Showing papers on "Influenza A virus published in 2007"

Influenza virus transmission is dependent on relative humidity and temperature.

Abstract: Using the guinea pig as a model host, we show that aerosol spread of influenza virus is dependent upon both ambient relative humidity and temperature. Twenty experiments performed at relative humidities from 20% to 80% and 5 °C, 20 °C, or 30 °C indicated that both cold and dry conditions favor transmission. The relationship between transmission via aerosols and relative humidity at 20 °C is similar to that previously reported for the stability of influenza viruses (except at high relative humidity, 80%), implying that the effects of humidity act largely at the level of the virus particle. For infected guinea pigs housed at 5 °C, the duration of peak shedding was approximately 40 h longer than that of animals housed at 20 °C; this increased shedding likely accounts for the enhanced transmission seen at 5 °C. To investigate the mechanism permitting prolonged viral growth, expression levels in the upper respiratory tract of several innate immune mediators were determined. Innate responses proved to be comparable between animals housed at 5 °C and 20 °C, suggesting that cold temperature (5 °C) does not impair the innate immune response in this system. Although the seasonal epidemiology of influenza is well characterized, the underlying reasons for predominant wintertime spread are not clear. We provide direct, experimental evidence to support the role of weather conditions in the dynamics of influenza and thereby address a long-standing question fundamental to the understanding of influenza epidemiology and evolution.

Aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus

Abstract: The 1918 'Spanish flu' influenza pandemic was unusually severe, causing about 50 million deaths. Why was it so destructive? The lack of antibiotics to fight secondary infections, and socioeconomic factors may be relevant. But experimental infection of nonhuman primates with reconstructed 1918 virus suggests that the lethal nature of the virus itself was a big factor. It is in fact the only influenza virus lethal to experimentally infected nonhuman primates, and the 1918 virus, unlike other strains, suppresses innate immune responses. The H5N1 viruses now circulating cause a severe lung infection similar to that caused by the 1918 virus and also suppress innate immunity, so therapies that protect this type of host immunity might reduce the severity of infection due to these influenza viruses. The 1918 influenza pandemic was unusually severe, resulting in about 50 million deaths worldwide1. The 1918 virus is also highly pathogenic in mice, and studies have identified a multigenic origin of this virulent phenotype in mice2,3,4. However, these initial characterizations of the 1918 virus did not address the question of its pathogenic potential in primates. Here we demonstrate that the 1918 virus caused a highly pathogenic respiratory infection in a cynomolgus macaque model that culminated in acute respiratory distress and a fatal outcome. Furthermore, infected animals mounted an immune response, characterized by dysregulation of the antiviral response, that was insufficient for protection, indicating that atypical host innate immune responses may contribute to lethality. The ability of influenza viruses to modulate host immune responses, such as that demonstrated for the avian H5N1 influenza viruses5, may be a feature shared by the virulent influenza viruses.

Avian Influenza Virus (H5N1): a Threat to Human Health

Abstract: Pandemic influenza virus has its origins in avian influenza viruses. The highly pathogenic avian influenza virus subtype H5N1 is already panzootic in poultry, with attendant economic consequences. It continues to cross species barriers to infect humans and other mammals, often with fatal outcomes. Therefore, H5N1 virus has rightly received attention as a potential pandemic threat. However, it is noted that the pandemics of 1957 and 1968 did not arise from highly pathogenic influenza viruses, and the next pandemic may well arise from a low-pathogenicity virus. The rationale for particular concern about an H5N1 pandemic is not its inevitability but its potential severity. An H5N1 pandemic is an event of low probability but one of high human health impact and poses a predicament for public health. Here, we review the ecology and evolution of highly pathogenic avian influenza H5N1 viruses, assess the pandemic risk, and address aspects of human H5N1 disease in relation to its epidemiology, clinical presentation, pathogenesis, diagnosis, and management.

