• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Nov 15, 2005; 102(46): 16825–16829.
Published online Nov 2, 2005. doi:  10.1073/pnas.0505587102
PMCID: PMC1283806
Microbiology

An improved reverse genetics system for influenza A virus generation and its implications for vaccine production

Abstract

The generation of vaccines for highly pathogenic avian influenza viruses, including those of the H5N1 subtype, relies on reverse genetics, which allows the production of influenza viruses from cloned cDNA. In the future, reverse genetics will likely be the method of choice for the generation of conventional influenza vaccine strains because gene reassortment by more traditional methods is cumbersome. Established systems for the artificial generation of influenza A viruses require transfection of cells with the eight to 12 plasmids that provide the eight influenza viral RNAs as well as the polymerase and nucleoproteins of the virus. However, cell lines appropriate for human vaccine production (e.g., Vero cells) cannot be transfected with high efficiencies. To overcome these problems, we established a reverse genetics system in which the eight RNA polymerase I transcription cassettes for viral RNA synthesis are combined on one plasmid. Similarly, two cassettes encoding the hemagglutinin and neuraminidase segments and six cassettes encoding the remaining proteins were combined. We also combined three RNA polymerase II transcription cassettes for the expression of the polymerase subunits. By combining these cassettes, we reduced the number of plasmids required for virus generation significantly and produced influenza A virus in Vero cells with higher efficiency than with the traditional 12 plasmid system. This new system is thus suitable for influenza virus vaccine production and may be applicable to other reverse genetics systems that rely on the introduction of several plasmids into eukaryotic cells.

Keywords: RNA polymerase I

Influenza epidemics and pandemics continue to claim human lives and impact the global economy. In the United States alone, influenza causes an estimated 50,000 deaths annually (1), whereas global pandemics can result in millions of influenza-related deaths. A classic example is the so-called “Spanish influenza,” which killed an estimated 40-50 million people worldwide in 1918-1919 (2). The threat imposed by influenza virus has been further elevated with the recent introductions of avian influenza viruses into the human population. Avian influenza viruses were long thought not to be directly transmissible to humans and cause lethal outcomes. However, this perception changed in 1997, when 18 Hong Kong residents were infected by a wholly avian influenza virus of the H5N1 subtype, that resulted in six deaths (3, 4). Over the next few years, several other cases of direct avian-to-human transmission were reported (5-7), including the ongoing outbreak of highly pathogenic H5N1 influenza viruses in several Asian countries that has claimed 41 lives of 54 infected individuals as of January 26, 2005 (8). The increasing numbers of human H5N1 virus infections, together with a high mortality rate and possible human-to-human transmission, make the development of vaccines to these viruses essential.

In the United States, two influenza vaccines are licensed for human use: an inactivated vaccine and a live-attenuated vaccine virus. The production of influenza virus vaccines relies on reassortment (9), which requires coinfection of cells with a circulating wild-type strain that provides the hemagglutinin (HA) and neuraminidase (NA) segments and either A/PR/8/34 (PR8) virus (an attenuated human virus that provides high-growth properties in eggs) or a live attenuated virus that provides the attenuated phenotype. The selection of the desired “six plus two” reassortants (i.e., those containing the HA and NA gene segments of the circulating wild-type strain in the genetic background of PR8 or live attenuated virus) is time-consuming and cumbersome. Moreover, the need for reassortment and selection, as well as the inability of some reassortant viruses to grow to high titers, have resulted in delays in vaccine production. For influenza A and B viruses, highly efficient reverse genetics systems are now in place that allow the generation of these viruses from cloned cDNA (10-13). In one system (10), eight plasmids encoding the eight influenza viral RNA segments under the control of the RNA polymerase I promoter and terminator sequences are transfected into eukaryotic cells together with four RNA polymerase II-driven plasmids for the expression of the three viral polymerase subunits and the nucleoprotein NP. These four proteins are required to initiate viral replication and transcription. An alternative system has also been developed (13) that relies on eight plasmids in which the viral cDNAs are flanked by an RNA polymerase I promoter on one site and an RNA polymerase II promoter on the other site, which permits the viral RNA (vRNA) and mRNA to be derived from the same template. These systems have allowed six plus two reassortants to be engineered without the need for reassortment and screening procedures.

