The Molecular Virology and Reverse Genetics of Influenza C Virus

Jpn. J. Infect. Dis., 63, 157­165, 2010

Review The Molecular Virology and Reverse Genetics of Influenza C Virus

Yasushi Muraki1,2* and Seiji Hongo2 1Department of Microbiology, Kanazawa Medical University School of Medicine, Ishikawa 920­0293; and 2Department of Infectious Diseases, Yamagata University Faculty of Medicine, Yamagata 990­9585, Japan (Received January 18, 2010. Accepted March 29, 2010) CONTENTS 1. Introduction 6. Analysis of the structure­function relationship of 2. Reverse genetics of influenza viruses the M1 protein 3. Molecular virology of influenza C virus 7. Comparison of the generation efficiencies of in­ 4. Epidemiology of influenza C virus fluenza C and A viruses 5. Generation of influenza C virus­like particles and a 8. Prospects for research on influenza C virology recombinant influenza C virus 9. Conclusion

SUMMARY: Influenza C virus, an enveloped virus containing seven single­stranded RNA segments of negative polarity, belongs to the genus Influenza C Virus of the family Orthomyxoviridae. A number of questions remain to be resolved with regard to the molecular virology and epidemiology of the virus. To address them, we have established a virus­like particle (VLP) generation system and reverse genetics of the virus and succeeded in clarifying the structure­function relationship of the M1 protein of the virus. Although the approach adopted was similar to that for influenza A virus reverse genetics, the number of infectious influenza C viruses generated was much lower than that for influenza A virus. Based on a comparison of the number of influenza C VLPs with that of influenza A VLPs generated using a similar system, we proposed a virion generation mechanism unique to influenza C virus.

for the generation of negative­sense RNA viruses, re­ 1. Introduction searchersfacedtheobstacleofprovidingtheviral Reverse genetics, as the term is used in molecular RNA (vRNA) with viral RNA polymerase and nucleo­ virology, describes the generation of viruses possessing protein. In the case of influenza A virus, the viral genome(s) derived from cloned cDNA(s). Of the viruses ribonucleoprotein (vRNP) complex, composed of belonging to the family Orthomyxoviridae,reverse three polymerase subunits (PB2, PB1, and PA), genetics has hitherto been reported for influenza A, in­ nucleoprotein (NP) and vRNA, is minimally required. fluenza B and Thogoto viruses. Recently, our research Initially, by transfecting an artificially reconstituted group, as well as Crescenzo­Chaigne and van der Werf, vRNP complex into eukaryotic cells followed by infec­ reported the successful reverse genetics of influenza C tion with an influenza helper virus, successful recovery virus. of a recombinant influenza virus containing a viral gene In this review, we will first provide an overview of segment derived from the cloned cDNA was reported research on reverse genetics of influenza A virus, and (1,2). then summarize the molecular virology and epidemiolo­ In 1996, Pleschka et al. reported the successful gener­ gy of influenza C virus, including a discussion of ation of a transfectant influenza virus (3). To synthesize presently unresolved issues of the virus. In the latter influenza vRNA in the nucleus, they made use of a part of this review, we will deal with a virus­like particle nucleolar enzyme, RNA polymerase I, in a system that (VLP) generation and reverse genetics of influenza C vi­ had been established by Zobel et al. (4). Cotransfection rus by our research group and provide a hypothesis to of the Pol Iplasmid encoding neuraminidase (NA) explain influenza C virion generation as observed in the vRNA together with viral protein­expressing plasmids established reverse­genetics system. for PB2, PB1, PA, and NP, followed by infection with an influenza helper virus, resulted in the recovery of a recombinant containing the NA gene of interest. Even 2. Reverse genetics of influenzaviruses in using this method, however, the helper virus­depen­ The genomes of negative­sense RNA viruses, includ­ dent system remained an obstacle to the efficient recov­ ing influenza viruses, are noninfectious. Therefore, ery of the recombinant virus. In 1999, a recombinant influenza A virus was report­ ed to be generated entirely from cloned cDNA for the *Corresponding author: Mailing address: Department of first time (5). The cDNAs of the eight vRNA segments Microbiology, Kanazawa Medical University School of of A/WSN/33 or A/PR/8/34 virus were each cloned Medicine, Uchinada, Ishikawa 920­0293, Japan. Tel: {81­ into the pHH21 vector in negative­sense orientation be­ 76­218­8097, Fax: {81­76­286­3961, E­mail: ymuraki— tween the RNA polymerase Ipromoter and terminator kanazawa­med.ac.jp sequences. The resulting eight plasmids were transfected

