Genotype Variation Of Chinese Infectious Bronchitis Virus And Construction Of Recombinant Vaccine Vector Of H120 Strain

Infectious bronchitis(IB)is an acute contact infectious disease caused by γ-coronavirus,which causes great harm to poultry industry and serious economic losses.Long-term epidemiological monitoring and development of vaccine against epidemic strains are of great significance for the prevention and control of this disease.The purpose of this study was to study Genotype variation of Chinese infectious bronchitis virus,to construct and modification infectious clone of H120 strain,and to quickly prepare vaccine against epidemic strain.The first part: the genotype variation of Chinese infectious bronchitis virusIn this study,203 strains of infectious bronchitis virus(IBV)were isolated and identified from yellow feather broiler and breeding chickens in 15 provinces from 2016 to2019,and S1 genes were sequenced.The obtained sequences together with 1700 genes of Chinese IBV S1 isolates downloaded from Gen Bank were divided into 5 time periods for evolutionary analysis and construct phylogenetic trees.By comparing and analyzing the genotypes of isolated strains at different periods,it was found that the IBV epidemic in mainland China began in 1980,and there were at least 13 evolutionary branches and a few mutant strains in the China IBV epidemic history.Among these evolutionary branches,GI-19,GI-22,GI-7 and GVI-1 branches originated from China.The GI-19 genotype strain emerged no later than 1993,and became the most dominant genotype in China after 2000,accounting for more than 50% of the isolated strains.It was distributed in all provinces,with no obvious tendency of time and geographical distribution.GI-22 branch is an early evolutionary branch in China,and the early strain appeared from 1995 to 1999.In recent years,the number and proportion of strains isolated from this branch showed a decreasing trend.The GI-7 branch originated in Taiwan,China,and the early strain can be traced back to 1964.After 1990,it divided into two subgenotypes,TW I and TW II.Before 2009,only a few TW II strains were isolated in mainland China,and after 2009,a number of TW I strains were isolated and identified,and became the main prevalent genotype.The GVI-1branch strain was isolated for the first time in 2007.The S1 genes of this type strains S1 are quite different from other genotype strains.Although it can be isolated continually,the total number of this genotype strains is small and the geographical distribution is limited.The branches of GI-1 and GI-13 are derived from the widely used Mass vaccine strains and 4/91 vaccine strains introduced from foreign country.These twogenotypes are widely distributed in China and are the main prevalent genotypes in China,including wild strains.Moreover,there are recombination of vaccine strains and local genotypes.Other genotype GI-4,GI-5,GI-6,GI-9,GI-16,GI-18 and GI-23 originated from abroad,and the number of Chinese isolates was small,only isolated in local areas or in a certain period and disappeared rapidly,without causing outbreak and epidemic.This study highlights the need to strengthen molecular epidemiology of IBV and to develop vaccines against prevalent strains.The second part: construction of infectious clone of H120 strainThe p BR322-H120 and its virus rescue system were successfully constructed by RED/ET recombinant technology.Using this system,the virus r H120 was successfully rescued,and the virus passage was stable.The third part: modification and rescue of infectious clone of H120 strainIn this study,RED/ET recombination combined with ccd B reverse screening was used to replace homologous gene S1,S and N genes,insert/replace exogenous EGFP,ILTV g D and H9N2 AIV HA genes in infectious clone p BR322-H120.In terms of homologous gene replacement,three S1 gene,two S gene and two N gene length and position were designed to replace.Replacement of GZ14 S1 and N together,and the chimeric S gene of GL15 strains and 4/91 strains of vaccine replacement were also performed in H120 infectious clones.14 recombinant plasmids were constructed and 9 recombinant viruses were successfully rescued.It was found that the 5’ terminator of S and N gene had a great influence on the rescue of recombinant virus.In terms of exogenous gene replacement,the replication not essential gene 3ab,5ab and IR were used for for exogenous gene replacement/inserts.The enhance green fluorescent protein(EGFP)gene,ILTV g D and truncated H9N2 AIV HA gene(900 bp)as gene donor,to replace/insert H120 infectious clone.We successful build 6 kinds of recombinant plasmid,and rescue 4 recombinant virus:r H120 –△5ab/EGFP,r H120 –△IR/EGFP,r H120 –△IR/Flag-ILTV g D,r H120 –△5ab/Flag-h H9HA(900 bp).Studies on the characteristics of the rescue virus showed that the9 recombinant virus with homologous gene replacement had stable passage and no significant difference in virus titer with the H120 strain.All the 4 recombinant virus that replaced/inserted exogenous genes could express exogenous proteins.Nine recombinant viruses with homologous gene replacement were tested in animals,and screened out two TW type vaccine candidate strains were: r H120-△S1p/GZ14 and r H120-△S1z-△Nz/GZ14,and three QX type vaccine candidate strains: r H120-△S1p/GL15,r H120-△Sp/GL15 and r H120-△Sp/GL15-4/91.