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What coronaviruses have been identified in racoon dogs?

What coronaviruses have been identified in racoon dogs?


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Christian Dorsten says in an (Apr 26) interview in the Guardian that racoon dogs as [intermediate] source of coronavirues have been overlooked (in China):

Q: What do we know about that intermediate host - is it the “poor pangolin”, as it's come to be known?

A: I don't see any reason to assume that the virus passed through pangolins on its way to humans. There is an interesting piece of information from the old Sars literature. That virus was found in civet cats, but also in raccoon dogs - something the media overlooked. Raccoon dogs are a massive industry in China, where they are bred on farms and caught in the wild for their fur. If somebody gave me a few hundred thousand bucks and free access to China to find the source of the virus, I would look in places where raccoon dogs are bred.

So what studies are there on coronavirues in racoon dogs up to now? (A quick search found a 1992 US paper that some coronavirus [unsurprisingly] infects racoons too. But I can't find much else. Is there more up-to-date survey? Also, duh, racoon dogs aren't even the same family [Canidae vs Procyonidae].)


Here's an abstract for a paper from 2006: https://www.ncbi.nlm.nih.gov/pubmed/16450706

An RT-nPCR assay was used for testing fecal samples of dogs, foxes, raccoon dogs and minks for the presence of canine coronavirus (CCV). The animals were raised in homes, dog schools or farms… Among 24 raccoon dogs samples, 22 were CCV type II-positive, and from those 16 were additionally type I positive.

Here's another paper from 2003: https://www.ncbi.nlm.nih.gov/pubmed/12958366

A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome (SARS)… Evidence of virus infection was also detected in other animals (including a raccoon dog, Nyctereutes procyonoides) and in humans working at the same market.

So at least a couple types of canine coronavirus and other strains closely related (but not identical) to the coronavirus that causes SARS.


Emergent virus

An emergent virus (or emerging virus) is a virus that is either newly appeared, notably increasing in incidence/geographic range or has the potential to increase in the near future. [1] Emergent viruses are a leading cause of emerging infectious diseases and raise public health challenges globally, given their potential to cause outbreaks of disease which can lead to epidemics and pandemics. [2] As well as causing disease, emergent viruses can also have severe economic implications. [3] Recent examples include the SARS-related coronaviruses, which have caused the 2002-2004 outbreak of SARS (SARS-CoV-1) and the 2019–20 pandemic of COVID-19 (SARS-CoV-2). [4] [5] Other examples include the human immunodeficiency virus which causes HIV/AIDS the viruses responsible for Ebola [6] the H5N1 influenza virus responsible for avian flu [7] and H1N1/09, which caused the 2009 swine flu pandemic [8] (an earlier emergent strain of H1N1 caused the 1918 Spanish flu pandemic). [9] Viral emergence in humans is often a consequence of zoonosis, which involves a cross-species jump of a viral disease into humans from other animals. As zoonotic viruses exist in animal reservoirs, they are much more difficult to eradicate and can therefore establish persistent infections in human populations. [10]

Emergent viruses should not be confused with re-emerging viruses or newly detected viruses. A re-emerging virus is generally considered to be a previously appeared virus that is experiencing a resurgence, [1] [11] for example measles. [12] A newly detected virus is a previously unrecognized virus that had been circulating in the species as endemic or epidemic infections. [13] Newly detected viruses may have escaped classification because they left no distinctive clues, and/or could not be isolated or propagated in cell culture. [14] Examples include human rhinovirus (a leading cause of common colds which was first identified in 1956), [15] hepatitis C (eventually identified in 1989), [16] and human metapneumovirus (first described in 2001, but thought to have been circulating since the 19th century). [17] As the detection of such viruses is technology driven, the number reported is likely to expand.


Study predicts where new coronaviruses might originate

The potential scale of novel coronavirus generation in wild and domesticated animals may have been highly underappreciated, suggests new University of Liverpool research.

Published in Nature Communications, the machine-learning study identifies mammals that are potential sources for generating new coronaviruses, including species implicated in previous outbreaks (such as horseshoe bats, palm civets and pangolins) and some novel candidates.

Predicting which animals could potentially be the source of a future coronavirus outbreak may guide approaches to reduce the risk of coronavirus emergence in animals and spill-over to human populations.

“New coronaviruses can emerge when two different strains co-infect an animal, causing the viral genetic material to recombine. Our understanding of how susceptible different mammals are to different coronaviruses has been limited, but such information could offer insights into where viral recombination might occur,” explained co-lead researcher Dr. Maya Wardeh from the Institute of Infection, Veterinary and Ecological Sciences.

The researchers sought to bridge this knowledge gap by using a machine-learning approach to predict relationships between 411 strains of coronavirus and 876 potential mammalian host species. They predict the mammals that are most likely to be co-infected, and therefore be potential recombination hosts for the production of novel coronaviruses.

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Their findings suggest that there are at least 11 times more associations between mammalian species and coronavirus strains than have been observed to date. In addition, they estimate that there are over 40 times more mammal species that can be infected with a diverse set of coronavirus strains than was previously known.

“Given that coronaviruses frequently undergo recombination when they co-infect a host, and that SARS-CoV-2 is highly infectious to humans, the most immediate threat to public health is recombination of other coronaviruses with SARS-CoV-2,” said Dr. Marcus Blagrove, co-lead of the study.

The researchers went on to identify hosts in which SARS-CoV-2 recombination could potentially occur and indicate there may be 30 times more host species than currently known. Notable new predicted hosts include the dormitory camel, African green monkey and the lesser Asiatic yellow bat.

Highlighting, as a specific example, the high-risk scenario of recombination occurring between the highly transmissible SARS-CoV-2 and the more deadly MERS-CoV, the researchers also identify 102 potential recombination hosts of the two viruses and recommend monitoring for this event.

The researchers note that their results draw on limited data on coronavirus genomes and virus-host associations, and that there are study biases for certain animal species, all of which present uncertainty in the predictions. However, recent testing of potential mammalian hosts for their susceptibility to SARS-CoV-2 has already confirmed a number of their predictions, such as the raccoon dog, the domestic goat and the alpaca.

