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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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Plaque phenotypes of parental virus strains and hybrid virus progenies in IEC-6 cells. Confluent IEC-6 cells were infected with the respective viruses and the HA expression was monitored at 36 hpi by immunoperoxidase staining of fixed cells. The panels show representative fields at approximately × 200 magnification. CPXV-NOHI (A), MVA-HANP (B), Rec 1(C), Rec 2 (D), Rec 3 (E), Rec 3a (F), Rec 3b (G).
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Figure 2: Plaque phenotypes of parental virus strains and hybrid virus progenies in IEC-6 cells. Confluent IEC-6 cells were infected with the respective viruses and the HA expression was monitored at 36 hpi by immunoperoxidase staining of fixed cells. The panels show representative fields at approximately × 200 magnification. CPXV-NOHI (A), MVA-HANP (B), Rec 1(C), Rec 2 (D), Rec 3 (E), Rec 3a (F), Rec 3b (G).

Mentions: CPXV-NOHI produced low, moderate and high CPE in one, seven and five cell lines respectively (Table 1). MVA-HANP gave low or no CPE in all the cell lines except BHK-21 and IEC-6 cells where it produced high and moderate CPE, respectively. Rec 1 had moderate to very high CPE in all the cell lines tested except Caco-2. Compared to parental CPXV-NOH1, Rec 1 showed enhanced CPE in seven cell lines (Table 1). Similarly, Rec 2 resulted in higher CPE in most cell lines compared to parental CPXV-NOHI. Conversely, Rec 3 resulted in lower CPE compared to CPXV-NOHI in most cell lines except RK-13 and CHO-KI cells. Interestingly, transgene negative derivatives of Rec 3 (Rec 3a and Rec 3b) produced higher CPE in many cell lines compared to the transgene positive Rec 3 (Table 1). In particular, Rec 3b is the most cytopathogenic of virus strains investigated in this study. The plaque phenotypes of parental and progeny viruses were examined in thirteen mammalian cell lines. Previously, we have reported the plaque phenotypes of these viruses in Vero cells [6]. However, MVA does not form distinct plaques in Vero cells and thus comparison of plaque phenotypes of hybrid viruses was made only with the parental CPXV-NOH1 [6]. Therefore, we re-examined plaque phenotypes of parental and hybrid viruses in rat IEC-6, a cell line in which MVA forms very clear plaques [7]. CPXV-NOH1 produced large lytic plaques in IEC-6 cells (Figure 2A) and the other twelve cell lines (data not shown). In permissive IEC-6 cells, MVA-HANP plaques were small, non-lytic with characteristic comet (satellite) formation (Figure 2B). The plaque phenotype of Rec 1 in IEC-6 cells (Figure 2C) and other cell lines (data not shown) was similar to CPXV-NOHI except that plaques were larger in size. Rec 2 produced small plaques and comets in IEC-6 cells (Figure 2D). Comet formation was enhanced in Rec 2 compared to MVA-HANP although the size of the primary plaque is larger in the latter. Rec 3 produced large semi-lytic plaques with some undetached cells in the center of the plaque (Figure 2E). Rec 3a plaques were very large and lytic (Figure 2F). Rec 3b produced the largest plaque size in IEC-6 cells (Fig. 2G) and other cell lines and its plaques were characterized by high level of cell detachment and syncytia formation. Taken in tandem, the progeny viruses displayed parental and non-parental characteristics with respect to in vitro host range, CPE and plaque phenotypes.


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)

Plaque phenotypes of parental virus strains and hybrid virus progenies in IEC-6 cells. Confluent IEC-6 cells were infected with the respective viruses and the HA expression was monitored at 36 hpi by immunoperoxidase staining of fixed cells. The panels show representative fields at approximately × 200 magnification. CPXV-NOHI (A), MVA-HANP (B), Rec 1(C), Rec 2 (D), Rec 3 (E), Rec 3a (F), Rec 3b (G).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Plaque phenotypes of parental virus strains and hybrid virus progenies in IEC-6 cells. Confluent IEC-6 cells were infected with the respective viruses and the HA expression was monitored at 36 hpi by immunoperoxidase staining of fixed cells. The panels show representative fields at approximately × 200 magnification. CPXV-NOHI (A), MVA-HANP (B), Rec 1(C), Rec 2 (D), Rec 3 (E), Rec 3a (F), Rec 3b (G).
Mentions: CPXV-NOHI produced low, moderate and high CPE in one, seven and five cell lines respectively (Table 1). MVA-HANP gave low or no CPE in all the cell lines except BHK-21 and IEC-6 cells where it produced high and moderate CPE, respectively. Rec 1 had moderate to very high CPE in all the cell lines tested except Caco-2. Compared to parental CPXV-NOH1, Rec 1 showed enhanced CPE in seven cell lines (Table 1). Similarly, Rec 2 resulted in higher CPE in most cell lines compared to parental CPXV-NOHI. Conversely, Rec 3 resulted in lower CPE compared to CPXV-NOHI in most cell lines except RK-13 and CHO-KI cells. Interestingly, transgene negative derivatives of Rec 3 (Rec 3a and Rec 3b) produced higher CPE in many cell lines compared to the transgene positive Rec 3 (Table 1). In particular, Rec 3b is the most cytopathogenic of virus strains investigated in this study. The plaque phenotypes of parental and progeny viruses were examined in thirteen mammalian cell lines. Previously, we have reported the plaque phenotypes of these viruses in Vero cells [6]. However, MVA does not form distinct plaques in Vero cells and thus comparison of plaque phenotypes of hybrid viruses was made only with the parental CPXV-NOH1 [6]. Therefore, we re-examined plaque phenotypes of parental and hybrid viruses in rat IEC-6, a cell line in which MVA forms very clear plaques [7]. CPXV-NOH1 produced large lytic plaques in IEC-6 cells (Figure 2A) and the other twelve cell lines (data not shown). In permissive IEC-6 cells, MVA-HANP plaques were small, non-lytic with characteristic comet (satellite) formation (Figure 2B). The plaque phenotype of Rec 1 in IEC-6 cells (Figure 2C) and other cell lines (data not shown) was similar to CPXV-NOHI except that plaques were larger in size. Rec 2 produced small plaques and comets in IEC-6 cells (Figure 2D). Comet formation was enhanced in Rec 2 compared to MVA-HANP although the size of the primary plaque is larger in the latter. Rec 3 produced large semi-lytic plaques with some undetached cells in the center of the plaque (Figure 2E). Rec 3a plaques were very large and lytic (Figure 2F). Rec 3b produced the largest plaque size in IEC-6 cells (Fig. 2G) and other cell lines and its plaques were characterized by high level of cell detachment and syncytia formation. Taken in tandem, the progeny viruses displayed parental and non-parental characteristics with respect to in vitro host range, CPE and plaque phenotypes.

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