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Construction of recombinant myxoma viruses expressing foreign genes from different intergenic sites without associated attenuation

Cooperative Research Centre for Biological Control of Vertebrate Pest Populations CSIRO Division of Wildlife and Ecology, PO Box 84, Lyneham, Canberra, ACT 2602, Australia

Two myxoma virus transient dominant selection vectors were constructed and used to generate recombinant viruses expressing single and double foreign gene insertions from intergenic sites. The intergenic insertion sites were located between the myxoma virus genes MJ2 (thymidine kinase) and MJ2a, and MA24 (-subunit RNA polymerase) and MA27 (fusion protein) located approximately 60 and 113 kb from the left-end of the viral genome, respectively. Recombinant myxoma viruses expressing the IacZ gene from either intergenic insertion site retained wild-type virulence. However, expression of the gus gene reduced the virulence of the recombinant viruses in vivo. Northern blot analysis indicated that the major late mRNAs encoding the viral RNA polymerase subunit and fusion protein are both of discrete size. Insertion of a foreign gene under the control of a synthetic late promoter between the MA24 and MA27 genes results in a specific-sized major late transcript for the inserted foreign gene. The MA27 gene transcripts directed by these recombinant viruses are heterogeneous in size, implying the typical pattern of poxvirus late transcription by random 3'-termination prior to polyadenylation. The transcription studies suggest signals located downstream of the insertion site direct 3'-processing of late transcripts irrespective of the gene immediately upstream.

Received 19 January 1996; accepted 26 February 1996.

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