Establishment Of An Infectious Full-length CDNA Clone Of Infectious Bronchitis Virus ZZ2004Strain

Infectious bronchitis virus (IBV) belongs to coronavirus family with the characteristics of chickenrespiratory symptoms, kidney damage, egg production drop and egg quality decline. Duck-derived IBVZZ2004strain was isolated from duck with the main symptoms of growth and immune suppression andbroilers infected with this virus had the same symptons. The aim of this study was to construct the IBVZZ2004full-length cDNA infectious clone based on the whole genome sequence and sequence analysis,which lay a new foundation for further investigation of IBV ZZ2004genome function, viral replicationmechanism, molecular pathogenesis and new vaccine development.Based on the complete genome sequence of IBV ZZ2004strain and DNA Star software analysis, asingle restriction enzyme cutting site of the full-length genome nucleotide sequence was selected.4pairs ofprimer were designed and synthesised, with the established long-distance RT-PCR method,4overlappingcDNA fragments of N1~N4were successfully amplified, they were N1(1nt~5954nt)、N2(5860nt~12491nt)、N3(12434nt~20930nt)、 N4(20717nt~27673nt)， which were then cloned into pGEM-T easy vectorrespectively, get4different recombinant plasmid, Using E.coli JM109competent cells chemical conversion,through the double digestion and sequencing identification, IBV ZZ2004strain cDNA library, including4cDNA fragments was established. Using restriction enzymes to cut4different recombinant and thepFastBacHTA vector, respectively, then connected in the22℃water bath pot for night, the connectedproduct were transformed into E.coli JM109competent cells chemical conversion, get2differentrecombinant plasmid, IBV ZZ2004strain cDNA library, including2cDNA fragments was established.Using restriction enzymes to cut2different recombinant, then connected in the22℃water bath pot fornight, constructed a full-length cDNA clone of IBV ZZ2004. According to the complete genome sequenceof IBV ZZ2004strain, In the front﹑middle﹑late of the sequence preceding designed three pairs ofprimers, were used to identify whether the full-length connection is successful. Position G1fragments:1-476; Length:476nt; position G2fragment:12154-12,941; Length:787nt; position G3fragments:27261-27673; Length:412nt. a full-length cDNA clone was constructed in this study.The linear IBV ZZ2004full-length cDNA was used as template to synthesis virus RNA by in-vitro transcription kits and the integrity of virus RNA was detected through PCR.9-day-oldSPF chicken embryos were directly inoculated by the virus RNA and then hatched in37℃for72hours.Collected allantoic fluid and then consecutively passaged3generations. Each generation was positive forIBV by the RT-PCR method and the EID50of the third generation virus was105.5/0.2mL. The IBV ZZ2004full-length cDNA infectious clone was successfully constructed based on abovedata.