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Neutralization-resistant variants of influenza C/Ann Arbor/1/50 virus were selected with monoclonal antibodies against four different antigenic sites on the hemagglutinin-esterase (HE) glycoprotein, and their HE genes were sequenced to identify amino acid residues important for the integrity of each site. Twelve different amino acid substitutions in a total of 18 antigenic variants were all located on the HE1 subunit. Although variants for antigenic site A-2 had a change at position 367, all substitutions in the variants for sites A-1, A-3, and A-4 occurred in the central region of the HE1 spanning amino acid positions 178 to 283. Furthermore, it was found that many of the substitutions in the variants selected with antibodies to sites A-1 and A-3 were clustered within or near one of the three variable regions revealed previously by comparing amino acid sequences of the HEs among various influenza C isolates (Buonagurio, D. A., Nakada, S., Fitch, W. M., and Palese, P., Virology 146, 221–232, 1985). The antigenic variants were also examined for their ability to agglutinate chicken and human erythrocytes in order to obtain information concerning the receptor-binding site on the HE molecule. The results suggested that the amino acid changes at residues 178, 186, 187, 190, 206, 212, and 226 decreased the hemagglutinating activity whereas those at residues 245, 266, and 283 produced an opposite effect.

Pre-mRNAs of the influenza A virus M and NS genes are poorly spliced in virus-infected cells. By contrast, in influenza C virus-infected cells, the predominant transcript from the M gene is spliced mRNA. The present study was performed to investigate the mechanism by which influenza C virus M gene-specific mRNA (M mRNA) is readily spliced. The ratio of M1 encoded by a spliced M mRNA to CM2 encoded by an unspliced M mRNA in influenza C virus-infected cells was about 10 times larger than that in M gene-transfected cells, suggesting that a viral protein(s) other than M gene translational products facilitates viral mRNA splicing. RNase protection assays showed that the splicing of M mRNA in infected cells was much higher than that in M gene-transfected cells. The unspliced and spliced mRNAs of the influenza C virus NS gene encode two nonstructural (NS) proteins, NS1(C/NS1) and NS2(C/NS2), respectively. The introduction of premature translational termination into the NS gene, which blocked the synthesis of the C/NS1 and C/NS2 proteins, drastically reduced the splicing of NS mRNA, raising the possibility that C/NS1 or C/NS2 enhances viral mRNA splicing. The splicing of influenza C virus M mRNA was increased by coexpression of C/NS1, whereas it was reduced by coexpression of the influenza A virus NS1 protein (A/NS1). The splicing of influenza A virus M mRNA was also increased by coexpression of C/NS1, though it was inhibited by that of A/NS1. These results suggest that influenza C virus NS1, but not A/NS1, can upregulate viral mRNA splicing.

To apply serological testing for human metapneumovirus (hMPV) to large-scale sera samples, an enzyme-linked immunosorbent assay (ELISA) was developed in which purified virions were used as the antigen. The ELISA was evaluated using 102 human sera specimens from patients aged 0-59 years. There was a positive association between the ELISA results and neutralization test titers, with the correlation coefficients being greater in children6 years old (rho=0.899, P0.0001), which is consistent with a primary infection, than in personsor=6 years old (rho=0.523, P0.0001). Fifty sera samples were subjected to radioimmunoprecipitation to measure the quantity of antibodies to the fusion protein (RIP-F) and the nucleoprotein (RIP-N). The results showed significant associations between the ELISA titers and the amount of RIP-F as determined by radioimmunoprecipitation in children6 years old (rho=0.804, P=0.0083) and in personsor=6 years old (rho=0.577, P=0.0009). The correlation between the ELISA titer and the amount of RIP-N determined by radioimmunoprecipitation was not significant in personsor=6 years old (rho=0.417, P=0.0829), although this correlation was significant in children6 years old (rho=0.764, P=0.0137). The ELISA titer correlated with the amount of antibodies to the F protein, but not to the N protein. This whole virus-based ELISA will be useful for the diagnosis of hMPV infection in clinical laboratories and is also useful for the large-scale investigations, such as seroprevalence among residents of a particular region.

