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Antigenic drift of the pandemic 2009 A(H1N1) influenza virus in A ferret model.

Guarnaccia T, Carolan LA, Maurer-Stroh S, Lee RT, Job E, Reading PC, Petrie S, McCaw JM, McVernon J, Hurt AC, Kelso A, Mosse J, Barr IG, Laurie KL - PLoS Pathog. (2013)

Bottom Line: In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties.This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection.Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance.

View Article: PubMed Central - PubMed

Affiliation: WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, Melbourne, Victoria, Australia.

ABSTRACT
Surveillance data indicate that most circulating A(H1N1)pdm09 influenza viruses have remained antigenically similar since they emerged in humans in 2009. However, antigenic drift is likely to occur in the future in response to increasing population immunity induced by infection or vaccination. In this study, sequential passaging of A(H1N1)pdm09 virus by contact transmission through two independent series of suboptimally vaccinated ferrets resulted in selection of variant viruses with an amino acid substitution (N156K, H1 numbering without signal peptide; N159K, H3 numbering without signal peptide; N173K, H1 numbering from first methionine) in a known antigenic site of the viral HA. The N156K HA variant replicated and transmitted efficiently between naïve ferrets and outgrew wildtype virus in vivo in ferrets in the presence and absence of immune pressure. In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties. The N156K escape mutant was antigenically distinct from wildtype virus as shown by binding of HA-specific antibodies. Glycan binding assays demonstrated the N156K escape mutant had altered receptor binding preferences compared to wildtype virus, which was supported by computational modeling predictions. The N156K substitution, and culture adaptations, have been detected in human A(H1N1)pdm09 viruses with N156K preferentially reported in sequences from original clinical samples rather than cultured isolates. This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection. Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance.

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Growth, detection and adaptation of N156K virus in cell cultures.(A) Virus underwent two passages in MDCK-SIAT1 cells (P1 and P2). Supernatant was collected during both passages, at 24, 48 and 72 h, and virus load was quantified by real time RT-PCR and hemagglutination (HAU). Infected cells were also harvested at 48 h and surface HA and M protein expression measured by flow cytometry. Cell culture adaptations detected by sequencing in the HA protein are shown in each graph. No adaptations in NA were detected. (B) Virus underwent two passages (P1 and P2) in the indicated cell lines. Virus load was quantified in supernatant as above, and cell culture adaptation was detected by sequencing the HA protein as indicated. Limit of detection is 103.8 copies. (C) Location of HA mutations in antigenic regions identified in this study. Visualization of mutation positions relative to classical antigenic sites [6] are shown as colored bubbles (Sa – red; Sb – green; Ca1 – cyan; Ca2 – blue; Cb – yellow). Pink balls indicate a bound host receptor ligand (α-2,6-linked); white balls indicate sugars on glycosylation sites. (D) Location of antigenic sites in HA trimer, top and side view, respectively.
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ppat-1003354-g003: Growth, detection and adaptation of N156K virus in cell cultures.(A) Virus underwent two passages in MDCK-SIAT1 cells (P1 and P2). Supernatant was collected during both passages, at 24, 48 and 72 h, and virus load was quantified by real time RT-PCR and hemagglutination (HAU). Infected cells were also harvested at 48 h and surface HA and M protein expression measured by flow cytometry. Cell culture adaptations detected by sequencing in the HA protein are shown in each graph. No adaptations in NA were detected. (B) Virus underwent two passages (P1 and P2) in the indicated cell lines. Virus load was quantified in supernatant as above, and cell culture adaptation was detected by sequencing the HA protein as indicated. Limit of detection is 103.8 copies. (C) Location of HA mutations in antigenic regions identified in this study. Visualization of mutation positions relative to classical antigenic sites [6] are shown as colored bubbles (Sa – red; Sb – green; Ca1 – cyan; Ca2 – blue; Cb – yellow). Pink balls indicate a bound host receptor ligand (α-2,6-linked); white balls indicate sugars on glycosylation sites. (D) Location of antigenic sites in HA trimer, top and side view, respectively.

Mentions: The N156K mutation arose in both passage lines of MIV+IFA-immunized ferrets (Table 2). Position 156 of HA is in antigenic site Sb [6] (Sa in [31]) (Figure 3C, D). The relative proportions of the N156K mutant and wildtype N156 viruses were quantified in samples from both passage lines of MIV+IFA-immunized ferrets using a pyrosequencing assay. The N156K mutant emerged in R4 of line A, persisted at a similar proportion in R5, and became dominant by R6. In line B, the N156K mutant emerged earlier, in R1, and became dominant in R2, persisting through to R7 (Figure 2D). The emergence of the N156K mutation did not result in a change of virus kinetics (peak viral load, growth rate or serial interval) compared to N156 wildtype virus (data not shown).


Antigenic drift of the pandemic 2009 A(H1N1) influenza virus in A ferret model.