A limited number of mammalian cell lines are available for the production of influenza virus vaccines. They include Madin-Darby canine kidney (MDCK) (14-16) and African green monkey kidney (Vero) (17-20) cells. These cell lines cannot be transfected with high efficiencies, which sometimes limits their use in reverse genetics systems for influenza virus vaccine production; however, the generation of influenza virus in Vero cells has been demonstrated (12, 21). To address this limitation, we established a reverse genetics system that reduces the number of plasmids required for virus generation. Rather than providing RNA polymerase I or II transcription cassettes from individual plasmids, we combined up to eight RNA polymerase I transcription cassettes for the synthesis of the respective viral RNAs on one plasmid. We, similarly, combined the transcription cassettes for the three viral polymerase subunits on one plasmid. This approach allowed the efficient and robust generation of influenza A virus in Vero cells.

Materials and Methods

Cells. 293T human embryonic kidney cells and African green monkey kidney (Vero) cells were maintained in DMEM supplemented with 10% fetal calf serum. For all experiments, we used Vero CCL-81 cells, which have been previously used to produce an inactivated Japanese encephalitis vaccine and have been screened for lack of tumorgenicity and adventitious infectious agents (22). MDCK cells were maintained in modified Eagle's medium containing 5% newborn calf serum. All cells were maintained at 37°C in 5% CO2.

Construction of Plasmids. To combine RNA polymerase I transcription cassettes for the synthesis of the influenza viral RNA segments, transcription cassettes comprising the human RNA polymerase I promoter, an influenza viral cDNA in negative-sense orientation, and the mouse RNA polymerase I terminator (10) were amplified by PCR with oligonucleotides that contained recognition sequences for restriction endonucleases that were not present in the viral genome. As templates, we used pPolI-WSN-PB2, -PB1, -PA, -HA, -NP, -NA, -M, -NS (all described in ref. 10), which contained the respective viral cDNA of A/WSN/33 (H1N1) virus positioned between RNA polymerase I promoter and terminator sequences. PCR products were cloned into standard vectors that contained the respective restriction sites and were sequenced to confirm that they lacked unwanted mutations. We also confirmed the functionality of the resulting plasmids by reverse genetics.

A modified pTM1 vector (23) with flanking, unique restriction sites was used to stepwise-combine individual RNA polymerase I transcription cassettes (this vector is described in more detail in Discussion). At each cloning step, we confirmed the functionality of the resulting plasmids, which contained two to seven RNA polymerase I transcription units, by reverse genetics. The final plasmid, pPolI-WSN-All (Fig. 1A), contained eight RNA polymerase I transcription cassettes for the synthesis of all eight influenza A/WSN/33 viral RNAs. This plasmid was stably maintained in Escherichia coli JM109 cells at room temperature; bacterial cultures were grown in Terrific Broth medium for ≈30 h.

Fig. 1.
Schematic diagrams of plasmids possessing multiple influenza viral genes. (A) pTM-PolI-WSN-All for the transcription of all eight influenza viral RNAs from one template. Transcription units comprising the human RNA polymerase I promoter (blue, Pol I promoter), ...

Using the same strategy, we generated two plasmids, pTM-PolI-WSN-HA-NA and pTM-PolI-WSN-PB2-PB1-PA-NP-M-NS (Fig. 1B), that contained RNA polymerase I transcription cassettes for the HA and NA segments and the remaining six viral segments (i.e., PB2, PB1, PA, NP, M, and NS), respectively.

