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Unfortunately, the inclusion of original names (in non-Latin script) of the following authors caused problems with author name indexing in PubMed. Therefore, these original names were removed from XML data to correct the PubMed record. Mengji Cao, Yuya Chiaki, Hideki Ebihara, Jingjing Fu, George Fú Gāo, Tong Han, Jiang Hong, Ni Hong, Seiji Hongo, Masayuki Horie, Dàohóng Jiāng, Fujio Kadono, Hideki Kondō, Kenji Kubota, Shaorong Li, Longhui Li, Jiànróng Lǐ, Huazhen Liu, Tomohide Natsuaki, Sergey V. Netesov, Anna Papa, Sofia Paraskevopoulou, Liying Qi, Takahide Sasaya, Mang Shi, Xiǎohóng Shí, Zhènglì Shí, Yoshifumi Shimomoto, Jin‑Won Song, Ayato Takada, Shigeharu Takeuchi, Yasuhiro Tomitaka, Keizō Tomonaga, Shinya Tsuda, Changchun Tu, Tomio Usugi, Nikos Vasilakis, Jiro Wada, Lin‑Fa Wang, Guoping Wang, Yanxiang Wang, Yaqin Wang, Tàiyún Wèi, Shaohua Wen, Jiangxiang Wu, Lei Xu, Hironobu Yanagisawa, Caixia Yang, Zuokun Yang, Lifeng Zhai, Yong‑Zhen Zhang, Song Zhang, Jinguo Zhang, Zhe Zhang, Xueping Zhou. In addition, the publication call-out in the supplementary material was updated from issue 11 to issue 12. The original article has been corrected.

Enterovirus 71 infections have become a major public issue in the Asia-Pacific region due to the large number of fatal cases. To clarify the longitudinal molecular epidemiology of enterovirus 71 (EV71) in a community, we isolated 240 strains from children, mainly with hand-foot-and-mouth diseases, between 1990 and 2013 in Yamagata, Japan. We carried out a sequence analysis of the VP1 region (891 bp) using 223 isolates and identified six subgenogroups (B2, B4, B5, C1, C2 and C4) during the study period. Subgenogroups C1 and B2 were found only between 1990 and 1993 and have not reappeared since. In contrast, strains in subgenogroups C2, C4 and B5 appeared repeatedly with genomic variations. Recent reports from several local communities in Japan have suggested that identical predominant subgenogroup strains, which have also been found in the Asia-Pacific region, have been circulating in a wide area in Japan. However, it is likely that there is a discrepancy between the major subgenogroups circulating in the Asia-Pacific region and those in Europe. It is necessary to continue the analysis of the longitudinal epidemiology of EV71 in local communities, as well as on regional and global levels, to develop strategies against severe EV71 infections.

Influenza C virus replicates more efficiently at 33°C than at 37°C. To determine whether hemagglutinin-esterase-fusion protein (HEF), a surface glycoprotein of influenza C virus, is a restricting factor for this temperature sensitivity, we analyzed the biological and biochemical properties of HEF at 33°C and 37°C. We found that HEF exhibits intrinsic temperature sensitivities for surface expression and fusion activity.

CM2 is the second membrane protein of influenza C virus. Although its biochemical characteristics, coding strategy, and properties as an ion channel have been extensively studied, the role(s) of CM2 in the virus replication cycle remains to be clarified. In order to elucidate this role, in the present study we generated CM2-deficient influenza C virus-like particles (VLPs) and examined the VLP-producing 293T cells, VLPs, and VLP-infected HMV-II cells. Quantification of viral RNA (vRNA) in the VLPs by real-time PCR revealed that the CM2-deficient VLPs contain approximately one-third of the vRNA found in wild-type VLPs although no significant differences were detected in the expression levels of viral components in VLP-producing cells or in the number and morphology of the generated VLPs. This finding suggests that CM2 is involved in the genome packaging process into VLPs. Furthermore, HMV-II cells infected with CM2-deficient VLPs exhibited significantly reduced reporter gene expression. Although CM2-deficient VLPs could be internalized into HMV-II cells as efficiently as wild-type VLPs, a smaller amount of vRNA was detected in the nuclear fraction of CM2-deficient VLP-infected cells than in that of wild-type VLP-infected cells, suggesting that the uncoating process of the CM2-deficient VLPs in the infected cells did not proceed in an appropriate manner. Taken together, the data obtained in the present study indicate that CM2 has a potential role in the genome packaging and uncoating processes of the virus replication cycle.

