key: cord-0792103-li1vzr0b authors: Sheng, Jie; Ren, Mengting; Han, Zongxi; Sun, Junfeng; Zhao, Yan; Liu, Shengwang title: Genetic and antigenic heterogeneity of GI-1/Massachusetts lineage infectious bronchitis virus variants recently isolated in China date: 2020-09-01 journal: Poult Sci DOI: 10.1016/j.psj.2020.08.037 sha: a5a1251ee5391599bd8b13c5899eb565e7cc89ef doc_id: 792103 cord_uid: li1vzr0b Four GI-1/Massachusetts-type (GI-1/Mass-type) infectious bronchitis virus (IBV) strains were isolated and the complete genomes of these isolates, coupled with the Mass-type live-attenuated vaccine H120 and the Mass-type pathogenic M41 strains, were sequenced in the present study. Our results show that isolates LJL/140820 and I0306/17 may be derived from the Ma5 (another Mass-type live-attenuated vaccine strain) and H120 vaccine strains, respectively. The I1124/16 strain was found to be a M41 variant that likely resulted from accumulated mutations in the genome. Consistently, the results of the virus neutralization (VN) test showed that isolate I1124/16 was antigenically related but slight different from the M41. Our results from the protection experiments pointed out that chickens immunized with H120 failed to eliminate viral shedding after infection with the isolate I1124/16, which was different from that of M41; this result was consistent to the field observation and further implicated that the variant IBV isolate I1124/16 was antigenic different from M41 strain. Furthermore, the I1124/16 was found to have comparable but slightly lower pathogenicity with M41 strain. More studies based on the reverse genetic techniques are needed to elucidate the amino acids in the S1 subunit of spike protein contributing to the altered antigenicity of the isolate I1124/16. In addition, an IBV isolate, LJL/130609, was found to be originated from recombination events between the I1124/16- and Connecticut-like strains. Our results from VN test also showed that isolates LJL/130609 and I1124/16 were antigenic closely related. Hence, there are at least three different genetic evolution patterns for the circulation of the GI-1/Mass-type IBV field strains in China. The differences of vaccines used, the field conditions and genetic pressures between different flocks, likely account for the emergence, evolution patterns and characteristics of the Mass-type IBV strains. days. All the birds from which the viruses were isolated suffered respiratory signs. 126 The Mass-type live-attenuated vaccine H120 strain and the Mass-type pathogenic this study were entered into the BLASTn program 155 (http://www.ncbi.nlm.nih.gov/BLAST/). All sequences with a genetic distance higher 156 than 95% were downloaded (equal or lower than 95% were Connecticut strains and 157 other IBV types). In total, 275 GI-1/Mass-type complete S1 gene sequences were 158 obtained, and were merged with those of our 4 field isolates, one vaccine strain and 159 one pathogenic M41 strain. The S1 sequence of ck/CH/LDL/091022 (LDL/091022) 160 was used as an outgroup. Of the 275 GI-1/Mass-type sequences, 140 Complete genomic sequence analysis 178 The BLASTn program was used to search GenBank for similar IBV sequences 179 using the complete genomic sequences of the H120 and M41 strains in this study. All 180 sequences with a genetic distance higher than 95% were downloaded (equal or lower 181 than 95% were Connecticut and other IBV types). A total of 69 complete genomic 182 sequences were obtained and merged with those of our six IBV strains and that of 183 strain LDL/091022, which was used as a reference strain. Of the 69 sequences, 46 184 were obtained from China, 10 from USA, four from Brazil, three from India, two 185 from Canada, and one from Egypt, Jordan, Pakistan, and UK. The MegAlign program 186 implemented in DNAStar was used to align sequences and calculate the percent 187 identity between the selected sequences. Phylogenetic trees were constructed using 188 the above-mentioned methods. To identify the genetic relationship among the GI-1/Mass-type viruses, the 190 complete genomic sequences of our six IBV strains were compared to those of strains The window width and step size in SimPlot were set to 500 bp and 50 bp, respectively. The complete genomic sequence of our H120 vaccine strain was used as a query. 214 To prepare hyperimmune sera for the VN assay, twenty-four one-month-old SPF 215 layers were randomly divided into six groups consisting of four birds per group, and ORF 5a, ORF 5b, N protein, 3′ UTR, and a poly (A) tail. The phylogenetic trees constructed with complete genomic sequences revealed 303 The results from MAFFT showed that the genomic sequence of isolate 304 LJL/140820 showed high identity to that of Ma5, however, the genomic sequence of 305 isolate I0603/17 showed high identity to that of the H120 vaccine strain (Figure 2 ). and 2447 nucleotide mutations were found when compared to H120, Ma5, H52, and 310 M41 strains, respectively. Taken together, these results show that the LJL/140820 311 isolate was possibly a re-isolated Ma5 strain and I0306/17 may derived from the 312 H120 vaccine strain. 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