Protection and compensation in the influenza virus-specific CD8+ T cell response.

Influenza virus-specific CD8+ T cells generally recognize peptides derived from conserved, internal proteins that are not subject to antibody-mediated selection pressure. Prior exposure to any one influenza A virus (H1N1) can prime for a secondary CD8+ T cell response to a serologically different influenza A virus (H3N2). The protection afforded by this recall of established CD8+ T cell memory, although limited, is not negligible. Key characteristics of primary and secondary influenza-specific host responses are probed here with recombinant viruses expressing modified nucleoprotein (NP) and acid polymerase (PA) genes. Point mutations were introduced into the epitopes derived from the NP and PA such that they no longer bound the presenting H2Db MHC class I glycoprotein, and reassortant H1N1 and H3N2 viruses were made by reverse genetics. Conventional (C57BL/6J, H2b, and Ig+/+) and Ig-/- (muMT) mice were more susceptible to challenge with the single NP [HKx31 influenza A virus (HK)-NP] and PA (HK-PA) mutants, but unlike the Ig-/- mice, Ig+/+ mice were surprisingly resistant to the HK-NP/-PA double mutant. This virus was found to promote an enhanced IgG response resulting, perhaps, from the delayed elimination of antigen-presenting cells. Antigen persistence also could explain the increase in size of the minor KbPB1703 CD8+ T cell population in mice infected with the mutant viruses. The extent of such compensation was always partial, giving the impression that any virus-specific CD8+ T cell response operates within constrained limits. It seems that the relationship between protective humoral and cellular immunity is neither simple nor readily predicted.

Swine influenza virus PA and neuraminidase gene reassortment into human H1N1iInfluenza Virus is associated with an altered pathogenic phenotype linked to increased MIP-2 expression

Swine are susceptible to infection by both avian and human influenza viruses, and this feature is thought to contribute to novel reassortant influenza viruses. In this study, the influenza virus reassortment rate in swine and human cells was determined. Coinfection of swine cells with 2009 pandemic H1N1 virus (huH1N1) and an endemic swine H1N2 (A/swine/Illinois/02860/09) virus (swH1N2) resulted in a 23% reassortment rate that was independent of α2,3- or α2,6-sialic acid distribution on the cells. The reassortants had altered pathogenic phenotypes linked to introduction of the swine virus PA and neuraminidase (NA) into huH1N1. In mice, the huH1N1 PA and NA mediated increased MIP-2 expression early postinfection, resulting in substantial pulmonary neutrophilia with enhanced lung pathology and disease. The findings support the notion that swine are a mixing vessel for influenza virus reassortants independent of sialic acid distribution. These results show the potential for continued reassortment of the 2009 pandemic H1N1 virus with endemic swine viruses and for reassortants to have increased pathogenicity linked to the swine virus NA and PA genes which are associated with increased pulmonary neutrophil trafficking that is related to MIP-2 expression. IMPORTANCE: Influenza A viruses can change rapidly via reassortment to create a novel virus, and reassortment can result in possible pandemics. Reassortments among subtypes from avian and human viruses led to the 1957 (H2N2 subtype) and 1968 (H3N2 subtype) human influenza pandemics. Recent analyses of circulating isolates have shown that multiple genes can be recombined from human, avian, and swine influenza viruses, leading to triple reassortants. Understanding the factors that can affect influenza A virus reassortment is needed for the establishment of disease intervention strategies that may reduce or preclude pandemics. The findings from this study show that swine cells provide a mixing vessel for influenza virus reassortment independent of differential sialic acid distribution. The findings also establish that circulating neuraminidase (NA) and PA genes could alter the pathogenic phenotype of the pandemic H1N1 virus, resulting in enhanced disease. The identification of such factors provides a framework for pandemic modeling and surveillance.

Pandemic (H1N1) 2009 influenza in Australia : absenteeism and redeployment of emergency medicine and nursing staff

Objective: The aim of the present study was to examine the impact of Pandemic (H1N1) 2009 Influenza on the Australian emergency nursing and medicine workforce, specifically absenteeism and deployment.

Methods: Data were collected using an online survey of 618 members of the three professional emergency medicine or emergency nursing colleges.

Results: Despite significant increases in emergency demand during the Pandemic (H1N1) 2009 Influenza, 56.6% of emergency nursing and medicine staff reported absenteeism of at least 1 day and only 8.5% of staff were redeployed. Staff illness with influenza-like illness was reported by 37% of respondents, and 87% of respondents who became ill were not tested for the Pandemic (H1N1) 2009 Influenza. Of the respondents who became ill, 43% (n = 79) reported missing no days of work and only 8% of respondents (n = 14) reported being absent for more than 5 days. The mean number of days away from work was 3.73 (standard deviation = 3.63). Factors anecdotally associated with staff absenteeism (caregiver responsibilities, concern about personal illness, concern about exposing family members to illness, school closures, risk of quarantine, stress and increased workload) appeared to be of little or no relevance. Redeployment was reported by 8% of respondents and the majority of redeployment was for operational reasons.