The purpose of this study was to evaluate the frequency of human metapneumovirus (hMPV) infection in adults and to determine the association between the levels of serum antibody titers and the susceptibility to reinfection. Serum samples collected at the periodic occupational medical checkup for employees of a hospital were subjected to an ELISA test. Of the 289 subjects, 288 (99.7%) had hMPV antibody titers that were more than 1:100 in May 2006. The percentage of subjects with a titer of ≥ 3,200 was significantly higher in adults aged 40-65 years old (30.2-31.5%) compared to young adults 20-39 years old (13.6%) (P < 0.05). To investigate the longitudinal course of the hMPV antibody titer, a total of 649 serum samples collected from 59 subjects who had participated in all biannual medical checkups between 2001 and 2006 were tested. We found that ten serum pairs showed a greater than fourfold increase in hMPV antibody titers. Additionally, the 5-year reinfection rate was estimated at 16.9% (10 of 59 subjects). The baseline titer before the fourfold increase ranged from 1:100 to 1:3,200, and the titer returned to baseline levels 2 or 3 years after the fourfold increase. The antibody titer of the person with the baseline titer of 1:100 showed a greater than fourfold increase twice within a year. Sixty to 80% of adults had an ELISA titer of 1:800 to 1:1,600, suggesting that such an antibody titer is not enough to protect from hMPV infection and that reinfection could occur among adults.

RNA segment 7 of influenza C virus encodes two non-structural (NS) proteins, NS1 and NS2. The influenza C virus NS2 protein has been proposed to possess nuclear export activity like that of influenza A and B virus NS2 proteins (NEP). In the present study, we investigated the kinetics and localization of the NS2 protein in influenza C virus-infected cells, and analysed whether NS2 is present in virions. Immunofluorescent staining analysis of the infected cells indicated that NS2 was localized in the nucleus immediately after synthesis and predominantly in the cytoplasm in the later stages of infection. Confocal microscopy revealed that a part of the NS2 protein was colocalized with nucleoprotein NP/vRNP in the cytoplasm and on the cell membrane in the late stages of infection. The NS2 protein was detected in influenza C virions purified by gradient centrifugations and/or affinity chromatography. Trypsin treatment demonstrated that the NS2 protein was present inside the viral envelope. Furthermore, glycerol gradient analysis of detergent-solubilized virions revealed that the NS2 protein cosedimented with vRNPs. These data suggest that the influenza C virus NS2 protein is incorporated into virions, where it associates with vRNP.

Reverse genetics has been documented for influenza A, B, and Thogoto viruses belonging to the family Orthomyxoviridae . We report here the reverse genetics of influenza C virus, another member of this family. The seven viral RNA (vRNA) segments of C/Ann Arbor/1/50 were expressed in 293T cells from cloned cDNAs, together with nine influenza C virus proteins. At 48 h posttransfection, the infectious titer of the culture supernatant was determined to be 2.51 × 10 3 50% egg infectious doses/ml, which is lower than the number of influenza C virus-like particles (VLPs) (10 6 /ml) generated using the same system. By generating influenza C VLPs containing a given vRNA segment, we showed that each of the vRNA segments was similarly synthesized in the plasmid-transfected cells but that some segments were less efficiently incorporated into the VLPs. This finding leads us to speculate that the differences in incorporation efficiency into VLPs between segments might be a reason for the inefficient production of infectious viruses. Second, we generated a mutant recombinant virus, rMG96A, which possesses an Ala→Thr mutation at residue 24 of the M1 protein, a substitution demonstrated to be involved in the morphology (filamentous or spherical) of the influenza C VLPs. As expected, rMG96A exhibited a spherical morphology, whereas recombinant wild-type of C/Ann Arbor/1/50, rWT, exhibited a mainly filamentous morphology. Membrane flotation analysis of the cells infected with rWT or rMG96A revealed a difference in the ratio of membrane-associated M1 proteins, suggesting that the affinity of M1 protein to the cell membrane is a determinant for virion morphology.

