Seasonal H1N1 Influenza Virus Research Articles

Protection of Ferrets from Infection by Swine-Origin 2009 A (H1N1) Influenza Virus by the Inactivated Vaccine

Swine-origin pandemic 2009 A (H1N1) influenza viruses are still infecting humans, and humans are currently being vaccinated with the inactivated vaccine of 2009 A (H1N1) influenza virus. We wanted to determine the efficacy of 2009 A (H1N1) inactivated vaccine in ferrets. Ferrets immunized with one dose (7.5 microg) of 2009 A (H1N1) inactivated vaccine were not protected from infections of either pandemic H1N1 or seasonal H1N1 influenza viruses, while ferrets immunized with two doses of 2009 A (H1N1) inactivated vaccine were protected from infections of pandemic H1N1, but not seasonal H1N1 influenza viruses. IgG subtype of antibody was dominantly detected in tissues of immunized ferrets. Our study suggests that pandemic H1N1 vaccine may not elicit the antibody cross-reactive to the seasonal H1N1 influenza virus.

Gene Expression Profiles Comparison between 2009 Pandemic and Seasonal H1N1 Influenza Viruses in A549 Cells

To perform gene expression profiles comparison so that to identify and understand the potential differences in pathogenesis between the pandemic and seasonal A (H1N1) influenza viruses. A549 cells were infected with A/California/07/09 (H1N1) and A/GuangdongBaoan/51/08 (H1N1) respectively at the same MOI of 2 and collected at 2, 4, 8, and 24 h post infection (p.i.). Gene expression profiles of A549 cells were obtained using the 22 K Human Genome Oligo Array, and differentially expressed genes were analyzed at selected time points. Microarrays results indicated that both of the viruses suppressed host immune response related pathways including cytokine production while pandemic H1N1 virus displayed weaker suppression of host immune response than seasonal H1N1 virus. Observation on similar anti-apoptotic events such as activation of apoptosis inhibitor and down-regulation of key genes of apoptosis pathways in both infections showed that activities of promoting apoptosis were different in later stage of infection. The immuno-suppression and anti-apoptosis events of pandemic H1N1 virus were similar to those seen by seasonal H1N1 virus. The pandemic H1N1 virus had an ability to inhibit biological pathways associated with cytokine responses, NK activation and macrophage recognition.

Oseltamivir–Resistant Pandemic H1N1/2009 Influenza Virus Possesses Lower Transmissibility and Fitness in Ferrets

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.

Monoclonal Antibody Kit for Identification of the Novel 2009 H1N1 Influenza A Virus

To develop an immunofluorescence assay for identification of the 2009 H1N1 influenza A virus, we generated a number of monoclonal antibodies (MAbs) by using inactivated H1N1 2009 virus (A/California/07/2009) as the immunogen. Two MAbs that target two different epitopes of the 2009 H1N1 hemagglutinin (HA) were selected to make the D(3) Ultra 2009 H1N1 Influenza A ID kit (2009 H1N1 ID kit; Diagnostic Hybrids, Inc., Athens, OH), which is intended for the identification of the 2009 H1N1 virus by indirect immunofluorescence assay (IFA). The kit does not detect any of 14 seasonal H1N1 or H3N2 prototype influenza virus strains and is also not reactive with seven other major respiratory viruses. Clinical respiratory specimens were evaluated using both the 2009 H1N1 ID kit and the CDC human influenza virus real-time reverse transcription-PCR swine flu panel (CDC rRT-PCR) and showed 100% agreement between the two assays. Four of these clinical specimens, however, were positive by the 2009 H1N1 ID kit but were identified as presumptively positive by the CDC rRT-PCR by virtue of showing threshold cycle (C(T)) values only with universal InfA and swInfA primers, not with swH1 primers. Sequence analysis of the HA genes of these four specimens revealed point mutations in both the primer and probe regions. In addition, unlike the CDC rRT-PCR, the 2009 H1N1 ID kit can differentiate the 2009 H1N1 virus from a swine-derived H1 influenza A virus (A/New Jersey/8/76). The 2009 H1N1 ID kit offers clinical laboratories an alternative to RT-PCR for the identification of the 2009 H1N1 influenza A virus.

