Highly pathogenic avian influenza A(H5N1) virus infections on fur farms connected to mass mortalities of black-headed gulls, Finland, July to October 2023.

Highly pathogenic avian influenza (HPAI) has caused widespread mortality in both wild and domestic birds in Europe 2020-2023. In July 2023, HPAI A(H5N1) was detected on 27 fur farms in Finland. In total, infections in silver and blue foxes, American minks and raccoon dogs were confirmed by RT-PCR. The pathological findings in the animals include widespread inflammatory lesions in the lungs, brain and liver, indicating efficient systemic dissemination of the virus. Phylogenetic analysis of Finnish A(H5N1) strains from fur animals and wild birds has identified three clusters (Finland I-III), and molecular analyses revealed emergence of mutations known to facilitate viral adaptation to mammals in the PB2 and NA proteins. Findings of avian influenza in fur animals were spatially and temporally connected with mass mortalities in wild birds. The mechanisms of virus transmission within and between farms have not been conclusively identified, but several different routes relating to limited biosecurity on the farms are implicated. The outbreak was managed in close collaboration between animal and human health authorities to mitigate and monitor the impact for both animal and human health.

First isolation and molecular characterization of canine parvovirus-type 2b (CPV-2b) from red foxes (Vulpes vulpes) living in the wild habitat of Turkey.

BACKGROUND: The canine parvovirus, with its many variants, is responsible for a pivotal and common viral infection affecting millions of dogs and other carnivore species worldwide, particularly the wild ones, which are considered as the main reservoir hosts. To that end, this study investigated the presence of canine parvovirus (CPV) in red foxes (Vulpes vulpes) living in wild habitats of several regions of Turkey. METHODS: We randomly collected 630 archival fox stool specimens from rural areas of 22 provinces and used real-time PCR to detect CPV. RESULTS: Two of the 630 (0.3%) stool samples were positive for CPV-DNA, named Tr-Fox/128(Aydin) and Tr-Fox/159(Manisa). We attempted to isolate the virus in a MDCK cell line, and cytopathic effects were observed four days post-inoculation. Three regions corresponding to the CPV capsid protein VP2 gene from extracted DNA of positive samples were amplified by conventional PCR, and the products were visualised, purified, and Sanger sequenced. Three overlapping DNA raw sequence fragments, were read, assembled, and aligned to obtain approximately 1.5 kb-long regions that cover most of the VP2 gene, then deposited in GenBank. After comparing the isolates with parvovirus sequences data of domestic and wild carnivores by BLAST processing, our isolates' similarity rate with each other was 99.40%, with base differences in 9 nucleotide positions. They were classified as 2b variant closely related to isolates from dogs in Turkey, Egypt, Iraq, Italy, Thailand, and China. CONCLUSION: This study presents evidence of interspecies transmission of CPV, of which there are no reports on prevalence in wildlife carnivores of our country. Identification of CPV in red foxes threatens local and hunting dogs, which may contract the infection or disseminate it to other wild animal species or vice-versa.

Divergent Rabies Virus Variant of Probable Bat Origin in 2 Gray Foxes, New Mexico, USA.

In the Western Hemisphere, bat-associated rabies viruses (RABVs) have established independent transmission cycles in multiple mammal hosts, forming genetically distinct lineages. In New Mexico, USA, skunks, bats, and gray foxes are rabies reservoir hosts and represent a public health risk because of encounters with humans. During 2015 and 2019, two previously undescribed RABVs were detected in 2 gray foxes (Urocyon cinereoargenteus) in Lincoln County, New Mexico. Phylogenetic analysis of the nucleoprotein gene indicated that the isolates are a novel RABV variant. These 2 cases probably represent repeated spillover events from an unknown bat reservoir to gray foxes. Molecular analysis of rabies cases across New Mexico identified that other cross-species transmission events were the result of viral variants previously known to be enzootic to New Mexico. Despite a robust rabies public health surveillance system in the United States, advances in testing and surveillance techniques continue to identify previously unrecognized zoonotic pathogens.

Comparison of intra- and inter-host genetic diversity in rabies virus during experimental cross-species transmission.

