Showing papers on "Influenza A virus published in 1998"
TL;DR: The results suggest transmission of the virus from infected chickens to the child without another intermediate mammalian host acting as a "mixing vessel" illustrates the importance of intensive global influenza surveillance.
1,366 citations
TL;DR: An avian H5N1 influenza A virus was isolated from a tracheal aspirate obtained from a 3-year-old child in Hong Kong with a fatal illness consistent with influenza, causing 87.5 to 100 percent mortality in experimentally inoculated White Plymouth Rock and White Leghorn chickens.
Abstract: An avian H5N1 influenza A virus (A/Hong Kong/156/97) was isolated from a tracheal aspirate obtained from a 3-year-old child in Hong Kong with a fatal illness consistent with influenza. Serologic analysis indicated the presence of an H5 hemagglutinin. All eight RNA segments were derived from an avian influenza A virus. The hemagglutinin contained multiple basic amino acids adjacent to the cleavage site, a feature characteristic of highly pathogenic avian influenza A viruses. The virus caused 87.5 to 100 percent mortality in experimentally inoculated White Plymouth Rock and White Leghorn chickens. These results may have implications for global influenza surveillance and planning for pandemic influenza.
1,291 citations
TL;DR: A structural basis for the hypothesis that pigs may serve as “mixing vessels” for the generation of human-avian influenza A virus reassortants, similar to those responsible for the 1957 and 1968 pandemics is demonstrated.
Abstract: Genetic and biologic observations suggest that pigs may serve as “mixing vessels” for the generation of human-avian influenza A virus reassortants, similar to those responsible for the 1957 and 1968 pandemics. Here we demonstrate a structural basis for this hypothesis. Cell surface receptors for both human and avian influenza viruses were identified in the pig trachea, providing a milieu conducive to viral replication and genetic reassortment. Surprisingly, with continued replication, some avian-like swine viruses acquired the ability to recognize human virus receptors, raising the possibility of their direct transmission to human populations. These findings help to explain the emergence of pandemic influenza viruses and support the need for continued surveillance of swine for viruses carrying avian virus genes.
1,010 citations
TL;DR: Avian Influenza A H5N1 virus causes human influenza-like illness with a high rate of complications in adults admitted to hospital, and rapid H5-subtype-specific laboratory diagnosis can be made by RT-PCR applied directly to clinical specimens.
1,010 citations
TL;DR: In this paper, a viable transfectant influenza A virus (delNS1) which lacks the NS1 gene has been generated through the use of reverse genetics, and it has been shown that the NS 1 protein plays a crucial role in inhibiting interferon-mediated antiviral responses of the host.
998 citations
TL;DR: The large proportion of influenza-related deaths during each pandemic and the following decade among persons <65 years old should be considered in planning for pandemics.
Abstract: Almost all deaths related to current influenza epidemics occur among the elderly. However, mortality was greatest among the young during the 1918-1919 pandemic. This study compared the age distribution of influenza-related deaths in the United States during this century's three influenza A pandemics with that of the following epidemics. Half of influenza-related deaths during the 1968-1969 influenza A (H3N2) pandemic and large proportions of influenza-related deaths during the 1957-1958 influenza A (H2N2) and the 1918-1919 influenza A (H1N1) pandemics occurred among persons <65 years old. However, this group accounted for decrementally smaller proportions of deaths during the first decade following each pandemic. A model suggested that this mortality pattern may be explained by selective acquisition of protection against fatal illness among younger persons. The large proportion of influenza-related deaths during each pandemic and the following decade among persons <65 years old should be considered in planning for pandemics.
684 citations
TL;DR: It is found that IL-6 and IFN-alpha levels in nasal lavage fluids peaked early (day 2) and correlated directly with viral titers, temperature, mucus production, and symptom scores, which implicate IL- 6 and IFn-alpha as key factors both in symptom formation and host defense in influenza.
