No abstract

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.

The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine. We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11. Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity. After metabolic labeling of NSP5 with 32 P i , only serine residues were phosphorylated. Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products. Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified. Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa. Moreover, there were polypeptides migrating between 28 and 35 kDa. Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28-to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation. Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A. The immunoprecipitated NSP5 possessed protein kinase activity. Incubation with [␥-32 P]ATP resulted in 32 P labeling of 28-to 35-kDa NSP5. The distribution of 32 P radioactivity between the components of the complex was similar to the phosphorylation in vivo. Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.

Fecal samples from 156 diarrheic piglets were collected from several herds located in two main breeding areas of Argentina. Rotaviruses were detected in 60 samples (38.4%) by polyacrylamide gel electrophoresis and in 55 samples by a group A-specific enzyme-linked immunosorbent assay (ELISA). All samples which were positive by polyacrylamide gel electrophoresis and negative by ELISA had elicited atypical electropherotypes resembling those of group B or C. ELISA-positive samples showing genome rearrangements were also detected

Two other strains from dairy herds were classified as bovine viruses related to the bovine B223 strain reported by Woode and co-workers (G.