Occurrence of mesocarnivores in montane sky islands: How spatial and temporal overlap informs rabies management in a regional hotspot.

Interspecific interactions among mesocarnivores can influence community dynamics and resource partitioning. Insights into these interactions can enhance understanding of local ecological processes that have impacts on pathogen transmission, such as the rabies lyssavirus. Host species ecology can provide an important baseline for disease management strategies especially in biologically diverse ecosystems and heterogeneous landscapes. We used a mesocarnivore guild native to the southwestern United States, a regional rabies hotspot, that are prone to rabies outbreaks as our study system. Gray foxes (Urocyon cinereoargenteus), striped skunks (Mephitis mephitis), bobcats (Lynx rufus), and coyotes (Canis latrans) share large portions of their geographic ranges and can compete for resources, occupy similar niches, and influence population dynamics of each other. We deployed 80 cameras across two mountain ranges in Arizona, stratified by vegetation type. We used two-stage modeling to gain insight into species occurrence and co-occurrence patterns. There was strong evidence for the effects of elevation, season, and temperature impacting detection probability of all four species, with understory height and canopy cover also influencing gray foxes and skunks. For all four mesocarnivores, a second stage multi-species co-occurrence model better explained patterns of detection than the single-species occurrence model. These four species are influencing the space use of each other and are likely competing for resources seasonally. We did not observe spatial partitioning between these competitors, likely due to an abundance of cover and food resources in the biologically diverse system we studied. From our results we can draw inferences on community dynamics to inform rabies management in a regional hotspot. Understanding environmental factors in disease hotspots can provide useful information to develop more reliable early-warning systems for viral outbreaks. We recommend that disease management focus on delivering oral vaccine baits onto the landscape when natural food resources are less abundant, specifically during the two drier seasons in Arizona (pre-monsoon spring and autumn) to maximize intake by all mesocarnivores.

An Opportunistic Survey Reveals an Unexpected Coronavirus Diversity Hotspot in North America.

In summer 2020, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was detected on mink farms in Utah. An interagency One Health response was initiated to assess the extent of the outbreak and included sampling animals from on or near affected mink farms and testing them for SARS-CoV-2 and non-SARS coronaviruses. Among the 365 animals sampled, including domestic cats, mink, rodents, raccoons, and skunks, 261 (72%) of the animals harbored at least one coronavirus. Among the samples that could be further characterized, 127 alphacoronaviruses and 88 betacoronaviruses (including 74 detections of SARS-CoV-2 in mink) were identified. Moreover, at least 10% (n = 27) of the coronavirus-positive animals were found to be co-infected with more than one coronavirus. Our findings indicate an unexpectedly high prevalence of coronavirus among the domestic and wild free-roaming animals tested on mink farms. These results raise the possibility that mink farms could be potential hot spots for future trans-species viral spillover and the emergence of new pandemic coronaviruses.

Local Surveillance and Control of Raccoon Rabies Virus in Striped Skunks (Mephitis mephitis) in Southwestern New Brunswick, Canada.

Targeted surveillance for raccoon rabies virus was conducted between February and May 2017, near Waweig, New Brunswick, Canada, in response to detection of a rabid striped skunk (Mephitis mephitis) on 8 February 2017. A total of six skunks, 11 raccoons (Procyon lotor), and two porcupines (Erethizon dorsatum) were live-trapped, euthanized, and tested for rabies virus antigens using the direct rapid immunohistochemical test. Of these, only two skunks tested positive for rabies. All three rabid skunks came from the same location, an abandoned barn used as a denning site. Four of five skunks removed from this barn were males. Feeding, aggression, extreme response to noise and light stimuli, and exposure to porcupine quills were observed in two rabid skunks. No additional cases of rabies in wildlife were detected in the area since 8 March 2017. A targeted surveillance approach that removed potentially infected wildlife followed by localized oral rabies vaccine distribution was implemented in this locality.

