Diverse amdoparvoviruses infection of farmed Asian badgers (Meles meles).

Amdoparvoviruses infect various carnivores, including mustelids, canids, skunks, and felids. Aleutian mink disease virus (AMDV) belongs to the prototypical species Amdoparvovirus carnivoran1. Here, we identified a novel amdoparvovirus in farmed Asian badgers (Meles meles), and we named this virus "Meles meles amdoparvovirus" (MMADV). A total of 146 clinical samples were collected from 134 individual badgers, and 30.6% (41/134) of the sampled badgers tested positive for amdoparvovirus by PCR. Viral DNA was detected in feces, blood, spleen, liver, lung, and adipose tissue from these animals. Viral sequences from eight samples were determined, five of which represented nearly full-length genome sequences (4,237-4,265 nt). Six serum samples tested positive by PCR, CIEP, and IAT, four of which had high antibody titers (> 512) against AMDV-G. Twenty-six of the 41 amdoparvovirus-positive badgers showed signs of illness, and necropsy revealed lesions in their organs. Sequence comparisons and phylogenetic analysis of the viral NS1 and VP2 genes of these badger amdoparvoviruses showed that their NS1 proteins shared 62.6%-88.8% sequence identity with known amdoparvoviruses, and they clustered phylogenetically into two related clades. The VP2 proteins shared 76.6%-97.2% identity and clustered into two clades, one of which included raccoon dog and arctic fox amdoparvovirus (RFAV), and the other of which did not include other known amdoparvoviruses. According to the NS1-protein-based criterion for parvovirus species demarcation, the MMADV isolate from farm YS should be classified as a member of a new species of the genus Amdoparvovirus. In summary, we have discovered a novel MMADV and other badger amdoparvoviruses that naturally infect Asian badgers and are possibly pathogenic in badgers.

Strong selection signatures for Aleutian disease tolerance acting on novel candidate genes linked to immune and cellular responses in American mink (Neogale vison).

Aleutian disease (AD) is a multi-systemic infectious disease in American mink (Neogale vison) caused by Aleutian mink disease virus (AMDV). This study aimed to identify candidate regions and genes underlying selection for response against AMDV using whole-genome sequence (WGS) data. Three case-control selection signatures studies were conducted between animals (N = 85) producing high versus low antibody levels against AMDV, grouped by counter immunoelectrophoresis (CIEP) test and two enzyme-linked immunosorbent assays (ELISA). Within each study, selection signals were detected using fixation index (FST) and nucleotide diversity (θπ ratios), and validated by cross-population extended haplotype homozygosity (XP-EHH) test. Within- and between-studies overlapping results were then evaluated. Within-studies overlapping results indicated novel candidate genes related to immune and cellular responses (e.g., TAP2, RAB32), respiratory system function (e.g., SPEF2, R3HCC1L), and reproduction system function (e.g., HSF2, CFAP206) in other species. Between-studies overlapping results identified three large segments under strong selection pressure, including two on chromosome 1 (chr1:88,770-98,281 kb and chr1:114,133-120,473) and one on chromosome 6 (chr6:37,953-44,279 kb). Within regions with strong signals, we found novel candidate genes involved in immune and cellular responses (e.g., homologous MHC class II genes, ITPR3, VPS52) in other species. Our study brings new insights into candidate regions and genes controlling AD response.

Genomic characterization and phylogenetic analysis of Aleutian mink disease virus identified in a sudden death mink case.

