Novel betaherpesviruses and gammaherpesviruses in bats from central China.

Herpesviruses are large double-stranded DNA viruses that cause infections in animals and humans with a characteristic of latent infectious within specific tissues. Bats are natural hosts of variety human-infecting viruses and recently have been described as hosts for herpesviruses in several countries around the world. In this study we collected 140 insectivorous bats in the neighboring urban areas of Wuhan City, Hubei Province in the central China between 2020 and 2021. Nested PCR targeting the dpol gene sequence indicated that a total of 22 individuals (15.7% of the sample) tested positive for herpesvirus with 4 strains belonging to the genus Betaherpesvirus and the remaining 18 strains classified as Gammahersvirus. Furthermore, the herpesvirus prevalence in Rhinolophus pusillus was higher at 26.3%, compared to 8.4% in Myotis davidii. The RP701 strain from R. pusillus was the predominant gammaherpesvirus strain detected in bats, accounting for 94.4% (17/18) of all strains. The variations in γ-herpesviruses genomic sequences was evident in phylogenetic tree, where RP701 strain was clustered together with ruminant γ-herpesviruses, while MD704 strain formed a distinct clade with a hedgehog γ-herpesvirus. Four betaherpesviruses exclusively identified from M. davidii, with nucleotide identities ranging from 79.7 to 82.6% compared to known betaherpesviruses. Our study provided evidence that M. davidii can sever as natural host for ß-herpesviruses, which extended the host species range. In conclusion, we found that bats from central China harbored novel ß-herpesviruses and γ-herpesviruses which were phylogenetically related to ruminant γ-herpesvirus and hedgehog γ-herpesvirus. Our study indicates that bats are natural hosts of ß- and γ-herpesviruses and further studies are needed to determine whether there is cross-species transmission of herpesviruses between bats and other animals, or humans.

Phylogenetic relationship between rabies virus (Lyssavirus rabies) variants from two different species / Relações filogenéticas do vírus da raiva (Lyssavirus rabies) em duas diferentes espécies / Relaciones filogenéticas del virus de la rabia (Lyssavirus rabies) en dos especies diferentes

Rabies is a fatal zoonotic disease that affects several mammals. Hematophagous bats are recognized hosts of the rabies virus, and their main food source is the blood of other mammals, particularly cattle. During feeding, bats transmit the virus to cattle, which are victims of the disease, contributing to economic losses and increasing the risk of infection for humans. Based on this affinity in the rabies cycle between bats and cattle, the objective of this study was to analyze the phylogenetic relationships of rabies virus samples in cattle and bats. The G gene of the rabies virus was chosen for this study because it is directly related to the infection process. Nucleotide sequences of the viral G gene were selected from GenBank for samples obtained from infected cattle and bats. Maximum parsimony analyses were conducted using the Molecular Evolutionary Genetics Analysis software. The Maxima Parsimony tree indicated a phylogenetic relationship between the G genes of both hosts, indicating that the virus evolved from bats to cattle. Analysis of parsimoniously informative sites revealed that the viral G gene carried specific mutations in each host. Knowledge of the evolutionary relationships between the rabies virus and its hosts is critical for identifying potential new hosts and the possible routes of infection for humans.