“It is important to note that viral recombination is distinct from mutations. Recombination occurs over longer periods of time and can generate completely new strains or species. Our work can help target surveillance programs to discover future strains before they spill-over to humans, giving us a head-start in combating them,” concluded Dr. Blagrove.

The researchers now plan to expand their model to include bird species, therefore, encompassing the full range of important coronavirus hosts, and a species-level contact network, accounting for behavior and habitat utilization of host species, to give a broader overview of potential coronavirus associations.

Provided by: University of Liverpool

More information: Maya Wardeh et al. Predicting mammalian hosts in which novel coronaviruses can be generated. Nature Communications (2021) DOI: 10.1038/s41467-021-21034-5


Brief overview of the SARS-CoV genome

Coronaviruses are subdivided into three groups based on genetic and serological markers [22]. Groups I, and II infect mammals while group III is specific for avian species. Group I members are the porcine transmissible gastroenteritis virus (TGEV) and epidemic diarrhea virus (PEDV), feline and canine coronavirus (FCoV and CCoV), and human coronavirus 229E (HCoV-229E). Group II includes porcine hemagglutinating encephalomyelitis virus (HEV), murine hepatitis virus (MHV), bovine, equine, and rat coronavirus (BCoV, ECoV, and RtCoV), and human coronavirus OC43 (HCoV-OC43). Group III includes the turkey coronavirus (TCoV), pheasant coronavirus and avian infectious bronchitis virus (IBV). Although most closely related to Group II coronaviruses, SARS-CoV, with some of its unique genetic features, represents a distinct phylogenetic group [22–24].

To date, approximately 61 SARS-CoV genomic sequences have been analyzed representing different phases of the epidemic (early, middle, and late) and two isolates obtained from palm civets [18]. The SARS-CoV genomic RNA is approximately 30 kb and is organized into 13 to 15 open reading frames (ORFs) [25–27]. The SARS CoV structural gene arrangement follows the same pattern as most coronavirus genomes: 5'- Replicase (ORF 1a)-Protease (ORF 1b)-Spike (S)-Envelope (E)-membrane (M)-Nucleocapsid (N)-3' [27]. However, in contrast to other coronaviruses, two ORFs of unknown function are located between the S and E ORFs and 3–5 ORFs are located between M and N. In addition, despite the evolutionary overlap between SARS-CoV and Group II coronavirus genome sequences, the SARS genome lacks a gene for hemagglutinin-esterase (HE) protein, which is common to a majority of Group II coronaviruses [25]. For an excellent pictorial representation of SARS-CoV genome with functions (or lack of) assigned to each ORF, please refer to the recent review by Tan et al [21]. A significant milestone in SARS-CoV molecular biology was the construction of a SARS-CoV full-length cDNA-containing plasmid from which infectious viral RNA can be produced [28]. This development facilitates the study of SARS-CoV gene functions and should promote the elucidation of function for ORFs whose function is still unknown [29]. Although it has been the perception that these ORFs are not essential for viral replication, they may play a role in the manifestation or severity of disease.


What coronaviruses have been identified in racoon dogs? - Biology

The epidemic of severe acute respiratory syndrome (SARS) was caused by a newly emerged coronavirus (SARS-CoV). Bats of several species in southern People’s Republic of China harbor SARS-like CoVs and may be reservoir hosts for them. To determine whether bats in North America also harbor coronaviruses, we used reverse transcription–PCR to detect coronavirus RNA in bats. We found coronavirus RNA in 6 of 28 fecal specimens from bats of 2 of 7 species tested. The prevalence of viral RNA shedding was high: 17% in Eptesicus fuscus and 50% in Myotis occultus. Sequence analysis of a 440-bp amplicon in gene 1b showed that these Rocky Mountain bat coronaviruses formed 3 clusters in phylogenetic group 1 that were distinct from group 1 coronaviruses of Asian bats. Because of the potential for bat coronaviruses to cause disease in humans and animals, further surveillance and characterization of bat coronaviruses in North America are needed.

Emerging diseases are frequently zoonoses caused by RNA viruses (1,2). Defense against emerging infectious diseases, identification of reservoirs for such viruses, surveillance for host-jumping events, and elucidation of viral and host factors that may facilitate such events are warranted. The epidemic of severe acute respiratory syndrome (SARS) in 2002–2003 was caused by a newly emerged zoonotic coronavirus (SARS-CoV) (order Nidovirales, family Coronaviridae, genus Coronavirus). Other coronaviruses have also jumped to new host species and caused emerging diseases. For example, porcine epidemic diarrhea virus emerged in European pigs from an unknown host species during the late 1970s and caused severe enteric disease (3). Human coronavirus OC43 is believed to have been derived from bovine coronavirus (4). In addition, the genomes of canine and feline coronaviruses can recombine in vivo and have developed into different biotypes that are serially transmissible in their new host species (5).

SARS-CoV entered the human population as a result of a zoonotic transmission in southern People’s Republic of China in 2002. Epidemiologic studies demonstrated that the first human cases of SARS were caused by coronaviruses closely related to viruses found in masked palm civets (Paguma larvata) and raccoon dogs (Nyctereutes procyonoides) in live animal markets (6). Subsequently, surveys of coronaviruses in domestic animals, livestock, poultry, and wildlife were conducted in Southeast Asia to identify the reservoir(s) of SARS-CoV. On the basis of low prevalence of SARS-like CoVs in wild and farmed masked palm civets, these animals are now believed to be an intermediate host rather than a primary reservoir for SARS-CoV (7). During these surveys, a wide variety of coronaviruses were detected in many bat species in Asia (811).

Horseshoe-nosed bats of several species (suborder Microchiroptera, family Rhinolophidae, genus Rhinolophus) from different locations in southern People’s Republic of China and the Hong Kong Special Administrative Region were found to be infected with SARS-like CoVs, and some of the bats had antibodies to these newly recognized coronaviruses (10,12). Phylogenetic analysis of the complete genome sequences of the bat SARS-like CoVs showed that they form a large and diverse clade within phylogenetic group 2b (also called group 4), which includes SARS-CoVs from palm civets and humans obtained during the 2002–2003 outbreak (10,12,13). Thus, the virus responsible for the SARS pandemic may have originated from bats, perhaps with the palm civet as an intermediate host. In addition to SARS-like CoVs, RNAs of many other viruses belonging to coronavirus groups 1 and 2a, and proposed new group 5, were detected in several species of Asian bats (8,9,14,15). To date, no infectious bat coronavirus has been isolated in cell culture.