Conclusion: Future research related to absenteeism, redeployment during actual pandemic events is urgently needed. Workforce data collection should be an integral part of organizational pandemic planning.

Pandemic (H1N1) Influenza 2009 and Australian emergency departments : implications for policy, practice and pandemic preparedness

Objective: To describe the reported impact of Pandemic (H1N1) 2009 on EDs, so as to inform future pandemic policy, planning and response management.

Methods: This study comprised an issue and theme analysis of publicly accessible literature, data from jurisdictional health departments, and data obtained from two electronic surveys of ED directors and ED staff. The issues identified formed the basis of policy analysis and evaluation.

Results: Pandemic (H1N1) 2009 had a significant impact on EDs with presentation for patients with ‘influenza-like illness’ up to three times that of the same time in previous years. Staff reported a range of issues, including poor awareness of pandemic plans, patient and family aggression, chaotic information flow to themselves and the public, heightened stress related to increased workloads and lower levels of staffing due to illness, family care duties and redeployment of staff to flu clinics. Staff identified considerable discomfort associated with prolonged times wearing personal protective equipment. Staff believed that the care of non-flu patients was compromised during the pandemic as a result of overwork, distraction from core business and the difficulties associated with accommodating infectious patients in an environment that was not conducive.

Conclusions: This paper describes the breadth of the impact of pandemics on ED operations. It identifies a need to address a range of industrial, management and procedural issues. In particular, there is a need for a single authoritative source of information, the re-engineering of EDs to accommodate infectious patients and organizational changes to enable rapid deployment of alternative sources of care.

The epidemiology of the first wave of H1N1 influenza pandemic in Australia : a population-based study

Objectives: Following the recent H1N1 influenza pandemic we were able to describe seropositivity in a repre-sentative sample of adults prior to the availability of a specific vaccine.

Methods: This cross-sectional serological study is set in the Barwon Statistical Division, Australia. Blood samples were collected from September 2009 through to May 2010, from 1184 individuals (569 men, 615 women; median age 61.7 years), randomly selected from electoral rolls. Serum was analysed for specific H1N1 immunity using a haemagglutina-tion inhibition test. A self-report provided information about symptoms, demographics and healthcare. Associations be-tween H1N1 infection, gender, households and occupation were determined using logistic regression, adjusting for age.

Results: Of 1184 individuals, 129 (58 men, 71 women) were seropositive. Gender-adjusted age-specific prevalence was: 8.3% 20-29 years, 13.5% 30-39, 10.4% 40-49, 6.5% 50-59, 9.7% 60-69, 10.3% 70-79, 18.8% 80+. Standardised preva-lence was 10.3% (95%CI 9.6-11.0). No associations were detected between seropositivity and gender (OR=0.82, 95%CI 0.57-1.19) or being a healthcare worker (OR=1.43, 95%CI 0.62-3.29). Smokers (OR=1.86, 95%CI 1.09-3.15) and those socioeconomically disadvantaged (OR=2.52, 95%CI 1.24-5.13) were at increased risk. Among 129 seropositive individu-als, 31 reported symptoms that were either mild (n = 13) or moderate (time off work, doctor visit, n = 18). For age <60, 39.6% of seropositive individuals reported symptoms, whereas the proportion was 13.2% for age 60+.

Conclusions: Following the pandemic, the proportion of seropositive adults was low, but significant subclinical infection was found. Social disadvantage increased the likelihood of infection. The low symptom rate for older ages may relate to pre-existing immunity.

Recommendations for and compliance with social restrictions during implementation of school closures in the early phase of the influenza A (H1N1) 2009 outbreak in Melbourne, Australia