Abstract: To further understand the role of cytokine responses in symptom formation and host defenses in influenza infection, we determined the levels of IL-1beta, IL-2, IL-6, IL-8, IFN-alpha, TGF-beta, and TNF-alpha in nasal lavage fluid, plasma, and serum obtained serially from 19 volunteers experimentally infected with influenza A/Texas/36/91 (H1N1) and correlated these levels with various measures of infection and illness severity. We found that IL-6 and IFN-alpha levels in nasal lavage fluids peaked early (day 2) and correlated directly with viral titers, temperature, mucus production, and symptom scores. IL-6 elevations were also found in the circulation at this time point. In contrast, TNF-alpha responses peaked later (day 3 in plasma, day 4 in nasal fluids), when viral shedding and symptoms were subsiding. Similarly, IL-8 peaked late in the illness course (days 4-6) and correlated only with lower respiratory symptoms, which also occurred late. None of IL-1beta, IL-2, or TGF-beta levels increased significantly. These data implicate IL-6 and IFN-alpha as key factors both in symptom formation and host defense in influenza.
670 citations
TL;DR: A small number of changes in hemagglutinin gene sequences defined two closely related subgroups, with both subgroups having human and chicken members, among the seven viruses examined from Hong Kong, and all seven viruses were highly pathogenic in chickens and caused similar lesions in experimental inoculations.
Abstract: Genes of an influenza A (H5N1) virus from a human in Hong Kong isolated in May 1997 were sequenced and found to be all avian-like (K. Subbarao et al., Science 279:393–395, 1998). Gene sequences of this human isolate were compared to those of a highly pathogenic chicken H5N1 influenza virus isolated from Hong Kong in April 1997. Sequence comparisons of all eight RNA segments from the two viruses show greater than 99% sequence identity between them. However, neither isolate’s gene sequence was closely (>95% sequence identity) related to any other gene sequences found in the GenBank database. Phylogenetic analysis demonstrated that the nucleotide sequences of at least four of the eight RNA segments clustered with Eurasian origin avian influenza viruses. The hemagglutinin gene phylogenetic analysis also included the sequences from an additional three human and two chicken H5N1 virus isolates from Hong Kong, and the isolates separated into two closely related groups. However, no single amino acid change separated the chicken origin and human origin isolates, but they all contained multiple basic amino acids at the hemagglutinin cleavage site, which is associated with a highly pathogenic phenotype in poultry. In experimental intravenous inoculation studies with chickens, all seven viruses were highly pathogenic, killing most birds within 24 h. All infected chickens had virtually identical pathologic lesions, including moderate to severe diffuse edema and interstitial pneumonitis. Viral nucleoprotein was most frequently demonstrated in vascular endothelium, macrophages, heterophils, and cardiac myocytes. Asphyxiation from pulmonary edema and generalized cardiovascular collapse were the most likely pathogenic mechanisms responsible for illness and death. In summary, a small number of changes in hemagglutinin gene sequences defined two closely related subgroups, with both subgroups having human and chicken members, among the seven viruses examined from Hong Kong, and all seven viruses were highly pathogenic in chickens and caused similar lesions in experimental inoculations.
354 citations
TL;DR: 4-Amino- and 4-guanidino-4H-pyran-6-carboxamides 4 and 5 related to zanamivir (GG167) are a new class of inhibitors of influenza virus sialidases and several of the most potent analogues displayed useful antiviral activity in vivo when evaluated in a mouse model of influenza A virus infection.
Abstract: 4-Amino- and 4-guanidino-4H-pyran-6-carboxamides 4 and 5 related to zanamivir (GG167) are a new class of inhibitors of influenza virus sialidases. Structure--activity studies reveal that, in general, secondary amides are weak inhibitors of both influenza A and B viral sialidases. However, tertiary amides, which contain one or more small alkyl groups, show much greater inhibitory activity, particularly against the influenza A virus enzyme. The sialidase inhibitory activities of these compounds correlate well with their in vitro antiviral efficacy, and several of the most potent analogues displayed useful antiviral activity in vivo when evaluated in a mouse model of influenza A virus infection. Carboxamides which were highly active sialidase inhibitors in vitro also showed good antiviral activity in the mouse efficacy model of influenza A infection when administered intranasally but displayed modest activity when delivered by the intraperitoneal route.
320 citations
TL;DR: The multiplex RT-PCR gave an excellent (100%) correlation with the results of conventional typing and subtyping with specific antisera for the typing andSubtyping of influenza A and B viruses and RSV types A andB.