The development of high-throughput genome sequencing enables accurate measurements of levels of sub-consensus intra-host virus genetic diversity and analysis of the role played by natural selection during cross-species transmission. We analysed the natural and experimental evolution of rabies virus (RABV), an important example of a virus that is able to make multiple host jumps. In particular, we (i) analyzed RABV evolution during experimental host switching with the goal of identifying possible genetic markers of host adaptation, (ii) compared the mutational changes observed during passage with those observed in natura, and (iii) determined whether the colonization of new hosts or tissues requires adaptive evolution in the virus. To address these aims, animal infection models (dog and fox) and primary cell culture models (embryo brain cells of dog and fox) were developed and viral variation was studied in detail through deep genome sequencing. Our analysis revealed a strong unidirectional host evolutionary effect, as dog-adapted rabies virus was able to replicate in fox and fox cells relatively easily, while dogs or neuronal dog cells were not easily susceptible to fox adapted-RABV. This suggests that dog RABV may be able to adapt to some hosts more easily than other host variants, or that when RABV switched from dogs to red foxes it lost its ability to adapt easily to other species. Although no difference in patterns of mutation variation between different host organs was observed, mutations were common following both in vitro and in vivo passage. However, only a small number of these mutations also appeared in natura, suggesting that adaptation during successful cross-species virus transmission is a complex, multifactorial evolutionary process.

The biological characteristics of the canine adenovirus type 1 from fox and the transcriptome analysis of the infected MDCK cell.

Canine adenovirus type 1 (CAdV-1) is the etiologic agent of fox encephalitis, and a virus strain from fox encephalitis is isolated and related research are conducted. In this experiment, the results showed that the F1301 strain was confirmed to be the CAdV-1. The whole genome of the CAdV-1 F1301 strain isolated from fox was 30,535 bp and had higher homology to the other reported CAdV-1 strains. After 0, 12, and 36 h of CAdV-1 infection, the difference gene of the 592 long noncoding RNA and 11,215 microRNA were involved in cell responses to CAdV-1 infection through the PI3K-AKT, Wnt, Herpes simplex, hepatitis C, and Epstein-Barr virus infection pathway in Madin-Darby canine kidney cell line (MDCK). The results indicate that the biological characterization of the CAdV-1 and the MDCK cell-CAdV-1 interaction are clarified.

Limited Intrahost Diversity and Background Evolution Accompany 40 Years of Canine Parvovirus Host Adaptation and Spread.

Canine parvovirus (CPV) is a highly successful pathogen that has sustained pandemic circulation in dogs for more than 40 years. Here, integrating full-genome and deep-sequencing analyses, structural information, and in vitro experimentation, we describe the macro- and microscale features that accompany CPV's evolutionary success. Despite 40 years of viral evolution, all CPV variants are more than ∼99% identical in nucleotide sequence, with only a limited number (<40) of substitutions becoming fixed or widespread during this time. Notably, most substitutions in the major capsid protein (VP2) gene are nonsynonymous, altering amino acid residues that fall within, or adjacent to, the overlapping receptor footprint or antigenic regions, suggesting that natural selection has channeled much of CPV evolution. Among the limited number of variable sites, CPV genomes exhibit complex patterns of variation that include parallel evolution, reversion, and recombination, compromising phylogenetic inference. At the intrahost level, deep sequencing of viral DNA in original clinical samples from dogs and other host species sampled between 1978 and 2018 revealed few subconsensus single nucleotide variants (SNVs) above ∼0.5%, and experimental passages demonstrate that substantial preexisting genetic variation is not necessarily required for rapid host receptor-driven adaptation. Together, these findings suggest that although CPV is capable of rapid host adaptation, a relatively low mutation rate, pleiotropy, and/or a lack of selective challenges since its initial emergence have inhibited the long-term accumulation of genetic diversity. Hence, continuously high levels of inter- and intrahost diversity are not necessarily required for virus host adaptation.IMPORTANCE Rapid mutation rates and correspondingly high levels of intra- and interhost diversity are often cited as key features of viruses with the capacity for emergence and sustained transmission in a new host species. However, most of this information comes from studies of RNA viruses, with relatively little known about evolutionary processes in viruses with single-stranded DNA (ssDNA) genomes. Here, we provide a unique model of virus evolution, integrating both long-term global-scale and short-term intrahost evolutionary processes of an ssDNA virus that emerged to cause a pandemic in a new host animal. Our analysis reveals that successful host jumping and sustained transmission does not necessarily depend on a high level of intrahost diversity nor result in the continued accumulation of high levels of long-term evolution change. These findings indicate that all aspects of the biology and ecology of a virus are relevant when considering their adaptability.

