Antigenic drift of the pandemic 2009 A(H1N1) influenza virus in a ferret model
- Authors: Guarnaccia, Teagan , Carolan, Louise , Maurer-Stroh, Sebastian , Lee, Raphael , Job, Emma , Reading, Patrick , Petrie, Stephen , McCaw, James , McVernon, Jodie , Hurt, Aeron , Kelso, Anne , Mosse, Jennifer , Barr, Ian , Laurie, Karen
- Date: 2013
- Type: Text , Journal article
- Relation: PLoS Pathogens Vol. 9, no. 5 (2013), p. 1-18
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- Description: 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.
Assessing the viral fitness of oseltamivir-resistant influenza viruses in ferrets, using a competitive-mixtures model
- Authors: Hurt, Aeron , Nor'e, Sit , McCaw, James , Fryer, Helen , Mosse, Jennifer , McLean, Angela , Barr, Ian
- Date: 2010
- Type: Text , Journal article
- Relation: Journal Of Virology Vol. 84, no. 18 (2010 2010), p. 9427-9438
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- Description: To determine the relative fitness of oseltamivir-resistant strains compared to susceptible wild-type viruses, we combined mathematical modeling and statistical techniques with a novel in vivo “competitive-mixtures” experimental model. Ferrets were coinfected with either pure populations (100% susceptible wild-type or 100% oseltamivir-resistant mutant virus) or mixed populations of wild-type and oseltamivir-resistant influenza viruses (80%:20%, 50%:50%, and 20%:80%) at equivalent infectivity titers, and the changes in the relative proportions of those two viruses were monitored over the course of the infection during within-host and over host-to-host transmission events in a ferret contact model. Coinfection of ferrets with mixtures of an oseltamivir-resistant R292K mutant A(H3N2) virus and a R292 oseltamivir-susceptible wild-type virus demonstrated that the R292K mutant virus was rapidly outgrown by the R292 wild-type virus in artificially infected donor ferrets and did not transmit to any of the recipient ferrets. The competitive-mixtures model was also used to investigate the fitness of the seasonal A(H1N1) oseltamivir-resistant H274Y mutant and showed that within infected ferrets the H274Y mutant virus was marginally outgrown by the wild-type strain but demonstrated equivalent transmissibility between ferrets. This novel in vivo experimental method and accompanying mathematical analysis provide greater insight into the relative fitness, both within the host and between hosts, of two different influenza virus strains compared to more traditional methods that infect ferrets with only pure populations of viruses. Our statistical inferences are essential for the development of the next generation of mathematical models of the emergence and spread of oseltamivir-resistant influenza in human populations.
Characterization of the localized immune response in the respiratory tract of ferrets following infection with influenza A and B viruses
- Authors: Carolan, Louise , Rockman, Steve , Borg, Kathryn , Guarnaccia, Teagan , Reading, Patrick , Mosse, Jennifer , Kelso, Anne , Barr, Ian , Laurie, Karen
- Date: 2016
- Type: Text , Journal article
- Relation: Journal of Virology Vol. 90, no. 6 (2016), p. 2838-2848
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- Description: The burden of infection with seasonal influenza viruses is significant. Each year is typically characterized by the dominance of one (sub)type or lineage of influenza A or B virus, respectively. The incidence of disease varies annually, and while this may be attributed to a particular virus strain or subtype, the impacts of prior immunity, population differences, and variations in clinical assessment are also important. To improve our understanding of the impacts of seasonal influenza viruses, we directly compared clinical symptoms, virus shedding, and expression of cytokines, chemokines, and immune mediators in the upper respiratory tract (URT) of ferrets infected with contemporary A(H1N1)pdm09, A(H3N2), or influenza B virus. Gene expression in the lower respiratory tract (LRT) was also assessed. Clinical symptoms were minimal. Overall cytokine/chemokine profiles in the URT were consistent in pattern and magnitude between animals infected with influenza A and B viruses, and peak expression levels of interleukin-1α (IL-1α), IL-1β, IL-6, IL-12p40, alpha interferon (IFN-α), IFN-β, and tumor necrosis factor alpha (TNF-α) mRNAs correlated with peak levels of viral shedding. MCP1 and IFN-γ were expressed after the virus peak. Granzymes A and B and IL-10 reached peak expression as the virus was cleared and seroconversion was detected. Cytokine/chemokine gene expression in the LRT following A(H1N1)pdm09 virus infection reflected the observations seen for the URT but was delayed 2 or 3 days, as was virus replication. These data indicate that disease severities and localized immune responses following infection with seasonal influenza A and B viruses are similar, suggesting that other factors are likely to modulate the incidence and impact of seasonal influenza. © 2016, American Society for Microbiology.