Plasmids pCAWSPB2, pCAWSPB1, and pCAWSPA comprised the chicken β-actin promoter, the coding sequence of the A/WSN/33 PB2, PB1, or PA protein, and a polyadenylation signal. These RNA polymerase II transcription units were flanked by recognition sequences for unique restriction endonucleases, either by PCR or by inserting short DNA linkers. PCR-amplified transcription cassettes were sequenced in their entirety before the three RNA polymerase II transcription cassettes were combined by using the unique restriction sites. The functionality of the resulting plasmid (pC-PolII-WSN-PB2-PB1-PA) (Fig. 1C) was verified by reverse genetics.

Generation of Virus from Plasmids. We transfected 293T cells (1 × 106) or Vero CCL-81 cells (5 × 105) by using Trans IT-LT1 (Mirus, Madison, WI) according to the manufacturer's instructions. Briefly, transfection reagent (2 μl of Trans IT-LT1 per μg of DNA for the transfection of 293T cells; 4 μl of Trans IT-LT1 per μg of DNA for the transfection of Vero cells) was diluted in 100 μl of OptiMEM (GIBCO/BRL), incubated for 5 min at room temperature, and added to premixed DNAs. For all transfection experiments, we used 0.1 μg of each of the single-unit plasmids for the synthesis of viral RNAs (i.e., pPolI-WSN-PB2, -PB1, -PA, -HA, -NP, -NA, -M, -NS; described in ref. 10), 1 μg of plasmids containing more than one RNA polymerase I transcription unit (i.e., pTM-PolI-WSN-All, pTM-PolI-WSN-HA-NA, or pTM-PolI-WSN-PB2-PB1-PA-NP-M-NS), and 1 μg of each of the protein expression plasmids. At times after transfection indicated in Tables Tables11 and and2,2, we determined the 50% tissue-culture infectious dose (TCID50) in MDCK cells.

Table 1.
Efficiency of virus generation in 293T cells
Table 2.
Efficiency of virus generation in Vero cells

Results

Plasmids Containing Multiple RNA Polymerase I or II Transcription Cassettes. To allow influenza virus generation from <8-12 plasmids (10, 12, 13), we combined RNA polymerase I transcription cassettes for vRNA synthesis, or RNA polymerase II transcription cassettes for mRNA synthesis on one plasmid. As a model, we used the A/WSN/33 (WSN) virus, for which parameters and efficiencies of viral generation are well established. Briefly, RNA polymerase I transcription cassettes comprising the human RNA polymerase I promoter, an influenza viral cDNA in negative-sense orientation, and the mouse RNA polymerase I terminator were amplified by PCR, cloned and sequenced, and then joined stepwise by the use of unique restriction sites. As a vector backbone, we used a modified pTM1 vector (23) that stably supported an Ebola viral cDNA of 20 kb (24) and is therefore suitable for the insertion of large DNA fragments. The generation of pTM-PolI-WSN-All (≈22.5 kb in length) (Fig. 1 A), which contains eight individual RNA polymerase I transcription units, did not present major obstacles; however, growth of E. coli JM109 bacteria at room temperature was required to prevent recombination of this plasmid.

For the annual generation of influenza virus vaccines, only two viral RNA segments, i.e., those encoding the HA and NA surface glycoproteins, need to be replaced. For this reason, we generated a plasmid in which the transcription units for the HA and NA segments were combined (pTM-PolI-WSN-HA-NA) (Fig. 1B), whereas a second plasmid combined the transcription units encoding the internal proteins (pTM-PolI-WSN-PB2-PB1-PA-NP-M-NS) (Fig. 1B). Both of these plasmids were stable during amplification in E. coli JM109 bacteria at 37°C.

To further reduce the number of plasmids required for virus generations, we combined the three RNA polymerase II transcription units for the WSN PB2, PB1, and PA proteins on one vector backbone, with the same strategy that allowed us to join the RNA polymerase I transcription units for vRNA synthesis. The resulting plasmid was stable in E. coli JM109 bacteria at 37°C and was designated pC-PolII-WSN-PB2-PB1-PA (Fig. 1C). Of note, we were unable to construct a plasmid combining the RNA polymerase II transcription unit for the polymerase proteins and for the nucleoprotein NP.

