vaccination strategies. In this review, bTB interactions with these wildlife reservoirs are discussed, illustrating the need to address bTB disease in wildlife hosts to achieve eradication in domestic livestock.

Bats host the most virulent-but not the most dangerous-zoonotic viruses.

SignificanceThe clear need to mitigate zoonotic risk has fueled increased viral discovery in specific reservoir host taxa. We show that a combination of viral and reservoir traits can predict zoonotic virus virulence and transmissibility in humans, supporting the hypothesis that bats harbor exceptionally virulent zoonoses. However, pandemic prevention requires thinking beyond zoonotic capacity, virulence, and transmissibility to consider collective "burden" on human health. For this, viral discovery targeting specific reservoirs may be inefficient as death burden correlates with viral, not reservoir, traits, and depends on context-specific epidemiological dynamics across and beyond the human-animal interface. These findings suggest that longitudinal studies of viral dynamics in reservoir and spillover host populations may offer the most effective strategy for mitigating zoonotic risk.

No evidence for persistent natural plague reservoirs in historical and modern Europe.

Caused by Yersinia pestis, plague ravaged the world through three known pandemics: the First or the Justinianic (6th-8th century); the Second (beginning with the Black Death during c.1338-1353 and lasting until the 19th century); and the Third (which became global in 1894). It is debatable whether Y. pestis persisted in European wildlife reservoirs or was repeatedly introduced from outside Europe (as covered by European Union and the British Isles). Here, we analyze environmental data (soil characteristics and climate) from active Chinese plague reservoirs to assess whether such environmental conditions in Europe had ever supported "natural plague reservoirs". We have used new statistical methods which are validated through predicting the presence of modern plague reservoirs in the western United States. We find no support for persistent natural plague reservoirs in either historical or modern Europe. Two factors make Europe unfavorable for long-term plague reservoirs: 1) Soil texture and biochemistry and 2) low rodent diversity. By comparing rodent communities in Europe with those in China and the United States, we conclude that a lack of suitable host species might be the main reason for the absence of plague reservoirs in Europe today. These findings support the hypothesis that long-term plague reservoirs did not exist in Europe and therefore question the importance of wildlife rodent species as the primary plague hosts in Europe.

Quantifying the effectiveness of betaherpesvirus-vectored transmissible vaccines.

Transmissible vaccines have the potential to revolutionize how zoonotic pathogens are controlled within wildlife reservoirs. A key challenge that must be overcome is identifying viral vectors that can rapidly spread immunity through a reservoir population. Because they are broadly distributed taxonomically, species specific, and stable to genetic manipulation, betaherpesviruses are leading candidates for use as transmissible vaccine vectors. Here we evaluate the likely effectiveness of betaherpesvirus-vectored transmissible vaccines by developing and parameterizing a mathematical model using data from captive and free-living mouse populations infected with murine cytomegalovirus (MCMV). Simulations of our parameterized model demonstrate rapid and effective control for a range of pathogens, with pathogen elimination frequently occurring within a year of vaccine introduction. Our results also suggest, however, that the effectiveness of transmissible vaccines may vary across reservoir populations and with respect to the specific vector strain used to construct the vaccine.

Multiple spillovers from humans and onward transmission of SARS-CoV-2 in white-tailed deer.

Many animal species are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and could act as reservoirs; however, transmission in free-living animals has not been documented. White-tailed deer, the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 is circulating in deer, 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through January of 2021 were assayed for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 (33.2%) deer samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, following the November 2020 peak of human cases in Iowa, and coinciding with the onset of winter and the peak deer hunting season, SARS-CoV-2 RNA was detected in 80 of 97 (82.5%) RPLN samples collected over a 7-wk period. Whole genome sequencing of all 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%) and B.1.311 (n = 19; 20%) accounting for ∼75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple human-to-deer transmission events followed by subsequent deer-to-deer spread. These discoveries have important implications for the long-term persistence of the SARS-CoV-2 pandemic. Our findings highlight an urgent need for a robust and proactive "One Health" approach to obtain enhanced understanding of the ecology, molecular evolution, and dissemination of SARS-CoV-2.

Effect of Rodent Control Program on Incidence of Zoonotic Cutaneous Leishmaniasis, Iran.

We report the effect of a rodent control program on the incidence of zoonotic cutaneous leishmaniasis in an endemic region of Iran. A 1-year interruption in rodent control led to 2 years of increased incidence of zoonotic cutaneous leishmaniasis. Restarting rodent control led to a decline of zoonotic cutaneous leishmaniasis.