Showing papers on "Influenza A virus published in 1996"
TL;DR: It is concluded that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice.
Abstract: The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2) Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L-arginine (L-NMMA) administration Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice
492 citations
TL;DR: This challenge provides an opportunity to develop, test, and have in place a strategy for control of interpandemic influenza before the next pandemic.
Abstract: The report of the Institute of Medicine's Committee on Emerging Microbial Threats to Health in the United States, published in 1992, defines influenza virus as the prototype emerging infection (1). Pandemics of influenza have been recognized since earliest recorded history and, because of the mutability of the virus, still represent a formidable threat to the health of the nation. Although much progress has been made in describing the molecular aspects of the virus, in elucidating the epidemiology and modes of spread, and in developing methods for prevention and treatment, a rational strategy for control has not been established. The trends of modern society, including the increasing availability of rapid human transportation and the urbanization of the rapidly expanding human population, tend to facilitate the spread of influenza and increase morbidity. Modern medicine can reduce the mortality that resulted from complications of infection with influenza virus during earlier epidemics, but the cost of medical interventions has increased to the point that effective methods of epidemic control should be considered. This challenge provides an opportunity to develop, test, and have in place a strategy for control of interpandemic influenza before the next pandemic. Pandemics result from the emergence of an influenza A virus that is novel for the human population. Evidence for recycling of subtypes of influenza A after intervals of 60 years or more has been derived by determining antibody prevalence in elderly populations prior to the emergence of subtypes H2N2 in 1957 and H3N2 in 1968 (2, 3). A more ominous threat is the reservoir of 14 influenza A subtypes that persist in avian hosts (4). An avian virus can reassort with a human virus, as occurred in 1957 and 1968, to allow the creation of progeny that possess novel surface antigens with the potential to spread in human popu-
414 citations
TL;DR: It was revealed that Ca-SP selectively inhibited the penetration of virus into host cells, and retention of molecular conformation by chelation of calcium ion with sulfate groups was suggested to be indispensable to its antiviral effect.
Abstract: Bioactivity-directed fractionation of a hot H2O extract from a blue-green alga Spirulina platensis led to the isolation of a novel sulfated polysaccharide named calcium spirulan (Ca-SP) as an antiviral principle. This polysaccharide was composed of rhamnose, ribose, mannose, fructose, galactose, xylose, glucose, glucuronic acid, galacturonic acid, sulfate, and calcium. Ca-SP was found to inhibit the replication of several enveloped viruses, including Herpes simplex virus type 1, human cytomegalovirus, measles virus, mumps virus, influenza A virus, and HIV-1. It was revealed that Ca-SP selectively inhibited the penetration of virus into host cells. Retention of molecular conformation by chelation of calcium ion with sulfate groups was suggested to be indispensable to its antiviral effect.
370 citations
TL;DR: These data show that intramuscular inoculation leads to Th1-like responses due to elevated IgG2a levels, production of gamma interferon, CTL activity, and lack of IL-4, consistent with the idea that the types of responses elicited following DNA immunization.
Abstract: Endpoint immunoglobulin G (IgG) titers and cytotoxic T-lymphocyte (CTL) activities were identical between mice immunized via the intramuscular and epidermal (gene gun) routes with 100 and 1 micrograms, respectively, of an influenza virus nucleoprotein (NP) expression vector. However, examination of the relative levels of two IgG subclasses demonstrated that muscle inoculation resulted in predominantly IgG2a responses, whereas gene gun immunization yielded a preponderance of IgG1 antibodies. Inasmuch as these data suggested that muscle inoculation and gene gun delivery elicited Th1-like and Th2-like responses, respectively, gamma interferon release profiles from antigen-stimulated splenocytes were remarkably similar between these groups. Interleukin-4 (IL-4) production assays, on the other hand, revealed qualitative differences that could be correlated with the divergent IgG subclass data. Waning gamma interferon production in gene gun-immunized animals was countered by a marked increase in IL-4 production following the third immunization, as was the case in control animals immunized with inactivated influenza virus formulated with Freund's adjuvant. In contrast, significant levels of IL-4 production were not observed in the intramuscular DNA inoculation group, despite similar decreases in gamma interferon production with increasing immunizations. These data show that intramuscular inoculation leads to Th1-like responses due to elevated IgG2a levels, production of gamma interferon, CTL activity, and lack of IL-4. However, gene gun responses are more difficult to categorize because of the presence of significant gamma interferon and CTL activity on the one hand and elevated IgG1 antibodies and increasing IL-4 production with successive immunizations on the other. In addition, there was a lack of correlation between IgG isotype ratios and cytokine production in all of the NP DNA-immunized animals, in that IgG subclass ratios remained fixed while cytokine production patterns fluctuated with successive immunizations. These data are consistent with the idea that the types of responses elicited following DNA immunization. are dependent on both the identity of the antigen and the route of DNA administration.
