Respiratory disease complex due to mixed viral infections in chicken in Jordan

Abstract

The global distribution of avian respiratory viruses highlights the need for effective surveillance programs and international collaboration to monitor viral circulation and implement timely control measures. In the current study, we aim to provide a comprehensive overview of avian respiratory viral infections in the poultry flocks in Jordan, focusing on the major viruses involved, their epidemiology, clinical manifestations, and evolution based on viroinformatics that will be helpful to improve the diagnostic methods, and control strategies including vaccines in the region. In this research, various poultry broiler groups in Jordan experiencing respiratory symptoms were tested for respiratory viral pathogens from January 2021 to February 2022. The mortality rates observed in the examined groups varied between 6% and 40%. The identified strains were authenticated using the RT-qPCR assay. Furthermore, they underwent in-depth characterisation through the sequencing of the complete spike (S1) gene for infectious bronchitis virus (IBV) and the haemagglutinin (HA) gene for avian influenza virus (AIV) subtype H9N2. Co-infection of IBV and AIV H9N2 viruses was detected through molecular analysis. The IBV strains showed affiliation with the variant groups GI-16 (3 strains) and GI-23 (9 strains) and exhibited numerous mutations. Meanwhile, H9N2 avian influenza viruses displayed various changes in amino acids within the HA gene, suggesting the influence of antibody-driven selection pressure. The phylogenetic analysis revealed that the H9N2 viruses identified in this investigation shared close genetic ties with EG3 (3 strains) and the Middle East group (ME1; 8 strains). These strains have been recently found in Jordan and nearby countries in the Middle East. Moreover, their HA genes exhibited similarities to viruses belonging to the G1-like lineage. In conclusion, avian respiratory viral infections remain a significant concern for the poultry industry, requiring constant vigilance and proactive measures to minimise their impact. Continued surveillance, robust diagnostic methods, effective vaccines, and international cooperation are essential components of a comprehensive approach to combat avian respiratory viral infections (AI, IBV, ND and ILT 'viruses) and safeguard avian health and global poultry production.

Keywords: Jordan; avian influenza viruses; infectious bronchitis virus; poultry health; respiratory infection.

Figure 1

The observed postmortem findings in the chickens were as follows: (A) The kidneys displayed signs of congestion, swelling, and distinctive lobes during necropsy examination, and (B) The trachea exhibited congestion, hemorrhaging of the tracheal mucosa, and the presence of a caseous plug within the tracheal lumen.

Figure 2

The pairwise identity plot of the spike (S1) (A) and haemagglutinin (HA) (B) glycoprotein sequences, concerning IBV and H9N2 AIV, respectively, were aligned using ClustalW and visualised through the Sequence Demarcation Tool (SDT) software.

Figure 3

WebLogo graphics were employed to visually depict the divergence in amino acid sequences between the IBV strains identified in this investigation, highlighting their differences in comparison to commercially utilised vaccines (H120/Ma5 and D274) as well as previously documented isolates in Jordan.

Figure 4

The collective dN/dS ratio was analysed, scrutinizing the average synonymous and non-synonymous substitutions, systematically examining codon by codon across the S1 (A) and HA (B) glycoproteins of IBV and H9N2 AIV strains in Jordan. This assessment incorporated the strains reported in this study, pinpointing the domains within the S1 and HA proteins most impacted by heightened selective pressure.

Figure 5

Phylogenetic investigation was conducted on the identified IBV strains to observe their clustering tendencies in relation to representative IBV lineages, genotypes, or serotypes, utilizing the complete S1 gene. The observed strains were classified into 2 distinct sublineages: nine strains were affiliated with variant 1 of the GI-23 lineage, while three strains fell within the GI-16 lineage. Unrooted phylogenetic trees were generated utilizing the distance-based maximum likelihood method and MEGA 7 software. To validate the branches within the tree, statistical analysis was performed through bootstrap analysis employing 1000 replications of bootstrap re-sampling, where numbers above branches represent neighbour-joining bootstrap values equal to or greater than 80%. The proportionate tree was depicted with branch lengths measured in substitutions per site. The IBV strains associated with variant 1 of the GI-23 lineage were highlighted within a light pink box, while those connected to the GI-16 lineage were enclosed in a light yellow box.

Figure 6

Phylogenetic examination was conducted on the studied H9N2 AIV strains, evaluating their clustering tendencies in relation to distinct subgroups or subclustering within the comprehensive HA gene. The identified strains were categorised into 2 separate subgroups: 3 strains were grouped within the sublineage EG3, while eight strains were clustered within the Middle East 1 (ME1) sublineage. The rooted phylogenetic trees were created using the distance-based maximum likelihood method and MEGA 6 software. Statistical validation for the tree branches was appraised through bootstrap analysis using 1000 replications of bootstrap resampling. The numbers displayed above branches signify neighbour-joining bootstrap values of ≥80%. The tree was drawn proportionally, depicting branch lengths measured in substitutions per site. The H9N2 AIV strains affiliated with the EG3 sublineage were highlighted within a light green box, whereas those linked to the ME1 sublineage were enclosed in a light-yellow box.