Antigenic variation among human strains of influenza C virus detected with monoclonal antibodies to gp88 glycoprotein.

Antigenic variation among influenza C virus strains was investigated with monoclonal antibodies against gp88 glycoprotein. Seven monoclonal antibodies obtained were tentatively classified into two groups, A and B. The group A antibodies had hemagglutination inhibition (HI), hemolysis inhibition and neutralization activities whereas the group B antibodies possessed none of them. A comparison of antigenicity among 15 human strains with these antibodies in radioimmunoprecipitation and HI tests showed that the regions recognized by the group A antibodies undergo considerable changes, whereas those by group B are conserved among the strains.

Genetic variation among human strains of influenza C virus isolated in Japan.

The RNA genomes of sixteen human strains of influenza C virus isolated in Japan between 1964 and 1983 were compared by SDS-polyacrylamide gel electrophoresis and oligonucleotide fingerprinting. A high degree of genetic variation was observed among the strains analysed. However, there were some strains with the genomes closely related to one another, and they could be divided into two groups. The first group consists of C/Shizuoka/79, C/Kanagawa/1/81 and four strains of C/Yamagata/81. The 1981 strains of this group were all isolated in March of the year. The second one consists of C/Kyoto/41/82, C/Nara/82 and C/Hyogo/1/83 that were isolated between February 1982 and December 1983. Little or no difference was observed in the genomes of the same group, while the difference was evident between two groups. The Aichi/1/81 strain isolated in November 1981 had a genome distantly related to either of these two groups. Thus three different types of influenza C virus were isolated during the period of 12 mth from March 1981 to February 1982, suggesting that multiple influenza C viruses with distant genetic relationship were circulating at the same time in Japan.

Isolation and characterization of influenza C virus inhibitor in rat serum.

Two hemagglutination inhibitors for influenza C virus were isolated from pooled sera of normal rats by sequential chromatography on Blue Sepharose CL 6B, Ultrogel AcA 22, and DEAE-cellulose. The two inhibitors were identified as alpha 1-macroglobulin and murinoglobulin by comparison with the authentic samples. These inhibitors abolished the hemagglutination by influenza C virus strains but did not affect the hemagglutination by influenza A and B virus strains. Hemagglutination inhibition activity of both inhibitors was completely destroyed by incubation with influenza C virus at 37 degrees C but not with the other types of influenza virus, indicating that the inhibitors are specific for influenza C virus. The inhibitory activity was also destroyed by incubation with neuraminidase from Arthrobacter ureafaciens. By contrast, no activity was lost after treatment with neuraminidase from Vibrio cholerae. These results suggest that the sialic acid residue(s) which is cleavable by the former neuraminidase but not by the latter is essential for the hemagglutination inhibition. The two inhibitors were inactivated by treating with sodium hydroxide and methylamine but not with sodium metaperiodate.

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.

Location of a linear epitope recognized by monoclonal antibody S16 on the hemagglutinin-esterase glycoprotein of influenza C virus.

We reported previously that monoclonal antibody S16, which had been raised against the hemagglutinin-esterase (HE) glycoprotein of influenza C/Ann Arbor/1/50 (AA/50) virus, recognizes a linear epitope present on the HE molecules of all influenza C viruses examined except for viruses belonging to a lineage represented by Aichi/1/81 (AI/81). Comparison of the deduced amino acid sequence of HE between viruses on the AI/81-related lineage and those on the others suggests that the epitope recognized by S16 is located in a region containing amino acid residue 403 and that a change from Glu to Lys at this position causes the loss of reactivity with the antibody. To prove it, the wild type (WT) HEs of AA/50 and AI/81 as well as their mutants with an amino acid substitution at residue 403 were expressed in CV-1 cells from the recombinant simian virus 40 (SV40) and tested for reactivity with S16 by immunoprecipitation. The results showed that the AA/50 virus WT and AI/81 virus mutant HEs (both having Glu at residue 403) were reactive with S16 whereas the AI/81 virus WT and AA/50 virus mutant HEs (both having Lys at residue 403) were not. Furthermore, we examined the reactivity of S16 with two synthetic peptides (corresponding to residues 397-409) that possess Glu and Lys at position 403, respectively, by enzyme-linked immunosorbent assays. The results demonstrated that the former peptide but not the latter was reactive with S16. These observations support strongly the notion described above. During this study it was also found that S16 cross-reacts with large T antigen of SV40.

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.

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.

Inhibition of sterol and DNA syntheses in phytohemagglutinin-stimulated human lymphocytes by 7 alpha-hydroxycholesterol.

Sterol and DNA syntheses were induced in human peripheral blood lymphocytes stimulated with phytohemagglutinin (PHA). DNA synthesis in the PHA-stimulated lymphocytes was suppressed by 7 alpha-hydroxycholesterol (7 alpha-HC). The maximum suppression of DNA synthesis was observed when 7 alpha-HC was added in the culture within 6 hr of PHA stimulation to the lymphocytes. However, as the concentration of fetal bovine serum (FBS) in the culture medium was increased, the inhibitory effect of 7 alpha-HC on the syntheses of sterol and DNA were decreased. Furthermore, low and high density lipoproteins completely reversed the inhibition of DNA synthesis by 7 alpha-HC. These results suggest that cholesterol is an essential requirement of lymphocyte blastogenesis regardless of whether the source of the sterol is exogenous or endogenous.