Spatial, Temporal, and Species Variation in Prevalence of Influenza A Viruses in Wild Migratory Birds

Abstract: Although extensive data exist on avian influenza in wild birds in North America, limited information is available from elsewhere, including Europe. Here, molecular diagnostic tools were employed for high-throughput surveillance of migratory birds, as an alternative to classical labor-intensive methods of virus isolation in eggs. This study included 36,809 samples from 323 bird species belonging to 18 orders, of which only 25 species of three orders were positive for influenza A virus. Information on species, locations, and timing is provided for all samples tested. Seven previously unknown host species for avian influenza virus were identified: barnacle goose, bean goose, brent goose, pink-footed goose, bewick's swan, common gull, and guillemot. Dabbling ducks were more frequently infected than other ducks and Anseriformes; this distinction was probably related to bird behavior rather than population sizes. Waders did not appear to play a role in the epidemiology of avian influenza in Europe, in contrast to the Americas. The high virus prevalence in ducks in Europe in spring as compared with North America could explain the differences in virus–host ecology between these continents. Most influenza A virus subtypes were detected in ducks, but H13 and H16 subtypes were detected primarily in gulls. Viruses of subtype H6 were more promiscuous in host range than other subtypes. Temporal and spatial variation in influenza virus prevalence in wild birds was observed, with influenza A virus prevalence varying by sampling location; this is probably related to migration patterns from northeast to southwest and a higher prevalence farther north along the flyways. We discuss the ecology and epidemiology of avian influenza A virus in wild birds in relation to host ecology and compare our results with published studies. These data are useful for designing new surveillance programs and are particularly relevant due to increased interest in avian influenza in wild birds.

Transmission of influenza A in human beings.

Abstract: Planning for the next influenza pandemic is occurring at many levels throughout the world, spurred on by the recent spread of H5N1 avian influenza in Asia, Europe, and Africa. Central to these planning efforts in the health-care sector are strategies to minimise the transmission of influenza to health-care workers and patients. The infection control precautions necessary to prevent airborne, droplet, and contact transmission are quite different and will need to be decided on and planned before a pandemic occurs. Despite vast clinical experience in human beings, there continues to be much debate about how influenza is transmitted. We have done a systematic review of the English language experimental and epidemiological literature on this subject to better inform infection control planning efforts. We have found that the existing data are limited with respect to the identification of specific modes of transmission in the natural setting. However, we are able to conclude that transmission occurs at close range rather than over long distances, suggesting that airborne transmission, as traditionally defined, is unlikely to be of significance in most clinical settings. Further research is required to better define conditions under which the influenza virus may transmit via the airborne route.

Inhibition of Retinoic Acid-Inducible Gene I-Mediated Induction of Beta Interferon by the NS1 Protein of Influenza A Virus

Abstract: The retinoic acid-inducible gene I product (RIG-I) has been identified as a cellular sensor of RNA virus infection resulting in beta interferon (IFN-β) induction. However, many viruses are known to encode viral products that inhibit IFN-β production. In the case of influenza A virus, the viral nonstructural protein 1 (NS1) prevents the induction of the IFN-β promoter by inhibiting the activation of transcription factors, including IRF-3, involved in IFN-β transcriptional activation. The inhibitory properties of NS1 appear to be due at least in part to its binding to double-stranded RNA (dsRNA), resulting in the sequestration of this viral mediator of RIG-I activation. However, the precise effects of NS1 on the RIG-I-mediated induction of IFN-β have not been characterized. We now report that the NS1 of influenza A virus interacts with RIG-I and inhibits the RIG-I-mediated induction of IFN-β. This inhibition was apparent even when a mutant RIG-I that is constitutively activated (in the absence of dsRNA) was used to trigger IFN-β production. Coexpression of RIG-I, its downstream signaling partner, IPS-1, and NS1 resulted in increased levels of RIG-I and NS1 within an IPS-1-rich, solubilization-resistant fraction after cell lysis. These results suggest that RIG-I, IPS-1, and NS1 become part of the same complex. Consistent with this idea, NS1 was also found to inhibit IFN-β promoter activation by IPS-1 overexpression. Our results indicate that, in addition to sequestering dsRNA, the NS1 of influenza A virus binds to RIG-I and inhibits downstream activation of IRF-3, preventing the transcriptional induction of IFN-β.