Most Drosophila mRNAs are actively translated in the early embryo, with the exception of the poorly translated ribosomal protein (r-protein) mRNAs. Two possible mechanisms for this translational discrimination were tested: (1) Translation of r-protein mRNAs is discriminated against by the limited activity of translational initiation factors in the early embryo and (2) translation of r-protein mRNAs is repressed by trans-acting factors that reversibly bind these mRNAs. Exogenously provided initiation factors promoted partial recruitment of r-protein mRNAs into polysomes, suggesting that modulation of initiation factor activity may play a role in the translational discrimination of r-protein mRNAs during embryogenesis. No evidence for involvement of reversibly binding trans-acting factors was obtained, although there are limitations in the interpretation of the latter experiments.

We isolated and identified six subgenogroups (B2, B4, B5, C1, C2, and C4) of enterovirus 71 (EV71) between 1990 and 2007 in Yamagata, Japan. We measured neutralizing antibody (NT Ab) titers against those subgenogroup strains and the BrCr reference strain for antigenic analysis. Serological analysis of 83 residents in Yamagata in 2004 showed that differences in the NT Ab titer of each individual against the different subgenogroups were mostly within 4-fold. Furthermore, sera from guinea pigs, immunized with the B2 and C1 strains indicated cross-antigenicity among the seven different subgenogroups. In conclusion, our results showed that cross-antigenicity exists among EV71 strains from different subgenogroups circulating in the community through genomic evolution. Our results also suggest that eliciting neutralizing antibodies against one genotype is likely to confer cross-neutralization against other genotypes.

Background: Noroviruses (NV) are one of the leading causes of gastroenteritis in young children; however, the duration of NV shedding in young children is not well known. Methods: Fecal specimens were collected from children with acute gastroenteritis at a pediatric clinic during the period from November to December 2002 and tested for NV by reverse transcription– polymerase chain reaction. Results: Of 71 children infected with NV, 60 (84.5%) were less than 3 years old. Among children aged 2 years and those aged 2 to 5 years, the duration of illness was longer (7 days versus 3.5 days, P 0.0069), the maximum number of stools in a 24-hour period was greater (7 versus 3, P 0.0078) and a 20-point severity score was higher (11 versus 8, P 0.0031) in patients aged 2 years than in patients aged 2 to 5 years. Among the 23 children whose follow-up specimens were obtained, the median duration of NV shedding was 16 days (range, 5– 47 days). Virus shedding for more than 2 weeks after onset was observed in 75% (6 of 8), 71.4% (5 of 7) and 25% (2 of 8) of children aged 1 year, 1 year and 2 to 3 years, respectively. Three infants aged 6 months continued to excrete NV for an extremely long period (more than 42, 44 and 47 days from onset) after recovery. Conclusion: Long-term virus shedding after the disappearance of clinical symptoms was observed. Caution should be exercised when handling the excrement of infants and young children infected with NV.

The influenza C virus genome consists of seven single-stranded RNA segments of negative polarity. The hemagglutinin-esterase (HE) glycoprotein of influenza C virus has three biological activities, i.e. receptor-binding activity for N-acetyl-9-O-acetylneuraminic acid, fusion activity, and receptor-destroying activity, which is a neuraminate-O-acetylesterase. Unspliced mRNA from RNA segment 6 is first translated into a 374-amino-acid protein, P42. P42 is cleaved by signal peptidase, producing M1' and CM2 proteins, composed of the N-terminal 259 amino acids and the C-terminal 115 amino acids, respectively. Xenopus laevis oocytes expressing influenza C virus CM2 protein demonstrated that CM2 protein forms a voltage-activated ion channel permeable to chloride ion.

Both transcription and replication of influenza virus genome take place in the nucleus of the infected cells. Initiation of mRNA synthesis requires the generation of capped primers cleaved from the 5' end of the host pre-mRNA by a cap-snatching mechanism. These transcripts are polyadenylated at their 3' ends when the viral polymerase stutters over a polyuridine stretch that is found 15-22 nucleotides from the 5' end of the vRNA. Replication of the viral genome is achieved through primer-independent synthesis of a full-length, positive-sense replicative intermediate, cRNA, that is copied into vRNA. The switch from mRNA to template RNA(cRNA) synthesis requires antitermination activity of NP proteins not associated with nucleocapsids. M gene expression differs among influenza A, B and C viruses.