Abstract: A multiplex reverse transcription (RT)-PCR method that has been developed is capable of detecting and subtyping influenza A (H1N1 and H3N2) and B viruses as well as respiratory syncytial virus (RSV) types A and B in respiratory clinical samples taken as part of a national community-based surveillance program of influenza-like illness in England and Wales. The detection of each different pathogen depended on distinguishing five amplification products of different sizes on agarose gels following RT-PCR with multiple primer sets. The multiplex RT-PCR was tested with 65 nasopharyngeal apirates from which RSV had been isolated and 237 combined nose and throat swabs from which influenza A (H1N1 and H3N2) or B virus had been detected by virus isolation, as well as 40 respiratory samples from which other viruses including cytomegalovirus, herpes simplex virus, enteroviruses, and parainfluenza viruses had been grown. For the typing and subtyping of influenza A and B viruses and RSV types A and B, the multiplex RT-PCR gave an excellent (100%) correlation with the results of conventional typing and subtyping with specific antisera. Multiplex RT-PCR can also be used to accurately detect more than one viral template in the same reaction mixture, allowing viral coinfections to be identified with the same respiratory specimen.
314 citations
TL;DR: Seasonal trends of viral respiratory tract infections in the tropics supported by a retrospective survey of laboratory virus isolation, serology and immunofluorescence microscopy in two large general hospitals in Singapore between September 1990 and September 1994.
Abstract: To evaluate the seasonal trends of viral respiratory tract infections in a tropical environment, a retrospective survey of laboratory virus isolation, serology and immunofluorescence microscopy in two large general hospitals in Singapore between September 1990 and September 1994 was carried out. Respiratory tract viral outbreaks, particularly among infants who required hospitalization, were found to be associated mainly with respiratory syncytial (RSV) infections (72%), influenza (11%) and parainfluenza viruses (11%). Consistent seasonal variations in viral infections were observed only with RSV (March-August) and influenza A virus (peaks in June, December-January). The RSV trends were associated with higher environmental temperature, lower relative humidity and higher maximal day-to-day temperature variation. Although the influenza A outbreaks were not associated with meteorological factors, influenza B isolates were positively associated with rainfall. These data support the existence of seasonal trends of viral respiratory tract infections in the tropics.
TL;DR: The results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro.
Abstract: We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1−/− animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1−/− mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT−/− mice, and mice lacking gamma interferon (IFNγ−/− mice) or the IFN-α receptor (IFNαR−/− mice). Influenza A/WSN/33 virus replicates to high titers in STAT1−/− or IFNαR−/− fibroblasts, while cells derived from WT or IFNγ−/− animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro.
Journal Article•
TL;DR: It is reported that cytokines secreted by influenza A virus-infected macrophages are able to induce IFN-gamma synthesis in human T cells and may play an important role for both of these cytokines in the development of Th1-type immune responses.
Abstract: T cells contribute significantly the to host's early defense against viral and bacterial infections and are essential for clearance of the pathogen. IFN-gamma, a product of activated T and NK cells, has, in addition to its direct antimicrobial activity, a major role in activating cell-mediated immunity. Here we report that cytokines secreted by influenza A virus-infected macrophages are able to induce IFN-gamma synthesis in human T cells. Influenza A virus-infected human peripheral macrophages secreted IFN-alpha/beta, TNF-alpha, IL-1beta, and a recently identified cytokine, IL-18 (or IFN-gamma-inducing factor), whereas the production of IL-12 was not detected. Supernatants collected from virus-infected macrophages induced rapid IFN-gamma mRNA expression and protein production in T cells. This was down-regulated by the addition of neutralizing anti-IFN-alpha/beta Abs, whereas neutralizing anti-IL-12 Abs had no effect on IFN-gamma gene expression. Exogenously added IFN-alpha/beta also rapidly stimulated the synthesis of IFN-gamma mRNA in T cells independently of protein synthesis. IL-18 synergized with IFN-alpha to up-regulate IFN-gamma gene expression and protein production. The data suggest that IFN-alpha/beta and IL-18 produced by macrophages during virus infection may act together to induce IFN-gamma synthesis and, consequently, may play an important role for both of these cytokines in the development of Th1-type immune responses.