Guarnaccia T, Carolan LA, Maurer-Stroh S, Lee RT, Job E, Reading PC, Petrie S, McCaw JM, McVernon J, Hurt AC, Kelso A, Mosse J, Barr IG, Laurie KL - PLoS Pathog. (2013)

Growth, detection and adaptation of N156K virus in cell cultures.(A) Virus underwent two passages in MDCK-SIAT1 cells (P1 and P2). Supernatant was collected during both passages, at 24, 48 and 72 h, and virus load was quantified by real time RT-PCR and hemagglutination (HAU). Infected cells were also harvested at 48 h and surface HA and M protein expression measured by flow cytometry. Cell culture adaptations detected by sequencing in the HA protein are shown in each graph. No adaptations in NA were detected. (B) Virus underwent two passages (P1 and P2) in the indicated cell lines. Virus load was quantified in supernatant as above, and cell culture adaptation was detected by sequencing the HA protein as indicated. Limit of detection is 103.8 copies. (C) Location of HA mutations in antigenic regions identified in this study. Visualization of mutation positions relative to classical antigenic sites [6] are shown as colored bubbles (Sa – red; Sb – green; Ca1 – cyan; Ca2 – blue; Cb – yellow). Pink balls indicate a bound host receptor ligand (α-2,6-linked); white balls indicate sugars on glycosylation sites. (D) Location of antigenic sites in HA trimer, top and side view, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3649996&req=5

ppat-1003354-g003: Growth, detection and adaptation of N156K virus in cell cultures.(A) Virus underwent two passages in MDCK-SIAT1 cells (P1 and P2). Supernatant was collected during both passages, at 24, 48 and 72 h, and virus load was quantified by real time RT-PCR and hemagglutination (HAU). Infected cells were also harvested at 48 h and surface HA and M protein expression measured by flow cytometry. Cell culture adaptations detected by sequencing in the HA protein are shown in each graph. No adaptations in NA were detected. (B) Virus underwent two passages (P1 and P2) in the indicated cell lines. Virus load was quantified in supernatant as above, and cell culture adaptation was detected by sequencing the HA protein as indicated. Limit of detection is 103.8 copies. (C) Location of HA mutations in antigenic regions identified in this study. Visualization of mutation positions relative to classical antigenic sites [6] are shown as colored bubbles (Sa – red; Sb – green; Ca1 – cyan; Ca2 – blue; Cb – yellow). Pink balls indicate a bound host receptor ligand (α-2,6-linked); white balls indicate sugars on glycosylation sites. (D) Location of antigenic sites in HA trimer, top and side view, respectively.
Mentions: The N156K mutation arose in both passage lines of MIV+IFA-immunized ferrets (Table 2). Position 156 of HA is in antigenic site Sb [6] (Sa in [31]) (Figure 3C, D). The relative proportions of the N156K mutant and wildtype N156 viruses were quantified in samples from both passage lines of MIV+IFA-immunized ferrets using a pyrosequencing assay. The N156K mutant emerged in R4 of line A, persisted at a similar proportion in R5, and became dominant by R6. In line B, the N156K mutant emerged earlier, in R1, and became dominant in R2, persisting through to R7 (Figure 2D). The emergence of the N156K mutation did not result in a change of virus kinetics (peak viral load, growth rate or serial interval) compared to N156 wildtype virus (data not shown).

Bottom Line: In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties.This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection.Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance.

View Article: PubMed Central - PubMed

Affiliation: WHO Collaborating Centre for Reference and Research on Influenza, Victorian Infectious Diseases Reference Laboratory, Melbourne, Victoria, Australia.

ABSTRACT
Surveillance data indicate that most circulating A(H1N1)pdm09 influenza viruses have remained antigenically similar since they emerged in humans in 2009. However, antigenic drift is likely to occur in the future in response to increasing population immunity induced by infection or vaccination. In this study, sequential passaging of A(H1N1)pdm09 virus by contact transmission through two independent series of suboptimally vaccinated ferrets resulted in selection of variant viruses with an amino acid substitution (N156K, H1 numbering without signal peptide; N159K, H3 numbering without signal peptide; N173K, H1 numbering from first methionine) in a known antigenic site of the viral HA. The N156K HA variant replicated and transmitted efficiently between naïve ferrets and outgrew wildtype virus in vivo in ferrets in the presence and absence of immune pressure. In vitro, in a range of cell culture systems, the N156K variant rapidly adapted, acquiring additional mutations in the viral HA that also potentially affected antigenic properties. The N156K escape mutant was antigenically distinct from wildtype virus as shown by binding of HA-specific antibodies. Glycan binding assays demonstrated the N156K escape mutant had altered receptor binding preferences compared to wildtype virus, which was supported by computational modeling predictions. The N156K substitution, and culture adaptations, have been detected in human A(H1N1)pdm09 viruses with N156K preferentially reported in sequences from original clinical samples rather than cultured isolates. This study demonstrates the ability of the A(H1N1)pdm09 virus to undergo rapid antigenic change to evade a low level vaccine response, while remaining fit in a ferret transmission model of immunization and infection. Furthermore, the potential changes in receptor binding properties that accompany antigenic changes highlight the importance of routine characterization of clinical samples in human A(H1N1)pdm09 influenza surveillance.

Show MeSH
Related in: MedlinePlus