H1N1 Influenza A Virus Research Articles

A Childhood Leukaemia Cluster in Milan: Possible Role of Pandemic AH1N1 Swine Flu Virus.

AbstractAbstract 2496Cases of childhood leukemia very rarely present as a significant time/space cluster. Clusters offer a unique opportunity to identify a point source causal exposure but, in practice, this is extremely difficult. We herewith report an apparent cluster of seven cases of childhood B cell precursor acute lymphoblastic leukaemia (ALL) occurring over a four week period (December 2009/January 2010) in Milan, Lombardy, Italy. Over the prior ten years (1999–2008), the mean number of cases per year in Milan was 7.3±1.3. The seven cases in the cluster were of varying ages at diagnosis (2–11 years); analysis of chromosomal abnormalities did not reveal recurrent features; inherited leukemia associated alleles did not show an augmented risk. Given the protracted natural history of ALL, initiated in utero, and the variable ages at diagnosis in this cluster, an exposure to a causal trigger or promotional factor focused proximally to the diagnostic time frame was suspected. Three cases attended one school and a fourth was the younger sibling of a student at this same school. No cases of leukemia were recorded in the previous ten years at the school. All four cases lived in the same small area of Milan. A space-time permutation scan statistic was applied to the whole series of data and revealed a clustering for the one month period of diagnosis and the small city area of the four cases (p-value=0.017). As the school had undergone extensive refurbishment 3–24 months prior to the diagnoses, exposure to chemicals and physical agents (including EMF) as well as leukemogenic paint solvents was considered but no supportive evidence for this was obtained. An outbreak of the AH1N1 influenza virus pandemic occurred in Milan 8–10 weeks prior to the diagnoses. All seven cases of ALL were later found to be AH1N1 antibody-positive, in contrast to 32.3% (95% confidence interval: 26,96% – 37,96%) of age-matched children in Milan. Interestingly, positivity was found in only one out of four children from Lombardy with kidney tumor for whom we could retrieve PB collected at that time. Additionally, all seven cases had features indicative of a likely paucity of infectious exposure in the first year of life (high birth order and lack of social contacts). These data do not establish causality but are compatible with the ‘delayed infection' hypothesis for childhood ALL in which an abnormal immune or inflammatory response to a common infection promotes ALL in susceptible individuals (Greaves M., Nat Rev Cancer, 2006;6:193–203).This is the first time that a significant cluster of childhood leukemia cases has been linked, albeit tentatively, to a known infectious agent. Prior, descriptive epidemiological data have however indicated an association between increased rates of childhood ALL and epidemics of seasonal influenza in the UK (Kroll ME et al, J Natl Cancer Inst, 2006;98:417-20). The credibility of flu viruses as potential triggers of ALL requires further exploration including functional assessment in model systems. Disclosures:No relevant conflicts of interest to declare.

Neutralizing DNA Aptamers against Swine Influenza H3N2 Viruses

Triple reassortant influenza A viruses (IAVs) of swine, particularly the North American H3N2 subtype, circulate in swine herds and may reassort and result in the emergence of novel zoonotic strains. Current diagnostic tools rely on isolation of the viruses, followed by serotyping by hemagglutination or genome sequencing, both of which can be expensive and time-consuming. Thus, novel subtype-specific ligands and methods are needed for rapid testing and subtyping of IAVs in the field. To address this need, we selected DNA aptamers against the recombinant HA protein from swine IAV H3 cluster IV using systematic evolution of ligands by exponential enrichment (SELEX). Four candidate aptamers (HA68, HA7, HA2a, and HA2b) were identified and characterized. The dissociation constants (K(d)) of aptamers HA68, HA7, HA2a, and HA2b against recombinant H3 protein were 7.1, 22.3, 16.0, and 3.7 nM, respectively. The binding site of HA68 to H3 was identified to be between nucleotide residues 8 and 40. All aptamers inhibited H3 hemagglutination. HA68 was highly specific to all four lineages within the North American H3N2 subtype. Further, the other three aptamers specifically identified live viruses belonging to the phylogenetic clusters I, II/III, and IV especially the virus that closely related to the recent H3N2 variant (H3N2v). Aptamer HA68 was also able to bind and detect H3N2v isolated from recent human cases. In conclusion, we provide subtype-specific aptamers against H3N2 IAVs of swine that can now be used in rapid detection and typing protocols for field applications.

