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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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Negatively stained purified virions of parental virus strains and hybrid progenies. CPXV-NOHI (A), MVA-HANP (B), Rec 1 (C), Rec 2 (D), Rec 3 (E), Rec 3a (F), Rec 3b (G). Arrows (round virions). Bars, 200 nm (A-G).
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Figure 5: Negatively stained purified virions of parental virus strains and hybrid progenies. CPXV-NOHI (A), MVA-HANP (B), Rec 1 (C), Rec 2 (D), Rec 3 (E), Rec 3a (F), Rec 3b (G). Arrows (round virions). Bars, 200 nm (A-G).

Mentions: The shape and size of negatively stained purified virions were determined in order to ascertain whether there are differences in the virion 2D architecture of the virus strains under study. The results are shown in Figure 5 and Table 2. The virions of CPXV-NOHI were brick shaped measuring 293 ± 27 nm × 229 ± 23 nm in size (Figure 5A, Table 2). Virions of CPXV-NOH1 were slightly smaller than what has been reported for strains of vaccinia virus [26-28]. Conversely, half of the virions of MVA-HANP were brick shaped (314 ± 23 nm × 256 ± 18 nm) while the other half were round shaped with dimensions measuring 255 ± 28 nm × 243 ± 29 nm (Figure 5B, Table 2). Virions obtained from Rec 1, Rec 3, Rec 3a and Rec 3b resemble that of CPXV-NOH1 in being mostly brick shaped. Unlike CPXV-NOH1, a small percentage of virions obtained from the aforementioned progeny viruses have round shape (Figure 5, Table 2). Apparently the virions of Rec 2 appear to be a mixture of what was obtained from the parental strains. Two thirds of Rec 2 virions were brick shaped and the remaining one third were round in shape (Figure 5D, Table 2). The results indicated that the brick shape is the major virion shape in all virus strains except MVA-HANP.


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)

Negatively stained purified virions of parental virus strains and hybrid progenies. CPXV-NOHI (A), MVA-HANP (B), Rec 1 (C), Rec 2 (D), Rec 3 (E), Rec 3a (F), Rec 3b (G). Arrows (round virions). Bars, 200 nm (A-G).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Negatively stained purified virions of parental virus strains and hybrid progenies. CPXV-NOHI (A), MVA-HANP (B), Rec 1 (C), Rec 2 (D), Rec 3 (E), Rec 3a (F), Rec 3b (G). Arrows (round virions). Bars, 200 nm (A-G).
Mentions: The shape and size of negatively stained purified virions were determined in order to ascertain whether there are differences in the virion 2D architecture of the virus strains under study. The results are shown in Figure 5 and Table 2. The virions of CPXV-NOHI were brick shaped measuring 293 ± 27 nm × 229 ± 23 nm in size (Figure 5A, Table 2). Virions of CPXV-NOH1 were slightly smaller than what has been reported for strains of vaccinia virus [26-28]. Conversely, half of the virions of MVA-HANP were brick shaped (314 ± 23 nm × 256 ± 18 nm) while the other half were round shaped with dimensions measuring 255 ± 28 nm × 243 ± 29 nm (Figure 5B, Table 2). Virions obtained from Rec 1, Rec 3, Rec 3a and Rec 3b resemble that of CPXV-NOH1 in being mostly brick shaped. Unlike CPXV-NOH1, a small percentage of virions obtained from the aforementioned progeny viruses have round shape (Figure 5, Table 2). Apparently the virions of Rec 2 appear to be a mixture of what was obtained from the parental strains. Two thirds of Rec 2 virions were brick shaped and the remaining one third were round in shape (Figure 5D, Table 2). The results indicated that the brick shape is the major virion shape in all virus strains except MVA-HANP.

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