Construction Of Neotype Aleutian Mink Disease Virus Infectious Clone And Identification Of Its Biological Characteristics

Aleutian mink disease (AD) is one of severe infectious disease caused by Aleutian mink disease virus (AMDV). A fatal acute pneumonia in the neonatal mink and a symptom of distinct hypergammaglobulinemia, plasmacytosis, immune-complex glomerulonephritis in adult were of its cheracteristics. A high economical loss was usually led by it.Because of 3’Y-shape and 5’U-shape hairpin termini in AMDV genome, only one full-length genome sequence of the non-pathogenic strain (ADV-G) had been reported. As to the pathogenic strains, just middle regions of their genomes were sequenced. This characteristics was an obstacle to declare the differences of genome between pathogenic AMDV strains and ADV-G. Furthermore, the ADV-G can replicate permissively in Crandell feline kidney (CRFK) cells, but wildtype isolates of AMDV could not be propagated in vitro. The molecular mechanism of AMDV infection is unclear, and the study on etiology and immunology of AD is progressed slowly.In this study, we designed a special method to amplify and clone the ITRs of AMDV genome. A full-length genome sequence of pathogenic strain (BJ) was completed. Based on the alignment results of coding regions between BJ and ADV-G, some functional residues which were relative to the replication in vitro and pathogenicity in vivo were speculated. An infectious clone of ADV-G was constructed and several mutants were rescued successfully. After analysis of the biological characteristics of these mutants, some functional residues of determining viral pathogenicity and host range were illustrated. Substitution of some amino acid residues in ADV-G VP2 with correlated residues in BJ was made and several chimera AMDVs were rescued. Finally the mink infection experiments with the chimera AMDVs were carried out smoothly. The main research results and conclusions are as follows.(1) The genome of a wild strain of Aleutian disease mink virus (BJ) was sequenced and compared with those of other strains reported. The results showed that 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. Moreover, we found that the positions of NS3 ORF stop codon in genome were not constant among AMDV strains.(2) Preparation of high titer monoclonal antibodies against NSla.a.36-52 and VP2 a.a.428-446 antigenic determinants of AMDV. Detection showed they were very specific and effective specifically.(3) A full-length genome clone of ADV-G, named as pADV-G, was constructed by using the In-Fusion technology to ligate all the fragments seamlessly. A restriction site of Sac I (A3875C) was introduced into pADV-G as a genetic marker to distinguish the rescue virus from normal ADV-G. Post transfection of pADV-G into CRFK cell line, identification of the rescued ADV-G with IFA, Western blot and absolute quantitative PCR was performed successfully.(4) Part sequences of four wild AMDV strains (BJ. SD, HLJ and HB) were analyzed and their VP2 a.a. alignment with other 17 AMDV isolates published in NCBI was made. Four a.a. sites (92,94,115 and 116) were very conservative among pathogenic strains but different with ADV-G. After a series of mutation on pADV-G infectious clone and identification of the recued mutant virues, the VP2 residues 92H and 94Q located on the surface of capsid were confirmed to be the critical elements determining ADV-G can survival in vitro.(5) On the basis of pADV-G, two chimera full-length genome clone (455-590 substitution) and VP2 (352/455-590 substitution) were constructed by replacing part of ADV-G VP2 ORF with corresponding BJ sequence and both of neotype AMDVs were rescued successfully. The transient viremia in minks was detected at the 10th day post infection of neotype AMDVs. No classic AD emerged in all infection time. But the specific antibody to AMDV could be still found in 30 days post infection.In this study, we located the critical functional residues on the surface of VP2 capsid in whole-viral sight. And it had been proved that substitution of of ADV-G partial VP2 with pathogenic strain could rescue chimera virus with infectivity to the natural host. The full-length genome comparison between BJ and ADV-G supply a solid basis and a new study direction in AMDV pathogenicity.