We investigated whether bats in North America also harbor coronaviruses. To our knowledge, we provide the first evidence of coronaviruses in bats in the Western Hemisphere. We studied oral, anal, and fecal specimens from 57 bats in the Rocky Mountain region and detected coronavirus RNA in 6 of 28 fecal specimens from 2 of 7 bat species tested. Limited sequence analysis showed that these viruses are in phylogenetic group 1 and that they differ from group 1 coronaviruses of Asian bats.

Materials and Methods

Sample Collection

Bats were sampled at 4 sites in the Rocky Mountain region in August 2006. At sites 1 and 2, bats of 2 species were sampled in colonies inhabiting 2 buildings 480 km apart on opposite sides of the continental divide of the Rocky Mountains. Eight occult myotis (Myotis occultus) and 1 Brazilian free-tailed bat (Tadarida brasiliensis) were captured in mist nets as they emerged from a roost in a building in Mancos in Montezuma County in southwestern Colorado (site 1) at dusk on August 19. This species was previously thought to be conspecific with the little brown bat (M. lucifugus) that is common throughout North America (16). Big brown bats (Eptesicus fuscus) were sampled at a roost in a building in Fort Collins in Larimer County in north-central Colorado (site 2) on August 7. Other bats (n = 27) were sampled at sites 3 and 4 incidental to ongoing, unrelated bat faunal surveys. One western small-footed myotis (M. ciliolabrum) and 1 long-eared myotis (M. evotis) were captured in mist nets over water on August 8 at Soapstone Prairie Natural Area in Larimer County (site 3). Four big brown bats, 3 long-eared myotis, 8 occult myotis, 1 Brazilian free-tailed bat, 7 long-legged myotis (M. volans), and 2 silver-haired bats (Lasionycteris noctivagans) were trapped in mist nets during the nights of August 14–20 as they drank or foraged near open water at 2 sewage treatment lagoons (9 km apart) (site 4) in Montezuma County, Colorado. Bats were captured under authority of a Colorado Division of Wildlife Scientific Collection License following procedures approved by the Institutional Animal Care and Use Committee of the US Geological Survey, Fort Collins Science Center. Typically, each bat was sampled within 5–10 minutes of capture and then released.

Whenever possible, 3 sample types were taken from each bat (Table). Sterile calcium alginate swabs were used for oral or anal area samples that were immediately placed into 2 mL of RNAlater (Ambion, Austin, TX, USA). Fecal samples were collected if the bat produced a fresh bolus during handling. Disposable latex gloves were changed between samples, and multiple forceps used to collect fecal boluses were rinsed, wiped in ethanol, and air-dried between samples. Samples were numbered, kept in a cooler in the field, stored at 4°C, and delivered to the laboratory on August 28.

RNA Extraction and Reverse Transcription (RT)

RNA from 140 μL of each of the 79 samples was extracted by using the QIamp viral RNA mini kit (QIAGEN, Valencia, CA, USA) following the manufacturer’s instructions. Extracted RNA was eluted in 50 μL of RNase-free water and stored at –80°C. We used Moloney murine leukemia virus reverse transcriptase (Invitrogen, Carlsbad, CA, USA) with random hexamers in a 20-μL reaction to generate cDNAs by using 10 μL of RNA as a template according to the manufacturer’s instructions. All samples were extracted and analyzed in triplicate. RT products were stored at –20°C.

PCR and Sequencing

All samples were screened by PCR and nested PCR. On the basis of previous reports, PCR with a pair of consensus primers that target a highly conserved region of coronavirus gene 1b was used to screen the cDNA samples (8). Three microliters of cDNA was amplified in a 50-μL reaction containing 1.5 mmol/L MgCl2, 0.2 mmol/L deoxynucleoside triphosophates, 2.5 U of HotStarTaq (QIAGEN), and 0.2 μmol/L of primers 1 and 2: 5′-GGTTGGGACTATCCTAAGTGTGA-3′ (primer 1) and 5′-CCATCATCAGATAGAATCATCATA-3′ (primer 2) by using the following PCR program: 15 min at 95°C 45 cycles for 1 min at 95°C, 1 min at 48°C, and 1 min at 72°C and 10 min at 72°C.

For nested PCR, 5 μL from each PCR was amplified in a 50-μL reaction with primer 2 and primer 3 (5′-GTTGTACTGCTAGTGACAGG-3′), an internal primer based on nucleotide sequences of the PCR amplicons by using 40 cycles of the same PCR program. All RT-PCRs were conducted in an enclosed nucleic acid workstation equipped with a UV light (Clone Zone USA Scientific, Ocala, FL, USA) in a room separate from the main laboratory. Water controls in all RT-PCRs did not show false-positive results. To overcome possible PCR inhibitors in fecal samples, PCR was performed both on the cDNA and on a 1:10 dilution of the cDNA. Amplicons were analyzed by agarose gel electrophoresis. For each positive specimen, amplicons from 2 independent RT-PCRs were sequenced on an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA, USA) at the University of Colorado Health Science Center Cancer Center DNA Sequencing and Analysis Core. Numbered specimens were then correlated with lists of bat samples.

Data Analysis

Viral sequences were analyzed and aligned by using ClustalW (http://workbench.sdsc.edu). Phylogenetic trees were constructed by using the neighbor-joining method in the program PAUP* version 4.0 (Sinauer Associates, Inc., Sunderland, MA, USA) rooted with porcine respiratory and reproductive syndrome virus (GenBank accession no. NC_001961). Sequences used for alignment were AF304460 (HCoV-229E), AY567487 (HCoV-NL63), DQ648858 (BtCoV 512), AY594268 (BtCoV HKU2), DQ249224 (BtCoV HKU6), DQ249226 (BtCoV HKU7), and DQ249228 (BtCoV HKU8). The deduced sequences from this study were deposited in GenBank under accession nos. EF544563–EF544568.