Phylodynamic analysis of two amino acid substitutions in the hemagglutinin protein of canine distemper virus strains detected in fur-bearing animals in China.

Canine distemper virus (CDV) causes a highly contagious disease in a wide range of carnivores. The hemagglutinin (H) protein of viruses shows the highest variability and plays an important role in modulation of viral antigenicity, virulence, and receptor recognition. Since 2012, canine distemper (CD) outbreaks in fur-bearing animals (minks, foxes, raccoon dogs) caused by CDV variants with I542N and Y549H substitutions in the H protein have been frequently reported in China. To characterize the molecular evolutionary dynamics and epidemiological dynamics of CDV, 235 H gene sequences of CDV wild-type strains collected from 22 countries between 1975 and 2015, including 44 strains predominant in fur-bearing animals in China, were analyzed. The phylogenetic relationships and evolutionary rates of the CDV strains were determined by Bayesian phylogenetics. The CDV strains clustered into distinct geographic genotypes, irrespective of the species of isolation. All the variant strains formed a distinct monophyletic cluster and belonged to the F sub-genotype within the Asia-1 genotype-currently the predominant sub-genotype in fur-bearing animals in China. Evolutionary analysis suggested that the variant strains originated in 2006. Furthermore, the selection pressure analysis revealed that the Y549H substitution was under positive selection pressure for adaptation toward the fur-bearing animals. The residue at position 549 also showed structural interaction with the V domain of the mink signaling lymphocyte-activation molecule (SLAM) receptor based on the homology modeling of the H-SLAM complex. Our results suggested that the Y549H substitution contributed to the molecular adaptation of CDV variants in the fur-bearing animals during the viral evolutionary phase in China.

Prioritisation of areas for early detection of southward movement of arctic fox rabies based on historical surveillance data in Quebec, Canada.

Arctic rabies virus variant (ARVV) is enzootic in Quebec (Canada) north of the 55th parallel. With climate change, increased risk of re-incursion of ARVV in more densely populated southern regions raises public and animal health concerns. The objective of this study was to prioritise geographical areas to target for an early detection of ARVV incursion south of the 55th parallel based on the historical spatio-temporal trends of reported rabies in foxes in Quebec. Descriptive analyses of fox rabies cases from 1953 to 2017 were conducted. Three periods show increases in the number of fox rabies cases in southern regions and indicate incursion from northern areas or neighbouring provinces. The available data, particularly in central and northern regions of the province, were scarce and of low spatial resolution, making it impossible to identify the path of spread with precision. Hence, we investigated the use of multiple criteria, such as historical rabies cases, human population density and red fox (Vulpes vulpes) relative abundance, to prioritise areas for enhanced surveillance. This study underscores the need to define and maintain new criteria for selecting samples to be analysed in order to detect rapidly ARVV cases outside the current enzootic area and any potential re-incursion of the virus into central and southern regions of the province.

Historical analysis of epizootiological situation of rabies on the territory of the Slovak Republic.

Currently, Slovakia is a rabies-free country, but the epizootiological situation of rabies was not always favorable. The main reservoir species of rabies virus in the first half of the last century was the domestic dog. Since 1906, hundreds of cases were reported, of which approximately 90% were infected dogs. The disease had a typical urban character. Since 1929, the number of rabid domestic animals decreased due to the implementation of dog vaccination campaigns in particular parts of Slovakia. From the second half of 1950s, red foxes (Vulpes vulpes) have become an important reservoir of the RABV. In this time period urban rabies in Slovakia changed into sylvatic form. One effective method of prevention and control of wildlife rabies is an oral rabies vaccination of red foxes. It is carried out in Slovakia since 1993. A detailed development of the rabies epizootiological situation on the territory of the Slovak Republic until the application of oral antirabies immunisation of foxes and the current situation after its performance is the main object of this review. Keywords: rabies; Lyssavirus; red fox; incidence; oral vaccination.

Highly Pathogenic Swine Getah Virus in Blue Foxes, Eastern China, 2017.