Powassan Virus Experimental Infections in Three Wild Mammal Species.

Powassan virus (POWV) is a tick-borne virus maintained in sylvatic cycles between mammalian wildlife hosts and ticks (primarily Ixodes spp.). There are two currently recognized lineages, POWV-lineage 1 (POWV-L1) and deer tick virus (DTV; lineage 2), both of which can cause fatal neurologic disease in humans. Increased numbers of human case reports in the northeastern and north central United States in recent years have fueled questions into POWV epidemiology. We inoculated three candidate wildlife POWV reservoir hosts, groundhogs (Marmota monax), striped skunks (Mephitis mephitis), and fox squirrels (Sciurus niger), with either POWV-L1 or DTV. Resulting viremia, tissue tropism, and pathology were minimal in most inoculated individuals of all three species, with low (peak titer range, 101.7-103.3 plaque-forming units/mL serum) or undetectable viremia titers, lack of detection in tissues except for low titers in spleen, and seroconversion in most individuals by 21 days postinoculation (DPI). Pathology was limited and most commonly consisted of mild inflammation in the brain of POWV-L1- and DTV-inoculated skunks on four and 21 DPI, respectively. These results reveal variation in virulence and host competence among wild mammalian species, and a likely limited duration of host infectiousness to ticks during enzootic transmission cycles. However, POWV can transmit rapidly from tick to host, and tick co-feeding may be an additional transmission mechanism. The rare and low-level detections of viremia in these three, common, wild mammal species suggest that vector-host dynamics should continue to be explored, along with eco-epidemiological aspects of enzootic POWV transmission in different regions and virus lineages.

Complete genome analysis confirms that the pygmy marmoset adenovirus is a variant of the skunk adenovirus 1 - Short communication.

The complete genomic sequence along with phylogenetic analyses of an adenovirus (AdV), isolated from a dead captive pygmy marmoset (Callithrix pygmaea) from a Hungarian zoo is reported. Earlier, based on the phylogenetic analysis of the sequence of a PCR-amplified fragment from the DNA polymerase gene, the pygmy marmoset AdV (PMAdV) has been reported to cluster closest to certain chiropteran AdVs. In the following years similar AdVs were discovered in additional mammalian hosts, including a skunk (Mephitis mephitis), African pygmy hedgehogs (Atelerix albiventris), North American porcupines (Erethizon dorsatum) and grey fox (Urocyon cinereoargenteus). After the full genome analysis of the skunk adenovirus (SkAdV-1), a novel species Skunk mastadenovirus A (SkAdV-A) has been established. The AdVs, originating from the African pygmy hedgehogs, have been found to belong to virus species SkAdV-A. Partial gene sequences from the porcupine AdVs have also implied their very close genetic relatedness to SkAdV-A. The complete genomic sequence of PMAdV, examined in this study, was found to share 99.83% nucleotide identity with SkAdV-1, thus unequivocally represents a genomic variant of SkAdV-1. The observation that viruses classifiable as SkAdV-A are able to infect and cause diseases in several, distantly related mammals seems to deserve further studies to elucidate the infection biology of this intriguing AdV.

Origins of the arctic fox variant rabies viruses responsible for recent cases of the disease in southern Ontario.

