Evidence for Borrelia bavariensis Infections of Ixodes uriae within Seabird Colonies of the North Atlantic Ocean.

The first report of members of the spirochete genus Borrelia in the seabird tick, Ixodes uriae, and seabird colonies occurred during the early 1990s. Since then, Borrelia spp. have been detected in these ticks and seabird colonies around the world. To date, the primary species detected has been Borrelia garinii, with rare occurrences of Borrelia burgdorferi sensu stricto and Borrelia lusitaniae. During our research on Borrelia and I. uriae in seabird colonies of Newfoundland and Labrador, Canada, we have identified Borrelia bavariensis in I. uriae To our knowledge, B. bavariensis has previously been found only in the Eurasian tick species Ixodes persulcatus and Ixodes ricinus, and it was believed to be a rodent-specific Borrelia ecotype. We found B. bavariensis within I. uriae from three seabird colonies over three calendar years. We also reanalyzed B. garinii sequences collected from I. uriae from Eurasian seabird colonies and determined that sequences from two Russian seabird colonies likely also represent B. bavariensis The Canadian B. bavariensis sequences from I. uriae analyzed in this study cluster with previously described sequences from Asia. Overall, this is an important discovery that illustrates and expands the range of hosts and vectors for B. bavariensis, and it raises questions regarding the possible mechanisms of pathogen dispersal from Asia to North America.IMPORTANCE To our knowledge, this is the first documentation of B. bavariensis outside Eurasia. Additionally, the bacterium was found in a marine ecosystem involving the seabird tick I. uriae and its associated seabird hosts. This indicates that the epizootiology of B. bavariensis transmission is much different from what had been described, with this species previously believed to be a rodent-specific ecotype, and it indicates that this pathogen is established, or establishing, much more widely.

Patterns of MHC-DRB1 polymorphism in a post-glacial island canid, the Newfoundland red fox (Vulpes vulpes deletrix), suggest balancing selection at species and population timescales.

As the only native insular Newfoundland canid between the extinction of the wolf in the 1930s and the recent arrival of coyotes, the red fox (Vulpes vulpes deletrix Bangs 1898) poses interesting questions about genetic distinctiveness and the post-glacial colonization history of the island's depauperate mammalian fauna. Here, we characterized genetic variability at the major histocompatibility complex (MHC) class II DR ß1 domain (DRB1) locus in 28 red foxes from six sampling localities island-wide and compared it with mitochondrial control region (CR) diversity and DRB1 diversity in other canids. Our goals were to describe novel DRB1 alleles in a new canid population and to make inferences about the role of selection in maintaining their diversity. As in numerous studies of vertebrates, we found an order-of-magnitude higher nucleotide diversity at the DRB1 locus compared with the CR and significantly positive nonsynonymous-to-synonymous substitution ratios, indicative of selection in the distant past. Although the evidence is weaker, the Ewens-Watterson test of neutrality and the geographical distribution of variation compared with the CR suggest a role for selection over the evolutionary timescale of populations. We report the first genetic data from the DRB1 locus in the red fox and establish baseline information regarding immunogenetic variation in this island canid population which should inform continued investigations of population demography, adaptive genetic diversity, and wildlife disease in red foxes and related species.

A cross sectional study of animal and human colonization with Methicillin-Resistant Staphylococcus aureus (MRSA) in an Aboriginal community.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) infections are common among humans in Aboriginal communities in Canada, for unknown reasons. METHODS: Cross sectional study of humans and dogs in an Aboriginal community of approximately 1200 persons. Our objectives were to measure community-based prevalence of nasal MRSA colonization among humans, use multivariable logistic regression to analyze risk factors for MRSA colonization, and perform molecular typing of Staphylococci isolated to investigate interspecies transmission. RESULTS: 461 humans were approached for consent and 442 provided complete data. 109/442 (24.7 %, 95 % C.I. = 20.7-28.7 %) of humans were colonized with MRSA. 169/442 (38.2 %) of humans had received antibiotics in the last 12 months. Only number of rooms in the house (OR 0.86, p = 0.023) and recreational dog use (OR 7.7, p = 0.002) were significant risk factors for MRSA colonization. 95/109 (87.1 %) of MRSA strains from humans were of the same spa type (CMRSA10/USA300). 8/157 (5.1 %, 95 % C.I. = 1.7-8.5 %) of dogs were colonized with methicillin-susceptible S. aureus, and no dogs were colonized with MRSA. CONCLUSIONS: Human MRSA colonization in this community is very common, and a single clone is predominant, suggesting local transmission. Antibiotic use is also very common. Crowding may partially explain high colonization, but most considered risk factors including animal exposure were not predictive. Very few dogs carried human Staphylococcal strains.