Sporadic Parkinson's disease (sPD) is a common neurodegenerative disorder, characterized by selective degeneration of dopaminergic neurons in the substantia nigra. Although the pathogenesis of the disease remains undetermined, phosphorylation of α-synuclein and its oligomer formation seem to play a key role. However, the protein kinase(s) involved in the phosphorylation in the pathogenesis of sPD has not been identified. Here, we found that G-protein-coupled receptor kinase 5 (GRK5) accumulated in Lewy bodies and colocalized with α-synuclein in the pathological structures of the brains of sPD patients. In cotransfected cells, GRK5 phosphorylated Ser-129 of α-synuclein at the plasma membrane and induced translocation of phosphorylated α-synuclein to the perikaryal area. GRK5-catalyzed phosphorylation also promoted the formation of soluble oligomers and aggregates of α-synuclein. Genetic association study revealed haplotypic association of theGRK5gene with susceptibility to sPD. The haplotype contained two functional single-nucleotide polymorphisms, m22.1 and m24, in introns of theGRK5gene, which bound to YY1 (Yin Yang-1) and CREB-1 (cAMP response element-binding protein 1), respectively, and increased transcriptional activity of the reporter gene. The results suggest that phosphorylation of α-synuclein by GRK5 plays a crucial role in the pathogenesis of sPD.

The conformational maturation of the influenza C virus nucleoprotein (NP) synthesized in infected cells was investigated. Monoclonal antibodies (mAbs) that have previously been characterized [Sugawara, K., Nishimura, H., Hongo, S., Kitame, F., Nakamura, K., 1991. Antigenic characterization of the nucleoprotein and matrix protein of influenza C virus with monoclonal antibodies. J. Gen. Virol. 72, 103-109] enabled this molecular maturation to be detected. Both pulse-labeled and chased NPs could equally retain high reactivity with H31 mAb recognizing a linear epitope on the NP molecule. However, pulse-labeled NP showed three- to four-fold lower reactivity with H27 mAb recognizing a conformational epitope, compared to chased NP. Sedimentation analyses by sucrose gradient centrifugation revealed that the mature NP could readily participate in nucleocapsid formation while the immature NP was free. The immature NP was rapidly transported into the nucleus and its maturation seemed to occur after or during translocation into the nucleus. A single expression of NP cDNA in COS-1 cells demonstrated that the NP maturation was an intrinsic feature of the NP molecule without relation to other viral components.

Phylogenetic analysis of 45 enterovirus 71 (EV71) isolates for 6 years in Yamagata, Japan, clarified that the annual outbreak of hand-foot-and-mouth disease was due to four genetically distinct subgenogroups, including a novel “B5.” Our results suggest that the importation of EV71 from surrounding countries has had a major epidemiological impact on the local community used in our study.

We investigated a measles virus (MV) outbreak that occurred at a junior high school in Yamagata, Japan between January and February, 2004. We received throat swab specimens from three patients at this school and carried out virus isolation with Vero/hSLAM cells and virus genome detection by reverse-transcription polymerase chain reaction. As a result, we isolated the virus from one patient and succeeded in amplifying the MV genome from the others. Further sequence analysis of the N gene revealed that these viruses were completely identical, and that their genotype could be characterized as type D9, which has not been reported in Japan previously. We also identified D9 viruses in two students at other junior high schools in Yamagata. These results suggested that D9 strains were imported from a region outside Japan. The genotypes of MVs found in Yamagata have changed in recent years, with D5 predominating in 2001 and H1 predominating in 2002 and 2003 as reported as national surveillance data. Therefore, we should monitor carefully to be sure that D9 strains do not become the next predominant virus. The more the number of measles cases decrease, the more important become the roles of public health laboratories, which genotype MVs and monitor their circulation and transmission pathways.

Publisher Summary This chapter demonstrates that the use of cross-well seismic tomography in an onshore aquifer. Results of seismic survey suggested that the injected CO2 may migrate preferentially along the formation direction. The geophysical loggings of induction and sonic showed the CO2 accession to the observation well CO2-2. The pore pressure buildup measured at the observation well CO2-4 was about 6 % caused by the CO2 injection. It is interesting to note that there was not any significant change in S-wave velocity at the observation well CO2-2. Such results may enable us to predict quantitatively the location and amount of CO2 in the cross-well image plane. A series of observations and measurements composed of cross-well seismic tomography, geophysical logging, pressure and temperature measurement, and seismicity monitoring have been conducted at the test site to understand the CO2 behavior in the underground aquifer.

