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Does pandemic A/H1N1 virus have the potential to become more pathogenic?

Ilyushina NA, Ducatez MF, Rehg JE, Marathe BM, Marjuki H, Bovin NV, Webster RG, Webby RJ - MBio (2010)

Bottom Line: When the pandemic virus was initially present at multiplicities of infection equal to or greater than those for the seasonal virus, only pandemic virus genotypes were detected.These adapted pandemic strains did, however, contain two nonsynonymous mutations (hemagglutinin K154Q and polymerase acidic protein L295P) that conferred a more virulent phenotype, both in cell cultures and in ferrets, than their parental strains.Our study demonstrates that the emergence of an A/H1N1 pandemic strain of higher virulence is possible and that, despite their lack of detection thus far in humans, viable seasonal/pandemic virus reassortants can be generated.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

ABSTRACT
Epidemiologic observations that have been made in the context of the current pandemic influenza virus include a stable virulence phenotype and a lack of propensity to reassort with seasonal strains. In an attempt to determine whether either of these observations could change in the future, we coinfected differentiated human airway cells with seasonal oseltamivir-resistant A/New Jersey/15/07 and pandemic A/Tennessee/1-560/09 (H1N1) viruses in three ratios (10:90, 50:50, and 90:10) and examined the resulting progeny viruses after 10 sequential passages. When the pandemic virus was initially present at multiplicities of infection equal to or greater than those for the seasonal virus, only pandemic virus genotypes were detected. These adapted pandemic strains did, however, contain two nonsynonymous mutations (hemagglutinin K154Q and polymerase acidic protein L295P) that conferred a more virulent phenotype, both in cell cultures and in ferrets, than their parental strains. The polymerase acidic protein mutation increased polymerase activity at 37°C, and the hemagglutinin change affected binding of the virus to α2,6-sialyl receptors. When the seasonal A/H1N1 virus was initially present in excess, the dominant progeny virus was a reassortant containing the hemagglutinin gene from the seasonal strain and the remaining genes from the pandemic virus. Our study demonstrates that the emergence of an A/H1N1 pandemic strain of higher virulence is possible and that, despite their lack of detection thus far in humans, viable seasonal/pandemic virus reassortants can be generated.

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Related in: MedlinePlus

Polymerase activity and receptor specificity of H1N1 influenza viruses. (A) Polymerase activity of ribonucleoprotein complexes of TN/560, G1, and G2 (H1N1) viruses as determined by a dual-luciferase reporter assay in three independent experiments. 293T cells were transfected in triplicate with luciferase and Renilla reporter plasmids, together with plasmids expressing PB2, PB1, PA, and NP from either TN/560, G1, or G2 viruses. Cells were incubated at 33°C (orange bars), 37°C (red bars), or 39°C (purple bars) for 24 h, and cell lysates were analyzed to measure firefly luciferase and Renilla activities. The latter was used to normalize transfection efficiency. Values shown represent the activities of each ribonucleoprotein (RNP) complex relative to that of TN/560. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA). (B) Receptor specificity of NJ/15, TN/560, G1, and G2 (H1N1) influenza viruses. Association constants (Kass) of virus complexes with “human-type” sialylglycopolymers conjugated to 6′-SL and 6′-SLN are shown. Higher Kass values indicate stronger binding. Values are the means from four independent experiments. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA).
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f3: Polymerase activity and receptor specificity of H1N1 influenza viruses. (A) Polymerase activity of ribonucleoprotein complexes of TN/560, G1, and G2 (H1N1) viruses as determined by a dual-luciferase reporter assay in three independent experiments. 293T cells were transfected in triplicate with luciferase and Renilla reporter plasmids, together with plasmids expressing PB2, PB1, PA, and NP from either TN/560, G1, or G2 viruses. Cells were incubated at 33°C (orange bars), 37°C (red bars), or 39°C (purple bars) for 24 h, and cell lysates were analyzed to measure firefly luciferase and Renilla activities. The latter was used to normalize transfection efficiency. Values shown represent the activities of each ribonucleoprotein (RNP) complex relative to that of TN/560. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA). (B) Receptor specificity of NJ/15, TN/560, G1, and G2 (H1N1) influenza viruses. Association constants (Kass) of virus complexes with “human-type” sialylglycopolymers conjugated to 6′-SL and 6′-SLN are shown. Higher Kass values indicate stronger binding. Values are the means from four independent experiments. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA).

Mentions: A luciferase minigenome assay was used to study the role of ribonucleoprotein (RNP) complex mutations in the viral replication, comparing G1 and G2 to TN/560. The G1 RNP complex, carrying the PA L295P mutation, showed ~20% more polymerase activity than TN/560 at 37°C (and comparable activities at 33°C and 39°C) (Fig. 3A). The G2 RNP complex also had a significantly higher polymerase activity at 33°C (+30%) but a slightly lower one at 39°C (−10%) than TN/560 RNP (P < 0.05) (Fig. 3A). Interestingly, growth curves at 33°C showed that both G1 and G2 grew faster than TN/560 at later time points (Fig. 2B), suggesting an advantage for the adapted viruses for replication in the upper respiratory tract. At least in the case of G2, it appears that the PB2 D87N mutation may play a role in viral replication and that this mutation functions by increasing polymerase activity. The discrepancy between comparative growth curve and polymerase assay data at 33°C for G1, however, clearly shows that polymerase activity is not the sole reason for increased viral production from human airway cells, at least at this temperature.


Does pandemic A/H1N1 virus have the potential to become more pathogenic?