335 citations
TL;DR: This method represents a convenient alternative to the previously established RNP transfection system and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs.
Abstract: A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system.
303 citations
TL;DR: Direct respiratory administration of the selective neuraminidase inhibitor GG167 appears safe and effective for both prevention and early treatment of experimental influenza.
Abstract: Objective. —The current study evaluated whether intranasal administration of the sialic acid analog 4-guanidino-Neu5Ac2en (GG167), an inhibitor of influenza virus neuraminidase, was effective and safe in either preventing or treating experimental human influenza. Methods. —Four randomized, double-blind, placebo-controlled trials involving three prophylaxis limbs, two early treatment limbs, and one delayed treatment limb were conducted. Setting. —Isolation in individual rooms. Participants. —Susceptible (serum hemagglutination-inhibition antibody titer ≤1:8) adult volunteers (n=166) were inoculated intranasally with 10 5 TCID 50 influenza A/Texas/91 (H1N1) virus. Intervention. —GG167, 3.6 to 16 mg, was administered intranasally two or six times daily beginning 4 hours before inoculation (prophylaxis) or 1 or 2 days afterward (early or delayed treatment). Main Outcomes. —Virological measures were frequency of infection based on viral shedding and/or seroconversion (prophylaxis) or quantitative viral shedding based on titers and duration of virus recovery (treatment). Clinical measures were the frequency of febrile illness and symptom severity scores. Results. —Intranasal GG167 was well tolerated for both prophylaxis and therapy. For all dose groups combined, GG167 prophylaxis was 82% effective in preventing laboratory evidence of infection and 95% effective in preventing febrile illness ( P P Conclusions. —Direct respiratory administration of the selective neuraminidase inhibitor GG167 appears safe and effective for both prevention and early treatment of experimental influenza. Influenza virus neuraminidase is important for viral replication in humans. ( JAMA . 1996;275:295-299)
280 citations
TL;DR: Molecular changes in the haemagglutinin (HA)-coding regions and proteolytic cleavage sites from multiple H5N2 subtype viruses isolated during a recent outbreak of avian influenza in central Mexico have been characterized.
Abstract: Molecular changes in the haemagglutinin (HA)-coding regions and proteolytic cleavage sites from multiple H5N2 subtype viruses isolated during a recent outbreak of avian influenza (AI) in central Mexico have been characterized. Eighteen isolates, collected during a 15 month period (October 1993 to January 1995) from six central states, were sequenced. None of the 18 predicted HA1 amino acid sequences were identical and changes were not restricted to a specific region of the sequence. Phylogenetic analyses of the HA1 sequences demonstrated two virus lineages, designated Puebla and Jalisco, with sequence variation as high as 10.5% for amino acid and 6.2% for nucleotide sequences. During the latter months of the surveillance period, highly pathogenic (HP) strains of Al emerged causing lethal disease in commercial poultry flocks. In each of the HP strains isolated, the HA protein was cleaved in chicken embryo fibroblast cells in the absence of trypsin, and two alterations not found in earlier non-HP isolates were detected. In the HA protein, HP strains all had a glutamic acid → lysine substitution at amino acid position 324 and an insertion of arginine and lysine as new residues 325 and 326. The insertion appears to be due to a duplication of the nucleotide sequence AAAGAA at nucleotide positions 965–970 of the HA1-coding region. Computer-assisted secondary structure analyses place the target for the insertion in a predicted RNA stem-loop structure. A mechanism is suggested by which the polymerase duplicates the sequence.
277 citations
TL;DR: It is shown that the neuraminidase glycoprotein of influenza A and B viruses directly activates latent TGF-beta in vitro, the first report showing direct activation of latent T GF-beta by a viral protein.
Abstract: Transforming growth factor beta (TGF-beta) is a family of proteins secreted by virtually all cells in a biologically inactive form. TGF-beta levels increase during many pathophysiological situations, including viral infection. The mechanism for increased TGF-beta activity during viral infection is not understood. We observed an increase in active TGF-beta levels within 1 day in mice infected with influenza virus. Further studies showed that the neuraminidase glycoprotein of influenza A and B viruses directly activates latent TGF-beta in vitro. There are sufficient levels of TGF-beta activated by virus to induce apoptosis in cells. In addition, influenza virus-induced apoptosis is partially inhibited by TGF-beta-specific antibodies. These novel findings suggest a potential role for activation of TGF-beta during the host response to influenza virus infection, specifically apoptosis. This is the first report showing direct activation of latent TGF-beta by a viral protein.