TL;DR: This work generates HAs with site-specific mutations of a human virus that contain different amino acid residues at these positions of hemagglutinin of the H3 subtype, and shows that the Ser-to-Gly mutation at position 228, in addition to the Leu- to-Gln mutation at location 226, is critical for human virus HA to support virus replication in duck intestine.
Abstract: Influenza A viruses can be isolated from a variety of animals, but their range of hosts is restricted For example, human influenza viruses do not replicate in duck intestine, the major replication site of avian viruses in ducks Although amino acids at positions 226 and 228 of hemagglutinin (HA) of the H3 subtype are known to be important for this host range restriction, the contributions of specific amino acids at these positions to restriction were not known Here, we address this issue by generating HAs with site-specific mutations of a human virus that contain different amino acid residues at these positions We also let ducks select replication-competent viruses from a replication-incompetent virus containing a human virus HA by inoculating animals with 10105 50% egg infectious dose of the latter virus and identified a mutation in the HA Our results showed that the Ser-to-Gly mutation at position 228, in addition to the Leu-to-Gln mutation at position 226 of the HA of the H3 subtype, is critical for human virus HA to support virus replication in duck intestine
TL;DR: An alternative cell culture system which allows rapid production of large volumes of vaccine and was demonstrated to be at least as immunogenic as a conventional egg-derived preparation in a mouse model.
TL;DR: The results indicate that GS 4104 is a novel, orally active antiviral agent with the potential to be used for the prophylaxis and treatment of influenza A and B virus infections.
Abstract: We have recently described GS 4071, a carbocyclic transition-state analog inhibitor of the influenza virus neuraminidase, which has potent inhibitory activity comparable to that of 4-guanidino-Neu5Ac2en (GG167; zanamivir) when tested against influenza A virus replication and neuraminidase activity in vitro. We now report that GS 4071 is active against several strains of influenza A and B viruses in vitro and that oral GS 4104, an ethyl ester prodrug which is converted to GS 4071 in vivo, is active in the mouse and ferret models of influenza virus infection. Oral administration of 10 mg of GS 4104 per kg of body weight per day caused a 100-fold reduction in lung homogenate viral titers and enhanced survival in mice infected with influenza A or B viruses. In ferrets, a 25-mg/kg dose of GS 4104 given twice daily reduced peak viral titers in nasal washings and eliminated constitutional responses to influenza virus infection including fever, increased nasal signs (sneezing, nasal discharge, mouth breathing), and decreased activity. Consistent with our demonstration that the parent compound is highly specific for influenza virus neuraminidases, no significant drug-related toxicity was observed after the administration of oral dosages of GS 4104 of up to 800 mg/kg/day for 14 days in nonclinical toxicology studies with rats. These results indicate that GS 4104 is a novel, orally active antiviral agent with the potential to be used for the prophylaxis and treatment of influenza A and B virus infections.
TL;DR: A reverse genetics system for the nonstructural (NS) gene segment of influenza A virus is established based on the use of the temperature-sensitive (ts) reassortant virus 25A-1, which allows the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein.
Abstract: We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40 degreesC selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.
TL;DR: It is demonstrated that neuraminidase, the second major protein on the virion surface, binds and sequesters plasminogen, leading to higher local concentrations of this ubiquitous protease precursor and thus to increased cleavage of the HA.
Abstract: Cleavage of the hemagglutinin (HA) molecule by proteases is a prerequisite for the infectivity of influenza A viruses. Here, we describe a novel mechanism of HA cleavage for a descendant of the 1918 pandemic strain of human influenza virus. We demonstrate that neuraminidase, the second major protein on the virion surface, binds and sequesters plasminogen, leading to higher local concentrations of this ubiquitous protease precursor and thus to increased cleavage of the HA. The structural basis of this unusual function of the neuraminidase molecule appears to be the presence of a carboxyl-terminal lysine and the absence of an oligosaccharide side chain at position 146 (N2 numbering). These findings suggest a means by which influenza A viruses, and perhaps other viruses as well, could become highly pathogenic in humans.
TL;DR: VSV-HA is an effective intranasal vaccine vector that raises high levels of neutralizing antibody to influenza virus and completely protects mice from bronchial pneumonia caused by challenge with a lethal dose of influenza A virus.