Background Localized reactive school and classroom closures were implemented as part of a suite of pandemic containment measures during the initial response to influenza A (H1N1) 2009 in Melbourne, Australia. Infected individuals, and those who had been in close contact with a case, were asked to stay in voluntary home quarantine and refrain from contact with visitors for seven days from the date of symptom onset or exposure to an infected person. Oseltamivir (Tamiflu®) was available for treatment or prophylaxis. Methods We surveyed affected families through schools involved in the closures. Analyses of responses were descriptive. We characterized recommendations made to case and contact households and quantified adherence to guidelines and antiviral therapy. Results Of the 314 respondent households, 51 contained a confirmed case. The prescribed quarantine period ranged from 1-14 days, reflecting logistic difficulties in reactive implementation relative to the stated guidelines. Household-level compliance with the requirement to stay at home was high (84.5%, 95% CI 79.3,88.5) and contact with children outside the immediate family infrequent. Conclusions Levels of compliance with recommendations in our sample were high compared with other studies, likely due to heightened public awareness of a newly introduced virus of uncertain severity. The variability of reported recommendations highlighted the difficulties inherent in implementing a targeted reactive strategy, such as that employed in Melbourne, on a large scale during a public health emergency. This study emphasizes the need to understand how public health measures are implemented when seeking to evaluate their effectiveness.

Characterization of H1N1 swine influenza viruses circulating in Canadian pigs in 2009

The 2009 pandemic H1N1 (pH1N1), of apparent swine origin, may have evolved in pigs unnoticed because of insufficient surveillance. Consequently, the need for surveillance of influenza viruses circulating in pigs has received added attention. In this study we characterized H1N1 viruses isolated from Canadian pigs in 2009. Isolates from May 2009 were comprised of hemagglutinin and neuraminidase (NA) genes of classical SIV origin in combination with the North American triple-reassortant internal gene (TRIG) cassette, here termed contemporary SIV (conSIV) H1N1. These conSIV H1N1 viruses were contiguous with the North American &alpha;H1 cluster, which was distinct from the pH1N1 isolates that were antigenically more related to the &gamma;H1 cluster. After the initial isolation of pH1N1 from an Alberta pig farm in early May 2009, pH1N1 was found several times in Canadian pigs. These pH1N1 isolates were genetically and antigenically homogeneous. In addition, H1N1 viruses bearing seasonal human H1 and N1 genes together with the TRIG cassette and an NA encoding an oseltamivir-resistance marker were isolated from pigs. The NS gene of one of these seasonal human-like SIV (shSIV) H1N1 isolates was homologous to pH1N1 NS, implicating reassortment between the two strains. Antigenic cross-reactivity was observed between pH1N1 and conSIV but not with shSIV H1N1. In summary, although there was cocirculation of pH1N1 with conSIV and shSIV H1N1 in Canadian pigs after May 2009, there was no evidence supporting the presence of pH1N1 in pigs prior to May 2009. The possibility for further reassortants being generated exists and should be closely monitored.

Consequences of immunodominant epitope deletion for minor influenza virus-specific CD8+-T-cell responses

The extent to which CD8+ T cells specific for other antigens expand to compensate for the mutational loss of the prominent DbNP366 and DbPA224 epitopes has been investigated using H1N1 and H3N2 influenza A viruses modified by reverse genetics. Significantly increased numbers of CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ T cells were found in the spleen and in the inflammatory population recovered by bronchoalveolar lavage from mice that were first given the -NP-PA H1N1 virus intraperitoneally and then challenged intranasally with the homologous H3N2 virus. The effect was less consistent when this prime-boost protocol was reversed. Also, though the quality of the response measured by cytokine staining showed some evidence of modification when these minor CD8+-T-cell populations were forced to play a more prominent part, the effects were relatively small and no consistent pattern emerged. The magnitude of the enhanced clonal expansion following secondary challenge suggested that the prime-boost with the -NP-PA viruses gave a response overall that was little different in magnitude from that following comparable exposure to the unmanipulated viruses. This was indeed shown to be the case when the total response was measured by ELISPOT analysis with virus-infected cells as stimulators. More surprisingly, the same effect was seen following primary challenge, though individual analysis of the CD8+ KbPB1703+ , CD8+ KbNS2114+, and CD8+ DbPB1-F262+ sets gave no indication of compensatory expansion. A possible explanation is that novel, as yet undetected epitopes emerge following primary exposure to the -NP-PA deletion viruses. These findings have implications for both natural infections and vaccines.

A gel-capture assay for characterizing the sialyl-glycan selectivity of influenza viruses