157 into 293T cells together with four (PB2, PB1, PA, and virus. Yamashita et al. cloned the three largest RNA NP) or nine (PB2, PB1, PA, HA, NP, NA, M1, M2, segments of C/JJ/50 (17). A comparison of the nucleo­ and NS2) viral protein­expressing plasmid DNAs. tide sequences of the segments with those of the influen­ Despite the need for the simultaneous transfection of as za A and B viruses revealed that RNA segments 1 and 2 many as 12 or 17 plasmids into the cells, approximately encode the proteins equivalent to PB2 and PB1 of the 106 to 107 infectious influenza A viruses/mL were pro­ influenza A and B viruses, respectively. The protein en­ duced at 48 to 72 h posttransfection. Thus, a recom­ coded by RNA segment 3 is referred to as P3 rather than binant influenza A virus in which all RNA segments PA, since it does not display any acid charge features at were derived from cDNA clones was generated. a neutral pH. Evidence has been obtained to suggest Improved approaches for the generation of the re­ that the transcription and replication of the influenza C combinant influenza A virus were later reported by virus genome follows the same strategy as that of in­ several research groups (6–8). Furthermore, influenza B fluenza A virus in that the three proteins form an RNA (9–11) and Thogoto viruses (12) were successfully gener­ ated in a similar way. Despite all these advances, however, no reverse genetics of influenza C virus was es­ tablished.

3. Molecular virology of influenza C virus Influenza C virus, which belongs to the genus In­ fluenza C Virus of the family Orthomyxoviridae,was first isolated from a patient with respiratory illness in 1947 (13). It is an enveloped virus containing seven sin­ gle­stranded RNA segments of negative polarity and en­ codes the following proteins: PB2, PB1, P3, hemag­ glutinin­esterase­fusion (HEF), NP, matrix (M1) pro­ tein, and CM2, as well as the non­structural proteins Fig. 1. Structure of influenza C virus. Influenza C virus has NS1 and NS2 (Figs. 1 and 2). The virus usually causes seven single­stranded RNA segments of negative polarity. The mild upper respiratory illness (14), but it can also cause viral ribonucleoprotein (vRNP) complex is composed of viral RNA (vRNA) and the PB2, PB1, P3, and NP proteins. HEF lower respiratory infections (15,16). forms a spike on the virion. M1 is present beneath the enve­ A number of research groups have studied the struc­ lope. CM2 is the second membrane protein of the virus. A ture and function of the genes and gene products of the small amount of NS2 is incorporated into the virions.

Fig. 2. Genome structure of influenza C/Ann Arbor/1/50 virus. RNA segments 1 (PB2 gene) to 7 (NS gene) are shown in positive­sense orientation. Numbers indicate the nucleotide positions along the genes. The lines at the 5? and 3? termini represent the noncoding regions. The V­shaped dotted lines in segments 6 and 7 indicate the intron. Viral proteins encoded by the genes are shown in boxes with the number of amino acids in parentheses. M1 is en­ coded by a spliced mRNA of RNA segment 6 (M gene), into which the TGA (stop codon) is introduced as a result of splicing. The P42 protein, encoded by a collinear transcript of the M gene, is cleaved by signal peptidase at an internal cleavage site (black triangle) to generate M1' and CM2. RNA segment 7 (NS gene) encodes the NS1 and NS2 proteins. NS1 is encoded by a collinear mRNA, and NS2 is encoded by a spliced mRNA. The C­terminal region of NS2 (gray area) contains {1 ORF. The C/Ann Arbor/1/50 strain contains a total of 12,906 nucleotides.

