Complete Genome Sequencing Of Raccoon Dog And Arctic Fox Amdoparvovirus And Construction Of Its Infectious Clone

Raccoon dog and arctic fox amdoparvovirus(RFAV)is a single-stranded negative-strand linear DNA virus,which belongs to the Parvoviridae,Parvovirinae,and Amdoparvovirus.RFAV is a new species of amdoparvovirus that naturally infects raccoon dogs and arctic foxes.Because of 3' Y-shape and 5' U-shape hairpin termini in amdoparvoviruses genome,it is difficult to amplify and sequence their terminal sequences.At present,the complete geneome sequence of amdoparvoviruses on Gen Bank is rarely released.In order to clarify its molecular pathogenic mechanism at the whole genome level,and to study the etiology and immunology of amdoparvoviruses in depth,the construction of infectious clone of RFAV is the research cornerstone and technical challenge.In this study,we designed a special method to amplify and clone the ITR of RFAV genome.Here,the whole genome of three RFAV strains of length 4 832 bp,4 827 bp and 4 830 bp was obtained,and named as “RFAV-Y9J” and “RFAV-RD15”,“RFAV-HS-R”,respectively.A homology comparison was conducted with the terminal sequences of the Aleutian mink disease virus(AMDV).Besides,the mfold Internet Server was used to predict the secondary structure of the terminal sequences of the RFAV.The3' end genome sequence had a 116-bp Y-shaped hairpin structure,which was highly conserved between and within the strains of the RFAV or AMDV;the 5' end genomic sequence of RFAV-Y9 J and RFAV-RD15 had 310-bp and 305-bp U-shaped hairpin structures,respectively.The 5' end genomic sequence was highly conserved within the strain of the RFAV or AMDV,whereas the inter-species 5'end genomic sequence showed a greater variation.The terminal sequence of RFAV is different from AMDV,forming a sequence feature of a new species of amdoparvovirus.Based on the consensus sequence of previously published AMDV-G and RFAV genomes,three primer pairs were designed to amplify the three overlapping fragments(M1,M2,M3)covering the whole genome of the middle of virus genome and identified them with gel electrophoresis.According to the obtained terminal sequence information,the 3' and 5' terminal sequences were artificially synthesized on the expression vector p BB,and the Stu I restriction site was introduced between the 3' and 5' ends.Then we use the infusion technology to insert the middle fragment into the p BB plasmid which contain3' and 5' ends.The full plasmid was identified by enzyme digestion to prove that it was successful constructed.The F81 cells were transected with Lip 3000 with the ratio of 3.75 ?L Lip3000: 3.5?g DNA and 7.5 ?L Lip3000: 3.5?g DNA.The r RFAV was passed to one generation in five days,which was passed to the sixth generation and identification of rescued r RFAV with CPE,PCR,electron microscope observation was performed successfully.We sequenced,for the first time,the 3'-and 5'-terminal sequences of the RFAV.In this way we provided an effective method for amplification of the 3'-and 5'-terminal sequences of other amdoparvoviruses.Constructed a full-genome infectious clone of RFAV and successfully rescued the virus,providing a platform for studying the pathogenic mechanism of amdoparvovirus at the full-gene level and vaccine development.