Abstract: Since the development of a system for generating vesicular stomatitis virus (VSV) from plasmid DNAs, our laboratory has reported the expression of several different glycoproteins from recombinant VSVs. In one of these studies, high-level expression of an influenza virus hemagglutinin (HA) from a recombinant VSV-HA and efficient incorporation of the HA protein into the virions was reported (E. Kretzschmar, L. Buonocore, M. J. Schnell, and J. K. Rose, J. Virol. 71:5982–5989, 1997). We report here that VSV-HA is an effective intranasal vaccine vector that raises high levels of neutralizing antibody to influenza virus and completely protects mice from bronchial pneumonia caused by challenge with a lethal dose of influenza A virus. Additionally, these recombinant VSVs are less pathogenic than wild-type VSV (serotype Indiana). This vector-associated pathogenicity was subsequently eliminated through introduction of specific attenuating deletions. These live attenuated recombinant VSVs have great potential as vaccine vectors.
TL;DR: In transgenic mice with a very high frequency of antiinfluenza CTL precursors, but without protective Abs, CD8+ CTLs conferred protection against low dose viral challenge, but exacerbated viral pathology and caused mortality at high viral dose.
Abstract: The ability of influenza virus to evade immune surveillance by neutralizing antibodies (Abs) directed against its variable surface antigens provides a challenge to the development of effective vaccines. CD8+ cytotoxic T lymphocytes (CTLs) restricted by class I major histocompatibility complex molecules are important in establishing immunity to influenza virus because they recognize internal viral proteins which are conserved between multiple viral strains. In contrast, protective Abs are strain-specific. However, the precise role of effector CD8+ CTLs in protection from influenza virus infection, critical for understanding disease pathogenesis, has not been well defined. In transgenic mice with a very high frequency of antiinfluenza CTL precursors, but without protective Abs, CD8+ CTLs conferred protection against low dose viral challenge, but exacerbated viral pathology and caused mortality at high viral dose. The data suggest a dual role for CD8+ CTLs against influenza, which may present a challenge to the development of effective CTL vaccines. Effector mechanisms used by CD8+ CTLs in orchestrating clearance of virus and recovery from experimental influenza infection, or potentiation of lethal pathology, are discussed.
TL;DR: RNA hybridization proved to be a sensitive assay for monitoring influenza virus replication in vitro besides direct measurement of infectious virus and immunofluorescence and the measurement of virus infectivity titers in culture supernatants.
TL;DR: Findings indicate that the human memory CTL response to influenza A virus is broadly directed to epitopes on a wide variety of proteins, unlike the limited response observed following infection of mice.
Abstract: The murine CD8+ cytotoxic-T-lymphocyte (CTL) repertoire appears to be quite limited in response to influenza A viruses. The CTL responses to influenza A virus in humans were examined to determine if the CTL repertoire is also very limited. Bulk cultures revealed that a number of virus proteins were recognized in CTL assays. CTL lines were isolated from three donors for detailed study and found to be specific for epitopes on numerous influenza A viral proteins. Eight distinct CD8+ CTL lines were isolated from donor 1. The proteins recognized by these cell lines included the nucleoprotein (NP), matrix protein (M1), nonstructural protein 1 (NS1), polymerases (PB1 and PB2), and hemagglutinin (HA). Two CD4+ cell lines, one specific for neuraminidase (NA) and the other specific for M1, were also characterized. These CTL results were confirmed by precursor frequency analysis of peptide-specific gamma interferon-producing cells detected by ELISPOT. The epitopes recognized by 6 of these 10 cell lines have not been previously described; 8 of the 10 cell lines were cross-reactive to subtype H1N1, H2N2, and H3N2 viruses, 1 cell line was cross-reactive to subtypes H1N1 and H2N2, and 1 cell line was subtype H1N1 specific. A broad CTL repertoire was detected in the two other donors, and cell lines specific for the NP, NA, HA, M1, NS1, and M2 viral proteins were isolated. These findings indicate that the human memory CTL response to influenza A virus is broadly directed to epitopes on a wide variety of proteins, unlike the limited response observed following infection of mice.
TL;DR: All eight "false-positive" Hexaplex results (in comparison with negative viral culture results) were for symptomatic patients with low numbers of virus RNA copies, suggesting that HexapLex may be more sensitive than virus culture.
