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Identification of mammalian-adapting mutations in the polymerase complex of an avian H5N1 influenza virus.

Taft AS, Ozawa M, Fitch A, Depasse JV, Halfmann PJ, Hill-Batorski L, Hatta M, Friedrich TC, Lopes TJ, Maher EA, Ghedin E, Macken CA, Neumann G, Kawaoka Y - Nat Commun (2015)

Bottom Line: Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population.Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus.The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans.

View Article: PubMed Central - PubMed

Affiliation: Influenza Research Institute, School of Veterinary Medicine, Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA.

ABSTRACT
Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population. The factors that enable avian H5N1 influenza viruses to replicate in humans are not completely understood. Here we use a high-throughput screening approach to identify novel mutations in the polymerase genes of an avian H5N1 virus that confer efficient polymerase activity in mammalian cells. Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus. The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans.

No MeSH data available.


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Schematic overview of HA gene-deficient influenza virus used for high-throughput screens.(a) Schematic diagram of influenza virus-like RNA encoding the reporter protein GFP. The GFP coding region is flanked by viral HA sequences that are required for efficient virion incorporation of the vRNA, and by viral regulatory sequences at both ends (indicated by small bars). (b) Generation of HA gene-deficient influenza virus. 293T cells were transfected with plasmids expressing HA, the viral polymerase (PB2, PB1 and PA) proteins and the NP. Cells were co-transfected with plasmids synthesizing all eight vRNAs; the plasmid for the synthesis of wild-type HA was replaced with that encoding GFP (a). Influenza virus encoding GFP can be propagated in MDCK cells expressing HA, but not in normal MDCK cells. (c) Summary of plasmid and virus libraries. AA seq., amino-acid sequence.
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f1: Schematic overview of HA gene-deficient influenza virus used for high-throughput screens.(a) Schematic diagram of influenza virus-like RNA encoding the reporter protein GFP. The GFP coding region is flanked by viral HA sequences that are required for efficient virion incorporation of the vRNA, and by viral regulatory sequences at both ends (indicated by small bars). (b) Generation of HA gene-deficient influenza virus. 293T cells were transfected with plasmids expressing HA, the viral polymerase (PB2, PB1 and PA) proteins and the NP. Cells were co-transfected with plasmids synthesizing all eight vRNAs; the plasmid for the synthesis of wild-type HA was replaced with that encoding GFP (a). Influenza virus encoding GFP can be propagated in MDCK cells expressing HA, but not in normal MDCK cells. (c) Summary of plasmid and virus libraries. AA seq., amino-acid sequence.

Mentions: First, we established a high-throughput screening system to test large numbers of viral polymerase mutants for their ability to confer efficient replication to avian H5N1 viral polymerase complexes in mammalian cells. We generated a biologically contained virus that possesses the viral polymerase (PB2, PB1 and PA) and NP RNA segments of the avian H5N1 A/Muscovy duck/Vietnam/TY93/2007 (TY93/H5N1) virus (Clade 2.3.4), the viral NA, M and NS RNA segments of the laboratory-adapted A/WSN/33 (WSN; H1N1) virus, and a modified WSN HA RNA segment in which most of the open reading frame of HA was replaced with that of the reporter protein green fluorescent protein (GFP; essentially as described before in ref. 23; Fig. 1a). The replication of this virus is restricted to a cell line that stably expresses the HA protein. The TY93/H5N1 PB2 protein of this virus (termed TY93/H5N1 GFP-627E) encodes glutamic acid at position 627, which restricts the replicative ability of avian H5N1 virus polymerase complexes in mammals13. As a control virus, we generated TY93/H5N1 GFP-627K encoding the PB2-E627K mutation known to increase the replicative ability of avian influenza virus polymerase complexes in mammalian cells13. The replication-incompetent TY93/H5N1 GFP-627E and -E627 K viruses can be propagated in cell lines expressing the WSN-HA protein, but do not replicate in wild-type cells (Fig. 1b). The University of Wisconsin-Madison Institutional Biosafety Committee (IBC) approved work with these viruses in BSL-2 containment.


Identification of mammalian-adapting mutations in the polymerase complex of an avian H5N1 influenza virus.