P115 Pathogenic Influenza A viruses and SARS-Coronaviruses modulate global interferon stimulated gene induction through diverse mechanisms

IntroductionSuccessful respiratory viruses have developed means to overcome specific IFN and ISG effector functions. Yet globally, ISGs as a class have broad functions and impede both pathogenesis and viral replication. Therefore, highly successful respiratory viruses including SARS-CoV and Influenza A virus (IAV), likely attempt to short-circuit global ISG induction, potentially through diverse mechanisms.MethodsIn this work, global RNA (microarray) and proteomics data (mass spectrometry) are utilized to compare and contrast ISG control strategies employed by IAV H5N1, H1N1 2009, and SARS-CoV. Calu-3 cells, a human lung epithelial cell line, are treated with type I IFN to define a consensus list of IFN responsive genes. These genes are then examined in the context of virus infections to evaluate the global ISG response.ResultsSARS-CoV and IAV H5N1 utilize contrasting approaches to interfere with the global host ISG response. Whereas IAV H5N1 actively manipulates the ISG response with up and down regulation of ISG transcripts, SARS-CoV successfully limits ISG induction until late times during infection. Global proteomic analysis confirms the absence of ISG proteins following H5N1 infection; in contrast, SARS-CoV induces increased abundance of several ISG proteins, but not until after peak viral titers have been achieved. Together, the data illustrate that these two virulent respiratory viruses control the global ISG response.Further examination reveals ISG manipulation is absent in a less virulent IAV strain. H1N1 2009 infection results in robust ISG transcript expression with minimal down regulation. Similarly, proteomics analysis finds increased abundance of ISG proteins absent in H5N1 infection. Transcriptional factor analysis found no direct antagonism; however, truncation of the C-terminal portion of NS1 ablated ISG down regulation in H5N1 infections. Meta-analysis finds a similar ablation is observed in A549 cells infected H1N1 1918 when NS1 is manipulated. Together, the data strongly implicate a role for NS1 in mediating control of the entire global ISG response following virulent IAV infections.For SARS-CoV, the delayed ISG induction is maintained in both mutant and precursor viruses. DORF6, a SARS-CoV mutant lacking a major IFN antagonist, fails to induce ISG induction any earlier. Similarly, Bat-SRBD, a synthetic precursor virus, maintains the ability control the global ISG response, suggesting this trait is maintained in pre-cursors of the epidemic SARS-CoV. Additional work examines the ability of the SARS-CoV to camouflage its viral intermediates as well as replication within double membrane vesicles as mechanisms mediating delayed ISG induction.ConclusionTogether, the work highlights the contrasting approaches used by SARS-CoV and IAV strains to limit global ISG induction. In addition, the results imply an important role for controlling ISG response in the context of virulent respiratory virus infection. Further examination of these results will likely produce novel approaches to treat important respiratory virus infection.

O020 Influenza a virus protein PB1-F2 activates the NLRP3 inflammasome to induce inflammation

