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Generation and characterization of influenza A viruses with altered polymerase fidelity.

Cheung PP, Watson SJ, Choy KT, Fun Sia S, Wong DD, Poon LL, Kellam P, Guan Y, Malik Peiris JS, Yen HL - Nat Commun (2014)

Bottom Line: We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses.However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice.Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Li Ka Shing Faculty of Medicine, Centre of Influenza Research, School of Public Health, The University of Hong Kong, No. 21 Sassoon Road, Pokfulam, Hong Kong SAR, China.

ABSTRACT
Genetic diversity of influenza A viruses (IAV) acquired through the error-prone RNA-dependent RNA polymerase (RdRP) or through genetic reassortment enables perpetuation of IAV in humans through epidemics or pandemics. Here, to assess the biological significance of genetic diversity acquired through RdRP, we characterize an IAV fidelity variant derived from passaging a seasonal H3N2 virus in the presence of ribavirin, a purine analogue that increases guanosine-to-adenosine mutations. We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses. The H5N1 PB1-V43I-recombinant virus replicates to comparable titres as the wild-type virus in vitro or in the mouse lungs. However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice. Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

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The mutagenic effect of ribavirin on IAV genome(a) Frequencies of nucleotide substitutions in the HA1 gene (nt 140 to 883, numbering from atg) of Wuhan95 viruses that were serial passaged four times in the absence (no drug control) or presence (N6) of 35 μM ribavirin in MDCK cells. Six clones for N6 and two clones for no drug control were analyzed. Nucleotide changes from letters in the first column to those in the first row were shown, with the most frequent changes highlighted in the black boxes. Increased G-to-A and C-to-T mutations were noted in N6 after 4 passages in the presence of ribavirin (Fisher’s exact test, P=0.0010). Fisher exact test was based on a 2×2 contingency table constructed to compare the relative number of G-to-A mutations to A-to-G mutations between wild-type and V43I viruses (b) Ribavirin competition assay with nucleoside guanosine was performed with the Wuhan95 virus at MOI=0.1 in MDCK cells under three different conditions: no drug treatment, 40 μM of ribavirin, and 40 μM ribavirin plus 20 μM guanosine. Culture supernatants were harvested at 48h post-infection to determine viral titers (log10 TCID50 per mL). Student’s t-test was performed to compare the difference in viral titres between different treatment groups. The mean viral titers ± standard deviation from triplicates derived from one out of two independent experiments are shown.
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Figure 1: The mutagenic effect of ribavirin on IAV genome(a) Frequencies of nucleotide substitutions in the HA1 gene (nt 140 to 883, numbering from atg) of Wuhan95 viruses that were serial passaged four times in the absence (no drug control) or presence (N6) of 35 μM ribavirin in MDCK cells. Six clones for N6 and two clones for no drug control were analyzed. Nucleotide changes from letters in the first column to those in the first row were shown, with the most frequent changes highlighted in the black boxes. Increased G-to-A and C-to-T mutations were noted in N6 after 4 passages in the presence of ribavirin (Fisher’s exact test, P=0.0010). Fisher exact test was based on a 2×2 contingency table constructed to compare the relative number of G-to-A mutations to A-to-G mutations between wild-type and V43I viruses (b) Ribavirin competition assay with nucleoside guanosine was performed with the Wuhan95 virus at MOI=0.1 in MDCK cells under three different conditions: no drug treatment, 40 μM of ribavirin, and 40 μM ribavirin plus 20 μM guanosine. Culture supernatants were harvested at 48h post-infection to determine viral titers (log10 TCID50 per mL). Student’s t-test was performed to compare the difference in viral titres between different treatment groups. The mean viral titers ± standard deviation from triplicates derived from one out of two independent experiments are shown.

Mentions: We first determined if ribavirin can induce mutagenesis in the IAV genome. To determine the effect of ribavirin on IAV genomic mutational frequencies, recombinant virus Wuhan95 (H3N2) was passaged four times in the presence (35 μM) or absence of ribavirin and the HA1 gene was analyzed by clonal sequencing. After four serial passages in vitro, ribavirin increased genomic mutation frequency from 3.69 (25 out of 67,704) to 15.74 (89 out of 56,544) per 104 nucleotide sequenced (Fisher’s exact test, P<0.0001; Supplementary Table 1), predominantly with the characteristic G-to-A, and complementary C-to-T mutations (Fisher’s exact test, P=0.001; Fig. 1a). This observation suggested that the purine analogue ribavirin can cause mutagenesis in the IAV genome. We then evaluated if addition of guanosine may compete with ribavirin and rescue viral replication. First, the 50% cytotoxic concentration (CC50) of guanosine and ribavirin in MDCK cells were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to be 122.00 ± 0.11 μM (mean CC50±standard error) (Supplementary Fig. 1a) and 352.00 ± 0.04 μM, respectively (Supplementary Fig. 1b). In the presence of 40 μM ribavirin, we observed that the addition of 20 μM guanosine reversed resistance of Wuhan 95 to ribavirin (Fig. 1b). This confirms previous observations that guanosine can reverse the antiviral activity of ribavirin at non-cytotoxic concentrations for influenza virus 15–18. Thus, although ribavirin can inhibit RdRP directly 16, our data confirms that ribavirin can also cause mutagenesis in the IAV genome as a purine analog.


Generation and characterization of influenza A viruses with altered polymerase fidelity.