Virus Generation in 293T Cells from Plasmids Containing Multiple Transcription Cassettes. To test the functionality of plasmids containing multiple transcription cassettes, we first transfected 293T cells with pTM-PolI-WSN-All (for the transcription of all eight vRNAs) (Table 1, experimental samples 2 and 3) or pTM-PolI-WSN-HA-NA and pTM-PolI-WSN-PB2-PB1-PA-NP-M-NS (for the transcription of two and six vRNAs) (Table 1, experimental samples 7 and 8). Cells were cotransfected with four plasmids for the expression of NP and the polymerase subunits from separate plasmids (Table 1, experimental samples 2 and 7), or with two plasmids that express NP or PB2, PB1, and PA, respectively (Table 1, experimental samples 3 and 8). We successfully generated viruses from these plasmids, demonstrating that RNA polymerase I or RNA polymerase II transcription units can be combined, thus reducing the number of plasmids required for the artificial generation of influenza virus. At 48 h after transfection, the efficiency of virus generation ranged from 2 × 107 to 2.7 × 108 TCID50/ml (mean, 1.1 × 108 TCID50/ml) (Table 1, experimental samples 2, 3, 7, and 8). These efficiencies were slightly higher (P = 0.17) than those obtained for control experiments in which cells were transfected with eight separate plasmids for the transcription of the influenza vRNAs and four or two plasmids for the synthesis of NP and the three polymerase subunits (yielding 6.3 × 106 to 1.3 × 108 TCID50/ml; mean, 5.5 × 107 TCID50/ml) (Table 1, experimental samples 9 and 10).

We also carried out a number of control experiments, including mock transfections (Table 1, experimental sample 14), cells transfected with protein expression plasmids only (Table 1, experimental samples 11 and 12), cells transfected with eight plasmids for vRNA synthesis only (Table 1, experimental sample 13), or cells transfected with plasmids for the synthesis of two or six vRNAs (Table 1, experimental samples 4 and 5, respectively). None of these controls yielded viruses. However, we consistently detected appreciable virus titers in cells transfected with pTM-PolI-WSN-All (Table 1, experimental sample 1) or with a combination of pTM-PolI-WSN-HA-NA and pTM-PolI-PB2-PB1-PA-NP-M-NS (Table 1, experimental sample 6). These plasmids were designed for the transcription of negative-sense viral RNAs, and synthesis of NP and the three polymerase proteins was not expected. Thus, virus generation with these plasmids alone was not expected either (for possible explanations, see Discussion).

Virus Generation in Vero Cells from Plasmids Containing Multiple Transcription Cassettes. Next, we tested the efficiency of virus generation in Vero cells, which are difficult to transfect to high efficiencies. At 48 h after transfection, virus generation from 12 plasmids was negligible in two experiments and low in one experiment (Table 2, experimental sample 9), whereas, at 72 h after transfection, virus was detected in all three experiments. The use of only one or two plasmids for the synthesis of vRNAs increased the efficiency of virus generation at 72 h after transfection, especially in combination with pC-PolII-WSN-PB2-PB1-PA, yielding up to 2.5 × 106 TCID50/ml [Table 2, experimental sample 9 vs. experimental sample 3 (P = 0.0017) and experimental sample 9 vs. experimental sample 8 (P = 0.0063)]. We consistently found that expression of the three polymerase proteins from plasmid pC-PolII-WSN-PB2-PB1-PA resulted in more efficient virus generation as compared to providing these proteins from separate plasmids [Table 2, compare experimental samples 2 and 3 (P = 0.0054), experimental samples 7 and 8 (P = 0.028), and experimental samples 9 and 10 (P = 0.2)]. We again detected virus from plasmid pTM-PolI-WSN-All only (Table 2, experimental sample 1) or from plasmids pTM-PolI-WSN-PB2-PB1-PA-NP-M-NS and pTM-PolI-WSN-HA-NA (Table 2, experimental sample 6); however, virus generation was not observed consistently, and the resulting virus titers were low, likely because of the lower transfection efficiency of the Vero cells. Taken together, these results show that plasmids containing multiple RNA polymerase I or II transcription units can be highly efficient at generating virus in Vero cells.