Introduction Characteristic of pandemic influenza A virus (IAV) strains is the hyper-inflammatory response associated with infections which can lead to severe clinical symptoms and death. PB1-F2 is a non-structural IAV protein that has gained attention in recent years mainly attributable via its ability to increase pathogenicity by disrupting macrophages. Critical to human health, most avian influenza and human H3N2 viruses carry the information for full-length PB1-F2 protein, whereas human non-pandemic H1N1 IAV strains harbour a truncated version. Importantly, PB1-F2 peptides form protein aggregates and the formation of PB1-F2 amyloid-like fibres are observed in infected cells. There is increasing awareness to the important role of PB1-F2 in enhanced pathogenesis, although the underlying mechanism for its role in inflammation and macrophage dysfunction is not understood. Methods We have elucidated the mechanism of PB1-F2 induced inflammation via the NRLP3-inflammasome using a series ofPB1-F2-deficient viral strains and PB1-F2 peptides combined with inflammasome-deficient macrophages. Results Mice treated with IAV lacking expression of PB1-F2 display decreased inflammation, neutrophil, macrophage and DC infiltration to the lung. Conversely, mice inoculated with PB1-F2 peptide display increased inflammatory cell infiltration and inflammation. Following on from this, we have found that PB1-F2 peptide induces maturation of IL-1 β in human PBMCs, which is reduced by inhibition of phagocytosis and Caspase-1. PB1-F2 also induced IL-1b secretion in murine macrophages that is phagocytosis-dependent. Importantly, cells deficient in caspase-1, ASC and NLRP3 display ablated IL-1 β maturation upon challenge with PB1-F2 indicating PB1-F2 induces inflammation via a NLRP3 inflammasome. These findings suggest a critical role for PB1-F2 in inducing the hyperinflammatory state characterised in pandemic IAV infections and its possible role in macrophage dysfunction via inflammasome-mediated pyroptosis. Conclusion Our findings identify a previously unknown role for PB1-F2-induced inflammation, via recognition by the NLRP3-inflammasome, leading to maturation of IL-1b. Activation of the inflammasome may also explain the observation of PB1-F2-mediated macrophage dysregulation, possibly via inflammasome-mediated pyroptosis. Elucidating mechanisms of the molecular basis for disease severity of emerging influenza viruses, especially those implicated in global outbreaks is essential to develop better treatment regimes to improve the clinical outcomes for patients with severe acute infections.

Caracterización epidemiológica y clínica de la infección por influenza A (H1N1) 2009 en San Felipe, Región de Valparaíso, Chile: Junio a agosto 2009

In order to understand the clinical and epidemiological aspects of infection with the AH1N1 influenza virus in Chile, a prospective study in the city of San Felipe was undertaken. It analyses the trends in consultations for respiratory causes in three primary care centers and hospital discharges by comparing data from years 2008 and 2009 until epidemiological week 37.It also includes a study of cases of ILI / SARI (influenza like disease/severe acute respiratory disease) in which viruses were detected by direct immunofluorescence (DIF) of nasopharyngeal aspirates and by real-time polymerase chain reaction in the case of influenza A (H1N1) 2009. A household survey was conducted in those cases with confirmed A (H1N1) infection, to identify contacts and history related to influenza virus transmission. The results indicate that the behavior of the pandemic was similar to that observed in the rest of the country, with an increase in emergency room visits for ILI. The most affected age group was from 5 to 14 years (26.5 per thousand inhabitants) and the least affected 60 years or older (1.2 per thousand). A 2.78% of the cases corresponded to SARI and the fatality rate was 0.11%.

Heightened adaptive immune responses following vaccination with a temperature-sensitive, live-attenuated influenza virus compared to adjuvanted, whole-inactivated virus in pigs

In the United States there are currently two influenza vaccine platforms approved for use in humans—conventional inactivated virus and live-attenuated influenza virus (LAIV). One of the major challenges for influenza A virus (IAV) vaccination is designing a platform that provides protection across strains. Pandemic H1N1 (pH1N1) IAV swept the globe in 2009 and crossed the species barrier, infecting swine in several countries. Pigs are a natural host for IAV and serve as a model for evaluating immune responses following vaccination and challenge. Recently, a temperature-sensitive (ts) LAIV was developed by introducing modifications in the polymerase genes of a swine-like triple reassortant (tr) virus and when paired with pandemic HA and NA, provided sterilizing immunity upon intratracheal challenge with virulent pH1N1 virus. The utility of a ts LAIV is expanded in this report to show vaccination of pigs induced a cell-mediated immune response characterized by an increased number of antigen-specific IFN-secreting cells and expanded T cell populations when compared to pigs vaccinated with a whole inactivated virus (WIV) vaccine. Following challenge, there was a significant increase in the percentage of proliferating lymphocytes in the LAIV group compared to the WIV group following restimulation with pH1N1 in vitro. Also, there was an increase in the percentage of CD4/CD8 double-positive memory T cells in LAIV vaccinated pigs compared to WIV vaccinated pigs. Hemagglutination inhibition and serum neutralization titers were significantly higher in the LAIV-vaccinated pigs compared to the WIV vaccinated pigs following the initial dose of vaccine. Taken together, these results indicate the ts LAIV vaccine, generated from a triple reassortant IAV, elicits greater cell-mediated and humoral immune responses in pigs.

