- Email: [email protected]
Generation of recombinant influenza virus using baculovirus delivery vector
Journal of Virological Methods 110 (2003) 111 /114 www.elsevier.com/locate/jviromet
Brief report
Generation of recombinant influenza virus using baculovirus delivery vector Kanokwan Poomputsa b, Christian Kittel a, Andrej Egorov a, Wolfgang Ernst a, Reingard Grabherr a,* b
a Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Muthgasse 18, Vienna 1190, Austria School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, 83 Moo 8 Thakham, Bangkhuntien, Bangkok 10150, Thailand
Received 28 November 2002; received in revised form 19 February 2003; accepted 20 February 2003
Abstract A recombinant baculovirus vector containing mammalian cell-active promoters and transcription terminators was used to deliver a mutated influenza NS gene into Vero cells. In addition to the influenza NS gene, the baculovirus contained a reporter gene expression cassette (Green fluorescent protein, GFP), allowing to monitor the Vero cell transduction efficiency. More than 90% of Vero cells were expressing GFP 24 /48 h post transduction. After infecting baculovirus transduced cells with influenza helper virus, progeny of attenuated influenza virus carrying the recombinant NS gene could be selected. Baculovirus delivery was highly reproducible and efficient in Vero cells. This new method for influenza gene delivery could contribute to influenza virus research and vaccine development. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Baculovirus; Influenza A virus; Non-structural protein
Influenza A viruses possess a genome of eight single viral RNA (vRNA) segments of negative polarity, coding for ten structural and one non-structural protein (NS1). Each segment is encapsulated by the nucleoprotein (NP) and associated with the trimeric polymerase (PB1, PB2 and PA), which forms the functional ribonucleoprotein complex (RNP). Several reverse-genetic systems have been developed to allow manipulation of the viral genome. One system is based on the principle that synthetic influenza vRNA transcribed in vitro and encapsulated with the purified ribonucleoprotein complex (RNP) can be transfected into cells infected with a special helper virus, e.g. a temperature sensitive mutant (Enami et al., 1990). This requires laborious selection procedures in order to select recombinant progeny from the helper virus. More recently, a plasmid-based transfection system using a mixture of
* Corresponding author. Tel.: /43-1-36006-6803; fax: /43-1-3697615. E-mail address: [email protected] (R. Grabherr).
plasmids expressing individual vRNA segments and proteins of influenza A and B virus was established for the rescue of infectious virus progeny (Fodor et al., 1999; Hoffmann et al., 2000, 2002; Neuman et al., 1994, 1999). In this helper virus-free system, transfection efficiency plays a major role in a successful rescue of recombinant influenza virus progeny. This method was highly effective in 293T and COS-1 cells, however it was less efficient in Vero cells (Fodor et al., 1999), which is a cell line suitable for vaccine production. In this study, the effectiveness of baculovirus transduction for gene delivery into Vero cells was demonstrated using green fluorescent protein (GFP) as a reporter gene and the successful rescue of a truncated NS1 gene in influenza A virus was shown. A recombinant baculovirus harboring a mutated influenza A/PR/8/34 NS gene, coding for a truncated NS1 protein of 38 amino acids, flanked by the human polymerase I (PolI) promoter and Hepatitis Delta Virus (HDV) genomic ribozyme sequences, was constructed (Fig. 1). Viral RNA from the influenza A/PR8/34 virus was extracted using Ultraspec RNA purification reagent
0166-0934/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0166-0934(03)00084-3
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Fig. 1. Construction of recombinant NS expressing cassette. The NS gene was cloned in reverse orientation between human polymerase I promoter (PolI) and hepatitis delta virus terminator (HDV) to transcribed NS vRNA. At nucleotide position 140, a multiple stop codon cassette was introduced, resulting in a truncated, 38 amino acid NS1 protein and a full size, 121 aa NEP. The non-translated part between the stop codon and the splicing signal of NEP (nucleotide position 283 /512) was deleted.
