Submitted to: International Symposium on Avian Corona and Pneumoviruses and Complicating Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/14/2006
Publication Date: 10/10/2006
Citation: Yu, Q., Estevez, C. 2006. Sequence analysis of the complete genome of avian metapneumovirus Subgroup C Colorado strain: development of a reverse genetics system for this virus. In: Proceedings of the International Symposium on Avian Corona and Pneumoviruses and Complicating Pathogens, May 14-16, 2006, Rauischholzhausen, Germany. p. 6-15. Interpretive Summary: Avian metapneumovirus (aMPV), previously known as turkey rhinotracheitis virus or avian pneumovirus, is the causative agent of an acute respiratory tract disease in turkeys and is also associated with swollen head syndrome in chickens. To study the molecular biology and eventually develop a genetically engineered vaccine to prevent this disease, the complete genomic sequence of aMPV Colorado strain propagated in our laboratory was determined. The results showed that, in addition to 23 nucleotide (nt) differences along the genome, the G gene of our lab virus stock is 1,015 nt shorter than that of the previously published sequences. Based on the genomic sequence of our lab virus stock, we developed a reverse genetics system using a green fluorescence protein (GFP) gene as a reporter to assess the effects of some of the nt differences on viral gene expression. It was found that one of three coding differences in the polymerase L gene was critical for the polymerase functionality. This reverse genetics system will allow us to generate a recombinant virus for vaccine development.
Technical Abstract: The availability of the complete genome information is essential for development of a reverse genetics system to study the molecular biology and rescue infectious avian metapneumovirus from cloned DNA. Therefore, we determined the nucleotide (nt) sequence of the complete genome of aMPV-C Colorado strain (aMPV-C-CO) propagated in Vero cells in our laboratory (here designated as SEPRL variant). The full-length genome is comprised of 13,136 nucleotides (nt) encoding eight genes, a 40 nt leader at its 3’ end and a 45 nt trailer at its 5’ end. It is two nt longer than the aMPV-C-CO strain propagated in the University of Minnesota (UMN variant, Lwamba et al., 2005), and 1,014 nt shorter than the same strain of virus propagated in the University of Maryland (UMD variant, Govindarajan and Samal, 2005). The significant difference in length between these variants was found in the coding region of the G gene, where the SEPRL and UMN variants were lack of 1,015 nt or 333 amino acids (aa) shorter when compared with the UMD variant. In addition, there were 23 nt differences scattered along the genome of the variants. Nine of them resulted in eight aa coding changes in five genes. Based on the genomic sequence of the SEPRL variant, we developed a reverse genetics system using a green fluorescence protein (GFP) gene as a reporter, which allowed us to assess the effects of coding differences in the trans-acting elements on viral gene expression. It was found that one of three coding differences at aa position 1371 (Leu vs Phe) in the RNA-dependent polymerase L gene was critical for the polymerase functionality.