New Geographic Records for Trichinella nativa and Echinococcus canadensis in Coyotes (Canis latrans) from Insular Newfoundland, Canada.

Coyotes (Canis latrans) rapidly expanded across North America during the 20th century and in 1987 colonized insular Newfoundland, Canada. Their arrival brought the potential for new predator-prey interactions and the potential for transmission of parasites to naïve populations. Trichinella spp. and Echinococcus spp. are zoonotic parasites not previously reported from the island of Newfoundland, Canada. Muscle samples (diaphragm and tongue) from 153 coyotes and feces from 35/153 coyotes were collected. Larvae of Trichinella spp. were recovered by muscle digestion from 6/153 coyotes (3.9%) and identified using multiplex PCR and Sanger sequencing as T. nativa. Fecal samples were screened for DNA of Echinococcus spp. using qPCR, and intestines from positive animals were examined for adult cestodes. No fecal samples were positive for DNA of E. multilocularis, and 2/35 (5.7%) samples were positive for E. canadensis, of which one was successfully genotyped as the G10 cervid strain. Echinococcus canadensis has not previously been reported on the island of Newfoundland, historically the only region of Canada where Echinococcus spp. was not known to occur. No species of Trichinella have previously been reported on the island. Both parasites are zoonotic, and hunters, trappers, dog owners, and the general public should be aware of these new risks for public health.

A 4-year study of avian influenza virus prevalence and subtype diversity in ducks of Newfoundland, Canada.

The island of Newfoundland, Canada, is at the eastern edge of North America and has migratory bird connections with the continental mainland as well as across the North Atlantic Ocean. Here, we report a 4-year avian influenza virus (AIV) epidemiological study in ducks in the St. John's region of Newfoundland. The overall prevalence of AIV detection in ducks during this study was 7.2%, with American Black Ducks contributing the vast majority of the collected samples and the AIV positives. The juvenile ducks showed a significantly higher AIV detection rate (10.6%) compared with adults (3.4%). Seasonally, AIV prevalence rates were higher in the autumn (8.4%), but positives were still detected in the winter (4.6%). Preliminary serology tests showed a high incidence of previous AIV infection (20/38, 52.6%). A total of 43 viruses were characterized for their HA-NA or HA subtypes, which revealed a large diversity of AIV subtypes and little recurrence of subtypes from year to year. Investigation of the movement patterns of ducks in this region showed that it is a largely non-migratory duck population, which may contribute to the observed pattern of high AIV subtype turnover. Phylogenetic analysis of 4 H1N1 and one H5N4 AIVs showed these viruses were highly similar to other low pathogenic AIV sequences from waterfowl in North America and assigned all gene segments into American-avian clades. Notably, the H1N1 viruses, which were identified in consecutive years, possessed homologous genomes. Such detection of homologous AIV genomes across years is rare, but indicates the role of the environmental reservoir in viral perpetuation.

Roles of host species, geographic separation, and isolation in the seroprevalence of Jamestown Canyon and snowshoe hare viruses in Newfoundland.