251 citations
TL;DR: The findings suggest that the emergence of mutants resistant to 4-guanidine-Neu5Ac2en is a multistep process requiring prolonged exposure to the inhibitor.
Abstract: The development of viral resistance to the neuraminidase (NA) inhibitor, 4-guanidino-Neu5Ac2en, of influenza viruses was studied by serial passage of A/Turkey/Minnesota/833/80 (H4N2) in Madin-Darby canine kidney cells in the presence of increasing concentrations of inhibitor. Resistant mutants selected after eight passages, had a 10,000-fold reduction in sensitivity to the inhibitor in plaque assays, but their affinity (1/Kd) to the inhibitor was similar to that of the parental virus. Electron microscopic analysis revealed aggregation of the mutant virus at the cell surface in the presence of the inhibitor. Sequence analysis established that a substitution had occurred in the NA (Arg-249 to Lys) and in the HA2 subunit of the hemagglutinin (Gly-75 to Glu), in the vicinity of the proposed second sialic acid binding site. The change of residue 249 appears to be a chance mutation, for we were unable to reisolate this mutant, whereas subsequent experiments indicate changes in the hemagglutinin. After 13 passages of the parental virus, mutants that were resistant to the high concentrations of inhibitor tested were obtained. These viruses retained their drug-resistant phenotype even after five passages without the inhibitor. Electron microscopic analysis revealed no aggregation of virus on the surface of infected cells in the presence of the inhibitor. Sequence analysis of the NA gene from these drug-resistant mutants revealed an additional substitution of Glu to Ala at the conserved amino acid residue 119. This substitution is responsible for reducing the affinity of the inhibitor to the NA. Our findings suggest that the emergence of mutants resistant to 4-guanidine-Neu5Ac2en is a multistep process requiring prolonged exposure to the inhibitor.
205 citations
TL;DR: The genetic analysis of avian influenza A H1N1 viruses recently isolated from pigs in southern China indicates that these genes form an Asian sublineage of the Eurasian avian lineage, suggesting that these viruses are an independent introduction into pigs in Asia.
Abstract: Avian influenza A viruses from Asia are recognized as the source of genes that reassorted with human viral genes to generate the Asian/57 (H2N2) and Hong Kong/68 (H3N2) pandemic strains earlier in this century. Here we report the genetic analysis of avian influenza A H1N1 viruses recently isolated from pigs in southern China, a host suspected to generate new pandemic strains through gene reassortment events. Each of the eight gene segments was of avian origin. Phylogenetic analysis indicates that these genes form an Asian sublineage of the Eurasian avian lineage, suggesting that these viruses are an independent introduction into pigs in Asia. The presence of avian influenza viruses in pigs in China places them in an optimal position for transmission to humans and may serve as an early warning of the emergence of the next human influenza virus pandemic.
202 citations
TL;DR: Monitoring of solid organ transplant recipients (SOTs) to influenza vaccine during consecutive influenza seasons found some SOTs have deficient responses to inactivated influenza vaccines.
Abstract: We monitored the responses of solid organ transplant recipients (SOTs) to influenza vaccine during consecutive influenza seasons. Standard 1993-1994 trivalent influenza vaccine was given to 68 SOTs and 29 healthy young adults, and hemagglutination-inhibition (HI) antibody titers were determined pre- and post-immunization. Significant rises in geometric mean antibody titers occurred post-immunization for all three antigens in both groups. However, the magnitude of the rise was lower in SOTs (1.5-2.3-fold vs. 8.7-10.4-fold, depending on the antigen) (P or = 1:40) for B/Panama/45/90 antigens (50% of SOTs vs. 76% of healthy subjects) and for A/Texas/36/91 (H1N1) antigens (60% vs. 90%). After exclusion of persons with high preimmunization titers, SOTs had significantly reduced frequencies of > or = 4-fold antibody responses compared with those of healthy subjects (23%-38% vs. 86%-100%) (P < .05 for each antigen). When a series of two injections of standard 1994-1995 vaccine was given to 23 SOTs, there was no significant improvement in vaccine response with the second dose. Some SOTs have deficient responses to inactivated influenza vaccines.
TL;DR: Results indicate that Vero cell lines could serve as an alternative host system for the cultivation of influenza A and B viruses, providing adequate quantities of either virus to meet the vaccine requirements imposed by an emerging pandemic.
