| Pseudorabies(PR)is a highly contagious animal infectious disease caused by Pseudorabies virus(PRV),which is characterized by invasion of the central nervous system.Pig is its only natural host.In the 1980 s,a live attenuated pseudorabies vaccine called Bartha-K61 from Hungary was introduced to China,and the disease had been effectively controlled because of the widely used of the vaccine.However,a PRV variant emerged and caused severe PRV outbreaks in many farms in 2011.The variant strain has once posed a great threat to the pig industry.To control and even eradicate the disease eventually,a more efficient and accurate antibody detection method for PRV is an urgent need in field practice.In this study,positive tissue samples from PRV infectious pigs collected in the farm in Heilongjiang Province in 2013 were first used for virus isolation,and a strain named PRV HLJ-2013 was obtained.Subsequently,the whole genome sequence of the virus was determined using high-throughput sequencing technology,and the evolutionary characteristics of the virus were analyzed at the level of genome,protein encoding gene,and amino acid sequence.The results showed that the full-length genomic sequence of HLJ-2013 was 142.56 kbp in length;on the phylogenetic tree based on the full-length genome sequences,HLJ-2013 was clustered with the previous Chinese isolates in the same branch belonging to Genotype II,but it was designated to a unique branch that phylogenetically occurred earlier than all the Chinese isolates;phylogenetic and evolutionary analysis showed that protein encoding genes of HLJ-2013 showed different evolutionary relationships with other reference strains,of which UL10(gM),UL44(gC),UL36(VP1/2)were phylogenetically closer to those of the foreign strains;while US6(gD),UL19(VP5)were similar to the earlier Chinese strains(such as SC,Fa,Ea strains).The above results suggested that HLJ-2013 strain was an early epidemic RPV strain in China.Furthermore,the infection and pathogenicity of HLJ-2103 in PK15 cells and mice showed that it was similar to that of SC and HeN1.The infections of HLJ-2013 in mice resulted in obvious injury in the brains and lungs,and the virulence was stronger than that of SC strain,but weaker than that of HeN1.A magnetic particle chemiluminescence assay for detecting antibodies against swine pseudorabies virus was established by purified and concentrated PRV HLJ-2013 virus antigen with a concentration of 5.2 mg/mL.The various reaction conditions affecting the results of the method were optimized and finally determined that the antigen concentration for coating magnetic beads was 400 ?g/mL,the dilution of the enzyme-labeled secondary antibody was 1:10000,the concentration of BSA was 2%,the concentration of PBST washing solution was 0.2%,the optimal time periods for the two-step immune reaction was 10 min+10 min,and the time period for luminescence value detection was 5 minutes post the enzymatic luminescence reaction.In order to further evaluate its application value,the sensitivity,specificity,cross-reactivity,repeatability,and compliance with a commercial kit of the method were analyzed.The results showed that the method established in this study was highly sensitive,it was more sensitive than a commercial PRV gB antibody ELISA kit;it has good stability,both of the coefficient of variations within and between batches were less than 10%,and there was no cross-reaction with the positive serum of ten other swine diseases.Using this method and the commercial ELISA kit to detect 126 clinical samples stored in the laboratory,the total coincidence rate of the two methods was 96.83%.And two methods reached a consistent result when they were used to detect antibodies against a PRV vaccine strain.In summary,a successfully isolated strain of PRV HLJ-2013 has certain genetic and biological characteristics that are different from the previously isolated domestic strains.By using it,we established a magnetic particle-based chemiluminescence assay to detect the antibodies against PRV.The assay showed good performance in terms of sensitivity,repeatability,specificity,and thermal stability,and has certain application value,which has the potential that could be used for monitoring the immune responses in pigs to the PRV infections and vaccination as well. |
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