Results

Identification of Rocky Mountain Bat Coronaviruses (RM-Bt-CoVs)

A total of 79 samples (28 fecal samples, 29 anal swab specimens, and 22 oral swab specimens) were collected from 57 bats of 7 species in 4 locations in the Rocky Mountain region during a 2-week period in August 2006 (Table). PCR amplicons that target a conserved region in gene 1b containing the RNA-dependent RNA polymerase common to all coronaviruses were detected in reversed-transcribed RNA from 6 of the 79 samples. All samples positive for coronavirus RNA were from the 28 fecal samples tested (Table). None of the anal region or oral swab specimens were positive for coronavirus RNA.

Despite the small number of bats sampled, there was a high prevalence of coronavirus RNA shedding in fecal samples of 2 species of bats. Five (50%) of 10 fecal samples from occult myotis and 1 (17%) of 6 fecal samples from big brown bats were positive for coronavirus in screening tests. The 1 coronavirus-positive sample from big brown bat (bat sample 65) was from feces of 1 (33%) of 3 big brown bats sampled at site 2 in north-central Colorado, whereas the positive samples from the occult myotis (bat samples 3, 6, 11, 27, and 48) were from sites 1 and 4 in southwestern Colorado, ≈480 km from site 2 (Table). Most of the fecal samples were only positive in the PCRs with cDNA diluted 1:10, which suggested that PCR inhibitors were present in feces. In addition, most of the samples were positive only in the nested PCRs, which indicated that either the RNA was present in small amounts or that the primers used were not an optimal match for these viruses.

Phylogenetic Analysis of RM-Bt-CoVs

Figure 1. Nucleotide sequence alignment of amplicons from a 440-nt region of gene 1b of Rocky Mountain bat coronaviruses (RM-Bt-CoVs) compared with group 1 coronaviruses of Asian bats (BtCoVs) and human coronavirus 229E.

Figure 2. Phylogenetic relationships based on a 440-nt sequence in a conserved region of gene 1b of Rocky Mountain bat coronaviruses (RM-Bt-CoVs) (shown in boldface), group 1 coronaviruses of Asian bats (BtCoVs), and.

A 440-nt sequence in the RNA-dependent RNA polymerase region of gene 1b was amplified by RT-PCR from the 6 positive samples. Analysis of nucleotide sequences of these amplicons showed that all 6 RM-Bt-CoVs are members of coronavirus group 1 (Figure 1). Although these sequences were similar to those published for Asian bat group 1 coronaviruses, there was enough dissimilarity in this highly conserved region to suggest that the Rocky Mountain specimens represent unique coronaviruses (8,9). Phylogenetic analysis of this region of gene 1b suggests that the RM-Bt-CoVs cluster in 3 subgroups within group 1. Three of the 5 specimens from the occult myotis (samples 6, 11, and 48) were in 1 cluster and the other 2 (samples 3 and 27) formed a second cluster within group 1 coronaviruses. The 1 specimen from the big brown bat (sample 65) was a more distantly related group 1 coronavirus (Figure 2).

Discussion

To our knowledge, this is the first report of coronaviruses in bats in the Western Hemisphere. With >1,100 species, bats are among the most divergent and widely distributed nonhuman mammals (17). Bats are reservoirs for rabies virus and other lyssaviruses and were recently shown to be reservoirs for other important emerging viruses. Old World fruit bats (family Pteropodidae) are reservoirs for Hendra virus, which caused small outbreaks of severe respiratory illnesses in horses and humans in Australia (1824) and Nipah virus, which caused large outbreaks of lethal encephalitis and respiratory illnesses in humans and pigs in Malaysia and Singapore (2528). Old World fruit bats may also be the long-sought reservoir hosts for Ebola and Marburg viruses (29,30). More than 60 different RNA viruses have been isolated from and detected in bats, which play important roles in maintaining and transmitting zoonotic viruses (3133).

The need for understanding the ecology and evolution of coronaviruses in wildlife was highlighted by the observation that SARS-CoVs that caused 4 sporadic human cases of SARS in 2003–2004 were more closely related to viruses from palm civets found in 2004 than to the human epidemic strain of SARS-CoV (34). The gene encoding the viral spike glycoprotein that binds the virus receptor human angiotensin-converting enzyme 2 was one of the fastest-adapting genes of SARS-CoV during the 2002–2003 epidemic. Nonsynonymous amino acid substitutions in the spike protein that were selected during the epidemic optimized binding of the spike to its human receptor and enhanced human-to-human transmission (34,35). Sequencing of SARS-CoV genomes during and after the epidemic suggests that multiple independent species-jumping events of SARS-CoV from animals to humans have occurred.

Although all samples we tested were from apparently healthy wild bats, a high prevalence of coronavirus RNA was detected in 2 of the 7 species of bats tested. Five (50%) of 10 occult myotis and 1 (17%) of 6 big brown bats tested contained low levels of coronavirus RNA in feces. No coronavirus RNA was detected in the oral or anal region swabs tested. Similarly in Asian bats, coronavirus RNA was found in a higher percentage of fecal samples than saliva samples (8,9,14). Thus, bats may be persistently infected carriers that shed low levels of coronaviruses in feces. Persistent fecal shedding of coronaviruses has also been detected in pigs, cats, dogs, and cattle (36). The mechanisms for persistent fecal shedding of viruses in bats without apparent disease have not yet been determined (32,33).

No bat of any species occurs in both the Eastern and Western Hemispheres (37). Therefore, it is of great interest that group 1 coronaviruses have now been found in bats in North America as well as in Asia. Comparison of the nucleotide sequences of related coronaviruses from different species of bats on different continents is likely to provide information about coronavirus evolution. Figure 2 shows the phylogeny of RM-Bt-CoVs in relation to group 1 coronaviruses from Asia on the basis of the 440-nt amplicon in gene 1b. Bats of the genera Myotis and Eptesicus are in the family Vespertilionidae, which has diversified into many different species in the Eastern and Western Hemispheres (17). Amplicons of 3 of the 5 coronaviruses (samples 6, 11, and 48) from occult myotis in Colorado have the highest nucleotide sequence identity with the HKU6 bat coronavirus found in an Asian bat of the same genus but a different species, Rickett’s big-footed myotis (M. ricketti, subfamily Myotinae) (11,17). The coronavirus RNA in the big brown bat (sample 65) from Colorado (subfamily Vespertilioninae) was most similar to HKU2 bat coronavirus found in Asian bats in the family Rhinolophidae (11) (Figure 2). Rhinolophid bats are not found in the Western Hemisphere and are phylogenetically far removed from the big brown bat (37,38).

