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Submitted to: Symposium on Avian Pneumovirus
Publication Type: Proceedings Publication Acceptance Date: 6/5/2005 Publication Date: 11/5/2005 Citation: Yu, Q. 2005. 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 2005 Turkey Research Review and Avian Pneumovirus Symposium, October 5, 2005, Willmar, MN. 2005 CDROM. Interpretive Summary: Not required. Technical Abstract: The availability of the complete genome information is essential for recovery of avian metapneumovirus subtype C (AMPV-C) from cloned cDNA through reverse genetics approach. To this end, we determined the nucleotide sequence of the complete genome of AMPV-C Colorado strain (AMPV-C-CO) propagated in Vero cells in our lab (here named as SEPRL variant). The full-length genome is comprised of 13,136 nucleotides (nt), with a 40 nt leader at its 3’ end and a 45 nt trailer at its 5’ end. It is two nt longer (a G residue insertion before the translation start codon AUG of the M2 gene and an extra U residue at the M2 gene polyA signal region) than the AMPV-C-CO strain propagated in the University of Minnesota (UMN variant, Lwamba et al., 2005), and 1,014 nt shorter than that of the same strain of virus propagated in the University of Maryland (UMD variant, Govindarajan and Samal, 2005). The UMD variant does not have the G insertion before the translation start codon of the M2 gene but it has extra 1,015 nt located to the end of the G gene ORF. This extra 1,015 nt in the UMD variant G gene resulted in 333 amino acids (aa) longer coding capacity (585 aa) than that of the SEPRL and UMN variants (252 aa). In addition, there were 26 nt differences among the variants. Nine of them resulted in eight aa coding changes in five genes. To develop a reverse genetics system for this virus, we used our sequence data and the consensus sequences to construct a T7-plasmid based minireplicon and plasmids expressing AMPV N, P, M2-1, and L proteins under the control of T7 promoter. This AMPV minireplicon contained a negative sense copy of the green fluorescence protein (GFP) reporter gene, flanked at its 3’ end with the leader together with the N gene start signal and at the 5’ end with the L gene end signal and the trailer region, followed by a hepatitis delta virus ribozyme (HDV) sequence and T7 terminator. We were able to detect the expression of GFP from the MVA-T7 virus-infected cells that were cotransfected with the AMPV-GFP minireplicon and the plasmids expressing the N, P, M2-1, and L proteins. Currently, we are optimizing the plasmid ratio and transfection conditions to improve the expression efficiency for rescue of infectious virus from cloned cDNAs. |