Detection of low pathogenicity influenza a(H7n3) virus during duck mortality event, Cambodia, 2017
- Authors: Suttie, Annika , Yann, Sokhoun , Phalla, Y. , Tum, Sothyra , Deng, Yi-Mo , Hul, Vibol , Horm, Viseth , Barr, Ian , Greenhill, Andrew , Horwood, Paul , Osbjer, Kristina , Karlsson, Erik , Dussart, Philippe
- Date: 2018
- Type: Text , Journal article
- Relation: Emerging Infectious Diseases Vol. 24, no. 6 (2018), p. 1103-1107
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- Description: In January 2017, an estimated 3,700 (93%) of 4,000 Khaki Campbell ducks (Anas platyrhynchos domesticus) died in Kampong Thom Province, Cambodia. We detected low pathogenicity avian influenza A(H7N3) virus and anatid herpesvirus 1 (duck plague) in the affected flock; however, the exact cause of the mortality event remains unclear.
In vitro generation and characterisation of an influenza B variant with reduced sensitivity to neuraminidase inhibitors
- Authors: Cheam, Ai Lee , Barr, Ian , Hampson, Alan , Mosse, Jennifer , Hurt, Aeron
- Date: 2004
- Type: Text , Journal article
- Relation: Antiviral Research Vol. 63, no. 3 (2004), p. 177-181
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- Description: A contemporary influenza type B virus was passaged in vitro in the presence of increasing concentrations of the neuraminidase inhibitors, zanamivir and oseltamivir carboxylate (0.1-1000 μM over nine passages). After the fifth passage in the presence of zanamivir (10 μM), the virus acquired a Glu 119 Asp neuraminidase mutation (influenza A N2 subtype numbering) in the enzyme active site. After a further three passages, in which growth occurred in 100 μM of zanamivir, a Gln 218 Lys mutation (A (H3) numbering) in the HA1 domain of the haemagglutinin was found. In a fluorescence-based neuraminidase inhibition assay, viruses with the Glu 119 Asp NA mutation had a 32,000-fold reduction in sensitivity to the NA inhibitor zanamivir compared to the wild-type virus, while the mutation resulted in a 105-fold reduction in sensitivity to oseltamivir carboxylate. Viruses grown in the presence of 1000 μM oseltamivir carboxylate did not acquire any neuraminidase mutations but did have a His 103 Gln substitution (A (H3) numbering) in the HA1 region of the haemagglutinin which was demonstrated to significantly reduce receptor binding strength in vitro. Tissue culture assays demonstrated that the HA mutation caused a seven-fold reduction in sensitivity to oseltamivir carboxylate, and a 90-fold reduction in sensitivity to zanamivir.