In our small, initial study of coronaviruses in North American bats, samples were restricted in size, location, and variety of bat species, and we found only group 1 coronaviruses. When larger numbers of bats and additional bat species in North America are studied, additional bat coronaviruses with complex phylogenetic attributes, biogeographic patterns, and perhaps epizootiologic attributes may be discovered. For example, determining if North American bat coronaviruses are species-specific will provide useful information. In Asia, different species of bats roosting in the same cave host different coronaviruses (9). However, bats of 1 species can also harbor different types of coronaviruses at different geographic locations (9).

A recent analysis of genome sequences of coronaviruses of bats, other animals, humans, and birds suggested that bats may be the original hosts from which all coronavirus lineages were derived (15). We find this hypothesis intriguing, in light of the high prevalence and diversity of coronaviruses in bats in North America found in our initial small survey. The North American species of bats found to harbor group 1 coronaviruses commonly roost in buildings inhabited by humans (39), which provides ecologic overlap between these bats and humans. Before the SARS epidemic of 2002–2003, only 2 coronaviruses, HCoV-229E and HCoV-OC43, were known to cause human disease, primarily mild upper respiratory tract infections. In contrast, SARS-CoV caused severe lower respiratory tract disease with a death rate of 10%. Recently, 2 additional human coronaviruses, HCoV-NL63 and HCoV-HKU1, were discovered and found to cause both upper and lower respiratory tract infections worldwide (40).

It is possible that another epidemic caused by an emerging coronavirus could occur in the future. As in the SARS epidemic, bats could play a role in future emergence of coronaviruses in humans or other species. Isolation of infectious bat coronaviruses and elucidation of their host ranges, receptor specificities, and genetic diversity will greatly aid in our understanding of their potential for emergence.

Dr Dominguez is a pediatric infectious disease fellow at The Children’s Hospital, Denver, Colorado, an affiliate of the University of Colorado Health Science Center. His research interests include emerging infectious diseases, Kawasaki syndrome, and pediatric respiratory viral infections.

Acknowledgments

We thank R. Pearce, L. Ellison, and E. Valdez for field assistance in capturing bats C. Calisher, P. Cryan, and S. Jeffers for critical evaluation of the manuscript E. Travanty for assistance in phylogenetic analysis and C. Calisher for providing field sampling supplies and advice.

This study was supported by National Institutes of Health grant AI-P01-059576 and a Pediatric Infectious Disease Society Fellowship Award from Roche Laboratories to S.R.D.

References

Figures
Table

1 These authors contributed equally to this article.

Please use the form below to submit correspondence to the authors or contact them at the following address:

Kathryn V. Holmes, Department of Microbiology, University of Colorado Health Sciences Center, MS 8333, 12800 E 19th Ave, Room P18-9117, PO Box 6511, Aurora, CO 80045, USA

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Study predicts where new coronaviruses might originate

Credit: University of Liverpool

The potential scale of novel coronavirus generation in wild and domesticated animals may have been highly underappreciated, suggests new University of Liverpool research.

Published in Nature Communications, the machine-learning study identifies mammals that are potential sources for generating new coronaviruses, including species implicated in previous outbreaks (such as horseshoe bats, palm civets and pangolins) and some novel candidates.

Predicting which animals could potentially be the source of a future coronavirus outbreak may guide approaches to reduce the risk of coronavirus emergence in animals and spill-over to human populations.

"New coronaviruses can emerge when two different strains co-infect an animal, causing the viral genetic material to recombine. Our understanding of how susceptible different mammals are to different coronaviruses has been limited, but such information could offer insights into where viral recombination might occur," explained co-lead researcher Dr. Maya Wardeh from the Institute of Infection, Veterinary and Ecological Sciences.

The researchers sought to bridge this knowledge gap by using a machine-learning approach to predict relationships between 411 strains of coronavirus and 876 potential mammalian host species. They predict the mammals that are most likely to be co-infected, and therefore be potential recombination hosts for the production of novel coronaviruses.

Their findings suggest that there are at least 11 times more associations between mammalian species and coronavirus strains than have been observed to date. In addition, they estimate that there are over 40 times more mammal species that can be infected with a diverse set of coronavirus strains than was previously known.

"Given that coronaviruses frequently undergo recombination when they co-infect a host, and that SARS-CoV-2 is highly infectious to humans, the most immediate threat to public health is recombination of other coronaviruses with SARS-CoV-2," said Dr. Marcus Blagrove, co-lead of the study.

The researchers went on to identify hosts in which SARS-CoV-2 recombination could potentially occur and indicate there may be 30 times more host species than currently known. Notable new predicted hosts include the dormitory camel, African green monkey and the lesser Asiatic yellow bat.

Highlighting, as a specific example, the high-risk scenario of recombination occurring between the highly transmissible SARS-CoV-2 and the more deadly MERS-CoV, the researchers also identify 102 potential recombination hosts of the two viruses and recommend monitoring for this event.

The researchers note that their results draw on limited data on coronavirus genomes and virus-host associations, and that there are study biases for certain animal species, all of which present uncertainty in the predictions. However, recent testing of potential mammalian hosts for their susceptibility to SARS-CoV-2 has already confirmed a number of their predictions, such as the raccoon dog, the domestic goat and the alpaca.

"It is important to note that viral recombination is distinct from mutations. Recombination occurs over longer periods of time and can generate completely new strains or species. Our work can help target surveillance programs to discover future strains before they spill-over to humans, giving us a head-start in combating them," concluded Dr. Blagrove.

The researchers now plan to expand their model to include bird species, therefore, encompassing the full range of important coronavirus hosts, and a species-level contact network, accounting for behavior and habitat utilization of host species, to give a broader overview of potential coronavirus associations.


