Kiyoto Nakamura

Interspecies transmission of influenza C virus between humans and pigs

The antigenic and genetic characteristics of the 18 human strains of influenza C virus isolated in Yamagata and Sendai Cities, Japan between January 1991 and February 1993 were investigated. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase glycoprotein showed that the isolates could be divided into three distinct groups closely related to C/Yamagata/26/81, C/Aichi/1/81 and C/Mississippi/80, respectively. T1-oligonucleotide fingerprinting of total vRNA revealed that the six isolates belonging to the C/Yamagata/26/81 virus group had the genomes greatly similar to one another but considerably different from those of the 1988/1990 isolates (except C/Yamagata/10/89) of the same antigenic group. Comparison of total or partial nucleotide sequences of the seven RNA segments of the three strains (C/Miyagi/3/91, C/Miyagi/9/91 and C/Miyagi/2/92) representative of the 1991/1993 strains of the C/Yamagata/26/81 virus group with those of the previous influenza C isolates obtained from humans and pigs during 1980/1989 showed that the 1991/1993 strains, like C/Yamagata/10/89, are more closely related to viruses isolated from pigs in Beijing, China in 1981/1982 than to any of the isolates from humans. This observation suggests strongly that interspecies transmission of influenza C virus between humans and pigs has occurred in nature, although it is not known whether the virus has been transmitted from pigs to humans or from humans to pigs.

EVOLUTION OF THE HAEMAGGLUTININ-ESTERASE GENE OF INFLUENZA C VIRUS

The nucleotide sequences of the haemagglutinin-esterase (HE) genes of 18 influenza C virus strains isolated in Japan during the period from 1964 to 1988 (11 published and 7 new sequences) were analysed to examine their evolutionary relationships. The phylogenetic tree constructed by the maximum parsimony method revealed the existence of four discrete lineages (I to IV), one of which (lineage III) may have died out in the late 1970s. Sequential evolution was demonstrated within seven strains of lineage I, which allowed estimation of an evolutionary rate of 0.49 x 10-3 nucleotide substitutions per site per year, a value corresponding to about one-ninth of the rates of human influenza A virus haemagglutinin genes. In the previously proposed immunodominant region on HE protein (positions 178 to 217), there was little or no amino acid sequence divergence among viruses on the same lineage although considerable divergence was seen among those on different lineages, raising the possibility that immune selection may not have played a significant role in the evolution of the glycoprotein, at least not after separation into lineages occurred. It was also found that the HE genes of the seven isolates obtained outside Japan during 1966-1983 could be each assigned to one of lineages I, II and IV, which suggests that influenza C virus is capable of spreading worldwide.

FREQUENT OCCURRENCE OF GENETIC REASSORTMENT BETWEEN INFLUENZA C VIRUS STRAINS IN NATURE

Previous studies of the haemagglutinin-esterase (HE) genes of various influenza C isolates suggested the existence of three distinct virus lineages (C/Yamagata/26/81-, C/Aichi/1/81- and C/Mississippi/80-related lineages) in Japan in the 1980s. Here we analysed the genetic properties of three strains (C/Yamagata/5/92, C/Miyagi/3/93 and C/Miyagi/4/93) isolated in Yamagata and Sendai Cities, Japan, in 1992/1993. Comparison of total or partial nucleotide sequences of the seven RNA segments of C/Yamagata/5/92 with those of 11 previous isolates suggested that the 1992 strain is a reassortant which inherited HE, P3, NP and M genes from a C/Mississippi/80-like virus and PB2, PB1 and NS genes from a C/pig/Beijing/115/81-like virus. Furthermore, it became evident that at least two (C/England/83 and C/Yamagata/9/88) of the 11 reference strains are also reassortants.

Evolutionary Analysis of Influenza C Virus M Genes

The previous study of the 25 hemagglutinin-esterase (HE) glycoprotein genes of influenza C viruses identified four discrete lineages represented by C/Yamagata/26/81, C/Aichi/1/81, C/Aomori/74 and C/Mississippi/80, respectively. Here we compared the M gene sequence among the 24 viruses isolated between 1964 and 1991. A phylogenetic analysis showed that these genes have evolved into three distinct lineages. Lineage I included most of viruses with the HE genes of C/Yamagata/26/81-related lineage. The predominant members of lineage II were viruses having the HE genes of either C/Aichi/1/81- or C/Mississippi/80-related lineage. Lineage III contained only C/Aomori/74. Phylogenetic positions of several strains (C/Yamagata/64, C/Kanagawa/1/76, C/Miyagi/77 and C/Nara/1/85) were different between the M and HE gene trees, suggesting that they are reassortants. Furthermore, phylogenetic relationships between C/Mississippi/80-like and C/Aichi/1/81-like viruses were much closer for the M gene than the HE gene, raising the possibility that these two virus groups are genetically related by a reassortment event. Nucleotide changes in the M genes occurred at about 7% positions with a uniform distribution throughout the molecules. However, the predicted amino acid sequence of the matrix protein (M1) was conserved almost completely among the isolates analyzed. The amino acid sequence of the second protein (CM2) encoded by M gene was also highly conserved, but was more divergent than the M1 protein sequence, suggesting that the two M gene products are evolving differently in response to selective pressures or structural and functional constraints.

