Isolation And Identification Of A Field Strain Of Infectious Bronchitis Virus, And S1 Glycoprotein Gene Sequence Analysis

Infectious Bronchitis(IB) is a severe acute respiratory disease of pouLtry, caused by Infectious bronchitis virus (IBV)。 Since broke out in USA in the 1930s, IB have been an important disease of poultry around the world. IBV is a sRNA virus, the gene of the virus always mutated owing to it's point-mutation and gene recombination, so the virus have many serotypes. The known serotypes can be classified into respiratory symptomatic serotypes such as Conn, Iowa97, JMK, Florida, Arkansas99 and nephritic symptomatic serotypes such as M41, Holte, Gray, Australia 'T'.Gough reported a new serotype of IBV named 793/B (also known as 4/91 and CR88) in UK in 1992, 793/B isolates have not any or a little serological relation with other serotypes of IBV, the whole S1 gene of 793/B isolates differed by 21% to 25% from those of other 17 European isolates. In recent years, the 793/B serotype of IBV, harmfuL to the healthy development of pouLtry breeding, prevailed and spread in Spain, Germany, Holland, Italy and Thailand, too.At the beginning of 2002, egg drop disease appeared in many layer flocks in Shandong province of China. We isolated and identified a IBV strain from the suffering pouLtry through Electron Microscopic Observation, Hamagglutination (HA) test and animal Regressive Research. The resuLts show that the isolate is a strain of IBV.Trachea, kidney and caecal tonsils samples collected from suspect IB outbreaks were submitted for virological investigation. Clarified suspensions (10% w/v) of tissue homogenates were inocuLated into the allantoic cavity of eggs of 9- to 11 -day-old embryonated specific pathogen free (SPF) chicken eggs., the virus was passaged three times with this method for the study. To know whether the allantoic fluid have NDV or AIV, HA test was detected and the resuLt showed that the allantoic fluid have not hemagglutination tochiken's 1% red blood cells.The allantoic fluid was centrifuged by hypervelocity centrifuge, Sediment was collected and examined by negative contrast electron microscopy for the presence of Coronavirus particles, the sediment can be observed typical Coronavirus particles which viral spikes surround. Then, we had dealed the allantoic fluid by I type of phosphoesterase C for HA test, the resuLt showed that the allantoic fluid have hemagglutination. So we can drew a primary conclusion that the virus we isolated is a strain of IBV.The following research including chiken embryo dwarfed test, NDV-B1 interference test and animal regressive research confirmed the presence of IBV in the allantoic fluid.The biologically character was determined in after research. Serum VN tests showed that that isolated strain is a mutation strain of IBV. gived fewer or no cross-reactions to the familiar serotype of IBV. For further research, the whole sequence of S1 gene was cloned and analyzed by RT-PCR using specific primer which can differentiate between 793/B IBV and other serotypes of IBV. the strain's serotype can be deduced as 793/B. the presence of 793/B was confirmed in China.Identity of the S1 gene sequence between the isolated strain and other serotypes of published IBV sequences is <79%. The S1 amino acid sequenced differed by 21 to 25% or more. Our analysis has shown that type 793/B which may came from foreign country has become a component of the IBV population in regions of China.