We isolated Getah virus from infected foxes in Shandong Province, eastern China. We sequenced the complete Getah virus genome, and phylogenetic analysis revealed a close relationship with a highly pathogenic swine epidemic strain in China. Epidemiologic investigation showed that pigs might play a pivotal role in disease transmission to foxes.

Development and evaluation of a direct TaqMan qPCR assay for the rapid detection of diverse carnivore amdoparvoviruses.

Amdoparvoviruses infect carnivore species, including mink, raccoon dog, fox, skunk, and red panda. Amdoparvovirus infection is a major cause of morbidity and mortality in farmed minks. Here, we developed a direct TaqMan qPCR assay for detection and quantification of carnivore amdoparvoviruses by using three primers and one probe based on the conserved VP2 gene. The detection limit for Aleutian mink disease virus (AMDV) and Raccoon dog and arctic fox amdoparvovirus (RFAV) were 4.06 × 101 copies/µl and 2.93 × 101 copies/µl, respectively. Both intra- and inter-assay variability were less than 2%. Among 74 carnivore samples, the positive rates for amdoparvoviruses were 62.2% (46/74) by direct TaqMan qPCR, while only 40.5% (30/74) by SYBR Green I qPCR. This result suggests that the direct TaqMan qPCR was more sensitive than the SYBR Green I qPCR. Additionally, the direct TaqMan qPCR is a rapid and sensitive method for liquid samples at microliter level as the assay employed the direct alkaline lysis method to obtain viral DNA and, therefore, eliminated the cumbersome steps in extracting DNA. Overall, the direct TaqMan qPCR assay possessed high specificity, sensitivity, and reproducibility, indicating that it can be used as a powerful tool for detection and quantification of various carnivore amdoparvoviruses in epidemiological and pathogenesis studies.

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.

Group A Rotavirus Associated with Encephalitis in Red Fox.

In 2011, a group A rotavirus was isolated from the brain of a fox with encephalitis and neurologic signs, detected by rabies surveillance in Italy. Intracerebral inoculation of fox brain homogenates into mice was fatal. Genome sequencing revealed a heterologous rotavirus of avian origin, which could provide a model for investigating rotavirus neurovirulence.

An Investigation of the First Case of Human Rabies Caused by a Fox in China in May 2016.

This study assesses the causes and prevention measures of rabies through epidemiological investigation and analysis. A field epidemiological survey was conducted to investigate a case of rabies by fox bite. The onset of symptoms began 50 days after the bite. The patient did not receive standard treatment, rabies vaccination, or rabies immunoglobulin injection. The fox was killed on the spot. Saliva and pre-death blood samples were collected at different periods, and only blood RT-PCR tests yielded positive results. Wild fox bite is a major risk factor of rabies infection in Xinjiang Province, China.

Population structure of two rabies hosts relative to the known distribution of rabies virus variants in Alaska.

For pathogens that infect multiple species, the distinction between reservoir hosts and spillover hosts is often difficult. In Alaska, three variants of the arctic rabies virus exist with distinct spatial distributions. We tested the hypothesis that rabies virus variant distribution corresponds to the population structure of the primary rabies hosts in Alaska, arctic foxes (Vulpes lagopus) and red foxes (Vulpes vulpes) to possibly distinguish reservoir and spillover hosts. We used mitochondrial DNA (mtDNA) sequence and nine microsatellites to assess population structure in those two species. mtDNA structure did not correspond to rabies virus variant structure in either species. Microsatellite analyses gave varying results. Bayesian clustering found two groups of arctic foxes in the coastal tundra region, but for red foxes it identified tundra and boreal types. Spatial Bayesian clustering and spatial principal components analysis identified 3 and 4 groups of arctic foxes, respectively, closely matching the distribution of rabies virus variants in the state. Red foxes, conversely, showed eight clusters comprising two regions (boreal and tundra) with much admixture. These results run contrary to previous beliefs that arctic fox show no fine-scale spatial population structure. While we cannot rule out that the red fox is part of the maintenance host community for rabies in Alaska, the distribution of virus variants appears to be driven primarily by the arctic fox. Therefore, we show that host population genetics can be utilized to distinguish between maintenance and spillover hosts when used in conjunction with other approaches.