Avian influenza A virus PB2 promotes interferon type I inducing properties of a swine strain in porcine dendritic cells

The 2009 influenza A virus (IAV) pandemic resulted from reassortment of avian, human and swine strains probably in pigs. To elucidate the role of viral genes in host adaptation regarding innate immune responses, we focussed on the effect of genes from an avian H5N1 and a porcine H1N1 IAV on infectivity and activation of porcine GM-CSF-induced dendritic cells (DC). The highest interferon type I responses were achieved by the porcine virus reassortant containing the avian polymerase gene PB2. This finding was not due to differential tropism since all viruses infected DC equally. All viruses equally induced MHC class II, but porcine H1N1 expressing the avian viral PB2 induced more prominent nuclear NF-κB translocation compared to its parent IAV. The enhanced activation of DC may be detrimental or beneficial. An over-stimulation of innate responses could result in either pronounced tissue damage or increased resistance against IAV reassortants carrying avian PB2.

505 Cytokine Profile Characterization in Patients Infected with AH1N1 Influenza Virus

BackgroundIn 2009, an outbreak of severe respiratory infection caused by influenza AH1N1 virus affected Mexican people, previously in 1996 in Guangdong province (China) an outbreak of H5N1 influenza started spreading throughout Asia and the western Palearctic in 2004 to 2006. Chinese patients were studied and it was observed a deleterious immune response related to cytokine storm, mainly characterized by higher levels of IFN and other proinflammatory cytokines causing fatal infections or severe clinical outcome. Until now, is unknown the cytokine profile involved during AH1N1 infection, thus it was the aim of this study.MethodsSerum samples were obtained from 39 infected patients and their close contacts from the first wave outbreak of influenza AH1N1 and were storage at –80°C until cytokine determination, Human Inflammatory and Th1/Th2/Th17 Cytometric Bead Array Kit was used to determine cytokine concentration in serum samples. Cytokine protein arrays were performed to know relative expression of 36 cytokines with the Profiler Array Membrane from R&D Systems. The results are reported on relative units (RU). It was performed with ANOVA test, considering P < 0.05 as statistically significant.ResultsWe did not find significative statistical differences between patients and their close contacts in IFN-γ, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, 12p70, IL-17A, and TNF evaluated with cytometry. However, the proteomic analysis showed a significant increased (P < 0.05) in G-CSF, I-309, sICAM-1, IL-1Ra. IL-16, IL-23, IL-27, I-TAC, MIP-1α, and E1-Serpin compared with the closed contacts.ConclusionsAH1N1 patients showed higher levels of IL-27. We observed more RU of sICAM expression in patients and closed contacts, possibly due to the IL-27 signaling. IL-23 were increased in AH1N1 patients, this cytokine is involved in the IFN-γ secretion and in the generation of memory CD4 T cells. These results suggest that AH1N1-patients developed an immune response characterized by IL-27 and IL-23. We need more studies to determine if this cytokines are related with the memory response induction.

Amino acid determinants conferring stable sialidase activity at low pH for H5N1 influenza A virus neuraminidase

Avian influenza A viruses (IAVs) and human 1918, 1957, and 1968 pandemic IAVs all have neuraminidases (NAs) that are stable at low pH sialidase activity, yet most human epidemic IAVs do not. We examined the pH stability of H5N1 highly pathogenic avian IAV (HPAI) NAs and identified amino acids responsible for conferring stability at low pH. We found that, unlike other avian viruses, most H5N1 IAVs isolated since 2003 had NAs that were unstable at low pH, similar to human epidemic IAVs. These H5N1 viruses are thus already human virus-like and, therefore, have the frequent infections of humans.