(Biotecx Laboratories) and served as a template for subsequent amplification of the viral NS gene by RTPCR. Plasmid pQBI 25/50-fc1 (Qbiogene, CA) was modified as follows. By PCR, the original GFP expressing cassette was replaced by PolI promoter which was PCR-amplified from the plasmid pPOLI-CAT-RT, a gift from Dr Garcia-Sastre (Department of Microbiology, Mount Sinai School of Medicine, New York). HDV ribozyme (HDV), a self-cleaving RNA sequence to ensure the correct 3? end (Fodor et al., 1999; Neuman et al., 1994), was synthetically produced (Invitrogen, Germany), blunt end ligated to the 5?-terminus of the NS gene and inserted into the modified vector downstream of the PolI promoter. A multiple stop codon cassette was introduced into the NS gene at position 140 by inverse PCR, using the primers 3?NS-140STOP and 5?NS-355 in order to delete the non-translated part between this stop codon cassette and the splicing signal of the nuclear export protein NEP primer sequences available on request). This plasmid, designated DNS38 was digested with StuI and the NS expressing cassette was inserted into a baculovirus transfer vector (pCMVGFP), a modified pBacPAK8 (Clontech), expressing a green fluorescence protein (GFP) under control of the cytomegalovirus promoter (CMV) and the bovine growth hormone polyadenylation site (BGH). The recombinant plasmid (pCMVGFPDNS38) was co-transfected into Spodoptera frugiperda (Sf9) insect cells with linear Ac NPV viral DNA (Baculogold, Becton Dickinson, PharMingen) and recombinant AcCMVGFP-DNS38 was generated. This virus was purified by plaque assay using standard procedure (Summers and Smith, 1987) and amplified in insect cells. Spodoptera Frugiperda (Sf9) insect cells were grown in IPL-41 insect medium (Sigma-Aldrich Chemical) containing yeastolate and a lipid/sterol cocktail with 3% FCS at 27 8C. Recombinant AcCMVGFP-DNS38 ba-
culoviruses were transduced into Vero cells at different MOIs in the presence of 0.01% DEAE-dextran sulfate. Vero cells (WHO-certified) were grown in DMEM/ Ham’s F12 (Biochrom) protein free medium (Kistner et al., 1999). After 48 h, GFP expressing cells were detected by FACS analysis. Transduction of Vero cells by the recombinant AcCMVGFP-DNS38 baculovirus was concentration dependent as shown in Fig. 2A. MOIs tested were 5, 50, 500 and 5000, yielding transfection rates of 2, 9, 62 and 91%, respectively. No changes in cell viability and no cytopathic effect of transduced Vero cells were observed. Vero cells were transduced with the baculovirus vector AcCMVGFP-DNS38 according to optimized transduction conditions described above. Recombinant baculovirus inoculum (MOI 1000) with DEAE-dextran sulfate was added to Vero cells and incubated for 2 h, inoculum was removed and cells were incubated with serum-free medium. After 24 h, the baculovirus transduced cells were infected with 25A-1 influenza helper viruses (Egorov et al., 1998) at MOI of 1 for 30 min. The inoculum was replaced by serum-free medium and further incubated at 37 8C for 24 h. The viral supernatant was passaged twice on Vero cells at 40 8C and analyzed by RT-PCR for the presence of the DNS38 gene (Fig. 3). Following three rounds of plaque purification on Vero cells, recombinant influenza virus containing the DNS38-vRNA was isolated and the correct sequence of the DNS38 gene was confirmed by nucleotide sequence analysis. Rescued DNS38 virus was fully attenuated in interferon competent systems like Madin Darby canine kidney (MDCK) cells, embryonated hen eggs or mice but gave almost equal titers, compared to the wild type variant in interferon deficient Vero cells (data not shown).
K. Poomputsa et al. / Journal of Virological Methods 110 (2003) 111 /114
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Fig. 2. Baculovirus concentration dependent expression of the reporter gene (GFP) in infected Vero cells. (A) Vero cells were transduced with baculovirus, carrying a GFP reporter gene at different MOIs. Using FACS analysis, the percentage of GFP expressing cells, reflecting cells transduced with baculovirus, could be measured. (B) Vero cells expressing GFP after 48 h transduction by recombinant AcCMVGFP-DNS38 baculovirus.
