Epidemiological Investigation And Construction Of Virus Infectious Clone Of Aluetian Mink Disease Virus

Aleutian Mink Disease(AD),also known as mink plasmacytosis,is a chronic infectious disease of immune system dysfunction in minks caused by Aleutian Mink Disease Virus(AMDV).AD is characterized by the progressive wasting,decreased fecundity,increased serum γ globulin,enlarged spleen,renal changes(from swelling and pestules to atrophy and depression),and enlarged mesenteric lymph nodes.AMDV can spread horizontally and vertically,causing spontaneous abortion of pregnant mink and death of young mink.It can significantly affect the reproductive performance and fur quality of mink.AD is one of the three major diseases affecting mink breeding industry.At present,there is no vaccine available,and there is no effective treatment strategy.The only way to control AD can be archived through regular quarantine,elimination,and gradually purifying the mink population.Therefore,it is necessary to establish sensitive,efficient and accurate diagnostic methods,and carry out epidemiological investigation and molecular biology research for AMDV.In this study,a pair of specific primers and a probe were designed,according to the conserved region of VP2 gene of AMDV.The reaction condition was optimized,and the real-time PCR detection method was established.The correlation correfficient(r2)of the standard curve was 0.994,and it presented a linear relationship in the concentration of the template DNA ranging from 102 copies/μL to108 copies /μL.The assay was specific for AMDV,but no amplification from CPV,MEV,CDV and CAV.The sensitivity of AMDV detection was 102 copies/μL,and the intra-group and inter-group repeatability tests showed that the coefficient of variation of the method was less than 5%.The established method was used to detect 417 clinical tissue samples.The positive rate in some areas of China was 81.5%.For further study of the genomic characteristics of AMDV,the whole genome was amplified and sequenced from the above positive samples.The sequencing results were compared with the reference sequences in GenBank to construct the evolutionary tree and conduct homology analysis.The results showed that the nucleotide similarity among the 14 sequenced strains was from 93.1% to 99.6%,and that of the AMDV reference sequence in GenBank was 92.0%-97.1%.All strains were divided into six branches in the evolutionary tree,and the AMDV strains in China showed obvious homology,indicating that the AMDV cross-propagation evolution into China formed complex genetic characteristics.The amino acid sequences of non-structural proteins NS1,NS2,NS3 and structural proteins VP1 and VP2 were compared and analyzed,and amino acid mutations at some special sites were found,indicating that AMDV has a high degree of genetic diversity.These differences provide a theoretical basis for the gene function and genetic characteristics of AMDV and provide an exploration direction for the development of AMDV vaccine.In order to prevent and control the harm of AD to mink breeding industry,vaccine development is the primary choice.Since only AMDV-G can be cultured on the Crandell feline kidney cells(CRFK),and none of the wild strains can be isolated.Here,the full-length of AMDV genomic DNA clonedplasmid p UC-AMDV-G and pUC-AMDV-Gluc were synthesized based on the complete sequence of ADV-G in the GenBank and the above sequence results.The vector was replaced by pBluescript SKⅡ(+)to construct plasmid pBlue-AMDV-G and pBlue-AMDV-Gluc.The enhanced green fluorescent protein(EGFP)gene,was inserted into the pBlue-AMDV-G,the new plasmid was named pBlue-AMDV-EGFP.The three plasmids were transfected into CRFK cells for blind transmission.Tests on luciferase and green fluorescent protein showed that the two plasmids failed to rescue the virus.pBlue-AMDV-G plasmid transfection can detect specific transcription patterns after blind transmission for three generations,but the transcription level is low,indicating that the virus has been rescued successfully but at very low level.The construction strategy of infectious clone of AMDV still needs to be improved.In summary,we established a reproducible,specific and sensitive AMDV real-time PCR detection method,which can be used for clinical detection and epidemiological investigation of AMDV.We analyzed the whole genome sequence of some strains to provide a theoretical basis for the gene function and genetic characteristics of AMDV in China.The AMDV infectious clone might lay a foundation for further study on the biological characteristics and pathogenic mechanism of AMDV and provided an exploration direction for the development of AMDV vaccine.