Aleutian Mink Disease Virus Strains Research Articles

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.

Seroprevalence and Molecular Epidemiology of Aleutian Disease in Various Countries during 1972-2021: A Review and Meta-Analysis.

Simple SummaryAleutian disease is caused by the Aleutian mink disease virus and is one of the most serious infectious diseases that affect the family Mustelidae, including the American mink, wild European mink, weasels, badgers and other animal species, such as skunks, raccoons, dogs, cats and mice, as well as humans. Effective treatments and vaccines against Aleutian disease have not been developed to date. Prophylactic programs that focus on the identification and elimination of infected mink are one of the methods of controlling the negative outcomes of Aleutian disease. This article analyses the seroprevalence of Aleutian mink disease virus infections in American and European mink and other species around the world, and reviews recent knowledge relating to the molecular epidemiology of the Aleutian mink disease virus.Aleutian disease (AD) poses a serious threat to both free-ranging and farmed mink around the world. The disease is caused by the Aleutian mink disease virus (AMDV), which also poses a health risk for other members of the family Mustelidae, including wild mink, weasels, badgers and other animal species. This article analyses the seroprevalence of AMDV infections in mink and other species around the world, and reviews recent knowledge relating to the molecular epidemiology of the AMDV. Depending on the applied diagnostic technique and the country, the prevalence of anti-AMDV antibodies or AMDV DNA was established at 21.60–100.00% in farmed American mink, 0.00–93.30% in free-ranging American mink and 0.00–25.00% in European mink. Anti-AMDV antibodies or AMDV DNA were also detected in other free-living fur-bearing animals in Europe and Canada, where their prevalence was determined at 0.00–32.00% and 0.00–70.50%, respectively. This may indicate a potential threat to various animal species. AMDV strains are not clustered into genotypes based on the geographic origin, year of isolation or pathogenicity. The isolates that were identified on mink farms around the world originated from North America because American mink were introduced to Europe and Asia for breeding purposes and to restock natural populations.

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

BackgroundAleutian 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.ObjectivesThis 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.MethodsThirty-seven strains from 17 farms were partially sequenced for the NS1 and VP2 genes and analyzed phylogenetically with other strains described worldwide.ResultsSpanish 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.ConclusionsThe 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.

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.

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.

A comparative molecular characterization of AMDV strains isolated from cases of clinical and subclinical infection

The Aleutian mink disease virus (AMDV) is one of the most serious threats to modern mink breeding. The disease can have various courses, from progressive to subclinical infections. The objective of the study was to provide a comparative molecular characterization of isolates of AMDV from farms with a clinical and subclinical course of the disease. The qPCR analysis showed a difference of two orders of magnitude between the number of copies of the viral DNA on the farm with the clinical course of the disease (105) and the farm with the subclinical course (103). The sequencing results confirm a high level of homogeneity within each farm and variation between them. The phylogenetic analysis indicates that the variants belonging to different farms are closely related and occupy different branches of the same clade. The in silico analysis of the effect of differences in the sequence encoding the VP2 protein between the farms revealed no effect of the polymorphism on its functionality. The close phylogenetic relationship between the isolates from the two farms, the synonymous nature of most of the polymorphisms and the potentially minor effect on the functionality of the protein indicate that the differences in the clinical picture may be due not only to polymorphisms in the nucleotide and amino acid sequences, but also to the stage of infection on the farm and the degree of stabilization of the pathogen–host relationship.

Generation of an infectious clone of AMDV and identification of capsid residues essential for infectivity in cell culture

Pathogenic strains of Aleutian mink disease virus (AMDV) such as Utah-1 do not replicate in cell culture (e.g., Crandell Rees feline kidney cells) while the in vitro-adapted AMDV strain ADV-Gorham (ADV-G) is not pathogenic. Here, we constructed a full-length infectious clone (pADV-G). Alignment of the VP2 gene of ADV-G with that of other AMDV strains revealed many amino acid (a.a.) residues conserved among pathogenic isolates that differed in ADV-G. Four virulence-associated, conserved residues of pADV-G VP2 were studied by site-directed mutagenesis (H92A, Q94S, Y115F, and I116L). Mutation of residue 92 or 94 decreased viral-transcription and viral-infectivity levels, whereas mutation of residue 115 or 116 did not affect viral-infectivity in CRFK cells. These results indicated that VP2 residues 92 and 94, both located on the surface of the viral capsid, are critical for AMDV infectivity in vitro.