Although, the Autographa californica nuclear polyhedrosis virus (AcNPV) has been used previously to produce mammalian or viral proteins in insect cells, more recently baculoviruses were used as a powerful vector to deliver various genes into a variety of mammalian cell types with high efficiency (Boyce and Bucher, 1996; Condreay et al., 1999; Shoji et al., 1997). Baculoviruses possess several advantages that make them an attractive tool for the delivery of foreign sequences into mammalian cells. First, multiple genes and large inserts (up to 38 kb) (Cheshenko et al., 2001) can be introduced into the baculovirus genome so that even the whole genome of some viruses could be cloned and delivered into mammalian cells at once (McCormick et al., 2002). Second, baculoviruses are inherently unable to replicate in mammalian cells, therefore they are not cytopathogenic and considered as biologically safe for therapeutic applications for humans (Kost, 2000). Recently, the formation of influenza virus like particles directed by a recombinant baculovirus was
demonstrated in insect cells (Latham and Galarza, 2001). Here we examined the potential of the baculovirus system for the purpose of influenza reverse genetics. We demonstrated the effective delivery and rescue of mutated influenza NS gene after transduction of Vero cells with a baculovirus vector. Although a high number (MOI/1000) of baculovirus particles was required, this method is considered feasible, since baculoviruses can be easily generated and grown in insect cells to high titers. Although MOI /5000 was determined to yield highest transduction rates in vero cells, 20% of this concentration was sufficient for viral rescue. Another advantage of this method was the possibility to monitor the transduction efficiency of the baculovirus in Vero cells by FACS analysis, using a GFP reporter gene, which was incorporated into the vector (Fig. 2). This cell line was selected for these experiments because of two reasons. First, Vero cells are well characterized and already accepted for the production of polio, rabies and human influenza virus vaccines. Second, because Vero cells are interferon deficient, influenza viruses containing a truncated NS1 protein could be rescued and grown on this cell line (Egorov et al., 1998). In this study, we have rescued an influenza virus containing a truncated NS1 protein consisting of only 38 amino acids. The truncation of NS1 protein mediates irreversible attenuation of influenza A virus and is considered to be a valuable tool for safe influenza vaccine development (Egorov et al., 1998; Palese et al., 1999).
Acknowledgements Fig. 3. Detection of the recombinant influenza NS gene. In comparison to the influenza 25A-1 helper virus (lane 1) NS, the DNS38 (lane 2) has a 215 nt deletion.
This work was funded by the Fonds zur Fo¨rderung der Wissenschaftlichen Forschung project P15759-
114
K. Poomputsa et al. / Journal of Virological Methods 110 (2003) 111 /114
MOB. K. Poomputsa was supported by the AseanEuropean University Network (ASEA-UNINET).
References Boyce, F.M., Bucher, N.L., 1996. Baculovirus-mediated gene transfer into mammalian cells. Proc. Natl. Acad. Sci. USA 93, 2348 /2352. Cheshenko, N., Krougliak, N., Eisensmith, R.C., Krougliak, V.A., 2001. A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. Gene Ther. 8, 846 /854. Condreay, J.P., Witherspoon, S.M., Clay, W.C., Kost, T.A., 1999. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proc. Natl. Acad. Sci. USA 96, 127 /132. Egorov, A., Brandt, S., Sereinig, S., Romanova, J., Ferko, B., Katinger, D., Grassauer, A., Alexandrova, G., Katinger, H., Muster, T., 1998. Transfectant influenza virus with long deletions in the NS1 protein grow efficiently in Vero cells. J. Virol. 72, 6437 / 6441. Enami, M., Luytjes, W., Krystal, M., Palese, P., 1990. Introduction of site specific mutations into the genome of influenza virus. Proc. Natl. Acad. Sci. USA 87, 3802 /3805. Fodor, E., Devenish, L., Engelhardt, O.G., Palese, P., Brownlee, G.G., Garcia-Sastre, A., 1999. Rescue of influenza A virus from recombinant DNA. J. Virol. 73, 9679 /9682. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G., Webster, R.G., 2000. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl. Acad. Sci. USA 97, 6108 / 6113.
