CSBV-SDLY Characteristic Analysis And Development Of Egg Yolk Antibody Against CSBV

Sacbrood virus(SBV)is one of the most pathogenic honey bee viruses that exhibits ho st specificity and regional variations.The SBV strains that infect the Chinese honey bee Apis cerana are called Chinese SBVs(CSBVs).CSBV infects Apis cerana larvae,resulting in the inability of the larvae to pupate and their consequent death,which may pose a serious threat to entire colonies.As there is no effective medical treatment for CSBV infections,further studies are necessary.In this study,31 CSBV strains were isolated from 2008 to 2017,and the homology of nucleotides and deduced amino acid and genetic evolution relationships were analyzed using VP1 gene as target gene.The results show that CSBV has several branches in China.However,the host specificity and regional differences of the newly isolated strains have changed.During this perio d,a strain of CSBV was isolated from A.mellifera larvae from an A.mellifera farm in Linyi,Shandong Province,which infecting naturally A.mellifera larvae and caused death.A strain CSBV(named as Am CSBV-SDLY)was identified by clinical observation,electron microscopy,Agar gel immunodiffusion test,SDS-PAGE and A.cerana larvae inoculated with CSBV.Next,genome composition and structure of Am CSBV-SDLY,and the homology of nucleotides and their deduced amino acids of Am CSBV-SDLY and other CSBVs/SBVs were analyzed.It was found that the nucleotide sequence of the genome consisted of 8794 bp(excluding poly A tail).The contents of base A,U,G and C were 29.95%,29.22%,24.30% and 16.52%,respectively.Nucleotide homology reached 86.8%-100%,and the homology from nucleotide to amino acid reached 86.2%-100%.And CSBV/SBV coding regions all gene as A target gene for genetic evolution analysis,the results show that all SBVs/CSBVs isolates form two distinct branches,A branch of the main separation east SBVs/CSBVs strain bees(A.cerana)(named AC genotype),another branch SBVs/CSBVs strain separation mainly in A.mellifera(named AM genotype)AC genotype further divided into four subtypes,They showed regional differences and host specificity.The genetic and evolutionary relationship of p Hylogenetic tree also indicated that Am CSBV-SDLY belongs to the same branch as CSBV and other Asian SBVs/CSBVs isolates,and that the genetic distance between Am CSBV-SDLY and CSBV-FZ isolates is the closest,which further proves that Am CSBV-SDLY belongs to CSBV.Similarity analysis and virus recombination analysis,found in Am CSBV-SDLY as A query sequence,originated from A.mellifera Am CSBV-SDLY isolates with previous CSBV isolates have high similarity,the similarity of more than 85%,with CSBV isolates formed A relatively independent of separation and Am SBV-UK isolates similarity is far lower than CSBV isolates,formed the obvious deviation,maximum deviation appeared in 2181 and 2221 bp,reached 19.3%,However,only CSBV-JL2014 had recombinant signal,and the recombinant fragments were 3 in total,one from Am CSBV-SDLY strain and two from CSBV-FZ strain,followed by the parent of CSBV-LN2009.Boot Scan analysis showed that the cross-recombination signals of am CSBV-SDLY and CSBV-FZ were detected at 1957-2162 and 6635-7110 respectively,using am CSBV-SDLY and CSBV-JL2014 as query sequences.It was proved for the first time that CSBV could break through the interspecific barrier and naturally infect the A.mellifera larvae and cause death,which had strong pathogenicity.The inactivated vaccine was produced by ultracentrifugation and formalin treatment,using CSBV purified from a natural outbreak.The specific Ig Y was produced by immunization of white leghorn hens with the vaccine.An enzyme-linked immunosorbent assay using purified CSBV as the coating antigen revealed that the anti-CSBV Ig Y titer began increasing in the egg yolk on the 14 th day post-immunization,reaching a peak on day 42,and anti-CSBV Ig Y remained at a high level until day 91.Ig Y isolated from the combinations of egg yolk collected between days 42-91 was purified by PEG and ammonium sulfate precipitation.In three repeated protection experiments using A.cerana larvae inoculated with CSBV,the survival rate of larvae was more than 80%,and the titer of anti-CSBV Ig Y was more than 25 and 24 when the larvae were fed Ig Y 24 h after and before inoculation with CSBV,respectively.Therefore,400 colonies infected with CSBV were treated by feeding sugar containing Ig Y solutions with an antibody titer of 25,and the cure rate was 95%-100%.Three hundred susceptible colonies were protected by feeding the larvae with sugar containing Ig Y solutions with an antibody titer of 24,and the protection rate was 97%.The results clearly suggest that a specific Ig Y was obtained from hens immunized with an inactivated-CSBV vaccine;this may be a novel method for controlling CSBV infection.