Influenza A/H3N2 viruses have developed an increased number of glycosylation sites on the globular head of the hemagglutinin (HA) protein since their appearance in 1968. Here, the effect of addition of oligosaccharide chains to the HA of A/H3N2 viruses on its biological activities was investigated. We constructed seven mutant HAs of A/Aichi/2/68 virus with one to six glycosylation sites on the globular head, as found in natural isolates, by site-directed mutagenesis and analyzed their intracellular transport, receptor binding, and cell fusion activities. The glycosylation sites of mutant HAs correspond to representative A/H3N2 isolates (A/Victoria/3/75, A/Memphis/6/86, or A/Sydney/5/97). The results showed that all the mutant HAs were transported to the cell surface as efficiently as wild-type HA. Although mutant HAs containing three to six glycosylation sites decreased receptor binding activity, their cell fusion activity was not affected. The reactivity of mutant HAs having four to six glycosylation sites with human sera collected in 1976 was much lower than that of wild-type HA. Thus, the addition of new oligosaccharides to the globular head of the HA of A/H3N2 viruses may have provided the virus with an ability to evade antibody pressures by changing antigenicity without an unacceptable defect in biological activity.

Between October 1999 and May 2000, a total of 28 strains of influenza C virus were isolated in four Japanese prefectures: Yamagata, Miyagi, Saitama and Hiroshima. Antigenic analysis showed that the 28 isolates were divided into three distinct antigenic groups, and viruses belonging to different antigenic groups were co-circulating in each of the four prefectures. Phylogenetic analysis of the seven protein genes demonstrated that the viruses having a similar genome composition spread in various areas of Japan during the same period. Furthermore, phylogenetic analysis showed that most of the influenza C viruses isolated in various areas of the world between the 1970s and 1980s were closely related to the contemporary Japanese viruses in all gene segments. These observations suggest that the influenza C viruses cause epidemics in some communities during the same season and that antigenically and genetically similar influenza C viruses spread throughout Japan and may be circulating worldwide.

Influenza C virus-like particles (VLPs) have been generated from cloned cDNAs. A cDNA of the green fluorescent protein (GFP) gene in antisense orientation was flanked by the 5′ and 3′ non-coding regions of RNA segment 5 of the influenza C virus. The cDNA cassette was inserted between an RNA polymerase I promoter and terminator of the Pol I vector. This plasmid DNA was transfected into 293T cells together with plasmids encoding virus proteins of C/Ann Arbor/1/50 or C/Yamagata/1/88. Transfer of the supernatants of the transfected 293T cells to HMV-II cells resulted in GFP expression in the HMV-II cells. The quantification of the GFP-positive HMV-II cells indicated the presence of approximately 106 VLPs (ml supernatant)−1. Cords 50−300 μm in length were observed on transfected 293T cells, although the cords were not observed when the plasmid for M1 protein of C/Ann Arbor/1/50 was replaced with that of C/Taylor/1233/47. A series of transfection experiments with plasmids encoding M1 mutants of C/Ann Arbor/1/50 or C/Taylor/1233/47 showed that an amino acid at residue 24 of the M1 protein is responsible for cord formation. This finding provides direct evidence for a previous hypothesis that M1 protein is involved in the formation of cord-like structures protruding from the C/Yamagata/1/88-infected cells. Evidence was obtained by electron microscopy that transfected cells bearing cords produced filamentous VLPs, suggesting the potential role of the M1 protein in determining the filamentous/spherical morphology of influenza C virus.

P42, encoded by a colinear transcript of Influenza C virus RNA segment 6 (M gene), is an integral membrane protein which is cleaved by signal peptidase to generate M1' and CM2 composed of N-terminal 259 amino acids and C-terminal 115 amino acids, respectively. Herein, the biochemical features of P42 were investigated. N-glycosylated form of P42, designated P44, forms disulphide-linked dimers and tetramers. P44 is transported to the Golgi apparatus, but not to the trans-Golgi, since P44 is completely sensitive to endoglycosidase H. P44 and P42 are unstable irrespective of N-glycosylation or oligomerization. 26S proteasome inhibitor, lactacystin prevented the degradation of P42 as well as M1', but not that of P44 efficiently, suggesting that P44 is degraded by another protease besides the 26S proteasome.