Aleutian mink disease (AMD) is one of the most serious diseases in minks worldwide, it brings tremendous financial losses in mink farming. AMD virus (AMDV) has unusually high genetic diversity, its genomic structure remains unclear. In 2014, sudden death of breeding minks was occurred in northeast China. After clinical signs evaluation and virus isolation, AMDV was identified in all sudden death minks, we investigated the complete genomic sequence of AMDV-LM isolated from the sudden death case. The full-genome sequence of AMDV-LM was 7 nucleotides (nts) or 8 nts longer than isolates AMDV-BJ and AMDV-G. AMDV-LM contained two unique nucleotide changes in VP2 (G79T, T710C), which led to two amino acid changes G27W and L237S. For NS1, some unique point mutations, such as A374C, A428C, A463C, and T476A were found and resulted in four unique amino acid mutations at N24V, H125P, V143P, K155Q, and V159N, respectively. The predicted secondary structure of the 5' terminal of AMDV-LM formed a large bubble formation near the 5' end, which affected the stability of the U-shaped hairpin. Phylogenetic analysis demonstrated that AMDV-LM was closely related to Chinese isolates and confirmed that AMDV strains circulating in China had different origins of ancestors. This study was first to investigate the association of sudden death of adult breeding minks with AMDV infection. Our findings provide useful suggestions for evaluation of the pathogenic potential of AMDV, additional details on AMDV genome characterization were also presented. Future work should focus on the importance of AMDV-LM strain in mink infection.

Long-term antibody production and viremia in American mink (Neovison vison) challenged with Aleutian mink disease virus.

BACKGROUND: Selecting American mink (Neovison vison) for tolerance to Aleutian mink disease virus (AMDV) has gained popularity in recent years, but data on the outcomes of this activity are scant. The objectives of this study were to determine the long-term changes in viremia, seroconversion and survival in infected mink. Mink were inoculated intranasally with a local isolate of Aleutian mink disease virus (AMDV) over 4 years (n = 1742). The animals had been selected for tolerance to AMDV for more than 20 years (TG100) or were from herds free of AMDV (TG0). The progenies of TG100 and TG0, and their crosses with 25, 50 and 75% tolerance ancestry were also used. Blood samples were collected from each mink up to 14 times until 1211 days post-inoculation (dpi) and were tested for viremia by PCR and for anti-AMDV antibodies by counter-immunoelectrophoresis (CIEP). Viremia and CIEP status were not considered when selecting replacements. Low-performing animals were pelted and the presence of antibodies in their blood and antibody titer were measured by CIEP, and viremia and viral DNA in seven organs (n = 936) were tested by PCR. RESULTS: The peak incidences of viremia (66.7%) and seropositivity (93.5%) were at 35 dpi. The incidence of viremia decreased over time while the incidence of seroconversion increased. The least-squares means of the incidence of PCR positive of lymph node (0.743) and spleen (0.656) were significantly greater than those of bone marrow, liver, kidneys, lungs and small intestine (0.194 to 0.342). Differences in tolerant ancestry were significant for every trait measured. Incidences of viremia over time, terminal viremia, seropositivity over time, AMDV DNA in organs and antibody titer were highest in the susceptible groups (TG0 or TG25) and lowest in the tolerant groups (TG100 or TG75). CONCLUSION: Previous history of selection for tolerance resulted in mink with reduced viral replication and antibody titer. Viremia had a negative effect and antibody production had a positive effect on survival and productivity.

Capsid Structure of Aleutian Mink Disease Virus and Human Parvovirus 4: New Faces in the Parvovirus Family Portrait.

Parvoviruses are small, single-stranded DNA viruses with non-enveloped capsids. Determining the capsid structures provides a framework for annotating regions important to the viral life cycle. Aleutian mink disease virus (AMDV), a pathogen in minks, and human parvovirus 4 (PARV4), infecting humans, are parvoviruses belonging to the genera Amdoparvovirus and Tetraparvovirus, respectively. While Aleutian mink disease caused by AMDV is a major threat to mink farming, no clear clinical manifestations have been established following infection with PARV4 in humans. Here, the capsid structures of AMDV and PARV4 were determined via cryo-electron microscopy at 2.37 and 3.12 Å resolutions, respectively. Despite low amino acid sequence identities (10-30%) both viruses share the icosahedral nature of parvovirus capsids, with 60 viral proteins (VPs) assembling the capsid via two-, three-, and five-fold symmetry VP-related interactions, but display major structural variabilities in the surface loops when the capsid structures are superposed onto other parvoviruses. The capsid structures of AMDV and PARV4 will add to current knowledge of the structural platform for parvoviruses and permit future functional annotation of these viruses, which will help in understanding their infection mechanisms at a molecular level for the development of diagnostics and therapeutics.