Abstract: A multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (Hexaplex; Prodesse, Milwaukee) was developed and used to rapidly detect and quantitate RNA of respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 in nasal wash specimens in a single test. Primers and probes originated from highly conserved regions of each viral genome. Six and a half primer pairs were mixed for the simultaneous detection and quantitation of RNA from seven different respiratory viruses. We tested 109 clinical samples with this assay. Twenty-nine virus culture-positive samples were all positive by Hexaplex. Samples from 40 symptomatic patients were negative by virus culture, but eight of these were positive by Hexaplex. Forty samples from asymptomatic children were negative by both virus culture and Hexaplex. No cross-reactions were noted among 17 different respiratory viruses with use of this assay. Hexaplex was 100% sensitive (95% confidence interval [CI], 0.88-1.0) and 98% specific (95% CI, 0.97-0.99). All eight "false-positive" Hexaplex results (in comparison with negative viral culture results) were for symptomatic patients with low numbers of virus RNA copies. This finding suggests that Hexaplex may be more sensitive than virus culture. Our data demonstrate that Hexaplex is a rapid, sensitive, and specific quantitative test for the diagnosis of infections with these seven common respiratory viruses.
TL;DR: The results of PCR suggest that at least part of the influenza type A genome existed in the central nervous system and Influenza-associated acute encephalopathy or encephalitis in young children deserves wider recognition.
TL;DR: Host NOS2 seems to contribute more significantly to the development of influenza pneumonitis in mice than the cytopathic effects of viral replication, and can suppress another IFN-γ–dependent antiviral mechanism during influenza infection.
Abstract: Viral infection often activates the interferon (IFN)-gamma-inducible gene, nitric oxide synthase 2 (NOS2). Expression of NOS2 can limit viral growth but may also suppress the immune system and damage tissue. This study assessed each of these effects in genetically deficient NOS2(-/-) mice after infection with influenza A, a virus against which IFN-gamma has no known activity. At inocula sufficient to cause consolidating pneumonitis and death in wild-type control mice, NOS2(-/-) hosts survived with little histopathologic evidence of pneumonitis. Moreover, they cleared influenza A virus from their lungs by an IFN-gamma-dependent mechanism that was not evident in wild-type mice. Even when the IFN-gamma-mediated antiviral activity was blocked in NOS2(-/-) mice with anti-IFN-gamma mAb, such mice failed to succumb to disease. Further evidence that this protection was independent of viral load was provided by treating NOS2(+/+) mice with the NOS inhibitor, Nomega-methyl-L-arginine (L-NMA). L-NMA prevented mortality without affecting viral growth. Thus, host NOS2 seems to contribute more significantly to the development of influenza pneumonitis in mice than the cytopathic effects of viral replication. Although NOS2 mediates some antiviral effects of IFN-gamma, during influenza infection it can suppress another IFN-gamma-dependent antiviral mechanism. This mechanism was observed only in the complete absence of NOS2 activity and appeared sufficient to control influenza A virus growth in the absence of changes in cytotoxic T lymphocyte activity.
TL;DR: Depleting the CD8+ T cells substantially reduced the capacity of these primed mice to deal with the infection, in spite of evidence for established CD4+ T-cell memory, and the control of this relatively mild influenza virus is relatively inefficient in the absence of B cells and CD8+.
Abstract: The primary CD8+ T-cell response protected most B-cell-deficient μMT mice against intranasal infection with the HKx31 influenza A virus. Prior exposure did not prevent reinfection upon homologous challenge, and the recall CD8+ T-cell response cleared the virus from the lung within 7 days. Depleting the CD8+ T cells substantially reduced the capacity of these primed mice to deal with the infection, in spite of evidence for established CD4+ T-cell memory. Thus, the control of this relatively mild influenza virus by both primary and secondary CD4+ T-cell responses is relatively inefficient in the absence of B cells and CD8+ T cells.
TL;DR: The porcine influenza A virus system is a relevant preclinical model for humans in terms of both disease and gene transfer to the epidermis and thus provides a basis for advancing the development of DNA-based vaccines.
