Detection and characterization of novel luchacoviruses, genus Alphacoronavirus, in saliva and feces of meso-carnivores in the northeastern United States.

IMPORTANCE: Several coronaviruses (CoVs) have been detected in domesticated, farmed, and wild meso-carnivores, causing a wide range of diseases and infecting diverse species, highlighting their important but understudied role in the epidemiology of these viruses. Assessing the viral diversity hosted in wildlife species is essential to understand their significance in the cross-species transmission of CoVs. Our focus here was on CoV discovery in meso-carnivores in the Northeast United States as a potential "hotspot" area with high density of humans and urban wildlife. This study identifies novel alphacoronaviruses circulating in multiple free-ranging wild and domestic species in this area and explores their potential epidemiological importance based on regions of the Spike gene, which are relevant for virus-host interactions.

GENETIC CHARACTERISTICS OF CANINE DISTEMPER VIRUSES CIRCULATING IN WILDLIFE IN THE UNITED STATES.

Canine distemper virus (CDV) is a highly contagious disease of wild and domestic mammals. Maintenance of CDV among wildlife plays an important role in the disease epidemiology. Wild animals, including raccoons (Procyon lotor) and gray foxes (Urocyon cinereoargenteus), serve as reservoirs of CDV and hamper the control of the disease. Recently, we discovered that at least three different CDV lineages (America-3 [Edomex], America-4, and America-5] that are genetically different from the available vaccine strains are circulating in domestic dogs in the United States. Because wildlife serve as a reservoir for the virus, it is important to determine if wildlife play a role in the maintenance and spread of these lineages. To determine the genetic characteristics of circulating strains of CDV in wildlife in various geographic regions in the United States, we studied the nucleotide sequences of the hemagglutinin (H) gene of 25 CDV strains detected in nondomestic species. The species included were free-ranging wildlife: three fishers (Martes pennanti), six foxes, one skunk (Mephitis mephitis), 10 raccoons, two wolves (Canis lupus), and one mink (Neovison vison). Strains from two species in managed care, one sloth (Choloepus didactylus) and one red panda (Ailurus fulgens), were also evaluated. Phylogenetic analysis of the H genes indicated that in addition to America-3, America-4, and America-5 lineages, there are at least two other lineages circulating in US wildlife. One of these includes CDV nucleotide sequences that grouped with that of a single CDV isolate previously detected in a raccoon from Rhode Island in 2012. The other lineage is independent and genetically distinct from other CDV strains included in the analysis. Additional genetically variable strains were detected, mainly in raccoons, suggesting that this species may be the host responsible for the genetic variability of newly detected strains in the domestic dog population.

Molecular detection of canine bufaviruses in wild canids.

Novel protoparvoviruses genetically related to human and non-human primate bufaviruses (BuVs) have been detected recently in respiratory and enteric specimens collected from dogs and cats. In this study, by molecular screening of archival collections of faecal samples from wolves and foxes, we detected BuVs with a rate of 17.1% (7/41) and 10.5% (9/86), respectively. Sequence analysis of a portion of the ORF2 gene region of nine positive samples showed that the viruses in these samples were closely related to BuVs (97.5-99.0% nucleotide sequence identity) found in domestic carnivores.

First molecular identification of kobuviruses in wolves (Canis lupus) in Italy.

Canine kobuviruses (CaKoVs) were first identified in diarrhoeic and asymptomatic dogs in 2011 in the USA. Subsequent studies have demonstrated a worldwide distribution of these viruses, but it is not clear if CaKoVs play a role as enteric pathogens of dogs. More recently, CaKoV RNA has been detected in wild carnivores, including red fox, golden jackal, side-striped jackal and spotted hyena. In this study, we addressed the hypothesis that wolves are susceptible to CaKoV infections. A total of 185 wolf stool samples were collected from necropsied animals and from transects in the Liguria, Piemonte and Valle D'Aosta regions of Italy, and CaKoV RNA was identified in two of these specimens. Both samples were obtained from necropsied wolves, with a prevalence rate of 4.9% (2/41). Sequence analysis of the full-length VP1 region showed that these strains displayed the highest nucleotide (nt) sequence identity (86.3-98.5%) to canine strains identified in the UK and Africa, and to kobuviruses that were previously detected in other African wild carnivores. This suggests that genetically related CaKoV strains circulate in domestic and wild carnivores, with interspecies transmission being not uncommon among carnivores of different ecosystems.