Influenza A(H5N1) viruses with A(H9N2) single gene (matrix or PB1) reassortment isolated from Cambodian live bird markets
- Authors: Suttie, Annika , Karlsson, Erik , Deng, Yi-Mo , Horm, Srey , Yann, Sokhoun , Tok, Songha , Sorn, San , Holl, Davun , Tum, Sothyra , Hurt, Aeron , Greenhill, Andrew , Barr, Ian , Horwood, Paul , Dussart, Philippe
- Date: 2018
- Type: Text , Journal article
- Relation: Virology Vol. 523, no. (2018), p. 22-26
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- Description: Live bird market surveillance for avian influenza viruses in Cambodia in 2015 has led to the detection of two 7:1 reassortant influenza A(H5N1) clade 2.3.2.1c viruses. These reassortant strains, designated A/duck/Cambodia/Z564W35M1/2015 and A/chicken/Cambodia/Z850W49M1/2015, both contained a single gene (PB1 and matrix gene, respectively) from concurrently circulating A(H9N2) influenza viruses. All other viral genes from both isolates clustered with A(H5N1) clade 2.3.2.1 viruses. Continued and prolonged co-circulation of influenza A(H5N1) and A(H9N2) viruses in Cambodian live bird markets may present a risk for the emergence of novel influenza reassortant viruses with negative agricultural and/or public health implications. © 2018
Influenza viruses with B/Yamagata- and B/Victoria-like neuraminidases are differentially affected by mutations that alter antiviral susceptibility
- Authors: Farrukee, Rubaiyea , Leang, Sookkwan , Butler, Jeff , Lee, Raphael , Maurer-Stroh, Sebastian , Tilmanis, Danielle , Sullivan, Sheena , Mosse, Jennifer , Barr, Ian , Hurt, Aeron
- Date: 2015
- Type: Text , Journal article
- Relation: Journal of Antimicrobial Chemotherapy Vol. 70, no. 7 (2015), p. 2004-2012
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- Description: Objectives: The burden of disease due to influenza B is often underestimated. Clinical studies have shown that oseltamivir, a widely used neuraminidase inhibitor (NAI) antiviral drug, may have reduced effectiveness against influenza B viruses. Therefore, it is important to study the effect of neuraminidase mutations in influenza B viruses that may further reduce NAI susceptibility, and to determine whether these mutations have the same effect in the two lineages of influenza B viruses that are currently circulating (B/Yamagata-like and B/Victoria-like). Methods: We characterized the effect of 16 amino acid substitutions across five framework residues and four monomeric interface residues on the susceptibility to four different NAIs (oseltamivir, zanamivir, peramivir and laninamivir). Results: Framework residue mutations E117A and E117G conferred highly reduced inhibition to three of the four NAIs, but substantially reduced neuraminidase activity, whereas other framework mutations retained a greater level of NA activity. Mutations E105K, P139S and G140R of the monomeric interface were also found to cause highly reduced inhibition, but, interestingly, their effect was substantially greater in a B/Victoria-like neuraminidase than in a B/Yamagata-like neuraminidase, with some susceptibility values being up to 1000-fold different between lineages. Conclusions: The frequency and the effect of key neuraminidase mutations on neuraminidase activity and NAI susceptibility can differ substantially between the two influenza B lineages. Therefore, future surveillance, analysis and interpretation of influenza B virus NAI susceptibility should consider the B lineage of the neuraminidase in the same manner as already occurs for different influenza A neuraminidase subtypes.
Interval between infections and viral hierarchy are determinants of viral interference following influenza virus infection in a ferret model
- Authors: Laurie, Karen , Guarnaccia, Teagan , Carolan, Louise , Yan, Aada , Aban, Malet , Petrie, Stephen , Cao, Pengxing , Heffernan, Jane , McVernon, Jodie , Mosse, Jennifer , Kelso, Anne , McCaw, James , Barr, Ian
- Date: 2015
- Type: Text , Journal article
- Relation: Journal of Infectious Diseases Vol. 212, no. 10 (2015), p. 1701-1710
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- Description: Background. Epidemiological studies suggest that, following infection with influenza virus, there is a short period during which a host experiences a lower susceptibility to infection with other influenza viruses. This viral interference appears to be independent of any antigenic similarities between the viruses. We used the ferret model of human influenza to systematically investigate viral interference. Methods. Ferrets were first infected then challenged 1-14 days later with pairs of influenza A(H1N1)pdm09, influenza A(H3N2), and influenza B viruses circulating in 2009 and 2010. Results. Viral interference was observed when the interval between initiation of primary infection and subsequent challenge was <1 week. This effect was virus specific and occurred between antigenically related and unrelated viruses. Coinfections occurred when 1 or 3 days separated infections. Ongoing shedding from the primary virus infection was associated with viral interference after the secondary challenge. Conclusions. The interval between infections and the sequential combination of viruses were important determinants of viral interference. The influenza viruses in this study appear to have an ordered hierarchy according to their ability to block or delay infection, which may contribute to the dominance of different viruses often seen in an influenza season.
