Strain Identification Of Potato Virus Y(PVY) Isolates From China And Preparation Of Antiserum Against PVY

Potato virus Y(PVY)is an important virus that mainly infects solanaceous crops including potato and tobacco.PVY is one of the most widespread and economically harmful plant virus and causes huge losses to potato production worldwide.To investigate the PVY strain is of great significance for early detection and effective control of PVY infecting potato.Planting virus-free seed potatoes is the most cost-effective way to control potato viral disease.Rapid,sensitive,and specific detection methods are the keys to ensure the quality of virus-free seed potatoes.Serological assays have the advantages of simple,rapid,and sensitive,and are widely used in the detection of plant viruses.However,some PVY isolates can escape the detection using monoclonal antibody for PVY.Therefore,improving the PVY detection system is of great significance for potato production.Here,we identified the strain of PVY isolates KS4 and KS7 from potato in Heilongjiang,analyzed the molecular characteristics of the Chinese PVY isolates available in Gen Bank,and obtained a combination of PVY antibody that can detect more PVY isolates.The main results are as follows.1.PVY isolates KS4 and KS7 belong to strains N:O and NTN-b,respectivelyThe genomic fragments of potato virus Y(PVY)isolates KS4 and KS7 were amplified using RT-PCR,cloned and sequenced.Recombination and phylogenetic analyses were conducted with Recombination Detection Package and the maximum-likelihood methods,respectively.Their open reading frames of isolates KS4 and KS7 were 9 186 nucleotides,encoding a polyprotein of 3 061 amino acids.Isolate KS4 shared the highest nucleotide and amino acid identities of 96.9%and 98.4%with isolate Mb112,while isolate KS7 had the highest nucleotide identity of 97.4%with isolate 12-94 and amino acid identity of 97.8%with isolate SYR-II-Be1.Recombination analysis indicated that isolates KS4 and KS7 were recombinants of isolates N-605 and Oz;the 5′-terminal 2 392 nucleotides of isolate KS4 were from isolate N-605,while the other genomic fragment was from isolate Oz;the 800-2 227nucleotides and 5 637-8 950 nucleotides of isolate KS7 were from isolate N-605,while the other genomic parts were from isolate Oz.The results of phylogenetic analysis showed that KS4 and KS7 isolates were clustered into groups of N:O and NTN-b,respectively.2.The strain composition and genetic structure of Potato virus Y(PVY)isolates from ChinaSequence recombination,phylogenetic relationship,and selection pressure were analyzed using all the 75 complete genomic sequences in PVY isolates available.The PVY isolates were divided into 11 strains including Eu-N,O,C,NA-N/NTN,N-Wi,N:O,NTN-a,NTN-b,SYR-I,and SYR-II.The PVY isolates from China were clustered into 9 strains except strains E and C.Recombination analysis showed that 69 of the 75 Chinese PVY isolates were recombinants.According to difference in recombination sites,the 69 isolates were divided into 11 recombination types,among which five were new recombination pattern.The results of selection pressure analysis showed that all the 11 proteins of PVY were under negative selection,with NIb gene under the highest selection pressure and PIPO under the least selection pressure.The gene flow was frequent among potato and tobacco PVY isolates from China,but infrequent among PVY isolates from other hosts.3.Combination of polyclonal antibodies that can detect more PVY isolatesSequence alignment using 1533 PVY CP sequences determined here and deposited in Gen Bank showed that there existed 90 types for the epitope covering amino acids 2 to 14 of PVY CP.Six types were selected for polyclonal antibodies preparation.Site-directed mutation was introduced into p Cam PVY~N605-GFP,an infectious clone of PVY N605,to produce 85 PVY mutants,which can express 85 of the 90 epitope types mentioned above.The results of indirect ELISA showed that MAb1130,the most widely used monoclonal antibody for PVY in the world could recognize 55 types,accounting to 64.71%of the epitope types,and detect 1433 isolates,accounting to 93.91%of the PVY isolates;while combination of PAb-3,PAb-5,and PAb-6 could recognize 68 types,accounting to 80.00%of the epitope types,and detect 1504 isolates,accounting to 98.56%of PVY isolates,indicating that the combination could detect 15.29%more types of the epitope and 4.65%more PVY isolates than MAb1130.