MODELING COVID-19 IN GENETICALLY ALTERED ANIMAL MODELS

Several approaches have been employed to increase susceptibility to SARS-CoV-2 infection in laboratory mice. These include genetic alteration of the virus so that it is able to bind mouse Ace2, expression of human ACE2 under a variety of promoters, and transfection of mice with human ACE2 cDNA using viral vectors. Murine models for SARS-CoV-2 are summarized in Table 3 .

Table 3

Clinical Signs, Pulmonary and Extrapulmonary Pathology, and Viral Distribution in Mouse Models of COVID-19

Viral Replication and Tissue TropismClinical SignsPulmonary PathologyExtrapulmonary PathologyImmunohistochemical Localization of SARS-CoV-2
Mouse-adapted SARS-CoV-2 23
Viral RNA: lung, nasal turbinate, trachea, feces, heart, and liver up to 7 DPI
Infectious virus: nasal turbinate, lung up to 2 DPI (young), 4 DPI (aged)
Age enhanced disease decrease in body weight by 3 dpi in aged mice onlyNot reportedFocal exudation and hemorrhage, interstitial pneumoniaLung: CC10 (club cell) bronchus and bronchioles, alveolar type 1 cells
Chimeric SARS-Co-V/SARS-CoV-2 82
Viral RNA: LungRemdesivir ameliorates loss of pulmonary function (whole-body plethysmography)Lung hemorrhage at 5 dpi reduced by remdesivir treatmentNoNot examined
Murine mAce2 exon 2-hACE2 knockin mouse81
Viral RNA: Predominantly in trachea, lung, brainMarked weight loss in older animalsInterstitial pneumoniaVascular system injuryLung: CC10+ Clara cells in airways, surfactant protein C positive (SPC+) alveolar type II cells
Murine mAce2 promoter-hACE2 transgenic mouse82
Viral RNA: Virus is shed from lung for 1 wk post infection and briefly from intestine 1𠄷 dpiTransient weight loss and recovery by 7 dpiModest interstitial pneumonia, interstitial and intra-alveolar mononuclear and granulocytic inflammationVasculitis in extrapulmonary organsLung: macrophages and T lymphocytes
HFH4/FOXJ1 promoter-hACE2 transgenic mouse83 , 84
Viral RNA: Lung, eyes, heart, brainBinary clinical phenotype: weight loss, respiratory distress, and neurological symptoms, die by 6 dpi others asymptomatic and surviveMild to severe interstitial inflammation with hyaline membrane formationCardiomyocyte edema and sporadic neuroinvasion in brains of deceased miceLung: bronchial epithelial cells and alveolar cells
K18 promoter-hACE2 transgenic mouse85�
Viral RNA: expressed at highest levels in lung and brain, evident in colon and serum in a subset of animals by 7 dpiSignificant weight loss by 4 dpi, some animals becoming moribund by 7𠄸 dpiEdema, alveolar, interstitial, and perivascular infiltration of neutrophils and mononuclear cells, and consolidationEncephalitis, vasculitis, and meningitis after 5 dpiLung: alveolar epithelial cells, macrophages, nasal epithelium Eye: inner nuclear layer Brain: olfactory bulb, extensive in neurons
Adenovirus 5- hACE2 transfected mouse57 , 78
Viral RNA: lung, low levels in spleen, heart, brainTransient weight loss in older BALB/c micePerivascular to interstitial inflammation, necrotic debris, alveolar edema and vascular congestion and hemorrhage, most severe at 5 dpiNot describedLung: alveolar and bronchiolar epithelial cells
Adeno-associated virus (AAV9)-hACE2 mouse93
Viral RNA: limited to respiratory tractNo weight loss or clinical illnessInterstitial pneumoniaNot describedLung: alveolar epithelial cells

Wild-Type and Immune-Compromised Mice

Wild-type mice do not support significant SARS-CoV-2 infection due to insufficient interaction between viral S protein and murine ACE2.86 This finding in consistent across several inbred strains, including C57BL/6 J,64 , 87 , 88 BALB/c, DBA/2J,87 and ICR88 mice. Viral binding to murine ACE2 is not entirely absent, however, and very limited viral replication is evident in lung in Stat1−/− mice,87  Rag1 −/− mice lacking mature B and T cells,87 SCID mice, Il28r−/− mice, Ifnar1−/− mice,77 and AG129 (type I and II interferon receptor-deficient mice87).

Mouse-Adapted SARS-CoV-2

Dinnon etਊl23 engineered Q498T/P499Y into the SARS-CoV-2 S gene to generate a recombinant virus (SARS-CoV-2 MA) that could utilize mouse ACE2 for entry. SARS-CoV-2 MA replicates in upper and, to a lesser extent, lower airways of both young adult and aged BALB/c mice. Compared with young mice, SARS-CoV-2 MA–infected old mice develop more severe interstitial pneumonia with greater impairment of pulmonary function in life.

Chimeric SARS-CoV/SARS-CoV-2 Viruses

A chimeric virus composed of mouse-adapted SARS-CoV (permitting infection via mACE2 binding) and SARS-CoV-2 RNA-dependent RNA polymerase allows testing of antiviral efficacy of remdesivir.89 Incorporation of active metabolite remdesivir triphosphate occurs preferentially to natural substrate ATP and results in chain termination 3 nucleotides downstream of incorporation. Mice produce a serum esterase, carboxyl esterase 1c (Ces1c), that is absent in humans and reduces half-life of remdesivir. Therefore mouse studies with remdesivir must be performed in transgenic C57Bl/6 Ces1c−/− mice. In this model,89 histopathology is not shown however, significant reduction of grossly observable lung hemorrhage at 5 dpi with remdesivir treatment is reported despite similar weight loss in vehicle and remdesivir-treated groups.