Recipients of Vaccine against the 1976 “Swine Flu” Have Enhanced Neutralization Responses to the 2009 Novel H1N1 Influenza Virus

BACKGROUND. The world is facing a novel H1N1 influenza pandemic. A pandemic scare with a similar influenza virus in 1976 resulted in the vaccination of nearly 45 million persons. We hypothesized that prior receipt of the 1976 "swine flu" vaccine would enhance immune responses to the 2009 novel H1N1 influenza strain. METHODS. A prospective, volunteer sample of employees aged > or = 55 years at a children's cancer hospital in August 2009 was assessed for antibody responses to the 2009 pandemic H1N1 influenza virus and the 2008-2009 seasonal H1N1 influenza virus. RESULTS. Antibody responses by hemagglutination-inhibition assay were high against both the seasonal influenza virus (89.7% had a titer considered seroprotective) and pandemic H1N1 influenza virus (88.8% had a seroprotective titer). These antibodies were effective at neutralizing the seasonal H1N1 influenza virus in 68.1% of participants (titer > or = 40), but only 18.1% had detectable neutralizing titers against the pandemic H1N1 influenza virus. Of 116 participants, 46 (39.7%) received the 1976 "swine flu" vaccine. Receipt of this vaccine significantly enhanced neutralization responses; 8 (17.4%) of 46 vaccine recipients had titers > or = 160, compared with only 3 (4.3%) of 70 who did not receive the vaccine (P = .018 by chi(2) test). CONCLUSIONS. In this cohort, persons aged > or = 55 years had evidence of robust immunity to the 2008-2009 seasonal H1N1 influenza virus. These antibodies were cross-reactive but nonneutralizing against the 2009 pandemic H1N1 influenza strain. Receipt of a vaccine to a related virus significantly enhanced the neutralization capacity of these responses, suggesting homologous vaccination against the 2009 pandemic H1N1 influenza virus would have a similar effect.

2009 Pandemic H1N1 Influenza Virus Replicates in Human Lung Tissues

The replication activity of 2009 pandemic H1N1 influenza virus in human lung cells was evaluated in this study. Twenty-two surgically removed human lung tissue samples were infected ex vivo with pandemic H1N1 influenza virus (A/California/04/2009), seasonal human H1N1 influenza virus (A/Shantou/92/09), or a highly pathogenic H5N1 influenza virus (A/Vietnam/1194/04). Examination of nucleoprotein expression and viral RNA replication in the infected human lung tissue samples showed that whereas the replication of pandemic H1N1 influenza virus varied between tissue samples, overall this virus replicated more efficiently than seasonal H1N1 influenza virus but less efficiently than H5N1 influenza virus. Double-immunostaining for viral antigens and cellular markers indicated that pandemic H1N1 influenza virus replicates in type 2 alveolar epithelial cells.

Co-infection With Pandemic H1N1 and Seasonal H3N2 Influenza Viruses

Co-infection With Pandemic H1N1 and Seasonal H3N2 Influenza Viruses

Genotyping assay for the identification of 2009–2010 pandemic and seasonal H1N1 influenza virus reassortants

New Zealand identified its first pandemic H1N1 influenza cases in late April 2009, immediately prior to the historical start of the New Zealand influenza season. Both pandemic and oseltamivir-resistant seasonal H1N1 viruses cocirculated in the population for a period of time. Thus, concerns were raised about the possibility of reassortment events between the two strains. An RT-PCR-based genotyping assay was developed so that H1N1 influenza coinfections and reassortants could be detected quickly. The assay differentiated effectively the seasonal and pandemic strains. It also confirmed the identification of the first reported coinfection of pandemic and seasonal H1N1 strains during the 2009 Southern Hemisphere influenza season in New Zealand.

