Limits...
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.

Show MeSH

Related in: MedlinePlus

Virus replication and transmission kinetics in ferret passage lines and emergence and persistence of the N156K mutant.(A–C) Nasal washes were collected daily from ferrets from passage lines (R0–R7) and assayed for viral RNA by real time RT-PCR as a measure of infection and transmission. (A) The peak viral load detected in the nasal wash from each ferret, (B) the viral growth rate ((peak virus load – first day virus load)/days to reach peak) and (C) the serial interval of virus transmission between ferrets (number of days between detection of infected and infecting animals) were determined for each immunization line. Ferrets artificially infected by intranasal virus inoculation are not included. Ferrets that failed to be naturally infected by contact transmission are indicated in white circles. Statistics do not include ferrets that failed to be naturally infected. (D–E) The proportion of N156 wildtype (black) and N156K (white) viruses in peak day nasal wash samples from all ferrets from the MIV+IFA (D) and N156K naïve (E) passage lines quantified by pyrosequencing. (F–G) Mixtures of (F) N156 wildtype and N156K or (G) N156K and K142N+N156K viruses were passaged by contact transmission through naïve ferrets. The proportion of N156 wildtype (black), N156K (white) or K142N+N156K (striped) in peak day nasal wash samples was quantified by pyrosequencing.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3649996&req=5

ppat-1003354-g002: Virus replication and transmission kinetics in ferret passage lines and emergence and persistence of the N156K mutant.(A–C) Nasal washes were collected daily from ferrets from passage lines (R0–R7) and assayed for viral RNA by real time RT-PCR as a measure of infection and transmission. (A) The peak viral load detected in the nasal wash from each ferret, (B) the viral growth rate ((peak virus load – first day virus load)/days to reach peak) and (C) the serial interval of virus transmission between ferrets (number of days between detection of infected and infecting animals) were determined for each immunization line. Ferrets artificially infected by intranasal virus inoculation are not included. Ferrets that failed to be naturally infected by contact transmission are indicated in white circles. Statistics do not include ferrets that failed to be naturally infected. (D–E) The proportion of N156 wildtype (black) and N156K (white) viruses in peak day nasal wash samples from all ferrets from the MIV+IFA (D) and N156K naïve (E) passage lines quantified by pyrosequencing. (F–G) Mixtures of (F) N156 wildtype and N156K or (G) N156K and K142N+N156K viruses were passaged by contact transmission through naïve ferrets. The proportion of N156 wildtype (black), N156K (white) or K142N+N156K (striped) in peak day nasal wash samples was quantified by pyrosequencing.

Mentions: The virus kinetics in all passage lines was assessed using real time RT-PCR to determine the relative copy number. There was no difference in the peak viral load achieved in naturally infected ferrets from any of the groups (Figure 2A). However growth rate of virus in the ferrets from the MIV+IFA passage lines was significantly lower compared to naïve and PBS+IFA-immunized ferrets (−0.83 log10 copy number/100 µl/day (p = 0.049) and −1.3 log10 copy number/100 µl/day (p<0.001), respectively). The growth rate of virus in the ferrets from the MIV passage line was also significantly lower compared to the PBS+IFA-immunized ferrets (−0.77 log10 copy number/100 µl/day (p = 0.049)) (Figure 2B). The time between transmission events (serial interval) was longer for the MIV+IFA line (5.3 days) compared to all other lines (naïve 3.6 days (p = 0.001); PBS+IFA 3.8 days (p = 0.010); MIV 3.9 days (p = 0.011)) (Figure 2C). Within each passage line, there was no significant change in any of the kinetics measurements as passaging progressed, suggesting that the growth characteristics and transmissibility of the virus remained stable over time (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)

Virus replication and transmission kinetics in ferret passage lines and emergence and persistence of the N156K mutant.(A–C) Nasal washes were collected daily from ferrets from passage lines (R0–R7) and assayed for viral RNA by real time RT-PCR as a measure of infection and transmission. (A) The peak viral load detected in the nasal wash from each ferret, (B) the viral growth rate ((peak virus load – first day virus load)/days to reach peak) and (C) the serial interval of virus transmission between ferrets (number of days between detection of infected and infecting animals) were determined for each immunization line. Ferrets artificially infected by intranasal virus inoculation are not included. Ferrets that failed to be naturally infected by contact transmission are indicated in white circles. Statistics do not include ferrets that failed to be naturally infected. (D–E) The proportion of N156 wildtype (black) and N156K (white) viruses in peak day nasal wash samples from all ferrets from the MIV+IFA (D) and N156K naïve (E) passage lines quantified by pyrosequencing. (F–G) Mixtures of (F) N156 wildtype and N156K or (G) N156K and K142N+N156K viruses were passaged by contact transmission through naïve ferrets. The proportion of N156 wildtype (black), N156K (white) or K142N+N156K (striped) in peak day nasal wash samples was quantified by pyrosequencing.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649996&req=5

ppat-1003354-g002: Virus replication and transmission kinetics in ferret passage lines and emergence and persistence of the N156K mutant.(A–C) Nasal washes were collected daily from ferrets from passage lines (R0–R7) and assayed for viral RNA by real time RT-PCR as a measure of infection and transmission. (A) The peak viral load detected in the nasal wash from each ferret, (B) the viral growth rate ((peak virus load – first day virus load)/days to reach peak) and (C) the serial interval of virus transmission between ferrets (number of days between detection of infected and infecting animals) were determined for each immunization line. Ferrets artificially infected by intranasal virus inoculation are not included. Ferrets that failed to be naturally infected by contact transmission are indicated in white circles. Statistics do not include ferrets that failed to be naturally infected. (D–E) The proportion of N156 wildtype (black) and N156K (white) viruses in peak day nasal wash samples from all ferrets from the MIV+IFA (D) and N156K naïve (E) passage lines quantified by pyrosequencing. (F–G) Mixtures of (F) N156 wildtype and N156K or (G) N156K and K142N+N156K viruses were passaged by contact transmission through naïve ferrets. The proportion of N156 wildtype (black), N156K (white) or K142N+N156K (striped) in peak day nasal wash samples was quantified by pyrosequencing.
Mentions: The virus kinetics in all passage lines was assessed using real time RT-PCR to determine the relative copy number. There was no difference in the peak viral load achieved in naturally infected ferrets from any of the groups (Figure 2A). However growth rate of virus in the ferrets from the MIV+IFA passage lines was significantly lower compared to naïve and PBS+IFA-immunized ferrets (−0.83 log10 copy number/100 µl/day (p = 0.049) and −1.3 log10 copy number/100 µl/day (p<0.001), respectively). The growth rate of virus in the ferrets from the MIV passage line was also significantly lower compared to the PBS+IFA-immunized ferrets (−0.77 log10 copy number/100 µl/day (p = 0.049)) (Figure 2B). The time between transmission events (serial interval) was longer for the MIV+IFA line (5.3 days) compared to all other lines (naïve 3.6 days (p = 0.001); PBS+IFA 3.8 days (p = 0.010); MIV 3.9 days (p = 0.011)) (Figure 2C). Within each passage line, there was no significant change in any of the kinetics measurements as passaging progressed, suggesting that the growth characteristics and transmissibility of the virus remained stable over time (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