Molecular and pathological investigation of avian reovirus (ARV) in Egypt with the assessment of the genetic variability of field strains compared to vaccine strains

Abstract

Avian orthoreovirus (ARV) is among the important viruses that cause drastic economic losses in the Egyptian poultry industry. Despite regular vaccination of breeder birds, a high prevalence of ARV infection in broilers has been noted in recent years. However, no reports have revealed the genetic and antigenic characteristics of Egyptian field ARV and vaccines used against it. Thus, this study was conducted to detect the molecular nature of emerging ARV strains in broiler chickens suffering from arthritis and tenosynovitis in comparison to vaccine strains. Synovial fluid samples (n = 400) were collected from 40 commercial broiler flocks in the Gharbia governorate, Egypt, and then pooled to obtain 40 samples, which were then used to screen ARV using reverse transcriptase polymerase chain reaction (RT-PCR) with the partial amplification of ARV sigma C gene. The obtained RT-PCR products were then sequenced, and their nucleotide and deduced amino acid sequences were analyzed together with other ARV field and vaccine strains from GenBank. RT-PCR successfully amplified the predicted 940 bp PCR products from all tested samples. The phylogenetic tree revealed that the analyzed ARV strains were clustered into six genotypic clusters and six protein clusters, with high antigenic diversity between the genotypic clusters. Surprisingly, our isolates were genetically different from vaccine strains, which aligned in genotypic cluster I/protein cluster I, while our strains were aligned in genotypic cluster V/protein cluster V. More importantly, our strains were highly divergent from vaccine strains used in Egypt, with 55.09-56.23% diversity. Sequence analysis using BioEdit software revealed high genetic and protein diversity between our isolates and vaccine strains (397/797 nucleotide substitutions and 148-149/265 amino acid substitutions). This high genetic diversity explains the vaccination failure and recurrent circulation of ARV in Egypt. The present data highlight the need to formulate a new effective vaccine from locally isolated ARV strains after a thorough screening of the molecular nature of circulating ARV in Egypt.

Keywords: ARV; avian orthoreovirus; histopathology; phylogenetic analysis; sigma C; vaccine.

Figure 1

Clinical signs of ARV infections in broiler chickens. Broiler chicken with unilateral arthritis, hock joint swelling, and ruffled wing's feather.

Figure 2

Avian orthoreovirus postmortem lesions in broiler chickens. (A) Broiler chicks show enlarged orange liver with numerous necrotic pale hepatic foci and hemorrhagic pericarditis (arrow). (B) Broiler chickens show hepatic necrosis. (C) Broiler chick with enlarged lemon-shaped proventriculus. (D) Broiler chicken with swelling, edema in the tendon, and full thickness tendon rupture (arrow).

Figure 3

Cardiac, hepatic, and splenic histopathological lesions in the ARV-infected broiler chickens. (A, B) The pericardium of broiler chicks shows pericarditis associated with intense inflammatory cells infiltration (arrowheads). (C) Myocardium of broiler chicks shows marked atrophy of the myocardial cells (white arrowheads) and interstitial fibrosis (black arrowhead). (D) Myocardium of broiler chicks show myxomatous changes (white arrowhead) and severe eosinophilic sarcoplasmic degeneration (black arrowhead). (F) Liver of broiler chicks show periportal heterophilic infiltration (white arrowheads). (E) Liver of broiler chicks show the congestion of red pulp (black arrowhead) and moderate degree of lymphoid depletion (white arrowhead), H&E, bar = 50 μm.

Figure 4

Synovial, tendon, and articular cartilaginous histopathological lesions in the ARV-infected broiler chickens. (A–C) Fibrin foci (black arrowhead) and marked heterophilic cell infiltration (white arrowheads) within the synovial membrane. (D) Necrosis o the synovial membrane (black arrowhead) and hypertrophy of synovial cells (white arrowhead). (E, F) Articular tissues showing multifocal necrosis on the surface or within the cartilage (white arrowhead), H&E, bar= 50 μm.

Figure 5

Phylogenetic tree of ARV sigma C gene nucleotide sequences constructed with MEGA X software in which the analyzed sequences are clustered into six genotyping clusters (genotypic cluster I–VI) according to Ayalew et al. (2017) ARV classification. Our isolates (red triangles) are clustered in genotypic cluster V away from all vaccine strains which are clustered in genotypic cluster I.

Figure 6

Phylogenetic tree of ARV sigma C gene deduced amino acid sequences constructed with MEGA X software in which the analyzed sequences are clustered into six antigenic clusters (antigenic cluster I–VI) according to Ayalew et al. (2017) ARV classification. Our isolates (red triangles) are clustered in the antigenic cluster V away from all vaccine strains which are clustered in the antigenic cluster I.

Figure 7

Avian orthoreovirus sigma C gene nucleotide sequences analysis using BioEdit software showing 397 nucleotide substitutions (out of 797 aligned nucleotides) in our isolates in comparison to vaccine strains used in Egypt.

Figure 8

Avian orthoreovirus sigma C gene deduced amino acid sequences analysis using BioEdit software showing 149 (Gharbia/1-20) and 150 (Gharbia/2-20) amino acid substitutions (out of 265 aligned amino acids) in our isolates in comparison to vaccine strains used in Egypt.