Abstract: The preparation of live, attenuated human influenza virus vaccines and of large quantities of inactivated vaccines after the emergence or reemergence of a pandemic influenza virus will require an alternative host cell system, because embryonated chicken eggs will likely be insufficient and suboptimal. Preliminary studies indicated that an African green monkey kidney cell line (Vero) is a suitable system for the primary isolation and cultivation of influenza A viruses (E. A. Govorkova, N. V. Kaverin, L. V. Gubareva, B. Meignier, and R. G. Webster, J. Infect. Dis. 172:250-253, 1995). We now demonstrate for the first time that Vero cells are suitable for isolation and productive replication of influenza B viruses and determine the biological and genetic properties of both influenza A and B viruses in Vero cells; additionally, we characterize the receptors on Vero cells compared with those on Madin-Darby canine kidney (MDCK) cells. Sequence analysis indicated that the hemagglutinin of Vero cell-derived influenza B viruses was identical to that of MDCK-grown counterparts but differed from that of egg-grown viruses at amino acid positions 196 to 198. Fluorescence-activated cell sorting analysis showed that although Vero cells possess predominantly alpha2,3 galactose-linked sialic acid, they are fully susceptible to infection with either human influenza A or B viruses. Moreover, all virus-specific polypeptides were synthesized in the same proportions in Vero cells as in MDCK cells. Electron microscopic and immunofluorescence studies confirmed that infected Vero cells undergo the same morphological changes as do other polarized epithelia] cells. Taken together, these results indicate that Vero cell lines could serve as an alternative host system for the cultivation of influenza A and B viruses, providing adequate quantities of either virus to meet the vaccine requirements imposed by an emerging pandemic.
TL;DR: It is concluded that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus.
Abstract: In this study, we investigated the role of the conserved neuraminidase (NA) cytoplasmic tail residues in influenza virus replication. Mutants of influenza A virus (A/WSN/33 [H1N1]) with deletions of the NA cytoplasmic tail region were generated by reverse genetics. The resulting viruses, designated NOTAIL, contain only the initiating methionine of the conserved six amino-terminal residues. The mutant viruses grew much less readily and produced smaller plaques than did the wild-type virus. Despite similar levels of NA cell surface expression by the NOTAIL mutants and wild-type virus, incorporation of mutant NA molecules into virions was decreased by 86%. This reduction resulted in less NA activity per virion, leading to the formation of large aggregates of progeny mutant virions on the surface of infected cells. A NOTAIL virus containing an additional mutation (Ser-12 to Pro) in the transmembrane domain incorporated three times more NA molecules into virions than did the NOTAIL parent but approximately half of the amount incorporated by the wild-type virus. However, aggregation of the progeny virions still occurred at the cell surface. All NOTAIL viruses were attenuated in mice. We conclude that the cytoplasmic tail of NA is not absolutely essential for virus replication but exerts important effects on the incorporation of NA into virions and thus on the aggregation and virulence of progeny virus. In addition, the relative abundance of long filamentous particles formed by the NOTAIL mutants, compared with the largely spherical wild-type particles, indicates a role for the NA cytoplasmic tail in virion morphogenesis.
TL;DR: The highly conserved cytoplasmic tails of the HA and NA play an important role in virus assembly in cells infected with the NA/TAIL(-) transfectant.
Abstract: We have analyzed the mechanism by which the matrix (M1) protein associates with cellular membranes during influenza A virus assembly. Interaction of the M1 protein with the viral hemagglutinin (HA) or neuraminidase (NA) glycoprotein was extensively analyzed by using wild-type and transfectant influenza viruses as well as recombinant vaccinia viruses expressing the M1 protein, HA, or NA. Membrane binding of the M1 protein was significantly stimulated at the late stage of virus infection. Using recombinant vaccinia viruses, we found that a relatively small fraction (20 to 40%) of the cytoplasmic M1 protein associated with cellular membranes in the absence of other viral proteins, while coexpression of the HA and the NA stimulated membrane binding of the M1 protein. The stimulatory effect of the NA (>90%) was significant and higher than that of the HA (>60%). Introduction of mutations into the cytoplasmic tail of the NA interfered with its stimulatory effect. Meanwhile, the HA may complement the defective NA and facilitate virus assembly in cells infected with the NA/TAIL(-) transfectant. In conclusion, the highly conserved cytoplasmic tails of the HA and NA play an important role in virus assembly.
TL;DR: Transfection with a PKR having a point mutation in the catalytic domain of K at 296 to R suppressed both the augmented expression of Fas and cell death by influenza virus infection, suggesting the involvement of PKR in influenza virus-induced cell death.