Identification of a 374 amino acid protein encoded by RNA segment 6 of influenza C virus.

Unspliced mRNA from RNA segment 6 of influenza C virus contains a single open reading frame that potentially encodes a polypeptide of 374 amino acids. This polypeptide, which has not been identified as yet, is predicted to contain the complete amino acid sequence of the matrix protein, M1, as well as that of a small integral membrane protein, CM2. Here, we found that small amounts of two previously unrecognized proteins with apparent molecular masses of 42 (P42) and 44 kDa (P44) were immunoprecipitated with immune serum against CM2. The electrophoretic mobilities of P42 and P44 varied depending on virus strain, indicating that they are virus-coded. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with various endoglycosidases revealed that P42 is modified by the addition of a high-mannose oligosaccharide chain to generate P44. A monoclonal antibody against M1, like anti-CM2 serum, was able to immunoprecipitate both the P42 and P44 proteins. Furthermore, the tryptic peptide map of either P42 or P44 was indistinguishable from the map of the mixture of M1 and CM2. These results, taken together, suggest strongly that P42 and P44 correspond to the 374 amino acid protein encoded by unspliced RNA segment 6 mRNA and its N-glycosylated form, respectively.

Phosphorylation of influenza C virus CM2 protein.

Labeling of influenza C virus-infected HMV-II cells with [32P]orthophosphate showed that the CM2 protein is posttranslationally modified by phosphorylation. The unglycosylated form of CM2 synthesized in the presence of tunicamycin was found to be highly phosphorylated. This result, together with the finding that digestion of CM2 with peptide-N-glycosidase F failed to remove the 32P label from the glycosylated form of CM2, indicated that phosphorylation occurs in the polypeptide backbone and not in the oligosaccharide chain. Furthermore, phospho-amino acid analysis revealed that phosphorylation occurs exclusively on serine residues. Treatment of infected cells with brefeldin A resulted in a complete inhibition of phosphorylation, showing that phosphorylation of CM2 occurs after its migration from the endoplasmic reticulum to the Golgi apparatus. Phosphorylation of CM2 was also inhibited strongly, although not completely, by monensin treatment, suggesting that CM2 is phosphorylated predominantly after its movement from medial to trans Golgi cisternae. Finally, we found that the CM2 protein incorporated into the progeny virion is phosphorylated, which indicates that there is no strictly selective incorporation of the unphosphorylated form of CM2 into the virion.

Comparison of receptor-binding properties among influenza C virus isolates.

A total of 10 influenza C virus strains isolated recently in Yamagata City, Japan and shown to belong to the same lineage was compared for the ability to agglutinate chicken and mouse erythrocytes under various conditions. C/Yamagata/10/89 was unique in lacking the ability to agglutinate chicken erythrocytes at a temperature > or = 4 degrees C. This isolate also agglutinated native mouse erythrocytes only very inefficiently, although the high agglutination titer was obtained with the glutaraldehyde-fixed cells. Furthermore, it was found that C/Yamagata/4/88, unlike the other isolates, agglutinated erythrocytes from chickens to lower titers than those from mice, even when assayed at 0 degree C. Comparison of the deduced amino acid sequence of hemagglutinin-esterase among the 6 representative strains including two older isolates, C/Yamagata/26/81 and C/Nara/2/85, suggested that the failures of C/Yamagata/10/89 to agglutinate chicken erythrocytes at > or = 4 degrees C and unfixed mouse erythrocytes to high titers may be due to amino acid changes at residues 337 (Glu-->Lys) and 340 (Thr-->Tyr), respectively, and that a change at residue 347 (Leu-->Ser) may be responsible for the decreased ability of C/Yamagata/4/88 to agglutinate chicken erythrocytes.

Identification of Influenza C Virus Phosphoproteins

The HMV‐II cells infected with influenza C virus were labeled with inorganic [32P]phosphate to identify phosphorylated proteins. Analysis by radioimmunoprecipitation with antiviral serum or monoclonal antibodies revealed that three major structural proteins of the virus, hemagglutinin‐esterase (HE), nucleoprotein (NP), and matrix protein (M1) are all phosphorylated in both infected cells and virions. It was also observed that, in the presence of trypsin (10 μg/ml), the unphosphorylated form of the HE glycoprotein was cleaved efficiently whereas the phosphorylated form was not, raising the possibility that phosphorylation of HE may influence its susceptibility to degradation by proteolytic enzymes.