A Raiva é uma zoonose fatal que infecta várias espécies de mamíferos. Os morcegos hematófagos são reconhecidos como hospedeiros do vírus da Raiva e sua principal fonte de alimento é o sangue de outros mamíferos, especialmente os bovinos. Quando se alimentam, os morcegos transmitem o vírus para o bovino os quais são vítimas da doença, contribuindo para perdas econômicas e riscos de infecção para humanos. Baseado nesta afinidade do ciclo da Raiva entre morcegos e bovinos, o objetivo deste estudo foi analisar as relações filogenéticas de amostras do vírus da Raiva em ambos os hospedeiros, bovinos e morcegos. O gene G do vírus da Raiva foi escolhido para esta pesquisa porque ele está diretamente relacionado ao processo de infecção. Sequências de nucleotídeos do gene G viral foram selecionadas no GenBank a partir de amostras obtidas de bovinos e morcegos infectados. Análises de Máxima Parcimônia foram conduzidas utilizando o software Molecular Evolutionary Genetics Analysis. A árvore de Máxima Parcimônia indicou uma relação filogenética entre o gene G de ambos os hospedeiros, indicando que o vírus evoluiu dos morcegos para os bovinos. A análise dos sítios parcimoniosamente informativos revelou que o gene G viral apresentou mutações específicas em cada hospedeiro. O conhecimento sobre as relações evolutivas do vírus da Raiva e seus hospedeiros é crucial para identificar nos hospedeiros potenciais e novas rotas possíveis de infecção para humanos.

La rabia es una zoonosis fatal que infecta a varias especies de mamíferos. Los murciélagos hematófagos son reconocidos como huéspedes del virus de la rabia y su principal fuente de alimentación es la sangre de otros mamíferos, especialmente del ganado. Al alimentarse, los murciélagos transmiten el virus al ganado que es víctima de la enfermedad, contribuyendo a pérdidas económicas y riesgos de infección para los humanos. Basado en esta afinidad del ciclo de la rabia entre murciélagos y ganado, el objetivo de este estudio fue analizar las relaciones filogenéticas de las muestras de virus de la rabia tanto en huéspedes, ganado y murciélagos. El gen G del virus de la rabia fue elegido para esta investigación porque está directamente relacionado con el proceso de infección. Las secuencias de nucleótidos del gen G viral se seleccionaron en GenBank a partir de muestras obtenidas de bovinos y murciélagos infectados. Los análisis de parsimonia máxima se realizaron utilizando el software Molecular Evolutionary Genetics Analysis. El árbol de Máxima Parsimônia indicó una relación filogenética entre el gen G de ambos huéspedes, indicando que el virus evolucionó de murciélagos a bovinos. El análisis de los sitios parsimoniosamente informativos reveló que el gen G viral presentaba mutaciones específicas en cada huésped. El conocimiento sobre las relaciones evolutivas del virus de la rabia y sus huéspedes es crucial para identificar huéspedes potenciales y nuevas posibles rutas de infección para humanos.

Pteropus vampyrus TRIM40 Is an Interferon-Stimulated Gene That Antagonizes RIG-I-like Receptors.

Nipah virus (NiV; genus: Henipavirus; family: Paramyxoviridae) naturally infects Old World fruit bats (family Pteropodidae) without causing overt disease. Conversely, NiV infection in humans and other mammals can be lethal. Comparing bat antiviral responses with those of humans may illuminate the mechanisms that facilitate bats' tolerance. Tripartite motif proteins (TRIMs), a large family of E3-ubiquitin ligases, fine-tune innate antiviral immune responses, and two human TRIMs interact with Henipavirus proteins. We hypothesize that NiV infection induces the expression of an immunosuppressive TRIM in bat, but not human cells, to promote tolerance. Here, we show that TRIM40 is an interferon-stimulated gene (ISG) in pteropodid but not human cells. Knockdown of bat TRIM40 increases gene expression of IFNß, ISGs, and pro-inflammatory cytokines following poly(I:C) transfection. In Pteropus vampyrus, but not human cells, NiV induces TRIM40 expression within 16 h after infection, and knockdown of TRIM40 correlates with reduced NiV titers as compared to control cells. Bats may have evolved to express TRIM40 in response to viral infections to control immunopathogenesis.

Characterization of CCoV-HuPn-2018 spike protein-mediated viral entry.

Canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) was recently isolated from a child with pneumonia. This novel human pathogen resulted from cross-species transmission of a canine coronavirus. It has been known that CCoV-HuPn-2018 uses aminopeptidase N (APN) from canines, felines, and porcines, but not humans, as functional receptors for cell entry. The molecular mechanism of cell entry in CCoV-HuPn-2018 remains poorly understood. In this study, we demonstrated that among the nine APN orthologs tested, the APN of the Mexican free-tailed bat could also efficiently support CCoV-HuPn-2018 spike (S) protein-mediated entry, raising the possibility that bats may also be an alternative host epidemiologically important for the transmission of this virus. The glycosylation at residue N747 of canine APN is critical for its receptor activity. The gain of glycosylation at the corresponding residues in human and rabbit APNs converted them to functional receptors for CCoV-HuPn-2018. Interestingly, the CCoV-HuPn-2018 spike protein pseudotyped virus infected multiple human cancer cell lines in a human APN-independent manner, whereas sialic acid appeared to facilitate the entry of the pseudotyped virus into human cancer cells. Moreover, while host cell surface proteases trypsin and TMPRSS2 did not promote the entry of CCoV-HuPn-2018, endosomal proteases cathepsin L and B are required for the entry of CCoV-HuPn-2018 in a pH-dependent manner. IFITMs and LY6E are host restriction factors for the CCoV-HuPn-2018 entry. Our results thus suggest that CCoV-HuPn-2018 has not yet evolved to be an efficient human pathogen. Collectively, this study helps us understand the cell tropism, receptor usage, cross-species transmission, natural reservoir, and pathogenesis of this potential human coronavirus. IMPORTANCE Viral entry is driven by the interaction between the viral spike protein and its specific cellular receptor, which determines cell tropism and host range and is the major constraint to interspecies transmission of coronaviruses. Aminopeptidase N (APN; also called CD13) is a cellular receptor for HCoV-229E, the newly discovered canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018), and many other animal alphacoronaviruses. We examined the receptor activity of nine APN orthologs and found that CCoV-HuPn-2018 utilizes APN from a broad range of animal species, including bats but not humans, to enter host cells. To our surprise, we found that CCoV-HuPn-2018 spike protein pseudotyped viral particles successfully infected multiple human hepatoma-derived cell lines and a lung cancer cell line, which is independent of the expression of human APN. Our findings thus provide mechanistic insight into the natural hosts and interspecies transmission of CCoV-HuPn-2018-like coronaviruses.

Characterization of Megabat-Favored, CA-Dependent Susceptibility to Retrovirus Infection.

The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.

Identification and Genetic Characterization of MERS-Related Coronavirus Isolated from Nathusius' Pipistrelle (Pipistrellus nathusii) near Zvenigorod (Moscow Region, Russia).

Being diverse and widely distributed globally, bats are a known reservoir of a series of emerging zoonotic viruses. We studied fecal viromes of twenty-six bats captured in 2015 in the Moscow Region and found 13 of 26 (50%) samples to be coronavirus positive. Of P. nathusii (the Nathusius' pipistrelle), 3 of 6 samples were carriers of a novel MERS-related betacoronavirus. We sequenced and assembled the complete genome of this betacoronavirus and named it MOW-BatCoV strain 15-22. Whole genome phylogenetic analysis suggests that MOW-BatCoV/15-22 falls into a distinct subclade closely related to human and camel MERS-CoV. Unexpectedly, the phylogenetic analysis of the novel MOW-BatCoV/15-22 spike gene showed the closest similarity to CoVs from Erinaceus europaeus (European hedgehog). We suppose MOW-BatCoV could have arisen as a result of recombination between ancestral viruses of bats and hedgehogs. Molecular docking analysis of MOW-BatCoV/15-22 spike glycoprotein binding to DPP4 receptors of different mammals predicted the highest binding ability with DPP4 of the Myotis brandtii bat (docking score -320.15) and the E. europaeus (docking score -294.51). Hedgehogs are widely kept as pets and are commonly found in areas of human habitation. As this novel bat-CoV is likely capable of infecting hedgehogs, we suggest hedgehogs can act as intermediate hosts between bats and humans for other bat-CoVs.