Discussion

The generation of vaccine viruses can now be achieved by reverse genetics. In fact, this approach is the only efficient way to produce vaccine strains to highly pathogenic avian influenza viruses. These viruses are lethal to humans and embryonated eggs (25); therefore, attenuation [for example, by altering their HA cleavage site sequence (26, 27)] is critical to ensure growth to high titers in embryonated eggs while protecting vaccine production staff against exposure to aerosolized virus. For human use, the production of vaccine strains will require cell lines that are certified for lack of tumorgenicity and adventitious infectious agents. One such cell line is a Vero cell line, which is currently used for the production of rabies and polio vaccines (28). Using a “12-plasmid” approach, Fodor et al. (12) reported the generation of 10-20 plaque-forming units from 107 Vero cells on day 4 after transfection. Wood and Robertson (29) generated an H5N1 reference vaccine strain in Vero cells by reverse genetics but did not report the rescue efficiency, whereas A/PR/8/34 (H1N1)- or A/PR/8/34-based viruses were generated in Vero cells with an efficiency of <103 plaque-forming units/ml (30). By combining RNA polymerase I and/or II transcription units and thus achieving virus rescue from fewer plasmids, we were able to produce ≈105 to 106 TCID50/ml from 5 × 105 Vero cells on day 3 after transfection. We, thereby, achieved more efficient virus generation in Vero cells with these systems as compared with the 12-plasmid approach (Table 2, compare experimental sample 3 or 8 with experimental sample 9). This robust and highly efficient reverse genetics system could, therefore, be an asset for the rapid preparation of vaccine strains in pandemic situations.

Influenza virus generation relies on the expression of the polymerase and NP proteins. We found that the combination of the polymerase subunits on one plasmid enhanced the efficiency of virus generation. This finding may be explained by the reduction in the number of plasmids used for virus rescue, or the combination of the three transcription units may more closely reflect the equimolar ratios of polymerase subunits found in infected cells.

The combination of identical promoter and terminator units on one plasmid is thought to cause recombination. However, here we demonstrated that eight RNA polymerase I or three RNA polymerase II promoter and terminator sequences can be combined on one vector backbone. Hoffmann et al. (13) demonstrated that a combination of RNA polymerase I and II promoters allows vRNA and mRNA synthesis from one template. One could therefore design a plasmid that contains four RNA polymerase I/II transcription units for the synthesis of PB2, PB1, PA, and NP vRNAs and mRNAs, and four RNA polymerase I transcription units for the synthesis of NA, HA, M, and NS vRNAs. Such a construct should allow for the efficient generation of influenza virus from one plasmid. Moreover, our success in combining transcription units on one plasmid may provide the incentive for others to apply this strategy to other reverse genetics systems that rely on the cotransfection of cells with several plasmids or to design vectors for the simultaneous expression of several proteins from one plasmid.

Surprisingly, we observed virus generation from a single plasmid, pTM-PolI-WSN-All. The expression of influenza viral proteins from this plasmid suggests protein synthesis from a (cryptic) RNA polymerase II promoter present in the vector or in the RNA polymerase I promoter or terminator region. To determine whether the RNA polymerase I promoter sequence harbors a promoter in the opposite direction that could potentially drive protein expression from the upstream transcription cassette, we cloned an inverted RNA polymerase I promoter in front of a reporter gene; however, we did not detect appreciable levels of reporter gene expression from this plasmid (data not shown). The generation of influenza virus relies on the expression of four different proteins (PB2, PB1, PA, and NP) and would therefore require several read-through events. Alternatively, protein expression may have resulted from another mechanism, such as internal initiation of translation. We used a modified pTM1 (23) vector that contains the f1 single-stranded DNA origin of replication, the ampicillin resistance gene, a multiple cloning site, and the T7 RNA polymerase transcriptional terminator. The strong T7 RNA polymerase promoter and parts of the encephalomyocarditis virus untranslated region and thymidine kinase sequences that are present in the original pTM1 cloning vector had been eliminated from this modified version. Protein synthesis of the polymerase and NP proteins from this vector was therefore not expected. Nonetheless, the generation of influenza virus from plasmids designed to produce only negative-strand RNAs is intriguing and deserves further study.

