Distinct epidemiological profiles of porcine circovirus 3 and fox circovirus in Canadian foxes (Vulpes spp.).

Circoviruses (genus Circovirus, family Circoviridae) are ssDNA viruses that infect mammals, and they sometimes can transmit among different species. We investigated the distribution and diversity of porcine circovirus 3 (PCV-3, species Porcine circovirus 3) and fox circovirus (species Canine circovirus 1) in different populations of foxes (Vulpes spp.) inhabiting the Canadian province of Newfoundland and Labrador to compare their epidemiological profiles. Of the 210 samples tested in this study 9 were positive for PCV-3 and 99 were positive for fox circovirus. Eight foxes were PCV-3-positive (8/128, 6.3%) and the virus was only found in the most human-populated areas. The PCV-3 positivity rate was significantly higher in stool (7/180, 8.8%) than in spleen (2/120, 1.7%: p < 0.05). Phylogenetic analyses showed that sequences from different animals were unrelated to each other. Fox circovirus was identified in 66 animals (51.6%) and positivity rates were the highest in the least human-populated areas. There were no significant differences between positivity rates in stool (32/80, 40.0%), spleen (59/120, 49.2%), or lymph nodes (8/10, 80.0%). Among 54 positive animals for which both spleen and stool samples were available, 25 (46.3%) had detectable virus in both samples. All fox circovirus sequences recovered in this study formed a monophyletic clade, and no geographic segregation of study strains was observed. The high prevalence and high genetic diversity observed for fox circovirus implies that the virus has been circulating in this population for a long time. PCV-3 cases were consistent with sporadic infections from multiple sources, possibly related to scavenging behavior and consumption of meat by-products and human waste, while fox circovirus was endemic, indicating that foxes are likely the maintenance host for this virus. To the best of our knowledge this is the first study demonstrating the presence of fox circovirus in North America and to show that PCV-3 can be detected in foxes. Future studies should evaluate the pathogenic potential of these viruses for wildlife.

Distribution and diversity of dog parvoviruses in wild, free-roaming and domestic canids of Newfoundland and Labrador, Canada.

Some parvoviruses of carnivorans can infect multiple host species. Since many canine parvoviruses were only discovered recently, their host-range is still unexplored. We examined the host distribution and diversity of five dog parvoviruses in four canine populations from Newfoundland and Labrador, Canada, and investigated the potential for these viruses to cross the species barriers. Canine bocavirus 2 (CBoV-2) and the minute virus of canines were detected in stool from free-roaming dogs from Labrador (5/48 [10.4%] and 3/48 [6.3%], respectively) and two different CBoV-2 variants were identified. Canine bufavirus was identified in stool from free-roaming dogs (1/48, 2.1%) and foxes (3/80, 3.8%) from Labrador, but two different variants were observed in the two host species. The variant found in foxes was highly divergent from previously identified strains. Two cachavirus 1 variants, genetically similar to those circulating in other Canadian wildlife, were found in spleens from Newfoundland coyotes (3/87, 3.5%). Canine parvovirus type 2 (CPV-2) was found in stool from free-roaming dogs from Labrador (2/48, 4.2%) and in spleens from Newfoundland coyotes (3/87, 3.5%). Comparing CPV-2 sequences from these hosts to those retrieved from local symptomatic domestic dogs revealed the presence of a highly heterogeneous viral population as detected strains belonged to five different clades. The close relationship between CPV-2a strains from a dog and a coyote suggests the occurrence of viral transfer between wild and domestic canids. The identification of highly related strains with a similar molecular signature characteristic of older CPV-2 strains in free-roaming and domestic dogs suggests a probable common ancestry and that older CPV-2 strains, which have not been identified in dogs since the 1990s, persist in this part of Canada. Follow-up studies should evaluate samples from a larger number of animals and host species to extensively investigate the possible occurrence of cross-species transmission for recently discovered parvoviruses.

Novel Divergent Members of the Kitrinoviricota Discovered through Metagenomics in the Intestinal Contents of Red-Backed Voles (Clethrionomys gapperi).

Metagenomic methods are powerful tools to investigate viral diversity in biological or environmental samples and to identify previously unknown viruses. We used RNA metagenomics to identify, in the gut of red-backed voles, the nearly complete genomes of two novel members of the Kitrinoviricota, a phylum including viruses with positive-sense ssRNA genomes encoding an RNA-directed RNA polymerase. The genome of a novel member of the Tombusviridae presented four open reading frames (ORFs); a -1 frameshift is potentially involved in generating the viral replicase. This sequence was part of a phylogenetic clade that did not include any officially classified species. The second genome presented a large ORF coding for a viral polyprotein containing the typical protein domains common to flexiviruses. The sequence clustered with currently known members of the Deltaflexiviridae. Both viruses appear to represent the first members of novel species in yet undefined genera. The identified viruses likely originated from the vole diet as members of the two viral families are known to infect plants and fungi, respectively. Investigating public databases demonstrated that a much higher richness than currently recognized exists for these two viral families, highlighting the need to update taxonomy systems and possibly also include genomes identified through metagenomics.

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.

Multi-host dispersal of known and novel carnivore amdoparvoviruses.