California serogroup viruses, including Jamestown Canyon virus (JCV) and snowshoe hare virus (SSHV), are mosquito-borne members of the Bunyaviridae family and are endemic across North America. These arboviruses are potential pathogens which occasionally cause neuroinvasive disease in humans and livestock. A neutralization assay was used to document JCV and SSHV seroprevalence using blood collected from a variety of domestic and wildlife host species. These species were sampled in an island setting, Newfoundland, which contains diverse ecoregions, ecological landscapes, and habitats. Seroprevalence rates for each virus differed significantly among host species and within certain species across different geographic areas. JCV was significantly associated with large mammals, and SSHV was significantly associated with snowshoe hares. Seroprevalence rates in the 5 species of animals tested for prior exposure to JCV ranged from 0% in snowshoe hares to 64% in horses. Seroprevalence rates for SSHV ranged from less than 1% in bovines to 55% in all snowshoe hares. The seroprevalence of SSHV differed significantly (P < 0.05) among hares occupying the discrete habitats of watersheds separated by 14 to 35 km. Cattle on farms in boreal forest landscapes displayed significantly higher JCV seroprevalence (P < 0.001) than those on farms located in seacoast landscapes. Lifelong geographic isolation of cattle to insular Newfoundland was associated with significantly lower JCV seroprevalence (P < 0.01) than that for cattle which had lived off-island.

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.

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.

Reassortment of American and Eurasian genes in an influenza A virus isolated from a great black-backed gull (Larus marinus), a species demonstrated to move between these regions.

The primary hosts for influenza A viruses are waterfowl, although gulls and shorebirds are also important in global avian influenza dynamics. Avian influenza virus genes are separated phylogenetically into two geographic clades, American and Eurasian, which is caused by the geographic separation of the host species between these two regions. We surveyed a gregarious and cosmopolitan species, the Great Black-backed Gull (Larus marinus), in Newfoundland, Canada, for the presence of avian influenza viruses. We have isolated and determined the complete genome sequence of an H13N2 virus, A/Great Black-backed Gull/Newfoundland/296/2008(H13N2), from one of these birds. Phylogenetic analysis revealed that this virus contained two genes in the American gull clade (PB1, HA), two genes in the American avian clade (PA, NA), and four genes in the Eurasian gull clade (PB2, NP, M, NS). We analyzed bird band recovery information and found the first evidence of trans-Atlantic migration from Newfoundland to Europe (UK, Spain and Portugal) for this species. Thus, great black-backed gulls could be important for movement of avian influenza viruses across the Atlantic Ocean and within North America.

Relationships between fox populations and rabies virus spread in northern Canada.

Rabies spreads in both Arctic (Vulpes lagopus) and red foxes (Vulpes vulpes) throughout the Canadian Arctic but limited wildlife disease surveillance, due to the extensive landmass of the Canadian north and its small widely scattered human population, undermines our knowledge of disease transmission patterns. This study has explored genetic population structure in both the rabies virus and its fox hosts to better understand factors that impact rabies spread. Phylogenetic analysis of 278 samples of the Arctic lineage of rabies virus recovered over 40 years identified four sub-lineages, A1 to A4. The A1 lineage has been restricted to southern regions of the Canadian province of Ontario. The A2 lineage, which predominates in Siberia, has also spread to northern Alaska while the A4 lineage was recovered from southern Alaska only. The A3 sub-lineage, which was also found in northern Alaska, has been responsible for virtually all cases across northern Canada and Greenland, where it further differentiated into 18 groups which have systematically evolved from a common predecessor since 1975. In areas of Arctic and red fox sympatry, viral groups appear to circulate in both hosts, but both mitochondrial DNA control region sequences and 9-locus microsatellite genotypes revealed contrasting phylogeographic patterns for the two fox species. Among 157 Arctic foxes, 33 mitochondrial control region haplotypes were identified but little genetic structure differentiating localities was detected. Among 162 red foxes, 18 control region haplotypes delineated three groups which discriminated among the Churchill region of Manitoba, northern Quebec and Labrador populations, and the coastal Labrador locality of Cartwright. Microsatellite analyses demonstrated some genetic heterogeneity among sampling localities of Arctic foxes but no obvious pattern, while two or three clusters of red foxes suggested some admixture between the Churchill and Quebec-Labrador regions but uniqueness of the Cartwright group. The limited population structure of Arctic foxes is consistent with the rapid spread of rabies virus subtypes throughout the north, while red fox population substructure suggests that disease spread in this host moves most readily down certain independent corridors such as the northeastern coast of Canada and the central interior. Interestingly the evidence suggests that these red fox populations have limited capacity to maintain the virus over the long term, but they may contribute to viral persistence in areas of red and Arctic fox sympatry.

