REVERSE GENETICS OF AVIAN METAPNEUMOVIRUS
REVERSE GENETICS OF AVIAN METAPNEUMOVIRUS
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Date
2005-12-06
Authors
Dhanasekaran, Govindarajan
Advisor
Samal, Siba K
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Abstract
Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses to the US turkey industry. With a long-term goal of developing a better vaccine for controlling AMPV in the US, a reverse genetics system to produce infectious AMPV entirely form cloned cDNA was established. To achieve this, the unpublished sequences of the G gene, the L gene, the leader and trailer region were first determined to complete the entire genome sequence of AMPV subgroup C strain Colorado (AMPV/CO). Our results showed that the full-length AMPV/CO genome was 14,150 nucleotides (nt) in length, denoting that AMPV/CO possessed the longest genome among known metapneumoviruses. Subsequently, a cDNA clone encoding the entire 14,150 nt genome was generated by assembling 5 cDNA fragments, representing the entire genome, between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a low-copy number transcription plasmid pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1 and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. The recovered virus was observed to contain the genetic markers that were artificially introduced during cloning. Characterization of the recombinant AMPV/CO showed that its growth characteristics in tissue culture were similar to those of the parental virus. These results demonstrate that infectious AMPV can be generated entirely from cloned DNA using reverse genetics techniques. The potential of AMPV/CO to serve as a viral-vector was examined using green fluorescent protein (GFP) as a reporter. The recovered rAMPV/CO-GFP virus stably expressed GFP for at least five serial passages and showed characteristics similar to that of the parental virus, except that there was a one-log reduction in the virus titer. These results demonstrated that the established reverse genetics system can be utilized effectively for various studies involving AMPV molecular biology, pathogenesis and vaccine development.