Human Infection with a Triple‐Reassortant Swine Influenza A(H1N1) Virus Containing the Hemagglutinin and Neuraminidase Genes of Seasonal Influenza Virus

A reassortant influenza A(H1N1) virus of swine origin distinct from the pandemic H1N1 2009 strain was isolated from 3 patients, all of whom worked at the same large hog operation in Saskatchewan, Canada. The genomic composition of the isolates has not been previously reported, to our knowledge, and was the product of a genetic reassortment between seasonal H1N1 and triple-reassortant influenza virus that emerged in North American swine during the late 1990s. The neuraminidase and hemagglutinin genes of A/Saskatchewan/5350/2009, A/Saskatchewan/5351/2009, and A/Saskatchewan/5131/2009 were derived from human H1N1 virus and were closely related to those of A/Brisbane/59/2007.

Severity of Pneumonia Due to New H1N1 Influenza Virus in Ferrets Is Intermediate between That Due to Seasonal H1N1 Virus and Highly Pathogenic Avian Influenza H5N1 Virus

Background. The newly emerged influenza A(H1N1) virus (new H1N1 virus) is causing the first influenza pandemic of this century. Three influenza pandemics of the previous century caused variable mortality, which largely depended on the development of severe pneumonia. However, the ability of the new H1N1 virus to cause pneumonia is poorly understood. Methods. The new H1N1 virus was inoculated intratracheally into ferrets. Its ability to cause pneumonia was compared with that of seasonal influenza H1N1 virus and highly pathogenic avian influenza (HPAI) H5N1 virus by using clinical, virological, and pathological analyses. Results. Our results showed that the new H1N1 virus causes pneumonia in ferrets intermediate in severity between that caused by seasonal H1N1 virus and by HPAI H5N1 virus. The new H1N1 virus replicated well throughout the lower respiratory tract and more extensively than did both seasonal H1N1 virus (which replicated mainly in the bronchi) and HPAI H5N1 virus (which replicated mainly in the alveoli). High loads of new H1N1 virus in lung tissue were associated with diffuse alveolar damage and mortality. Conclusions. The new H1N1 virus may be intrinsically more pathogenic for humans than is seasonal H1N1 virus.

Clinical Management of Pandemic 2009 Influenza A(H1N1) Infection

Antiviral therapy and vaccination are important strategies for controlling pandemic 2009 influenza A(H1N1) but efficacy depends on the timing of administration and is often limited by supply shortage. Patients with dyspnea, tachypnea, evidence of hypoxemia, and pulmonary infiltrates on chest radiograph should be hospitalized. Patients with severe illness or underlying medical conditions that increase the risk of more severe disease should be treated with oseltamivir or zanamivir as soon as possible, without waiting for the results of laboratory tests. Lung-protective ventilation strategy with a low tidal volume and adequate pressure, in addition to a conservative fluid management approach, is recommended when treating adult patients with ARDS. Extracorporeal membrane oxygenation has emerged as an important rescue therapy for critically ill patients. Use of systemic steroids was associated with delayed viral clearance in severe acute respiratory syndrome and H3N2 infection. Low-dose corticosteroids may be considered in the treatment of refractory septic shock. Passive immunotherapy in the form of convalescent plasma or hyperimmune globulin may be explored as rescue therapy. More data are needed to explore the potential role of IV gamma globulin and other drugs with immunomodulating properties, such as statins, gemfibrozil, and N-acetyl-cysteine. Health-care workers must apply strict standard and droplet precautions when dealing with suspected and confirmed case and upgrade to airborne precautions when performing aerosol-generating procedures. Nonpharmacologic measures, such as early case isolation, household quarantine, school/workplace closure, good community hygiene, and restrictions on travel are useful measures in controlling an influenza pandemic at its early phase.

Prior infection with classical swine H1N1 influenza viruses is associated with protective immunity to the 2009 pandemic H1N1 virus.

