| Infectious bursal disease(IBD)is an acute,highly contagious infection and immunosuppressive disease caused by infectious bursal disease virus(IBDV).It seriously endangers the poultry health and brings great economic losses to the poultry industry.In addition to the prevalence of the virulent IBDV(vvIBDV),classical IBDV(c IBDV)and attenuated IBDV(attIBDV)strains in China,the emerging of novel variant IBDV(nvIBDV)in recent years has also brought new challenges to the diagnosis,prevention,and control of the disease.To investigate the prevalence of IBD and the molecular characteristics of epidemic IBDV strains,bursal tissues were collected from the chickens with the suspected IBDV infection during 2020-2021.IBDV was isolated by the inoculation of the samples into the chicken embryos and then the molecular identification and analysis on the isolates were performed.The results showed that a total of 9 IBDV strains were successfully isolated.Nucleotide similarities of the important genes vVP2 and VP1-b of the isolates with the reference strains,and the comparison on the key amino acid sites were also evaluated.Based on the segment A,there were two,two and five isolates belonging to types A1a,A2d and A3,respectively.While based on the segment B,there were four,three,one and one isolates belonging to B1a type,B1b type,B2 type and B3 type,respectively.By constructing phylogenetic tree and using the latest genotyping scheme,the 9 isolates belonged to six genotypes:two A1a B1a,one A2d B1b,one A2d B3,two A3B1a,two A3B1b and one A3B2.Using the traditional classification scheme,5 of the 9 isolates belonged to vvIBDV(55.56%),two belonged to nvIBDV,and two belonged to c IBDV(22.22%).The results of the recombination and selection pressure analysis showed that there were no recombination found in the vVP2 and VP1-b genes of all the strains and they were mainly subjected to purifying selection.Based on the finding of the co-epidemic multiple pathotypes of IBDV and for the purpose of rapid differential diagnosis and quantification in clinical practice.A novel technique so called RT-ARMS-qPCR assay,specific for the three major pathotypes vvIBDV,attIBDV and nvIBDV,based on the amplification refractory mutation system PCR(ARMS-PCR)and combined with the quantitative real-time PCR(qPCR),was developed in the study.The test of the developed assay showed that it had a good linear relationship.The standard equations of vvIBDV,attIBDV and nvIBDV were y=-3.2254x+42.93,y=-3.3183x+39.07 and y=-3.4296x+42.14,respectively.R~2 was all higher than0.99,the intra-group coefficient of variation was less than 0.51%,and the inter-group coefficient of variation was less than 1.15%,indicating that the repeatability of the assays was good.The minimal quantities of the assay on vvIBDV,attIBDV,and nvIBDV were 65.6 copies/μL,70.2 copies/μL and 57.8copies/μL,respectively,indicating that the sensitivity of the assay was good.The type-specific primers used in the detection could only amplify the target fragment of the corresponding pathotype,but not to other pathotypes of IBDV and other common avian pathogens,indicating that the specificity of the assay was good.To study the virus proliferation and pathogenicity of the mixed infection of different pathotypes of IBDV in chickens,an experimental model of the mixed infection of the three pathotypes was simulated by the challenge of the birds with the combination of the viruses.The observations of the clinical symptoms,gross lesions and the histopathological the bursal tissue were recorded,and the immune organ index and the viral load in bursal tissue were detected.The results showed that the birds infected with attIBDV and nvIBDV strains did not show typical IBD symptoms,while the co-infection of vvIBDV and nvIBDV and the triple mixed infection of attIBDV,vvIBDV and nvIBDV strains could significantly increase the pathogenicity and resulted in significant disease.When infected with a single pathotype of virus,the proliferation ability of vvIBDV and nvIBDV strains was all higher than that of the attIBDV strain,and the proliferation speed of vvIBDV strain was higher than those of the attIBDV and nvIBDV strains at the early stage of infection.The proliferation speed of attIBDV strain was significantly inhibited by either with the mixed infection of vvIBDV or nvIBDV strain,while the vvIBDV or nvIBDV strains were not affected in the cases.The results of the study demonstrated that the prevalence of IBD in Guangxi showed the characteristics of the co-prevalence of multiple pathotypes and genotypes,and vvIBDV was still the dominant type.A novel RT-ARMS-qPCR assay that can differentiate vvIBDV,attIBDV and nvIBDV was successfully developed,which can be used both for the qualitative diagnosis and the quantitative detection.The biggest advantage is that each single pathotype in the mixed infection cases can be qualified and quantified.The co-infection of different pathotype viruses not only can enhance the pathogenicity,but also affect the proliferation of the virus. |