Mechanisms behind the varying severity of Aleutian mink disease virus: Comparison of three farms with a different disease status.

Aleutian mink disease virus (AMDV) is distributed widely among mink farms and wild mustelids despite ongoing attempts to stop the spread. The severity of Aleutian disease (AD) varies from subclinical to fatal but the reasons for its varying severity are complex and unclear. Recently, breeding of tolerant mink has drawn attention as the possible solution to reduce the effects of AD in farms. The aim of this study was to gather information on the effects of breeding based on overall health, production traits, and antibody titer on AD severity by comparing a positive farm (farm 1) that has been breeding for tolerance in mink to an infected farm without tolerance selection, and an AMDV-free farm. During the 2.5-year follow-up, the mink in farm 1 remained mostly free of clinical AD, had normal pelt quality and litter size, and had low virus copy numbers in tissues and low antibody titers in ELISA. In histopathological studies, most of the farm 1 mink had no/mild lesions in their kidneys. 29-43% of the mink were ELISA negative but PCR positive throughout the follow-up and frequent changes in virus strains and coinfections were observed. Several differences in gene expression between animals from different farms were also detected. These results indicate that the disease burden of AMDV can be reduced, with seemingly normal health and production rates, despite continual circulation of ADMV in cases where eradication attempts are unsuccessful.

Impact of viral features, host jumps and phylogeography on the rapid evolution of Aleutian mink disease virus (AMDV).

Aleutian mink disease virus (AMDV) is one the most relevant pathogens of domestic mink, where it can cause significant economic losses, and wild species, which are considered a threat to mink farms. Despite their relevance, many aspects of the origin, evolution, and geographic and host spreading patterns of AMDV have never been investigated on a global scale using a comprehensive biostatistical approach. The present study, benefitting from a large dataset of sequences collected worldwide and several phylodynamic-based approaches, demonstrates the ancient origin of AMDV and its broad, unconstrained circulation from the initial intercontinental spread to the massive among-country circulation, especially within Europe, combined with local persistence and evolution. Clear expansion of the viral population size occurred over time until more effective control measures started to be applied. The role of frequent changes in epidemiological niches, including different hosts, in driving the high nucleotide and amino acid evolutionary rates was also explored by comparing the strengths of selective pressures acting on different populations. The obtained results suggest that the viral passage among locations and between wild and domesticated animals poses a double threat to farm profitability and animal welfare and health, which is particularly relevant for endangered species. Therefore, further efforts must be made to limit viral circulation and to refine our knowledge of factors enhancing AMDV spread, particularly at the wild-domestic interface.

Genetic and phenotypic parameters for Aleutian disease tests and their correlations with pelt quality, reproductive performance, packed-cell volume, and harvest length in mink.

Aleutian disease (AD), caused by the Aleutian mink disease virus (AMDV), is a major health concern that results in global economic losses to the mink industry. The unsatisfactory outcome of the culling strategy, immunoprophylaxis, and medical treatment in controlling AD have urged mink farmers to select AD resilient mink based on several detection tests, including enzyme-linked immunosorbent assay (ELISA), counterimmunoelectrophoresis (CIEP), and iodine agglutination test (IAT). However, the genetic analysis of these AD tests and their correlations with pelt quality, reproductive performance, packed-cell volume (PCV), and harvest length (HL) have not been investigated. In this study, data on 5,824 mink were used to estimate the genetic and phenotypic parameters of four AD tests, including two systems of ELISA, CIEP, and IAT, and their genetic and phenotypic correlations with two pelt quality, five female reproductive performance, PCV, and HL traits. Significances (P < 0.05) of fixed effects (sex, year, dam age, and color type), covariates (age at harvest and blood sampling), and random effects (additive genetic, permanent environmental, and maternal effects) were determined under univariate models using ASReml 4.1 software. The genetic and phenotypic parameters for all traits were estimated under bivariate models using ASReml 4.1 software. Estimated heritabilities (±SE) were 0.39 ± 0.06, 0.61 ± 0.07, 0.11 ± 0.07, and 0.26 ± 0.05 for AMDV antigen-based ELISA (ELISA-G), AMDV capsid protein-based ELISA, CIEP, and IAT, respectively. The ELISA-G also showed a moderate repeatability (0.58 ± 0.04) and had significant negative genetic correlations (±SE) with reproductive performance traits (from -0.41 ± 0.16 to -0.49 ± 0.12), PCV (-0.53 ± 0.09), and HL (-0.45 ± 0.16). These results indicated that ELISA-G had the potential to be applied as an indicator trait for genetic selection of AD resilient mink in AD endemic ranches and therefore help mink farmers to reduce the adverse effects caused by AD.