Please cite this paper as: Kash et al. (2010) Prior infection with classical swine H1N1 influenza viruses is associated with protective immunity to the 2009 pandemic H1N1 virus. Influenza and Other Respiratory Viruses 4(3), 121–127. Background The 2009 H1N1 pandemic emerged even though seasonal H1N1 viruses have circulated for decades. Epidemio‐logical evidence suggested that the current seasonal vaccine did not offer significant protection from the novel pandemic, and that people over the age of 50 might were less susceptible to infection. Objectives In a mouse challenge study with the 2009 pandemic H1N1 virus, we evaluated protective immune responses elicited by prior infection with human and swine influenza A viruses. Results Mice infected with A/Mexico/4108/2009 (Mex09) showed significant weight loss and 40% mortality. Prior infection with a 1976 classical swine H1N1 virus resulted in complete protection from Mex09 challenge. Prior infection with either a 2009 or a 1940 seasonal H1N1 influenza virus provided partial protection and a >100‐fold reduction in viral lung titers at day 4 post‐infection. Conclusions These findings indicate that in experimental animals recently induced immunity to 1918‐derived H1N1 seasonal influenza viruses, and to a 1976 swine influenza virus, afford a degree of protection against the 2009 pandemic virus. Implications of these findings are discussed in the context of accumulating data suggesting partial protection of older persons during the 2009 pandemic.

Influenza H5N1 and H1N1 Virus Replication and Innate Immune Responses in Bronchial Epithelial Cells Are Influenced by the State of Differentiation

Influenza H5N1 virus continues to be enzootic in poultry and transmits zoonotically to humans. Although a swine-origin H1N1 virus has emerged to become pandemic, its virulence for humans remains modest in comparison to that seen in zoonotic H5N1 disease. As human respiratory epithelium is the primary target cells for influenza viruses, elucidating the viral tropism and host innate immune responses of influenza H5N1 virus in human bronchial epithelium may help to understand the pathogenesis. Here we established primary culture of undifferentiated and well differentiated normal human bronchial epithelial (NHBE) cells and infected with highly pathogenic influenza H5N1 virus (A/Vietnam/3046/2004) and a seasonal influenza H1N1 virus (A/Hong Kong/54/1998), the viral replication kinetics and cytokine and chemokine responses were compared by qPCR and ELISA. We found that the in vitro culture of the well differentiated NHBE cells acquired the physiological properties of normal human bronchi tissue which express high level of α2-6-linked sialic acid receptors and human airway trypsin-like (HAT) protease, in contrast to the low expression in the non-differentiated NHBE cells. When compared to H1N1 virus, the H5N1 virus replicated more efficiently and induced a stronger type I interferon response in the undifferentiated NHBE cells. In contrast, in well differentiated cultures, H5N1 virus replication was less efficient and elicited a lower interferon-beta response in comparison with H1N1 virus. Our data suggest that the differentiation of bronchial epithelial cells has a major influence in cells' permissiveness to human H1N1 and avian H5N1 viruses and the host innate immune responses. The reduced virus replication efficiency partially accounts for the lower interferon-beta responses in influenza H5N1 virus infected well differentiated NHBE cells. Since influenza infection in the bronchial epithelium will lead to tissue damage and associate with the epithelium regeneration, the data generated from the undifferentiated NHBE cultures may also be relevant to disease pathogenesis.

Systems-Level Comparison of Host-Responses Elicited by Avian H5N1 and Seasonal H1N1 Influenza Viruses in Primary Human Macrophages

