Genetic and antigenic heterogeneity of GI-1/Massachusetts lineage infectious bronchitis virus variants recently isolated in China

Abstract

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 nucleotide accumulated mutations in the genome. Consistently, the results of the virus neutralization 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 the M41 strain. Furthermore, the I1124/16 was found to have comparable but slightly lower pathogenicity with the 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 the virus neutralization test also showed that isolates LJL/130609 and I1124/16 were antigenic closely related. Hence, there are at least 3 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.

Keywords: GI-1/Massachusetts type; antigenicity; evolution patterns; infectious bronchitis virus; pathogenicity.

Figure 1

Phylogenetic trees based on the complete S1 amino acid sequences of 275 GI-1/Mass-type strains, including our 6 strains and 269 reference strains (A) and the complete genomic sequences of 69 IBV strains, including our 6 strains and 63 reference strains (B). The sequence of the IBV strain LDL/091022 was used as an outgroup. The phylogenetic trees were constructed using the maximum-likelihood with 1000 bootstrap replicates using MEGA 6.0 (http://www.megasoftware.net/). The stars indicate the vaccine strains. Our strains used in this study are shown in bold. The black squares indicate IBV strains with available complete genomes. Abbreviations: IBV, infectious bronchitis virus.

Figure 1

Phylogenetic trees based on the complete S1 amino acid sequences of 275 GI-1/Mass-type strains, including our 6 strains and 269 reference strains (A) and the complete genomic sequences of 69 IBV strains, including our 6 strains and 63 reference strains (B). The sequence of the IBV strain LDL/091022 was used as an outgroup. The phylogenetic trees were constructed using the maximum-likelihood with 1000 bootstrap replicates using MEGA 6.0 (http://www.megasoftware.net/). The stars indicate the vaccine strains. Our strains used in this study are shown in bold. The black squares indicate IBV strains with available complete genomes. Abbreviations: IBV, infectious bronchitis virus.

Figure 2

Analysis of the complete genomic sequences of our 4 IBV strains with those of the H120, Ma5, and M41 strains by using MAFFT. Nucleotide sequence disagreement at the indicated positions is shown in black, whereas the nucleotide sequence agreement is shown in gray. The GenBank accession numbers are shown at the end of the name of the viruses. The numbers indicate the genomic position at the top of the figure. Abbreviations: IBV, infectious bronchitis virus; MAFFT, Multiple Alignment with Fast Fourier Transformation.

Figure 3

MAFFT, SimPlot, and bootscan analyses were used to detect the genetic relationship of the complete genomes of isolates I1124/16 (A) and LJL/130906 (B). Nucleotide sequence disagreement at the indicated positions is shown in black, whereas the nucleotide sequence agreement is shown in gray in the MAFFT. The complete genomic sequence of the H120 strain (GenBank no. MK937831) was used as the query in the SimPlot and bootscan analysis. The window width and step size in SimPlot were set to 500 bp and 50 bp, respectively. Different regions in the genome of the isolates I1124/16 (I-IV) and LJL/130906 (I-III) showed different degrees of diversity. The hollow boxes indicate the different fragments in the bootscan analysis. Abbreviations: MAFFT, Multiple Alignment with Fast Fourier Transformation; SimPlot, Similarity plot.

Figure 3

MAFFT, SimPlot, and bootscan analyses were used to detect the genetic relationship of the complete genomes of isolates I1124/16 (A) and LJL/130906 (B). Nucleotide sequence disagreement at the indicated positions is shown in black, whereas the nucleotide sequence agreement is shown in gray in the MAFFT. The complete genomic sequence of the H120 strain (GenBank no. MK937831) was used as the query in the SimPlot and bootscan analysis. The window width and step size in SimPlot were set to 500 bp and 50 bp, respectively. Different regions in the genome of the isolates I1124/16 (I-IV) and LJL/130906 (I-III) showed different degrees of diversity. The hollow boxes indicate the different fragments in the bootscan analysis. Abbreviations: MAFFT, Multiple Alignment with Fast Fourier Transformation; SimPlot, Similarity plot.

Figure 4

The vaccination-challenge test. The mean score was calculated from individual scores that were based on severity of respiratory signs detected in individual chickens (A). The virus recovery from the tracheal swabs collected from H120-vaccinated chickens at different time points after challenge with isolate I1124/16 and strain M41, comparing with those of the nonvaccinated and challenge chickens (B).