MDCK-SIAT1 cells show improved isolation rates for recent human influenza viruses compared to conventinal MDCK cells
- Authors: Oh, Ding , Barr, Ian , Mosse, Jennifer , Laurie, Karen
- Date: 2008
- Type: Text , Journal article
- Relation: Journal of Clinical Microbiology Vol. 46, no. 7 (2008), p. 2189-2194
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- Description: The ability to isolate and propagate influenza virus is an essential tool for the yearly surveillance of circulating virus strains and to ensure accurate clinical diagnosis for appropriate treatment. The suitability of MDCK-SIAT1 cells, engineered to express increased levels of alpha-2,6-linked sialic acid receptors, as an alternative to conventional MDCK cells for isolation of circulating influenza virus was assessed. A greater number of influenza A (H1N1 and H3N2) and B viruses from stored human clinical specimens collected between 2005 and 2007 were isolated following inoculation in MDCK-SIAT1 cells than in MDCK cells. In addition, a higher titer of virus was recovered following culture in MDCK-SIAT1 cells. All A(H1N1) viruses recovered from MDCK-SIAT1 cells were able to agglutinate both turkey and guinea pig red blood cells (RBC), while half of the A(H3N2) viruses recovered after passage in MDCK-SIAT1 cells lost the ability to agglutinate turkey RBC. Importantly, the HA-1 domain of the hemagglutinin gene was genetically stable after passaging in MDCK-SIAT1 cells, a feature not always seen following MDCK cell or embryonated chicken egg passage of human influenza virus. These data indicate that the MDCK-SIAT1 cell line is superior to conventional MDCK cells for isolation of human influenza virus from clinical specimens and may be used routinely for the isolation and propagation of current human influenza viruses for surveillance, diagnostic, and research purposes.
Neuraminidase inhibitor drug susceptibility differs between influenza N1 and N2 neuraminidase following mutagenesis of two conserved residues
- Authors: Ho, Hui-Ting , Hurt, Aeron , Mosse, Jennifer , Barr, Ian
- Date: 2007
- Type: Text , Journal article
- Relation: Antiviral Research Vol. 76, no. 3 (2007), p. 263-266
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- Description: Neuraminidase (NA) inhibitors are a class of antivirals designed to target the conserved residues of the influenza NA active site. While there are many conserved residues in the NA active site that are involved in NA inhibitor binding, only a few have been demonstrated to confer resistance. As such, little is known regarding the potential of the other conserved residues in the NA active site to cause NA inhibitor resistance. Two conserved residues (E227 and E276) of an N1 NA that have not previously been associated with resistance to NA inhibitors were investigated. Site-directed mutagenesis was used to generate three alternative amino acids at each residue. Reverse genetics was used to generate recombinant mutant viruses which were characterized for growth, NA activity and NA inhibitor sensitivity. Of the six recombinant viruses expressing NA with mutations at either E227 or E276, only the E227D and E276D viruses were able to grow without supplementary NA activity, and all mutant viruses had a significant reduction in NA activity. The E227D virus demonstrated significantly reduced sensitivity to zanamivir while the E276D virus did not demonstrate any significant changes in NA inhibitor sensitivity. Interestingly, the resistance profiles of E227D and E276D in N1 NA were significantly different from these sites that have been reported for N2 NA. This study confirmed the essential role of NA active site residues in viral fitness, and identified clear differences in the role of residues E227 and E276 in NA inhibitor resistance with N1 and N2 neuraminidases.