Close relatives of MERS-CoV in bats use ACE2 as their functional receptors.

Middle East respiratory syndrome coronavirus (MERS-CoV) and several bat coronaviruses use dipeptidyl peptidase-4 (DPP4) as an entry receptor1-4. However, the receptor for NeoCoV-the closest known MERS-CoV relative found in bats-remains unclear5. Here, using a pseudotype virus entry assay, we found that NeoCoV and its close relative, PDF-2180, can efficiently bind to and use specific bat angiotensin-converting enzyme 2 (ACE2) orthologues and, less favourably, human ACE2 as entry receptors through their receptor-binding domains (RBDs) on the spike (S) proteins. Cryo-electron microscopy analysis revealed an RBD-ACE2 binding interface involving protein-glycan interactions, distinct from those of other known ACE2-using coronaviruses. We identified residues 337-342 of human ACE2 as a molecular determinant restricting NeoCoV entry, whereas a NeoCoV S pseudotyped virus containing a T510F RBD mutation efficiently entered cells expressing human ACE2. Although polyclonal SARS-CoV-2 antibodies or MERS-CoV RBD-specific nanobodies did not cross-neutralize NeoCoV or PDF-2180, an ACE2-specific antibody and two broadly neutralizing betacoronavirus antibodies efficiently inhibited these two pseudotyped viruses. We describe MERS-CoV-related viruses that use ACE2 as an entry receptor, underscoring a promiscuity of receptor use and a potential zoonotic threat.

Functional binding dynamics relevant to the evolution of zoonotic spillovers in endemic and emergent Betacoronavirus strains.

Comparative functional analysis of the dynamic interactions between various Betacoronavirus mutant strains and broadly utilized target proteins such as ACE2 and CD26, is crucial for a more complete understanding of zoonotic spillovers of viruses that cause diseases such as COVID-19. Here, we employ machine learning to replicated sets of nanosecond scale GPU accelerated molecular dynamics simulations to statistically compare and classify atom motions of these target proteins in both the presence and absence of different endemic and emergent strains of the viral receptor binding domain (RBD) of the S spike glycoprotein. A multi-agent classifier successfully identified functional binding dynamics that are evolutionarily conserved from bat CoV-HKU4 to human endemic/emergent strains. Conserved dynamics regions of ACE2 involve both the N-terminal helices, as well as a region of more transient dynamics encompassing residues K353, Q325 and a novel motif AAQPFLL 386-92 that appears to coordinate their dynamic interactions with the viral RBD at N501. We also demonstrate that the functional evolution of Betacoronavirus zoonotic spillovers involving ACE2 interaction dynamics are likely pre-adapted from two precise and stable binding sites involving the viral bat progenitor strain's interaction with CD26 at SAMLI 291-5 and SS 333-334. Our analyses further indicate that the human endemic strains hCoV-HKU1 and hCoV-OC43 have evolved more stable N-terminal helix interactions through enhancement of an interfacing loop region on the viral RBD, whereas the highly transmissible SARS-CoV-2 variants (B.1.1.7, B.1.351 and P.1) have evolved more stable viral binding via more focused interactions between the viral N501 and ACE2 K353 alone.Communicated by Ramaswamy H. Sarma.

Genomic Characterization of Diverse Bat Coronavirus HKU10 in Hipposideros Bats.