Murine mAce2 Exon 2-hACE2 Knockin Mouse

Using CRISPR-Cas9 technology, Sun etਊl 52 inserted hACE2 cDNA into Exon 2 of the mAce2 gene to disrupt mAce2 gene expression and drive expression of hACE2 under control of the mAce2 promoter. hACE2 expression occurs in lung, small intestine, spleen, and kidney. In lung, hACE2 is expressed in the CC10+ Clara cells in airways as well as a subpopulation of surfactant protein C positive alveolar type II cells. High viral loads of SARS-CoV-2 are evident in lung, trachea, and brain (despite low hACE2 expression) on intranasal infection. Young inoculated animals do not display obvious clinical symptoms but develop interstitial pneumonia and vascular system injury. More severe disease is seen in aged hACE2. These exhibit more marked weight loss, more prolonged viral shedding (including from feces), and more severe pneumonia accompanied by stronger cytokine responses. Intragastric inoculation of SARS-CoV-2 induces productive infection and pulmonary disease.

HACE2 Transgenic Mice

Mice transgenic for hACE2 were developed over a decade ago to study SARS-CoV and to allow creation of significantly greater disease than that induced in wild-type mice. The same models also permit infection with SARS-CoV-2, and their effects differ by the promoter driving expression of hACE2.

Murine mAce2 Promoter-hACE2 Transgenic Mouse

Originally described by Yang etਊl,90 SARS-CoV–infected mice expressing hACE2 in lung, heart, kidney, and gut permit greater viral replication and develop more severe pulmonary lesions than wild-type mice. Vasculitis in extrapulmonary organs and viral antigen in brain is noted, but encephalitis does not occur. ICR-hACE2 transgenic mice inoculated with SARS-CoV-288 experience transient weight loss and recovery by 7 dpi. Virus is shed from lung for 1 week post infection and briefly from intestine 1𠄷 dpi. Relatively mild interstitial pneumonia develops and is characterized by interstitial and intra-alveolar mononuclear and granulocytic inflammation. SARS-CoV-2 is evident immunohistochemically in macrophages and T lymphocytes.

HFH4/FOXJ1 Promoter-hACE2 Transgenic Mice

Originally developed in 2016,91 lung ciliated epithelial cell-specific HFH4/FOXJ1 promoter drives hACE2 expression at high levels of hACE2 in the lung and to varying degrees in brain, liver, kidney, and gastrointestinal tract. SARS-CoV-2–inoculated mice92 experience binary clinical phenotypes, in which some lose weight, display respiratory distress and neurological symptoms, and die by 6 dpi, whereas others survive without evidence of clinical distress. Interestingly, the former are male, possibly recapitulating male susceptibility to COVID-19 mortality. A spectrum of pulmonary pathology ranging from mild to severe interstitial inflammation with hyaline membrane formation is evident. Cardiomyocyte edema and sporadic neuroinvasion in brains of deceased mice is evident. Dying mice experience lymphopenia, 1 of the key clinical hallmarks of severe COVID-19 disease.

K18 Promoter-hACE2 Transgenic Mice

Mice expressing hACE2 under control of the K18 promoter were developed to study SARS-CoV.93 , 94 When infected with SARS-CoV-2,95 , 96 pulmonary pathology is among the most severe of that described in murine models96 , 97 and is accompanied by profound perivascular mononuclear inflammation with minimal viral endothelial infection.97 Significant lethality and male bias in disease severity are noted.97 In hemizygous K18-hACE2 mice,96 expression is highest in lung, followed by heart, brain, colon, and kidney with lower levels in duodenum, ileum, and spleen. Pulmonary levels of hACE2 decline over the disease course.96 K18-hACE2 mice given intranasal SARS-CoV-2 exhibit significant weight loss by 4 dpi,95 , 96 with many animals becoming moribund by 7 dpi.96 , 97 Viral RNA is expressed at highest levels in lung and is evident in colon and serum only in a subset of animals by 7 dpi. Pulmonary histopathology is severe and characterized by consolidation, edema, alveolar, and interstitial and perivascular infiltration of neutrophils and mononuclear cells. Viral RNA is evident by in situ hybridization in pulmonary alveolar epithelial cells and macrophages97 as well as nasal epithelia. K18-hACE2 TG mice experience significant central nervous system infection following infection with both SARS-CoV93 , 94 and SARS-Cov-2.53 , 54 , 97 Similar cytokine (lung) and chemokine (brain) elevations are described in SARS-CoV-2–infected K18 mice.54 Individual mice express viral RNA in the retina97 and olfactory bulb and throughout the brain,53 , 97accompanied by encephalitis, vasculitis, and meningitis by 5਍pi.96� By in situ hybridization, viral RNA is present in NeuN + cells, implicating neuronal infection in mice97 and human COVID-19 patients.53 Rare thromboses are noted in murine brain.97 Viral presence in murine endothelial cells within brain is lacking53 however, vascular remodeling is noted in regions of neuronal viral infection. In contrast, endothelial invasion by virus is evident in COVID-19 patients experiencing ischemic and hemorrhagic infarcts.53 Neurologic lesions in SARS-Cov-2–infected mice are consistent with original studies using SARS-Co-V in this model in which extrapulmonary virus spreads to the brain via the olfactory tract and killed all mice by 7 dpi.93 , 94 Medullary infection is associated with aspiration pneumonia that complicates interpretation of pulmonary pathology. Both lung and brain express increased inflammatory cytokines (chemokine (C-X-C motif) ligand 1 (CXCL-1), chemokine (C-X-C motif) ligand 10 (CXCL-10), Interleukin-6 (IL-6), and Interleukin-61 beta (IL-1beta)). This is accompanied by significant inflammation in the lung but neuronal death without inflammation in the brain.94 Interestingly, SARS-CoV-2–infected regions of human brain similarly lack inflammation.53

Viral Transduction of hACE2 Using Adenovirus 5

Adenovirally transduced hACE2 BALB/c and C57BL/6 mice infected with SARS-CoV-264 experience acute but transient disease duration of 7�ꃚys. Pulmonary lesions include perivascular to interstitial inflammation, necrotic debris, alveolar edema, and vascular congestion and hemorrhage, most severe at 5 dpi. Upregulation of genes encoding inflammatory mediators and components of adaptive and innate responses occurs by 2 dpi and is followed by appearance of virus-neutralizing antibodies and CD4 and CD8 responses to N and S viral proteins, respectively, by 10 dpi. Viral clearance is impaired and lung phenotype worsened by CD4/CD8 depletion or by impaired interferon signaling conferred by Stat1−/− genotype. Conversely, viral clearance is accelerated, and phenotype improved by vaccination, type I IFN induction using poly I:C, passive transfer of antibodies from recovered mice, or transfer of serum from COVID-19 patients. In a similar model, lung pathology in AdV-hACE2–transduced, SARS-CoV-2–infected mice can be worsened given anti-Ifnar1 mAb or improved by pretreatment with an anti-SARS-CoV-2 mAb.87

Adeno-associated Virus-hACE2 Mice

Mice transduced with adeno-associated virus-hACE2 and subsequently infected by SARS-CoV-2 experience no weight loss or clinical illness.99 Interstitial pneumonia is accompanied by upregulation of cytokines and interferon stimulated genes and infiltration by monocyte-derived macrophages and activated lymphoid cells. Neutralizing antibodies appear by 7 dpi.