A Two-Amino Acid Change in the Hemagglutinin of the 1918 Influenza Virus Abolishes Transmission

Abstract: The 1918 influenza pandemic was a catastrophic series of virus outbreaks that spread across the globe. Here, we show that only a modest change in the 1918 influenza hemagglutinin receptor binding site alters the transmissibility of this pandemic virus. Two amino acid mutations that cause a switch in receptor binding preference from the human α-2,6 to the avian α-2,3 sialic acid resulted in a virus incapable of respiratory droplet transmission between ferrets but that maintained its lethality and replication efficiency in the upper respiratory tract. Furthermore, poor transmission of a 1918 virus with dual α-2,6 and α-2,3 specificity suggests that a predominant human α-2,6 sialic acid binding preference is essential for optimal transmission of this pandemic virus. These findings confirm an essential role of hemagglutinin receptor specificity for the transmission of influenza viruses among mammals.

Surveillance of Resistance to Adamantanes among Influenza A(H3N2) and A(H1N1) Viruses Isolated Worldwide

Abstract: Our previous reports demonstrated an alarming increase in resistance to adamantanes among influenza A(H3N2) viruses isolated in 2001-2005. To continue monitoring drug resistance, we conducted a comprehensive analysis of influenza A(H3N2) and A(H1N1) viruses isolated globally in 2005-2006. The results obtained by pyrosequencing indicate that 96.4% (n=761) of A(H3N2) viruses circulating in the United States were adamantane resistant. Drug resistance has reached 100% among isolates from some Asian countries. Analysis of correlation between the appearance of drug resistance and the evolutionary pathway of the hemagglutinin (HA) gene suggests at least 2 separate introductions of resistance into circulating populations that gave rise to identifiable subclades. It also indicates that resistant A(H3N2) viruses may have emerged in Asia in late 2001. Among A(H1N1) viruses isolated worldwide, resistance reached 15.5% in 2005-2006; in the United States alone, it was 4.0%. Phylogenetic analysis of the HA and M genes indicates that the acquisition of resistance in A(H1N1) viruses can be linked to a specific genetic group and was not a result of reassortment between A(H3N2) and A(H1N1) viruses. The results of the study highlight the necessity of close monitoring of resistance to existing antivirals as wells as the need for new therapeutics.

Mortality benefits of influenza vaccination in elderly people: an ongoing controversy.

Abstract: Summary Influenza vaccination policy in most high-income countries attempts to reduce the mortality burden of influenza by targeting people aged at least 65 years for vaccination. However, the effectiveness of this strategy is under debate. Although placebo-controlled randomised trials show influenza vaccine is effective in younger adults, few trials have included elderly people, and especially those aged at least 70 years, the age-group that accounts for three-quarters of all influenza-related deaths. Recent excess mortality studies were unable to confirm a decline in influenza-related mortality since 1980, even as vaccination coverage increased from 15% to 65%. Paradoxically, whereas those studies attribute about 5% of all winter deaths to influenza, many cohort studies report a 50% reduction in the total risk of death in winter—a benefit ten times greater than the estimated influenza mortality burden. New studies, however, have shown substantial unadjusted selection bias in previous cohort studies. We propose an analytical framework for detecting such residual bias. We conclude that frailty selection bias and use of non-specific endpoints such as all-cause mortality have led cohort studies to greatly exaggerate vaccine benefits. The remaining evidence base is currently insufficient to indicate the magnitude of the mortality benefit, if any, that elderly people derive from the vaccination programme.

