[show abstract][hide abstract] ABSTRACT: Oseltamivir is relied upon worldwide as the drug of choice for the treatment of human influenza infection. Surveillance for oseltamivir resistance is routinely performed to ensure the ongoing efficacy of oseltamivir against circulating viruses. Since the emergence of the pandemic 2009 A(H1N1) influenza virus (A(H1N1)pdm09), the proportion of A(H1N1)pdm09 viruses that are oseltamivir resistant (OR) has generally been low. However, a cluster of OR A(H1N1)pdm09 viruses, encoding the neuraminidase (NA) H275Y oseltamivir resistance mutation, was detected in Australia in 2011 amongst community patients that had not been treated with oseltamivir. Here we combine a competitive mixtures ferret model of influenza infection with a mathematical model to assess the fitness, both within and between hosts, of recent OR A(H1N1)pdm09 viruses. In conjunction with data from in vitro analyses of NA expression and activity we demonstrate that contemporary A(H1N1)pdm09 viruses are now more capable of acquiring H275Y without compromising their fitness, than earlier A(H1N1)pdm09 viruses circulating in 2009. Furthermore, using reverse engineered viruses we demonstrate that a pair of permissive secondary NA mutations, V241I and N369K, confers robust fitness on recent H275Y A(H1N1)pdm09 viruses, which correlated with enhanced surface expression and enzymatic activity of the A(H1N1)pdm09 NA protein. These permissive mutations first emerged in 2010 and are now present in almost all circulating A(H1N1)pdm09 viruses. Our findings suggest that recent A(H1N1)pdm09 viruses are now more permissive to the acquisition of H275Y than earlier A(H1N1)pdm09 viruses, increasing the risk that OR A(H1N1)pdm09 will emerge and spread worldwide.
[show abstract][hide abstract] ABSTRACT: Modulation of CD8 coreceptor levels can profoundly affect T-cell sensitivity to antigen. Here we show that the heritable downregulation of CD8 during type 2 polarization of murine CD8(+) effector T cells in vitro and in vivo is associated with CpG methylation of several regions of the Cd8a locus. These epigenetic modifications are maintained long-term in vivo following adoptive transfer. Even after extended type 2 polarization, however, some CD8(low) effector cells respond to interferon-γ by re-expressing CD8 and a type 1 cytokine profile in association with partial Cd8a demethylation. Cd8a methylation signatures in naive, polarized and repolarized cells are distinct from those observed during the initiation, maintenance and silencing of CD8 expression by developing T cells in the thymus. This persistent capacity for epigenetic reprogramming of coreceptor levels on effector CD8(+) T cells enables the heritable tuning of antigen sensitivity in parallel with changes in type 1/type 2 cytokine balance.
[show abstract][hide abstract] ABSTRACT: The divergence of the hemagglutinin gene of A/goose/Guangdong/1/1996-lineage H5N1 viruses during 2011 and 2012 (807 new sequences collected through December 31, 2012) was analyzed by phylogenetic and p-distance methods to define new clades using the pre-established nomenclature system. Eight new clade designations were recommended based on division of clade 1.1 (Mekong River Delta), 188.8.131.52 (Indonesia), 2.2.2 (India/Bangladesh), 184.108.40.206 (Egypt/Israel), and 220.127.116.11 (Asia). A simplification to the previously defined criteria, which adds a letter rather than number to the right-most digit of fifth-order clades, was proposed to facilitate this and future updates.
[show abstract][hide abstract] ABSTRACT: Historical records of influenza pandemics demonstrate variability in incidence and severity between waves. The influenza A(H1N1)pdm09 pandemic was the first in which many countries implemented strain-specific vaccination to mitigate subsequent seasons. Serosurveys provide opportunity to examine the constraining influence of antibody on population disease experience.
Changes in the proportion of adults seropositive to influenza A(H1N1)pdm09over the 2009/10 (summer) interepidemic period and 2010 (winter) influenza season were measured to determine whether there was a temporal relationship with vaccine distribution and influenza activity, respectively.