Spectre of Sars coronavirus

The Wuhan coronavirus outbreak bears similarity to the 2002-03 epidemic of Sars (severe acute respiratory syndrome) coronavirus (Sars-CoV). The Sars-CoV outbreak, which started in south China, lasted for over nine months. It spread to 37 countries, causing 8,098 people to become ill and 774 to die.

Nearly 10% of those confirmed to be infected went on to die. The deadly nature of the disease, the frequent human-to-human spread, and infection of front-line clinical staff, contributed to the seriousness of the outbreak.

Sars-CoV was traced to several animals, including civet cats and raccoon dogs, being sold as food in markets. The infected animals had no symptoms. Closure of the markets with animal culling alongside treatment and containment of patients led to the outbreak being halted.

Raccoon dogs might have spread Sars. Stanislav Duben/Shutterstock

Further investigations traced Sars-like viruses to horseshoe bats found in a cave in China. It is thought that civet cats could have picked up the infection from bats and then spread it to humans in city markets.

Sars has not been seen since 2003 and it is thought that the virus is now extinct. The new Wuhan coronavirus is not the Sars-CoV, but it is similar to viruses thought to be precursors of Sars in bats.


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Animal origins of HCoVs

All four community-acquired HCoVs causing mild symptoms have been well adapted to humans. From another perspective, it might also be true that humans have been well adapted to these four HCoVs. In other words, both could be the survivors of ancient HCoV pandemics. HCoVs that cause severe diseases in humans and humans who developed severe HCoV diseases have been eliminated. For this to happen, HCoVs have to replicate in humans to sufficient extent to allow the accumulation of adaptive mutations that counteract host restriction factors. In this sense, the longer the SARS-CoV-2 outbreak persists and the more people that it infects, the greater chance that it will fully adapt to humans. If it adapts well, its transmission in humans would be difficult to stop by quarantine or other infection control measures.

For many years, the four community-acquired CoVs circulate in human populations, triggering common cold in immunocompetent subjects. These viruses do not need an animal reservoir. In contrast, highly pathogenic SARS-CoV and MERS-CoV have not adapted to humans well and their transmission within humans cannot be sustained. They need to maintain and propagate in their zoonotic reservoirs and seek the chance to spillover to susceptible human targets, possibly via one or more intermediate and amplifying hosts. SARS-CoV-2 has features that are similar to both SARS-CoV/MERS-CoV and the four community-acquired HCoVs. It is highly transmissible like community-acquired HCoVs, at least for the time being. However, it is more pathogenic than community-acquired HCoVs and less pathogenic than SARS-CoV or MERS-CoV. It remains to be seen whether it will adapt fully to humans and circulate within humans without a reservoir or intermediate animal host.

Before discussing the animal origins of HCoVs, it will serve us well to discuss the definitions and characteristics of evolutionary, natural, reservoir, intermediate and amplifying hosts of HCoVs. An animal serves as the evolutionary host of an HCoV if it harbours a closely related ancestor sharing high homology at the level of nucleotide sequence. The ancestral virus is usually well adapted and non-pathogenic in this host. Likewise, a reservoir host harbours HCoV continuously and for long term. In both cases, the hosts are naturally infected and are the natural hosts of HCoV or its parental virus. In contrast, if the HCoV is newly introduced to an intermediate host right before or around its introduction to humans, it is not well adapted to the new host and is often pathogenic. This intermediate host can serve as the zoonotic source of human infection and play the role of an amplifying host by allowing the virus to replicate transiently and then transmitting it to humans to amplify the scale of human infection. An HCoV can undergo a dead-end infection if it cannot sustain its transmission within the intermediate host. On the contrary, HCoVs can also adapt to the intermediate host and even establish long-term endemicity. In this case, the intermediate host becomes a natural reservoir host.


We thank the Genome Technology Center (RGTC) at Radboudumc for the use of the Sequencing Core Facility (Nijmegen, The Netherlands), which provided the PacBio SMRT sequencing service on the Sequel II platform. We also thank Damian Baranski for help with the DNA isolation and library preparations, and Norbert Peter and Dorian D. Dörge for providing samples.

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Keywords: genome assembly and annotation, SARS-CoV-2, Carnivora, raccoon dog (Nyctereutes procyonoides), B chromosome

Citation: Chueca LJ, Kochmann J, Schell T, Greve C, Janke A, Pfenninger M and Klimpel S (2021) De novo Genome Assembly of the Raccoon Dog (Nyctereutes procyonoides). Front. Genet. 12:658256. doi: 10.3389/fgene.2021.658256

Received: 25 January 2021 Accepted: 24 March 2021
Published: 29 April 2021.

Gabriele Bucci, San Raffaele Hospital (IRCCS), Italy

Andrea Spitaleri, San Raffaele Hospital (IRCCS), Italy
Shilpa Garg, Harvard Medical School, United States

Copyright © 2021 Chueca, Kochmann, Schell, Greve, Janke, Pfenninger and Klimpel. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.


Watch the video: Noisy Raccoon Dogs (May 2022).


Comments:

  1. Tearlach

    It doesn't come close to me. Can the variants still exist?

  2. Raidon

    not very impressive

  3. Kagaran

    Thank you very much.

  4. Kara

    Good article :) Just haven't found a link to the RSS blog?



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