In a 9-year survey from December 1990 to December 1999 in Sendai City, Japan, we succeeded in isolating a total of 45 strains of influenza C virus. These 45 strains were isolated in clusters within 4 months in a year, especially from winter to early summer. Previous studies of the hemagglutinin-esterase genes of various influenza C virus isolates revealed the existence of five distinct virus lineages (Aichi/1/81-, Yamagata/26/81-, Mississippi/80-, Sao Paulo/82-, and Kanagawa/1/76-related lineage) in Japan between 1970 and the early 1990s (Y. Matsuzaki, K. Mizuta, H. Kimura, K. Sugawara, E. Tsuchiya, H. Suzuki, S. Hongo, and K. Nakamura, J. Gen. Virol. 81: 1447-1452, 2000). Antigenic and genetic analyses of the 45 strains showed that they could be divided into these five virus lineages and a few antigenic groups were cocirculating in Sendai City. In 1990 and 1991 the dominant antigenic group was the Aichi/1/81 virus group, and in 1992 it was Yamagata/26/81 virus group. The Mississippi/80 virus group was isolated from 1993 to 1996, and the Yamagata/26/81 virus group reemerged in 1996 and continued to circulate until 1999. This finding led us to a speculation that the replacement of the dominant antigenic groups had occurred by immune selection within the human population in the restricted area. Phylogenetic analysis of seven RNA segments showed that 44 viruses among the 45 strains isolated in our surveillance work were reassortant viruses that have various genome compositions distinguishable from those of the reference strains of the each lineage. This observation suggests that the reassortment between two different influenza C virus strains occurs frequently in nature and the genome composition of influenza C viruses may influence their ability to spread in humans.

The haemagglutinin (HA) protein of influenza A/H2N2 virus possesses five oligosaccharide attachment sites, two of which have overlapping glycosylation sequons at positions 20–23 (NNST) and 169–172 (NNTS). Here, the role of these two oligosaccharide attachment sites is investigated with regard to antigenic property, intracellular transport and biological activity of the HA protein. Glycosylation-site HA mutants with mutation(s) in their overlapping glycosylated sequons, each of which had one or two oligosaccharide attachment sites removed, were constructed. Comparison of electrophoretic mobility between the wt and mutant HA proteins showed that both Asn residues 20 and 21 and Asn residues 169 and 170 could be used for glycosylation. Analysis of reactivity of the mutants with anti-HA monoclonal antibodies suggested that amino acid changes at these two positions result in a conformational change of the HA molecule. Even if oligosaccharide chains linked to Asn 20 or 21 and Asn 169 or 170 are eliminated, the antigenic properties, intracellular transport and biological activities are not influenced strongly. Thus it is reasonable to conclude that the two overlapping glycosylation sequons at positions 20–23 and 169–172 are conserved among all of the HAs of influenza A/H2N2 viruses because conservation of the amino acid sequence itself rather than that ofN-glycosylation is essential for the formation of the proper conformation, intracellular transport and biological activities of the H2 subtype HA.

The haemagglutinin (HA) of influenza A/H2N2 virus possesses six antigenic sites (I-A to I-D, II-A and II-B), and sites I-A, I-B and I-C are located in the regions corresponding to sites A, B and D on the H3 HA. We demonstrated previously that most escape mutants selected by mAbs to site I-A, I-B or I-C had acquired a new oligosaccharide at position 160, 187 or 131, respectively, but this has never occurred during circulation of A/H2N2 virus in humans. Here, to examine whether the H2 HA has the potential to gain two new oligosaccharides on its tip, 31 double escape mutants were isolated by using a single escape mutant with an oligosaccharide at position 160, 187 or 131 as a parental virus and a mAb to an antigenic site different from that to which the mAb used for selection of the parental virus was directed as a selecting antibody, but there were no mutants with two new oligosaccharides. Glycosylation-site HA mutants containing one to three oligosaccharides at positions 160, 187 and 131 were also constructed and their intracellular transport and biological activities were analysed. The results showed that all of the mutant HAs were transported to the cell surface but exhibited a decrease in both receptor-binding and cell-fusing activities. Thus, influenza A/H2N2 virus may have failed to increase the number of oligosaccharides on the HA because, if this happens, the biological activities of the HA are reduced, decreasing the ability of the virus to replicate in humans.