A subpopulation of the arctic fox lineage of rabies virus has circulated extensively in red fox populations of Ontario, Canada, between the 1960s and 1990s. An intensive wildlife rabies control program, in which field operations were initiated in 1989, resulted in elimination of the disease in eastern Ontario. However in southwestern Ontario, as numbers of rabid foxes declined the proportion of skunks confirmed to be infected with this rabies virus variant increased and concerted control efforts targeting this species were employed to eliminate the disease. Since 2012 no cases due to this viral variant were reported in southwestern Ontario until 2015 when a single case of rabies due to the arctic fox variant was reported in a bovine. Several additional cases have been documented subsequently. Since routine antigenic typing cannot discriminate between the variants which previously circulated in Ontario and those from northern Canada it was unknown whether these recent cases were the result of a new introduction of this variant or a continuation of the previous enzootic. To explore the origins of this new outbreak whole genome sequences of a collection of 128 rabies viruses recovered from Ontario between the 1990s to the present were compared with those representative of variants circulating in the Canadian north. Phylogenetic analysis shows that the variant responsible for current cases in southwestern Ontario has evolved from those variants known to circulate in Ontario previously and is not due to a new introduction from northern regions. Thus despite ongoing passive surveillance the persistence of wildlife rabies went undetected in the study area for almost three years. The apparent adaptation of this rabies virus variant to the skunk host provided the opportunity to explore coding changes in the viral genome which might be associated with this host shift. Several such changes were identified including a subset for which the operation of positive selection was supported. The location of a small number of these amino acid substitutions in or close to protein motifs of functional importance suggests that some of them may have played a role in this host shift.

Endemic Skunk amdoparvovirus in free-ranging striped skunks (Mephitis mephitis) in California.

The genus Amdoparvovirus includes the newly discovered skunk amdoparvovirus and the well-characterized Aleutian disease virus which causes significant health impacts in farmed mink worldwide. In 2010-2013, an outbreak of fatal amdoparvovirus-associated disease was documented in free-ranging striped skunks (Mephitis mephitis) from the San Francisco Bay Area of California. To characterize the geographic distribution, earliest occurrence and abundance of this virus, as well as possible impacts on sympatric mustelids of conservation concern, we tested blood samples from skunks throughout California and fishers (Pekania pennanti) from northern California for amdoparvovirus DNA. Amdoparvovirus DNA was detected in 64.8% of sampled skunks (140/216), and test-positive skunks were distributed widely throughout the state, from as far north as Humboldt County and south to San Diego County. The first test-positive skunks were detected from 2004, prior to the 2010-2013 outbreak. No significant spatial or temporal clustering of infection was detected. Although healthy and clinically ill animals tested positive for amdoparvovirus DNA, histopathologic evaluation of a subset from clinically ill skunks indicated that positive PCR results were associated with pneumonia as well as there being more than one inflammatory type lesion. None of 38 fishers were PCR-positive. Given the widespread geographic distribution and lack of a clear epizootic centre, our results suggest the presence of an endemic skunk-associated amdoparvovirus strain or species. However, if the virus is not host-specific, skunks' ubiquitous presence across rural and urban habitats may pose a risk to susceptible domestic and wild species including mustelids of conservation concern such as fishers and Pacific martens (Martes caurina).

Probable reverse zoonosis of influenza A(H1N1)pdm 09 in a striped skunk (Mephitis mephitis).

Striped skunks (skunks) are susceptible to respiratory infection by influenza A viruses (IAV). As they are common synanthropes, maintenance of IAV in skunks could provide a source of infection for humans. We previously studied the nasal turbinates, lungs and faeces of 50 free-ranging skunks for the presence of IAV and identified two individuals with influenza A(H1N1)pdm09 infection during the 2009/2010 and 2013/2014 flu seasons. Subsequent to publication of that study, ferrets were shown to preferentially replicate and harbour A(H1N1)pdm09 in the soft palate, a site which had not been investigated in the skunks. From March 2015 to May 2016, we surveyed a convenience sample of 80 free-ranging urban skunks for IAV in soft palate, nasal turbinates and lungs. The newly emergent influenza A(H1N1)pdm09 clade 6B.1 was detected at all three sites in one skunk with acute rhinitis in February 2016. Clade 6B.1 was the dominant clade in circulation during the 2015/2016 flu season. As the skunk was detected at the height of flu season, reverse zoonosis was considered the most probable source of infection.

The Role of Mammalian Reservoir Hosts in Tick-Borne Flavivirus Biology.