In summary, here we present an improved system for the generation of influenza viruses that allows the easy and reproducible production of vaccine viruses in Vero cells. Application of this system may be especially advantageous in situations of outbreaks of highly pathogenic avian influenza viruses.

Acknowledgments

We thank Krisna Wells and Martha McGregor for excellent technical assistance and Susan Watson for editing the manuscript. This work was supported by Public Health Service research grants from the National Institute of Allergy and Infectious Diseases, by Grants-in-Aid for Scientific Research on Priority Areas from the Ministries of Education, Culture, Sports, Science, and Technology of Japan, and by the Core Research for Evolutional Science and Technology.

Notes

Author contributions: G.N. and Y. Kawaoka designed research; G.N. and K.F. performed research; Y. Kino contributed new reagents/analytic tools; G.N. analyzed data; and G.N. wrote the paper.

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: vRNA, viral RNA; HA, hemagglutinin; NA, neuraminidase; MDCK, Madin-Darby canine kidney; TCID50, 50% tissue-culture infectious dose.

References

1. Thompson, W. W., Shay, D. K., Weintraub, E., Brammer, L., Cox, N., Anderson, L. J. & Fukuda, K. (2003) J. Am. Med. Assoc. 289, 179-186. [PubMed]
2. Potter, C. W. (1998) in Textbook of Influenza, eds. Nicholson, K. G., Webster, R. G. & Hay, A. J. (Blackwell Scientific, Malden, U.K.), pp. 3-18.
3. Subbarao, K., Klimov, A., Katz, J., Regnery, H., Lim, W., Hall, H., Perdue, M., Swayne, D., Bender, C., Huang, J., et al. (1998) Science 279, 393-396. [PubMed]
4. Claas, E. C., Osterhaus, A. D., Van Beek, R., de Jong, J. C., Rimmelzwaan, G. F., Senne, D. A., Krauss, S., Shortridge, K. F. & Webster, R. G. (1998) Lancet 351, 472-477. [PubMed]
5. Peiris, J. S., Yu, W. C., Leung, C. W., Cheung, C. Y., Ng, W. F., Nicholls, J. M., Ng, T. K., Chan, K. H., Lai, S. T., Lim, W. L., et al. (2004) Lancet 363, 617-619. [PubMed]
6. Fouchier, R. A., Schneeberger, P. M., Rozendaal, F. W., Broekman, J. M., Kemink, S. A., Munster, V., Kuiken, T., Rimmelzwaan, G. F., Schutten, M., Van Doornum, G. J., et al. (2004) Proc. Natl. Acad. Sci. USA 101, 1356-1361. [PMC free article] [PubMed]
7. Koopmans, M., Wilbrink, B., Conyn, M., Natrop, G., van der Nat, H., Vennema, H., Meijer, A., van Steenbergen, J., Fouchier, R., Osterhaus, A., et al. (2004) Lancet 363, 587-593. [PubMed]
8. World Health Organization (2005) Cumulative Number of Confirmed Human Cases of Avian Influenza A (H5N1) (WHO, Geneva). Available at www.who.int/csr/disease/avian_influenza/country/en. Accessed January 28, 2005.
9. Gerdil, C. (2003) Vaccine 21, 1776-1779. [PubMed]
10. Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., et al. (1999) Proc. Natl. Acad. Sci. USA 96, 9345-9350. [PMC free article] [PubMed]