Identification of a novel Aleutian mink disease virus B-cell epitope using a monoclonal antibody against VP2 protein

Aleutian mink disease virus (AMDV) is a parvovirus that causes an immune complex-mediated disease in minks. Capsid protein VP2 is a major structural viral protein and can be used to diagnose AMDV. In this study, a specific monoclonal antibody, 1M13, was produced against the AMDV VP2 protein (amino acids 291–502). A linear VP2-protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to be enzyme-linked immunosorbent assay (ELISA) analysis. The results indicated that 386HLQQNFSTRYIYD398 was the minimal linear epitope that could be recognized by mAb 1M13. ELISA assays revealed that mink anti-AMDV sera could also recognize the minimal linear epitope. Sequence alignments demonstrated that the linear epitope is highly conserved among AMDV strains except 386H and is less conserved among Raccoon dog amdovirus, Gray fox amdovirus, Red fox amdovirus, Bat parvovirus and Mink enteritis parvovirus. Taken together, the generation of this VP2-specific mAb with a defined linear peptide epitope may have potential applications in the development of suitable diagnostic techniques for AMDV.

Progression of experimental chronic Aleutian mink disease virus infection.

BackgroundAleutian mink disease virus (AMDV) is found world-wide and has a major impact on mink health and welfare by decreasing reproduction and fur quality. In the majority of mink, the infection is subclinical and the diagnosis must be confirmed by serology or polymerase chain reaction (PCR). Increased knowledge based on a systematically description of clinical signs, pathology and histopathology might be a tool to reduce the risk of infection from subclinically infected mink to AMDV free herds. The aim of this study was to give a histopathological description of the progression of a chronic experimental infection with a currently circulating Danish strain of AMDV, Saeby/DEN/799.1/05. These results were compared with the pathogenesis of previously published AMDV stains.ResultsThis experimental AMDV infection resulted in only decreased appetite and soft or discolored feces, primarily within the first 8 weeks after AMDV inoculation. Gross pathology revealed few and inconsistent findings mainly associated with the liver, spleen and kidneys. The majority of the AMDV inoculated wild type mink (n = 41) developed various histopathological changes consistent with AMDV infection in one or more organs: infiltrations of mononuclear cells in liver, kidney and brain, reduced density of lymphocytes and increased numbers of plasma cells in lymph nodes and spleen. Natural infection, as occurred in the sentinel sapphire mink (four of six mink), progressed similar to the experimentally inoculated mink.ConclusionsExperimental AMDV inoculation mainly resulted in subclinical infection with unspecific clinical signs and gross pathology, and more consistent histopathology appearing at any time after AMDV inoculation during the 24 weeks of observation. Thus, the observed histopathology substantiates AMDV infection and no correlation to time of inoculation was found. This confirms that diagnosing AMDV infection requires serology and/or PCR and the Saeby/DEN/799.1/05 AMDV strain results in histopathology consistent with other AMDV strains.

Aleutian mink disease virus in free-ranging mustelids in Finland - a cross-sectional epidemiological and phylogenetic study.

Aleutian mink disease virus (AMDV) can cause severe immune-complex-mediated disease in American mink. AMDV has also been detected in several other mustelid species with potential negative impact on their health and population. A molecular and cross-sectional epidemiological study was conducted to obtain data on the prevalence, distribution, transmission and diversity of AMDV strains in Finnish free-ranging mustelids and risk factors associated with infection. The presence of anti-AMDV antibodies and/or AMDV DNA was tested from 308 samples representing eight mustelid species and 17 administrative regions. Positive samples were detected across Finland, and in 54 % (31/57) of feral American mink, 27 % (7/26) of European badgers and 7 % (1/14) of European polecats. Samples from Eurasian otters, European pine martens, least weasels, stoat and wolverine were negative. Major risk factors for infection were the species American mink with 335 and badger with 74 times higher odds than other species, and the years 2006-2009 with five times higher odds than the years 2010-2014. No clustering according to species, geographical origin or year was evident in phylogeny, except for four divergent sequences from Estonian badgers that formed a separate phylogroup distinct from other AMDV strains. This study showed that AMDV was prevalent in certain species of Finnish free-ranging mustelids and widely distributed across Finland. Furthermore, the free-ranging mustelids carried both strains similar to those found in farmed mink, but also distinct strains that may represent novel amdoparvoviruses.

Molecular epidemiology of Aleutian mink disease virus in China

Aleutian mink disease (mink plasmacytosis) is a very severe immune-complex-mediated disease affecting minks. It is caused by the Aleutian mink disease virus (AMDV). To obtain a better understanding of the molecular epidemiology of AMDV in China, a total of 420 samples were collected from mink farms in five major mink-farming provinces in China. After testing serum antibodies using counterimmunoelectrophoresis (CIEP), 23 of the 340 positive samples were randomly selected and analyzed. The full length of the major structural protein gene (VP2) from all the samples was amplified and sequenced. The sequences in the twenty-three samples from 5 farms in 5 provinces were phylogenetically analyzed, and eleven were found to have homologous sequences in GenBank. A rooted phylogenetic tree was constructed using the unweighted pair-group method with arithmetic (UPGMA) method. Phylogenetic analysis showed that the AMDV strains formed five groups (I–VI), and four of them contained Chinese strains. The tree showed that the two AMVD lineages had been introduced to China independently. Over 70% of the Chinese isolates were classified into two groups, all of which contained Chinese strains. The results of the analysis suggested that the distribution of the AMDV strains was not based on geographical origin, and both indigenous AMDV and imported AMDV were prevalent in the primary mink production areas in China.