Plasma samples were collected from healthy blood donors from seven cities at the end of the first wave (November 2009), and before (March/April 2010) and after (November 2010) the subsequent influenza season.
Haemagglutination inhibition (HI) assays were performed to assess reactivity of plasma against A(H1N1)pdm09, and the proportion seropositive (HI titre ≥ 40) compared over time, by age group and location.
Between the 2009 and 2010 influenza seasons, the seropositive proportion rose from 22% to 43%, an increase observed across all ages and sites. Brisbane alone recorded a significant rise in seropositivity over the 2010 influenza season - from a baseline of 35% to 53%. The seropositive proportion elsewhere was ≥40% pre-season, and did not rise over winter.
A vaccine-associated increase in seropositive proportion preceding the influenza season correlated with low levels of disease activity in winter 2010. These observations support the role of immunisation in mitigating the 'second wave' of A(H1N1)pdm09, with timing critical to ensure sustained herd protection.
Influenza and Other Respiratory Viruses 12/2013; · 1.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: Influenza A virus-specific CD8(+) cytotoxic T lymphocytes (CTLs) provide a degree of cross-strain protection that is potentially subverted by mutation. Here we describe the sequential emergence of such variants within CTL epitopes for a persistently infected, immunocompromised infant. Further analysis in immunodeficient and wild-type mice supports the view that CTL escape variants arise frequently in influenza, accumulate with time and revert in the absence of immune pressure under MHCI-mismatched conditions. Viral fitness, the abundance of endogenous CD8(+) T cell responses and T cell receptor repertoire diversity influence the nature of these de novo mutants. Structural characterization of dominant escape variants shows how the peptide-MHCI interaction is modified to affect variant-MHCI stability. The mechanism of influenza virus escape thus looks comparable to that recognized for chronic RNA viruses like HIV and HCV, suggesting that immunocompromised patients with prolonged viral infection could have an important part in the emergence of influenza quasispecies.
[show abstract][hide abstract] ABSTRACT: Influenza viruses collected from regions of Asia, Africa and Oceania between 2009 and 2012 were tested for their susceptibility to two new neuraminidase inhibitors, peramivir and laninamivir. All viruses tested had normal laninamivir inhibition. However, 3·2% (19/599) of A(H1N1)pdm09 viruses had highly reduced peramivir inhibition (due to H275Y NA mutation) and <1% (6/1238) of influenza B viruses had reduced or highly reduced peramivir inhibition, with single occurrence of variants containing I221T, A245T, K360E, A395E, D432G and a combined G145R+Y142H mutation. These data demonstrate that despite an increase in H275Y variants in 2011, there was no marked change in the frequency of peramivir- or laninamivir-resistant variants following the market release of the drugs in Japan in 2010.
Influenza and Other Respiratory Viruses 10/2013; · 1.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: Objective: Vaccination is the most effective way to prevent seasonal influenza and its severe outcomes. The objective of our study was to synthesize information on seasonal influenza vaccination policies, recommendations and practices in place in 2011 for all countries and areas in the Western Pacific Region of the World Health Organization (WHO).
Methods: Data were collected via a questionnaire on seasonal influenza vaccination policies, recommendations and practices in place in 2011.
Results: Thirty-six of the 37 countries and areas (97%) responded to the survey. Eighteen (50%) reported having established seasonal influenza vaccination policies, an additional seven (19%) reported having recommendations for risk groups for seasonal influenza vaccination only and 11 (30%) reported having no policies or recommendations in place. Of the 25 countries and areas with policies or recommendations, health-care workers and the elderly were most frequently recommended for vaccination; 24 (96%) countries and areas recommended vaccinating these groups, followed by pregnant women (19 [76%]), people with chronic illness (18 [72%]) and children (15 [60%]). Twenty-six (72%) countries and areas reported having seasonal influenza vaccines available through public funding, private market purchase or both. Most
of these countries and areas purchased only enough vaccine to cover 25% or less of their populations.