Abstract: We reported previously that influenza virus infection induces the apoptotic death of HeLa cells associated with activation of the Fas gene. In this report, we show that transfection with a PKR having a point mutation in the catalytic domain of K at 296 to R suppressed both the augmented expression of Fas and cell death by influenza virus infection. These results suggested the involvement of PKR in influenza virus-induced cell death.
TL;DR: It is suggested that the virus-specific induction of mononuclear cell-attracting chemokines accounts for the preferential influx ofmononuclear leukocytes into virus-infected tissue.
Abstract: It is characteristic for virus infections that monocytes/macrophages and lymphocytes infiltrate infected tissue while neutrophils are absent. To understand the mechanisms selectively attracting mononuclear cells in viral diseases, we examined in an influenza A virus model the expression and regulation of chemokines as candidate molecules responsible for the immigration of leukocytes into inflamed tissue. After influenza A virus infection of human monocytes, a rapid expression of the mononuclear cell attracting CC-chemokine genes MIP-1, MCP-1, and RANTES occurred which was followed by the release of chemokine proteins. In striking contrast to CC-chemokines, the expression of the prototype neutrophil CXC-chemoattractants IL-8 and GRO-alpha was completely suppressed after influenza A infection. The release of other neutrophil chemotactic factors was excluded by microchemotaxis assays. These results suggest that the virus-specific induction of mononuclear cell-attracting chemokines accounts for the preferential influx of mononuclear leukocytes into virus-infected tissue.
TL;DR: Results indicate that collectin-mediated viral aggregation per se may be an important host defense mechanism not only by virtue of reducing the number of infectious viral particles, but also by promoting phagocyte responsiveness.
TL;DR: A novel anti-influenza virus compound, flutimide, was identified in extracts of a recently identified fungal species, Delitschia confertaspora, and selectively inhibited the cap-dependent transcriptase of influenza A and B viruses and had no effect on the activities of other polymerases.
Abstract: A novel anti-influenza virus compound, flutimide, was identified in extracts of a recently identified fungal species, Delitschia confertaspora (F. Pelaez, J.D. Polishook, M. Valldosera, and J.Guarro, Mycotaxon 50:115-122, 1994). The compound, a substituted 2,6-diketopiperazine, selectively inhibited the cap-dependent transcriptase of influenza A and B viruses and had no effect on the activities of other polymerases. Similar to the 4-substituted 2,4-dioxobutanoic acids, a series of transcriptase inhibitors which we described previously (J. Tomassini, H. Selnick, M.E. Davies, M.E. Armstrong, J. Baldwin, M. Bourgeois, J.Hastings, D. Hazuda, J. Lewis, W. McClements, G. Ponticello, E. Radzilowski, G. Smith, A. Tebben, and A. Wolfe, Antimicrob. Agents Chemother. 38:2827-2837, 1994), this inhibitor, which is a natural product, affected neither the initiation nor the elongation of influenza virus mRNA synthesis, but it specifically targeted the cap-dependent endonuclease of the transcriptase. Additionally, the compound was inhibitory to the replication of influenza A and B viruses in cell culture. The selective antiviral properties of this compound further demonstrate the utility of influenza virus endonuclease as a target of antiviral agents.
TL;DR: It is demonstrated that IFN-gamma affects the local cellular response in the respiratory tract as well as the systemic humoral response to influenza virus infection.
Abstract: Influenza virus infection induces the local production of gamma interferon (IFN-gamma) by T cells and non-T cells in the respiratory tract. To elucidate the possible functions of this cytokine, the humoral and local cellular immune responses to influenza virus were studied in BALB/c mice with or without in vivo neutralization of IFN-gamma by using monoclonal antibodies. Neutralization of IFN-gamma led to a significant reduction in virus-specific titers of immunoglobulins G2a and G3 in serum but had little effect on other isotypes. Studies on cells isolated from the lung parenchyma itself revealed that at the height of the immune response the ability of these cells to produce cytokines after antigen or T-cell receptor/CD3 stimulation was not affected. Ex vivo cytolytic activity by lung parenchyma cells, which is induced by infection with this virus in normal mice, was also found to be undisturbed by this treatment, even though anti-IFN-gamma antibody activity was recovered from lung lavage samples and sera at all days studied. Surprisingly, in vivo neutralization of IFN-gamma led to a significant reduction in the magnitude of the cellular infiltrate in the lung tissue which followed infection, suggesting an involvement of IFN-gamma in the mechanisms that regulate increased leucocyte traffic in the inflamed lung parenchyma. This conclusion was supported by findings of differences between mock-treated and anti-IFN-gamma-treated mice in the number of CD8+ lung T cells expressing CD49d (alpha4-integrin) and CD62L at various times after influenza virus infection. This study therefore demonstrates that IFN-gamma affects the local cellular response in the respiratory tract as well as the systemic humoral response to influenza virus infection.