Small-to-medium sized mammals and large animals are lucrative sources of blood meals for ixodid ticks that transmit life-threatening tick-borne flaviviruses (TBFVs). TBFVs have been isolated from various organs obtained from wild-caught Myodes and Apodemus species in Europe and Asia. Thus, these rodents are well-established reservoirs of TBFVs. Wild-caught Peromyscus species have demonstrated seropositivity against Powassan virus, the only TBFV known to circulate in North America, suggesting that they may play an important role in the biology of the virus in this geographic region. However, virus isolation from Peromyscus species is yet to be demonstrated. Wild-caught medium-sized mammals, such as woodchucks (Marmota monax) and skunks (Mephitis mephitis) have also demonstrated seropositivity against POWV, and virus was isolated from apparently healthy animals. Despite the well-established knowledge that small-to-medium sized animals are TBFV reservoirs, specific molecular biology addressing host-pathogen interactions remains poorly understood. Elucidating these interactions will be critical for gaining insight into the mechanism(s) of viral pathogenesis and/or resistance.

Challenges to controlling rabies in skunk populations using oral rabies vaccination: A review.

Controlling rabies in skunk populations is an important public health concern in many parts of the United States due to the potential for skunk rabies outbreaks in urban centres and the possible role for skunks in raccoon rabies variant circulation. Oral rabies vaccination (ORV) programmes have supported wildlife rabies control efforts globally but using ORV to control rabies in skunk populations has proven more challenging than with other target species, like foxes, coyotes and raccoons. A review of published studies found that some ORV constructs are immunogenic in skunks and protect against virulent rabies virus challenges, especially when delivered by direct installation into the oral cavity. However, in field ORV programmes using currently available vaccine-bait formats and distribution methods targeting other rabies reservoir species, skunks often fail to seroconvert. Field effectiveness of ORV in skunks appears to be limited by poor bait uptake or inadequate ingestion of vaccine rather than from poor vaccine efficacy. Observations of captive skunks revealed vaccine spillage when handling and biting into baits such that modification of bait formats might improve field effectiveness. In addition, a dose-response relationship between bait distribution density and post-baiting seroconversion among skunks was observed across the limited number of field studies. Additional research is needed to identify opportunities to modify ORV baits and distribution strategies to improve the viability of ORV as a rabies control strategy in skunks.

Frequency of Virus Coinfection in Raccoons ( Procyon lotor) and Striped Skunks ( Mephitis mephitis) During a Concurrent Rabies and Canine Distemper Outbreak.

Rabies and canine distemper virus infections in wildlife share similar presenting signs. Canine distemper virus was detected using real-time PCR of conjunctival swabs in rabies positive raccoons (22/32) and skunks (7/34) during a concurrent rabies and canine distemper outbreak in Ontario, Canada in 2015-16. Coinfections with both viruses should be considered, particularly in distemper endemic areas that are at risk of rabies incursion.

ALEUTIAN DISEASE VIRUS-LIKE VIRUS ( AMDOPARVOVIRUS SP.) INFECTING FREE-RANGING STRIPED SKUNKS ( MEPHITIS MEPHITIS) IN THE MIDWESTERN USA.

: Aleutian disease virus (ADV) and closely related (ADV-like) viruses are Parvoviridae members (genus Amdoparvovirus) that primarily infect farmed mustelids and have been detected in humans and free-ranging Carnivora from North America. We describe ADV-like/ Amdoparvovirus sp. infection in four free-ranging striped skunks ( Mephitis mephitis) from the Midwestern US.

Molecular characterization of atypical antigenic variants of canine rabies virus reveals its reintroduction by wildlife vectors in southeastern Mexico.