Previous studies of the hemagglutinin (HA) genes of various influenza B virus isolates demonstrated the existence of two antigenically distinct virus lineages represented by B/Victoria/2/87 and B/Yamagata/16/88, respectively. Here, we investigated the antigenic and genetic characteristics of influenza B viruses isolated from children living in Lusaka, Zambia between January and May 1999. Antigenic analysis with chicken antiviral sera showed that all the Zambian isolates had the HA protein belonging to B/Yamagata/16/88-related lineage. Furthermore, phylogenetic analyses of the eight RNA segments performed by using the total or partial nucleotide sequences of the two representative Zambian strains (B/Lusaka/270/99 and B/Lusaka/432/99) as well as the previously reported sequences suggested that the Zambian viruses are closely related to the recently circulating reassortants represented by B/Shiga/T30/98 and B/Yamanashi/166/98 which acquired the genes coding for three polymerase proteins (PB2, PB1, and PA), HA, nucleoprotein, and matrix protein from a B/Yamagata/16/88-like parent and the gene encoding nonstructural proteins (NS1 and NS2) from a B/Guandong/8/93-like parent.

The nucleotide sequences of RNA segment 7 (nonstructural protein gene; NS) were compared among 34 influenza C virus strains isolated between 1947 and 1992. The results showed that all the NS genes analysed had the potential to encode NS1 and NS2 proteins of 246 and 182 amino acids, respectively. The deduced amino acid sequence of the previously unidentified NS2 was fairly well conserved, although it was more divergent than the NS1 protein sequence. Moreover, immunoprecipitation experiments with rabbit immune serum against a glutathione S-transferase fusion protein containing the C-terminal region of the 182 amino acid NS2 protein revealed synthesis of a protein with an apparent molecular mass of ∼22 kDa in infected cells. A phylogenetic analysis showed that the 34 NS genes were split into two distinct groups, A and B. Comparison of the phylogenetic positions of the individual isolates in the NS gene tree with those in the haemagglutinin–esterase (HE) gene tree suggested that most of the influenza C viruses currently circulating in Japan, irrespective of their HE gene lineage, had acquired group B NS genes through reassortment events that presumably occurred either in the 1970s or in the early 1980s.

Three influenza C virus strains (C/Yamagata/1/92, C/Yamagata/1/93 and C/Miyagi/5/93) isolated in Yamagata and Sendai Cities, Japan, between June 1992 and May 1993 were found to possess haemagglutinin–esterase glycoproteins that were antigenically indistinguishable from one another but were clearly different from any previous Japanese isolates. To investigate the origin of the 1992/1993 strains, their antigenic and genetic properties were compared with those of eight strains isolated outside Japan between 1967 and 1982. The results showed that the 1992/1993 isolates were closely related to a virus isolated in Brazil in 1982 (C/SaoPaulo/378/82) and that these viruses (including C/SaoPaulo/378/82) are reassortants that had obtained PB1 and NP genes from a C/Yamagata/26/81-like parent and the other genes from another as yet unidentified parent.

Although unspliced mRNA from influenza C virus RNA segment 6 (M gene) has a single open reading frame capable of encoding a 374-amino-acid protein (M r , 42,000), the major polypeptide synthesized from this mRNA species is the CM2 protein, with an M r of 18,000. The present study was performed to investigate the mechanism by which CM2 is generated from the unspliced mRNA. It was reported previously that the 374-amino-acid protein (P42) is an integral membrane protein having two internal hydrophobic domains, one of which (residues 241 to 252) is followed by two sequences (252 Ile-Thr-Ser and 257 Ala-Ser-Ala) favorable for cleavage by signal peptidase. To examine the possibility that P42 is cleaved by signal peptidase after Ser residue 254 or Ala residue 259 to yield CM2, we constructed three mutated M gene cDNAs in which either or both of the two sequences were eliminated and tested their ability to synthesize CM2 in the transfected COS cells. The results showed that CM2 synthesis was blocked completely when the second recognition motif for signal peptidase was removed. It was also found that when the mRNA transcript of the wild-type M gene was translated in vitro, P42, but not CM2, was synthesized in the absence of dog pancreas microsomal membranes, whereas CM2, in addition to a polypeptide (designated M1′) slightly larger than matrix protein (M1), was synthesized in the presence of microsomes. When the same experiment was done with the transcript of the mutated M gene in which the second recognition motif was removed, synthesis of CM2 could not be seen, even in the presence of microsomes. From these results, we conclude that cleavage of P42 by signal peptidase after Ala residue 259 produces CM2, composed of the C-terminal 115 amino acids, in addition to M1′, composed of the N-terminal 259 amino acids.

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.