Human disease caused by highly pathogenic avian influenza (HPAI) H5N1 can lead to a rapidly progressive viral pneumonia leading to acute respiratory distress syndrome. There is increasing evidence from clinical, animal models and in vitro data, which suggests a role for virus-induced cytokine dysregulation in contributing to the pathogenesis of human H5N1 disease. The key target cells for the virus in the lung are the alveolar epithelium and alveolar macrophages, and we have shown that, compared to seasonal human influenza viruses, equivalent infecting doses of H5N1 viruses markedly up-regulate pro-inflammatory cytokines in both primary cell types in vitro. Whether this H5N1-induced dysregulation of host responses is driven by qualitative (i.e activation of unique host pathways in response to H5N1) or quantitative differences between seasonal influenza viruses is unclear. Here we used microarrays to analyze and compare the gene expression profiles in primary human macrophages at 1, 3, and 6 h after infection with H5N1 virus or low-pathogenic seasonal influenza A (H1N1) virus. We found that host responses to both viruses are qualitatively similar with the activation of nearly identical biological processes and pathways. However, in comparison to seasonal H1N1 virus, H5N1 infection elicits a quantitatively stronger host inflammatory response including type I interferon (IFN) and tumor necrosis factor (TNF)-α genes. A network-based analysis suggests that the synergy between IFN-β and TNF-α results in an enhanced and sustained IFN and pro-inflammatory cytokine response at the early stage of viral infection that may contribute to the viral pathogenesis and this is of relevance to the design of novel therapeutic strategies for H5N1 induced respiratory disease.

Subtype identification of the novel A H1N1 and other human influenza A viruses using an oligonucleotide microarray

A novel strain of influenza A (H1N1) virus was isolated in Mexico and the US in March and April 2009. This novel virus spread to many countries and regions in a few months, and WHO raised the level of pandemic alert from phase 5 to phase 6 on June 11, 2009. The accurate identification of H1N1 virus and other human seasonal influenza A viruses is very important for further treatment and control of their infections. In this study, we developed an oligonucleotide microarray to subtype human H1N1, H3N2 and H5N1 influenza viruses, which could distinguish the novel H1N1 from human seasonal H1N1 influenza viruses and swine H1N1 influenza viruses. The microarray utilizes a panel of primers for multiplex PCR amplification of the hemagglutinin (HA), neuraminidase (NA) and matrix (MP) genes of human influenza A viruses. The 59-mer oligonucleotides were designed to distinguish different subtypes of human influenza A viruses. With this microarray, we accurately identified and correctly subtyped the reference virus strains. Moreover, we confirmed 4 out of 39 clinical throat swab specimens from suspected cases of novel H1N1.

The Effects of Influenza A Virus PB1-F2 Protein on Polymerase Activity Are Strain Specific and Do Not Impact Pathogenesis

The influenza A virus PB1-F2 protein has been implicated as a virulence factor, but the mechanism by which it enhances pathogenicity is not understood. The PB1 gene segment of the H1N1 swine-origin influenza virus pandemic strain codes for a truncated PB1-F2 protein which terminates after 11 amino acids but could acquire the full-length form by mutation or reassortment. It is therefore important to understand the function and impact of this protein. We systematically assessed the effect that PB1-F2 expression has on viral polymerase activity, accumulation and localization of PB1, and replication in vitro and in mice. We used both the laboratory strain PR8 and a set of viruses engineered to study clinically relevant PB1-F2 proteins. PB1-F2 expression had modest effects on polymerase activity, PB1 accumulation, and replication that were cell type and virus strain dependent. Disruption of the PB1-F2 reading frame in a recent, seasonal H3N2 influenza virus strain did not affect these parameters, suggesting that this is not a universal function of the protein. Disruption of PB1-F2 expression in several backgrounds or expression of PB1-F2 from the 1918 pandemic strain or a 1956 H1N1 strain had no effect on viral lung loads in mice. Alternate mechanisms besides alterations to replication are likely responsible for the enhanced virulence in mammalian hosts attributed to PB1-F2 in previous studies.

