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The H275Y neuraminidase mutation of the pandemic A/H1N1 influenza virus lengthens the eclipse phase and reduces viral output of infected cells, potentially compromising fitness in ferrets.

ABSTRACT: The H275Y amino acid substitution of the neuraminidase gene is the most common mutation conferring oseltamivir resistance in the N1 subtype of the influenza virus. Using a mathematical model to analyze a set of in vitro experiments that allow for the full characterization of the viral replication cycle, we show that the primary effects of the H275Y substitution on the pandemic H1N1 (H1N1pdm09) strain are to lengthen the mean eclipse phase of infected cells (from 6.6 to 9.1 h) and decrease (by 7-fold) the viral burst size, i.e., the total number of virions produced per cell. We also find, however, that the infectious-unit-to-particle ratio of the H275Y mutant strain is 12-fold higher than that of the oseltamivir-susceptible strain (0.19 versus 0.016 per RNA copy). A parallel analysis of the H275Y mutation in the prior seasonal A/Brisbane/59/2007 background shows similar changes in the infection kinetic parameters, but in this background, the H275Y mutation also allows the mutant to infect cells five times more rapidly. Competitive mixed-strain infections in vitro, where the susceptible and resistant H1N1pdm09 strains must compete for cells, are characterized by higher viral production by the susceptible strain but suggest equivalent fractions of infected cells in the culture. In ferrets, however, the mutant strain appears to suffer a delay in its infection of the respiratory tract that allows the susceptible strain to dominate mixed-strain infections.

SUBMITTER: Pinilla LT

PROVIDER: S-EPMC3457267 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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The H275Y neuraminidase mutation of the pandemic A/H1N1 influenza virus lengthens the eclipse phase and reduces viral output of infected cells, potentially compromising fitness in ferrets.

Pinilla Lady Tatiana LT Holder Benjamin P BP Abed Yacine Y Boivin Guy G Beauchemin Catherine A A CA

Journal of virology 20120725 19

The H275Y amino acid substitution of the neuraminidase gene is the most common mutation conferring oseltamivir resistance in the N1 subtype of the influenza virus. Using a mathematical model to analyze a set of in vitro experiments that allow for the full characterization of the viral replication cycle, we show that the primary effects of the H275Y substitution on the pandemic H1N1 (H1N1pdm09) strain are to lengthen the mean eclipse phase of infected cells (from 6.6 to 9.1 h) and decrease (by 7- ...[more]

PMID: 22837199

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Project description:BackgroundResistance of pandemic A(H1N1)2009 (H1N1pdm09) virus to neuraminidase inhibitors (NAIs) has remained limited. A new mutation I223R in the neuraminidase (NA) of H1N1pdm09 virus has been reported along with H275Y in immunocompromised patients. The aim of this study was to determine the impact of I223R on oseltamivir and zanamivir susceptibility.MethodsThe NA enzymatic characteristics and susceptibility to NAIs of viruses harbouring the mutations I223R and H275Y alone or in combination were analyzed on viruses produced by reverse genetics and on clinical isolates collected from an immunocompromised patient with sustained influenza H1N1pdm09 virus shedding and treated by oseltamivir (days 0-15) and zanamivir (days 15-25 and 70-80).ResultsCompared with the wild type, the NA of recombinant viruses and clinical isolates with H275Y or I223R mutations had about two-fold reduced affinity for the substrate. The H275Y and I223R isolates showed decreased susceptibility to oseltamivir (246-fold) and oseltamivir and zanamivir (8.9- and 4.9-fold), respectively. Reverse genetics assays confirmed these results and further showed that the double mutation H275Y and I223R conferred enhanced levels of resistance to oseltamivir and zanamivir (6195- and 15.2-fold). In the patient, six days after initiation of oseltamivir therapy, the mutation H275Y conferring oseltamivir resistance and the I223R mutation were detected in the NA. Mutations were detected concomitantly from day 6-69 but molecular cloning did not show any variant harbouring both mutations. Despite cessation of NAI treatment, the mutation I223R persisted along with additional mutations in the NA and the hemagglutinin.ConclusionsReduced susceptibility to both oseltamivir and zanamivir was conferred by the I223R mutation which potentiated resistance to both NAIs when associated with the H275Y mutation in the NA. Concomitant emergence of the I223R and H275Y mutations under oseltamivir treatment underlines the importance of close monitoring of treated patients especially those immunocompromised.

Project description:The neuraminidase (NA) inhibitor oseltamivir offers an important immediate option for the control of influenza, and its clinical use has increased substantially during the recent H1N1 pandemic. In view of the high prevalence of oseltamivir-resistant seasonal H1N1 influenza viruses in 2007-2008, there is an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant H1N1/2009 viruses, although resistant variants have been isolated at a low rate. Here we studied the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, one oseltamivir-sensitive and one resistant, in the ferret model. The resistant H275Y mutant was derived from a patient on oseltamivir prophylaxis and was the first oseltamivir-resistant isolate of the pandemic virus. Full genome sequencing revealed that the pair of viruses differed only at NA amino acid position 275. We found that the oseltamivir-resistant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets (0/2), while it retained efficient transmission via direct contact (2/2). The sensitive H1N1/2009 virus was efficiently transmitted via both routes (2/2 and 1/2, respectively). The wild-type H1N1/2009 and the resistant mutant appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. We compared viral fitness within the host by co-infecting a ferret with oseltamivir-sensitive and -resistant H1N1/2009 viruses and found that the resistant virus showed less growth capability (fitness). The NA of the resistant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of possible evolving viral proteins during the pandemic.

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The H275Y neuraminidase mutation of the pandemic A/H1N1 influenza virus lengthens the eclipse phase and reduces viral output of infected cells, potentially compromising fitness in ferrets.

Publications

The H275Y neuraminidase mutation of the pandemic A/H1N1 influenza virus lengthens the eclipse phase and reduces viral output of infected cells, potentially compromising fitness in ferrets.

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