TL;DR: M1's role as the inhibitor of premature nuclear import and as the main regulator of nuclear transport of vRNPs is confirmed and vRNP shuttled between the nucleus and the cytoplasm in ts51-infected cells is confirmed.
Abstract: The influenza virus nucleoprotein (NP), matrix protein (M1), and ribonucleoproteins (vRNPs) undergo regulated nuclear import and export during infection. Their trafficking was analyzed by using interspecies heterokaryons containing nuclei from infected and uninfected cells. Under normal conditions, it was demonstrated that the vRNPs which were assembled in the nucleus and transported to the cytosol were prevented from reimport into the nucleus. To be import competent, they must first assemble into virions and enter by the endosomal entry pathway. In influenza virus mutant ts51, in which M1 is defective, direct reimport took place but was inhibited by heterologous expression of wild-type M1. These data confirm M1's role as the inhibitor of premature nuclear import and as the main regulator of nuclear transport of vRNPs. In addition to this vRNP shuttling, M1 also shuttled between the nucleus and the cytoplasm in ts51-infected cells. When NP was expressed in the absence of virus infection, it was also found to be a shuttling protein.
TL;DR: RT-PCR is an effective alternative to virus isolation for the detection of influenza A virus in clinical specimens and has a sensitivity, specificity, and efficiency higher than the best diagnostic kit.
Abstract: We applied a reverse transcription (RT)-PCR assay for influenza A virus to combined nasal wash-throat swab specimens previously obtained from an outpatient pediatric population with acute respiratory illness during concurrent epidemics of influenza A virus and respiratory syncytial virus. The results of the RT-PCR assay were compared with those previously reported with virus cultivation and commercially available rapid diagnostic kits (E.A. Dominguez, L.H. Taber, and R.B. Couch, J. Clin. Microbiol. 31:2286-2290, 1993). With virus cultivation as the "gold standard", the RT-PCR assay had a sensitivity, specificity, and efficiency of 95, 98, and 97%, respectively, compared with 75, 100, and 93%, respectively, for the best diagnostic kit (Becton Dickinson Directigen). RT-PCR is an effective alternative to virus isolation for the detection of influenza A virus in clinical specimens.
TL;DR: It is found that the vaccine enhanced recovery from infection caused by a shifted (H3N2) influenza virus, probably through the induction of nucleoprotein-specific cytotoxic T-lymphocyte activity.
Abstract: Mice immunized with two intragastrically administered doses of a replication-deficient recombinant vaccinia virus containing the hemagglutinin and nucleoprotein genes from H1N1 influenza virus developed serum anti-H1 immunoglobulin G (IgG) antibody that completely protected the lungs from challenge with H1N1. Almost all of the mice given two intragastric doses also developed mucosal anti-H1 IgA antibody, and those with high anti-H1 IgA titers had completely protected noses. Intramuscular injection of the vaccine protected the lungs but not the noses from challenge. We also found that the vaccine enhanced recovery from infection caused by a shifted (H3N2) influenza virus, probably through the induction of nucleoprotein-specific cytotoxic T-lymphocyte activity. A replication-deficient, orally administered, enteric-coated, vaccinia virus-vectored vaccine might safely protect humans against influenza.
TL;DR: The results show that the induction and magnitude of the primary CD8+ T cell response closely depends on the viral dose used for immunization, while it is not affected by the route of immunization.
TL;DR: It is demonstrated that by supplying the vTF7-3-infected cells with plasmids containing cDNAs of all 10 influenza virus-encoded proteins, the transfected CAT RNA can be expressed and rescued into particles that are budded into the supernatant fluids.
