Genome Sequence Variations of Infectious Bronchitis Virus Serotypes From Commercial Chickens in Mexico

Abstract

New variants of infectious bronchitis viruses (IBVs; Coronaviridae) continuously emerge despite routine vaccinations. Here, we report genome sequence variations of IBVs identified by random non-targeted next generation sequencing (NGS) of vaccine and field samples collected on FTA cards from commercial flocks in Mexico in 2019-2021. Paired-ended sequencing libraries prepared from rRNA-depleted RNAs were sequenced using Illumina MiSeq. IBV RNA was detected in 60.07% (n = 167) of the analyzed samples, from which 33 complete genome sequences were de novo assembled. The genomes are organized as 5'UTR-[Rep1a-Rep1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b]-3'UTR, except in eight sequences lacking non-structural protein genes (accessory genes) 4b, 4c, and 6b. Seventeen sequences have auxiliary S2' cleavage site located 153 residues downstream the canonically conserved primary furin-specific S1/S2 cleavage site. The sequences distinctly cluster into lineages GI-1 (Mass-type; n = 8), GI-3 (Holte/Iowa-97; n = 2), GI-9 (Arkansas-like; n = 8), GI-13 (793B; n = 14), and GI-17 (California variant; CAV; n = 1), with regional distribution in Mexico; this is the first report of the presence of 793B- and CAV-like strains in the country. Various point mutations, substitutions, insertions and deletions are present in the S1 hypervariable regions (HVRs I-III) across all 5 lineages, including in residues 38, 43, 56, 63, 66, and 69 that are critical in viral attachment to respiratory tract tissues. Nine intra-/inter-lineage recombination events are present in the S proteins of three Mass-type sequences, two each of Holte/Iowa-97 and Ark-like sequence, and one each of 793B-like and CAV-like sequences. This study demonstrates the feasibility of FTA cards as an attractive, adoptable low-cost sampling option for untargeted discovery of avian viral agents in field-collected clinical samples. Collectively, our data points to co-circulation of multiple distinct IBVs in Mexican commercial flocks, underscoring the need for active surveillance and a review of IBV vaccines currently used in Mexico and the larger Latin America region.

Keywords: NGS; hypervariable region; lineage; mutation; recombination; vaccine.

Figure 1

Schematic and amino acid alignment of the overall features of the S glycoprotein. The scheme was constructed in Geneious Prime based on the S protein sequence of the field sequence 2602/21; all 33 S protein sequences in this study have similar general features (see Supplementary Table 4). Sequence IDs are shown on the alignments; the positions indicated at the top of the sequence alignments are in reference to ungapped amino acid residues; dots and dashes indicate identical and deleted amino acid residues, respectively. Features of subunit S1 include N-terminal signal peptide (SP), N-and C-terminal domains (S1-NTD/CTD), which harbor the receptor-binding domains (RBD), and hypervariable regions (HVR I-III). Shown is a 20- residue (running from positions P14–P6') motif furin S1/S2 cleavage site, consisting of a core region (8-residues; position P6–P2'), which harbors the canonical 4-residue motif (Rx[K/R]-R↓S); the core region is flanked by two solvent accessible regions (8-residue; P7 to P14, and a 4-residue; P6 to P2'). Note the backward and forward numbering of P1-P14 and P1'-P6', respectively, starting at the conserved R immediately upstream of the cleavage S1/S2 site. The auxiliary S2′ (PS(G)SPRxR↓S) cleavage site positioned 153-residues downstream the primary S1/S2 cleavage site is also shown. Subunit S2 domains include fusion peptide (FP), heptad repeat regions (HR1 and HR2), transmembrane domain (TM), and intracellular (IC) tail. Purple vertical bars represent predicted N-linked glycosylation sites.

Figure 2

Maximum likelihood phylogenetic tree of nt sequences of subunit S1-gene, hypervariable regions I-III (HVRs I-III) of the S1-gene, and complete S-gene using GTR model in MEGA 6. The 33 sequences in this study (3 vaccine sequences highlighted in blue; 30 field sequences; color-coded based on sampling regions in Mexico) clustered with serotypes in 5 lineages of IBVs, i.e., lineages GI-1 (Mass-type serotypes; n = 8 sequences), GI-3 (Holte/Iowa/97 serotypes; n = 2 sequences), GI-9 (Ark-like serotypes; n = 8 sequences), GI-13 (793B also known as 4/91 serotypes; n = 14 sequences), and GI-17 (CAVs; n = 1 sequence). The analysis involved 94 sequences. All positions with <95% site coverage were eliminated. The final datasets had 1,576, 757, and 3,431 positions for the complete S1-gene, HVRs I-III, and complete S-gene, respectively.

Figure 3

Maximum likelihood phylogenetic tree nt sequences of complete genome, Rep1a, and Rep1b using GTR model in MEGA 6. The 3 vaccine sequences are highlighted in blue color; the 30 field sequences are color-coded based on sampling regions in Mexico. The analysis involved 83 sequences. All positions with <95% site coverage were eliminated. The final dataset had 26,895 (complete genome), 11,782 (Rep1a), and 8,037 (Rep1b) positions.

Figure 4

Analyses of the S1 glycoprotein HVRs I- III (amino acid residues 37-301). The phylogenetic tree was constructed using the JTT matrix-based model in MEGA and involved 67 sequences and a final dataset consisting of 253 positions. The 33 sequences in this study (3 vaccine sequences highlighted in blue; 30 field sequences; color-coded based on sampling regions in Mexico) assembled in this study are aligned with representatives of serotypes belonging to 5 IBV lineages. Dots and dashes in the alignments indicate identical and deleted amino acid residues, respectively. Amino acid residues critical for attachment of the Mass-type prototype (M41; GenBank accession No. AY851295) to chicken respiratory tract tissues are boxed in black [i.e., N38, H43, S56, P63, I66, and T69 (14)]; red and blue boxes indicate deleted and inserted amino acid residues, respectively.

Figure 5

Reticulate network using complete S-gene sequences constructed using SplitsTree5 v 5.0.0 (53). The network predicts putative evolutionary histories of the IBVs, where the internal nodes and the edges correspond to ancestral taxa and patterns of descents, respectively. Nodes with more than two parents represent reticulate events (e.g., recombination, horizontal gene transfer, or hybridization). “Split” is a partition of the taxa into two subsets with the edges separating the taxa subsets of the split from those on the other side of the split (the length of the edge in the network is proportional to the weight of the split it is associated with, which is analogous to branches in conventional phylogenetic trees). (A) The input contained 70 taxa and 101 trees constructed using Splits Network Algorithm with default options to obtain a splits network with 153 nodes and 165 edges. In the figure inset (B), the input consisted of a subset of 28 taxa and 101 trees (from a splits network with 66 nodes and 72 edges). The recombination events of the sequences in panel B of this figure as determined using RDP4 are shown in Table 4.