Molecular characterization of H9N2 influenza viruses: were they the donors of the "internal" genes of H5N1 viruses in Hong Kong? (57/4894)

The origin of the H5N1 influenza viruses that killed six of eighteen infected humans in 1997 and were highly pathogenic in chickens has not been resolved. These H5N1 viruses transmitted directly to humans from infected poultry. In the poultry markets in Hong Kong, both H5N1 and H9N2 influenza viruses were cocirculating, raising the possibility of genetic reassortment. Here we analyze the antigenic and genetic features of H9N2 influenza viruses with different epidemiological backgrounds. The results suggest that the H9N2 influenza viruses of domestic ducks have become established in the domestic poultry of Asia. Phylogenetic and antigenic analyses of the H9N2 viruses isolated from Hong Kong markets suggest three distinct sublineages. Among the chicken H9N2 viruses, six of the gene segments were apparently derived from an earlier chicken H9N2 virus isolated in China, whereas the PB1 and PB2 genes are closely related to those of the H5N1 viruses and a quail H9N2 virus-A/quail/Hong Kong/G1/97 (Qa/HK/G1/97)-suggesting that many of the 1997 chicken H9 isolates in the markets were reassortants. The similarity of the internal genes of Qa/HK/G1/97 virus to those of the H5N1 influenza viruses suggests that the quail virus may have been the internal gene donor. Our findings indicate that the human and poultry H5N1 influenza viruses in Hong Kong in 1997 were reassortants that obtained internal gene segments from Qa/HK/G1/97. However, we cannot be certain whether the replicate complex of H5N1 originated from Qa/HK/G1/97 or whether the reverse transfer occurred; the available evidence supports the former proposal.  (+info)

Th2 responses to inactivated influenza virus can Be converted to Th1 responses and facilitate recovery from heterosubtypic virus infection. (58/4894)

Immunization with live influenza virus expands Th1 memory cells and facilitates more rapid recovery after heterosubtypic virus challenge. Immunization with inactivated virus generates a Th2 response and does not lead to heterosubtypic immunity. Creation of a Th1 priming environment by the inclusion of interleukin (IL)-12 with antibodies to IL-4 converted the response against inactivated virus to a Th1 response that was able to facilitate virus clearance upon heterosubtypic virus challenge. Evaluation of memory responses of mice immunized by the various protocols demonstrated that the type of immunization imprints T cell memory, dictating the nature of the response to subsequent infection. After live virus challenge, expansion of Th1 cells seems to facilitate the generation of cytotoxic T lymphocytes from naive precursors. This latter finding may be the mechanism by which inactivated virus immunization in a Th1 cytokine context mediates heterosubtypic immunity.  (+info)

Nasal cytokine and chemokine responses in experimental influenza A virus infection: results of a placebo-controlled trial of intravenous zanamivir treatment. (59/4894)

The local immune response to influenza virus infection was characterized by determining cytokine and chemokine levels in serial nasal lavage fluid samples from 15 volunteers experimentally infected with influenza A/Texas/36/91 (H1N1). The study was part of a randomized double-blind placebo-controlled trial to determine the prophylactic effect of intravenous zanamivir (600 mg 2x/day for 5 days), a highly selective inhibitor of influenza A and B virus neuraminidases, on the clinical symptoms of influenza infection. Nasal lavage fluid levels of interleukin (IL)-6, tumor necrosis factor-alpha, interferon-gamma, IL-10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1alpha and -1beta increased in response to influenza virus infection and correlated statistically with the magnitude and time course of the symptoms. Treatment with zanamivir prevented the infection and abrogated the local cytokine and chemokine responses. These results reveal a complex interplay of cytokines and chemokines in the development of symptoms and resolution of influenza.  (+info)

Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein. (60/4894)

The negative-sense segmented RNA genome of influenza virus is transcribed into capped and polyadenylated mRNAs, as well as full-length replicative intermediates (cRNAs). The mechanism that regulates the two forms of transcription remains unclear, although several lines of evidence imply a role for the viral nucleoprotein (NP). In particular, temperature-shift and biochemical analyses of the temperature-sensitive viruses A/WSN/33 ts56 and A/FPV/Rostock/34/Giessen tsG81 containing point mutations within the NP coding region have indicated specific defects in replicative transcription at the nonpermissive temperature. To identify the functional defect, we introduced the relevant mutations into the NP of influenza virus strain A/PR/8/34. Both mutants were temperature sensitive for influenza virus gene expression in transient-transfection experiments but localized and accumulated normally in transfected cells. Similarly, the mutants retained the ability to self-associate and interact with the virus polymerase complex whether synthesized at the permissive or the nonpermissive temperatures. In contrast, the mutant NPs were defective for RNA binding when expressed at the nonpermissive temperature but not when expressed at 30 degrees C. This suggests that the RNA-binding activity of NP is required for replicative transcription.  (+info)

