Charles R. Penn

Evidence for the involvement of influenza A (fowl plague Rostock) virus protein P2 in ApG and mRNA primed in vitro RNA synthesis.

Eleven temperature-sensitive (ts) mutants of influenza A (fowl plague, Rostock) virus were analysed for in vitro RNA transcriptase activity in reactions primed by ApG or globin mRNA at 31 degrees C or at 40.5 degrees C, the restrictive temperature for ts mutant growth. Only those ts mutants studied which were defective in RNA segment 1, coding for the virion P2 protein, were defective in RNA transcriptase activity when compared to wild-type virus. Mutants having a defect in the P2 protein had no significant RNA transcriptase activity in reactions at 40.5 degrees C primed by globin mRNA. However, one mutant showed RNA transcriptase activity similar to wild-type virus at 40.5 degrees C when ApG (0.3 mM) was used as primer. The results suggest that influenza (fowl plague, Rostock) P2 protein is directly involved in the mRNA priming reaction, as well as in the RNA transcription reaction in vitro.

The critical cut-off temperature of avian influenza viruses.

We have measured the pathogenicity for 6-week-old chicks of infection by H7 avian influenza viruses. One virus, strain S3 from A/FPV/Rostock/34(H7N1) showed a temperature sensitive phenotype at 41.5 degrees C and reduced pathogenicity. By analysis of reassortants made between virus S3 and A/FPV/Dobson/27(H7N7), a fully pathogenic virus, two conclusions arise. (1) The critical cut-off temperature for avian influenza virus in 6-week-old chicks is 41.5 degrees. (2) RNA segment 1 of virus S3 is responsible for the lack of pathogenicity in reassortant viruses. Nucleotide sequencing of RNA segment 1 from S3 and its parent, A/FPV/Rostock/34 has revealed a single mutation at nucleotide 1561. This results in a substitution of isoleucine for leucine at amino acid position 512 in the cap-binding protein, PB2.

The role of RNA segment 1 in an in vitro host restriction occurring in an avian influenza virus mutant

A temperature sensitive mutant, ts C47, derived from A/FPV/Rostock/34 and with a ts mutation in RNA segment 8, fails to form plaques in MDCK cells. From data obtained with reassortant viruses using the human influenza isolate A/FM/1/47 it was apparent that more than one mutation contributed to the temperature-sensitive (ts) and host range (hr) phenotypes of ts C47, and the phenotype of reassortants containing RNA segment 1 from A/FM/1/47 indicated that this segment was involved. A single nucleotide substitution at nucleotide 1961, resulting in valine instead of methionine in the predicted amino acid sequence of polypeptide PB2, was found in RNA segment 1 of ts C47, but this mutation did not segregate with the attenuated phenotype on gene reassortment. The following conclusions are drawn: (a) that ts C47 has at least two mutations in addition to that already known to exist in RNA segment 8, one of which (that in RNA segment 1) does not contribute to the observed ts hr phenotypes and (b) that the hr phenotype can be suppressed by substitution of RNA segment 1 by that of another strain.