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.

Investigating the Diversity and Host Range of Novel Parvoviruses from North American Ducks Using Epidemiology, Phylogenetics, Genome Structure, and Codon Usage Analysis.

Parvoviruses are small single-stranded DNA viruses that can infect both vertebrates and invertebrates. We report here the full characterization of novel viruses we identified in ducks, including two viral species within the subfamily Hamaparvovirinae (duck-associated chapparvovirus, DAC) and a novel species within the subfamily Densovirinae (duck-associated ambidensovirus, DAAD). Overall, 5.7% and 21.1% of the 123 screened ducks (American black ducks, mallards, northern pintail) were positive for DAC and DAAD, respectively, and both viruses were more frequently detected in autumn than in winter. Genome organization and predicted transcription profiles of DAC and DAAD were similar to viruses of the genera Chaphamaparvovirus and Protoambidensovirus, respectively. Their association to these genera was also demonstrated by subfamily-wide phylogenetic and distance analyses of non-structural protein NS1 sequences. While DACs were included in a highly supported clade of avian viruses, no definitive conclusions could be drawn about the host type of DAAD because it was phylogenetically close to viruses found in vertebrates and invertebrates and analyses of codon usage bias and nucleotide frequencies of viruses within the family Parvoviridae showed no clear host-based viral segregation. This study highlights the high parvoviral diversity in the avian reservoir with many avian-associated parvoviruses likely yet to be discovered.

The genome sequence of an H11N2 avian influenza virus from a Thick-billed Murre (Uria lomvia) shows marine-specific and regional patterns of relationships to other viruses.

Influenza A viruses infect a range of host species, including various mammals and more than 100 species of birds. For avian influenza viruses (AIV), prevalence varies between different groups of birds, with waterfowl showing the highest prevalence. We have sequenced the complete genome of A/Thick-billed Murre/Newfoundland/031/2007(H11N2), an AIV identified in the pelagic seabird, Thick-billed Murre (Uria lomvia). This represents the first complete genome sequence of an AIV from this host species, and only the second complete genome sequence from a seabird in the alcid group. All of the virus segments fall within the American avian lineage. Several of the segments show a close relationship to AIV identified in other marine host species, and also a strong geographic association with other AIV sequences from the northeastern coast of North America from recent years. The identification of this virus, and the growing number of AIV identified in seabird species, indicates these marine birds could be underappreciated host species. This has potential consequences for global influenza dynamics because of the seasonal distributions and migratory patterns of this group of birds.

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.

Gastrointestinal parasites in dogs from the Island of St. Pierre off the south coast of Newfoundland.

The present work was performed to survey the gastrointestinal parasites of domestic dogs (Canis lupus familiaris) and also to determine if any were infected with Angiostrongylus vasorum (French Heartworm), on the French Island of St. Pierre off the south coast of Newfoundland. A total of 57 fecal samples were collected and examined for intestinal parasites. The overall prevalence of parasitism was 57.9% and the six species found were: Uncinaria stenocephala/Ancylostoma caninum (47.4%), Toxocara canis (22.8%), Isospora canis (8.8%), Trichuris vulpis (7.0%), and Alaria canis (1.8%). There was no significant difference in overall prevalence between genders, except for T. canis, which was more common in female dogs than male dogs (p<0.05).

Genetic diversity of Borrelia garinii from Ixodes uriae collected in seabird colonies of the northwestern Atlantic Ocean.

The occurrence of Borrelia garinii in seabird ticks, Ixodes uriae, associated with different species of colonial seabirds has been studied since the early 1990s. Research on the population structure of this bacterium in ticks from seabird colonies in the northeastern Atlantic Ocean has revealed admixture between marine and terrestrial tick populations. We studied B. garinii genetic diversity and population structure in I. uriae collected from seabird colonies in the northwestern Atlantic Ocean, in Newfoundland and Labrador, Canada. We applied a multi-locus sequence typing (MLST) scheme to B. garinii found in ticks from four species of seabirds. The B. garinii strains found in this seabird colony ecosystem were diverse. Some were very similar to strains from Asia and Europe, including some obtained from human clinical samples, while others formed a divergent group specific to this region of the Atlantic Ocean. Our findings highlight the genetic complexity of B. garinii circulating in seabird ticks and their avian hosts but also demonstrate surprisingly close connections between B. garinii in this ecosystem and terrestrial sources in Eurasia. Genetic similarities among B. garinii from seabird ticks and humans indicate the possibility that B. garinii circulating within seabird tick-avian host transmission cycles could directly, or indirectly via connectivity with terrestrial transmission cycles, have consequences for human health.