Pathogenesis, humoral immune responses, and transmission between cohoused animals in a ferret model of human respiratory syncytial virus infection
- Authors: Chan, Kok Fei , Carolan, Louise , Druce, Julian , Chappell, Keith , Watterson, Daniel , Young, Paul , Korenkov, Daniil , Subbarao, Kanta , Barr, Ian , Laurie, Karen , Reading, Patrick
- Date: 2018
- Type: Text , Journal article
- Relation: Journal of Virology Vol. 92, no. 4 (2018), p.
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- Description: Small-animal models have been used to obtain many insights regarding the pathogenesis and immune responses induced following infection with human respiratory syncytial virus (hRSV). Among those described to date, infections in cotton rats, mice, guinea pigs, chinchillas, and Syrian hamsters with hRSV strains Long and/or A2 have been well characterized, although clinical isolates have also been examined. Ferrets are also susceptible to hRSV infection, but the pathogenesis and immune responses elicited following infection have not been well characterized. Here, we describe the infection of adult ferrets with hRSV Long or A2 via the intranasal route and characterized virus replication, as well as cytokine induction, in the upper and lower airways. Virus replication and cytokine induction during the acute phase of infection (days 0 to 15 postinfection) were similar between the two strains, and both elicited high levels of F glycoprotein-specific binding and neutralizing antibodies following virus clearance (days 16 to 22 postinfection). Importantly, we demonstrate transmission from experimentally infected donor ferrets to cohoused naive recipients and have characterized virus replication and cytokine induction in the upper airways of infected contact animals. Together, these studies provide a direct comparison of the pathogenesis of hRSV Long and A2 in ferrets and highlight the potential of this animal model to study serological responses and examine interventions that limit transmission of hRSV. Ferrets have been widely used to study pathogenesis, immunity, and transmission following human influenza virus infections however, far less is known regarding the utility of the ferret model to study hRSV infections. Following intranasal infection of adult ferrets with the well-characterized Long or A2 strain of hRSV, we report virus replication and cytokine induction in the upper and lower airways, as well as the development of virus-specific humoral responses. Importantly, we demonstrate transmission of hRSV from experimentally infected donor ferrets to cohoused naive recipients. Together, these findings significantly enhance our understanding of the utility of the ferret as a small-animal model to investigate aspects of hRSV pathogenesis and immunity.
Rapid diagnostic tests for influenza
- Authors: Hurt, Aeron , Barr, Ian
- Date: 2019
- Type: Text , Book chapter
- Relation: Revolutionizing Tropical Medicine: Point-of-Care Tests, New Imaging Technologies and Digital Health p. 191-201
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- Description: Rapid diagnostic tests, or point-of-care tests, generally refer to simple tests that can either be carried out at the bedside or in a non-specialized local facility without the need to transport the sample to a modern laboratory for testing. Rapid influenza diagnostic tests (RIDTs) are primarily carried out in developed countries such as Japan and the USA in doctor's surgeries where they are used to confirm influenza prior to the prescription of influenza antiviral drugs. The current influenza RIDTs can be broadly separated based on the detection of either influenza viral antigen or influenza nucleic acid. In their simplest form, antigen detection RIDTs (AD RIDTs) typically use chromatographic or fluorescence-based immunoassays to detect the viral nucleoprotein of either influenza A or B viruses and these tests can be further divided into those that are read by eye and those that are read by an analyzer. © 2019 John Wiley & Sons Inc.
SARS-CoV-2 does not replicate in embryonated hen's eggs or in MDCK cell lines
- Authors: Barr, Ian , Rynehart, Cleve , Whitney, Paul , Druce, Julian
- Date: 2020
- Type: Text , Journal article
- Relation: Euro surveillance : bulletin Europeen sur les maladies transmissibles = European communicable disease bulletin Vol. 25, no. 25 (2020), p.
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- Description: The advent of COVID-19, has posed a risk that human respiratory samples containing human influenza viruses may also contain SARS-CoV-2. This potential risk may lead to SARS-CoV-2 contaminating conventional influenza vaccine production platforms as respiratory samples are used to directly inoculate embryonated hen's eggs and continuous cell lines that are used to isolate and produce influenza vaccines. We investigated the ability of these substrates to propagate SARS-CoV-2 and found that neither could support SARS-CoV-2 replication.