Bats have been identified as natural reservoirs of a variety of coronaviruses. They harbor at least 19 of the 33 defined species of alpha- and betacoronaviruses. Previously, the bat coronavirus HKU10 was found in two bat species of different suborders, Rousettus leschenaultia and Hipposideros pomona, in south China. However, its geographic distribution and evolution history are not fully investigated. Here, we screened this viral species by a nested reverse transcriptase PCR in our archived samples collected over 10 years from 25 provinces of China and one province of Laos. From 8004 bat fecal samples, 26 were found to be positive for bat coronavirus HKU10 (BtCoV HKU10). New habitats of BtCoV HKU10 were found in the Yunnan, Guangxi, and Hainan Provinces of China, and Louang Namtha Province in Laos. In addition to H. pomona, BtCoV HKU10 variants were found circulating in Aselliscus stoliczkanus and Hipposideros larvatus. We sequenced full-length genomes of 17 newly discovered BtCoV HKU10 strains and compared them with previously published sequences. Our results revealed a much higher genetic diversity of BtCoV HKU10, particularly in spike genes and accessory genes. Besides the two previously reported lineages, we found six novel lineages in their new habitats, three of which were located in Yunnan province. The genotypes of these viruses are closely related to sampling locations based on polyproteins, and correlated to bat species based on spike genes. Combining phylogenetic analysis, selective pressure, and molecular-clock calculation, we demonstrated that Yunnan bats harbor a gene pool of BtCoV HKU10, with H. pomona as a natural reservoir. The cell tropism test using spike-pseudotyped lentivirus system showed that BtCoV HKU10 could enter cells from human and bat, suggesting a potential interspecies spillover. Continuous studies on these bat coronaviruses will expand our understanding of the evolution and genetic diversity of coronaviruses, and provide a prewarning of potential zoonotic diseases from bats.

The host range restriction of bat-associated no-known-vector flaviviruses occurs post-entry.

Most flaviviruses are transmitted horizontally between vertebrate hosts by haematophagous arthropods. Others exhibit host ranges restricted to vertebrates or arthropods. Vertebrate-specific flaviviruses are commonly referred to as no-known-vector (NKV) flaviviruses and can be separated into bat- and rodent-associated NKV flaviviruses. Rio Bravo virus (RBV) is one of eight recognized bat-associated NKV (B-NKV) flaviviruses. Studies designed to identify the genetic determinants that condition the host range restriction of B-NKV flaviviruses have never been performed. To investigate whether the host range restriction occurs at the level of attachment or entry, chimeric flaviviruses were created by inserting the pre-membrane and envelope protein genes of RBV into the genetic backbones of yellow fever virus (YFV) and Zika virus (ZIKV), two mosquito-borne flaviviruses associated with human disease. The chimeric viruses infected both vertebrate and mosquito cells. In vertebrate cells, all viruses produced similar mean peak titres, but the chimeric viruses grew more slowly than their parental viruses during early infection. In mosquito cells, the chimeric virus of YFV and RBV grew more slowly than YFV at early post-inoculation time points, but reached a similar mean peak titre. In contrast, the chimeric virus of ZIKV and RBV produced a mean peak titre that was approximately 10-fold lower than ZIKV. The chimeric virus of YFV and RBV produced an intermediate plaque phenotype, while the chimeric virus of ZIKV and RBV produced smaller plaques than both parental viruses. To conclude, we provide evidence that the structural glycoproteins of RBV permit entry into both mosquito and vertebrate cells, indicating that the host range restriction of B-NKV flaviviruses is mediated by a post-attachment/entry event.

Retroviruses of Bats: a Threat Waiting in the Wings?

Bats are infamous reservoirs of deadly human viruses. While retroviruses, such as the human immunodeficiency virus (HIV), are among the most significant of virus families that have jumped from animals into humans, whether bat retroviruses have the potential to infect and cause disease in humans remains unknown. Recent reports of retroviruses circulating in bat populations builds on two decades of research describing the fossil records of retroviral sequences in bat genomes and of viral metagenomes extracted from bat samples. The impact of the global COVID-19 pandemic demands that we pay closer attention to viruses hosted by bats and their potential as a zoonotic threat. Here we review current knowledge of bat retroviruses and explore the question of whether they represent a threat to humans.

A potential bat adenovirus-based oncolytic virus targeting canine cancers.