Assessment of the Antiviral Properties of Recombinant Porcine SP-D against Various Influenza A Viruses In Vitro

The emergence of influenza viruses resistant to existing classes of antiviral drugs raises concern and there is a need for novel antiviral agents that could be used therapeutically or prophylacticaly. Surfactant protein D (SP-D) belongs to the family of C-type lectins which are important effector molecules of the innate immune system with activity against bacteria and viruses, including influenza viruses. In the present study we evaluated the potential of recombinant porcine SP-D as an antiviral agent against influenza A viruses (IAVs) in vitro. To determine the range of antiviral activity, thirty IAVs of the subtypes H1N1, H3N2 and H5N1 that originated from birds, pigs and humans were selected and tested for their sensitivity to recombinant SP-D. Using these viruses it was shown by hemagglutination inhibition assay, that recombinant porcine SP-D was more potent than recombinant human SP-D and that especially higher order oligomeric forms of SP-D had the strongest antiviral activity. Porcine SP-D was active against a broad range of IAV strains and neutralized a variety of H1N1 and H3N2 IAVs, including 2009 pandemic H1N1 viruses. Using tissue sections of ferret and human trachea, we demonstrated that recombinant porcine SP-D prevented attachment of human seasonal H1N1 and H3N2 virus to receptors on epithelial cells of the upper respiratory tract. It was concluded that recombinant porcine SP-D holds promise as a novel antiviral agent against influenza and further development and evaluation in vivo seems warranted.

Identification of a Highly Conserved H1 Subtype-Specific Epitope with Diagnostic Potential in the Hemagglutinin Protein of Influenza A Virus

Subtype specificity of influenza A virus (IAV) is determined by its two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). For HA, 16 distinct subtypes (H1–H16) exist, while nine exist for NA. The epidemic strains of H1N1 IAV change frequently and cause annual seasonal epidemics as well as occasional pandemics, such as the notorious 1918 influenza pandemic. The recent introduction of pandemic A/H1N1 IAV (H1N1pdm virus) into humans re-emphasizes the public health concern about H1N1 IAV. Several studies have identified conserved epitopes within specific HA subtypes that can be used for diagnostics. However, immune specific epitopes in H1N1 IAV have not been completely assessed. In this study, linear epitopes on the H1N1pdm viral HA protein were identified by peptide scanning using libraries of overlapping peptides against convalescent sera from H1N1pdm patients. One epitope, P5 (aa 58–72) was found to be immunodominant in patients and to evoke high titer antibodies in mice. Multiple sequence alignments and in silico coverage analysis showed that this epitope is highly conserved in influenza H1 HA [with a coverage of 91.6% (9,860/10,767)] and almost completely absent in other subtypes [with a coverage of 3.3% (792/23,895)]. This previously unidentified linear epitope is located outside the five well-recognized antigenic sites in HA. A peptide ELISA method based on this epitope was developed and showed high correlation (χ2 = 51.81, P<0.01, Pearson correlation coefficient R = 0.741) with a hemagglutination inhibition test. The highly conserved H1 subtype-specific immunodominant epitope may form the basis for developing novel assays for sero-diagnosis and active surveillance against H1N1 IAVs.

Outbreak of swine influenza in Argentina reveals a non-contemporary human H3N2 virus highly transmissible among pigs

Sporadic outbreaks of human H3N2 influenza A virus (IAV) infections in swine populations have been reported in Asia, Europe and North America since 1970. In South America, serological surveys in pigs indicate that IAVs of the H3 and H1 subtypes are currently in circulation; however, neither virus isolation nor characterization has been reported. In November 2008, an outbreak of respiratory disease in pigs consistent with swine influenza virus (SIV) infection was detected in Argentina. The current study describes the clinical epidemiology, pathology, and molecular and biological characteristics of the virus. Phylogenetic analysis revealed that the virus isolate shared nucleotide identities of 96-98 % with H3N2 IAVs that circulated in humans from 2000 to 2003. Antigenically, sera from experimentally inoculated animals cross-reacted mainly with non-contemporary human-origin H3N2 influenza viruses. In an experimental infection in a commercial swine breed, the virus was of low virulence but was transmitted efficiently to contact pigs and caused severe disease when an infected animal acquired a secondary bacterial infection. This is the first report of a wholly human H3N2 IAV associated with clinical disease in pigs in South America. These studies highlight the importance of two-way transmission of IAVs and SIVs between pigs and humans, and call for enhanced influenza surveillance in the pig population worldwide.

