Detection and Molecular Characterization of GI-1 and GI-23 Avian Infectious Bronchitis Virus in Broilers Indicate the Emergence of New Genotypes in Bolivia

Abstract

Infectious Bronchitis Virus (IBV) is a major threat to the poultry industry worldwide, causing significant economic losses. While the virus's genetic structure is well understood, the specific strains circulating in Bolivia have remained uncharacterized until now. This study aimed to identify and characterize new IBV strains in Bolivia. Tissue samples from broilers exhibiting clinical signs of Infectious Bronchitis were screened to detect IBV using real-time RT-PCR (RT-qPCR). Positive samples with low cycle threshold (Ct) values were selected for sequencing the full S1 gene. Of the 12 samples analyzed, 10 were determined to be positive for IBV. However, only four samples yielded sufficient genetic material for sequencing and subsequent phylogenetic analysis. The results revealed the presence of GI-1 and GI-23 lineages, both belonging to genotype I (GI). The GI-1 lineage showed >99% sequence identity to the H120 and Massachusetts vaccine strains, suggesting a close relationship. In contrast, the GI-23 lineage clustered with other IBV strains, showing a distinct subclade that is genetically distant from Brazilian strains. No evidence of recombination was found. Furthermore, amino acid substitution analysis identified specific mutations in the S1 subunit, particularly in the hypervariable regions 1, 2, and 3. These mutations could potentially alter the virus's antigenicity, leading to reduced vaccine efficacy. The findings of this study highlight the importance of continued and broad genomic surveillance of circulating IBV strains and the need to improve vaccination strategies in Bolivia.

Keywords: Bolivia; GI-1 lineage; GI-23 lineage; Infectious Bronchitis Virus; S1; broiler; phylogeny.

Figure 1

Detection of IBV from samples with suspected Infectious Bronchitis. (a) Melting curve analysis demonstrated specific amplification of the N gene in 10 of the 12 samples tested, indicating IBV positivity. Each sample is identified with a different thin line color; a Positive Control and a No Template Control were included. (b) Agarose gel electrophoresis of RT-PCR-amplified products confirmed the full S1 gene in the four positive samples with Ct values below 25. The arrow in the gel image indicates the amplified full S1 gene size (1750 bp). Lane 1: GeneRuler 100 bp Plus DNA Ladder, ready-to-use (Thermo Scientific, Waltham, MA, USA); lane 2: VFAR-187 (PQ140481.1); lane 3: VFAR-188 (PQ140483.1); lane 4: VFAR-189 (PQ140480.1); lane 5: VFAR-190 (PQ140484.1); lane 6: Positive Control; and lane 7: No Template Control.

Figure 2

Phylogenetic tree based on full S1 gene sequences of Infectious Bronchitis Virus. The diagram was constructed using the Neighbor-Joining method with 1000 bootstrap replicates in MEGA version 11.0.13. The tree illustrates the evolutionary relationships among various IBV isolates, highlighting the GI-1 and GI-23 lineages. South American strains are indicated with different colored dots as follows: Bolivian strains (red), Brazilian strain (blue), Mexican strain (purple), Trinidad and Tobago strain (yellow), and Peruvian strain (green). Branch lengths correspond to evolutionary distances calculated using the Tajima–Nei method. The bootstrap support for each cluster is indicated on the respective branches.

Figure 3

Comparative analysis of amino acid changes detected in the S1 gene of Infectious Bronchitis Virus strains belonging to the GI-1 and GI-23 lineages. HVR1 (purple), HVR2 (blue), and HVR3 (green) in the S1 gene amino acid sequence were compared among the Bolivian isolates and a representative strain and the closest relatives of each GI-1 and GI-23 lineage. (a) Comparison of the Bolivian VFAR-187 and VFAR-189 strains (lineage GI-23) with the Israeli strain (reference strain) and other related strains (Poland and Brazil). (b) Comparison of the Bolivian VFAR-188 and VFAR-190 strains (GI-1 lineage) with the M41 vaccine strain from the USA (reference strain) and other related strains from Trinidad and Tobago and Mexico. Amino acid changes are represented by different letters; those conserved are presented with dots, and deletions are presented with dashes. The receptor-binding domain (RBD) is indicated with a red line.