Human and avian influenza viruses target different cells in the lower respiratory tract of humans and other mammals

Abstract: Viral attachment to the host cell is critical for tissue and species specificity of virus infections. Recently, pattern of viral attachment (PVA) in human respiratory tract was determined for highly pathogenic avian influenza virus of subtype H5N1. However, PVA of human influenza viruses and other avian influenza viruses in either humans or experimental animals is unknown. Therefore, we compared PVA of two human influenza viruses (H1N1 and H3N2) and two low pathogenic avian influenza viruses (H5N9 and H6N1) with that of H5N1 virus in respiratory tract tissues of humans, mice, ferrets, cynomolgus macaques, cats, and pigs by virus histochemistry. We found that human influenza viruses attached more strongly to human trachea and bronchi than H5N1 virus and attached to different cell types than H5N1 virus. These differences correspond to primary diagnoses of tracheobronchitis for human influenza viruses and diffuse alveolar damage for H5N1 virus. The PVA of low pathogenic avian influenza viruses in human respiratory tract resembled that of H5N1 virus, demonstrating that other properties determine its pathogenicity for humans. The PVA in human respiratory tract most closely mirrored that in ferrets and pigs for human influenza viruses and that in ferrets, pigs, and cats for avian influenza viruses.

Cases of Swine Influenza in Humans: A Review of the Literature

Abstract: As the threat of a pandemic looms, improvement in our understanding of interspecies transmission of influenza is necessary. Using the search terms “swine,” “influenza,” and “human,” we searched the PubMed database in April 2006 to identify publications describing symptomatic infections of humans with influenza viruses of swine origin. From these reports, we extracted data regarding demographic characteristics, epidemiological investigations, and laboratory results. We found 50 cases of apparent zoonotic swine influenza virus infection, 37 of which involved civilians and 13 of which involved military personnel, with a case-fatality rate of 14% (7 of 50 persons). Most civilian subjects (61%) reported exposure to swine. Although sporadic clinical cases of swine influenza occur in humans, the true incidence of zoonotic swine influenza virus infection is unknown. Because prior studies have shown that persons who work with swine are at increased risk of zoonotic influenza virus infection, it is prudent to include them in pandemic planning efforts.

A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence.

Abstract: The proapoptotic PB1-F2 protein of influenza A viruses has been shown to contribute to pathogenesis in the mouse model. Expression of full-length PB1-F2 increases the pathogenesis of the influenza A virus, causing weight loss, slower viral clearance, and increased viral titers in the lungs. After comparing viruses from the Hong Kong 1997 H5N1 outbreak, one amino acid change (N66S) was found in the PB1-F2 sequence at position 66 that correlated with pathogenicity. This same amino acid change (N66S) was also found in the PB1-F2 protein of the 1918 pandemic A/Brevig Mission/18 virus. Two isogenic recombinant chimeric viruses were created with an influenza A/WSN/33 virus background containing the PB1 segment from the HK/156/97: WH and WH N66S. In mice infected with WH N66S virus there was increased pathogenicity as measured by weight loss and decreased survival, and a 100-fold increase in virus replication when compared to mice infected with the WH virus. The 1918 pandemic strain A/Brevig Mission/18 was reconstructed with a pathogenicity-reducing mutation in PB1-F2 (S66N). The resultant 1918 S66N virus was attenuated in mice having a 3-log lower 50% lethal dose and caused less morbidity and mortality in mice than the wild-type virus. Viral lung titers were also decreased in 1918 S66N–infected mice compared with wild-type 1918 virus–infected mice. In addition, both viruses with an S at position 66 (WH N66S and wt 1918) induced elevated levels of cytokines in the lungs of infected mice. Together, these data show that a single amino acid substitution in PB1-F2 can result in increased viral pathogenicity and could be one of the factors contributing to the high lethality seen with the 1918 pandemic virus.