Experimental infections of dogs with type C influenza virus.

A study was performed to determine if type C influenza infection could be established in dogs as a model for human cases. Mongrel dogs were infected with the Ann Arbor/1/50 strain of type C influenza virus and were examined for clinical symptoms, virus isolation and antibody response. After the first exposure to the virus, all infected animals developed nasal discharge and some of them also showed swelling of the eyelids, and suffusion of the eyes with tears and eye mucus, within 1 to 4 days. The animals showed an increase in hemagglutination-inhibiting (HI) serum antibody, and recovery of the agent from the nasal swabs was successful. The symptoms lasted for as long as 10 days in most infected dogs, which was comparable to our human cases reported previously (Katagiri, S., Ohizumi, A., and Homma, M. 1983. J. Infect. Dis. 48: 51-56). After the second and third virus exposures at intervals of 50 days, all animals developed the same symptoms as those described above and the rise in antibody titer was evident. The virus could be recovered from four of the six dogs 2 to 5 days after the second exposure and from one dog as late as 10 days after the third exposure. Increases in antibody titer in the IgM fraction were observed after every infection. In control dogs which were mock-infected with UV-inactivated virus, no symptoms were evident and recovery of the virus was not successful although an increase in HI serum antibody titer was seen. These results show that mongrel dogs are sensitive to type C influenza virus and that repeated infections characteristic of human influenza C can be experimentally produced in dogs.

A comparison of proteins among various influenza B virus strains by one-dimensional peptide mapping.

The major virus-specific proteins (HA, NA, NP, NS1 and M) of five different isolates of influenza B virus (B/Lee/40, B/Osaka/2/70, B/Yamagata/1/73, B/Aomori/1/76 and B/Yamagata/26/77) were compared by limited proteolysis with Staphylococcus aureus V8 protease and subsequent polyacrylamide gel electrophoresis. The peptide patterns of matrix (M) proteins from all five strains were virtually identical. The nucleoproteins (NP) as well as the non-structural proteins (NS1) were also very similar among strains although the peptides of B/Lee/40 could be distinguished from those of the strains isolated from 1970 to 1977. In contrast, the peptides from haemagglutinin (HA) glycoproteins were largely different even among the strains isolated later than 1970. It therefore appears that the HA glycoproteins of influenza B virus are more changeable than any of the non-glycosylated proteins. Furthermore, it was found that the maps of HA1 were markedly different among strains while the maps of HA2 were very similar, which suggests that the structural changes in the HA polypeptide occur preferentially in the HA1 portion. The neuraminidase (NA) glycoproteins also showed strain-dependent differences in their mapping patterns.

The use of the anti-Acholeplasma activities of Streptomyces culture filtrates for the screening of new antibiotics.

A screening method for new antimicrobial agents from streptomyces culture filtrates was developed. Three different types of test organisms were used for determining the antimicrobial activities of the culture filtrates: they were (a) an antibacterial activity test against Staphylococcus aureus, (b) an antimycoplasmal activity test against Acholeplasma laidlawii, and (c) a cytotoxicity test against HeLa-S3 cells. The active filtrates showing antimicrobial spectra (anticellogram) which did not match those of known antimicrobial agents were used for further purification to obtain new antimicrobial agents. Of these screening methods, antimycoplasmal activity against Acholeplasma laidawii was the most sensitive for discriminating new compounds contained in the filtrates of known antibiotics.

Operational and topological analyses of antigenic sites on influenza C virus glycoprotein and their dependence on glycosylation.

In our previous study, seven monoclonal antibodies specific for influenza C virus glycoprotein (gp88) were prepared and tentatively classified into two groups: group A (J14, J9, Q5, K16) has neutralization activity whereas group B (S16, J6, J15) does not. These antibodies were used to analyse the antigenic structure of gp88 and to examine the effect of glycosylation on the antigenicity of the glycoprotein. Operational analysis with a panel of antigenic variants selected with each of the group A antibodies identified two non-overlapping antigenic sites on the gp88 molecules, site A-1 recognized by J14, J9 and Q5 and site A-2 by K16. Sites A-1 and A-2 were shown, however, to be topographically overlapping by competitive binding assays. Competitive binding analysis with group B antibodies identified two additional non-over-lapping antigenic sites, site B-1 recognized by S16 and site B-2 by J6 and J15. It was found in radioimmunoprecipitation experiments that antibodies to sites B-1 and B-2 were reactive not only with gp88 but with its non-glycosylated form (T76) synthesized in the presence of tunicamycin. Antibodies to sites A-1 and A-2, in contrast, immunoprecipitated the T76 polypeptide in only trace amounts or not at all. Additionally, Western blot analysis showed that denatured gp88 blotted on nitrocellulose was reactive with antibodies to sites B-1 and B-2 but not with those to sites A-1 and A-2. These observations suggest that glycosylation of gp88 selectively influences the integrity of antigenic sites A-1 and A-2 which are composed of conformation-dependent epitopes.