Hoffmann, E., Mahmood, K., Yang, C.F., Webster, R.G., Greenberg, H.B., Kemble, G., 2002. Rescue of influenza B virus from eight plasmids. Proc. Natl. Acad. Sci. USA 99, 11411 /11416. Kistner, O., Barrett, P.N., Mundt, W., Reiter, M., Schober-Bendixen, S., Eder, G., Dorner, F., 1999. Development of a Vero cell-derived influenza whole virus vaccine. Dev. Biol. Stand. 98, 101 /110. Kost, T.A., 2000. Biosafety and viral gene transfer vectors. In: Fleming, D.O., Hunt, D.L. (Eds.), Biological Safety, Principles and Practices, 3rd edition. ASM Press, New York, pp. 579 /597. Latham, T., Galarza, J.M., 2001. Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins. J. Virol. 75, 6154 /6165. McCormick, C.J., Rowlands, D.J., Harris, M., 2002. Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors. J. Gen. Virol. 83, 383 /394. Neuman, G., Zobel, A., Hobom, G., 1994. RNA polymerase I mediates expression of influenza viral RNA molecules. Virology 202, 477 /479. Neuman, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Goa, P., Huges, M., Perez, D.R., Donis, R., Hoffmann, E., Hobom, M., Kawaoka, Y., 1999. Generation of influenza A viruses entirely from cloned cDNA. Proc. Natl. Acad. Sci. USA 96, 9345 /9350. Palese, P., Muster, T., Zheng, H., O’Neill, R., Garcia-Sastre, A., 1999. Learning from our foes: a novel vaccine concept for influenza virus. Arch. Virol. Suppl. 15, 131 /138. Shoji, H., Aizaki, H., Tani, H., Ishii, K., Chiba, T., Saito, I., Miyamura, T., Mutsuura, T., 1997. Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors. J. Gen. Virol. 78, 2657 /2664. Summers, M.D., Smith, G.E., 1987. Texas Agriculture Experiment Station Bulletin No.1555.
Brief report
Generation of recombinant influenza virus using baculovirus delivery vector Kanokwan Poomputsa b, Christian Kittel a, Andrej Egorov a, Wolfgang Ernst a, Reingard Grabherr a,* b
a Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Vienna, Muthgasse 18, Vienna 1190, Austria School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, 83 Moo 8 Thakham, Bangkhuntien, Bangkok 10150, Thailand
Received 28 November 2002; received in revised form 19 February 2003; accepted 20 February 2003
Abstract A recombinant baculovirus vector containing mammalian cell-active promoters and transcription terminators was used to deliver a mutated influenza NS gene into Vero cells. In addition to the influenza NS gene, the baculovirus contained a reporter gene expression cassette (Green fluorescent protein, GFP), allowing to monitor the Vero cell transduction efficiency. More than 90% of Vero cells were expressing GFP 24 /48 h post transduction. After infecting baculovirus transduced cells with influenza helper virus, progeny of attenuated influenza virus carrying the recombinant NS gene could be selected. Baculovirus delivery was highly reproducible and efficient in Vero cells. This new method for influenza gene delivery could contribute to influenza virus research and vaccine development. # 2003 Elsevier Science B.V. All rights reserved. Keywords: Baculovirus; Influenza A virus; Non-structural protein
Influenza A viruses possess a genome of eight single viral RNA (vRNA) segments of negative polarity, coding for ten structural and one non-structural protein (NS1). Each segment is encapsulated by the nucleoprotein (NP) and associated with the trimeric polymerase (PB1, PB2 and PA), which forms the functional ribonucleoprotein complex (RNP). Several reverse-genetic systems have been developed to allow manipulation of the viral genome. One system is based on the principle that synthetic influenza vRNA transcribed in vitro and encapsulated with the purified ribonucleoprotein complex (RNP) can be transfected into cells infected with a special helper virus, e.g. a temperature sensitive mutant (Enami et al., 1990). This requires laborious selection procedures in order to select recombinant progeny from the helper virus. More recently, a plasmid-based transfection system using a mixture of
* Corresponding author. Tel.: /43-1-36006-6803; fax: /43-1-3697615. E-mail address: [email protected] (R. Grabherr).
