GENOMIC CONCATEMERIZATION DELETION IN ROTAVIRUSES - A NEW MECHANISM FOR GENERATING RAPID GENETIC CHANGE OF POTENTIAL EPIDEMIOLOGIC IMPORTANCE/

Three variants of group A rotavirus with large changes in their gene 5 structures have been analyzed at the molecular level. The first of th ese, P9DELTA5, was obtained during plaque purification undertaken as p art of the biological cloning of a field isolate of virus. The gene 5 homolog in this isolate migrated just ahead of the normal segment 6 RN A, giving an estimated size of 1,300 bp. Molecular cloning and sequenc ing of this homolog revealed it to have a single 308-bp deletion in th e center of the normal gene 5 sequence extending between nucleotides 4 60 and 768 of the normal gene sequence. This deletion caused a framesh ift in the gene such that a stop codon was encountered 8 amino acids d ownstream of the deletion point, giving a predicted size for the prote in product of this gene of 150 amino acids compared with the 490 amino acids of its normal-size counterpart. Attempts to detect this shorten ed protein in virus-infected cells were not successful, indicating tha t it was much less stable than the full-length protein and/or had suff ered a large change in its antigenicity. The second two variants, brvA and brvE, were generated in an earlier study following the high-multi plicity passage of the UKtc strain of bovine rotavirus. Polyacrylamide gel electrophoresis analysis of these nondefective variants showed th at brvA had a gene 5 homolog approximately equal in size to the normal RNA segment 2 (approximately 2,700 bp) and that brvE had a size of ap proximately 2,300 bp. Both variants showed changes in their gene 5 pro tein products, with brvA mimicking P9DELTA5 in failing to produce a de tectable product whereas brvE produced a new virus-specific protein ap proximately 80 kDa in size. Full-length cDNA clones of the brvE gene 5 homolog were isolated, and analysis of their structure revealed a hea d-to-tail concatemerization of the normal gene 5 sequence with the fir st copy of the concatemer covering nucleotides 1 to 808 and the second covering nucleotides 92 to 1579, giving a total length of 2,296 bp. S equencing across the junction region of the two copies of the gene sho wed that they were joined in frame to give a predicted combined open r eading frame of 728 amino acids with the amino-terminal region consist ing of amino acids 1 to 258 fused at the carboxy terminus to amino aci ds 21 to 490. The biological phenotypes, in terms of virus yield and p laque size, of brvA and brvE were compared with those of the parent UK tc strain. This showed that both variants gave a somewhat reduced viru s yield and that brvE had plaques covering an average 2.5-fold smaller area whereas brvA gave plaques whose area was reduced by approximatel y 50-fold. These results demonstrate that in rotaviruses rapid genomic change through both deletion and concatemerization can generate nonde fective virus variants whose protein coding potential has been altered , leading to changes in biological phenotype. This new mechanism for e ffecting rapid genomic change has now to be considered one by which ro taviruses are able to rapidly change the nature of the proteins they e ncode, which may in turn lead to important epidemiological changes.