Rabies is an infectious viral disease that is practically always fatal following the onset of clinical signs. In Mexico, the last case of human rabies transmitted by dogs was reported in 2006 and canine rabies has declined significantly due to vaccination campaigns implemented in the country. Here we report on the molecular characterization of six rabies virus strains found in Yucatan and Chiapas, remarkably, four of them showed an atypical reaction pattern when antigenic characterization with a reduced panel of eight monoclonal antibodies was performed. Phylogenetic analyses on the RNA sequences unveiled that the three atypical strains from Yucatan are associated with skunks. Analysis using the virus entire genome showed that they belong to a different lineage distinct from the variants described for this animal species in Mexico. The Chiapas atypical strain was grouped in a lineage that was considered extinct, while the others are clustered within classic dog variants.

Predicting spatial spread of rabies in skunk populations using surveillance data reported by the public.

BACKGROUND: Prevention and control of wildlife disease invasions relies on the ability to predict spatio-temporal dynamics and understand the role of factors driving spread rates, such as seasonality and transmission distance. Passive disease surveillance (i.e., case reports by public) is a common method of monitoring emergence of wildlife diseases, but can be challenging to interpret due to spatial biases and limitations in data quantity and quality. METHODOLOGY/PRINCIPAL FINDINGS: We obtained passive rabies surveillance data from dead striped skunks (Mephitis mephitis) in an epizootic in northern Colorado, USA. We developed a dynamic patch-occupancy model which predicts spatio-temporal spreading while accounting for heterogeneous sampling. We estimated the distance travelled per transmission event, direction of invasion, rate of spatial spread, and effects of infection density and season. We also estimated mean transmission distance and rates of spatial spread using a phylogeographic approach on a subsample of viral sequences from the same epizootic. Both the occupancy and phylogeographic approaches predicted similar rates of spatio-temporal spread. Estimated mean transmission distances were 2.3 km (95% Highest Posterior Density (HPD95): 0.02, 11.9; phylogeographic) and 3.9 km (95% credible intervals (CI95): 1.4, 11.3; occupancy). Estimated rates of spatial spread in km/year were: 29.8 (HPD95: 20.8, 39.8; phylogeographic, branch velocity, homogenous model), 22.6 (HPD95: 15.3, 29.7; phylogeographic, diffusion rate, homogenous model) and 21.1 (CI95: 16.7, 25.5; occupancy). Initial colonization probability was twice as high in spring relative to fall. CONCLUSIONS/SIGNIFICANCE: Skunk-to-skunk transmission was primarily local (< 4 km) suggesting that if interventions were needed, they could be applied at the wave front. Slower viral invasions of skunk rabies in western USA compared to a similar epizootic in raccoons in the eastern USA implies host species or landscape factors underlie the dynamics of rabies invasions. Our framework provides a straightforward method for estimating rates of spatial spread of wildlife diseases.

Full genetic characterization and epidemiology of a novel amdoparvovirus in striped skunk (Mephitis mephitis).

Amdoparvovirus is a newly defined parvoviral genus that contains four species (Carnivore amdoparvovirus 1-4), including the well-known Aleutian mink disease virus (AMDV). Amdoparvoviruses cause an immune-associated and often lethal wasting syndrome in Mustelidae and Caninae hosts. In this study, we molecularly investigated amdoparvoviruses detected in 44 striped skunks (Mephitis mephitis) found dead in and around Vancouver, British Columbia, Canada. Some of the animals exhibited pathological changes compatible with amdoparvovirus-associated disease. The nearly complete genomic sequence was obtained for seven different strains and our analyses show how this virus, which we named skunk amdoparvovirus (SKAV), should be classified as a separate species within the genus (proposed Carnivore amdoparvovirus 5). We detected co-infections, recombinant genomes, at least three separate viral lineages, and preliminary evidence for geographic segregation of lineages. Furthermore, we proved that similar viruses, only partially characterized in previous studies and labeled as AMDV, circulate in skunks from other distant areas of North America (Ontario and California) and found evidence for spillover events in mink (Neovison vison). Although SKAVs are capable of causing disease in infected animals, a high proportion of sub-clinical infections has been observed, suggesting these animals might act as asymptomatic carriers and pose a threat to wild and captive carnivores. Finally, we highlight the need for more specific diagnostic tests and further molecular investigations to clarify the epidemiology and host- and geographical distributions of amdoparvoviruses in terrestrial carnivores, especially because the whole spectrum of viral diversity in this group is likely still unknown.

