Genotyping and Classification of Tunisian Strains of Avian Reovirus using RT-PCR and RFLP Analysis.

Since 1998, avian reovirus (ARV) infection has been detected in broiler and breeding chicken flocks in Tunisia. The genotype of avian reoviruses was established using simple and rapid approaches. Reverse transcription PCR (RT-PCR) on both sigma C (σC) and sigma B (σB)-encoding genes followed by restriction fragment length polymorphism (RFLP) analyses were used to better characterize Tunisian isolated strains. The RT-PCR amplified fragments of 738 and 540 bp for σC- and σB-encoding genes, respectively, of 15 ARV Tunisian strains. DNA fragments amplified from S 1133 vaccine and isolated strains were digested with different restrictions enzymes. RFLP on the σC gene indicated that the field isolates and the S 1133 vaccine strain have identical profiles when separately digested with TaqI, PstI, DdeI, and HincII. Considering the σB gene, RFLP profiles were identical with RsaI, BclI, DpnII, and NciI restriction enzymes for all the strains. However, using MseI and AciI enzymes, it was shown that all tested isolates could be clearly distinguished from the vaccine strain. ARV strains could be classified in groups with strong relatedness. Strain-typing based on cleavage site results are in agreement with ARV clustering based on nucleotide sequences of both the σC and σB genes. RT-PCR-RFLP provides a simple and a rapid approach for genotyping ARV isolates, especially when a large number of isolates are being studied. Additionally, this approach may also determine whether a new variant strain has been introduced into a flock or if a given virus strain is being spread from one flock to another.

Evaluation of protection conferred by a vaccination program based on the H120 and CR88 commercial vaccines against a field variant of avian infectious bronchitis virus.

Due to serotype variations among different avian infectious bronchitis viruses isolated in Tunisia since 2000, protection of chicks, especially broiler flocks, with Mass H120 vaccine often fails. Therefore, association of CR88 (793B type) with H120 vaccines was used for better response. Challenge experiments were then conducted to evaluate tracheal and renal cross-protection in chickens immunized via nasal and eye drops. Conferred protection was measured by clinical signs and macroscopic lesions observed, based on scores attributed according to their severities. The results showed a low protection conferred by H120 alone, as vaccination did not reduce tracheal and kidney lesions (70% scored as 3) after TN20/00 virus challenge, which also led to 10% mortality. Conversely, the challenge results indicated that the combination of the 2 strains (H120/CR88) allow high protection. Based on the results of the challenge experiments, a vaccination protocol coupling CR88 to H120 was applied for industrial broiler flocks. Clinical observations and serological results confirmed that association of heterologous serotypes (H120 and CR88 vaccines) increased the levels of protection against infectious bronchitis viruses compared with the H120 vaccine given alone.

Molecular characterization of avian reovirus isolates in Tunisia.

BACKGROUND: Genotype analyses of avian reoviruses isolated from organ samples collected from chickens with suspicious clinical symptoms, between 1997-2008, was based on sequences for both σC and σB genes and aligned with those published in the Genbank, making it possible to carry out studies of molecular classification and relationships. METHODS: The full length of the known variable protein σC and part of the σB encoding genes, were amplified with RT-PCR, using conserved primers. PCR products were sequenced and the sequences were analyzed and aligned with avian reovirus sequences from the Genbank database. RESULTS: The sequences of σC-encoding genes of all the isolated strains indicated their close relationship with the American, Chinese and Indian strains. Taking the American strain S1133 as a reference, the two Tunisian isolates 97.1 and 97.2 showed some nucleotide substitutions. For isolate 97.1, the substitution was silent whereas for strain 97.2 the mutation was at the first position of the corresponding codon and induced the substitution of the amino acid encoded. For the σB-encoding gene, the sequences of the Tunisian strains showed mutations at positions two or three of the corresponding codons, inducing substitutions of amino acids at these positions. The phylogenic trees based on σC and σB encoding genes indicated closer relationship between Tunisian, American and Taiwanese isolates of genotype I. CONCLUSION: Our study describes the genotype of avian reoviruses that are not yet well characterized genetically. The characterization and classification of these viruses might be significant for understanding the epidemiology of malabsorption syndrome and viral arthritis, and improving our knowledge of the genotype of strains circulating in Tunisian flocks. Furthermore, the study of their variable pathogenicity could be extremely important in the choice of the appropriate vaccine strain to control disease.

S1 gene sequence analysis of new variant isolates of avian infectious bronchitis virus in Tunisia.

PURPOSE: Tissue samples were collected from suspected broiler flocks showing respiratory signs to identify infectious bronchitis virus (IBV), characterize emerging field strains, and study their relationships with the Massachusetts H120 strain, the only IB vaccine used in Tunisia. SAMPLES AND METHODS: Several IBV isolates were identified from field samples collected from flocks located in different regions in the northeast of Tunisia. The IBV isolates were characterized and compared to commonly used vaccine strains (including 793B, D274, and H120 types), other reference IBV strains from Europe, and the recently characterized Tunisian field variants TN20/00, TN200/01, and TN335/01. Reverse transcription-polymerase chain reaction and nucleotide sequencing analyses of the hypervariable regions of the S1 gene were carried out. RESULTS: Four new IBV variants were isolated during the period 2007-10 and were designated TN295/07, TN296/07, TN556/07, and TN557/07. The amino acid sequence data showed 100% similarity between TN295/07 and TN296/07, suggesting that these two isolates are identical and belong to the same genotype. Similar results were demonstrated for TN556/07 and TN557/07. Sequence identity values indicated that TN296/07 and TN556/07 share 55% amino acid homologies between each other, but are very different from the reference IBV serotypes, in particular the H120 strain. It was also shown that they have 50%-77% similarities with the Tunisian virus isolated between 2000 and 2001. Phylogenetic clustering allowed classification of these Tunisian isolates as new genotypes that are closer to TN200/01, TN335/01 Tunisian field variants, and Italy02 variant than MassH120 vaccine strain. CONCLUSION: S1 sequence analyses confirmed the cocirculation of H120 vaccine strain with novel IBV variants isolated from Tunisian field.

Characterization of new variants of avian infectious bronchitis virus in Tunisia.

Three infectious bronchitis virus (IBV) strains, isolated from suspected Tunisian broiler flocks, were characterized as variant viruses using genotyping and serotyping techniques. They were compared with commonly used vaccine strains, including 793/B, D274, and Massachusetts types. Reverse transcription-PCR-restriction fragment length polymorphism, nucleotide sequencing, and GenBank BLAST database analyses of the hypervariable region of the S1 subunit of the virus spike gene showed that the three isolates, designated TN20/00, TN200/01, and TN335/01, share from 64% to 82% homologies between each other but are very different from the H120 strain, the only infectious bronchitis vaccine used in Tunisia. In addition, they showed from 57% to 78% similarities with the European genotypes, including D274 and 793/B. Phylogenetic data allowed classification of the three Tunisian isolates as new genotypes placed inside the same genetic group as the CR88121 and D274 genotypes but very distant from the Massachusetts genotype. Cross-virus neutralization tests confirmed the genotyping results and showed that both TN200/01 and TN335/01 isolates are serologically related, whereas the TN20/00 is closer to TN335/01 than to TN200/01. Moreover, all three Tunisian isolates are closely related to the European variant serotypes, including the CR88121 and the D274 strains, but none is serologically related to the H120 vaccine strain. These data demonstrated, for the first time in Tunisia, the cocirculation of IBV variant serotypes along with the Massachusetts type, causing severe clinical diseases and high economic losses to the poultry industry.