The biochemical properties of a second protein (CM2) encoded by RNA segment 6 of influenza C virus were investigated. Three forms of CM2 with different electrophoretic mobilities (CM2(0), CM2a, and CM2b) were detected in infected cells by immunoprecipitation with antiserum to the glutathione S-transferase (GST)-CM2 fusion protein. Treatment of infected cells with tunicamycin and digestion of immunoprecipitated proteins with endoglycosidase H or peptide-N-glycosidase F suggested that a mannose-rich oligosaccharide core is added to unglycosylated CM2(0) (Mr, approximately 16,000) to form CM2a (Mr, approximately 18,000) and that the processing of the carbohydrate chain from the high-mannose type to the complex type converts CM2a into CM2b, which is heterogeneous in electrophoretic mobility (Mr, approximately 22,000 to 30,000). Labeling of infected cells with [3H]palmitic acid showed that CM2 is fatty acylated. The fatty acid bond was sensitive to treatment with hydroxylamine and mercaptoethanol, which indicates a labile thioester-type linkage. The CM2 protein was also found to form disulfide-linked dimers and tetramers on sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions. Trypsin treatment of infected cell surfaces as well as of microsome vesicles from infected cells followed by immunoprecipitation with antiserum to the GST fusion protein containing the 56 C-terminal amino acid residues of CM2 suggested that this C-terminal domain is intracellular and exposed to the cytoplasms of microsomes. Furthermore, evidence that a small amount of CM2 is incorporated into progeny virus particles was obtained by Western blot analysis. These results, altogether, suggest that CM2 is an integral membrane protein with biochemical properties similar to those of influenza A virus M2 and influenza B virus NB proteins.

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 micrograms/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.

Influenza C virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 6. Unspliced mRNA from this RNA segment, which has not been previously identified, can potentially encode a polypeptide that contains an additional 132 amino acids on the carboxy terminus of the M1 protein. Here the nucleotide sequences of RNA segment 6 of four influenza C strains, isolated in Japan between 1964 and 1988, were compared with the previously determined sequence of C/Ann Arbor/1/50. The results indicated that the deduced amino acid sequence of the carboxy-terminal 132 amino acid domain is conserved fairly well although it is more divergent than the M1 protein sequence. Examination of RNA segment 6-specific mRNAs also showed that unspliced mRNA is present, although in small quantities (approximately 13% of spliced mRNA), in influenza C virus-infected cells. To search for a polypeptide encoded by the unspliced mRNA, the extra carboxy-terminal domain was expressed in Escherichia coli as the glutathione S-transferase fusion protein, and rabbit immune serum was raised against the purified fusion protein. Immunoprecipitation experiments with this antiserum revealed that a previously unrecognized protein of apparent M(r) approximately 18,000, designated CM2, is synthesized in influenza C virus-infected cells.

Five strains of influenza C virus were isolated and passaged in the amniotic sacs of embryonated hens' eggs, or in the HMV-II line of human malignant melanoma cells, and were tested for reactivity with a panel of monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein. It was observed with two strains (C/Yamagata/4/88, C/Yamagata/7/88) that the HE of virus passaged in HMV-II cells was antigenically distinguishable from that of virus cultivated in eggs. Virus clones obtained after repeated passages of these two strains in HMV-II cells all showed a significant increase in the ability to replicate in the cell culture compared to clones derived from viruses grown in eggs. No difference was seen, by contrast, in the ability to grow in eggs between HMV-II- and egg-derived virus clones. It was also found that HMV-II-grown viruses but not egg-grown viruses could agglutinate glutaraldehyde-fixed chicken erythrocytes at 23°. These observations, taken together, suggest that isolation and passage of influenza C virus in HMV-II cells sometimes result in selection of antigenically distinct variants which have an advantage in binding to the cell surface receptors. Sequence analyses of the HE genes revealed that compared to egg-grown viruses, HMV-II-adapted variant of the Yamagata/4/88 strain had a single amino acid substitution in the HE molecule at position 283 (Asp → Asn) and that of the Yamagata/7/88 strain had two substitutions at positions 212 (Glu → Lys) and 519 (Asn → Asp).