Detection of selection signatures for response to Aleutian mink disease virus infection in American mink.

Aleutian disease (AD) is the most significant health issue for farmed American mink. The objective of this study was to identify the genomic regions subjected to selection for response to infection with Aleutian mink disease virus (AMDV) in American mink using genotyping by sequencing (GBS) data. A total of 225 black mink were inoculated with AMDV and genotyped using a GBS assay based on the sequencing of ApeKI-digested libraries. Five AD-characterized phenotypes were used to assign animals to pairwise groups. Signatures of selection were detected using integrated measurement of fixation index (FST) and nucleotide diversity (θπ), that were validated by haplotype-based (hap-FLK) test. The total of 99 putatively selected regions harbouring 63 genes were detected in different groups. The gene ontology revealed numerous genes related to immune response (e.g. TRAF3IP2, WDR7, SWAP70, CBFB, and GPR65), liver development (e.g. SULF2, SRSF5) and reproduction process (e.g. FBXO5, CatSperß, CATSPER4, and IGF2R). The hapFLK test supported two strongly selected regions that contained five candidate genes related to immune response, virus-host interaction, reproduction and liver regeneration. This study provided the first map of putative selection signals of response to AMDV infection in American mink, bringing new insights into genomic regions controlling the AD phenotypes.

Aptamer-targeting of Aleutian mink disease virus (AMDV) can be an effective strategy to inhibit virus replication.

Aleutian mink disease (AMD), which is caused by Aleutian mink disease virus (AMDV), is an important contagious disease for which no effective vaccine is yet available. AMD causes major economic losses for mink farmers globally and threatens some carnivores such as skunks, genets, foxes and raccoons. Aptamers have exciting potential for the diagnosis and/or treatment of infectious viral diseases, including AMD. Using a magnetic beads-based systemic evolution of ligands by exponential enrichment (SELEX) approach, we have developed aptamers with activity against AMDV after 10 rounds of selection. After incubation with the ADVa012 aptamer (4 µM) for 48 h, the concentration of AMDV in the supernatant of infected cells was 47% lower than in the supernatant of untreated cells, whereas a random library of aptamers has no effect. The half-life of ADVa012 was ~ 32 h, which is significantly longer than that of other aptamers. Sequences and three dimensions structural modeling of selected aptamers indicated that they fold into similar stem-loop structures, which may be a preferred structure for binding to the target protein. The ADVa012 aptamer was shown to have an effective and long-lasting inhibitory effect on viral production in vitro.

Molecular epidemiology of Aleutian mink disease virus causing outbreaks in mink farms from Southwestern Europe: a retrospective study from 2012 to 2019.

BACKGROUND: Aleutian mink disease virus (AMDV) causes major economic losses in fur-bearing animal production. The control of most AMDV outbreaks is complex due to the difficulties of establishing the source of infection based only on the available on-farm epidemiological data. In this sense, phylogenetic analysis of the strains present in a farm may help elucidate the origin of the infection and improve the control and biosecurity measures. OBJECTIVES: This study had the following aims: characterize the AMDV strains from most outbreaks produced at Spanish farms between 2012-2019 at the molecular level, and assess the utility of the combined use of molecular and epidemiological data to track the possible routes of infection. METHODS: Thirty-seven strains from 17 farms were partially sequenced for the NS1 and VP2 genes and analyzed phylogenetically with other strains described worldwide. RESULTS: Spanish AMDV strains are clustered in four major clades that generally show a good geographical correlation, confirming that most had been established in Spain a long time ago. The combined study of phylogenetic results and epidemiological information of each farm suggests that most of the AMDV outbreaks since 2012 had been produced by within-farm reservoirs, while a few of them may have been due to the introduction of the virus through international trade. CONCLUSIONS: The combination of phylogenetic inference, together with epidemiological data, helps assess the possible origin of AMDV infections in mink farms and improving the control and prevention of this disease.

