Identification Of Biological Characteristics And Establishment Of RT-PCR Protocol BD Strain Of Infectious Bronchitis Virus

Infectious bronchitis (IB) is characterized by coughing,tracheal rales,sneezing,and lesions in kidney and reproductive tract.This disease can cause mortality in young chickens and result in economic losses due to poor weight and reduction in egg quality and quantity. Infectious bronchitis virus (IBV) ,the causative agent of IB,is the prototype of the Coronaviridae family.The virus has a worldwide distribution and many different variant have been isolated.IBV is an important pathogen of poultry industry . Despited of routine use of vaccines,variant stains continue to outbreak in field situations. Now , IB had spread all over most provinces,city and municipality in china, it result in enormous losses. One avian infectious bronchitis (IB) viruses (IBV) was isolated named IBV BD from outbreaks in chickens in baoding of Hebei province . The biological characteristics of the BD isolate strain was analyzed . Polymerize chain reaction (PCR) assay was developed for detection of IBV base on specific test, sensitive test and clinic diagnosis.One field strain of infectious bronchitis virus (IBV) was isolated from chicken kidney, trachea and lung of the young layer flocks .Biological characteristic chicken embryo changes such as stunting , dawrfing or curling of embryos were observed between 2 and 9 days post-inoculation , and typical round coronavirus morphology with sparse spikes was observed by electron microscopy. Haemagglutination assays were performed on all the allantoic fluids without trypsinase treatment, all the results were negative. Chickens challenged with BD showed mild clinical signs, overt diseases including respiratory distress, shuffledness and depression .Death occurred at 4 days after challenge with duration from 5 to 11 days. Bronchial hemorrhage and oedema were observed almost in all dead chickens. The kidneys from the dead chickens showed the predominant gross lesions which were pale,mottled, and swollen. The renal tubules and ureters were distended with excess urates. With the disease development, the kidneys became further swollen. Biological characteristics of the isolate were studied by interference with NDV, dwarfing embryonated chicken eggs, these data demonstrated that the virus has the biological characteristics of IBV. We get the specific bands of 405bp through RT-PCR with cDNA of isolated virus, which testified that it was IBV.These data indicate that the isolated virus could be IBV. In this study, Physicochemical affection of IBV-BD strain was mensurated. The results shows,BD strain EID50 has remarkable changed after deal with chloroform and ether,it showed that BD strain was sensitive to chloroform and ether; Thermal stability tests showed that BD strain is the heat-sensitive strains, however, the Mg²⁺ can improve this virus on the thermal tolerance;BD strain has strong resist in PH3.0 and PH12.0.AGP double diffusion test showed that the titre of antiserum is 1:64;Serotype of BD strain was identificated by virus neutralization test in chicken embryo,the results showed that BD strain has a certain cross-reaction with M41 standard strain,R-value is 67%,it showed that BD strain and M41 standard strain were the different subtypes of the same serotype.According to the sequences of thirty strains of IBV M gene published in GenBank, a pair of primers was designed and synthesized to specifically amplify a 405bp nucleotide fragment,the RT-PCR technique to detect IBV was established.The 405bp special strip was found only in the PCR amplification of the M41 and BD serotypes of IBV in the specificity assay,there were no the same strip appeared in the other viruses and strains of IBV. The negative results were achieved from avian relativity disease virus ,IBDV ,NDV and JEV .The sensitivity result indicated that as little as 5×10^-3EID₅₀ of sample were detected in PCR.Twelve uncertain isolates detected by the established RT-PCR technique were 83.3% positive.The above results showed that the technique provided a more sensitive,specific and rapid method for diagnosis and epidemiological survey of IBV.