158 polymerase complex (18–20), although the precise the 374­amino­acid protein, P42, which is cleaved by role(s) of the respective proteins remains to be elucidat­ signal peptidase at an internal cleavage site to give M1' ed. and CM2 (43,44). The biochemical features of CM2, the RNA segment 4 encodes the HEF glycoprotein, which second membrane protein of the virus, are closely simi­ forms a spike on the virus envelope (Fig. 1). The HEF lar to those of M2 (45,46), a proton channel of influenza protein has three biological activities: receptor binding, A virus. Although CM2 expressed in Xenopus laevis oo­ receptor destroying (acetylesterase), and membrane cytes forms an ion channel permeable to Cl| (47) and fusing activities. The receptor of the virus is the 9­O­ CM2 has the ability to modulate the pH of the exocytic acetyl­N­acetylneuraminic acid (Neu5,9Ac2), and the pathway (48), the role of CM2 in the virus replication acetylesterase activity of HEF inactivates the virus cycle remains unclear. M1', composed of the N­terminal receptor by releasing the O­acetyl residues from the C­9 259aminoacidsofP42,isdegradedshortlyafter position of Neu5,9Ac2 (21–23). The fusion activity of cleavage through the signal located in the C­terminal 17­ HEF is dependent on the proteolytic cleavage of a amino acid region of the protein (49). The half­life of precursor (HEF0)intoHEF1 and HEF2, and it requires P42 is approximately 30 min (50), although a part of activation at a low pH upon internalization of the vi­ P42 is transported to the cis­Golgi apparatus (51). The ral particle through the endosome pathway (24,25). significance of M1' and P42 in the viral life cycle also Sugawara et al. produced 37 monoclonal antibodies remains to be determined. (MAbs) against HEF and indicated the presence of nine Analysis of the NS gene of the C/California/78 strain antigenicsites(A­1toA­5andB­1toB­4)onthemole­ initially showed that the gene contained 934 nucleotides cule (26). The amino acid(s) recognized by the MAbs and was capable of encoding both the 286­amino­acid were identified by isolating the escape mutants of the NS1 and 121­amino­acid NS2 proteins (52,53). Based on MAbs directed against A­1 to A­4 (27). In 1998, Rosen­ a comparison of the nucleotide sequences from a larger thal et al. revealed the three­dimensional structure of number of influenza C virus isolates, including C/ HEF and showed that the three biological functions California/78, the gene was later demonstrated to were attributable to distinct domains on the molecule potentially encode NS1 (246 amino acids) from un­ (28). Together with a report by Matsuzaki et al. (27), spliced mRNA and NS2 (182 amino acids) from spliced this demonstrated that the antigenic sites A­1, A­2, and mRNA (54). In fact, the 246­amino­acid NS1 and 182­ A­4 were located on the receptor­binding domain and amino­acid NS2 proteins were identified in virus­infect­ A­2 on the acetylesterase domain, indicating that, like ed cells (54,55). The involvement of NS1 in persistent in­ the influenza A virus hemagglutinin (HA), the main an­ fection (55) and viral mRNA splicing (56) was also tigenic region of HEF is located on the globular head of reported. Like the influenza A virus NS2/NEP, the in­ the molecule. fluenza C virus NS2 possesses nuclear export activity HEF has a number of unique characteristics that are and is incorporated into virions (57,58). different from those of the influenza A virus HA. There Crescenzo­Chaigne et al. investigated the functions of are only three amino acids in the cytoplasmic region of the NCR of an influenza C virus RNA segment (19,20). HEF, whereas the corresponding region of HA contains In their study, the model­type RNA flanked by NCR of 11 to 12 amino acids. The short cytoplasmic region may the C/Johannesburg/1/66 strain NS gene was expressed or may not affect the transportation of HEF to the cell in COS–1 cells from the Pol I plasmid­based system, surface (29–32). The fatty acid attached to the cysteine together with the PB2, PB1, P3, and NP proteins, and at residue 642 of HEF is a stearic acid (33), whereas the RNA template was shown to be transcribed and palmitic acid is mainly attached to the influenza A virus replicated. Thus, the involvement of the NCR of the in­ HA (34). Whether these facts are significant to the in­ fluenza C viral genome in its replication and transcrip­ fluenza C virus replication remains to be elucidated. tion was demonstrated. Nakada et al. determined the entire sequence of RNA segment 5 of C/California/78 and suggested that the 4. Epidemiology of influenza C virus segment codes for NP (35). Sugawara et al. constructed a panel of MAbs against NP and reported that (i) there The epidemiology of influenza C virus has also been are at least two antigenic sites (I and II) on the molecule, extensively investigated. In the 1980s, on the basis of the (ii) the localization of NP in virus­infected cells is con­ results obtained from a limited number of influenza C sistent with that of the influenza A virus NP, and (iii) virus isolates, the virus was considered to be antigenical­ NP exhibits molecular maturation during transport to ly stable, divided into several antigenic groups, and the nucleus in virus­infected cells (36,37). NP was also evolved more slowly than influenza A virus (59–61). shown to be essential to the transcription/replication Moriuchi et al. developed a tissue culture method for processes of an artificial vRNA flanked by the noncod­ primary virus isolation that is convenient for routine ing regions (NCRs) of C/Johannesburg/1/66 (19,20). work with a large number of clinical specimens (15,62), Thus, although the features of the NP protein clarified and they then initiated surveillance for influenza C virus to date are consistent with those of the influenza A virus infection in Sendai and Yamagata Cities in Japan. The NP protein, the functional domain(s) of NP remains to established system has enabled us to systematically ana­ be analyzed. lyze influenza C virus epidemiology and thereby find The RNA segments 6 and 7 are bicistronic genes (Fig. that (i) influenza C virus is divided into six antigenic and 2). The spliced mRNA of the RNA segment 6 encodes genetic groups (Taylor/1233/47­, Aichi/1/81­, Sao M1 (38). M1 gives rigidity to the virion and is involved Paulo/378/82­, Kanagawa/1/76­, Yamagata/26/81­, in the budding and morphogenesis processes of the virus and Mississippi/80­related lineages) (Fig. 3) (63,64), (ii) (39–42). The unspliced mRNA of the segment encodes the evolutionary rate of the virus is lower than that of