A rabies virus vampire bat variant shows increased neuroinvasiveness in mice when compared to a carnivore variant.

Rabies is one of the most important zoonotic diseases and is caused by several rabies virus (RABV) variants. These variants can exhibit differences in neurovirulence, and few studies have attempted to evaluate the neuroinvasiveness of variants derived from vampire bats and wild carnivores. The aim of this study was to evaluate the neuropathogenesis of infection with two Brazilian RABV street variants (variant 3 and crab-eating fox) in mice. BALB/c mice were inoculated with RABV through the footpad, with the 50% mouse lethal dose (LD50) determined by intracranial inoculation. The morbidity of rabies in mice infected with variant 3 and the crab-eating fox strain was 100% and 50%, respectively, with an incubation period of 7 and 6 days post-inoculation (dpi), respectively. The clinical disease in mice was similar with both strains, and it was characterized initially by weight loss, ruffled fur, hunched posture, and hind limb paralysis progressing to quadriplegia and recumbency at 9 to 12 dpi. Histological lesions within the central nervous system (CNS) characterized by nonsuppurative encephalomyelitis with neuronal degeneration and necrosis were observed in mice infected with variant 3 and those infected with the crab-eating fox variant. However, lesions and the presence of RABV antigen, were more widespread within the CNS of variant-3-infected mice, whereas in crab-eating fox-variant-infected mice, RABV antigens were more restricted to caudal areas of the CNS, such as the spinal cord and brainstem. In conclusion, the results shown here demonstrate that the RABV vampire bat strain (variant 3) has a higher potential for neuroinvasiveness than the carnivore variant.

Disease outbreaks caused by steppe-type rabies viruses in China.

While rabies is a significant public health concern in China, the epidemiology of animal rabies in the north and northwest border provinces remains unknown. From February 2013 to March 2014, seven outbreaks of domestic animal rabies caused by wild carnivores in Xinjiang (XJ) and Inner Mongolia (IM) Autonomous Regions, China were reported and diagnosed in brain samples of infected animals by the fluorescent antibody test (FAT) and RT-PCR. Ten field rabies viruses were obtained. Sequence comparison and phylogenetic analysis based on the complete N gene (1353 bp) amplified directly from the original brain tissues showed that these ten strains were steppe-type viruses, closely related to strains reported in Russia and Mongolia. None had been identified previously in China. The viruses from XJ and IM clustered separately into two lineages showing their different geographical distribution. This study emphasizes the importance of wildlife surveillance and of cross-departmental cooperation in the control of transboundary rabies transmission.

Frequent cross-species transmission of parvoviruses among diverse carnivore hosts.

Although parvoviruses are commonly described in domestic carnivores, little is known about their biodiversity in nondomestic species. A phylogenetic analysis of VP2 gene sequences from puma, coyote, gray wolf, bobcat, raccoon, and striped skunk revealed two major groups related to either feline panleukopenia virus ("FPV-like") or canine parvovirus ("CPV-like"). Cross-species transmission was commonplace, with multiple introductions into each host species but, with the exception of raccoons, relatively little evidence for onward transmission in nondomestic species.

Molecular inferences suggest multiple host shifts of rabies viruses from bats to mesocarnivores in Arizona during 2001-2009.