Identification of amino acid residues of influenza virus nucleoprotein essential for RNA binding. (61/4894)

The influenza virus nucleoprotein (NP) is a single-strand-RNA-binding protein associated with genome and antigenome RNA and is one of the four virus proteins necessary for transcription and replication of viral RNA. To better characterize the mechanism by which NP binds RNA, we undertook a physical and mutational analysis of the polypeptide, with the strategy of identifying first the regions in direct contact with RNA, then the classes of amino acids involved, and finally the crucial residues by mutagenesis. Chemical fragmentation and amino acid sequencing of NP that had been UV cross linked to radiolabelled RNA showed that protein-RNA contacts occur throughout the length of the polypeptide. Chemical modification experiments implicated arginine but not lysine residues as important for RNA binding, while RNA-dependent changes in the intrinsic fluorescence spectrum of NP suggested the involvement of tryptophan residues. Supporting these observations, single-codon mutagenesis identified five tryptophan, one phenylalanine, and two arginine residues as essential for high-affinity RNA binding at physiological temperature. In addition, mutants unable to bind RNA in vitro were unable to support virus gene expression in vivo. The mutationally sensitive residues are not localized to any particular region of NP but instead are distributed throughout the protein. Overall, these data are inconsistent with previous models suggesting that the NP-RNA interaction is mediated by a discrete N-terminal domain. Instead, we propose that high-affinity binding of RNA by NP requires the concerted interaction of multiple regions of the protein with RNA and that the individual protein-RNA contacts are mediated by a combination of electrostatic interactions between positively charged residues and the phosphate backbone and planar interactions between aromatic side chains and bases.  (+info)

Association of influenza virus matrix protein with ribonucleoproteins. (62/4894)

To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.  (+info)

Phase 1 evaluation of intranasal virosomal influenza vaccine with and without Escherichia coli heat-labile toxin in adult volunteers. (63/4894)

Virosomal vaccines were prepared by extracting hemagglutinin (HA) and neuraminidase from influenza virus and incorporating it in the membranes of liposomes composed of phosphatidylcholine. Two intranasal spray vaccine series were prepared: one series comprised 7.5 micrograms of HA of each of three strains recommended by the World Health Organization and 1 microgram of Escherichia coli heat-labile toxin (HLT), and the other contained the HA without HLT. In addition, a third vaccine preparation contained 15 micrograms of HA and 2 micrograms of HLT. The parenteral virosomal vaccine contained 15 micrograms of HA without additional adjuvant. The immunogenicity of a single spray vaccination (15 micrograms of HA and 2 micrograms of HLT) was compared with that of two vaccinations (7.5 micrograms of HA with or without 1 microgram of HLT) with an interval of 1 week in 60 healthy working adults. Twenty volunteers received one parenteral virosomal vaccine. Two nasal spray vaccinations with HLT-adjuvanted virosomal influenza vaccine induced a humoral immune response which was comparable to that with a single parenteral vaccination. A significantly higher induction of influenza virus-specific immunoglobulin A was noted in the saliva after two nasal applications. The immune response after a single spray vaccination was significantly lower. It could be shown that the use of HLT as a mucosal adjuvant is necessary to obtain a humoral immune response comparable to that with parenteral vaccination. All vaccines were well tolerated.  (+info)

Human mannan-binding lectin inhibits the infection of influenza A virus without complement. (64/4894)

Mannan-binding lectin (MBL) is a C-type serum lectin that is believed to play an important role in innate immunity. It is one of the collectin family, which is characterized by having a collagen-like sequence and a carbohydrate recognition domain. MBL can bind to sugar determinants of several micro-organisms, neutralize them and inhibit infection by complement activation through the lectin pathway and opsonization by collectin receptors. Bovine conglutinin and mouse MBL inhibit the infective and haemagglutinating activities of influenza A viruses. To identify the direct antiviral activity of human MBL against influenza A viruses that does not depend on complement activation or opsonization, we isolated native MBL from human serum and produced a recombinant MBL in Chinese hamster ovary (CHO) cells using a pNOW/CMV-A expression vector system. Native and recombinant human MBL exhibited neutralization activity against A/Ibaraki/1/90 (H3N2), with the plaque focus reduction assay at the viral attachment phase. Their activities were inhibited by EDTA, mannose and anti-human MBL antibody. Furthermore, at the viral expansion phase both MBL in culture medium prevented viral spreading from primary infected cells to neighbour cells. A virus recovery study using EDTA indicated that interaction between MBL and virus was reversible and non-damaging to the virus. Lectin blot and immunohistochemistry assays showed that these antiviral activities involved binding between MBL and two viral envelope proteins, haemagglutinin and neuraminidase. These findings suggest that human MBL can play an important role in innate immunity by direct viral neutralization and inhibition of viral spread, as well as an indirect role through opsonization and complement activation.  (+info)