159 Fig. 3. Phylogenetic tree of influenza C virus HEF genes. The region from nucleotides 64 to 1,989 of the HEF gene was analyzed. Horizontal distances are proportional to the minimum number of nucleotide differences needed to join the sequences. Numbers above the branches are the bootstrap probabilities (z) of each branch. A total of 74 strains were divided into six lineages as indicated on the right of the figure (adapted from reference 64 with permis­ sion; kindly provided by Yoko Matsuzaki). influenza A virus (63,65); e.g., the rate of the HEF gene advantage in spreading among the human population is 0.49 ~ 10|3 nucleotides per site per year, which is (54,64,68,69). only one­ninth of that of the influenza A virus HA gene, There is evidence that influenza C virus has the poten­ (iii) viruses belonging to different genetic and antigenic tial to infect animals, as the presence of specific anti­ groups cocirculate within a limited geographical area bodies was demonstrated in the serum of pigs (70,71) during a given period, and reassortment events between and dogs (72–74). Furthermore, a number of influenza different viruses frequently occur (64,66,67), and (iv) C viruses were isolated from abattoir pigs in Beijing, viruses containing a proper combination of genetic China, and pig­to­pig transmission of the virus on ex­ elements through reassortment events may have an perimental infection has been demonstrated (75). Hu­

160 man influenza C virus isolates were found to be geneti­ cally and antigenically related to pig isolates, suggesting that interspecies transmission between humans and pigs has occurred in nature (76). Ohwada et al. showed that experimental infection resulted in clinical symptoms and viral replication in dogs (77). The potential role of animals as a reservoir for human influenza C remains to be elucidated, although influenza C viruses seem to be maintained among the human population.