Abstract: Particle-mediated delivery of a DNA expression vector encoding the hemagglutinin (HA) of an H1N1 influenza virus (A/Swine/Indiana/1726/88) to porcine epidermis elicits a humoral immune response and accelerates the clearance of virus in pigs following a homotypic challenge. Mucosal administration of the HA expression plasmid elicits an immune response that is qualitatively different than that elicited by the epidermal vaccination in terms of inhibition of the initial virus infection. In contrast, delivery of a plasmid encoding an influenza virus nucleoprotein from A/PR/8/34 (H1N1) to the epidermis elicits a strong humoral response but no detectable protection in terms of nasal virus shed. The efficacy of the HA DNA vaccine was compared with that of a commercially available inactivated whole-virus vaccine as well as with the level of immunity afforded by previous infection. The HA DNA and inactivated viral vaccines elicited similar protection in that initial infection was not prevented, but subsequent amplification of the infection is limited, resulting in early clearance of the virus. Convalescent animals which recovered from exposure to virulent swine influenza virus were completely resistant to infection when challenged. The porcine influenza A virus system is a relevant preclinical model for humans in terms of both disease and gene transfer to the epidermis and thus provides a basis for advancing the development of DNA-based vaccines.
TL;DR: Results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.
Abstract: An oral prodrug of GS 4071, a potent and selective inhibitor of influenza neuraminidases, is currently under clinical development for the treatment and prophylaxis of influenza virus infections in humans. To investigate the potential development of resistance during the clinical use of this compound, variants of the human influenza A/Victoria/3/75 (H3N2) virus with reduced susceptibility to the neuraminidase inhibitor GS 4071 were selected in vitro by passaging the virus in MDCK cells in the presence of inhibitor. After eight passages, variants containing two amino acid substitutions in the hemagglutinin (A28T in HA1 and R124M in HA2) but no changes in the neuraminidase were isolated. These variants exhibited a 10-fold reduction in susceptibility to GS 4071 and zanamivir (GG167) in an in vitro plaque reduction assay. After 12 passages, a second variant containing these hemagglutinin mutations and a Lys substitution for the conserved Arg292 of the neuraminidase was isolated. The mutant neuraminidase enzyme exhibited high-level (30,000-fold) resistance to GS 4071, but only moderate (30-fold) resistance to zanamivir and 4-amino-Neu5Ac2en, the amino analog of zanamivir. The mutant enzyme had weaker affinity for the fluorogenic substrate 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and lower enzymatic activity compared to the wild-type enzyme. The viral variant containing the mutant neuraminidase did not replicate as well as the wild-type virus in culture and was 10,000-fold less infectious than the wild-type virus in a mouse model. These results suggest that although the R292K neuraminidase mutation confers high-level resistance to GS 4071 in vitro, its effect on viral virulence is likely to render this mutation of limited clinical significance.
TL;DR: It is concluded that development of antiviral resistance in immunocompromised individuals should be considered when they have been treated with antivirals and have shed influenza virus for a prolonged period and isolates from five of six patients were resistant.
Abstract: The importance and significance of amantadine- or rimantadine-resistant influenza viruses in immunocompromised patients was studied in a population of adult bone marrow transplant (BMT) recipients and patients with leukemia prospectively cultured for respiratory viruses. Influenza A viruses were isolated from 29 patients with acute respiratory illness (14 BMT recipients and 15 patients with leukemia). Fifteen patients (52%) received amantadine (n = 4) or rimantadine (n = 11) therapy. All influenza isolates recovered from six patients shedding virus for > or = 3 days were screened for antiviral susceptibility; resistant isolates were further genetically characterized. Initial influenza isolates were susceptible to amantadine or rimantadine, but subsequent isolates from five of six patients were resistant. Influenza-associated mortality was similar among patients with and without documented antiviral resistance (2 of 5 vs. 5 of 24). We conclude that development of antiviral resistance in immunocompromised individuals should be considered when they have been treated with antivirals and have shed influenza virus for a prolonged period. Isolation procedures should be instituted for all immunocompromised patients with influenza, both during and after therapy with amantadine or rimantadine.
TL;DR: The role of NS1 in PKR modulation during viral infection that is mediated through a complex formation between the two proteins is supported, supported by a coprecipitation assay.
Abstract: The interferon (IFN)-induced protein kinase (PKR) functions as a gatekeeper of mRNA translation initiation and is, therefore, a key mediator of the host IFN-induced antiviral defense syste...