plasmids expressing individual vRNA segments and proteins of influenza A and B virus was established for the rescue of infectious virus progeny (Fodor et al., 1999; Hoffmann et al., 2000, 2002; Neuman et al., 1994, 1999). In this helper virus-free system, transfection efficiency plays a major role in a successful rescue of recombinant influenza virus progeny. This method was highly effective in 293T and COS-1 cells, however it was less efficient in Vero cells (Fodor et al., 1999), which is a cell line suitable for vaccine production. In this study, the effectiveness of baculovirus transduction for gene delivery into Vero cells was demonstrated using green fluorescent protein (GFP) as a reporter gene and the successful rescue of a truncated NS1 gene in influenza A virus was shown. A recombinant baculovirus harboring a mutated influenza A/PR/8/34 NS gene, coding for a truncated NS1 protein of 38 amino acids, flanked by the human polymerase I (PolI) promoter and Hepatitis Delta Virus (HDV) genomic ribozyme sequences, was constructed (Fig. 1). Viral RNA from the influenza A/PR8/34 virus was extracted using Ultraspec RNA purification reagent
0166-0934/03/$ - see front matter # 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0166-0934(03)00084-3
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K. Poomputsa et al. / Journal of Virological Methods 110 (2003) 111 /114
Fig. 1. Construction of recombinant NS expressing cassette. The NS gene was cloned in reverse orientation between human polymerase I promoter (PolI) and hepatitis delta virus terminator (HDV) to transcribed NS vRNA. At nucleotide position 140, a multiple stop codon cassette was introduced, resulting in a truncated, 38 amino acid NS1 protein and a full size, 121 aa NEP. The non-translated part between the stop codon and the splicing signal of NEP (nucleotide position 283 /512) was deleted.
(Biotecx Laboratories) and served as a template for subsequent amplification of the viral NS gene by RTPCR. Plasmid pQBI 25/50-fc1 (Qbiogene, CA) was modified as follows. By PCR, the original GFP expressing cassette was replaced by PolI promoter which was PCR-amplified from the plasmid pPOLI-CAT-RT, a gift from Dr Garcia-Sastre (Department of Microbiology, Mount Sinai School of Medicine, New York). HDV ribozyme (HDV), a self-cleaving RNA sequence to ensure the correct 3? end (Fodor et al., 1999; Neuman et al., 1994), was synthetically produced (Invitrogen, Germany), blunt end ligated to the 5?-terminus of the NS gene and inserted into the modified vector downstream of the PolI promoter. A multiple stop codon cassette was introduced into the NS gene at position 140 by inverse PCR, using the primers 3?NS-140STOP and 5?NS-355 in order to delete the non-translated part between this stop codon cassette and the splicing signal of the nuclear export protein NEP primer sequences available on request). This plasmid, designated DNS38 was digested with StuI and the NS expressing cassette was inserted into a baculovirus transfer vector (pCMVGFP), a modified pBacPAK8 (Clontech), expressing a green fluorescence protein (GFP) under control of the cytomegalovirus promoter (CMV) and the bovine growth hormone polyadenylation site (BGH). The recombinant plasmid (pCMVGFPDNS38) was co-transfected into Spodoptera frugiperda (Sf9) insect cells with linear Ac NPV viral DNA (Baculogold, Becton Dickinson, PharMingen) and recombinant AcCMVGFP-DNS38 was generated. This virus was purified by plaque assay using standard procedure (Summers and Smith, 1987) and amplified in insect cells. Spodoptera Frugiperda (Sf9) insect cells were grown in IPL-41 insect medium (Sigma-Aldrich Chemical) containing yeastolate and a lipid/sterol cocktail with 3% FCS at 27 8C. Recombinant AcCMVGFP-DNS38 ba-
culoviruses were transduced into Vero cells at different MOIs in the presence of 0.01% DEAE-dextran sulfate. Vero cells (WHO-certified) were grown in DMEM/ Ham’s F12 (Biochrom) protein free medium (Kistner et al., 1999). After 48 h, GFP expressing cells were detected by FACS analysis. Transduction of Vero cells by the recombinant AcCMVGFP-DNS38 baculovirus was concentration dependent as shown in Fig. 2A. MOIs tested were 5, 50, 500 and 5000, yielding transfection rates of 2, 9, 62 and 91%, respectively. No changes in cell viability and no cytopathic effect of transduced Vero cells were observed. Vero cells were transduced with the baculovirus vector AcCMVGFP-DNS38 according to optimized transduction conditions described above. Recombinant baculovirus inoculum (MOI 1000) with DEAE-dextran sulfate was added to Vero cells and incubated for 2 h, inoculum was removed and cells were incubated with serum-free medium. After 24 h, the baculovirus transduced cells were infected with 25A-1 influenza helper viruses (Egorov et al., 1998) at MOI of 1 for 30 min. The inoculum was replaced by serum-free medium and further incubated at 37 8C for 24 h. The viral supernatant was passaged twice on Vero cells at 40 8C and analyzed by RT-PCR for the presence of the DNS38 gene (Fig. 3). Following three rounds of plaque purification on Vero cells, recombinant influenza virus containing the DNS38-vRNA was isolated and the correct sequence of the DNS38 gene was confirmed by nucleotide sequence analysis. Rescued DNS38 virus was fully attenuated in interferon competent systems like Madin Darby canine kidney (MDCK) cells, embryonated hen eggs or mice but gave almost equal titers, compared to the wild type variant in interferon deficient Vero cells (data not shown).