Use of plethysmography in assessing the efficacy of antivirals in a mouse model of pandemic influenza A virus

The recently emerged swine-origin H1N1 influenza A virus (IAV) caused a pandemic outbreak in 2009 with higher risk of severe disease among children and pregnant women in their third trimester ( Van Kerkhove et al., 2011), and is continuing to be important seasonal IAV strain. Mice are commonly used in antiviral studies as models of influenza disease, which utilize morbidity and mortality to assess the efficacy of a test compound. Here, we investigated the utility of unrestrained plethysomography to quantify the lung function of IAV-infected BALB/c mice. Administration of a lethal dose (∼30X LD 50) of pandemic H1N1 IAV resulted in a rapid decline in breath volume, as determined by a significant ( P < 0.001) decrease in the pressure associated with inspiration and expiration detected as early as 2 days after virus challenge. Severe disease was also accompanied by a significant ( P < 0.05) increase in breath time on 8 dpi. Plethysmography parameters correlated with weight loss and other parameters of disease such as gross pathology and the weight of the lung. Breath time was reduced in surviving mice challenged with a sublethal dose of virus as compared with normal controls, and is a predictive indicator of outcome in these mice. In antiviral studies, the use of plethysmography resulted in the detection of a clear and rapid treatment response, which was similar to other non-invasive parameters, such as weight change. Oseltamivir and ribavirin significantly ( P < 0.001) improved parameters of lung function, particularly mean breath volume, as early as 2 dpi and in a dose-dependent manner. Moreover, a combination of these two drugs further improved these parameters. Plethysmography provides a sensitive evaluation of lung function in IAV-infected mice in response to antiviral therapy.

Modeling the epidemic waves of AH1N1/09 influenza around the world

The 2009 swine flu pandemic was a global outbreak of a new strain of H1N1 influenza virus and there are more than 14,000 confirmed deaths worldwide. The aim of this paper is to propose new mathematical models to study different dynamics of H1N1 influenza virus spread in selected regions around the world. Spatial and temporal elements are included in these models to reproduce the dynamics of AH1N1/09 virus. Different models are used since H1N1 influenza virus spread in regions with different contact structures are not the same. We rely on time series notifications of individuals to estimate some of the parameters of the models. We find that, in order to reproduce the time series data and the spread of the disease, it is convenient to suggest spatio-temporal models. Regions with only one wave are modeled with the classical SEIR model and regions with multiple waves using models with spatio-temporal elements. These results help to explain and understand about potential mechanisms behind the spread of AH1N1 influenza virus in different regions around the world.

Infección por virus pandémico (H1N1) 2009 en Andalucía

In April 2009, in response to the WHO's alert due to the existence of human infection cases with a new AH1N1 influenza virus, known as swine flu, Andalusian Health Authorities trigger an specific action plan. The surveillance actions developped provided us with appropriate clinical, epidemiological and virological characteristics of the disease. During the first few days, contingency plans were set up based on epidemiological surveillance and outbreak control measures were adopted through early alert and rapid response systems. After phase 6 was declared, influenza sentinel and severe cases surveillance were used in order to plan healthcare services, to reduce transmission and to identify and protect the most vulnerable population groups. Behaviour of pandemic influenza in Andalusia was similar to that observed in the rest of the world. Atack rate was similar to a seasonal flu and the peak was reached at the 46th/2009 week. Most of them were mild cases and affected particularly to young people. The average age of hospitalised patients was 32. Prior pulmonary disease, smoking and morbid obesity (BMI > 40) were the most common pathologies and risk factors in severe cases. An impact scenario of pandemic wave in Andalusia, with an expected attack rate from 2 to 5%, was prepared considering watt observed in the southern hemisphere. Characteristics of the epidemic concerning its extent, severity and mortality rate were adjusted to this scenario.

Development of a safe and convenient neutralization assay for rapid screening of influenza HA-specific neutralizing monoclonal antibodies

