Molecular Epidemiology Of Infectious Bursal Disease Virus Circulating In Pakistan

Infectious bursal disease(IBD),caused by infectious bursal disease virus(IBDV),is one of the most devastating and immunosuppressive diseases of the poultry and has been a constraint on the sustainable food security around the globe including Pakistan.Poultry industry is the second biggest industry in Pakistan while IBD is an important disease seriously threatening poultry farming.Despite the use of mass and intense vaccination regimens,the disease continues to sustain in many countries around the globe including Pakistan.However,to some extent,the epidemic strains,epidemic patterns and predominant strains of IBDV in Pakistan are unclear.Even no complete genome of Pakistan IBDV is available in GenBank.This highlights the need to characterize field strains of IBDV to ascertain the evidences of vaccine failure and to establish foundations for the development of vaccine matching with the epidemic strains.IBDV is non-enveloped virus with bi-segmented,linear,and double-stranded RNA genome with 3.2 kb long segment A and 2.8 kb long segment B.Segment A encodes two open reading frames(ORF)where ORF1 encodes the protein VP5 whereas ORF2 encodes a longer precursor polyprotein(PP).This PP is further cleavage into VP2,VP3,and VP4.VP2 is the major structural protein,virulent determinant,and protective immunogen.Segment B encodes VP1 which is an RNA dependant RNA polymerase(RdRp).In this study,an epidemiology research was performed covering 29 broiler flocks in the main poultry farming regions(Punjab,Sindh,Baluchistan,and Islamabad)in Pakistan from 2017 to 2018.RT-PCR results showed that 87% samples were positive for IBDV.For the first time,the batch of 37 Pakistan IBDV sequences covering both VP2 and VP1 were amplified and analysed.The VP2 and VP1 genes are representative genes of the two segments of the IBDV genome,respectively,and their sequence analysis is very important for the genetic evolution research of IBDV.Previous research revealed only a few partial VP2 but not any VP1 sequences of Pakistan IBDVs had been published.Our data discovered the characteristic molecular features of Pakistan IBDVs.The unique segment-reassortant IBDVs(vv-A/Uniq-B),carrying segment A from vvIBDV and segment B from one unique ancestor,were firstly identified as the predominant strains in Pakistan.To further understand the epidemic characteristics of IBDV predominant strains in Pakistan,six selected IBDV strains(PK2,PK12,PK19,PK28,PK33,and PK47)from different places covering Punjab,Sindh,Balochistan,and Islamabad of Pakistan were isolated and the full-length genomes were cloned and determined.To our knowledge,it is the first batch of full-length genome sequences of Pakistan IBDVs appeared in GenBank.The full-length genome analysis further confirmed the unique reassortant characteristics(vv-A / Uniq-B)with regional characters of Pakistan IBDVs.Furthermore,the pathogenicity of Pakistan IBDVs was evaluated using animal experiment of SPF chickens.All six Pakistan IBDVs induced high morbidity and high mortality.The present study clearly discovered the main epidemic characteristics of Pakistan IBDVs.To further understand the antigen matching of currently available vaccines with Pakistan IBDVs.The efficiency of two vaccines against Pakistan IBDVs was evaluated using SPF chickens.The challenge protection experiment showed that vaccines available could provide clinical protection against Pakistan IBDVs.Furthermore,one questionnaire survey discovered that various factors including antigen matching of vaccine,biosecurity,and feeding management might be involved in avian disease control.In conclusion,one epidemiological survey of IBDV covering the main poultry farming regions of Pakistan was performed.For the first time,the genome of Pakistan IBDVs covering both segments were determined and analyzed.The unique segment-reassortant IBDVs(vv-A/Uniq-B)with regional characters were firstly identified as the predominant strains in Pakistan.Pakistan IBDVs showed high morbidity and mortality.Vaccines selected could provide clinical protection against Pakistan IBDVs while various factors including feeding management might be involved in disease control.This study will contribute to the scientific prevention of IBD.