A number of epidemiological studies have shown human metapneumovirus (hMPV) to be one of the most important viral agents associated with acute respiratory infections in humans. However, due to the difficulty in growing the virus, all epidemiological studies of hMPV infection have been performed on the basis of the molecular method. Thus, the development of a cell line suitable for the isolation of hMPV from clinical specimens is a crucial step for further research. Using the Vero E6 cell line, which could be stably maintained for 1 month without passage or medium change, we succeeded in isolating 79 strains from 4,112 specimens obtained in Yamagata, Japan, in 2004 and 2005. The total isolation rate was 1.9% (79/4,112). The monthly distribution revealed that hMPV infections occurred between February and April in 2004 and throughout most of the year in 2005. Phylogenetic analysis indicated that subgenogroup B2 was predominant in 2004, whereas three subgenogroups, A2, B1, and B2, had cocirculated in 2005. Although multiple subgenogroups cocirculated in 2005, each individual subgenogroup strain was found to predominate at specific sites. An infectivity assay of hMPV strains also indicated that the infection efficiency in Vero E6 cells was better than that in LLC-MK2 cells. Finally, we found that Vero E6 cells are useful for the isolation of hMPVs and that this utility might aid further research into hMPVs beyond the epidemiological data shown in this study.

During the period from January to July 2004, a total of 131 influenza C viruses were detected by cell culture or reverse transcription-PCR (RT-PCR) from specimens that were obtained from children with acute respiratory symptoms in 10 prefectures across Japan. Influenza C virus was identified most frequently in the Miyagi (1.4%, 45 of 3,226 specimens) and Yamagata (2.5%, 31 of 1,263 specimens) prefectures, and the frequency in this year was the highest since 1990. Phylogenetic analysis of the hemagglutinin esterase gene of the 13 strains isolated in nine prefectures revealed that genetically similar strains belonging to the Kanagawa/1/76-related lineage dominantly spread throughout Japan. During the 2004 influenza season, influenza C virus coexisted with epidemics of influenza A virus (H3 strain), and 12 cases were identified from patients who had been diagnosed with influenza-like illness (7 were detected by RT-PCR, and 5 were detected by culture). A comparison of specimens that were found positive by culture with those found positive only by RT-PCR shows that the amount of virus in PCR-positive specimens tended to be lower than in isolation-positive specimens. Although the mean peak temperature in patients in the PCR-positive group was slightly lower, there were no significant differences in characteristics between specimens (i.e., kind of specimen, period from onset to specimen collection, age distribution of patients, and severity of illness). These results suggest that an epidemic of influenza C virus occurred on a national scale during this period and that RT-PCR can be an effective supplemental tool for the evaluation of clinical and epidemiological information.

To characterize the genetic diversity of influenza B viruses isolated during one influenza season, the antigenic and genetic relationships among 20 strains of influenza B virus isolated in February and March 2001 at one pediatric clinic in Yamagata City, Japan, were investigated. The HA gene and seven other gene segments were phylogenetically divided into three distinct sublineages (Harbin/7/94-, Tokyo/6/98-, and Shiga/T30/98-related lineage) of the Yamagata/16/88-like lineage. The NS genes of the viruses belonging to the Harbin/7/94-related lineage have additional three nucleotides at positions 439–447, and were phylogenetically distinguishable from those of the currently circulating Yamagata/16/88- and Victoria/2/87-like lineages, but were closely related to that of the Yamagata/16/88-like lineage isolated before 1994. Moreover, four strains of influenza B virus isolated in the same community between 2002 and 2003 were further examined. Phylogenetic analysis revealed that a virus of Victoria/2/87-like lineage isolated in 2003 had acquired the NA, NS, M, and PA gene segments from a Shiga/T30/98-like virus, and two strains of Harbin/7/94-related lineage had acquired the various gene segments from Shiga/T30/98-like virus through a reassortment event. These results indicate that genetically distinct multiple viruses can combine to cause an influenza B epidemic in a community and that the frequent reassortment among these viruses plays a role in generating the genetic diversity of influenza B viruses. J. Med. Virol. 74:132–140, 2004. © 2004 Wiley-Liss, Inc.

An influenza C virus was isolated from a Japanese traveler who had visited Malaysia in April 1999. Phylogenetic analysis indicated that the genome composition of this virus was distinct from that of any other strain isolated in Japan. The possibility that a genetically unique influenza C virus was introduced into Japan by a traveler is shown.

