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.

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.

Dietary supplementation of Ascophylum nodosum improved kidney function of mink challenged with Aleutian mink disease virus.

BACKGROUND: Feed additives which can ease the negative effects of infection by the Aleutian mink disease virus (AMDV) are of interest to mink farmers. The effects of kelp meal (Ascophylum nodosum) supplementation on immune response, virus replication and blood parameters of mink inoculated with AMDV were assessed. AMDV-free black mink (n = 75) were intranasally inoculated with a local strain of AMDV and fed a commercial pellet supplemented with kelp meal at the rates of 1.5% or 0.75% of the feed or were kept as controls (no kelp) for 451 days. Blood was collected on days 0 (pre-inoculation), 31, 56, 99, 155, 366 and 451 post-inoculation (dpi). RESULTS: No significant difference was observed among the treatments for the proportion of animals positive for antibodies against the virus measured by the counter-immunoelectrophoresis (CIEP), viremia measured by PCR, antibody titer measured by quantitative ELISA, total serum protein measured by a refractometer or elevated levels of gamma globulin measured by iodine agglutination test at the sampling occasions. At the termination of the experiment on 451 dpi, there were no differences among treatments for antibody titer measured by CIEP, total serum protein, albumin, globulins, albumin:globulin ratio, alkaline phosphatase, gamma-glutamyl transferase, and proportions of PCR positive spleen, lymph node or bone marrow samples, but blood urea nitrogen and creatine levels were significantly lower in the 1.5% kelp supplemented group than in the controls. CONCLUSION: Kelp supplementation improved kidney function of mink infected with AMDV with no effect on liver function, immune response to infection by AMDV or virus replication.

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.

A comparison between intraperitoneal injection and intranasal and oral inoculation of mink with Aleutian mink disease virus.

Intranasal, with (INS) and without (IN) sedation, and oral inoculation were compared with intraperitoneal (IP) injection for establishing infection with a local isolate of Aleutian mink disease virus (AMDV) in 35 American mink. Blood samples were collected on 0, 21, 36 and 56 day post-inoculation (dpi). Antiviral-antibodies and viral DNA in plasma and tissues were measured by counter-immunoelectrophoresis (CIEP) and PCR, respectively. The presence of AMDV DNA was tested by PCR in saliva, rectal and fecal samples collected on 0, 6, 10, 15, 21, 28, 36 and 56 dpi. Animals were killed at 56 dpi, samples of six organs were tested for antibody and AMDV DNA, and samples of the lungs, liver, kidneys and heart were subjected to histology. Viral DNA was detected in the spleen, lungs and lymph nodes of all inoculated mink on 56 dpi, indicating that all inoculation routes caused infection in mink. Viral DNA and antibodies were detected in plasma of all IP and INS inoculated mink by 36 dpi, but some animals which were inoculated orally or via IN remained seronegative by 56 dpi. It was concluded that INS route was the most effective method for establishing infection in mink without breaking the integrity of the animals' anatomical barriers. Viremia was short-lived in some mink, whereas antibody production persisted in seroconverted animals during the duration of the experiment. Saliva, rectal and fecal samples did not accurately detect infection. Histologic lesions of AD were observed on the four organs of most mink.

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.

Genetic diversity and phylogenetic analysis of Aleutian mink disease virus isolates in north-east China.

Aleutian mink disease is the most important disease in the mink-farming industry worldwide. So far, few large-scale molecular epidemiological studies of AMDV, based on the NS1 and VP2 genes, have been conducted in China. Here, eight new Chinese isolates of AMDV from three provinces in north-east China were analyzed to clarify the molecular epidemiology of AMDV. The seroprevalence of AMDV in north-east China was 41.8% according to counterimmuno-electrophoresis. Genetic variation analysis of the eight isolates showed significant non-synonymous substitutions in the NS1 and VP2 genes, especially in the NS1 gene. All eight isolates included the caspase-recognition sequence NS1:285 (DQTD↓S), but not the caspase recognition sequence NS1:227 (INTD↓S). The LN1 and LN2 strains had a new 10-amino-acid deletion in-between amino acids 28-37, while the JL3 strain had a one-amino-acid deletion at position 28 in the VP2 protein, compared with the AMDV-G strain. Phylogenetic analysis based on most of NS1 (1755 bp) and complete VP2 showed that the AMDV genotypes did not cluster according to their pathogenicity or geographic origin. Local and imported ADMV species are all prevalent in mink-farming populations in the north-east of China. This is the first study to report the molecular epidemiology of AMDV in north-east China based on most of NS1 and the complete VP2, and further provides information about polyG deletions and new variations in the amino acid sequences of NS1 and VP2 proteins. This report is a good foundation for further study of AMDV in China.

Global phylogenetic analysis of contemporary aleutian mink disease viruses (AMDVs).