Screening red foxes (Vulpes vulpes) for possible viral causes of encephalitis.

BACKGROUND: Next to various known infectious and non-infectious causes, the aetiology of non-suppurative encephalitis in red foxes (Vulpes vulpes) often remains unclear. Known causes in foxes imply rabies, canine distemper, toxoplasmosis, Aujeszky's disease, as well as parvovirus, adenovirus, circovirus and flavivirus infections. In this study, particular attention was paid on bornaviruses, since red foxes are predators of bicoloured white-toothed shrews, a reservoir of Borna disease virus 1 (BoDV-1). In addition, foxes are known to be highly susceptible for viruses of the order Mononegavirales. METHODS: Analyses for the presence of anti-BoDV-1 antibodies, BoDV-1-RNA and antigen were performed on 225 blood and 59 brain samples, from a total of 232 red foxes. Foxes originated from BoDV-1 endemic and non-endemic German areas. Additional investigations for the presence of rabies, canine distemper, toxoplasmosis, Aujeszky's disease, parvovirus, adenovirus and flavivirus infections were carried out on 16 red foxes with non-suppurative (meningo-) encephalitis. A metagenomic analysis was used on three representative brain samples displaying encephalitis. RESULTS: Among 225 foxes, 37 displayed anti-BoDV-1 antibodies with titres ranging between 1:40 and 1:2560, regardless of geographic origin. In 6 out of 16 foxes with encephalitis, canine distemper virus was detected. No evidence of any of the other investigated agents was found in the 16 fox brains with encephalitis. Metagenomics revealed no infectious agents, except for one already known canine distemper case. CONCLUSION: Red foxes can exhibit BoDV-1 specific antibodies without association with geographic origin or encephalitis due to bornavirus infection. The encephalitis pattern was highly conspicuous for a viral infection, but remained unclear in 10 out of 16 foxes. Thus, presently unknown infectious and non-infectious causes need to be considered and further investigated, especially since foxes also tend to occur in human proximity.

Pseudorabies in farmed foxes fed pig offal in Shandong province, China.

Pseudorabies (PR, Aujeszky's disease) is an acute, highly contagious viral disease resulting in major economic losses to the swine industry. PR is endemic in wild and domestic animals, although its natural host is the pig. Here, we report an outbreak of PR in foxes on a fur-producing farm in Yuncheng county, Shandong, China, that were fed pig offal. The diagnosis of PR was based on nervous signs and standard PCR methods and by isolation of PRV from fox brain tissue in Vero cells. The diagnosis was confirmed by an indirect immunofluorescence assay and electron microscopy. Phylogenetic analysis of a partial (804 nt) viral glycoprotein gC gene sequence indicated that it was likely to be a field strain closely related to a cluster of PRV previously identified in China.

Development and evaluation of the RT-PCR kit for the rabies virus diagnosis.

To improve the diagnosis, surveillance, and control for the rabies virus, a kit for hybridization-triggered fluorescence detection of rabies virus DNA by the RT-PCR technique was developed and evaluated. The analytical sensitivity of the kit was 4*10 GE per ml. High specificity of the kit was shown using representative sampling of viral, bacterial, and human nucleic acids.

Detection of Circovirus in Foxes with Meningoencephalitis, United Kingdom, 2009-2013.

A fox circovirus was identified in serum samples from foxes with unexplained neurologic signs by using viral metagenomics. Fox circovirus nucleic acid was localized in histological lesions of the cerebrum by in situ hybridization. Viruses from the family Circoviridae may have neurologic tropism more commonly than previously anticipated.

Genetic characterisation of the rabies virus vaccine strains used for oral immunization of foxes in Poland to estimate the effectiveness of vaccination.

The main reservoir of rabies virus in Poland has been the red fox. To control rabies in wildlife, oral immunization of foxes was introduced in 1993. The vaccine is effective when it confers immunity against the virus circulating in the environment. To assess the above issue, a study of the molecular characteristics of 570-bp fragments of the N and G genes of vaccine strains SAD B19 and SAD Bern against street virus strains was performed. The results confirmed the similarity of the vaccine strains and rabies virus strains circulating in the environment and also demonstrate the genetic stability of vaccine strains that have been distributed in Poland for 20 years.