Abstract: We have shown previously that COS-1 cells infected with a vaccinia virus recombinant (vTF7-3) which expresses the T7 RNA polymerase gene and then transfected with four pGEM-derived plasmids encoding the influenza A virus core proteins (nucleoprotein, PB1, PB2, and PA polypeptides) can express a synthetic influenza virus-like chloramphenicol [correction of chloramphenical] acetyltransferase (CAT) RNA (I. Mena, S. de la Luna, C. Albo, J. Martin, A. Nieto, J. Ortin, and A. Portela, J. Gen. Virol. 75:2109-2114, 1994). Here we demonstrate that by supplying the vTF7-3-infected cells with plasmids containing cDNAs of all 10 influenza virus-encoded proteins, the transfected CAT RNA can be expressed and rescued into particles that are budded into the supernatant fluids. The released particles can transfer the enclosed CAT RNA to MDCK cultures and resemble true influenza virions in that they require trypsin treatment to deliver the RNA to fresh cells and are neutralized by a monoclonal antibody specific for the influenza A virus hemagglutinin. Moreover, analysis by electron microscopy showed that the culture medium harvested from the transfected cells contained vesicles that could be labeled with an anti-HA monoclonal antibody and that were similar in size and morphology to authentic influenza virus particles. It is also shown that detection of recombinant particles capable of transmitting the CAT RNA does not require expression of the influenza virus nonstructural protein NS1. All of these data indicate that influenza virus-like particles enclosing a synthetic virus-like RNA can be assembled in cells expressing all viral structural components from recombinant plasmids.
TL;DR: In this paper, the authors showed that CD4+ T-cell-dependent effector mechanisms can eliminate an H3N2 influenza A virus from lung cells that are unable to express class II major histocompatibility complex (MHC) glycoproteins.
Abstract: The experiments described establish that CD4+ T-cell-dependent effector mechanisms can eliminate an H3N2 influenza A virus from lung cells that are unable to express class II major histocompatibility complex (MHC) glycoproteins. Radiation chimeras were made by using CD4+ T cells and bone marrow from CD8-depleted, MHC class II +/+ mice and irradiated (950 rads) MHC class II -/- recipients. The influenza virus-specific CD4+ T-cell responses in these +/+-->-/- mice were not obviously different from those in the +/+-->+/+ controls: the cytokine profiles, the spectra of plasma cells producing the various immunoglobulin isotypes, and the frequencies of virus-specific CD4+ T cells were similar for the two groups. Expression of class II MHC glycoproteins on stimulator cells, B lymphocytes, and monocytes/macrophages is apparently sufficient for CD4+ T cells to terminate influenza virus infection of MHC class II -/- respiratory epithelium. A possible explanation is that the local spread of this lytic virus in the lung is limited by cytokines and/or antibody.
TL;DR: Interestingly, except for one mutant virus (CAC), all of the rescued mutant viruses were restricted for replication in the upper respiratory tract but much less restricted in the lungs, so the HA cytoplasmic tail may play a very important role in the generation of virus that can replicate in multiple cell types.
Abstract: The C terminus of the influenza virus hemagglutinin (HA) contains three cysteine residues that are highly conserved among HA subtypes, two in the cytoplasmic tail and one in the transmembrane domain. All of these C-terminal cysteine residues are modified by the covalent addition of palmitic acid through a thio-ether linkage. To investigate the role of HA palmitylation in virus assembly, we used reverse genetics technique to introduce substitutions and deletions that affected the three conserved cysteine residues into the H3 subtype HA. The rescued viruses contained the HA of subtype H3 (A/Udorn/72) in a subtype H1 helper virus (A/WSN/33) background. Rescued viruses which do not contain a site for palmitylation (by residue substitution or substitution combined with deletion of the cytoplasmic tail) were obtained. Rescued virions had a normal polypeptide composition. Analysis of the kinetics of HA low-pH-induced fusion of the mutants showed no major change from that of virus with wild-type (wt) HA. The PFU/HA ratio of the rescued viruses grown in eggs ranged from that of virus with wt HA to 16-fold lower levels, whereas the PFU/HA ratio of the rescued viruses grown in MDCK cells varied only 2-fold from that of virus with wt HA. However, except for one rescued mutant virus (CAC), the mutant viruses were attenuated in mice, as indicated by a > or = 400-fold increase in the 50% lethal dose. Interestingly, except for one mutant virus (CAC), all of the rescued mutant viruses were restricted for replication in the upper respiratory tract but much less restricted in the lungs. Thus, the HA cytoplasmic tail may play a very important role in the generation of virus that can replicate in multiple cell types.
TL;DR: The conservation of the RNA-binding domain of the NS1 protein among influenza A and B viruses strongly suggests that this domain is required for the replication of all these influenza viruses.
TL;DR: It is found that influenza A virus isolates bind to sulphatide (HSO3-Gal beta 1-->1'Cer), which has no sialic acid residue, and that the infection of Madin-Darby canine kidney cells with the human influenza virus A/Memphis/1/71 (H3N2) is inhibited by sulphatides.