In nature, rabies virus (RABV; genus Lyssavirus, family Rhabdoviridae) represents an assemblage of phylogenetic lineages, associated with specific mammalian host species. Although it is generally accepted that RABV evolved originally in bats and further shifted to carnivores, mechanisms of such host shifts are poorly understood, and examples are rarely present in surveillance data. Outbreaks in carnivores caused by a RABV variant, associated with big brown bats, occurred repeatedly during 2001-2009 in the Flagstaff area of Arizona. After each outbreak, extensive control campaigns were undertaken, with no reports of further rabies cases in carnivores for the next several years. However, questions remained whether all outbreaks were caused by a single introduction and further perpetuation of bat RABV in carnivore populations, or each outbreak was caused by an independent introduction of a bat virus. Another question of concern was related to adaptive changes in the RABV genome associated with host shifts. To address these questions, we sequenced and analyzed 66 complete and 20 nearly complete RABV genomes, including those from the Flagstaff area and other similar outbreaks in carnivores, caused by bat RABVs, and representatives of the major RABV lineages circulating in North America and worldwide. Phylogenetic analysis demonstrated that each Flagstaff outbreak was caused by an independent introduction of bat RABV into populations of carnivores. Positive selection analysis confirmed the absence of post-shift changes in RABV genes. In contrast, convergent evolution analysis demonstrated several amino acids in the N, P, G and L proteins, which might be significant for pre-adaptation of bat viruses to cause effective infection in carnivores. The substitution S/T242 in the viral glycoprotein is of particular merit, as a similar substitution was suggested for pathogenicity of Nishigahara RABV strain. Roles of the amino acid changes, detected in our study, require additional investigations, using reverse genetics and other approaches.

Viral metagenomic analysis of feces of wild small carnivores.

BACKGROUND: Recent studies have clearly demonstrated the enormous virus diversity that exists among wild animals. This exemplifies the required expansion of our knowledge of the virus diversity present in wildlife, as well as the potential transmission of these viruses to domestic animals or humans. METHODS: In the present study we evaluated the viral diversity of fecal samples (n = 42) collected from 10 different species of wild small carnivores inhabiting the northern part of Spain using random PCR in combination with next-generation sequencing. Samples were collected from American mink (Neovison vison), European mink (Mustela lutreola), European polecat (Mustela putorius), European pine marten (Martes martes), stone marten (Martes foina), Eurasian otter (Lutra lutra) and Eurasian badger (Meles meles) of the family of Mustelidae; common genet (Genetta genetta) of the family of Viverridae; red fox (Vulpes vulpes) of the family of Canidae and European wild cat (Felis silvestris) of the family of Felidae. RESULTS: A number of sequences of possible novel viruses or virus variants were detected, including a theilovirus, phleboviruses, an amdovirus, a kobuvirus and picobirnaviruses. CONCLUSIONS: Using random PCR in combination with next generation sequencing, sequences of various novel viruses or virus variants were detected in fecal samples collected from Spanish carnivores. Detected novel viruses highlight the viral diversity that is present in fecal material of wild carnivores.

Species loss on spatial patterns and composition of zoonotic parasites.

Species loss can result in the subsequent loss of affiliate species. Though largely ignored to date, these coextinctions can pose threats to human health by altering the composition, quantity and distribution of zoonotic parasites. We simulated host extinctions from more than 1300 host-parasite associations for 29 North American carnivores to investigate changes in parasite composition and species richness. We also explored the geography of zoonotic parasite richness under three carnivore composition scenarios and examined corresponding levels of human exposure. We found that changes in parasite assemblages differed among parasite groups. Because viruses tend to be generalists, the proportion of parasites that are viruses increased as more carnivores went extinct. Coextinction of carnivore parasites is unlikely to be common, given that few specialist parasites exploit hosts of conservation concern. However, local extirpations of widespread carnivore hosts can reduce overall zoonotic richness and shift distributions of parasite-rich areas. How biodiversity influences disease risks remains the subject of debate. Our results make clear that hosts vary in their contribution to human health risks. As a consequence, so too does the loss (or gain) of particular hosts. Anticipating changes in host composition in future environments may help inform parasite conservation and disease mitigation efforts.

Emergence of canine distemper virus strains with modified molecular signature and enhanced neuronal tropism leading to high mortality in wild carnivores.