The worldwide outbreak of the swine-origin 2009 H1N1 influenza A virus (IAV) and an increasing number of influenza cases caused by a highly pathogenic avian influenza (HPAI) H5N1 have accelerated the need to develop vaccines and antiviral agents against IAVs. Among various antivirals, neutralizing monoclonal antibodies (mAbs) are considered important passive therapeutics having an immediate effect against viral pathogens. Here we report a pseudovirus neutralization assay for rapid screening of neutralizing mAbs targeting hemagglutinin (HA) of H5N1 and H1N1 IAV. In this study, we generated six pseudoviruses with an HIV-1 backbone, respectively, expressing HA of four clades of H5N1 IAV and the 2009 epidemic H1N1 IAV. The resulting pseudoviruses were able to infect a variety of human and non-human cells, with 293T cells from human kidney as the most susceptible target cells. Using the established pseudovirus neutralization assay, we showed that three of ten selected mAbs specific to HA could potently neutralize infection of a pseudovirus bearing HA from the homologous IAV A/VietNam/1194/2004(H5N1) strain. This was highly consistent with the result of a microneutralization assay testing the same strain of a live IAV. Since the pseudovirus neutralization assay does not involve an infectious virus and can be performed without the requirement of a biosafety-3 laboratory, it may be applied for safe and rapid screening of neutralizing mAbs and antiviral agents targeting HA of IAVs.

Quantifying the early immune response and adaptive immune response kinetics in mice infected with influenza A virus.

Seasonal and pandemic influenza A virus (IAV) continues to be a public health threat. However, we lack a detailed and quantitative understanding of the immune response kinetics to IAV infection and which biological parameters most strongly influence infection outcomes. To address these issues, we use modeling approaches combined with experimental data to quantitatively investigate the innate and adaptive immune responses to primary IAV infection. Mathematical models were developed to describe the dynamic interactions between target (epithelial) cells, influenza virus, cytotoxic T lymphocytes (CTLs), and virus-specific IgG and IgM. IAV and immune kinetic parameters were estimated by fitting models to a large data set obtained from primary H3N2 IAV infection of 340 mice. Prior to a detectable virus-specific immune response (before day 5), the estimated half-life of infected epithelial cells is approximately 1.2 days, and the half-life of free infectious IAV is approximately 4 h. During the adaptive immune response (after day 5), the average half-life of infected epithelial cells is approximately 0.5 days, and the average half-life of free infectious virus is approximately 1.8 min. During the adaptive phase, model fitting confirms that CD8(+) CTLs are crucial for limiting infected cells, while virus-specific IgM regulates free IAV levels. This may imply that CD4 T cells and class-switched IgG antibodies are more relevant for generating IAV-specific memory and preventing future infection via a more rapid secondary immune response. Also, simulation studies were performed to understand the relative contributions of biological parameters to IAV clearance. This study provides a basis to better understand and predict influenza virus immunity.

Modeling gene sequences over time in 2009 H1N1 Influenza A Virus populations

BackgroundA sudden emergence of Influenza A Virus (IAV) infections with a new pandemic H1N1 IAV is taking place since April of 2009. In order to gain insight into the mode of evolution of these new H1N1 strains, we performed a Bayesian coalescent Markov chain Monte Carlo (MCMC) analysis of full-length neuraminidase (NA) gene sequences of 62 H1N1 IAV strains (isolated from March 30th to by July 28th, 2009).ResultsThe results of these studies revealed that the expansion population growth model was the best to fit the sequence data. A mean of evolutionary change of 7.84 × 10-3 nucleotide substitutions per site per year (s/s/y) was obtained for the NA gene. A significant contribution of first codon position to this mean rate was observed. Maximum clade credibility trees revealed a rapid diversification of NA genes in different genetic lineages, all of them containing Oseltamivir-resistant viruses of very recent emergence. Mapping of naturally occurring amino acid substitutions in the NA protein from 2009 H1N1 IAV circulating in 62 different patients revealed that substitutions are distributed all around the surface of the molecule, leaving the hydrophobic core and the catalytic site essentially untouched.ConclusionHigh evolutionary rates and fast population growth have contributed to the initial transmission dynamics of 2009 H1N1 IAV. Naturally occurring substitutions are preferentially located at the protein surface and do not interfere with the NA active site. Antigenic regions relevant for vaccine development can differ from previous vaccine strains and vary among patients.