The antigenic and genetic relationships among 15 strains of influenza C virus isolated between August, 1988, and May, 1990, in Yamagata City, Japan, were investigated. Based on the results of antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoproteins and oligonucleotide mapping of total vRNA, the isolates were divided into two distinct groups closely related to either C/Yamagata/26/81 or C/Aichi/1/81. Antigenic differences between two groups could be detected clearly with heterogeneous antiviral sera. Comparison of the HE gene sequences between the representative Yamagata isolates and the previous isolates from other countries showed that C/Yamagata/26/81-like and C/Aichi/1/81-like viruses had high degrees of nucleotide sequence homology with C/pig/Beijing/115/81 and C/Johannesburg/1/66, respectively. These observations suggest that two lineages of influenza C virus, markedly different from each other in both antigenic and genetic structures, were simultaneously present in Yamagata City during 1988-1990 and that viruses belonging to each of the two lineages may also be prevalent in other areas of Japan and even in other countries.

We observed previously that cord-like structures which had lengths up to 500 μm or greater were protruding from the surface of HMV-II cells infected with influenza C/Yamagata/1/88 virus (Nishimura et al., 1990, Virology 179, 179-188). Comparison of the cord-forming ability among a number of influenza C isolates revealed that the C/Taylor/1233/47 strain was unique in lacking the ability. It was also found that the major structural proteins, hemagglutinin-esterase (HE), nucleoprotein (NP), and matrix (M), could each be distinguished between C/Yamagata/1/88 and C/Taylor/1233/47 viruses by SDS-polyacrylamide gel electrophoresis. To determine the genes involved in the cord formation, a series of reassortant viruses were prepared between C/Yamagata/1/88 and C/Taylor/1233/47, and parental derivation of genes coding for HE, NP, and M was determined by gel electrophoresis. All reassortants which derived M gene from C/Yamagata/1/88, irrespective of derivation of genes coding for HE and NP, had the ability to generate cords, whereas none of reassortants which derived M gene from C/Taylor/1233/47 were capable of producing cords. With respect to several representative reassortants, the origins of genes encoding the polymerase proteins (PB2, PB1, P3) and the nonstructural proteins (NS1, NS2) were determined by T1-oligonucleotide fingerprinting of the isolated RNA segments. The results suggested that none of genes coding for these proteins are exclusively associated with the cord formation. Thus, it is likely that the M gene is the key determinant of the cord-forming ability of influenza C viruses. Nucleotide sequence analysis of the M genes revealed that compared to C/Yamagata/1/88, C/Taylor/1233/47 had two amino acid substitutions in the M molecule at positions 24 (Ala → Thr) and 133 (Asp → Asn).

Four different antigenic sites (A-1, A-2, B-1, B-2) have been identified previously on the haemagglutinin-esterase (HE) glycoprotein of influenza C/Ann Arbor/1/50 virus with seven monoclonal antibodies (MAbs). In this study we produced 30 additional anti-HE MAbs, nine of which demonstrated at least one of the following activities: haemagglutination inhibition, receptor-destroying enzyme inhibition, haemolysis inhibition, and neutralization (group A). The remaining had none of these activities (group B). These antibodies, and those previously isolated, were used to construct a more complete antigenic map of the HE molecule. Operational and topological analyses showed that a minimum of nine non-overlapping or partially overlapping antigenic sites were present on the HE protein, five recognized by group A MAbs (A-1 to A-5) and four by group B (B-1 to B-4). Among these antigenic sites, site A-5 was unique in that MAbs to this site inhibited the receptor-destroying activity without influencing the receptor-binding activity, which supports the idea that the sites responsible for these two functions are separate. It was also found that several group B MAbs were cross-reactive with host cell antigens.

To clarify a longitudinal epidemiology, we isolated 280 hMPV strains from patients with acute respiratory infections in Yamagata, Japan, between 2004 and 2009. We observed that the high season for hMPV was from winter to spring (between January and May) and the low season was in the fall (around September and October). A further molecular analysis revealed that subgenogroup A2 (A2) strains were the most commonly isolated (151/280; 53.9%), followed by B2 (108/280; 38.6%) and B1 (19/280; 6.8%). Our results suggested that A2 and B2 have been endemically in circulation as the major types almost every year, whereas other subgenogroups have appeared less frequently.

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.

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.

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.

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.

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.

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 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.

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.