Abstract: We found, by using a virus overlay assay, that influenza A virus isolates bind to sulphatide (HSO3-Gal beta 1-->1'Cer), which has no sialic acid residue, and that the infection of Madin-Darby canine kidney cells with the human influenza virus A/Memphis/1/71 (H3N2) is inhibited by sulphatide. A/Memphis/1/71 (H3N2) causes obvious haemagglutination and low-pH haemolysis of asialoerythrocytes reconstituted with sulphatide. All influenza A virus isolates from the species of animals so far tested bound to sulphatide. The sulphatide-binding specificity of the isolates was different from the viral sialyl-linkage specificity. Influenza A virus isolates also bound to galactosyl ceramide (GalCer; Gal beta 1-->1'Cer), as well as sulphatide, in the virus overlay assays. In contrast, the influenza virus did not bind to N-deacyl, a derivative of sulphatide, glucosyl ceramide or the other neutral glycolipids tested. These results indicate that the linkage of galactose, or sulphated galactose, to ceramide is important for viral binding.
TL;DR: A mobile-cart influenza vaccination program was associated with a significant increase in compliance among healthcare workers, but a majority still remained unvaccinated.
Abstract: OBJECTIVE To study compliance with preventive strategies at a university hospital during an outbreak of nosocomial influenza A during the winter of 1988, and the rates of vaccination of healthcare workers and of nosocomial influenza following changes in vaccine practices after the outbreak. DESIGN Retrospective review of employee health, hospital epidemiology, hospital computing; and clinical microbiology records. SETTING A university hospital. INTERVENTIONS Unvaccinated personnel with exposure within the previous 72 hours to an unisolated case of influenza were offered influenza vaccine and 14 days of amantadine hydrochloride prophylaxis. Personnel with exposure more than 72 hours before evaluation were offered vaccine. A mobile cart was introduced for vaccinating personnel after the 1988 outbreak. RESULTS An outbreak of influenza with 10 nosocomial cases occurred in 1988. Only 4% of exposed employees had been vaccinated previously and 23% of exposed, unvaccinated employees agreed to take vaccine, amantadine, or both. A mobile-cart vaccination program was instituted, and annual vaccination rates steadily increased from 26.3% in 1989 to 1990 to 38% in 1993 to 1994 (P < .0001). The relative frequency of documented cases of influenza in employees with symptoms of influenza decreased significantly during this period (P = .025), but nosocomial influenza rates among patients did not change significantly. CONCLUSION A mobile-cart influenza vaccination program was associated with a significant increase in compliance among healthcare workers, but a majority still remained unvaccinated. The rate of nosocomial influenza among patients was not reduced by the modest increase in the vaccination rate, but influenza rates remained acceptably low, perhaps due to respiratory isolation of patients and furlough of employees with influenza.
TL;DR: Serologic responses to the N1Texas/91 and N2Beijing/92 NA components of trivalent inactivated influenza virus vaccine were measured by NA inhibition and enzyme-linked immunosorbent assay (ELISA), and the results for adults aged 18 to 45 or > or = 65 (elderly) years were compared.
Abstract: Little information is available on the potential role of antibody to influenza virus neuraminidase (NA) in vaccine-induced immunity. In the present study, serologic responses to the N1Texas/91 and N2Beijing/92 NA components of trivalent inactivated influenza virus vaccine were measured by NA inhibition (NI) and enzyme-linked immunosorbent assay (ELISA), and the results for adults aged 18 to 45 (young) or > or = 65 (elderly) years were compared. The two age groups had comparable rates (32 to 50%) of NI response. In contrast, ELISA immunoglobulin G (IgG) antibody responses to N1 and N2 NAs occurred in 70 to 71 and 67 to 83%, respectively, of young subjects but in only 3 to 18 and 18 to 35%, respectively, of elderly subjects. prevaccination mean ELISA IgG and IgA NA antibody titers were generally lower for the young adults than they were for the elderly, whereas the corresponding NI titers were comparable. In young adults, plaque size-reducing NA antibody increases were positively associated with ELISA but not with NI antibody increases. There were no apparent age-related differences in the immunoglobulin isotype distribution of the anti-NA response, with IgG being the dominant class and IgG1 the dominant subclass of serum antibody. Anti-hemagglutinin antibody responses to H1Texas/91 and H3Beijing/92 were greater in magnitude and frequency than the corresponding NA-specific responses to N1Texas/91 and N2Beijing/92 when measured by hemagglutination inhibition and NI, respectively, but not when measured by ELISA. The discordance between NI and ELISA for measurement of NA-specific vaccine responses may reflect the relative insensitivity of NI in discriminating differences when initial antibody titers are low.