Discussion: In light of the new WHO position paper on influenza vaccines published in 2012 and the increasing availability of country-specific data, countries and areas should consider reviewing or developing their seasonal influenza vaccination policies to reduce morbidity and mortality associated with annual epidemics and as part of ongoing efforts for pandemic preparedness.
Western Pacific Surveillance and Response Journal. 09/2013; 4(3).
[show abstract][hide abstract] ABSTRACT: Previous influenza pandemics had second and on occasion third waves in many countries that were at times more severe than the initial pandemic waves.
This study aims to determine the seroepidemiology of successive waves of H1N1pdm09 infections in Singapore and the overall risks of infection.
We performed a cohort study amongst 838 adults, with blood samples provided upon recruitment and at 5 points from 2009 to 2011 and tested by haemagglutination inhibition (HI) with A/California/7/2009 (H1N1pdm09). Surveys on key demographic and clinical information were conducted at regular intervals, and associations between seroconversion and these variables were investigated.
After the initial wave from June to September 2009, second and third waves occurred from November 2009 to February 2010 and April to June 2010, respectively. Seroconversion was 13·5% during the first wave and decreased to 6·2% and 6·8% in subsequent waves. Across the three waves, the elderly and those with higher starting HI titres were at lower risk of seroconversion, while those with larger households were at greater risk. Those with higher starting HI titres were also less likely to have an acute respiratory infection.
The second and third waves in Singapore had lower serological attack rates than the first wave. The elderly and those with higher HI titres had lower risk, while those in larger households had higher risk of seroconversion.
Influenza and Other Respiratory Viruses 07/2013; · 1.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Influenza B viruses belong to two antigenically and genetically distinct lineages which co-circulate in varying proportions in many countries. OBJECTIVE: To develop simple, rapid, accurate and robust methods to detect and differentiate currently circulating B-lineage viruses in respiratory samples and virus isolates. STUDY DESIGN: Haemagglutinin (HA) gene sequences from more than 6300 influenza B strains were analysed to identify signature sequences that could be used to distinguish between B-lineages and sublineages. RESULTS: Pyrosequencing and a real time PCR assays were developed to detect the major B-lineages (B/Victoria/2/87 or B/Yamagata/16/88) and pyrosequencing for a unique mutation was used to further differentiate the B/Yamagata viruses into two currently co-circulating subgroups. More than 300 influenza virus-containing samples, including original specimens, cell and egg grown viruses, were tested with a 100% accuracy. Furthermore, when the same PCR primers were used in an rRT-PCR assay, the two lineages could be differentiated by their distinct ranges of melting temperature with an overall accuracy of 99% for 158 samples tested. CONCLUSIONS: These new pyrosequencing and rRT-PCR methods have the potential to aid the rapid identification of influenza B-lineages for surveillance purposes and to increase the available data for bi-annual selection of viruses for updating influenza vaccines.
Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 05/2013; · 3.12 Impact Factor
[show abstract][hide abstract] 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 abstract][hide abstract] ABSTRACT: A minor viral population of oseltamivir-resistant A(H3N2) viruses (E119V neuraminidase mutation) was selected and maintained in a continually infected immunocompromised child following initial oseltamivir treatment. A subsequent course of oseltamivir given 7 weeks later rapidly selected for the E119V variant resulting in a near-pure population of the resistant virus. The study highlights the challenges of oseltamivir treatment of immunocompromised patients that are continually shedding virus and demonstrates the ability of the E119V oseltamivir-resistant virus to be maintained for prolonged periods even in the absence of drug-selective pressure.