Bird Feeders as Locations for Skunk Uptake of Oral Rabies Vaccine Baits.

Significantly more (54%, P=0.003) placebo baits placed under 26 bird feeders in Arizona, US were removed by striped skunks ( Mephitis mephitis ) than at paired, nonfeeder locations (19%). Baiting at bird feeders could supplement traditional oral rabies vaccine bait placement in urban-suburban areas while engaging the public in rabies control efforts.

Unusual Necrotizing Encephalitis in Raccoons and Skunks Concurrently Infected With Canine Distemper Virus and Sarcocystis sp.

Canine distemper virus commonly infects free-ranging, terrestrial mesopredators throughout the United States. Due to the immunosuppressive effects of the virus, concurrent opportunistic infections are also common. Among these, secondary systemic protozoal infections have been described in a number of species. We report an unusual presentation of necrotizing encephalitis associated withSarcocystissp in four raccoons and one skunk concurrently infected with canine distemper virus. Lesions were characterized by variably sized necrotizing cavitations composed of abundant mineral admixed with inflammatory cells and protozoa.Sarcocystissp was confirmed via immunohistochemistry using a monoclonal antibody toSarcocystis neurona The pathologic changes are similar to lesions in human AIDS patients infected withToxoplasma gondii.

Experimental infection of peridomestic mammals with emergent H7N9 (A/Anhui/1/2013) influenza A virus: Implications for biosecurity and wet markets.

During 2013, a novel avian-origin H7N9 influenza A virus (IAV) emerged in China and subsequently caused large economic and public health burdens. We experimentally infected three common peridomestic wild mammals with H7N9 (A/Anhui/1/2013) IAV. Striped skunks exhibited the highest burden of disease followed by raccoons and cottontail rabbits. Striped skunks also produced the highest levels of viral shedding (up to 10(6.4)PFU/mL nasal flush) followed by cottontail rabbits (up to 10(5.8)PFU/mL nasal flush) and raccoons (up to 10(5.2)PFU/mL nasal flush). Thus, various mammalian species, especially those that are peridomestic, could play a role in the epidemiology of emergent H7N9 IAV. Mammals should be accounted for in biosecurity plans associated with H7N9 and their presence in wet markets, dependent on species, could lead to increased transmission among interspecific species aggregations and may also pose an elevated zoonotic disease risk to visitors and workers of such markets.

Diabolical effects of rabies encephalitis.

Rabies is an acute encephalomyelitis in humans and animals caused by rabies virus (RABV) infection. Because the neuropathological changes are very mild in rabies, it has been assumed that neuronal dysfunction likely explains the severe clinical disease. Recently, degenerative changes have been observed in neuronal processes (dendrites and axons) in experimental rabies. In vitro studies have shown evidence of oxidative stress that is caused by mitochondrial dysfunction. Recent work has shown that the RABV phosphoprotein (P) interacts with mitochondrial Complex I leading to overproduction of reactive oxygen species, which results in injury to axons. Amino acids at positions 139 to 172 of the P are critical in this process. Rabies vectors frequently show behavioral changes. Aggressive behavior with biting is important for transmission of the virus to new hosts at a time when virus is secreted in the saliva. Aggression is associated with low serotonergic activity in the brain. Charlton and coworkers performed studies in experimentally infected striped skunks with skunk rabies virus and observed aggressive behavioral responses. Heavy accumulation of RABV antigen was found in the midbrain raphe nuclei, indicating that impaired serotonin neurotransmission from the brainstem may account for the aggressive behavior. We now have an improved understanding of how RABV causes neuronal injury and how the infection results in behavioral changes that promote viral transmission to new hosts.