Sialic acids (SA) usually linked to galactose (Gal) in an &alpha;2,6- or &alpha;2,3-configuration are considered the main cell receptors for influenza viruses, in particular for their hemagglutinins (HA). The typing of influenza virus HA receptor selectivity is relevant for understanding the transmissibility of avian and swine viruses to the human population. In this study we developed a simple and inexpensive gel-capture assay (GCA) of the influenza virus HA receptor-binding selectivity. Its principle is the binding of soluble influenza virus to pentasaccharide analogs, representatives of receptors of human and avian influenza viruses, immobilized on a gel resin. The human and avian analogs consisted of a sialyllactose-N-tetraose c (LSTc) [Neu5Ac(&alpha;2,6)Gal(&beta;1-3)GlcNAc(&beta;1-3)Gal(&beta;1-4)Glc] and a sialyllactose-N-tetraose a (LSTa) [Neu5Ac(&alpha;2,3)Gal(&beta;1-3)GlcNAc(&beta;1-3)Gal(&beta;1-4)Glc], respectively. Following equilibration, the unbound virus is washed away and the bound one is assayed via HA by densitometry as a function of the analog concentration. Using GCA, the receptor selectivity of three influenza viruses of different HA subtype was investigated. The results showed that the egg-adapted A/California/07/2009 (H1N1) virus exhibited an avian &alpha;2,3-linked LSTa selectivity, however, it retained the ability to bind to the &alpha;2,6-linked LSTc human receptor analog. Influenza B virus B/Florida/4/2006 showed &alpha;2,6-linked LSTc selectivity and a poor &alpha;2,3-linked LSTa avidity. The H3N2 virus A/Wisconsin/15/2009 displayed almost comparable avidity for both receptor analogs with a marginally greater &alpha;2,3-linked LSTa avidity. The described assay protocol provides a simple and rapid method for the characterization of influenza virus HA receptor binding selectivity. Keywords: influenza virus; hemagglutinin; receptor; sialyllactose-N-tetraose; gel-capture assay.

Design of multiligand inhibitors for the swine flu H1N1 neuraminidase binding site

Viral neuraminidase inhibitors such as oseltamivir and zanamivir prevent early virus multiplication by blocking sialic acid cleavage on host cells. These drugs are effective for the treatment of a variety of influenza subtypes, including swine flu (H1N1). The binding site for these drugs is well established and they were designed based on computational docking studies. We show here that some common natural products have moderate inhibitory activity for H1N1 neuraminidase under docking studies. Significantly, docking studies using AutoDock for biligand and triligand forms of these compounds (camphor, menthol, and methyl salicylate linked via methylene bridges) indicate that they may bind in combination with high affinity to the H1N1 neuraminidase active site. These results also indicate that chemically linked biligands and triligands of these natural products could provide a new class of drug leads for the prevention and treatment of influenza. This study also highlights the need for a multiligand docking algorithm to understand better the mode of action of natural products, wherein multiple active ingredients are present.

Sources, perceived usefulness and understanding of information disseminated to families who entered home quarantine during the H1N1 pandemic in Victoria, Australia: a cross-sectional study

Background Voluntary home quarantine of cases and close contacts was the main non-pharmaceutical intervention used to limit transmission of pandemic (H1N1) 2009 influenza (pH1N1) in the initial response to the outbreak of the disease in Australia. The effectiveness of voluntary quarantine logically depends on affected families having a clear understanding of what they are being asked to do. Information may come from many sources, including the media, health officials, family and friends, schools, and health professionals. We report the extent to which families who entered home quarantine received and used information on what they were supposed to do. Specifically, we outline their sources of information; the perceived usefulness of each source; and associations between understanding of recommendations and compliance. Methods Cross-sectional survey administered via the internet and computer assisted telephone interview to families whose school children were recommended to go into home quarantine because they were diagnosed with H1N1 or were a close contact of a case. The sample included 314 of 1157 potentially eligible households (27% response rate) from 33 schools in metropolitan Melbourne. Adjusting for clustering within schools, we describe self-reported 'understanding of what they were meant to do during the quarantine period'; source of information (e.g. health department) and usefulness of information. Using logistic regression we examine whether compliance with quarantine recommendations was associated with understanding and the type of information source used. Results Ninety per cent understood what they were meant to do during the quarantine period with levels of understanding higher in households with cases (98%, 95% CI 93%-99% vs 88%, 95% CI 84%-91%, P = 0.006). Over 87% of parents received information about quarantine from the school, 63% from the health department and 44% from the media. 53% of households were fully compliant and there was increased compliance in households that reported that they understood what they were meant to do (Odds Ratio 2.27, 95% CI 1.35-3.80). Conclusions It is critical that public health officials work closely with other government departments and media to provide clear, consistent and simple information about what to do during quarantine as high levels of understanding will maximise compliance in the quarantined population.

Inhibition of reactive oxygen species production ameliorates inflammation induced by influenza A viruses via upregulation of SOCS1 and SOCS3.

Highly pathogenic avian influenza virus infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression, and acute lung injury. Proinflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesized that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low-pathogenicity avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low-pathogenicity avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (interleukin-6, beta interferon, CXCL10, and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro. Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with antiviral strategies (targeting viruses) as therapeutic agents to ameliorate disease severity in susceptible species.