Although a canine adenovirus (CAdV)-based oncolytic virus (OV) candidate targeting canine tumors has been reported, its oncolytic effect could be attenuated by CAdV vaccine-induced neutralizing antibodies in dog patients. To circumvent this issue, we focused on the bat adenovirus (BtAdV) strain, which was previously isolated from healthy microbats. We previously showed that this virus replicated efficiently in canine cell lines and did not serologically cross-react with CAdVs, suggesting that it may offer the possibility of an OV candidate for canine tumors. Here, we tested the growth properties and cytotoxicity of the BtAdV Mm32 strain in a panel of canine tumor cells and found that its characteristics were equivalent to those of CAdVs. To produce an Mm32 construct with enhanced tumor specificity, we established a novel reverse genetics system for BtAdV based on bacterial artificial chromosomes, and generated a recombinant virus, Mm32-E1Ap + cTERTp, by inserting a tumor-specific canine telomerase reverse transcriptase promoter into its E1A regulatory region. The growth and cytotoxicity of this recombinant were superior to those of wild-type Mm32 in canine tumor cells, unlike in normal canine cells. These data suggest that Mm32-E1Ap + cTERTp could be a promising OV for alternative canine cancer therapies.

Regulation of the Dimerization and Activity of SARS-CoV-2 Main Protease through Reversible Glutathionylation of Cysteine 300.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for coronavirus disease 2019 (COVID-19), encodes two proteases required for replication. The main protease (Mpro), encoded as part of two polyproteins, pp1a and pp1ab, is responsible for 11 different cleavages of these viral polyproteins to produce mature proteins required for viral replication. Mpro is therefore an attractive target for therapeutic interventions. Certain proteins in cells under oxidative stress undergo modification of reactive cysteines. We show Mpro is susceptible to glutathionylation, leading to inhibition of dimerization and activity. Activity of glutathionylated Mpro could be restored with reducing agents or glutaredoxin. Analytical studies demonstrated that glutathionylated Mpro primarily exists as a monomer and that modification of a single cysteine with glutathione is sufficient to block dimerization and inhibit its activity. Gel filtration studies as well as analytical ultracentrifugation confirmed that glutathionylated Mpro exists as a monomer. Tryptic and chymotryptic digestions of Mpro as well as experiments using a C300S Mpro mutant revealed that Cys300, which is located at the dimer interface, is a primary target of glutathionylation. Moreover, Cys300 is required for inhibition of activity upon Mpro glutathionylation. These findings indicate that Mpro dimerization and activity can be regulated through reversible glutathionylation of a non-active site cysteine, Cys300, which itself is not required for Mpro activity, and provides a novel target for the development of agents to block Mpro dimerization and activity. This feature of Mpro may have relevance to the pathophysiology of SARS-CoV-2 and related bat coronaviruses. IMPORTANCE SARS-CoV-2 is responsible for the devastating COVID-19 pandemic. Therefore, it is imperative that we learn as much as we can about the biochemistry of the coronavirus proteins to inform development of therapy. One attractive target is the main protease (Mpro), a dimeric enzyme necessary for viral replication. Most work thus far developing Mpro inhibitors has focused on the active site. Our work has revealed a regulatory mechanism for Mpro activity through glutathionylation of a cysteine (Cys300) at the dimer interface, which can occur in cells under oxidative stress. Cys300 glutathionylation inhibits Mpro activity by blocking its dimerization. This provides a novel accessible and reactive target for drug development. Moreover, this process may have implications for disease pathophysiology in humans and bats. It may be a mechanism by which SARS-CoV-2 has evolved to limit replication and avoid killing host bats when they are under oxidative stress during flight.

Full-Genome Sequences of Alphacoronaviruses and Astroviruses from Myotis and Pipistrelle Bats in Denmark.

