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In vitro host range, multiplication and virion forms of recombinant viruses obtained from co-infection in vitro with a vaccinia-vectored influenza vaccine and a naturally occurring cowpox virus isolate.

Okeke MI, Nilssen Ø, Moens U, Tryland M, Traavik T - Virol. J. (2009)

Bottom Line: Poxvirus-vectored vaccines against infectious diseases and cancer are currently under development.Analysis of the subcellular localization of the transgenic HA protein showed that neither virus strain nor cell line have effect on the subcellular targets of the HA protein.The influenza virus HA protein was targeted to enveloped virions, plasma membrane, Golgi apparatus and cytoplasmic vesicles.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø, Tromsø, Norway. malachy.okeke@uit.no

ABSTRACT

Background: Poxvirus-vectored vaccines against infectious diseases and cancer are currently under development. We hypothesized that the extensive use of poxvirus-vectored vaccine in future might result in co-infection and recombination between the vaccine virus and naturally occurring poxviruses, resulting in hybrid viruses with unpredictable characteristics. Previously, we confirmed that co-infecting in vitro a Modified vaccinia virus Ankara (MVA) strain engineered to express influenza virus haemagglutinin (HA) and nucleoprotein (NP) genes with a naturally occurring cowpox virus (CPXV-NOH1) resulted in recombinant progeny viruses (H Hansen, MI Okeke, Ø Nilssen, T Traavik, Vaccine 23: 499-506, 2004). In this study we analyzed the biological properties of parental and progeny hybrid viruses.

Results: Five CPXV/MVA progeny viruses were isolated based on plaque phenotype and the expression of influenza virus HA protein. Progeny hybrid viruses displayed in vitro cell line tropism of CPXV-NOH1, but not that of MVA. The HA transgene or its expression was lost on serial passage of transgenic viruses and the speed at which HA expression was lost varied with cell lines. The HA transgene in the progeny viruses or its expression was stable in African Green Monkey derived Vero cells but became unstable in rat derived IEC-6 cells. Hybrid viruses lacking the HA transgene have higher levels of virus multiplication in mammalian cell lines and produced more enveloped virions than the transgene positive progenitor virus strain. Analysis of the subcellular localization of the transgenic HA protein showed that neither virus strain nor cell line have effect on the subcellular targets of the HA protein. The influenza virus HA protein was targeted to enveloped virions, plasma membrane, Golgi apparatus and cytoplasmic vesicles.

Conclusion: Our results suggest that homologous recombination between poxvirus-vectored vaccine and naturally circulating poxviruses, genetic instability of the transgene, accumulation of non-transgene expressing vectors or hybrid virus progenies, as well as cell line/type specific selection against the transgene are potential complications that may result if poxvirus vectored vaccines are extensively used in animals and man.

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Relative amount of mature virus forms produced by respective virus strains in Vero cells. Virion forms produced at different times post infection were quantified by electron microscopy as described in methods. IMV, IEV and CEV were counted in 50 randomly chosen sections of infected cells. The values represent percentage of each virus form relative to the total number of virus forms counted. IMV, intracellular mature virus; CEV, cell associated enveloped virus; EEV, extracellular enveloped virus. CPXV-NOHI (A), Rec 1 (B), Rec 2 (C), Rec 3 (D), Rec 3a (E), Rec 3b (F).
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Figure 6: Relative amount of mature virus forms produced by respective virus strains in Vero cells. Virion forms produced at different times post infection were quantified by electron microscopy as described in methods. IMV, IEV and CEV were counted in 50 randomly chosen sections of infected cells. The values represent percentage of each virus form relative to the total number of virus forms counted. IMV, intracellular mature virus; CEV, cell associated enveloped virus; EEV, extracellular enveloped virus. CPXV-NOHI (A), Rec 1 (B), Rec 2 (C), Rec 3 (D), Rec 3a (E), Rec 3b (F).

Mentions: We carried out detailed analysis of the morphogenesis of the virus strains under study by electron microscopy. Relative and absolute numbers of various mature and immature viral forms were determined at different times post infection. The kinetics of mature virus production in Vero cells is depicted in Figure 6. MVA-HANP results were not included because a very low level of mature virus forms were produced in Vero cells [7]. Assembly of CPXV-NOHI and hybrid progenies was similar to what have been reported for strains of vaccinia virus [7,29,30]. Differences existed in the abundance of mature virus forms produced by CPXV-NOHI and progeny viruses. The intracellular mature virus (IMV) is the major mature virus type produced in CPXV-NOH1 infected cells accounting for 87% of virion forms (IMV, IEV, CEV) at 24 hpi (Figure 6A). Intracellular enveloped virus (IEV) and cell associated enveloped virus (CEV) represented 4% and 9% respectively of virion forms produced in CPXV-NOH1 infected cells (Figure 6A). Similarly, 95% of virions produced by Rec 1 at 24 hpi were IMVs (Figure 6B). However in Rec 2, a higher proportion of IMVs were converted to enveloped forms such that at 24 hpi, CEV is the predominant form representing 55% of virion forms produced (Figure 6C). Rec 3 appeared defective in the production of enveloped virions. At 18 and 24 hpi, less than 1% of Rec 3 virions were IEV or CEV (Figure 6D). Transgene negative derivatives of Rec 3 (Rec 3a and Rec 3b) were efficient in the production of enveloped virions. The enveloped forms (IEV and CEV) accounted for approximately 50% of virion types produced by Rec 3a and Rec 3b respectively at 24 hpi (Figures 6E, F).


