[Show abstract][Hide abstract] ABSTRACT: Influenza is an infectious disease that primarily attacks the respiratory system. Innate immunity provides both a very early defense to influenza virus invasion and an effective control of viral growth. Previous modelling studies of virus-innate immune response interactions have focused on infection with a single virus and, while improving our understanding of viral and immune dynamics, have been unable to effectively evaluate the relative feasibility of different hypothesised mechanisms of antiviral immunity. In recent experiments, we have applied consecutive exposures to different virus strains in a ferret model, and demonstrated that viruses differed in their ability to induce a state of temporary immunity or viral interference capable of modifying the infection kinetics of the subsequent exposure. These results imply that virus-induced early immune responses may be responsible for the observed viral hierarchy. Here we introduce and analyse a family of within-host models of re-infection viral kinetics which allow for different viruses to stimulate the innate immune response to different degrees. The proposed models differ in their hypothesised mechanisms of action of the non-specific innate immune response. We compare these alternative models in terms of their abilities to reproduce the re-exposure data. Our results show that 1) a model with viral control mediated solely by a virus-resistant state, as commonly considered in the literature, is not able to reproduce the observed viral hierarchy; 2) the synchronised and desynchronised behaviour of consecutive virus infections is highly dependent upon the interval between primary virus and challenge virus exposures and is consistent with virus-dependent stimulation of the innate immune response. Our study provides the first mechanistic explanation for the recently observed influenza viral hierarchies and demonstrates the importance of understanding the host response to multi-strain viral infections. Re-exposure experiments provide a new paradigm in which to study the immune response to influenza and its role in viral control.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
The Journal of Infectious Diseases 05/2015; DOI:10.1093/infdis/jiv260 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The molecular mechanisms that regulate the rapid transcriptional changes that occur during cytotoxic T lymphocyte (CTL) proliferation and differentiation in response to infection are poorly understood. We have utilized ChIP-seq to assess histone H3 methyl-ation dynamics within naive, effector, and memory virus-specific T cells isolated directly ex vivo after influenza A virus infection. Our results show that within naive T cells, codeposition of the permissive H3K4me3 and repressive H3K27me3 modifications is a signature of gene loci associated with gene tran-scription, replication, and cellular differentiation. Upon differentiation into effector and/or memory CTLs, the majority of these gene loci lose repressive H3K27me3 while retaining the permissive H3K4me3 modification. In contrast, immune-related effector gene promoters within naive T cells lacked the permissive H3K4me3 modification, with acquisition of this modification occurring upon differentiation into effector/memory CTLs. Thus, coordinate tran-scriptional regulation of CTL genes with related func-tions is achieved via distinct epigenetic mechanisms. INTRODUCTION
[Show abstract][Hide abstract] ABSTRACT: Background
The Cambodian National Influenza Center (NIC) monitored and characterized circulating influenza strains from 2009 to 2011.
Sentinel and study sites collected nasopharyngeal specimens for diagnostic detection, virus isolation, antigenic characterization, sequencing and antiviral susceptibility analysis from patients who fulfilled case definitions for influenza-like illness, acute lower respiratory infections and event-based surveillance. Each year in Cambodia, influenza viruses were detected mainly from June to November, during the rainy season. Antigenic analysis show that A/H1N1pdm09 isolates belonged to the A/California/7/2009-like group. Circulating A/H3N2 strains were A/Brisbane/10/2007-like in 2009 before drifting to A/Perth/16/2009-like in 2010 and 2011. The Cambodian influenza B isolates from 2009 to 2011 all belonged to the B/Victoria lineage represented by the vaccine strains B/Brisbane/60/2008 and B/Malaysia/2506/2004. Sequences of the M2 gene obtained from representative 2009–2011 A/H3N2 and A/H1N1pdm09 strains all contained the S31N mutation associated with adamantanes resistance except for one A/H1N1pdm09 strain isolated in 2011 that lacked this mutation. No reduction in the susceptibility to neuraminidase inhibitors was observed among the influenza viruses circulating from 2009 to 2011. Phylogenetic analysis revealed that A/H3N2 strains clustered each year to a distinct group while most A/H1N1pdm09 isolates belonged to the S203T clade.
In Cambodia, from 2009 to 2011, influenza activity occurred throughout the year with peak seasonality during the rainy season from June to November. Seasonal influenza epidemics were due to multiple genetically distinct viruses, even though all of the isolates were antigenically similar to the reference vaccine strains. The drug susceptibility profile of Cambodian influenza strains revealed that neuraminidase inhibitors would be the drug of choice for influenza treatment and chemoprophylaxis in Cambodia, as adamantanes are no longer expected to be effective.