Molecular epidemiology of Aleutian mink disease virus from fecal swab of mink in northeast China.

BACKGROUND: Aleutian mink disease parvovirus (AMDV) causes Aleutian mink disease (AMD), which is a serious infectious disease of mink. The aim of this study was to get a better understanding of the molecular epidemiology of AMDV in northeast China to control and prevent AMD from further spreading. This study for the first time isolated AMDV from fecal swab samples of mink in China. RESULTS: A total of 157/291 (54.0%) of the fecal swab samples were positive for AMDV. Of these, 23 AMDV positive samples were randomly selected for sequence alignment and phylogenetic analysis based on the acquired partial fragments of VP2 gene with the hypervariable region. Comparative DNA sequence analysis of 23 AMDV isolates with a reference nonpathogenic (AMDV-G) strain revealed 8.3% difference in partial VP2 nucleotide sequences. Amino acid alignment indicated the presence of several genetic variants, as well as one single amino acid residue deletion. The most concentrated area of variation was located in the hypervariable region of VP2 protein. According to phylogenetic analysis, the Chinese AMDV strains and the other reference AMDV strains from different countries clustered into three groups (clades A, B and C). Most of the newly sequenced strains were found to form a Chinese-specific group, which solely consisted of Chinese AMDV strains. CONCLUSION: These findings indicated that a high genetic diversity was found in Chinese AMDV strains and the virus distribution were not dependent on geographical origin. Both local and imported AMDV positive species were prevalent in the Chinese mink farming population. The genetic evidence of AMDV variety and epidemic isolates have importance in mink farming practice.

Dose response of black American mink to Aleutian mink disease virus.

INTRODUCTION: Aleutian mink disease virus (AMDV) causes a serious health problem for mink globally. The disease has no cure nor an effective vaccine and selection for tolerance using antibody titer is adopted by many mink farmers. The objective of this study was to investigate the effects of various doses of a local AMDV isolate on the response of black American mink to infection with AMDV. METHODS: Eight black American mink were each inoculated intranasally with 0.5 mL of eight serial 10-fold dilutions (100 to 10-7 ) of a 10% spleen homogenate containing a local AMDV isolate. Blood samples were collected on days 0, 20, 35, 56, 84, 140, and 196 postinoculation (dpi). Anti-AMDV antibodies and viral DNA were tested by counter-immunoelectrophoresis (CIEP) and PCR, respectively. Animals that were PCR or CIEP positive at 196 dpi (n = 41) were killed at 218 dpi, and samples of blood and seven organs were tested by CIEP and PCR. RESULTS: Antibody production persisted in all seroconverted mink until the termination of the experiment, whereas 71.1% of the mink showed short-lived viremia. Significant associations were observed between inoculum dose and the incidence of viremia until 84 dpi which disappeared thereafter, whereas associations between inoculum dose and the incidence of seropositive mink were significant on all sampling occasions. Antibody titer at 218 dpi significantly decreased with decreasing inoculum dose. AMDV DNA was detected in the bone marrow, lymph nodes, and spleen samples of almost all mink inoculated at every dose but was not detected in other organs of some mink. CONCLUSIONS: CIEP is more accurate than PCR for detecting AMDV infection in mink. Using antibody titer in naturally infected mink may not be accurate for the identification of tolerant mink.

Mink Aleutian disease seroprevalence in China during 1981-2017: A systematic review and meta-analysis.