In vitro host range, multiplication and virion forms of recombinant viruses obtained from co-infection in vitro with a vaccinia-vectored influenza vaccine and a naturally occurring cowpox virus isolate.

Okeke MI, Nilssen Ø, Moens U, Tryland M, Traavik T - Virol. J. (2009)

Relative amount of mature virus forms produced by respective virus strains in Vero cells. Virion forms produced at different times post infection were quantified by electron microscopy as described in methods. IMV, IEV and CEV were counted in 50 randomly chosen sections of infected cells. The values represent percentage of each virus form relative to the total number of virus forms counted. IMV, intracellular mature virus; CEV, cell associated enveloped virus; EEV, extracellular enveloped virus. CPXV-NOHI (A), Rec 1 (B), Rec 2 (C), Rec 3 (D), Rec 3a (E), Rec 3b (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690591&req=5

Figure 6: Relative amount of mature virus forms produced by respective virus strains in Vero cells. Virion forms produced at different times post infection were quantified by electron microscopy as described in methods. IMV, IEV and CEV were counted in 50 randomly chosen sections of infected cells. The values represent percentage of each virus form relative to the total number of virus forms counted. IMV, intracellular mature virus; CEV, cell associated enveloped virus; EEV, extracellular enveloped virus. CPXV-NOHI (A), Rec 1 (B), Rec 2 (C), Rec 3 (D), Rec 3a (E), Rec 3b (F).
Mentions: We carried out detailed analysis of the morphogenesis of the virus strains under study by electron microscopy. Relative and absolute numbers of various mature and immature viral forms were determined at different times post infection. The kinetics of mature virus production in Vero cells is depicted in Figure 6. MVA-HANP results were not included because a very low level of mature virus forms were produced in Vero cells [7]. Assembly of CPXV-NOHI and hybrid progenies was similar to what have been reported for strains of vaccinia virus [7,29,30]. Differences existed in the abundance of mature virus forms produced by CPXV-NOHI and progeny viruses. The intracellular mature virus (IMV) is the major mature virus type produced in CPXV-NOH1 infected cells accounting for 87% of virion forms (IMV, IEV, CEV) at 24 hpi (Figure 6A). Intracellular enveloped virus (IEV) and cell associated enveloped virus (CEV) represented 4% and 9% respectively of virion forms produced in CPXV-NOH1 infected cells (Figure 6A). Similarly, 95% of virions produced by Rec 1 at 24 hpi were IMVs (Figure 6B). However in Rec 2, a higher proportion of IMVs were converted to enveloped forms such that at 24 hpi, CEV is the predominant form representing 55% of virion forms produced (Figure 6C). Rec 3 appeared defective in the production of enveloped virions. At 18 and 24 hpi, less than 1% of Rec 3 virions were IEV or CEV (Figure 6D). Transgene negative derivatives of Rec 3 (Rec 3a and Rec 3b) were efficient in the production of enveloped virions. The enveloped forms (IEV and CEV) accounted for approximately 50% of virion types produced by Rec 3a and Rec 3b respectively at 24 hpi (Figures 6E, F).

Bottom Line: Poxvirus-vectored vaccines against infectious diseases and cancer are currently under development.Analysis of the subcellular localization of the transgenic HA protein showed that neither virus strain nor cell line have effect on the subcellular targets of the HA protein.The influenza virus HA protein was targeted to enveloped virions, plasma membrane, Golgi apparatus and cytoplasmic vesicles.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø, Tromsø, Norway. malachy.okeke@uit.no

ABSTRACT

Background: Poxvirus-vectored vaccines against infectious diseases and cancer are currently under development. We hypothesized that the extensive use of poxvirus-vectored vaccine in future might result in co-infection and recombination between the vaccine virus and naturally occurring poxviruses, resulting in hybrid viruses with unpredictable characteristics. Previously, we confirmed that co-infecting in vitro a Modified vaccinia virus Ankara (MVA) strain engineered to express influenza virus haemagglutinin (HA) and nucleoprotein (NP) genes with a naturally occurring cowpox virus (CPXV-NOH1) resulted in recombinant progeny viruses (H Hansen, MI Okeke, Ø Nilssen, T Traavik, Vaccine 23: 499-506, 2004). In this study we analyzed the biological properties of parental and progeny hybrid viruses.

Results: Five CPXV/MVA progeny viruses were isolated based on plaque phenotype and the expression of influenza virus HA protein. Progeny hybrid viruses displayed in vitro cell line tropism of CPXV-NOH1, but not that of MVA. The HA transgene or its expression was lost on serial passage of transgenic viruses and the speed at which HA expression was lost varied with cell lines. The HA transgene in the progeny viruses or its expression was stable in African Green Monkey derived Vero cells but became unstable in rat derived IEC-6 cells. Hybrid viruses lacking the HA transgene have higher levels of virus multiplication in mammalian cell lines and produced more enveloped virions than the transgene positive progenitor virus strain. Analysis of the subcellular localization of the transgenic HA protein showed that neither virus strain nor cell line have effect on the subcellular targets of the HA protein. The influenza virus HA protein was targeted to enveloped virions, plasma membrane, Golgi apparatus and cytoplasmic vesicles.

Conclusion: Our results suggest that homologous recombination between poxvirus-vectored vaccine and naturally circulating poxviruses, genetic instability of the transgene, accumulation of non-transgene expressing vectors or hybrid virus progenies, as well as cell line/type specific selection against the transgene are potential complications that may result if poxvirus vectored vaccines are extensively used in animals and man.

Show MeSH
Related in: MedlinePlus