Preliminary Study On Molecular Mechanism Of A Chicken Embryo-attenuated QX Type Infectious Bronchitis Virus Vaccine Strain

Infectious bronchitis (IB) is an acute and highly contagious disease caused by infectious bronchitis virus (IBV). Due to the variability of IBV, prevalent strains are various in different eras and regions. Since QX type IBV was first isolated in China in 1996, it had spread quickly and found in many countries. Now it has become the advantage genotype in our country. Because little or no cross-protection occurred between different serotypes, and the Mass-type vaccine being widely used, which was genetically different with the QX type strain, the failure of vaccination happened quite often and brought a huge loss. Therefore, it is important to develop the vaccine which is in accordance with antigenicity of prevalent strains. Our laboratory had attenuated QX type strain CK/CH/JS/2010/12 through passing continuously in embryonated eggs. And got the attenuated virus, named QXL strain, showing good immunogenicity and stable genetic properties. QXL strain was safe and could provide a satisfactory immune protection. In order to identify the molecular orbitals of attenuation of QX type IBV, full-length genome sequencing of IBV strain QXL-5、QXL-40、QXL-60、QXL-80、QXL-100 and construction of strain QXL-5 infectious clone were described.1. Sequence analysis of the genomes of pathogenic and attenuated infectious bronchitis virus CK/CH/JS/2010/12 strainsThe complete genome sequences of QXL strains in our study were obtained through fragment cloning, sequencing and segments aligning, joining with biology software. Results showed that the genome organization as follows: 5’ UTR-1a-1b-S-3a-3b-M-ORFX-5a-5b-N-UTR3’. The full-length of QXL-5 and QXL-40 was 27681bp and the others 27682bp, and QXL-60、QXL-80. QXL-100 each had one nucleotide insertion in the intergenic untranslated region between M gene and gene 5,named ORFX. Comparing the genome sequences of these strains revealed that, QXL-40 and QXL-60 sequences had some variation compared with QXL-5, and the rate was 28.21%,66.67%, respectively; But QXL-80、QXL-100 sequences tend to be stable compared with QXL-40. Which was consistent with animal experiment results. Forty-four nucleotide mutations between QXL-40 and QXL-5 were observed, resulting in 23 amino acids changes. QXL-60 had 108 nucleotide mutations compared with QXL-40, resulting in 31 amino acids changes. There were two and six nucleotide mutations between QXL-80 and QXL-60, QXL-100 and QXL-80, respectively, resulting in two and three amino acids changes, respectively. Mutations existed dispersedly in different parts of the genome, and nsp2 had the highest number of amino acid differences, followed by nsp3 and S glycoprotein. Our study showed that the highly pathogenic IBV virulence factors might not be determined by a single amino acid mutation, and might be determined by multiple genes. Moreover, not only the S protein, but the non-structural proteins might play an important role in the pathogenicity of IBV.2. Construction of a cDNA clone of infectious bronchitis virus strain QXL-5In order to further study IBV variation mechanism, the genome of strain QXL-5 was divided into 13 fragments to amplify and construct the infectious clone. For getting infectious transcript, we added T7 RNA polymerase promoter at 51 terminal, and polyA tails at 3’ terminal. Meanwhile, introducing a silent mutation in ORF5 served as a genetic marker. The fragments were firstly cloned into pEASY-Blunt vector to sequence the whole genome. Then the fragments were digested by BsmB I or Bsa I,and purified, ligated orderly with T4 DNA ligase to get the full-length cDNA. Simultaneously obtain the cDNA of N gene to improve the rescue efficiency of the virus. The recombined virus was obtained after transcription in vitro and transfection of viral mRNAs based on the full-length infectious cDNA clone. The transfected cells were collected after 48 h post-transfection and freeze thawing for 3 times. The mixtures of the collected liquids were incubated into SPF embryos and passed several generations. And the recused virus, named rQXL-5, was confirmed by RT-PCR and gene sequencing analysis. Test EID50 of the parent strain QXL and rQXL-5. The virus titers were 106-3EID50/0.1mL and 106.0EID50/0.1mL, respectively. The result indicated that QXL reverse genetic system was successfully constructed, which provided an advanced platform for future research of IBV and the associated IBV-based vector vaccines development.