Amdoparvoviruses (family Parvoviridae) are ssDNA viruses that cause an immune complex-mediated wasting syndrome in carnivores. They are multi-host pathogens and cross-species infection is facilitated by the fact that viral entry is mediated by cellular Fc receptors recognizing antibody-coated viruses. We developed a pan-amdoparvovirus PCR and screened tissue samples from 666 wild carnivores (families Felidae, Canidae, and Mustelidae) from Newfoundland or Labrador (Canada) and molecularly characterized the identified strains. Fifty-four out of 666 (8.1%) animals were amdoparvovirus-positive. Infection rate was the highest in American mink (34/47, 72.3%), followed by foxes (Arctic and red foxes, 13/311, 4.2%), lynx (2/58, 3.5%), and American martens (5/156, 3.4%). No virus was detected in samples from 87 coyotes and 17 ermines. Viruses from Newfoundland were classified as Aleutian mink disease virus (AMDV). Mink harvested near AMDV-affected fur farms had higher prevalence (24/24, 100%) than other mink (10/23, 43.5%; P < 0.001) and their viruses were phylogenetically closely related to those from farms, while most viruses from other mink were in other clades. Strains from three foxes and two lynx were highly related to mink strains. This proves that farms disperse AMDV that subsequently spreads among wild mink (maintenance host) and transmits to other spillover carnivore hosts. In Labrador two novel viruses were identified, Labrador amdoparvovirus 1 (LaAV-1) found in foxes (9/261, 3.5%) and martens (5/156, 3.4%), and LaAV-2 found in one fox (0.4%). LaAV-1 fulfills all requirements to be classified as a novel species. LaAV-1 was most similar to viruses of mink and skunks (AMDV and skunk amdoparvovirus (SKAV)) while LaAV-2 was more closely related to other viruses infecting canids. LaAV-1 capsid proteins were almost indistinguishable from those of AMDV in some regions, suggesting that LaAV-1 could be a virus of mustelids that can infect foxes. While intensive farming practices provide occasions for inter-species transmission in farms, niche overlap or predation could explain cross-species transmission in the wild, but competition among sympatric species reduces the chances of direct contacts, making this an infrequent event. Pan-amdoparvovirus detection methods in wide epidemiological investigations can play a crucial role in defining amdoparvoviral ecology and evolution and discovering novel viruses.

Identification of a Novel Gammaherpesvirus in Canada lynx (Lynx canadensis).

Gammaherpesviruses (GHVs) infect many animal species and are associated with lymphoproliferative disorders in some. Previously, we identified several novel GHVs in North American felids; however, a GHV had never been identified in Canada lynx (Lynx canadensis). We, therefore, hypothesized the existence of an unidentified GHV in lynx. Using degenerate nested and subsequently virus-specific PCR, we amplified and sequenced 3.4 kb of DNA from a novel GHV in lynx, which we named Lynx canadensis gammaherpesvirus 1 (LcaGHV1). Phylogenetic analysis determined that LcaGHV1 is a distinct GHV species belonging to the genus Percavirus. We then estimated the prevalence of LcaGHV1 in lynx by developing a PCR-based assay and detected LcaGHV1 DNA in 36% (95% CI: 22-53%) of lynx spleen DNA samples from Maine, USA and 17% (95% CI: 8-31%) from Newfoundland, Canada. The LcaGHV1 DNA sequences from Maine and Newfoundland lynx were nearly identical to each other (two nucleotide substitutions in 3.4 kb), suggesting that the unique lynx subspecies present on the island of Newfoundland (Lynx canadensis subsolanus) is infected with virus that very closely resembles virus found in mainland lynx. The potential ecologic and pathologic consequences of this novel virus for Canada lynx populations warrant further study.

Introduction of canine parvovirus 2 into wildlife on the Island of Newfoundland, Canada.

Canine parvovirus-2 (CPV-2) and feline panleukopenia virus (FPV) (species Carnivore protoparvovirus 1, family Parvoviridae) cause a severe gastrointestinal disease associated with immune depression in a broad range of terrestrial carnivores. We report here the first molecular epidemiological investigation of protoparvoviruses on the Island of Newfoundland, Canada. In particular, we investigated red foxes (Vulpes vulpes deletrix) and lynx (Lynx canadensis subsolanus), two autochthonous species, and coyotes (Canis latrans), which immigrated onto the island during the 1980s. CPV-2 was identified in coyotes (3/85, 3.5%), while no viruses were found in lynx (0/38) or foxes (0/22). Based on complete genome analyses, two of the identified viruses (which were 99.98% identical to each other) were variant CPV-2b, while the third strain was a CPV-2a variant. Phylogenetic analyses showed that the CPV-2b viruses were part of a group that also included viruses identified in wildlife in the USA (including coyotes) while the CPV-2a virus clustered with viruses identified in dogs. We conclude that the CPV-2b viruses could have been introduced into Newfoundland during the immigration of coyotes, while the CPV-2a virus was possibly introduced into the coyote population from an infected dog. Although a more extended screening effort is required, our preliminary data suggest that FPV is not circulating in Newfoundland and that CPV-2 viruses have not spread from coyotes to the other investigated autochthonous wild carnivores.