SUMMARYEight strains of influenza C virus isolated in various areas of Japan between January 1985 and January 1989 were compared using monoclonal antibodies to the haemagglutinin-esterase (HE) glycoproteins and by oligonucleotide mapping of total vRNA. Five of six strains isolated during 1986–9 were closely related to one another and also resembled the virus, C/Aichi/1/81, isolated in 1981 in Aichi prefecture. This suggests that the C/Aichi/l/81-related viruses had an epidemiological advantage over any co-circulating viruses at least during that period. One of two 1985 isolates (C/Nara/1/85) was antigenically indistinguishable from the C/Mississippi/1/80 strain though their oligonucleotide patterns were markedly different from each other. This raises the possibility that C/Nara/1/85 may be a recombinant virus which receives its HE gene from the C/Mississippi/l/80-related parent.

It was previously shown that the shortest RNA of influenza C/California/78 virus contains 934 nucleotides and codes for two nonstructural proteins of 286 amino acids (NS 1) and 121 amino acids (NS 2). In this report, we determined the nucleotide sequence of the NS gene of the recently isolated influenza C/Yamagata/1/88 strain by using cloned cDNA derived from the viral RNA. Compared with the NS gene of C/California/78, one nucleotide insertion has occurred in the NS gene of C/Yamagata/1/88. This caused frame shifts of both the NS 1 and NS 2 reading frames, directing the synthesis of the NS 1 and NS 2 proteins consisting of 246 and 182 amino acids, respectively.

The effects of serum antibody on the replication of influenza C virus in the nose and lung were evaluated in rats challenged with the virus by the intranasal and endotracheal routes, respectively. Convalescent rat serum administered intraperitoneally prior to infection suppressed virus replication significantly in both the nasal and pulmonary tissues. Resistance achieved was however much greater in the lung than in the nose. Rats with a serum neutralizing antibody titer of 1:800 showed almost complete resistance to pulmonary virus infection, and virus yield from the lung was reduced 10- to 100-fold in animals with the antibody titer of 1:80-160 or less. In contrast, significant decrease in virus shedding from the nose was observed only in animals with a serum antibody titer of 1:800 or greater. The effect of adoptive transfer of monoclonal antibodies (MAbs) to haemagglutinin-esterase (HE) glycoprotein and matrix (M) protein on pulmonary virus replication was also examined. Anti-HE MAbs with neutralization activity prevented virus shedding from the lung almost completely whereas non-neutralizing anti-HE MAb and anti-M Mab showed no inhibitory effect.

The antigenic properties of influenza C viral glycoprotein gp88 were compared with those of its nonglycosylated counterpart T76 synthesized in infected cells treated with tunicamycin. Radioimmunoprecipitation experiments with three different monoclonal antibodies against gp88 revealed that an antibody designated Q-5 precipitated gp88 but not T76, indicating the requirement for glycosylation for the binding of this antibody to gp88. It is unlikely, however, that the antigenic determinant recognized by Q-5 is carbohydrate moiety since the ability of the antibody to bind to gp88 varied depending on the virus strain, and trypsin-treatment of gp88 eliminated its reactivity with Q-5. Gel electrophoretic analysis under nonreducing conditions showed that T76 underwent the formation of disulfide-linked multimers in the absence of reducing agent while gp88 behaved as monomers, suggesting that glycosylation is required for gp88 molecules to attain an appropriate conformation. These observations, altogether, suggests that glycosylation is important in determining the immunological specificity of gp88 presumably by influencing the folding of this glycoprotein.

The antigenicity of influenza C viral glycoprotein gp88 was compared with that of its non-glycosylated counterpart T76 by immunoprecipitation utilizing monoclonal antibodies against gp88. Of the three monoclonal antibodies tested, an antibody designated Q-5 was found to precipitate gp88 but not T76, indicating the requirement for glycosylation for the binding of Q-5 to gp88. However, the antigenic determination recognized by Q-5 did not appear to be carbohydrates since trypsin-treatment of gp88 eliminated its reactivity with this antibody. These results suggest that glycosylation is important in determining the antigenicity of gp88 presumably by influencing the folding of the glycoproteins.

To assess the functional importance of ribosomal protein rpA1 gene expression during development of Drosophila melanogaster, we have transformed into the fly's genome an antisense rpA1 gene driven by a heat shock promoter. Antisense rpA1 expression severely disrupted oogenesis and produced a "small egg" female-sterile phenotype. The severities of these defects were proportional to the level of antisense rpA1 expression. Anti-rpA1 expression did not affect larval or pupal development. Quantitative RNA analysis suggested that high anti-rpA1 expression results in a general decrease of mRNA in the ovary.