K. Poomputsa et al. / Journal of Virological Methods 110 (2003) 111 /114
113
Fig. 2. Baculovirus concentration dependent expression of the reporter gene (GFP) in infected Vero cells. (A) Vero cells were transduced with baculovirus, carrying a GFP reporter gene at different MOIs. Using FACS analysis, the percentage of GFP expressing cells, reflecting cells transduced with baculovirus, could be measured. (B) Vero cells expressing GFP after 48 h transduction by recombinant AcCMVGFP-DNS38 baculovirus.
Although, the Autographa californica nuclear polyhedrosis virus (AcNPV) has been used previously to produce mammalian or viral proteins in insect cells, more recently baculoviruses were used as a powerful vector to deliver various genes into a variety of mammalian cell types with high efficiency (Boyce and Bucher, 1996; Condreay et al., 1999; Shoji et al., 1997). Baculoviruses possess several advantages that make them an attractive tool for the delivery of foreign sequences into mammalian cells. First, multiple genes and large inserts (up to 38 kb) (Cheshenko et al., 2001) can be introduced into the baculovirus genome so that even the whole genome of some viruses could be cloned and delivered into mammalian cells at once (McCormick et al., 2002). Second, baculoviruses are inherently unable to replicate in mammalian cells, therefore they are not cytopathogenic and considered as biologically safe for therapeutic applications for humans (Kost, 2000). Recently, the formation of influenza virus like particles directed by a recombinant baculovirus was
demonstrated in insect cells (Latham and Galarza, 2001). Here we examined the potential of the baculovirus system for the purpose of influenza reverse genetics. We demonstrated the effective delivery and rescue of mutated influenza NS gene after transduction of Vero cells with a baculovirus vector. Although a high number (MOI/1000) of baculovirus particles was required, this method is considered feasible, since baculoviruses can be easily generated and grown in insect cells to high titers. Although MOI /5000 was determined to yield highest transduction rates in vero cells, 20% of this concentration was sufficient for viral rescue. Another advantage of this method was the possibility to monitor the transduction efficiency of the baculovirus in Vero cells by FACS analysis, using a GFP reporter gene, which was incorporated into the vector (Fig. 2). This cell line was selected for these experiments because of two reasons. First, Vero cells are well characterized and already accepted for the production of polio, rabies and human influenza virus vaccines. Second, because Vero cells are interferon deficient, influenza viruses containing a truncated NS1 protein could be rescued and grown on this cell line (Egorov et al., 1998). In this study, we have rescued an influenza virus containing a truncated NS1 protein consisting of only 38 amino acids. The truncation of NS1 protein mediates irreversible attenuation of influenza A virus and is considered to be a valuable tool for safe influenza vaccine development (Egorov et al., 1998; Palese et al., 1999).
Acknowledgements Fig. 3. Detection of the recombinant influenza NS gene. In comparison to the influenza 25A-1 helper virus (lane 1) NS, the DNS38 (lane 2) has a 215 nt deletion.