11. Hoffmann, E., Mahmood, K., Yang, C. F., Webster, R. G., Greenberg, H. B. & Kemble, G. (2002) Proc. Natl. Acad. Sci. USA 99, 11411-11416. [PMC free article] [PubMed]
12. Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G. & Garcia-Sastre, A. (1999) J. Virol. 73, 9679-9682. [PMC free article] [PubMed]
13. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G. & Webster, R. G. (2000) Proc. Natl. Acad. Sci. USA 97, 6108-6113. [PMC free article] [PubMed]
14. Brands, R., Visser, J., Medema, J., Palache, A. M. & van Scharrenburg, G. J. (1999) Dev. Biol. Stand. 98, 93-100. [PubMed]
15. Palache, A. M., Scheepers, H. S., de Regt, V., van Ewijk, P., Baljet, M., Brands, R. & van Scharrenburg, G. J. (1999) Dev. Biol. Stand. 98, 115-125. [PubMed]
16. Halperin, S. A., Smith, B., Mabrouk, T., Germain, M., Trepanier, P., Hassell, T., Treanor, J., Gauthier, R. & Mills, E. L. (2002) Vaccine 20, 1240-1247. [PubMed]
17. Kistner, O., Barrett, P. N., Mundt, W., Reiter, M., Schober-Bendixen, S. & Dorner, F. (1998) Vaccine 16, 960-968. [PubMed]
18. Kistner, O., Barrett, P. N., Mundt, W., Reiter, M., Schober-Bendixen, S., Eder, G. & Dorner, F. (1999) Dev. Biol. Stand. 98, 101-110. [PubMed]
19. Kistner, O., Barrett, P. N., Mundt, W., Reiter, M., Schober-Bendixen, S., Eder, G. & Dorner, F. (1999) Wien. Klin. Wochenschr. 111, 207-214. [PubMed]
20. Bruhl, P., Kerschbaum, A., Kistner, O., Barrett, N., Dorner, F. & Gerencer, M. (2000) Vaccine 19, 1149-1158. [PubMed]
21. Nicolson, C., Major, D., Wood, J. M. & Robertson, J. S. (2005) Vaccine 23, 2943-2952. [PubMed]
22. Sugawara, K., Nishiyama, K., Ishikawa, Y., Abe, M., Sonoda, K., Komatsu, K., Horikawa, Y., Takeda, K., Honda, T., Kuzuhara, S., et al. (2002) Biologicals 30, 303-314. [PubMed]
23. Moss, B., Elroy-Stein, O., Mizukami, T., Alexander, W. A. & Fuerst, T. R. (1990) Nature 348, 91-92. [PubMed]
24. Neumann, G., Feldmann, H., Watanabe, S., Lukashevich, I. & Kawaoka, Y. (2002) J. Virol. 76, 406-410. [PMC free article] [PubMed]
25. Shortridge, K. F., Zhou, N. N., Guan, Y., Gao, P., Ito, T., Kawaoka, Y., Kodihalli, S., Krauss, S., Markwell, D., Murti, K. G., et al. (1998) Virology 252, 331-342. [PubMed]
26. Horimoto, T. & Kawaoka, Y. (1994) J. Virol. 68, 3120-3128. [PMC free article] [PubMed]
27. Subbarao, K., Chen, H., Swayne, D., Mingay, L., Fodor, E., Brownlee, G., Xu, X., Lu, X., Katz, J., Cox, N., et al. (2003) Virology 305, 192-200. [PubMed]
28. Montagnon, B. J., Vincent-Falquet, J. C. & Saluzzo, J. F. (1999) Dev. Biol. Stand. 98, 137-140. [PubMed]
29. Wood, J. M. & Robertson, J. S. (2004) Nat. Rev. Microbiol. 2, 842-847. [PubMed]
30. Ozaki, H., Govorkova, E. A., Li, C., Xiong, X., Webster, R. G. & Webby, R. J. (2004) J. Virol. 78, 1851-1857. [PMC free article] [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

    Your browsing activity is empty.

    Activity recording is turned off.

    Turn recording back on

    See more...