Influenza and Other Respiratory Viruses 03/2013; · 1.47 Impact Factor
[show abstract][hide abstract] ABSTRACT: Despite greater than 99% of influenza A viruses circulating in the Asia-Pacific region being resistant to the adamantane antiviral drugs in 2011, the large majority of influenza A (>97%) and B strains (∼99%) remained susceptible to the neuraminidase inhibitors oseltamivir and zanamivir. However, compared to the first year of the 2009 pandemic, cases of oseltamivir-resistant A(H1N1)pdm09 viruses with the H275Y neuraminidase mutation increased in 2011, primarily due to an outbreak of oseltamivir-resistant viruses that occurred in Newcastle, as reported in Hurt et al. (2011c, 2012a), where the majority of the resistant viruses were from community patients not being treated with oseltamivir. A small number of influenza B viruses with reduced oseltamivir or zanamivir susceptibility were also detected. The increased detection of neuraminidase inhibitor resistant strains circulating in the community and the detection of novel variants with reduced susceptibility are reminders that monitoring of influenza viruses is important to ensure that antiviral treatment guidelines remain appropriate.
[show abstract][hide abstract] ABSTRACT: In February and September each year the World Health Organisation (WHO) recommends influenza viruses to be included in influenza vaccines for the forthcoming winters in the Northern and Southern Hemispheres respectively. These recommendations are based on data collected by National Influenza Centres (NIC) through the Global Influenza Surveillance and Response System (GISRS) and a more detailed analysis of representative and potential antigenically variant influenza viruses from the WHO Collaborating Centres for Influenza (WHO CCs) and Essential Regulatory Laboratories (ERLs). This article provides a detailed summary of the antigenic and genetic properties of viruses and additional background data used by WHO experts during development of the recommendations for the 2012 Southern Hemisphere influenza vaccine composition.
[show abstract][hide abstract] ABSTRACT: Oseltamivir resistance in A(H1N1)pdm09 influenza is rare, particularly in untreated community cases. Sustained community transmission has not previously been reported.
Influenza specimens from the Asia-Pacific region were collected through sentinel surveillance, hospital, and general practitioner networks. Clinical and epidemiological information was collected on patients infected with oseltamivir-resistant viruses.
Twenty-nine (15%) of 191 A(H1N1)pdm09 viruses collected between May and September 2011 from Hunter New England (HNE), Australia, contained the H275Y neuraminidase substitution responsible for oseltamivir resistance. Only 1 patient had received oseltamivir before specimen collection. The resistant strains were genetically very closely related, suggesting the spread of a single variant. Ninety percent of cases lived within 50 kilometers. Three genetically similar oseltamivir-resistant variants were detected outside of HNE, including 1 strain from Perth, approximately 4000 kilometers away. Computational analysis predicted that neuraminidase substitutions V241I, N369K, and N386S in these viruses may offset the destabilizing effect of the H275Y substitution.
This cluster represents the first widespread community transmission of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza. These cases and data on potential permissive mutations suggest that currently circulating A(H1N1)pdm09 viruses retain viral fitness in the presence of the H275Y mutation and that widespread emergence of oseltamivir-resistant strains may now be more likely.
The Journal of Infectious Diseases 05/2012; 206(2):148-57. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pandemic (H1N1) 2009 influenza spread through the Northern Territory, Australia, during June-August 2009. We performed 2 cross-sectional serologic surveys on specimens from Northern Territory residents, with 445 specimens obtained prepandemic and 1,689 specimens postpandemic. Antibody titers were determined by hemagglutination inhibition against reference virus A/California/7/2009 on serum samples collected opportunistically from outpatients. All specimens had data for patients' gender, age, and address, with patients' indigenous status determined for 94.1%. Protective immunity (titer >40) was present in 7.6% (95% confidence interval [CI] 5.2%-10.1%) of prepandemic specimens and 19.5% (95% CI 17.6%-21.4%) of postpandemic specimens, giving a population-standardized attack rate of 14.9% (95% CI 11.0%-18.9%). Prepandemic proportion of immune persons was greater with increasing age but did not differ by other demographic characteristics. Postpandemic proportion of immune persons was greater in younger groups and around double in indigenous persons. Postpandemic proportion immune was geographically heterogeneous, particularly among remote-living and indigenous groups.