BACKGROUND: Aleutian mink disease has major economic consequences on the mink farming industry worldwide, as it causes a disease that affects both the fur quality and the health and welfare of the mink. The virus causing this disease is a single-stranded DNA virus of the genus Amdoparvovirus belonging to the family of Parvoviridae. In Denmark, infection with AMDV has largely been restricted to a region in the northern part of the country since 2001, affecting only 5% of the total Danish mink farms. However, in 2015 outbreaks of AMDV were diagnosed in all parts of the country. Initial analyses revealed that the out breaks were caused by two different strains of AMDV that were significant different from the circulating Danish strains. To track the source of these outbreaks, a major investigation of global AMDV strains was initiated. METHODS: Samples from 13 different countries were collected and partial NS1 gene was sequenced and subjected to phylogenetic analyses. RESULTS: The analyses revealed that AMDV exhibited substantial genetic diversity. No clear country wise clustering was evident, but exchange of viruses between countries was revealed. One of the Danish outbreaks was caused by a strain of AMDV that closely resembled a strain originating from Sweden. In contrast, we did not identify any potential source for the other and more widespread outbreak strain. CONCLUSION: To the authors knowledge this is the first major global phylogenetic study of contemporary AMDV partial NS1 sequences. The study proved that partial NS1 sequencing can be used to distinguish virus strains belonging to major clusters. The partial NS1 sequencing can therefore be a helpful tool in combination with epidemiological data, in relation to outbreak tracking. However detailed information on farm to farm transmission requires full genome sequencing.

Comparative molecular analysis of strains of the Aleutian Disease Virus isolated from farmed and wild mink.

INTRODUCTION AND OBJECTIVE: Aleutian Disease is a significant biological factor causing substantial losses in mink farming. The virus inducing the disease also infects wild populations which may constitute an asymptomatic reservoir. To compare genetic variants of the AMD virus occurring in wild and farmed mink populations, an analysis was performed on a fragment of the VP2 protein sequence of the virus infecting both populations, taken from different living environments. MATERIAL AND METHODS: Genetic material was isolated from 11 farmed animals in which anti-AMDV antibodies had been detected and from 20 wild animals. The DNA obtained was amplified using primers specific for the fragment encoding the VP2 protein. The product obtained was sequenced and bioinformatic analysis was performed. RESULTS: Viral material was detected in 11 farmed and 7 free-living animals. Similarity of sequences averaged 99% within groups and 94% between groups. The sequencing results made it possible to identify characteristic changes for each group. In the isolates from the wild animals, the following changes were observed in the epitope region with respect to the reference sequence: C3704T, G3710A, T3722C, T3746C and A3749G. In the isolates from the farmed animals a G3779A transition was noted. Phylogenetic analysis showed that the variants infecting the two groups occupy separate branches of the phylogenetic tree. CONCLUSIONS: The variants of the virus infecting the two groups may have a common origin, but at present they constitute two separate groups, with characteristic differences making it possible to recognize their genotype.

Evolutionary analysis of whole-genome sequences confirms inter-farm transmission of Aleutian mink disease virus.

Aleutian mink disease virus (AMDV) is a frequently encountered pathogen associated with mink farming. Previous phylogenetic analyses of AMDV have been based on shorter and more conserved parts of the genome, e.g. the partial NS1 gene. Such fragments are suitable for detection but are less useful for elucidating transmission pathways while sequencing entire viral genomes provides additional informative sites and often results in better-resolved phylogenies. We explore how whole-genome sequencing can benefit investigations of AMDV transmission by reconstructing the relationships between AMDV field samples from a Danish outbreak. We show that whole-genome phylogenies are much better resolved than those based on the partial NS1 gene sequences extracted from the same alignment. Well-resolved phylogenies contain more information about the underlying transmission trees and are useful for understanding the spread of a pathogen. In the main case investigated here, the transmission path suggested by the tree structure was supported by epidemiological data. The use of molecular clock models further improved tree resolution and provided time estimates for the viral ancestors consistent with the proposed direction of spread. It was however impossible to infer transmission pathways from the partial NS1 gene tree, since all samples from the case farms branched out from a single internal node. A sliding window analysis showed that there were no shorter genomic regions providing the same phylogenetic resolution as the entire genome. Altogether, these results suggest that phylogenetic analyses based on whole-genome sequencing taking into account sampling dates and epidemiological data is a promising set of tools for clarifying AMDV transmission.

Distribution of Aleutian mink disease virus contamination in the environment of infected mink farms.

Control and eradication of Aleutian Mink Disease Virus (AMDV) are a major concern for fur-bearing animal production. Despite notably reducing disease prevalence, current control programs are unable to prevent the reinfection of farms, and environmental AMDV persistence seems to play a major role regarding this issue. In this study 114 samples from different areas and elements of seven infected mink farms were analyzed by qPCR in order to evaluate the environmental distribution of AMDV load. Samples were classified into nine categories, depending on the type of sample and degree of proximity to the animals, the main source of infection. Two different commercial DNA extraction kits were employed in parallel for all samples. qPCR analysis showed 69.3% positive samples with one kit and 81.6% with the other, and significant differences between the two DNA extraction methods were found regarding AMDV DNA recovery. Regarding sample categorization, all categories showed a high percentage of AMDV positive samples (31%-100%). Quantification of positive samples showed a decrease in AMDV load from animal barns to the periphery of the farm. In addition, those elements in direct contact with animals, the street clothes and vehicles of farm workers and personal protective equipment used for sampling showed a high viral load, and statistical analysis revealed significant differences in AMDV load between the first and last categories. These results indicate high environmental contamination of positive farms, which is helpful for future considerations about cleaning and disinfection procedures and biosecurity protocols.