An ongoing canine distemper epidemic was first detected in Switzerland in the spring of 2009. Compared to previous local canine distemper outbreaks, it was characterized by unusually high morbidity and mortality, rapid spread over the country, and susceptibility of several wild carnivore species. Here, the authors describe the associated pathologic changes and phylogenetic and biological features of a multiple highly virulent canine distemper virus (CDV) strain detected in and/or isolated from red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), stone (Martes foina) and pine (Martes martes) martens, from a Eurasian lynx (Lynx lynx), and a domestic dog. The main lesions included interstitial to bronchointerstitial pneumonia and meningopolioencephalitis, whereas demyelination--the classic presentation of CDV infection--was observed in few cases only. In the brain lesions, viral inclusions were mainly in the nuclei of the neurons. Some significant differences in brain and lung lesions were observed between foxes and mustelids. Swiss CDV isolates shared together with a Hungarian CDV strain detected in 2004. In vitro analysis of the hemagglutinin protein from one of the Swiss CDV strains revealed functional and structural differences from that of the reference strain A75/17, with the Swiss strain showing increased surface expression and binding efficiency to the signaling lymphocyte activation molecule (SLAM). These features might be part of a novel molecular signature, which might have contributed to an increase in virus pathogenicity, partially explaining the high morbidity and mortality, the rapid spread, and the large host spectrum observed in this outbreak.

Canine Distemper Outbreaks in Wild Carnivores in Northern Italy.

Canine distemper (CD) is a fatal, highly contagious disease of wild and domestic carnivores. In the Alpine territory, several outbreaks have occurred in the past few decades within wild populations. This study investigated the presence of canine distemper virus (CDV) infections in wild carnivores in Lombardy, relating to the different circulating genotypes. From 2018 to 2020, foxes, badgers, and martens collected during passive surveillance were subjected to necropsy and histological examination, showing classical signs and microscopic lesions related to CDV. Pools of viscera from each animal were analysed by molecular methods and immunoelectron microscopy. Total prevalences of 39.7%, 52.6%, and 14.3% were recorded in foxes, badgers, and stone martens, respectively. A phylogenetic analysis showed that the sequences obtained belonged to the European 1 lineage and were divided into two different clades (a and b) according to the geographical conformation of alpine valleys included in the study. Clade a was related to the European outbreaks originating from Germany in 2006-2010, while clade b was closely related to the CDV sequences originating from northeastern Italy during the 2011-2018 epidemic wave. Our results suggest that CDV is currently well adapted to wild carnivores, mostly circulating with subclinical manifestations and without severe impact on the dynamics of these populations.

Newlavirus, a Novel, Highly Prevalent, and Highly Diverse Protoparvovirus of Foxes (Vulpes spp.).

The genus Protoparvovirus (family Parvoviridae) includes several viruses of carnivores. We describe a novel fox protoparvovirus, which we named Newlavirus as it was discovered in samples from Newfoundland and Labrador, Canada. Analysis of the full non-structural protein (NS1) sequence indicates that this virus is a previously uncharacterized species. Newlavirus showed high prevalence in foxes from both the mainland (Labrador, 54/137, 39.4%) and the island of Newfoundland (22/50, 44%) but was not detected in samples from other carnivores, including coyotes (n = 92), lynx (n = 58), martens (n = 146), mink (n = 47), ermines (n = 17), dogs (n = 48), and ringed (n = 4), harp (n = 6), bearded (n = 6), and harbor (n = 2) seals. Newlavirus was found at similar rates in stool and spleen (24/80, 30% vs. 59/152, 38.8%, p = 0.2) but at lower rates in lymph nodes (2/37, 5.4%, p < 0.01). Sequencing a fragment of approximately 750 nt of the capsid protein gene from 53 samples showed a high frequency of co-infection by more than one strain (33.9%), high genetic diversity with 13 genotypes with low sequence identities (70.5-87.8%), and no geographic segregation of strains. Given the high prevalence, high diversity, and the lack of identification in other species, foxes are likely the natural reservoir of Newlavirus, and further studies should investigate its distribution.

Characterization of phylogenetically diverse astroviruses of marine mammals.