PLoS ONE 10/2014; 9(10):e110713. DOI:10.1371/journal.pone.0110713 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives
The emergence of the pandemic influenza A(H1N1)pdm09 virus in 2009 saw a significant increase in the therapeutic and prophylactic use of neuraminidase inhibitors (NAIs) to mitigate the impact of this highly transmissible virus. Prior to the pandemic, many countries stockpiled NAIs and developed pandemic plans for the use of antiviral drugs, based on either treatment of high-risk individuals and/or prophylaxis of contacts. However, to date there has been a lack of in vivo models to test the efficacy of treatment or prophylaxis with NAIs, for influenza-infected individuals or exposed contacts, in a household setting.
A ferret model of household contact was developed to study the efficacy of different prophylaxis regimens in preventing infection in contact ferrets exposed to influenza A(H1N1)pdm09-infected index ferrets.
Among the different prophylactic regimens, contact ferrets receiving oseltamivir prophylaxis twice daily showed better outcomes than those receiving oseltamivir once daily. Benefits included a significant delay in the time to secondary infection, lower weight loss and higher activity levels. The treatment of index ferrets at 36 h post-infection did not influence either secondary infection rates or clinical symptoms in exposed contact ferrets. Neither prophylaxis nor treatment prevented infection or reduced the duration of viral shedding, although clinical symptoms did improve in infected animals receiving prophylaxis.
Different oseltamivir prophylaxis regimens did not prevent infections, but consistently resulted in a reduction in symptoms in infected ferrets. However, oseltamivir prophylaxis failed to reduce viral titres, which warrants further investigation in humans.
[Show abstract][Hide abstract] ABSTRACT: The ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states.
[Show abstract][Hide abstract] ABSTRACT: Distinct lineages of avian influenza viruses (AIVs) are harbored by spatially segregated birds, yet significant surveillance gaps exist around the globe. Virtually nothing is known from the Antarctic. Using virus culture, molecular analysis, full genome sequencing, and serology of samples from Adélie penguins in Antarctica, we confirmed infection by H11N2 subtype AIVs. Their genetic segments were distinct from all known contemporary influenza viruses, including South American AIVs, suggesting spatial separation from other lineages. Only in the matrix and polymerase acidic gene phylogenies did the Antarctic sequences form a sister relationship to South American AIVs, whereas distant phylogenetic relationships were evident in all other gene segments. Interestingly, their neuraminidase genes formed a distant relationship to all avian and human influenza lineages, and the polymerase basic 1 and polymerase acidic formed a sister relationship to the equine H3N8 influenza virus lineage that emerged during 1963 and whose avian origins were previously unknown. We also estimated that each gene segment had diverged for 49 to 80 years from its most closely related sequences, highlighting a significant gap in our AIV knowledge in the region. We also show that the receptor binding properties of the H11N2 viruses are predominantly avian and that they were unable to replicate efficiently in experimentally inoculated ferrets, suggesting their continuous evolution in avian hosts. These findings add substantially to our understanding of both the ecology and the intra- and intercontinental movement of Antarctic AIVs and highlight the potential risk of an incursion of highly pathogenic AIVs into this fragile environment.
[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: 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 03/2014; 8(2):135-9. DOI:10.1111/irv.12187 · 2.20 Impact Factor
[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), 18.104.22.168 (Indonesia), 2.2.2 (India/Bangladesh), 22.214.171.124 (Egypt/Israel), and 126.96.36.199 (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.
Influenza and Other Respiratory Viruses 01/2014; DOI:10.1111/irv.12230 · 2.20 Impact Factor
[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; 8(2). DOI:10.1111/irv.12225 · 2.20 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: 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.
[Show abstract][Hide abstract] ABSTRACT: Cells of both the innate and the adaptive immune systems contribute to the clearance of influenza viruses. The focus of this chapter is the adaptive cellular response mediated by T lymphocytes. Influenza-specific CD4+ and CD8+ T cells recognize that host cells have been infected with this intracellular pathogen, provide help to the developing antibody response, kill the infected, virus-producing cellular “factories,”f and secrete cytokines and chemokines that modulate other characteristics of the immune response. Most important of all, this arm of the immunity has the property of memory, so that T cells activated by earlier infections can accelerate and shape the response to a future challenge. As T cells generally recognize peptide+ MHC class I glycoprotein (pMHC) epitopes derived from the more conserved internal proteins of the virus, memory T cells activated by influenza A viruses of one subtype frequently display reactivity against serologically distinct subtypes. While these memory T cells cannot prevent influenza virus infection, they can reduce the severity of disease by promoting more rapid clearance. For these reasons, there is interest in developing vaccines that induce both heterosubtypic T cell-mediated immunity and strain-specific antibodies in order to provide reduce the impact of novel influenza A virus strains and subtypes.
Textbook of Influenza, 08/2013: pages 298-310; , ISBN: 9780470670484
[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; 7(6). DOI:10.1111/irv.12129 · 2.20 Impact Factor