Identification And Isolation Of The Muscovy Duck IBV And The Sequence Analysis Of The N Gene

Infectious bronchitis(IB) is an acute infectious disease caused by infectious bronchitis virus(IBV) in chicken. The serotypes of viruses are complex, leading to big differences in clinical symptom. The cardinal symptom of IB is hard breathing, laying drop, nephritis and proventriculitis. IB is more prevalent in the recent years, with more and more report of variant strain. With wide viral tropism, IBV mainly invades breathing, digesting and urogenital system. With complex serotype, the viruses vary frequently, causing big difference in clinical feature.Genome of viruses is ameristic linear RNA of positive sub-thread, of which the overall length is 27.6kb. It's main structural proteins are spike (S) proteins, membrane (M) proteins and nucleoproteid (N) proteins. The only one within membrane composed of 409 amino aids is N proteins, which could specially couple on leading RNA, and participates in replication, tanscription and translation of RNA in virus. During the ancient study, people concentrated mainly on gene S1, they considered gene N to be high conservation in the evolution of IBV. But recent study shows that gene N is of extensive variations too, what'more, the variations spead over epitope and relevant functional region, which indicate that variation of gene N in different strains may lead to changes of some biological traits of viruses.In December 2008, some diseases happened in a specific Muscovy duck farm in putian as follows: Laying descent of muscovy ducks happened during the laying fastiqium. The laying rate dropped from 85% to 30-40%, which could recover to 60% gradually in the next two months, with quality reduction of eggs; soft shelled eggs and malformed aggs increased; hatchability decreased. There are temporal respiratory symptom in the muscovy ducks with diseases, and few muscovy ducks died. Through gross examination of muscovy ducks with diseases we found out that ovary was swollen and conqestive, though there are no obvious pathological changes within other solid organs. We use the ovary of muscovy ducks with diseases to do virus isolation (the strain was named strain PTFY). After passages of chicken embryos, examination of physical and chemical properties, Hemagglutination test, electron microscope survy and sequencing comprison, the results showed as follow:First, through passages of SPF chicken embryos at nine days, dwarf embryos come up and there are lots of white flocks in allantoic cavity. Examination of physical and chemical properties about viruses showed the infectivity of viruses decreased after using chloroform, aether and deoxysodium cholate to dispose the viruses. through negative staining of Electron microscope ,we found out the diameter of virus particles was 100nm, which was sphere, covered with cyst membrane. On the cyst membrane we could see the petaloid spikes. All these characters accorded with the coronavirus.second, we utilized pancreatin of 1% to dispose the collecting allantoic fluid of the fifth generation, after which the hemagglutination titer was teseted. We found out that the longer time pancreatin digested, the higher hemagglutination titer would be to some extent. The hemagglutination titer would be highest at four hours, after which it would be stable. Collect allantoic fluid of chicken embryos at differernt cultured time(36h,48h,72h,96h,120h), after which the hemagglutination titer was teseted. The results showed that the longer time cultured,the higher hemagglutination titer would be.Third, we use viruses to do eye droppings and nose droppings to chickens, hard breathing and sluggishness heppened from the third day. Through gross examination of muscovy ducks with diseases we didn't found out obvious symptom. The control group are normal. We do RT-PCR test after geting kidney tissue, which proved that the muscovy ducks were infected with IBV.Fourth, interferential test of NDV using strain PEFY showed strain PTFY could inhibit the replication of NDV.Fifth , collect allantoic fluid of the fifth generation and do test of virus infectivity using cultures of tracheal ring, from which TOC-ID50=10-5.413/0.1ml were showed. We used strain PTFY and strain PT from our laboratory to do serum neutralization test, from which we got that the two antigens had low relevance.Sixth, refer to strain IBV(GenBank:DQ001338.1)in GeneBank, we design primers and get our products of 600bp through RT-PCR amplification. Sequencing comparison showed the homology of strain PTFY and Mass41 of American respiratory velogenstrain is 93.7%, the homology of strain PTFY and CK/CH/LSD/03I of shandong reniform strain is 99%, and the homology of strain PTFY and H52 is 97.8%, which showed the strain PTFY was IBV.Seventh, using DNAstar and DNAman, we comprison the nucleotide sequence and drthology of amplificated products of gene N within strain PTFY to other referrences interiorly, after which we drewed the Phylogenetic tree of nucleotide sequence. We reached a conclusion that the homology of nucleotide sequence and branching of evolutiontree didn't have some to do with the viral tropism and pathogenicity.Our test seperated a strain of IBV virus (strain PTFY)from ovaries of moscovy ducks. After passages of chicken embryos, examination of physical and chemical properties, Hemagglutination test, electron microscope survy and sequencing comprision, the results showed the strain PTFY was IBV, which stated the IBV could infect waterfowls. We get the conclusion that IBV infect not only land fowls such as chicken,peacock and so on,but also waterfowls.