High Prevalence of Aleutian Mink Disease Virus in Free-ranging Mink on a Remote Danish Island

Aleutian mink disease virus (AMDV) causes severe disease in farmed mink (Neovison vison) worldwide. In Denmark, AMDV in farmed mink has been confined to the northern part of the mainland since 2002. From 1998 to 2009, samples from 396 free-ranging mink were collected from mainland Denmark, and a low AMDV antibody prevalence (3% of 296) was found using countercurrent immune electrophoresis. However, on the island of Bornholm in the Baltic Sea, a high prevalence (45% of 142 mink) was detected in the free-ranging mink. Aleutian mink disease virus was detected by polymerase chain reaction in 32 of 49 antibody-positive free-ranging mink on Bornholm, but not in mink collected from other parts of Denmark. Sequence analysis of 370 base pairs of the nonstructural gene of the AMDV of 17 samples revealed two clusters with closest similarity to Swedish AMDV strains.

Genetic characterization of Aleutian mink disease viruses isolated in China

Aleutian mink disease virus (AMDV) is a parvovirus that causes an immune complex mediated disease in minks. To understand the genetic characterization of AMDV in China, the genomic sequences of three isolates, ADV-LN1, ADV-LN2, and ADV-LN3, from different farms in the Northern China were analyzed. The results showed that the lengths of genomic sequences of three isolates were 4,543, 4,566, and 4,566 bp, respectively. They shared only 95.5-96.3 % nucleotide identity with each other. The nucleotide and amino acid homology of genome sequence between the Chinese isolates and European or American strains (ADV-G, ADV-Utah1, and ADV-SL3) were 92.4-95.0 % and 92.1-93.8 %, respectively. The amino acid substitutions randomly distributed in the genome, especially NS gene. ADV-LN1 strain had a 9-amino-acid deletion at amino acid positions 70 and 72-79 in the VP1 gene, comparing with ADV-G strain; ADV-LN2 and ADV-LN3 strains had 1-amino-acid deletion at amino acid positions 70 in the VP1. Some potential glycosylation site mutations in VP and NS genes were also observed. Phylogenetic analysis results showed that the three strains belonged to two different branches based on the complete coding sequence of VP2 gene. However, they all were in the same group together with the strains from United States based on the NS1 sequence. It indicated that Chinese AMDV isolates had genetic diversity. The origin of the ancestors of the Chinese AMDV strains might be associated with the American strains.

Implementation and validation of a sensitive PCR detection method in the eradication campaign against Aleutian mink disease virus

Aleutian mink disease virus (AMDV) is a severe progressive disease causing multiple different clinical syndromes in mink. In Denmark, the disease is notifiable and under official control. The control programme, based on serological screening, has confined successfully AMDV to the northern part of Denmark. However, re-infections and new introductions of virus into farms require a confirmatory virological test to verify the positive test results of single animals and ultimately to investigate disease transmission. A one step PCR amplifying a 374-base fragment of the NS1 gene of AMDV was compared to the counter-current immune electrophoresis (CIE) routinely used in the serological screening programme. Mink organs ( n = 299) obtained from 55 recently infected farms and 8 non-infected farms from 2008 to 2010 were tested by PCR, and the results were found to have a high correlation with the serological status of the mink. The relative diagnostic sensitivity of the PCR was 94.7%, and the relative diagnostic specificity was 97.9% when read in parallel with the CIE. PCR positive samples were sequenced and phylogenetic analysis revealed high similarity within the analysed AMDV strains and to AMDV strains described previously.

Molecular epidemiology of Aleutian mink disease virus in Finland

Aleutian mink disease virus (AMDV) is a parvovirus that causes an immune complex-mediated disease in minks. To gain a more detailed view of the molecular epidemiology of mink AMDV in Finland, we phylogenetically analysed 14 new Finnish strains from 5 farms and all 40 strains with corresponding sequences available in GenBank. A part of the major non-structural (NS1) protein gene was amplified and analysed phylogenetically. A rooted nucleotide tree was constructed using the maximum parsimony method. The strains described in this study showed 86–100% nucleotide identity and were nearly identical on each farm. The ratio of synonymous to non-synonymous substitutions was approximately 2.7, indicating a mild purifying selection. Phylogenetic analysis confirmed that AMDV strains form three groups (I–III), all of which contained Finnish strains. The tree inferred that the three lineages of AMDV have been introduced to Finland independently. The analysis suggested that AMDV strains do not cluster into genotypes based on geographical origin, year of isolation or pathogenicity. Based on these data, the molecular clock is not applicable to AMDV, and within this gene area no recombination was detected.

Identification of breakpoints in intergenotypic recombinants of HIV type 1 by bootscanning.

Identification of breakpoints in intergenotypic recombinants of HIV type 1 by bootscanning.