This work was funded by the Fonds zur Fo¨rderung der Wissenschaftlichen Forschung project P15759-
114
K. Poomputsa et al. / Journal of Virological Methods 110 (2003) 111 /114
MOB. K. Poomputsa was supported by the AseanEuropean University Network (ASEA-UNINET).
References Boyce, F.M., Bucher, N.L., 1996. Baculovirus-mediated gene transfer into mammalian cells. Proc. Natl. Acad. Sci. USA 93, 2348 /2352. Cheshenko, N., Krougliak, N., Eisensmith, R.C., Krougliak, V.A., 2001. A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. Gene Ther. 8, 846 /854. Condreay, J.P., Witherspoon, S.M., Clay, W.C., Kost, T.A., 1999. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Proc. Natl. Acad. Sci. USA 96, 127 /132. Egorov, A., Brandt, S., Sereinig, S., Romanova, J., Ferko, B., Katinger, D., Grassauer, A., Alexandrova, G., Katinger, H., Muster, T., 1998. Transfectant influenza virus with long deletions in the NS1 protein grow efficiently in Vero cells. J. Virol. 72, 6437 / 6441. Enami, M., Luytjes, W., Krystal, M., Palese, P., 1990. Introduction of site specific mutations into the genome of influenza virus. Proc. Natl. Acad. Sci. USA 87, 3802 /3805. Fodor, E., Devenish, L., Engelhardt, O.G., Palese, P., Brownlee, G.G., Garcia-Sastre, A., 1999. Rescue of influenza A virus from recombinant DNA. J. Virol. 73, 9679 /9682. Hoffmann, E., Neumann, G., Kawaoka, Y., Hobom, G., Webster, R.G., 2000. A DNA transfection system for generation of influenza A virus from eight plasmids. Proc. Natl. Acad. Sci. USA 97, 6108 / 6113.
Hoffmann, E., Mahmood, K., Yang, C.F., Webster, R.G., Greenberg, H.B., Kemble, G., 2002. Rescue of influenza B virus from eight plasmids. Proc. Natl. Acad. Sci. USA 99, 11411 /11416. Kistner, O., Barrett, P.N., Mundt, W., Reiter, M., Schober-Bendixen, S., Eder, G., Dorner, F., 1999. Development of a Vero cell-derived influenza whole virus vaccine. Dev. Biol. Stand. 98, 101 /110. Kost, T.A., 2000. Biosafety and viral gene transfer vectors. In: Fleming, D.O., Hunt, D.L. (Eds.), Biological Safety, Principles and Practices, 3rd edition. ASM Press, New York, pp. 579 /597. Latham, T., Galarza, J.M., 2001. Formation of wild-type and chimeric influenza virus-like particles following simultaneous expression of only four structural proteins. J. Virol. 75, 6154 /6165. McCormick, C.J., Rowlands, D.J., Harris, M., 2002. Efficient delivery and regulable expression of hepatitis C virus full-length and minigenome constructs in hepatocyte-derived cell lines using baculovirus vectors. J. Gen. Virol. 83, 383 /394. Neuman, G., Zobel, A., Hobom, G., 1994. RNA polymerase I mediates expression of influenza viral RNA molecules. Virology 202, 477 /479. Neuman, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Goa, P., Huges, M., Perez, D.R., Donis, R., Hoffmann, E., Hobom, M., Kawaoka, Y., 1999. Generation of influenza A viruses entirely from cloned cDNA. Proc. Natl. Acad. Sci. USA 96, 9345 /9350. Palese, P., Muster, T., Zheng, H., O’Neill, R., Garcia-Sastre, A., 1999. Learning from our foes: a novel vaccine concept for influenza virus. Arch. Virol. Suppl. 15, 131 /138. Shoji, H., Aizaki, H., Tani, H., Ishii, K., Chiba, T., Saito, I., Miyamura, T., Mutsuura, T., 1997. Efficient gene transfer into various mammalian cells, including non-hepatic cells, by baculovirus vectors. J. Gen. Virol. 78, 2657 /2664. Summers, M.D., Smith, G.E., 1987. Texas Agriculture Experiment Station Bulletin No.1555.