Bat species worldwide are receiving increased attention for the discovery of emerging viruses, cross-species transmission, and zoonoses, as well as for characterizing virus infections specific to bats. In a previous study, we investigated the presence of coronaviruses in faecal samples from bats at different locations in Denmark, and made phylogenies based on short, partial ORF1b sequences. In this study, selected samples containing bat coronaviruses from three different bat species were analysed, using a non-targeted approach of next-generation sequencing. From the resulting metagenomics data, we assembled full-genome sequences of seven distinct alphacoronaviruses, three astroviruses, and a polyomavirus, as well as partial genome sequences of rotavirus H and caliciviruses, from the different bat species. Comparisons to published sequences indicate that the bat alphacoronaviruses belong to three different subgenera-i.e., Pedacovirus, Nyctacovirus, and Myotacovirus-that the astroviruses may be new species in the genus Mamastrovirus, and that the polyomavirus could also be a new species, but unassigned to a genus. Furthermore, several viruses of invertebrates-including two Rhopalosiphum padi (aphid) viruses and a Kadipiro virus-present in the faecal material were assembled. Interestingly, this is the first detection in Europe of a Kadipiro virus.

Translation from bats to humans beyond infectious diseases.

Bats are attracting the greatest attention recently as a putative reservoir of SARS-CoV-2 responsible for the COVID-19 pandemic. However, less known to the public, bats also have several unique traits of high value to human health. The lessons we learn from bats can potentially help us fight many human diseases, including infection, aging, and cancer.

Metagenomic analysis of fecal and tissue samples from 18 endemic bat species in Switzerland revealed a diverse virus composition including potentially zoonotic viruses.

Many recent disease outbreaks in humans had a zoonotic virus etiology. Bats in particular have been recognized as reservoirs to a large variety of viruses with the potential to cross-species transmission. In order to assess the risk of bats in Switzerland for such transmissions, we determined the virome of tissue and fecal samples of 14 native and 4 migrating bat species. In total, sequences belonging to 39 different virus families, 16 of which are known to infect vertebrates, were detected. Contigs of coronaviruses, adenoviruses, hepeviruses, rotaviruses A and H, and parvoviruses with potential zoonotic risk were characterized in more detail. Most interestingly, in a ground stool sample of a Vespertilio murinus colony an almost complete genome of a Middle East respiratory syndrome-related coronavirus (MERS-CoV) was detected by Next generation sequencing and confirmed by PCR. In conclusion, bats in Switzerland naturally harbour many different viruses. Metagenomic analyses of non-invasive samples like ground stool may support effective surveillance and early detection of viral zoonoses.

Evaluation of bat adenoviruses suggests co-evolution and host roosting behaviour as drivers for diversity.

Adenoviruses (AdVs) are diverse pathogens of humans and animals, with several dozen bat AdVs already identified. Considering that over 100 human AdVs are known, and the huge diversity of bat species, many bat AdVs likely remain undiscovered. To learn more about AdV prevalence, diversity and evolution, we sampled and tested bats in Cameroon using several PCR assays for viral and host DNA. AdV DNA was detected in 14 % of the 671 sampled animals belonging to 37 different bat species. There was a correlation between species roosting in larger groups and AdV DNA detection. The detected AdV DNA belonged to between 28 and 44 different, mostly previously unknown, mastadenovirus species. The novel isolates are phylogenetically diverse and while some cluster with known viruses, others appear to form divergent new clusters. The phylogenetic tree of novel and previously known bat AdVs does not mirror that of the various host species, but does contain structures consistent with a degree of virus-host co-evolution. Given that closely related isolates were found in different host species, it seems likely that at least some bat AdVs have jumped species barriers, probably in the more recent past; however, the tree is also consistent with such events having taken place throughout bat AdV evolution. AdV diversity was highest in bat species roosting in large groups. The study significantly increased the diversity of AdVs known to be harboured by bats, and suggests that host behaviours, such as roosting size, may be what limits some AdVs to one species rather than an inability of AdVs to infect other related hosts.

The virome of German bats: comparing virus discovery approaches.

