Analysis Of BCR H Chain CDR3 Difference Expression From Mice Infected With PRV Variant And Attenuated Vaccine Strains By HTS

Pseudorabies virus(PRV),also called Aujeszky disease virus(ADV)or suid alpha-herpesvirus 1(SuHV-1),can infect many kinds of animals.Since 2011 late,so many PRV variants have appeared in our contry that PRV outbreaks agin,which has been well controlled in China by immunizing introduced vaccine Bartha-K61.Pregnant sows showed abortion,stillbirth,and mummy fetus,suckling piglets showed neurological disorders,diarrhea,and high mortality,growing and fattening pigs showed persistent diarrhea,adult pigs showed seriously respiratory disorders,and sows showed reproductive failure.The results of virus islation and identification,bioinformatics analysis,and animal challenge experiments all reavled that the immune effect of Bartha-K61 vaccine has been slightly reduced.In this work,we identified the virulence of variant PRV strain XJ and low virulence PRV stain Bartha-K61 by 50%tissue culture infective dose in BHK-21 cells and median lethal dose in BALB/c mice at first.The TCID50 in BHK-21 of strains XJ and Batha-K61 were 10-7.27/0.1mL and 10-6.11/0.1mL,respectively.The virulence in mice of strains XJ and Batha-K61 were 10-5.16/0.1mL?10-2.32/0.1mL,respectively.Based on these results,strains XJ and Batha-K61 were used 10-6 and 10-3 virus to inject mice from hypodermic of neck.Then we collected the serums before and after 1,4,7,9,11,14,17,19,21,28 day post inoculation(dpi),and monitored the changes of PRV gB antibody level by enzyme linked immunosorbent assay to find the second injection time and sample collected time.The results displayed that average antibody change was similar between groups X and B.Antibody of gB was first discovered at 7 dpi,up to peak at 9 dpi,and began to slow down until 14 dpi,which point we chosed to inject again.Then,gB antibody up to the most higher point at 17 dpi,began to slow down at 19 dpi,tend to be gentle at 21 dpi,and could still be detected at 28 dpi.According to these results,we collected sample at 17 dpi.Total RNA were extracted respectively from three mixed samples belonging to groups X,B,and,C,and used to amplify the B-cell receptor(BCR)H chain genes V,D,and J by Multiplex-PCR,which contained complete complementarity-determining region 3(CDR3).The PCR products,also called sequencing libraries,were prepared to sequence by Illumina Miseq,and datas were analyzed by IMGT/V-QUEST,IMGT/HighV-QUEST and other softwares of bioinformatics.The basic statistics of high throughput sequencing(HTS)showed that datas had a good quality.Analysis results revealed that the specific CDR3 nt and aa sequence expression from high to low were groups X,B,and C,which were supposed to be related to PRV infection,and the highly expended clones were produced by variant and low virulence PRV strains.Compared with group C,groups X and B up-regulated genes IgHV2-9?IgHD1-1?IgHJ1?IgHV1-14-IgHJ2?IgHV3-2-IgHJ2,and down-regulated genes IgHV14-3?IgHD4-1?IgHJ3?IgHV1-14-IgHJ2?IgHV1-47-IgHJ3?IgHV1-5-IgHJ3,lost genes IgHV 1-11-IgHJ1?IgHV 1-50-IgHJ 1?IgHV1S61-IgHJ1,the unique expression CDR3 nt and aa might be related to PRV infection.Compared with group B,groups X showed difference usages of genes IgHV14-3?IgHV3-2?IgHV2-9?IgHD1-1?IgHD1-2?IgHJ4?IgHV8-12-IgHJ1?IgHV12-3-IgHJ4,and expression genes IgHV1-16?IgHV1-48?IgHV1-62-1?IgHV1S15?IgHV1-12-IgHJ1?IgHV1-22-IgHJ1,also the unique expression CDR3 nt and aa might be related to variant and low virulence PRV strains infection.In this paper,we successfully established a BCR sequencing model of mice infected with PRV variant and attenuated vaccine strains.We found the different BCR clones before and after PRV infection,and between different PRV strains infection,and comprehensive analysed the complete genes of CDR3 highly expended clone of groups X,B,and C,which were helpful for research of newly security and efficient BCR vaccines of PRV.