5. Generation of influenza C virus­like particles and a recombinant influenza C virus For a number of negative­sense RNA viruses, the es­ tablishment of a reverse genetics has been preceded by the construction of a mini­replicon system. Therefore, we attempted to establish an influenza C VLP genera­ tion system, as the packaging of the artificial genome into influenza C virions and gene transfer to susceptible cells by VLPs had not yet been reported at that time. The cDNA of the green fluorescent protein (GFP) gene flanked by the NCR sequences of RNA segment 5 (NP gene) of C/Ann Arbor/1/50 was cloned into a pHH21 vector in an anti­sense orientation between the Pol I promoter and the terminator sequences so that the artificial RNA (GFP­vRNA) was expressed under the control of the human Pol I promoter. The resulting plasmid DNA, pPolI/NP­AA.GFP(|), was transfect­ ed into 293T cells, a human embryonic kidney cell line Fig. 4. Reverse genetics of influenza C virus. Seven Pol I plas­ mids for the expression of vRNAs were transfected into 293T constitutively expressing simian virus 40 large T antigen, cells together with viral protein­expressing plasmids for PB2, together with viral protein­expressing plasmids for PB2, PB1,P3,HEF,NP,M1,CM2,NS1,andNS2.Recombinant PB1, P3, HEF, NP, M1, CM2, NS1, and NS2, and in­ influenza C viruses produced from the transfected 293T cells cubated for up to 72 h. Using electron microscopy, we were then titrated and propagated in the amniotic cavity of em­ confirmed that the supernatant of the 293T cells con­ bryonated chicken eggs. tained influenza C VLPs, and, by the quantification of GFP­positive HMV­II cells infected with the VLPs and helper virus (C/Ann Arbor/1/50), we determined that expression in the transfected 293T cells was found to be the number of VLPs generated was approximately 106/ required for efficient gene transfer to HMV­II cells (84). mL (41). On the other hand, even without NS1 expression, in­ The findings by several groups suggest that influenza fluenza A VLPs were successfully generated and found C virus proteins function more efficiently at 339Cthan capable of efficiently transmitting the synthetic reporter at 379C. Nagele and Meier­Ewert showed that the maxi­ RNA to susceptible cells (85). This discrepancy may sug­ mum RNA polymerase activity of influenza C virus was gest that the influenza C virus NS1 has a unique fun­ detected at 339C (18). We also observed that, in the Pol ction(s) in the virus replication cycle that differs from I­based system, a higher amount of luciferase was ex­ those reported for influenza A virus. pressed at 339Cthanat379C (data not shown). There­ To establish the reverse genetics of influenza C virus, fore, our experiment was carried out at 339C, not 379C, we constructed Pol I plasmid DNAs for the seven RNA including incubation of the transfected 293T cells and segments of C/Ann Arbor/1/50, a prototype strain of infection of the HMV­II cells with the VLPs (41). influenza C virus. The resulting Pol I plasmids were ``Cap snatching'' is a unique feature of the influenza then transfected into 293T cells, together with four A virus transcription initiation process, and the roles of (PB2, PB1, P3, and NP) or nine (PB2, PB1, P3, HEF, the three RNA polymerase subunits involved in that NP, M1, CM2, NS1, and NS2) viral protein­expressing process have been extensively analyzed (78–83). In our plasmids (Fig. 4). The resultant supernatant was deter­ 1 3 study, in determining the sequence of the 3? end of the mined to contain 10 to 10 EID50/mL of infectious NP gene, we found that 12 nucleotides were added to recombinant viruses (42), the titer of which was much the 5? end of the NP gene mRNA (unpublished results). lower than that of influenza A virus (106 to 107 Similar results were obtained for the other six gene seg­ PFU/mL) (see below). ments (data not shown, see below). These findings pro­ Crescenzo­Chaigne and van der Werf reported the vide evidence that the cap snatching machinery by in­ reverse genetics of influenza C virus using a similar ap­ fluenza C virus polymerases is in fact activated in a simi­ proach by which, at 10 days posttransfection, they ob­ lar manner as for influenza A virus, although the roles tained 104 PFU/mL of the recombinant virus (86). This of the respective polymerase subunits in the cap snatch­ finding suggests that, as in our observation, a limited ing process remain to be clarified. number of infectious recombinants existed in the super­ In the influenza C VLP generation experiment, NS1 natant of the transfected 293T cells. Thus helper virus