Bats are known to be reservoirs of several highly pathogenic viruses. Hence, the interest in bat virus discovery has been increasing rapidly over the last decade. So far, most studies have focused on a single type of virus detection method, either PCR, virus isolation or virome sequencing. Here we present a comprehensive approach in virus discovery, using all three discovery methods on samples from the same bats. By family-specific PCR screening we found sequences of paramyxoviruses, adenoviruses, herpesviruses and one coronavirus. By cell culture we isolated a novel bat adenovirus and bat orthoreovirus. Virome sequencing revealed viral sequences of ten different virus families and orders: three bat nairoviruses, three phenuiviruses, one orbivirus, one rotavirus, one orthoreovirus, one mononegavirus, five parvoviruses, seven picornaviruses, three retroviruses, one totivirus and two thymoviruses were discovered. Of all viruses identified by family-specific PCR in the original samples, none was found by metagenomic sequencing. Vice versa, none of the viruses found by the metagenomic virome approach was detected by family-specific PCRs targeting the same family. The discrepancy of detected viruses by different detection approaches suggests that a combined approach using different detection methods is necessary for virus discovery studies.

SARS-CoV-2 infection is effectively treated and prevented by EIDD-2801.

All coronaviruses known to have recently emerged as human pathogens probably originated in bats1. Here we use a single experimental platform based on immunodeficient mice implanted with human lung tissue (hereafter, human lung-only mice (LoM)) to demonstrate the efficient in vivo replication of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as well as two endogenous SARS-like bat coronaviruses that show potential for emergence as human pathogens. Virus replication in this model occurs in bona fide human lung tissue and does not require any type of adaptation of the virus or the host. Our results indicate that bats contain endogenous coronaviruses that are capable of direct transmission to humans. Our detailed analysis of in vivo infection with SARS-CoV-2 in human lung tissue from LoM showed a predominant infection of human lung epithelial cells, including type-2 pneumocytes that are present in alveoli and ciliated airway cells. Acute infection with SARS-CoV-2 was highly cytopathic and induced a robust and sustained type-I interferon and inflammatory cytokine and chemokine response. Finally, we evaluated a therapeutic and pre-exposure prophylaxis strategy for SARS-CoV-2 infection. Our results show that therapeutic and prophylactic administration of EIDD-2801-an oral broad-spectrum antiviral agent that is currently in phase II/III clinical trials-markedly inhibited SARS-CoV-2 replication in vivo, and thus has considerable potential for the prevention and treatment of COVID-19.

Dispersal history of Miniopterus fuliginosus bats and their associated viruses in east Asia.

In this study, we examined the role of the eastern bent-winged bat (Miniopterus fuliginosus) in the dispersion of bat adenovirus and bat alphacoronavirus in east Asia, considering their gene flows and divergence times (based on deep-sequencing data), using bat fecal guano samples. Bats in China moved to Jeju Island and/or Taiwan in the last 20,000 years via the Korean Peninsula and/or Japan. The phylogenies of host mitochondrial D-loop DNA was not significantly congruent with those of bat adenovirus (m2XY = 0.07, p = 0.08), and bat alphacoronavirus (m2XY = 0.48, p = 0.20). We estimate that the first divergence time of bats carrying bat adenovirus in five caves studied (designated as K1, K2, JJ, N2, and F3) occurred approximately 3.17 million years ago. In contrast, the first divergence time of bat adenovirus among bats in the 5 caves was estimated to be approximately 224.32 years ago. The first divergence time of bats in caves CH, JJ, WY, N2, F1, F2, and F3 harboring bat alphacoronavirus was estimated to be 1.59 million years ago. The first divergence time of bat alphacoronavirus among the 7 caves was estimated to be approximately 2,596.92 years ago. The origin of bat adenovirus remains unclear, whereas our findings suggest that bat alphacoronavirus originated in Japan. Surprisingly, bat adenovirus and bat alphacoronavirus appeared to diverge substantially over the last 100 years, even though our gene-flow data indicate that the eastern bent-winged bat serves as an important natural reservoir of both viruses.