Facilitated leaky scanning and atypical ribosome shunting direct downstream translation initiation on the tricistronic S1 mRNA of avian reovirus.
Racine Trina,Duncan Roy
Nucleic acids research
The S1 mRNA of avian reovirus is functionally tricistronic, encoding three unrelated proteins, p10, p17 and σC, from three sequential, partially overlapping open reading frames (ORFs). The mechanism of translation initiation at the 3'-proximal σC ORF is currently unknown. Transient RNA transfections using Renilla luciferase reporter constructs revealed only a modest reduction in reporter expression upon optimization of either the p10 or p17 start sites. Insertion of multiple upstream AUG (uAUG) codons in a preferred start codon sequence context resulted in a substantial retention of downstream translation initiation on the S1 mRNA, but not on a heterologous mRNA. The S1 mRNA therefore facilitates leaky scanning to promote ribosome access to the σC start codon. Evidence also indicates that σC translation is mediated by a second scanning-independent mechanism capable of bypassing upstream ORFs. This alternate mechanism is cap-dependent and requires a sequence-dependent translation enhancer element that is complementary to 18S rRNA. Downstream translation initiation of the tricistronic S1 mRNA is therefore made possible by two alternate mechanisms, facilitated leaky scanning and an atypical form of ribosome shunting. This dual mechanism of downstream translation initiation ensures sufficient expression of the σC cell attachment protein that is essential for infectious progeny virus production.
Evidence that avian reovirus σNS is an RNA chaperone: implications for genome segment assortment.
Borodavka Alexander,Ault James,Stockley Peter G,Tuma Roman
Nucleic acids research
Reoviruses are important human, animal and plant pathogens having 10-12 segments of double-stranded genomic RNA. The mechanisms controlling the assortment and packaging of genomic segments in these viruses, remain poorly understood. RNA-protein and RNA-RNA interactions between viral genomic segment precursors have been implicated in the process. While non-structural viral RNA-binding proteins, such as avian reovirus σNS, are essential for virus replication, the mechanism by which they assist packaging is unclear. Here we demonstrate that σNS assembles into stable elongated hexamers in vitro, which bind single-stranded nucleic acids with high affinity, but little sequence specificity. Using ensemble and single molecule fluorescence spectroscopy, we show that σNS also binds to a partially double-stranded RNA, resulting in gradual helix unwinding. The hexamer can bind multiple RNA molecules and exhibits strand-annealing activity, thus mediating conversion of metastable, intramolecular stem-loops into more stable heteroduplexes. We demonstrate that the ARV σNS acts as an RNA chaperone facilitating specific RNA-RNA interactions between genomic precursors during segment assortment and packaging.
Development of a recombinant σB protein based dot-ELISA for the diagnosis of avian reovirus (ARV).
Majumder Saurav,Chauhan Tapan K S,Nandi Sukdeb,Goswami Purushottam P,Tiwari Ashok K,Dhama Kuldeep,Mishra Bishnu P,Kumar Deepak
Journal of virological methods
Avian reovirus (ARV) causes significant economic losses to the poultry industry worldwide. The ARV proteins fall into three different classes based on their sizes:λ (large); μ (medium) and σ (small). σB, an outer capsid protein of the ARV contains group specific neutralizing epitopes and induces strong immune response in naturally infected chickens. This study describes the development of a rapid dot-enzyme linked immunosorbent assay (dot-ELISA) using recombinant σB protein antigen of 54 kDa (approx). The assay is rapid (4-5 h) and results can be read by the naked eye. Sixteen ARV positive serum samples (group A) produced strong reaction in the dot-ELISA while twenty of the ARV negative serum samples (group B) collected from SPF chickens showed no reaction. Seventy six randomly collected serum samples were tested with a commercial indirect ELISA kit and the in-house developed dot-ELISA. A total of sixty eight serum samples were found to be positive by indirect ELISA and sixty five serum samples were found to be positive by dot-ELISA. Therefore, using the commercial ELISA as the reference test, the dot-ELISA had a diagnostic sensitivity of 83.8% and specificity of 88.6%. This dot-ELISA can be used as a simple, reliable and inexpensive alternative to commercial ELISA kits for serodiagnosis of ARV where the facilities for standard ELISA are not available.
Genetic and pathogenic characteristics of newly emerging avian reovirus from infected chickens with clinical arthritis in China.
Zhang Xiaohui,Lei Xiangdong,Ma Lifang,Wu Jiaxin,Bao Endong
In recent years, emerging avian reovirus (ARV) strains causing viral arthritis have become a challenge to the worldwide chicken industry, and were responsible for significant economic losses. In this study, we characterized emerging variant ARV strains and examined their genetic relationship and pathogenicity variation with reference strains. A total of 18 emerging variant ARV strains were isolated from tendon and capsular synovial fluid of broiler chickens with clinical cases of arthritis/tenosynovitis at commercial farms in China. Comparative analysis based on σC sequence showed that 4/18 isolates were in the same cluster (Cluster 1) as vaccine strains (S1133), whereas 14 of 18 isolates were in Clusters 2, 3, and 6. The field isolates shared a rather low identity (38.1 to 81.9%) with S1133 in Cluster 1, especially for those from Cluster 6 (38.1 to 67.2%). A higher ARV isolation rate was observed in chicken embryos (47/61) compared to cell culture (37/61) through PCR with a detection primer. A total of 3 isolates were selected to infect specific-pathogen-free (SPF) chickens, showing that the tested isolates, especially that from Cluster 6, displayed greater pathogenicity than S1133 strain, characterized by higher incidence. These findings suggest that the virulence of Chinese ARVs has been increasing rapidly in recent years, and the vaccine need to be updated correspondingly.