Astroviruses are small, non-enveloped, positive-stranded RNA viruses. Previously studied mammalian astroviruses have been associated with diarrhoeal disease. Knowledge of astrovirus diversity is very limited, with only six officially recognized astrovirus species from mammalian hosts and, in addition, one human and some bat astroviruses were recently described. We used consensus PCR techniques for initial identification of five astroviruses of marine mammals: three from California sea lions (Zalophus californianus), one from a Steller sea lion (Eumetopias jubatus) and one from a bottlenose dolphin (Tursiops truncatus). Bayesian and maximum-likelihood phylogenetic analysis found that these viruses showed significant diversity at a level consistent with novel species. Astroviruses that we identified from marine mammals were found across the mamastrovirus tree and did not form a monophyletic group. Recombination analysis found that a recombination event may have occurred between a human and a California sea lion astrovirus, suggesting that both lineages may have been capable of infecting the same host at one point. The diversity found amongst marine mammal astroviruses and their similarity to terrestrial astroviruses suggests that the marine environment plays an important role in astrovirus ecology.

Truncation of TRIM5 in the Feliformia explains the absence of retroviral restriction in cells of the domestic cat.

TRIM5alpha mediates a potent retroviral restriction phenotype in diverse mammalian species. Here, we identify a TRIM5 transcript in cat cells with a truncated B30.2 capsid binding domain and ablated restrictive function which, remarkably, is conserved across the Feliformia. Cat TRIM5 displayed no restriction activity, but ectopic expression conferred a dominant negative effect against human TRIM5alpha. Our findings explain the absence of retroviral restriction in cat cells and suggest that disruption of the TRIM5 locus has arisen independently at least twice in the Carnivora, with implications concerning the evolution of the host and pathogen in this taxon.

Unveiling patterns of viral pathogen infection in free-ranging carnivores of northern Portugal using a complementary methodological approach.

Pathogen surveillance in free-ranging carnivores presents challenges due to their low densitie and secretive nature. We combined molecular and serological assays to investigate infections by viral pathogens (Canine parvovirus (CPV), Canine distemper virus (CDV) and Canine coronavirus (CCoV)) in Portuguese carnivores (Canis lupus, Vulpes vulpes, Lutra lutra, Martes foina, M. martes, Meles meles, and Genetta genetta) over a period of 16 years. Additionally we explored spatio-temporal patterns of virus occurrence in Canis lupus. Our study identified CPV DNA in all carnivore species with an overall prevalence of 91.9 %. CPV was detected in all sampled years and seasons in Canis lupus, supporting its enzootic nature. CDV RNA was mainly detected in the Canidae family, with viral nucleic acid recorded between 2005 and 2008 with a peak prevalence of 67 % among the wolf population, followed by a sharp decline, suggesting an epizootic behaviour of the virus. Antibodies show that mustelids and viverrids were often exposed to CDV. CCoV was first recorded by molecular methods in wolf samples in 2002, remaining in the wolf populations with marked fluctuations over time. The dual serological and molecular approach provided important epidemiological data on pathogens of wild carnivores in Portugal. These programmes should also include monitoring of other potential reservoir hosts such as domestic cats and dogs.

Genetic characterization of Carnivore Parvoviruses in Spanish wildlife reveals domestic dog and cat-related sequences.

The impact of carnivore parvovirus infection on wild populations is not yet understood; disease signs are mainly developed in pups and assessing the health of litters in wild carnivores has big limitations. This study aims to shed light on the virus dynamics among wild carnivores thanks to the analysis of 213 samples collected between 1994 and 2013 in wild ecosystems from Spain. We determined the presence of carnivore parvovirus DNA by real-time PCR and sequenced the vp2 gen from 22 positive samples to characterize the strains and to perform phylogenetic analysis. The presence of carnivore parvovirus DNA was confirmed in 18% of the samples, with a higher prevalence detected in wolves (Canis lupus signatus, 70%). Fourteen sequences belonging to nine wolves, three Eurasian badgers (Meles meles), a common genet (Genetta genetta) and a European wildcat (Felis silvestris) were classified as canine parvovirus 2c (CPV-2c); five sequences from three wolves, a red fox (Vulpes vulpes) and a stone marten (Martes foina) as CPV-2b; and three sequences from a badger, a genet and a stone marten as feline parvovirus (FPV). This was the first report of a wildcat infected with a canine strain. Sequences described in this study were identical or very close related to others previously found in domestic carnivores from distant countries, suggesting that cross-species transmission takes place and that the parvovirus epidemiology in Spain, as elsewhere, could be influenced by global factors.

