Establishment and application of multiplex PCR for rapid detection of three mink diarrhea-associated viruses.

In this report, a multiplex PCR method was developed for the detection of three diarrhea-associated viruses in mink, including circovirus (MCV), bocavirus (MBoV), and enteritis virus (MEV). Three compatible sets of primers specific for each virus were designed respectively based on their conserved sequences. After optimization of the crucial factors such as primer concentration and annealing temperature in single and multiple amplification, three specific fragments were simultaneously amplified with the highest sensitivity and specificity in one PCR reaction. The fragments amplified were 259 bp (MCV)，455 bp (MBoV) and 671 bp (MEV). The sensibility of this one-step multiplex PCR is about 10 times lower than that of regular singleplex PCR. There were no cross-reactions with some relevant pathogens like mink coronavirus, canine distemper virus, and aleutian mink disease virus. In our study we analyzed viral DNA in mink fecal samples by multiplex PCR assay from China, which revealed the occurrence of MCV, MBoV, and MEV as 3.1 %, 5.7 %, and 9.8 %, respectively. The testing results of multiplex PCR agreed with the singleplex PCR results with a coincidence rate of 100 %. These results indicated that the method could provide technical support for rapid detection of the three diarrhea-associated viruses, and epidemiological investigation of mink viral diarrhea.

Loop-mediated Isothermal Amplification-Single Nucleotide Polymorphism Analysis for Detection and Differentiation of Wild-type and Vaccine Strains of Mink Enteritis Virus.

Broad coverage of mink enteritis virus (MEV) vaccination program in northeast of China has provided effective protection from mink viral enteritis. Nevertheless, MEV vaccine failures were reported due to continually evolving and changing virulence of field variants or wild-type MEV. In this study, a combined loop-mediated isothermal amplification (LAMP) and single nucleotide polymorphism (SNP) method, named LAMP-SNP assay, was developed for detection and differentiation of wild-type and vaccine strains of MEV. Four primers in MEV-VP2-LAMP were used to detect both wild-type and vaccine strains of MEV in our previous publication, and other four primers in LAMP-SNP were designed to amplify the NS1 gene in wild-type MEV and only used to detect wild-type viruses. The LAMP-SNP assay was performed in a water bath held at a constant temperature of 65 °C for 60 min. LAMP-SNP amplification can be judged by both electrophoresis and visual assessment with the unaided eyes. In comparison with virus isolation as the gold standard in testing 171 mink samples, the percentage of agreement and relative sensitivity and specificity of the LAMP-SNP assay were 97.1, 100%, and 94.0%, respectively. There were no cross-reactions with other mink viruses. The LAMP-SNP assay was found to be a rapid, reliable and low-cost method to differentiate MEV vaccine and field variant strains.

Comparison of biological and genomic characteristics between a newly isolated mink enteritis parvovirus MEV-LHV and an attenuated strain MEV-L.

A virus isolated from mink showing clinical signs of enteritis was identified as a high virulent mink enteritis parvovirus (MEV) based on its biological characteristics in vivo and in vitro. Mink, challenged with this strain named MEV-LHV, exhibited severe pathological lesions as compared to those challenged with attenuated strain MEV-L. MEV-LHV also showed higher infection and replication efficiencies in vitro than MEV-L. Sequence of the complete genome of MEV-LHV was determined and analyzed in comparison with those in GenBank, which revealed that MEV-LHV shared high homology with virulent strain MEV SD12/01, whereas MEV-L was closely related to Abashiri and vaccine strain MEVB, and belonged to a different branch of the phylogenetic tree. The genomes of the two strains differed by insertions and deletions in their palindromic termini and specific unique mutations (especially VP2 300) in coding sequences which may be involved in viral replication and pathogenicity. The results of this study provide a better understanding of the biological and genomic characteristics of MEV and identify certain regions and sites that may be involved in viral replication and pathogenicity.

Development of a nanoparticle-assisted PCR (nanoPCR) assay for detection of mink enteritis virus (MEV) and genetic characterization of the NS1 gene in four Chinese MEV strains.

BACKGROUND: Mink enteritis virus (MEV) causes mink viral enteritis, an acute and highly contagious disease whose symptoms include violent diarrhea, and which is characterized by high morbidity and mortality. Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a recently developed technique for the rapid detection of bacterial and viral DNA. Here we describe a novel nanoPCR assay for the clinical detection and epidemiological characterization of MEV. RESULTS: This assay is based upon primers specific for the conserved region of the MEV NS1 gene, which encodes nonstructural protein 1. Under optimized conditions, the MEV nanoPCR assay had a detection limit of 8.75 × 10(1) copies recombinant plasmids per reaction, compared with 8.75 × 10(3) copies for conventional PCR analysis. Moreover, of 246 clinical mink samples collected from five provinces in North-Eastern China, 50.8% were scored MEV positive by our nanoPCR assay, compared with 32.5% for conventional PCR. Furthermore no cross reactivity was observed for the nanoPCR assay with respect to related viruses, including canine distemper virus (CDV) and Aleutian mink disease parvovirus (AMDV). Phylogenetic analysis of four Chinese wild type MEV isolates using the nanoPCR assay indicated that they belonged to a small MEV clade, named "China type", in the MEV/FPLV cluster, and were closely clustered in the same location. CONCLUSIONS: Our results indicate that the MEV China type clade is currently circulating in domestic minks in China. We anticipate that the nanoPCR assay we have described here will be useful for the detection and epidemiological and pathological characterization of MEV.

