Susumu Nakada

Noncumulative sequence changes in the hemagglutinin genes of influenza C virus isolates

Sequence analysis and comparison of hemagglutinin (HA) genes of different influenza C viruses isolated between 1947 and 1983 reveals that (1) the extent of difference among the HA genes is independent of the year in which these viruses were isolated and that (2) changes in the HA genes do not appear to accumulate with time. These results suggest that epidemiologically dominant variants of influenza C viruses do not emerge successively with time and that C virus variants derived from multiple evolutionary pathways cocirculate at any one time. Thus the epidemiology of influenza C viruses differs markedly from that of influenza A viruses, which is characterized by the emergence of successive variants. Based on the nucleotide sequence data, we propose different evolutionary models for influenza A and influenza C viruses.

Epidemiology of influenza C virus in man: Multiple evolutionary lineages and low rate of change

The nucleotide sequences of nonstructural protein (NS) genes of human influenza C viruses isolated between 1947 and 1983 were determined and compared. Assuming constant evolutionary rates, the extent of nucleotide differences among NS genes does not correspond to the isolation years of the strains. This suggests that more than one gene lineage is present in the population. Furthermore, examination of the eight C virus NS gene sequences by the maximum parsimony method (W. M. Fitch, 1971, Syst. Zool. 20, 406-416) yielded phylogenetic trees that were grossly different from those obtained using the hemagglutinin genes for the same eight isolates. This result is compatible with the idea of reassortment of genes in nature across lineages of influenza C viruses. The sequence analysis also suggests that nucleotide substitutions occur at a lower rate in the C virus NS genes than in influenza A virus NS genes.

Infectious influenza A and B virus variants with long carboxyl terminal deletions in the NS1 polypeptides.

An influenza A virus, A/turkey/Oregon/71, was shown by protein gel analysis to code for an NS1 protein approximately half the size of those of other influenza A viruses. Sequence analysis of the NS gene of this virus revealed a 10 nucleotide deletion resulting in an NS1 protein of only 124 amino acids. This truncated NS1 polypeptide retained its karyophilic pattern as detected by indirect immunofluorescence analysis of virus infected cells. Also, A/turkey/Oregon/71 virus grew to high titer in embryonated chicken eggs comparable to other influenza A viruses. We also identified a laboratory variant of an influenza B virus, clone 201, which codes for a truncated NS1 protein. Sequence analysis revealed a 13 nucleotide deletion resulting in a shortened NS1 protein of only 127 amino acids as compared to other influenza B virus NS1 proteins possessing a length of 281 amino acids. Again as shown for the NS1 proteins of other influenza B viruses the NS1 polypeptide of B virus clone 201 was found to localize in the nucleus of infected cells. It appears that large deletions in the carboxyl terminus of the NS1 proteins of influenza A and B viruses can be tolerated without affecting the functional integrity of the NS1 polypeptide.

Complete nucleotide sequence of the influenza C/California/78 virus nucleoprotein gene

The complete nucleotide sequence of RNA segment 5 of the influenza C/California/78 (C/Cal/78) virus was determined by using cloned cDNA derived from viral RNA. The gene contains 1809 nucleotides and can code for a protein of 565 amino acids with a molecular weight of 63 525. The RNA 5 protein of the influenza C/Cal/78 virus possesses two short regions which share a high degree (60-83%) of sequence homology with the nucleoproteins of influenza A and B viruses. These and other structural features of the RNA 5 protein suggest that RNA 5 of influenza C viruses codes for the nucleoprotein. The data also suggest that influenza C viruses are orthomyxoviruses, but that they are more distantly related to either type A or type B viruses than are influenza A and B viruses to each other.

Molecular dissection of influenza virus RNA polymerase: PB1 subunit alone is able to catalyze RNA synthesis

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. Enzyme reconstitution experiments indicated that all three P proteins are required for RNA synthesis in vitro (Kobayashi, et al. Virus Res22, 235–245, 1992). Nuclear extracts of HeLa cells infected with three kinds of the recombinant vaccinia virus, each carrying one of the three P protein cDNAs, exhibited the activity of complete replication, that is, vRNA-sense RNA-directed and cRNA-sense RNA-directed RNA synthesis in the absence of primers. The nuclear extract from cells singly infected with the virus carrying PB1 cDNA exhibited a significant level of model v-sense RNA-directed RNA synthesis activity. Thus we conclude that PB1 is the catalytic subunit of influenza virus RNA polymerase and that under certain conditions, PB1 alone is able to catalyze RNA synthesis in vitro.

THE NONSTRUCTURAL GENE SEGMENT OF INFLUENZA A VIRUS: EXPRESSION OF NS1 PROTEIN IN MAMMALIAN CELLS; ANALYSIS OF A DELETION MUTANT

Publisher Summary The RNA segment 8 of influenza A virus codes for two overlapping polypeptides that are synthesized during viral infection and are not present in mature virions. A number of influenza Viral proteins, including hemagglutinin (HA), neuraminidase, nucleoprotein (NP), and matrix (M) protein, have been successfully expressed in mammalian cells by viral vectors containing cloned influenza viral cDNA copies. This chapter discusses the analysis of a host range mutant of influenza virus with a deletion in the NS gene segment and the successful expression of NSl protein from cloned cDNA in CV-1 cells using an SV40 viral vector. In the analysis, each plasmid was digested to remove the pBR322 sequences and relegated to produce circular SV40-NS recombinant molecules. Each DNA molecule was then co-transfected into CV-1 cells along with a helper SV40 recombinant. This recombinant pSV-r-INS-7 is an SV40 vector that has the insulin gene replacing portions of the early region of SV40. After a two week incubation period, cells were freeze thawed three times and the supernatant was used as the viral stock.