Taft AS, Ozawa M, Fitch A, Depasse JV, Halfmann PJ, Hill-Batorski L, Hatta M, Friedrich TC, Lopes TJ, Maher EA, Ghedin E, Macken CA, Neumann G, Kawaoka Y - Nat Commun (2015)

Schematic overview of HA gene-deficient influenza virus used for high-throughput screens.(a) Schematic diagram of influenza virus-like RNA encoding the reporter protein GFP. The GFP coding region is flanked by viral HA sequences that are required for efficient virion incorporation of the vRNA, and by viral regulatory sequences at both ends (indicated by small bars). (b) Generation of HA gene-deficient influenza virus. 293T cells were transfected with plasmids expressing HA, the viral polymerase (PB2, PB1 and PA) proteins and the NP. Cells were co-transfected with plasmids synthesizing all eight vRNAs; the plasmid for the synthesis of wild-type HA was replaced with that encoding GFP (a). Influenza virus encoding GFP can be propagated in MDCK cells expressing HA, but not in normal MDCK cells. (c) Summary of plasmid and virus libraries. AA seq., amino-acid sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4557292&req=5

f1: Schematic overview of HA gene-deficient influenza virus used for high-throughput screens.(a) Schematic diagram of influenza virus-like RNA encoding the reporter protein GFP. The GFP coding region is flanked by viral HA sequences that are required for efficient virion incorporation of the vRNA, and by viral regulatory sequences at both ends (indicated by small bars). (b) Generation of HA gene-deficient influenza virus. 293T cells were transfected with plasmids expressing HA, the viral polymerase (PB2, PB1 and PA) proteins and the NP. Cells were co-transfected with plasmids synthesizing all eight vRNAs; the plasmid for the synthesis of wild-type HA was replaced with that encoding GFP (a). Influenza virus encoding GFP can be propagated in MDCK cells expressing HA, but not in normal MDCK cells. (c) Summary of plasmid and virus libraries. AA seq., amino-acid sequence.
Mentions: First, we established a high-throughput screening system to test large numbers of viral polymerase mutants for their ability to confer efficient replication to avian H5N1 viral polymerase complexes in mammalian cells. We generated a biologically contained virus that possesses the viral polymerase (PB2, PB1 and PA) and NP RNA segments of the avian H5N1 A/Muscovy duck/Vietnam/TY93/2007 (TY93/H5N1) virus (Clade 2.3.4), the viral NA, M and NS RNA segments of the laboratory-adapted A/WSN/33 (WSN; H1N1) virus, and a modified WSN HA RNA segment in which most of the open reading frame of HA was replaced with that of the reporter protein green fluorescent protein (GFP; essentially as described before in ref. 23; Fig. 1a). The replication of this virus is restricted to a cell line that stably expresses the HA protein. The TY93/H5N1 PB2 protein of this virus (termed TY93/H5N1 GFP-627E) encodes glutamic acid at position 627, which restricts the replicative ability of avian H5N1 virus polymerase complexes in mammals13. As a control virus, we generated TY93/H5N1 GFP-627K encoding the PB2-E627K mutation known to increase the replicative ability of avian influenza virus polymerase complexes in mammalian cells13. The replication-incompetent TY93/H5N1 GFP-627E and -E627 K viruses can be propagated in cell lines expressing the WSN-HA protein, but do not replicate in wild-type cells (Fig. 1b). The University of Wisconsin-Madison Institutional Biosafety Committee (IBC) approved work with these viruses in BSL-2 containment.

Bottom Line: Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population.Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus.The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans.

View Article: PubMed Central - PubMed

Affiliation: Influenza Research Institute, School of Veterinary Medicine, Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53711, USA.

ABSTRACT
Avian influenza viruses of the H5N1 subtype pose a serious global health threat due to the high mortality (>60%) associated with the disease caused by these viruses and the lack of protective antibodies to these viruses in the general population. The factors that enable avian H5N1 influenza viruses to replicate in humans are not completely understood. Here we use a high-throughput screening approach to identify novel mutations in the polymerase genes of an avian H5N1 virus that confer efficient polymerase activity in mammalian cells. Several of the identified mutations (which have previously been found in natural isolates) increase viral replication in mammalian cells and virulence in infected mice compared with the wild-type virus. The identification of amino-acid mutations in avian H5N1 influenza virus polymerase complexes that confer increased replication and virulence in mammals is important for the identification of circulating H5N1 viruses with an increased potential to infect humans.

No MeSH data available.


Related in: MedlinePlus