Ilyushina NA, Ducatez MF, Rehg JE, Marathe BM, Marjuki H, Bovin NV, Webster RG, Webby RJ - MBio (2010)

Polymerase activity and receptor specificity of H1N1 influenza viruses. (A) Polymerase activity of ribonucleoprotein complexes of TN/560, G1, and G2 (H1N1) viruses as determined by a dual-luciferase reporter assay in three independent experiments. 293T cells were transfected in triplicate with luciferase and Renilla reporter plasmids, together with plasmids expressing PB2, PB1, PA, and NP from either TN/560, G1, or G2 viruses. Cells were incubated at 33°C (orange bars), 37°C (red bars), or 39°C (purple bars) for 24 h, and cell lysates were analyzed to measure firefly luciferase and Renilla activities. The latter was used to normalize transfection efficiency. Values shown represent the activities of each ribonucleoprotein (RNP) complex relative to that of TN/560. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA). (B) Receptor specificity of NJ/15, TN/560, G1, and G2 (H1N1) influenza viruses. Association constants (Kass) of virus complexes with “human-type” sialylglycopolymers conjugated to 6′-SL and 6′-SLN are shown. Higher Kass values indicate stronger binding. Values are the means from four independent experiments. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2993010&req=5

f3: Polymerase activity and receptor specificity of H1N1 influenza viruses. (A) Polymerase activity of ribonucleoprotein complexes of TN/560, G1, and G2 (H1N1) viruses as determined by a dual-luciferase reporter assay in three independent experiments. 293T cells were transfected in triplicate with luciferase and Renilla reporter plasmids, together with plasmids expressing PB2, PB1, PA, and NP from either TN/560, G1, or G2 viruses. Cells were incubated at 33°C (orange bars), 37°C (red bars), or 39°C (purple bars) for 24 h, and cell lysates were analyzed to measure firefly luciferase and Renilla activities. The latter was used to normalize transfection efficiency. Values shown represent the activities of each ribonucleoprotein (RNP) complex relative to that of TN/560. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA). (B) Receptor specificity of NJ/15, TN/560, G1, and G2 (H1N1) influenza viruses. Association constants (Kass) of virus complexes with “human-type” sialylglycopolymers conjugated to 6′-SL and 6′-SLN are shown. Higher Kass values indicate stronger binding. Values are the means from four independent experiments. *, P < 0.05; °, P <0.01 compared with the value for TN/560 virus (one-way ANOVA).
Mentions: A luciferase minigenome assay was used to study the role of ribonucleoprotein (RNP) complex mutations in the viral replication, comparing G1 and G2 to TN/560. The G1 RNP complex, carrying the PA L295P mutation, showed ~20% more polymerase activity than TN/560 at 37°C (and comparable activities at 33°C and 39°C) (Fig. 3A). The G2 RNP complex also had a significantly higher polymerase activity at 33°C (+30%) but a slightly lower one at 39°C (−10%) than TN/560 RNP (P < 0.05) (Fig. 3A). Interestingly, growth curves at 33°C showed that both G1 and G2 grew faster than TN/560 at later time points (Fig. 2B), suggesting an advantage for the adapted viruses for replication in the upper respiratory tract. At least in the case of G2, it appears that the PB2 D87N mutation may play a role in viral replication and that this mutation functions by increasing polymerase activity. The discrepancy between comparative growth curve and polymerase assay data at 33°C for G1, however, clearly shows that polymerase activity is not the sole reason for increased viral production from human airway cells, at least at this temperature.

Bottom Line: When the pandemic virus was initially present at multiplicities of infection equal to or greater than those for the seasonal virus, only pandemic virus genotypes were detected.These adapted pandemic strains did, however, contain two nonsynonymous mutations (hemagglutinin K154Q and polymerase acidic protein L295P) that conferred a more virulent phenotype, both in cell cultures and in ferrets, than their parental strains.Our study demonstrates that the emergence of an A/H1N1 pandemic strain of higher virulence is possible and that, despite their lack of detection thus far in humans, viable seasonal/pandemic virus reassortants can be generated.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.

ABSTRACT
Epidemiologic observations that have been made in the context of the current pandemic influenza virus include a stable virulence phenotype and a lack of propensity to reassort with seasonal strains. In an attempt to determine whether either of these observations could change in the future, we coinfected differentiated human airway cells with seasonal oseltamivir-resistant A/New Jersey/15/07 and pandemic A/Tennessee/1-560/09 (H1N1) viruses in three ratios (10:90, 50:50, and 90:10) and examined the resulting progeny viruses after 10 sequential passages. When the pandemic virus was initially present at multiplicities of infection equal to or greater than those for the seasonal virus, only pandemic virus genotypes were detected. These adapted pandemic strains did, however, contain two nonsynonymous mutations (hemagglutinin K154Q and polymerase acidic protein L295P) that conferred a more virulent phenotype, both in cell cultures and in ferrets, than their parental strains. The polymerase acidic protein mutation increased polymerase activity at 37°C, and the hemagglutinin change affected binding of the virus to α2,6-sialyl receptors. When the seasonal A/H1N1 virus was initially present in excess, the dominant progeny virus was a reassortant containing the hemagglutinin gene from the seasonal strain and the remaining genes from the pandemic virus. Our study demonstrates that the emergence of an A/H1N1 pandemic strain of higher virulence is possible and that, despite their lack of detection thus far in humans, viable seasonal/pandemic virus reassortants can be generated.

Show MeSH
Related in: MedlinePlus