Reduced severity of histopathological lesions in mink selected for tolerance to Aleutian mink disease virus infection.

The objective of this study was to measure the effect of selection for tolerance on the severity of the Aleutian disease (AD) lesions in mink. Sensitivity and specificity of antibody detection in the blood by counter-immunoelectrophoresis (CIEP) relative to the presence of Aleutian mink disease virus (AMDV) in the spleen by PCR in naturally infected farmed mink were also estimated. Carcasses of 680 sero-positive (CIEP-P) black mink from 28 farms in Nova Scotia, Canada, and from 132 sero-negative (CIEP-N) mink from 14 of these farms were collected at pelting time. A total of 116 of the CIEP-P mink were from three farms where animals have been selected for tolerating AD for almost 20years. The severity of the AD lesions was assessed by histopathological examination of kidneys, lungs, heart, brain and liver on a scale of 0 to 4. Sensitivity and specificity of CIEP relative to PCR were 0.97 and 0.85, respectively, and 16.5% of CIEP-N mink were PCR positive, which could be one of the reasons for the failure of virus eradication by CIEP in Canada. The CIEP-N and tolerant CIEP-P animals had 9.39 and 6.23 greater odds of showing lower lesion severity, respectively, than the CIEP-P animals (P<0.01). The CIEP-N mink had a slightly higher chance (P=0.07) of showing lower lesion severity (odds ratio 1.51) compared with tolerant CIEP-P mink. The results suggested that tolerant mink had significantly reduced severity of AD lesions despite having anti-viral antibodies and carrying the virus.

A fast and accurate method of detecting Aleutian mink disease virus in blood and tissues of chronically infected mink.

The objective of this study was to assess the sensitivity of the Omni Klentaq-LA DNA polymerase for detecting Aleutian mink disease virus (AMDV) in mink blood and tissues by PCR without DNA extraction. The presence of AMDV DNA was directly tested by Klentaq in the plasma, serum, whole blood, and spleen homogenates of 188 mink 4 and 16 months after inoculation with the virus. Samples from bone marrow, small intestine, liver, lungs, kidneys, and lymph nodes of 20 of the same mink were also tested by Klentaq. DNA was extracted from paired samples of plasma and the aforesaid tissues by a commercial nucleic acid extraction kit (Dynabeads Silane) and tested by PCR. Compared with the extracted DNA, Klentaq detected a significantly greater number of samples in the whole blood, serum, plasma, spleen, and small intestine. It was concluded that Klentaq is a preferred system for directly detecting AMDV DNA in mink blood and tissues. The lower success rate of extracted DNA compared with Klentaq could be the result of DNA losses during the extraction process. This is an important factor in chronically infected mink, which have a low AMDV copy number in the bloodstream. Direct AMDV detection also reduces the cost of PCR amplification and lowers the risk of sample contamination.

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 (386)HLQQNFSTRYIYD(398) 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 (386)H 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.

Genetic characterization of the complete genome of an Aleutian mink disease virus isolated in north China.

The genome of a highly pathogenic strain of Aleutian disease mink virus (AMDV-BJ) isolated from a domestic farm in North China has been determined and compared with other strains. Alignment analysis of the major structural protein VP2 revealed that AMDV-BJ is unique among 17 other AMDV strains. Compared with the nonpathogenic strain ADV-G, the 3' end Y-shaped hairpin was highly conserved, while a 4-base deletion in the 5' U-shaped terminal palindrome resulted in a different unpaired "bubble" group near the NS1-binding region of the 5' end hairpin which may affect replication efficiency in vivo. We also performed a protein analysis of the NS1, NS2, and new-confirmed NS3 of AMDV-BJ with some related AMDV DNA sequence published, providing information on evolution of AMDV genes. This study shows a useful method to obtain the full-length genome of AMDV and some other parvoviruses.

Development of an Enzyme-Linked Immunosorbent Assay Based on Fusion VP2332-452 Antigen for Detecting Antibodies against Aleutian Mink Disease Virus.

For detection of Aleutian mink disease virus (AMDV) antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed using the recombinant VP2332-452 protein as an antigen. Counterimmunoelectrophoresis (CIEP) was used as a reference test to compare the results of the ELISA and Western blotting (WB); the specificity and sensitivity of the VP2332-452 ELISA were 97.9% and 97.3%, respectively, which were higher than those of WB. Therefore, this VP2332-452 ELISA may be a preferable method for detecting antibodies against AMDV.