Que sera, sera: evolution of the swine H1N1 influenza A virus

A new human influenza A virus (IAV) strain of swine-like origin emerged in Mexico and the United States in March and April of 2009 [1]. The virus disseminated globally with sufficient celerity that on June 11, 2009 the World Health Organization (WHO) raised the Flu Pandemic Alert Level to Phase 6, officially designating a new global pandemic. At this time there were 30,000 confirmed cases in 74 countries. Less than a month later (as of July 1, 2009), the WHO confirmed over 77,000 cases with 332 deaths. The collaborative nature of the global response to the outbreak has been remarkable. Within days of the initial reporting of the virus, researchers completely sequenced the genome of several viral isolates and posted the sequences on the internet. Sequences of new isolates continue to be uploaded daily on websites such as the Global Initiative on Sharing Avian Influenza Data (http:platform.gisaid.org) and the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/genomes/FLU/SwineFlu/html). Nearly in real time, phylogeneticists identified the complex origins of the virus. Within weeks, a vaccine seed stock was isolated, and the private and public sector began intense collaborative efforts to produce a vaccine post-haste [2]. This is the first major introduction of a pandemic influenza strain since 1977, and the first to benefit from the biotechnology revolution. Those looking to defend the investment of world governments into biomedical research have another shining example of the enormous payoff in both lives and money gained from achieving a better understanding of biology. Retrospectively, concerns over the pandemic potential of avian IAVs prepared society well for the current pandemic, as recently upgraded resources have played critical roles in monitoring and responding rapidly. Scientists, physicians, administrators, and politicians who fought for these resources deserve the gratitude of their nations. Clearly, appropriate actions have been taken to limit disease and death associated with the new virus. The contrast with the 1918 pandemic, whose causative agent was not known for 13 years, could not be starker. Despite our advances, it is critical to recognize that today, as in 1918, humanity can neither control nor predict the genetic and antigenic evolution of this virus. Here we discuss factors in the evolution of 2009 swine-origin H1N1 IAV (hereafter referred to as S-IAV-2009).

Swine flu virus H1N1: a threat to human health

The sudden outburst of headlines fl ashing news about Infl uenza-A virus H1N1, causing “Swine Flu” since April this year prompts us to think yet again, what makes a virus seek alternate hosts? Is it nature’s way of making humans realise of the atrocities they subject on farm animals-rearing them in confi nement, fattening and killing them for mass production? On a serious note, the capability of these smallest known parasitic microbes to become virulent by developing new strains is just amazing. The novel Infl uenza-A subtype H1N1 is an apt example of how rapidly viruses evolve. All infl uenza viruses contain two surface antigens namely-hemagglutinin (H) and neuraminidase (N) and the viral strains are assigned an HxNx nomenclature based on which forms of these two proteins are present [1]. There are 16 H and 9 N subtypes known in birds, of which only H-1, 2 and 3, and N-1 and 2 are commonly found in humans [2]. The viral structure differs from strain-to-strain due to rapid genetic mutation in the genome 1 and newly identifi ed strain H1N1 responsible for swine fl u in humans is the latest add-on to the list. The viral genome of H1N1 is believed to have been constituted by the reassortment of four known strains of Infl uenza-A virus, one endemic in humans and birds each, and two endemic in pigs (swine) [3]. This kind of quadruple reassortment is a cause of worry as the virus picks up newer genes, can develop new resistance to drugs and strike even more severely. Human immune system combats such a viral attack with β-lymphocytes that undergo rearrangement at a high frequency and generate a repertoire of antibodies against the viral antigens. But the speed of molecular evolution in β-lymphocytes is no match for high mutation rates among viruses. That is, the virus could mutate again, leading to a potentially more dangerous fl u outbreak, keeping researchers and pharmaceutical companies on toes to predict the virus’s behaviour that would strike next season. More so, since the clinical features of infl uenza infection overlap with other respiratory infections, the diagnosis is often delayed due to low suspicion and the limited use of specifi c diagnostic tests. There is currently no vaccine against H1N1 and seasonal infl uenza vaccines available do not offer any protection against it. Contrary to these beliefs, some virologists are of the view that the current situation is not problematic since H1N1 lacks PB1-F2, a protein which has been linked to virulence [4]. Researchers are digging past data for clues about the seasonality and geography of pandemic infl uenza fl u. There is a good amount of information available on the four pandemics that have occurred since 1890, and they all followed different seasonal patterns [4]. It is vital to predict the path and pace of the virus causing this outbreak so that decisions about mitigation strategies, antiviral therapy and vaccination can be pursued. Although the threat seems to have subsided now but making predictions about H1N1’s next move is not easy, as is aptly said, “There’s nothing more predictable about fl u than its unpredictability [4].”