Isolation And Preliminary Characterization Of Proventriculopathogenic Isolates Of Inrectious Bronchitis Virus.

Biological and physiochemical characteristics of the virus isolates2/97,3/97 and 1/98Enlarged Proventriculi of clinically affected chickens from 2 chicken farms one atJiande city(named 2/97 and 3/97) in march, 1997 and the other chicken farm at thesuburb of hangzhou city(named 1/98) in April, 1998 all in Zhejiang province werecollected, homogenized and purified. Suspected virus sample was adapted in SPFchicken embryos for viral isolation and identification. The virus as revealed bytransmission electron microscope is about 100-120 in diameter.The virus had several biological characteristics similar to IBV by agglutinating red bloodcells after treatment with trypsin. It also interfered with the reproduction of Newcastledisease virus in chicken embryos. Virus isolates also exhibited lower specificneutralizing reaction with reference positive IBV antiserum. Detection of virus infectionwas also possible through Embryo death. Embryos that die and survive through the l8^thday of incubation are dwarfed and are seen curled into sphericai form with the feetdeformed and compressed over the head. Dead embryos were abnormally smaller thanthe normai chicken embryos of the same age. Lesion of the embryo such as stumting, andabnormal development of muscle were observed too. The physiochemical propertiesshowed tha infectivity of the virus isolates 2/97, 3/97 and 1/98 was decreased at 45℃ for90 min and 56℃ for 15 min in chicken embryos as well as to alkaline (pH12). Theirinfectivity to chicken embryos was also lost after exposure to chloroform for 10 min.Repeated freezing and thawing for 5 and 10 times or exposure to acidic (pH3) for 3 hr at37℃ did not affect its infectivity.Cloning and sequence analysis of the S1 gene of the virus isolates2/97,3/97 and 1/98cDNA of S1 gene of the virus isolates 2/97,3/97 and 1/98 was amplified by reversetranscriptase polymerase chain reaction with special primers and were then cloned intopBS SK(+). Recombinants of each of the isolates were ideatified, sequenced andanalysed. Sequence analysis showed that the S1 gene of each of the isolates havenucleotides of 1930 residues with a capacity to code a polypeptide of 620 amin acids.The nucleotide sequences of IBV-2/97, 3/97 and 1/98 S1 gen were compared with eachof the 10 IBV strains in the Genbank. It was found that the homlogies between 2/97,3/97, 1/98, H52, M41, Beaudette and ZJ971 S1 gene are high but low in other IBVstrains used in this comparison. Further analysis showed that 2/97, 3/97, ZJ971 and H52are closely related while 1/98 is less related to any of these four or even when comparedwith other strains. At position 1239-1244, 1/98 isolate has six different residues from2/97, 3/97, 1/98, H52, M41, Beaudette, 6/82, Connecticut,Gray and ZJ971 and fiveresidues different from Ark99, Cut2 and Holte. There was no any insertion nor deletionof S1 gene except several point mutations.Pop wise cOmParisons of the deduced amin acids seqUenCe of the Sl gen of thesepe isolates with othe I0 IBV twe strains used in nds ndes reVealed highhopology ranging from 93.4%-99.25% among 2/97, 3/97, l/98, H52, M4li BeaUdeheand ZJ971 S1 SUtw bu low homlogy with other IBV shas. Point mndons whichresulted in wt acid subsitutiOn ocrmd in the S l SUbuni ofdfor shas. Notablyat POsihon l62, there is tyrosine m in l/98, 2/97, 3/97 and ZJ971 but aSPaxagin oo inobo IBV bons. ttis difference is believed to be the caase of high hydroPhObic indexin I/98, 2/97, 3/97 and ZJ97l Sl subunit and Inay be the faCtor leading to dmerent IBVtroPismThe8e fmding8 coupled with the biologlcal and phy8iockemica cboracteristics pointto tbe very po88ibnity that these are novel variant8 of infectious brouckthe viru8Laboratory trial of inactivated oil emulsion Vaccine against proventri-culoPathogenic strain of infectious brochitis virus ZJ97lIBM97l a proVentricuIopathogenic otn is the cause ofproventriculitis, a ProbIem deing theprocessing of br...