Isolation And Identification Of IBDV In Guangxi And The Full-length Genome Sequence Analysis And Pathogenicity Study Of Two Variant Strains

Infectious bursal disease(IBD)is an acute,serious and highly contagious infectious disease caused by the infectious bursal disease virus(IBDV).It is one of the most important immunosuppressive diseases in the world poultry industry.There are four main pathotypes of IBDV,including classical IBDV(c IBDV),variant IBDV(vIBDV),very virulent IBDV(vvIBDV)and novel variant IBDV(nvIBDV),and the latter is the first newly discovered pathotype in China recently.There are differences in pathogenicity and antigenicity among different pathotypes,which might bring difficulties to the industry for the prevention and control of IBD.Therefore,it is of great significance to track and understand the prevalence of IBD,the pathotype and characteristics of the prevalent strains in time for the effective control of the disease.IBDV was isolated and identified by sampling from suspected clinical cases of IBD in Guangxi during the years 2017-2019 and inoculating into the chicken embryos,and 13 IBDV isolates were successfully isolated.Based on the amplification and analysis of vVP2gene in segment A and VP1-b gene in segment B of the isolates,the prevalence of vvIBDV(9 strains),attenuated strain(1 strain)and nvIBDV(3 strains)was confirmed in Guangxi during 2017-2019.These13 isolates belong to four different genotypes:genotype?(segment A with the that of the attenuated strain,segment B with that of the vv strain),genotype?(segment A with that of the vv strain,segment B with that of the Uniq-B strain),genotype?(segment A with that of the novel variant strain,segment B with that of the Uniq-B)and genotype?(segment A with that of the novel variant strain,segment B with that of the novel variant strain).For the first time,the prevalence of the nvIBDV strains(QZ191002 and QZ191003)and the novel variant reassortant strain(YL190623)was confirmed in Guangxi,of which the nvrIBDV strain was reported for the first time in China.The nvIBDV strains QZ191002 and QZ191003,and nvrIBDV strains YL160304 and YL190623,iaolated during 2016-2019 were firstly purified by plaque technology and then the full-length genome sequence of these strains was sequenced and analyzed.The results indicated that the segment A of all these strains have the highest nucleotide similarity(94.4%-98.9%)with the nvIBDV reference strain SHG19.Phylogenetic tree analysis showed that they belonged to the same clade and had the highest similarity(85%-100%)on the antigen-related amino acid residues in segment A with SHG19.The segment B of nvrIBDV strains YL160304and YL190623 have the highest nucleotide similarity(97.6%-97.9%)with the dominant prevalent vvIBDV strain HLJ-0504 in China in recent years.Phylogenetic tree analysis showed that they belong to the same branch and have the highest similarity(100%)of the key amino acid residues in segment B with HLJ-0504 strain.The segment B of nvIBDV strains QZ191002 and QZ191003 has the highest nucleotide similarity(97.2%-97.9%)with the nvIBDV reference strain SHG19.Phylogenetic tree analysis showed that they belong to the same branch and have the highest similarity(88%)of the key amino acid residues in segment B with SHG19.The nvIBDV strain QZ191002 and the nvrIBDV strain YL160304were selected to study the pathogenicity in Three-yellow chickens.A total of 30 commercial day-old Three-yellow chicks were used in this experiment,which were kept separately in the isolation condition in the whole 35 days of the experiment and were free to eat and drink.The serum of birds was collected every week and the antibody titres were detected by ELISA.When the serum antibody level turned negative at the age of 28 days,the birds were randomly divided into three groups with 10chickens for each,groups A and B were challenged with the isolates QZ191002 and YL160304 via the oral infection with the doses of 10^5.0TCID₅₀/0.2 ml,respectively,and group C was inoculated the same volume of PBS in the same route and kept as the uninfected control.The performance and death of the birds were recorded daily.At the end of the experiment(35 days of age),birds in each group were weighed and serum was collected.All the survived birds and the birds that died during the experiment were necropsed.Spleen and bursa of fabricius were collected respectively for the detections and calculations of the IBDV viral load,bursal/body weight ratio,BBIX and spleen/body weight ratio.The results showed that the nvIBDV strain QZ191002 did not cause typical symptoms of IBD and death,but could cause severe atrophy of the bursa of fabricius(BBIX was 0.46),while the nvrIBDV strain YL160304caused typical symptoms of IBD with a mortality rate of 10%(1/10)and more severe atrophy of the bursa(BBIX was 0.35).The viral load tests of the bursa and spleen,as well as the histopathological observation of the bursa showed that the virulence of nvrIBDV strain YL160304 was significantly higher than that of nvIBDV strain QZ191002.The results of the study showed that vvIBDV,attenuated strain and nvIBDV were prevalent in chickens in Guangxi during 2017-2019,and the prevalence of nvIBDV and nvrIBDV strain were first began in 2016.The full-length genome sequence analysis also revealed the difference of the evolutionary relationship between the segment A and segment B of these two novel variant strains.The results of the pathogenicity study on Three-yellow chickens found that the pathological characteristics of these two novel strains was the severe atrophy of bursa and that of the nvrIBDV strain was significantly worse than that of the nvIBDV strain.