Transmission of Pandemic H1N1 Influenza Virus and Impact of Prior Exposure to Seasonal Strains or Interferon Treatment

Novel swine-origin influenza viruses of the H1N1 subtype were first detected in humans in April 2009. As of 12 August 2009, 180,000 cases had been reported globally. Despite the fact that they are of the same antigenic subtype as seasonal influenza viruses circulating in humans since 1977, these viruses continue to spread and have caused the first influenza pandemic since 1968. Here we show that a pandemic H1N1 strain replicates in and transmits among guinea pigs with similar efficiency to that of a seasonal H3N2 influenza virus. This transmission was, however, partially disrupted when guinea pigs had preexisting immunity to recent human isolates of either the H1N1 or H3N2 subtype and was fully blocked through daily intranasal administration of interferon to either inoculated or exposed animals. Our results suggest that partial immunity resulting from prior exposure to conventional human strains may blunt the impact of pandemic H1N1 viruses in the human population. In addition, the use of interferon as an antiviral prophylaxis may be an effective way to limit spread in at-risk populations.

Prior infection with an H1N1 swine influenza virus partially protects pigs against a low pathogenic H5N1 avian influenza virus

Most humans lack virus neutralizing (VN) and haemagglutination inhibition (HI) antibodies to H5N1 avian influenza viruses (AIVs), but cross-reactive neuraminidase inhibition (NI) antibodies and cell-mediated immune (CMI) responses are common. These immune responses result largely from infections with seasonal human H1N1 influenza viruses, but the protective effect of H1N1 infection-immunity against H5N1 infection has never been examined. To this purpose, we have used the pig model of influenza and a low pathogenic (LP) H5N1 AIV. Pigs were inoculated intranasally with sw/Belgium/1/98 (H1N1) 4 weeks before challenge with duck/Minnesota/1525/81 (H5N1). While the viruses failed to cross-react in HI and VN tests, the H1N1 infection induced high levels of H5N1 cross-reactive NI antibodies. Cross-reactive CMI was demonstrated by measurements of lymphoproliferation and IFN-γ secretion after in vitro restimulation of peripheral blood mononuclear cells. All control pigs showed clinical signs and H5N1 virus isolation from the respiratory tract post-challenge. The H1N1-immune pigs, in contrast, showed a complete clinical protection and only 3 pigs out of 10 were H5N1 virus-positive. In a second and smaller experiment, H1N1 virus infection also conferred cross-protection against a LP H5N2 AIV, while cross-reactive immunity was solely detected in tests for CMI. Our data further support the notion that immunity induced by seasonal human H1N1 influenza virus infection may provide some protection against H5N1 or other H5 AIVs in the absence of neutralizing H5 antibodies. Further studies should reveal whether cross-protection holds against H5N1 viruses that are better adapted to replicate in mammals or with a more distantly related N1.

Transmission and Pathogenesis of Swine-Origin 2009 A(H1N1) Influenza Viruses in Ferrets and Mice

Recent reports of mild to severe influenza-like illness in humans caused by a novel swine-origin 2009 A(H1N1) influenza virus underscore the need to better understand the pathogenesis and transmission of these viruses in mammals. In this study, selected 2009 A(H1N1) influenza isolates were assessed for their ability to cause disease in mice and ferrets and compared with a contemporary seasonal H1N1 virus for their ability to transmit to naïve ferrets through respiratory droplets. In contrast to seasonal influenza H1N1 virus, 2009 A(H1N1) influenza viruses caused increased morbidity, replicated to higher titers in lung tissue, and were recovered from the intestinal tract of intranasally inoculated ferrets. The 2009 A(H1N1) influenza viruses exhibited less efficient respiratory droplet transmission in ferrets in comparison with the highly transmissible phenotype of a seasonal H1N1 virus. Transmission of the 2009 A(H1N1) influenza viruses was further corroborated by characterizing the binding specificity of the viral hemagglutinin to the sialylated glycan receptors (in the human host) by use of dose-dependent direct receptor-binding and human lung tissue-binding assays.

Likelihood that an Unsubtypeable Influenza A Virus Result Obtained with the Luminex xTAG Respiratory Virus Panel Is Indicative of Infection with Novel A/H1N1 (Swine-Like) Influenza Virus

With the sudden emergence of novel A/H1N1 (swine-like) influenza virus against a background of concurrently circulating seasonal H1N1 and H3N2 influenza viruses, diagnostic laboratories have been placed in a difficult situation regarding sensitive and accurate detection. This situation has been