Mink Aleutian disease (AMD) is the first of the three major diseases of fur animals. It is a common immunosuppressive disease in mink farms worldwide, which seriously endangers the development of the mink farming industry. Strengthening the understanding of the positive serum rate and spatial distribution of AMD is of great significance for the prevention and control of disease caused by the Aleutian virus. Therefore, we conducted a systematic review and meta-analysis to assess the seroprevalence of AMD in China. We extracted 45 studies related to the seroprevalence of Chinese AMD, with samples taken between 1981 and 2017. Our systematic review and meta-analysis results show that, during the selected period, the overall positive rate of AMD in China was 55.3% (95% CI 48.5-62.0). The results from subgroups analysis of the potential risk factors showed that the seroprevalence rate of AMD in China in the past 36 years rose from 48% (95% CI 37.0-60.5) in 1981-2009 to 61.4% (95% CI 43.6-79.3) in 2010-2017. The date of the spatial difference in AMD seroprevalence indicated that AMD seroprevalence was unevenly distributed in different regions: the number of mink in eastern China and northeastern China was relatively high, and the seroprevalence rates were 57.9%, (95% CI 46.2-69.7) and 61.3% (95% CI 53.1-69.5), respectively. Central China had the highest seroprevalence rate of AMD at 69.8% (95% CI 64.4-75.2). At the provincial level, the AMD seroprevalence rate in Jiangsu was as high as 96% (95% CI 94.1-97.8), and the AMD seroprevalence rate in Shaanxi was the lowest at 22.1% (95% CI 20.3-23.9). This suggested that the AMD seroprevalence rate in China was unevenly distributed. In other subgroups, the positive rate of AMD in adult mink was higher than in juvenile mink. This implied that the high prevalence of AMD in China was caused by multiple factors. The meta-regression results indicated that the detection method subgroup (P = 0.008) may be the source of heterogeneity. Our data system evaluated the prevalence of Aleutian disease in China in the last 37 years and a preliminary discussion on the risk factors of AMD. It may help prevent and control AMD in China. It is recommended to conduct further epidemiological testing and develop a comprehensive testing plan to determine the risk factors associated with Aleutian disease and improve the Aleutian disease control strategy.

Development of an EvaGreen-based real-time PCR assay for detection of Aleutian mink disease virus.

The objective of this study was to develop a rapid, sensitive and specific EvaGreen (EG)-based real-time PCR assay capable of detecting Aleutian mink disease virus (AMDV) and to evaluate the reliability of the assay for analysis of blood or tissue samples. For this assay, a pair of primers was designed based on a nonstructural protein (NS)-encoding gene of AMDV, and the identity of PCR products was identified based on a melting temperature of 82.8°C. The EG-based real-time PCR assay did not detect canine distemper virus or mink enteritis virus, and the assay could be used to detect Chinese and American AMDV strains, in contrast to a commercial TaqMan kit that could only detect American AMDV strains. The amplification efficiencies of the EG assay were 104.8% for the Chinese strain and 94.4% for the American strain, and the detection limit was 1 copy/µL of AMDV plasmid or 3 pg/µL of viral DNA (Chinese strain). The intra- and inter-assay variation coefficients of melting temperature were all lower than 0.15%, confirming the high reproducibility of the assay. Forty-five clinical blood samples were simultaneously analyzed using the EG real-time PCR, TaqMan kit and conventional PCR, and the detection rates were 91.1%, 0.0% and 86.7%, respectively. Serum samples were also collected from the corresponding blood samples and tested using the counterimmunoelectrophoresis (CIEP) assay, where positive samples accounted for 24.4% of the 45 samples. In conclusion, EG-based real-time PCR is a rapid, sensitive, universal assay that can be effectively utilized as a reliable and specific tool for detection and quantitation of AMDV.

Development and validation of nucleic acid tests to diagnose Aleutian mink disease virus.

Aleutian disease (AD), caused by Aleutian mink disease virus (AMDV), causes significant welfare problems to mink, and financial losses to the farmers. As there is no vaccine or treatment available, reliable diagnostics is important for disease control. Here, we set up a probe-based real-time PCR (NS1-probe-PCR) to detect all strains of AMDV. PCR was validated and compared to two other real-time PCR methods (pan-AMDV- and pan-AMDO-PCR) currently used for AMDV diagnostics in Finland. The NS1-probe-PCR had a similar detection limit of 20 copies/reaction based on plasmid dilution series, and similar or better diagnostic sensitivity, when evaluated using spleen samples from mink, and stool samples from mink and foxes. None of the three PCR tests cross-reacted with other parvoviruses. The NS1-probe-PCR also showed a significantly higher specificity than the pan-AMDO-PCR with spleen samples and the best specificity with stool samples. Furthermore, it produced the results more rapidly than the other two PCRs making it a promising tool for both diagnostic and research purposes.