Discovery and Characterization of Novel RNA Viruses in Aquatic North American Wild Birds.

Wild birds are recognized viral reservoirs but our understanding about avian viral diversity is limited. We describe here three novel RNA viruses that we identified in oropharyngeal/cloacal swabs collected from wild birds. The complete genome of a novel gull metapneumovirus (GuMPV B29) was determined. Phylogenetic analyses indicated that this virus could represent a novel avian metapneumovirus (AMPV) sub-group, intermediate between AMPV-C and the subgroup of the other AMPVs. This virus was detected in an American herring (1/24, 4.2%) and great black-backed (4/26, 15.4%) gulls. A novel gull coronavirus (GuCoV B29) was detected in great black-backed (3/26, 11.5%) and American herring (2/24, 8.3%) gulls. Phylogenetic analyses of GuCoV B29 suggested that this virus could represent a novel species within the genus Gammacoronavirus, close to other recently identified potential novel avian coronaviral species. One GuMPV-GuCoV co-infection was detected. A novel duck calicivirus (DuCV-2 B6) was identified in mallards (2/5, 40%) and American black ducks (7/26, 26.9%). This virus, of which we identified two different types, was fully sequenced and was genetically closest to other caliciviruses identified in Anatidae, but more distant to other caliciviruses from birds in the genus Anas. These discoveries increase our knowledge about avian virus diversity and host distributions.

New Insight Into Avian Papillomavirus Ecology and Evolution From Characterization of Novel Wild Bird Papillomaviruses.

Viruses in the family Papillomaviridae have circular dsDNA genomes of approximately 5.7-8.6 kb that are packaged within non-enveloped, icosahedral capsids. The known papillomavirus (PV) representatives infect vertebrates, and there are currently more than 130 recognized PV species in more than 50 genera. We identified 12 novel avian papillomavirus (APV) types in wild birds that could represent five distinct species and two genera. Viruses were detected in paired oropharyngeal/cloacal swabs collected from six bird species, increasing the number of avian species known to harbor PVs by 40%. A new duck PV (DuPV-3) was found in mallard and American black duck (27.6% estimated prevalence) that was monophyletic with other known DuPVs. A single viral type was identified in Atlantic puffin (PuPV-1, 9.8% estimated prevalence), while a higher genetic diversity was found in other Charadriiformes. Specifically, three types [gull PV-1 (GuPV-1), -2, and -3] were identified in two gull species (estimated prevalence of 17% and 2.6% in American herring and great black-backed gull, respectively), and seven types [kittiwake PV-1 (KiPV-1) through -7] were found in black-legged kittiwake (81.3% estimated prevalence). Significantly higher DuPV-3 circulation was observed in spring compared to fall and in adults compared to juveniles. The studied host species' tendencies to be in crowded environments likely affect infection rates and their migratory behaviors could explain the high viral diversity, illustrating how host behavior can influence viral ecology and distribution. For DuPV-3, GuPV-1, PuPV-1, and KiPV-2, we obtained the complete genomic sequences, which showed the same organization as other known APVs. Phylogenetic analyses showed evidence for virus-host co-divergence at the host taxonomic levels of family, order, and inter-order, but we also observed that host-specificity constraints are relaxed among highly related hosts as we found cross-species transmission within ducks and within gulls. Furthermore, the phylogeny of viruses infecting the Charadriiformes did not match the host phylogeny and gull viruses formed distinct monophyletic clades with kittiwake viruses, possibly reflecting past host-switching events. Considering the vast PV genotype diversity in other hosts and the large number of bird species, many more APVs likely remain to be discovered.