THE EFFECT OF DELETING OR INVERTING THE 132 BP REPEATS IN A PATHOGENIC MDV

Authors: Silva, Robert; REDDY, SANJAY - TEXAS A&M; LUPIANI, BLANCA - TEXAS A&M

Submitted to: Workshop on Molecular Pathogenesis of Marek's Disease and Avian Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/12/2003
Publication Date: 11/26/2003
Citation: Silva, R.F., Reddy, S., Lupiani, B. 2003. The effect of deleting or inverting the 132 bp repeats in a pathogenic mdv [CD-ROM]. Workshop on Molecular Pathogenesis of Marek's Disease and Avian Immunology. Limassol, Cyprus, BARD.

Interpretive Summary: One of the most economically devastating infectious diseases of poultry is Marek's disease (MD), a cancer-like disease caused by a virus (MDV). Although it is not known how MDV causes disease, it is easy to develop a non-pathogenic MDV by simply passing the virus repeatedly in cell culture. Researchers have known for years that passing MDV in cell culture also induces a specific change in the genome of the virus. The generation of non-pathogenic viruses and the genomic modification always occur together. Consequently, researchers have hypothesized that the changes in the genome are responsible for the loss of pathogenicity. To test this hypothesis, we used a new technique to construct recombinant MDVs in which we made the same specific genetic changes (mutations) in the MDV genome that are seen after the virus is repeatedly passed in cell culture. We found the recombinant viruses were still pathogenic. Thus, the genomic modifications seen in viruses in cell culture are not sufficient to cause the loss of pathogenicity. Contrary to previous speculations, this portion of the MDV genome is not a suitable region to modify in order to make recombinant MDV vaccines. This information should be valuable to vaccine manufacturers who are looking for new methods to improve vaccines against the disease.

Technical Abstract: Pathogenic Marek's disease viruses (MDVs) generally have two head-to-tail copies of a 132 base pair (bp) repeat. As the virus is serially passed in cell culture, the number of copies increases from two to more than 15 copies. Concurrently, the pathogenic virus becomes attenuated. To determine the role of the 132 bp repeats in pathogenesis, we used five overlapping cosmid clones that spanned the Md5 genome to reconstitute infectious virus (rMd5). By mutating two of these cosmids, we generated infectious virus that had all copies of the 132 bp repeats deleted (rMd5(?132)). In a second recombinant MDV, we replaced the two repeats with 9 copies of the 132 bp repeat (rMd5(9-132)). A third recombinant was created by replacing the two repeats with 9 copies of the 132 bp repeat, but inserted in the reverse orientation (rMd5(rev 9-132)). After two passages in cell culture, rMd5 and rMd5(?132) were stable. However, the 9 copies of the 132 bp repeats in rMd5(9-132) and rMd5(rev 9-132) were unstable, rapidly giving rise to a population of viruses that had anywhere from 3 to over 15 copies of the repeats, similar to what is seen in attenuated MDVs. A major 1.8 kb mRNA present in wild-type Md5 and rMd5, was not present in rMd5(?132), rMd5(9-132), or rMd5(rev 9-132). Instead, the RNAs from the 132 bp repeat region in rMd5(9-132), and rMd5(rev 9-132) closely resembled the RNAs found in attenuated MDVs. When the viruses were inoculated into maternal antibody positive, susceptible day-old chicks, wild-type Md5, rMd5, rMd5(?132), rMd5(9-132) and rMd5(rev 9-132) all produced various lesions, including tumors, nerve lesions, and thymic atrophy. Thus, expansion of the number of copies of 132 bp repeats, that accompanies attenuation, is not sufficient in and of itself to attenuate pathogenic MDVs. Additional mutations, crucial to the attenuation process, must also be occurring as the pathogenic MDVs are serially passed in cell culture. We hypothesize that the 132 bp repeats and the 1.8 kb mRNA are involved in determining how efficiently MDV replicates in the chicken and cell culture.