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.

Breeding parameters on a mink farm infected with Aleutian mink disease virus following the use of methisoprinol.

Aleutian mink disease virus is one of the greatest threats to modern mink farming. The disease reduces fecundity and causes high mortality among kits. The aim of this study was to evaluate the effectiveness of methisoprinol in counteracting the effects of Aleutian disease, both by inhibiting replication of the virus and by mitigating the harmful effects of the disease on the fecundity and weight of infected animals. The study included 300 individuals with confirmed infection, divided according to antibody titres into three experimental groups, which received a 20% methisoprinol solution, and three control groups, which did not receive the immunostimulant. In the mink from the experimental groups, the number of copies of the genetic material of the virus in the spleens and lymph nodes was one order of magnitude lower than in the case of the control groups. Mink receiving the supplement also showed higher fecundity (on average 5.83 in the experimental groups and 4.83 in the control groups), and the weight of their offspring before slaughter was over 200 g higher. Given the lack of effective methods for immunoprophylaxis and treatment, methisoprinol supplementation can be an effective means of counteracting the effects of AMDV on persistently infected farms.

Aleutian Mink Disease Virus in the breeding environment in Poland and its place in the global epidemiology of AMDV.

Aleutian mink disease (AMD) leads to an increase in mortality of animals and causes losses in mink farming. The study investigated the presence of AMDV in tissue and environmental samples from farmed mink in Poland, and selected samples were genetically characterized. Blood, spleens and swabs from the breeding environment were collected on 27 farms in seven voivodeships in Poland (n = 250). DNA was isolated, amplified by PCR and subsequently subjected to sequencing to reveal information on the molecular epidemiology of the samples. A qPCR method was used to determine the viral load in test samples. The presence of AMDV was confirmed in tissues and the farm environment on 26 of the 27 farms. The average viral load in spleens was 108 copies. The virus was also present in the blood (average - 105 copies) and the farm environment (average - 103 copies). Isolates from the West Pomeranian Voivodeship showed high similarity within the voivodeship (over 99%). Variants from the Lublin and Podlaskie Voivodeships differed 5% from any of the AMDV isolates present in the NCBI database. Isolates from the Greater Poland, Pomeranian, Podkarpackie and Lesser Poland Voivodeships formed heterogeneous clades, showing over 97% similarity to variants previously isolated in Poland, the Netherlands and Lithuania. A high degree of genetic variation was identified among the majority of the samples, which indicates that AMDV has been introduced to Poland multiple times. However, the results within one area showed high identity between isolates, suggesting that one common ancestor was the source of these outbreaks.

Co-circulation of highly diverse Aleutian mink disease virus strains in Finland.

Aleutian mink disease virus (AMDV) is the causative agent of Aleutian disease (AD), which affects mink of all genotypes and also infects other mustelids such as ferrets, martens and badgers. Previous studies have investigated diversity in Finnish AMDV strains, but these studies have been restricted to small parts of the virus genome, and mostly from newly infected farms and free-ranging mustelids. Here, we investigated the diversity and evolution of Finnish AMDV strains by sequencing the complete coding sequences of 31 strains from mink originating from farms differing in their virus history, as well as from free-ranging mink. The data set was supplemented with partial genomes obtained from 26 strains. The sequences demonstrate that the Finnish AMDV strains have considerable diversity, and that the virus has been introduced to Finland in multiple events. Frequent recombination events were observed, as well as variation in the evolutionary rate in different parts of the genome and between different branches of the phylogenetic tree. Mink in the wild carry viruses with high intra-host diversity and are occasionally even co-infected by two different strains, suggesting that free-ranging mink tolerate chronic infections for extended periods of time. These findings highlight the need for further sampling to understand the mechanisms playing a role in the evolution and pathogenesis of AMDV.