Development of a PCR-RFLP assay for the detection and differentiation of canine parvovirus and mink enteritis virus.

A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay has been developed to detect and differentiate between canine parvovirus (CPV) and mink enteritis virus (MEV). Eight CPV and three MEV epidemic strains isolated from 28 pathological samples from dogs and minks suspected of being infected with parvovirus were amplified by PCR using a pair of specific primers designed based on the CPV-N strain (M19296). PCR amplified a fragment of 1016bp from the genomic DNA of both MEV and CPV. The MEV-derived fragment could be digested with the restriction enzyme BSP1407I into three fragments of 102bp, 312bp and 602bp, while the fragment amplified from the CPV genomic DNA was digested into only two fragments of 414bp and 602bp. The lowest DNA concentration of CPV and MEV that could be detected using this assay was 0.004µg/ml and 0.03µg/ml, respectively. The PCR-RFLP assay developed in the present study can, therefore, be used to detect and differentiate MEV from CPV with high specificity and sensitivity.

Detection of mink enteritis virus by loop-mediated isothermal amplification (LAMP).

Loop-mediated isothermal amplification (LAMP) method was discovered in the last decade but only used for the first time in the diagnosis of mink enteritis virus (MEV) infection in this study. The amplification could be completed within 60 min, under isothermal condition at 65°C, by employing a set of four primers targeting the VP2 gene of MEV. The LAMP was more sensitive than the conventional PCR, with a detection limit of 10(-1) median tissue culture infective doses (TCID(50))/ml per reaction, compared with 10 TCID(50)/ml for PCR analysis. No cross reactivity was observed for other related viruses, including canine distemper virus (CDV) and Aleutian mink disease parvovirus (AMDV). Eighty four of 230 clinical samples were found to be positive for MEV, which is higher than that determined by using the conventional PCR method (68). The results indicate the LAMP can be potentially used to determine MEV as a simple, rapid procedure. This assay would be an available alternative to PCR analysis for the diagnosis of MEV infection in mink, particularly in less well-equipped laboratories and in rural settings where resources are limited.

Evidence for natural recombination between mink enteritis virus and canine parvovirus.

A virus was isolated from mink showing clinical and pathological signs of enteritis in China. This virus, designated MEV/LN-10, was identified as mink enteritis virus (MEV) based on its cytopathic effect in the feline F81 cell line, the hemagglutination (HA) and hemagglutination inhibition (HI) assay, electron microscopy (EM) and animal infection experiments. The complete viral genome was cloned and sequenced. Phylogenetic and recombination analyses on the complete MEV/LN-10 genome showed evidence of recombination between MEV and canine parvovirus (CPV). The genome was composed of the NS1 gene originating from CPV while the VP1 gene was of MEV origin. This is the first demonstration of recombination between a CPV and MEV in nature. Our findings not only provide valuable evidence indicating that recombination is an important genetic mechanism contributing to the variation and evolution of MEV, but also that heterogeneous recombination can occur in the feline parvovirus subspecies.

Analysis of the vp2 gene sequence of a new mutated mink enteritis parvovirus strain in PR China.

BACKGROUND: Mink enteritis virus (MEV) causes a highly contagious viral disease of mink with a worldwide distribution. MEV has a linear, single-stranded, negative-sense DNA with a genome length of approximately 5,000 bp. The VP2 protein is the major structural protein of the parvovirus encoded by the vp2 gene. VP2 is highly antigenic and plays important roles in determining viral host ranges and tissue tropisms. This study describes the bionomics and vp2 gene analysis of a mutated strain, MEV-DL, which was isolated recently in China and outlines its homologous relationships with other selected strains registered in Genbank. RESULTS: The MEV-DL strain can infect F81 cells with cytopathic effects. Pig erythrocytes were agglutinated by the MEV-DL strain. The generation of MEV-DL in F81 cells could infect mink within three months and cause a disease that was similar to that caused by wild-type MEV. A comparative analysis of the vp2 gene nucleotide (nt) sequence of MEV-DL showed that this was more than 99% homologous with other mink enteritis parvoviruses in Genbank. However, the nucleotide residues at positions 1,065 and 1,238 in the MEV-DL strain of the vp2 gene differed from those of all the other MEV strains described previously. It is noteworthy that the mutation at the nucleotide residues position 1,238 led to Asp/Gly replacement. This may lead to structural changes. A phylogenetic tree and sequence distance table were obtained, which showed that the MEV-DL and ZYL-1 strains had the closest inheritance distance. CONCLUSIONS: A new variation of the vp2 gene exists in the MEV-DL strain, which may lead to structural changes of the VP2 protein. Phylogenetic analysis showed that MEV-DL may originate from the ZYL-1 strain in DaLian.