161 independent­reverse genetics has currently become cy between the HA and infectious virus titers could be available for application to influenza C virology, there­ explained by the presence of large and limited numbers by affording the opportunity to resolve a number of of noninfectious and infectious virus particles, respec­ questions regarding the virus. At the moment, however, tively, in the supernatant of the plasmid­transfected wemustadmitthatthecurrentsystemhasadisadvan­ 293T cells. tage in that recombinants with severe growth defect(s) Neumann et al. reported that in the plasmid­driven may not be obtained due to the low generation efficien­ reverse­genetics system, one in 102.8 to 103.3 293 T cells cy, a problem that needs to be overcome. produce the recombinant infectious influenza A virus (5). This means that approximately one in 1,000 cells ex­ presses one set (eight segments) of vRNA, leading to the 6. Analysis of the structure­function relationship generation of infectious influenza A viruses, and that of the M1 protein the remaining 999 in 1,000 cells express less than seven Nishimura et al. reported that cord­like structures vRNA segments. This suggestion seems to apply in the (CLSs), composed of numerous filamentous particles in case of influenza C virus reverse genetics: one in 1,000 the process of budding, were found to extrude from cells expresses one set (seven segments) of vRNA, and C/Yamagata/1/88­infected HMV­II cells (39). Each of the remaining 999 cells express fewer than six RNA seg­ these particles was covered with a layer of surface ments. Therefore, the above­mentioned discrepancy be­ projections and aggregated with their long axes. Further tween the HA and infectious virus titers in the super­ analysis of a series of reassortant viruses between natant suggests that the remaining 999 cells efficiently C/Yamagata/1/88 and C/Taylor/1233/47, the latter of produce noninfectious particles. which is a unique strain incapable of forming CLS, The hypothesis that the 293T cells expressing less than showed that reassortants with the M gene from C/Tay­ six RNA segments efficiently produce influenza C parti­ lor/1233/47 could not form CLS on infected cells (40). cles is supported by the previous observations that (i) Upon establishment of the influenza C­VLP genera­ interaction of HEF with M1 may be sufficient to lead to tion system, we identified CLSs extruding from the budding at the cell surface (39, our unpublished result) VLP­generating 293T cells. By expressing a series of M1 and (ii) nucleocapsids may not be required to initiate the mutants in the 293T cells together with the other plas­ budding process of the virus (40,88). In other words, in mids required for VLP generation, we demonstrated the case of influenza C virion formation, coexpression that the M1 protein is a determinant for CLS formation of M1 and HEF in a transfected cell, even if the ratio of and residue 24 of M1 (Ala or Thr) is responsible for M1 to HEF in the cell is different from that in virus­in­ CLS formation as well as VLP morphology (filamen­ fected cells, may readily lead to particle formation tous or spherical) (41). Furthermore, the generation of regardless of the presence or absence of nucleocapsids. an infectious recombinant virus with an M1 mutation Furthermore, it is possible that inefficient interaction of revealed that residue 24 of the M1 protein (Ala or Thr) M1 with nucleocapsid (39) facilitates the production of also affects CLS formation on the infected cells and noninfectious particles. Alternatively, as a driving force virion morphology (filamentous or spherical). Mem­ for virion formation, the influenza C virus M1 protein brane flotation analysis of recombinant virus­infected may function more efficiently than the influenza A virus cells revealed that the wild­type M1 protein (possessing M1 protein. Ala at residue 24) showed higher affinity to the plasma ThereisasignificantdifferencebetweentheVLP membrane than mutant M1 (possessing Thr at residue generation efficiencies of influenza C (106/mL) and A 24), suggesting that an amino acid on M1 affects the (104/mL) viruses (41,87). A comparison of the number virion morphology through the membrane affinity of of VLPs with that of infectious virus particles between M1 to the plasma membrane (42). Thus, using the re­ influenza A and C viruses suggests that in the case of in­ verse­genetics system of influenza C virus, we could fluenza C virus generation, cells expressing only one demonstrate the structure­function relationship of the M1 protein in the context of viral replication.

7. Comparison of the generation efficiencies of influenza C and A viruses Initially, we expected that the adoption of a similar approach would allow the recombinant influenza C virus to be generated as efficiently as influenza A virus (106 to 107 PFL/mL), since (i) the number of RNA seg­ ments in influenza C virus is seven, which is smaller by Fig. 5. Hypothesized virion generation mechanism for influenza one segment than that in influenza A virus, and (ii) the C and A viruses. 293T cells transfected with plasmid DNAs for reverse genetics are divided into seven (influenza C virus) or number of influenza C VLPs generated using a similar eight (influenza A virus) groups according to the number of system was approximately 106/mL, which is 100­fold vRNAs expressed in each cell. The length of the white (influen­ higher than that of influenza A VLPs (104/mL) (41,87). za C virus) and black (influenza A virus) arrows indicates the In fact, the supernatant of the plasmid­transfected 293T generation efficiencies of the viruses from the respective cells. 5 In the case of influenza C virus, 293T cells expressing one RNA cells showed 4 HAU/mL, a titer corresponding to 10 segment are the most efficient in the production of virions. In PFU/mL of the egg­grown influenza C virus (data not contrast, for influenza A virus, cells expressing one set (eight shown). However, the supernatant contained only 101 to segments) of vRNA are the most efficient in the production of 3 infectious virions. 10 EID50/mL of infectious viruses (42). The discrepan­

162 RNA segment may be the most efficient in the produc­ questions regarding influenza C molecular virology and tion of virus particles. Based on these findings, we epidemiology, particularly those associated with the formed a hypothesis explaining the generation of in­ type­specificity requirements of influenza viruses. fluenza C virus, which is shown in Fig. 5. In the case of influenza A virus, 293T cells expressing one set (eight Acknowledgments We thank Drs. Y. Kawaoka and H. Goto for segments) of vRNA produce infectious virions most ef­ providing plasmid DNAs and for their helpful comments. We also ficiently. This is consistent with the results reported by thank Drs. K. Sugawara, Y. Matsuzaki and E. Takashita for their ex­ Fujii et al., in which cells expressing seven and six seg­ cellent advice in establishing the reverse genetics of influenza C virus. ments are less efficient in the production of influenza A viruses (89). 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