Multiple anti-interferon actions of the influenza A virus NS1 protein

Abstract: The replication and pathogenicity of influenza A virus (FLUAV) are controlled in part by the alpha/beta interferon (IFN-α/β) system. This virus-host interplay is dependent on the production of IFN-α/β and on the capacity of the viral nonstructural protein NS1 to counteract the IFN system. Two different mechanisms have been described for NS1, namely, blocking the activation of IFN regulatory factor 3 (IRF3) and blocking posttranscriptional processing of cellular mRNAs. Here we directly compare the abilities of NS1 gene products from three different human FLUAV (H1N1) strains to counteract the antiviral host response. We found that A/PR/8/34 NS1 has a strong capacity to inhibit IRF3 and activation of the IFN-β promoter but is unable to suppress expression of other cellular genes. In contrast, the NS1 proteins of A/Tx/36/91 and of A/BM/1/18, the virus that caused the Spanish influenza pandemic, caused suppression of additional cellular gene expression. Thus, these NS1 proteins prevented the establishment of an IFN-induced antiviral state, allowing virus replication even in the presence of IFN. Interestingly, the block in gene expression was dependent on a newly described NS1 domain that is important for interaction with the cleavage and polyadenylation specificity factor (CPSF) component of the cellular pre-mRNA processing machinery but is not functional in A/PR/8/34 NS1. We identified the Phe-103 and Met-106 residues in NS1 as being critical for CPSF binding, together with the previously described C-terminal binding domain. Our results demonstrate the capacity of FLUAV NS1 to suppress the antiviral host defense at multiple levels and the existence of strain-specific differences that may modulate virus pathogenicity.

A question of self-preservation: immunopathology in influenza virus infection.

Abstract: Influenza A viruses that circulate normally in the human population cause a debilitating, though generally transient, illness that is sometimes fatal, particularly in the elderly. Severe complications arising from pandemic influenza or the highly pathogenic avian H5N1 viruses are often associated with rapid, massive inflammatory cell infiltration, acute respiratory distress, reactive hemophagocytosis and multiple organ involvement. Histological and pathological indicators strongly suggest a key role for an excessive host response in mediating at least some of this pathology. Here, we review the current literature on how various effector arms of the immune system can act deleteriously to initiate or exacerbate pathological damage in this viral pneumonia. Generally, the same immunological factors mediating tissue damage during the anti-influenza immune response are also critical for efficient elimination of virus, thereby posing a significant challenge in the design of harmless yet effective therapeutic strategies for tackling influenza virus.

Cutting Edge: Influenza A virus activates TLR3-dependent inflammatory and RIG-I-dependent antiviral responses in human lung epithelial cells.

Abstract: Influenza A virus (IAV) triggers a contagious acute respiratory disease that causes considerable mortality annually. Recently, we established a role for the pattern-recognition TLR3 in the response of lung epithelial cells to IAV-derived dsRNA. However, additional nucleic acid-recognition proteins have lately been implicated as key viral sensors, including the RNA helicases retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene (MDA)-5. In this study, we investigated the respective role of TLR3 vs RIG-I/MDA-5 signaling in human respiratory epithelial cells infected by IAV using BEAS-2B cells transfected with vectors encoding either a dominant-negative form of TLR3 or of mitochondrial antiviral signaling protein (MAVS; a signaling intermediate of RIG-I and MDA-5), or with plasmids overexpressing functional RIG-I or MDA-5. We demonstrate that the sensing of IAV by TLR3 primarily regulates a proinflammatory response, whereas RIG-I (but not MDA-5) mediates both a type I IFN-dependent antiviral signaling and a proinflammatory response.

Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay

Abstract: Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test. The assay employs multiplex PCR using 14 virus-specific primer pairs, followed by a multiplexed target-specific primer extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified by using a fluid microsphere-based array (Universal Array; TmBioscience Corporation, Toronto, Canada) and the Luminex x-MAP system. The assay detected influenza A and B viruses; influenza A virus subtypes H1, H3, and H5 (including subtype H5N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and B; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKU1. In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 112 positives, for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that either were not detected by DFA/culture or were positive for viruses not tested for by DFA/culture. After resolution of discordant results by using a second unique PCR assay and by using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives, for a sensitivity of 98.5%, whereas DFA and culture detected only 126 of 183 true positives, for a sensitivity of 68.8%. The RVP test should improve the capabilities of hospital and public health laboratories for diagnosing viral respiratory tract infections and should assist public health agencies in identifying etiologic agents in respiratory tract infection outbreaks.

Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract.

Abstract: Poor human-to-human transmission of influenza A H5N1 virus has been attributed to the paucity of putative sialic acid alpha2-3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site. We now demonstrate that ex vivo cultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.