Infection of eight mesocarnivores in New Hampshire and Vermont with a distinct clade of canine distemper virus in 2016-2017.

Three fishers (Martes pennanti), 2 gray foxes (Urocyon cinereoargenteus), 1 mink (Neovison vison), 1 skunk (Mephitis mephitis), and 1 raccoon (Procyon lotor), from Vermont and New Hampshire, had lesions on autopsy consistent with canine distemper virus (CDV) infections diagnosed in a 12-mo period in 2016-2017. Lesions of CDV infection were most commonly noted in the lungs (8 of 8 animals), urothelium (5 of 8), biliary tract (5 of 8), gastrointestinal tract (4 of 7), and brain (4 of 6). Splenic lesions were seen in 3 animals. The diagnosis was confirmed via immunohistochemistry and virus isolation. Viral genotyping indicated that all 8 animals were infected with a distinct clade of CDV that has only been reported in wildlife in New England, and this clade of viruses is distinct from vaccine strains. During the 12 mo when these cases occurred, no other CDV clade was identified in any other wildlife or domesticated animal submitted from the 2 states.

Canine distemper in Nepal's Annapurna Conservation Area - Implications of dog husbandry and human behaviour for wildlife disease.

Dogs are often commensal with human settlements. In areas where settlements are adjacent to wildlife habitat, the management of dogs can affect risk of spillover of disease to wildlife. We assess dog husbandry practices, and measure the prevalence of Canine Distemper Virus (CDV) in dogs, in 10 villages in Nepal's Annapurna Conservation Area (ACA), an important region for Himalayan wildlife. A high proportion (58%) of owned dogs were allowed by their owners to roam freely, and many village dogs originated from urban areas outside the region. CDV antibodies, indicating past exposure, were detected in 70% of dogs, and 13% were positive for P-gene, suggesting current circulation of CDV. This is the first detection of canine distemper virus in a National Park in Nepal Himalaya. Dogs were generally in good condition, and none exhibited clinical signs of CDV infection, which suggests that infections were asymptomatic. CDV exposure varied with village location and age of dogs, but this variation was minor, consistent with high rates of movement of dogs across the region maintaining high seroprevalence. Residents reported the occurrence of several species of wild carnivores in or close to villages. These results suggest a high potential for transmission of CDV from village dogs to wild carnivores in ACA. We suggest that control of dog immigration, along with vaccination and neutering of dogs could mitigate the risk of CDV spillover into wild carnivore populations.

Carnivore Protoparvovirus 1 at the Wild-Domestic Carnivore Interface in Northwestern Mexico.

Eighty-three wild and domestic carnivores of nine species from Janos Biosphere Reserve (JBR), Mexico, were tested by serologic and molecular assays to determine exposure and infection rates of carnivore protoparvovirus 1. Overall, 50.8% (33/65) of the wild carnivores and 100% (18/18) of the domestic dogs tested were seropositive for Canine protoparvovirus 1 (CPV), while 23% (15/65) of the wild carnivores and 22.2% (4/18) of the domestic dogs were PCR positive for CPV. Phylogenetic analysis confirmed circulation of CVP-2 with residues 426 Asn (CPV2a = 1/19) and 426 Glu (CPV-2c = 18/19) among carnivores in JBR. The prevalence of both PCR positivity and antibodies to CPV varied significantly among wild host species. Of the six identified haplotypes, three were unique to kit foxes (Vulpes macrotis) (the species with higher haplotype richness) and two to striped skunks (Mephitis mephitis). The remaining haplotype was shared among all carnivore species including dogs suggesting non-host specificity and bidirectional and continuous viral transmission cycle in the JBR. The phylogenetic similarity of CPV strains from dogs and wild carnivores and the higher prevalence of CPV in wild carnivores captured near towns relative to those captured far from towns suggest that dogs might be an important source of CPV infection for wild carnivores in the JBR. We provide evidence that cross-species transmission occurs at the domestic-wildlife interface in JBR.