Genetic stability of a porcine rotavirus RNA segment during repeated plaque isolation

The porcine group A rotavirus CC86 was characterized to explore its utility as a tool for mutation analysis. It has a semiduplication of the gene 11 RNA segment. Nucleotide sequence determination of cDNA confirmed that the NSP5 coding sequence and the conserved nontranslated termini of the RNA segment were retained. A comparison of the NSP5 genes of CC86 and CN86 that were isolated from the same fecal specimen showed eight base pair changes, suggesting that CN86 was not the immediate progenitor of CC86. Synthesis of NSP5 in monkey MA104 cells infected with CC86, CN86, or simian rotavirus SA11 was compared by one- and two-dimensional polyacrylamide gel electrophoresis. NSP5 from all three viruses had similar posttranslational modifications, and no difference in the expression levels was observed. To experimentally address the genetic stability of CC86 segment 11, the virus was passaged by serial plaque to plaque transfer. The repeated genetic bottlenecking led to a gradual loss of fitness. This effect is not observed when virus is passaged by the standard method of moderate dilution. Nucleotide sequence analysis of cDNA clones isolated from viral segment 11 RNA of virus from plaque-to-plaque passage numbers 0, 1, 4, and 8 showed occasional base substitutions, mostly in the NSP5 coding sequence. Two mutations, leading to His-to-Arg and Lys-to-Arg replacements, respectively, in NSP5 were established in the virus population. Forward and reverse base pair changes (A-U<-->G-C) at the two sites appeared to be concerted and take place at a very high frequency, suggesting that a mechanism equivalent to RNA editing might operate. The overall mutation rate of segment 11 was much lower, having a calculated maximal value of 5 x 10(-5) per replicated base.