Influenza Virus Hemagglutinin and Neuraminidase, but Not the Matrix Protein, Are Required for Assembly and Budding of Plasmid-Derived Virus-Like Particles

Abstract: For influenza virus, we developed an efficient, noncytotoxic, plasmid-based virus-like particle (VLP) system to reflect authentic virus particles. This system was characterized biochemically by analysis of VLP protein composition, morphologically by electron microscopy, and functionally with a VLP infectivity assay. The VLP system was used to address the identity of the minimal set of viral proteins required for budding. Combinations of viral proteins were expressed in cells, and the polypeptide composition of the particles released into the culture media was analyzed. Contrary to previous findings in which matrix (M1) protein was considered to be the driving force of budding because M1 was found to be released copiously into the culture medium when M1 was expressed by using the vaccinia virus T7 RNA polymerase-driven overexpression system, in our noncytotoxic VLP system M1 was not released efficiently into the culture medium. Additionally, hemagglutinin (HA), when treated with exogenous neuraminidase (NA) or coexpressed with viral NA, could be released from cells independently of M1. Incorporation of M1 into VLPs required HA expression, although when M1 was omitted from VLPs, particles with morphologies similar to those of wild-type VLPs or viruses were observed. Furthermore, when HA and NA cytoplasmic tail mutants were included in the VLPs, M1 failed to be efficiently incorporated into VLPs, consistent with a model in which the glycoproteins control virus budding by sorting to lipid raft microdomains and recruiting the internal viral core components. VLP formation also occurred independently of the function of Vps4 in the multivesicular body pathway, as dominant-negative Vps4 proteins failed to inhibit influenza VLP budding.

Virus-Like Particle Vaccine Induces Protective Immunity against Homologous and Heterologous Strains of Influenza Virus

Abstract: Recurrent outbreaks of highly pathogenic avian influenza virus pose the threat of pandemic spread of lethal disease and make it a priority to develop safe and effective vaccines. Influenza virus-like particles (VLPs) have been suggested to be a promising vaccine approach. However, VLP-induced immune responses, and their roles in inducing memory immune responses and cross-protective immunity have not been investigated. In this study, we developed VLPs containing influenza virus A/PR8/34 (H1N1) hemagglutinin (HA) and matrix (M1) proteins and investigated their immunogenicity, long-term cross-protective efficacy, and effects on lung proinflammatory cytokines in mice. Intranasal immunization with VLPs containing HA induced high serum and mucosal antibody titers and neutralizing activity against PR8 and A/WSN/33 (H1N1) viruses. Mice immunized with VLPs containing HA showed little or no proinflammatory lung cytokines and were protected from a lethal challenge with mouse-adapted PR8 or WSN viruses even 5 months postimmunization. Influenza VLPs induced mucosal immunoglobulin G and cellular immune responses, which were reactivated rapidly upon virus challenge. Long-lived antibody-secreting cells were detected in the bone marrow of immunized mice. Immune sera administered intranasally were able to confer 100% protection from a lethal challenge with PR8 or WSN, which provides further evidence that anti-HA antibodies are primarily responsible for preventing infection. Taken together, these results indicate that nonreplicating influenza VLPs represent a promising strategy for the development of a safe and effective vaccine to control the spread of lethal influenza viruses.

Persistence of h5 and h7 avian influenza viruses in water

Abstract: Although fecal-oral transmission of avian influenza viruses (AIV) via contaminated water represents a recognized mechanism for transmission within wild waterfowl populations, little is known about viral persistence in this medium. In order to provide initial data on persistence of H5 and H7 AIVs in water, we evaluated eight wild-type low-pathogenicity H5 and H7 AIVs isolated from species representing the two major influenza reservoirs (Anseriformes and Charadriiformes). In addition, the persistence of two highly pathogenic avian influenza (HPAI) H5N1 viruses from Asia was examined to provide some insight into the potential for these viruses to be transmitted and maintained in the environments of wild bird populations. Viruses were tested at two temperatures (17 C and 28 C) and three salinity levels (0, 15, and 30 parts per thousand sea salt). The wild-type H5 and H7 AIV persistence data to date indicate the following: 1) that H5 and H7 AIVs can persist for extended periods of time in water, with a duration of infectivity comparable to AIVs of other subtypes; 2) that the persistence of H5 and H7 AIVs is inversely proportional to temperature and salinity of water; and 3) that a significant interaction exists between the effects of temperature and salinity on the persistence of AIV, with the effect of salinity more prominent at lower temperatures. Results from the two HPAI H5N1 viruses from Asia indicate that these viruses did not persist as long as the wild-type AIVs.

Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice.

Abstract: Highly pathogenic avian H5N1 influenza A viruses have spread throughout Asia, Europe, and Africa, raising serious worldwide concern about their pandemic potential. Although more than 250 people have been infected with these viruses, with a consequent high rate of mortality, the molecular mechanisms responsible for the efficient transmission of H5N1 viruses among humans remain elusive. We used a mouse model to examine the role of the amino acid at position 627 of the PB2 viral protein in efficient replication of H5N1 viruses in the mammalian respiratory tract. Viruses possessing Lys at position 627 of PB2 replicated efficiently in lungs and nasal turbinates, as well as in cells, even at the lower temperature of 33 °C. Those viruses possessing Glu at this position replicated less well in nasal turbinates than in lungs, and less well in cells at the lower temperature. These results suggest that Lys at PB2–627 confers to avian H5N1 viruses the advantage of efficient growth in the upper and lower respiratory tracts of mammals. Therefore, efficient viral growth in the upper respiratory tract may provide a platform for the adaptation of avian H5N1 influenza viruses to humans and for efficient person-to-person virus transmission, in the context of changes in other viral properties including specificity for human (sialic acid α-2,6-galactose containing) receptors.

Differential role of TLR- and RLR-signaling in the immune responses to influenza A virus infection and vaccination.

Abstract: The innate immune system recognizes influenza A virus via TLR 7 or retinoic acid-inducible gene I in a cell-type specific manner in vitro, however, physiological function(s) of the MyD88- or interferon-β promoter stimulator 1 (IPS-1)-dependent signaling pathways in antiviral responses in vivo remain unclear. In this study, we show that although either MyD88- or IPS-1-signaling pathway was sufficient to control initial antiviral responses to intranasal influenza A virus infection, mice lacking both pathways failed to show antiviral responses, resulting in increased viral load in the lung. By contrast, induction of B cells or CD4 T cells specific to the dominant hemagglutinin or nuclear protein Ags respectively, was strictly dependent on MyD88 signaling, but not IPS-1 signaling, whereas induction of nuclear protein Ag-specific CD8 T cells was not impaired in the absence of either MyD88 or IPS-1. Moreover, vaccination of TLR7- and MyD88-deficient mice with inactivated virus failed to confer protection against a lethal live virus challenge. These results strongly suggest that either the MyD88 or IPS-1 signaling pathway is sufficient for initial antiviral responses, whereas the protective adaptive immune responses to influenza A virus are governed by the TLR7-MyD88 pathway.

Influenza A Virus NS1 Protein Activates the PI3K/Akt Pathway To Mediate Antiapoptotic Signaling Responses

Abstract: Recently we have shown that influenza A virus infection leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway and that this cellular reaction is dependent on the expression of the viral nonstructural protein 1 (NS1). These data also suggested that PI3K activation confers a virus-supporting activity at intermediate stages of the infection cycle. So far it is not known which process is regulated by the kinase that supports virus replication. It is well established that upon infection with influenza A virus, the expression of the viral NS1 keeps the induction of beta interferon and the apoptotic response within a tolerable limit. On a molecular basis, this activity of NS1 has been suggested to preclude the activation of cellular double-stranded RNA receptors as well as impaired modulation of mRNA processing. Here we present a novel mode of action of the NS1 protein to suppress apoptosis induction. NS1 binds to and activates PI3K, which results in the activation of the PI3K effector Akt. This leads to a subsequent inhibition of caspase 9 and glycogen synthase-kinase 3β and limitation of the virus-induced cell death program. Thus, NS1 not only blocks but also activates signaling pathways to ensure efficient virus replication.