Cheung PP, Watson SJ, Choy KT, Fun Sia S, Wong DD, Poon LL, Kellam P, Guan Y, Malik Peiris JS, Yen HL - Nat Commun (2014)

The mutagenic effect of ribavirin on IAV genome(a) Frequencies of nucleotide substitutions in the HA1 gene (nt 140 to 883, numbering from atg) of Wuhan95 viruses that were serial passaged four times in the absence (no drug control) or presence (N6) of 35 μM ribavirin in MDCK cells. Six clones for N6 and two clones for no drug control were analyzed. Nucleotide changes from letters in the first column to those in the first row were shown, with the most frequent changes highlighted in the black boxes. Increased G-to-A and C-to-T mutations were noted in N6 after 4 passages in the presence of ribavirin (Fisher’s exact test, P=0.0010). Fisher exact test was based on a 2×2 contingency table constructed to compare the relative number of G-to-A mutations to A-to-G mutations between wild-type and V43I viruses (b) Ribavirin competition assay with nucleoside guanosine was performed with the Wuhan95 virus at MOI=0.1 in MDCK cells under three different conditions: no drug treatment, 40 μM of ribavirin, and 40 μM ribavirin plus 20 μM guanosine. Culture supernatants were harvested at 48h post-infection to determine viral titers (log10 TCID50 per mL). Student’s t-test was performed to compare the difference in viral titres between different treatment groups. The mean viral titers ± standard deviation from triplicates derived from one out of two independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4155405&req=5

Figure 1: The mutagenic effect of ribavirin on IAV genome(a) Frequencies of nucleotide substitutions in the HA1 gene (nt 140 to 883, numbering from atg) of Wuhan95 viruses that were serial passaged four times in the absence (no drug control) or presence (N6) of 35 μM ribavirin in MDCK cells. Six clones for N6 and two clones for no drug control were analyzed. Nucleotide changes from letters in the first column to those in the first row were shown, with the most frequent changes highlighted in the black boxes. Increased G-to-A and C-to-T mutations were noted in N6 after 4 passages in the presence of ribavirin (Fisher’s exact test, P=0.0010). Fisher exact test was based on a 2×2 contingency table constructed to compare the relative number of G-to-A mutations to A-to-G mutations between wild-type and V43I viruses (b) Ribavirin competition assay with nucleoside guanosine was performed with the Wuhan95 virus at MOI=0.1 in MDCK cells under three different conditions: no drug treatment, 40 μM of ribavirin, and 40 μM ribavirin plus 20 μM guanosine. Culture supernatants were harvested at 48h post-infection to determine viral titers (log10 TCID50 per mL). Student’s t-test was performed to compare the difference in viral titres between different treatment groups. The mean viral titers ± standard deviation from triplicates derived from one out of two independent experiments are shown.
Mentions: We first determined if ribavirin can induce mutagenesis in the IAV genome. To determine the effect of ribavirin on IAV genomic mutational frequencies, recombinant virus Wuhan95 (H3N2) was passaged four times in the presence (35 μM) or absence of ribavirin and the HA1 gene was analyzed by clonal sequencing. After four serial passages in vitro, ribavirin increased genomic mutation frequency from 3.69 (25 out of 67,704) to 15.74 (89 out of 56,544) per 104 nucleotide sequenced (Fisher’s exact test, P<0.0001; Supplementary Table 1), predominantly with the characteristic G-to-A, and complementary C-to-T mutations (Fisher’s exact test, P=0.001; Fig. 1a). This observation suggested that the purine analogue ribavirin can cause mutagenesis in the IAV genome. We then evaluated if addition of guanosine may compete with ribavirin and rescue viral replication. First, the 50% cytotoxic concentration (CC50) of guanosine and ribavirin in MDCK cells were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to be 122.00 ± 0.11 μM (mean CC50±standard error) (Supplementary Fig. 1a) and 352.00 ± 0.04 μM, respectively (Supplementary Fig. 1b). In the presence of 40 μM ribavirin, we observed that the addition of 20 μM guanosine reversed resistance of Wuhan 95 to ribavirin (Fig. 1b). This confirms previous observations that guanosine can reverse the antiviral activity of ribavirin at non-cytotoxic concentrations for influenza virus 15–18. Thus, although ribavirin can inhibit RdRP directly 16, our data confirms that ribavirin can also cause mutagenesis in the IAV genome as a purine analog.

Bottom Line: We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses.However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice.Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Li Ka Shing Faculty of Medicine, Centre of Influenza Research, School of Public Health, The University of Hong Kong, No. 21 Sassoon Road, Pokfulam, Hong Kong SAR, China.

ABSTRACT
Genetic diversity of influenza A viruses (IAV) acquired through the error-prone RNA-dependent RNA polymerase (RdRP) or through genetic reassortment enables perpetuation of IAV in humans through epidemics or pandemics. Here, to assess the biological significance of genetic diversity acquired through RdRP, we characterize an IAV fidelity variant derived from passaging a seasonal H3N2 virus in the presence of ribavirin, a purine analogue that increases guanosine-to-adenosine mutations. We demonstrate that a single PB1-V43I mutation increases selectivity to guanosine in A/Wuhan/359/95 (H3N2) and A/Vietnam/1203/04 (H5N1) viruses. The H5N1 PB1-V43I-recombinant virus replicates to comparable titres as the wild-type virus in vitro or in the mouse lungs. However, a decrease in viral population diversity at day 3 post inoculation is associated with a tenfold reduced lethality and neurotropism in mice. Applying a fidelity variant with reduced mutational frequency, we provide direct experimental evidence for the role of genetic diversity in IAV pathogenesis.

Show MeSH
Related in: MedlinePlus