Semiannual versus annual influenza vaccination in older adults in the tropics : An observer-blind, active-comparator-controlled, randomized superiority trial
- Authors: Young, Barnaby , Sadarangani, Sapna , Haur, Sen , Yung, Chee , Barr, Ian , Connolly, John , Chen, Mark , Wilder-Smith, Annelies
- Date: 2019
- Type: Text , Journal article
- Relation: Clinical Infectious Diseases Vol. 69, no. 1 (2019), p. 121-129
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- Description: Background. Antibody titres and vaccine effectiveness decline within 6 months after influenza vaccination in older adults. Biannual vaccination may be necessary to provide year-round protection in the tropics, where influenza circulates throughout the year. Methods. Tropical Influenza Control Strategies (TROPICS1) was a single-center, 1:1 randomized, observer-blinded, active-comparator- controlled, superiority study in 200 community-resident adults aged ≥65 years. Participants received a standard-dose trivalent inactivated influenza vaccination (IIV3) at enrollment, and either tetanus-diphtheria-pertussis vaccination or IIV3 6 months later. The primary outcome was the proportion of participants with haemagglutination-inhibition (HI) geometric mean titre (GMT) ≥1:40 1 month after the second vaccination (month 7). Secondary outcomes included GMTs to month 12, the incidence of influenza- like illness (ILI), and adverse reactions after vaccination. Results. At month 7, the proportion of participants with an HI tire ≥1:40 against A/H1N1 increased by 21.4% (95% confidence interval [CI] 8.6-33.4) in the semiannual vaccination group. This proportion was not significantly higher for A/H3N2 (4.3, 95% CI -1.1-10.8) or B (2.1, 95% CI -2.0-7.3). Semiannual vaccination significantly increased GMTs against A/H1N1 and A/H3N2, but not B, at month 7. Participants receiving a repeat vaccination of IIV3 reported a significantly lower incidence of ILI in the 6 months after the second vaccination (relative vaccine effectiveness 57.1%, 95% CI 0.6-81.5). The frequency of adverse events was similar after the first and second influenza vaccinations. Conclusions. Semiannual influenza vaccination in older residents of tropical countries has the potential to improve serological measures of protection against infection. Alternative vaccination strategies should also be studied.
Zanamivir-resistant influenza viruses with Q136K or Q136R neuraminidase residue mutations can arise during MDCK cell culture creating challenges for antiviral susceptibility monitoring
- Authors: Little, Karen , Leang, Sookkwan , Butler, Jeff , Baas, Chantal , Harrower, Bruce , Mosse, Jennifer , Barr, Ian , Hurt, Aeron
- Date: 2015
- Type: Text , Journal article
- Relation: Eurosurveillance Vol. 20, no. 45 (2015), p.
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- Description: Surveillance of circulating influenza strains for antiviral susceptibility is important to ensure patient treatment guidelines remain appropriate. Influenza A(H3N2) and A(H1N1)pdm09 virus isolates containing mutations at the Q136 residue of the neuraminidase (NA) that conferred reduced susceptibility to the NA inhibitor (NAI) zanamivir were detected during antiviral susceptibility monitoring. Interestingly, the mutations were not detectable in the viruses from respective clinical specimens, only in the cultured isolates. We showed that variant viruses containing the Q136K and Q136R NA mutations were preferentially selected in Madin-Darby canine kidney epithelial (MDCK) cells, but were less well supported in MDCK-SIAT1 cells and embryonated eggs. The effect of Q136K, Q136R, Q136H and Q136L substitutions in NA subtypes N1 and N2 on NAI susceptibility and in vitro viral fitness was assessed. This study highlights the challenges that cell culture derived mutations can pose to the NAI susceptibility analysis and interpretation and reaffirms the need to sequence viruses from respective clinical specimens to avoid misdiagnosis. However, we also demonstrate that NA mutations at residue136 can confer reduced zanamivir, peramivir or laninamivir susceptibility, and therefore close monitoring of viruses for mutations at this site from patients being treated with these antivirals is important.