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Defective influenza A virus RNAs analyzed in two studies so far possess at least 80–90 nucleotides from the 5′ end of the virion RNA segment and more typically around 200 nucleotides, whereas the 3′ sequence could be as short as 25 nucleotides (P. A. Jennings et al., Cell 34, 619–627; 1983; S. D. Duhaut and N. J. Dimmock, Virology 247, 241–253, 1998). To determine the biological significance of the...
Phylogenetically informative amino acid positions (PIPs) were identified in influenza A neuraminidases of subtypes N1 and N2. Neuraminidase evolves in a lineage-specific way as the virus adapts to a new host or changes to evade the host's immune system. Thus, many PIPs undoubtedly identify positions involved in virus–host interactions. Phylogenetically important regions (PIRs) are defined as several...
Mabs H36 (IgG2a) and H37 (IgG3) recognize epitopes in antigenic sites Sb and Ca2, respectively, in the HA1 subunit of influenza virus A/PR/8/34 (H1N1). Their neutralization was complex. Our aim here was to investigate the mechanism of neutralization by the IgGs and their Fabs. In MDCK and BHK cells, both IgGs neutralized primarily by inhibiting virus–cell fusion, although at higher IgG concentrations...
The N-terminal domains of the NS1 protein of influenza B virus (NS1B protein) and the NS1 protein of influenza A virus (NS1A protein) share one function: binding double-stranded RNA (dsRNA). Here we show that the N-terminal domain of the NS1B protein possesses an additional function that is not shared by its NS1A counterpart: binding the ubiquitin-like ISG15 protein that is induced by influenza B...
FluMist influenza A vaccine strains contain the PB1, PB2, PA, NP, M, and NS gene segments of ca A/AA/6/60, the master donor virus-A strain. These gene segments impart the characteristic cold-adapted (ca), attenuated (att), and temperature-sensitive (ts) phenotypes to the vaccine strains. A plasmid-based reverse genetics system was used to create a series of recombinant hybrids between the isogenic...
The influenza A virus NS1 protein (NS1A protein) binds and inhibits the function of the 30-kDa subunit of CPSF, a cellular factor that is required for the 3'-end processing of cellular pre-mRNAs. Here we generate a recombinant influenza A/Udorn/72 virus that encodes an NS1A protein containing a mutated binding site for the 30-kDa subunit of CPSF. This mutant virus is substantially attenuated, indicating...
Based on the observation that an internally located 3' promoter sequence can be functional (R. Flick and G. Hobom, Virology, 1999, 262(1), 93-103), we generated transfectant influenza A viruses harboring a dicistronic segment containing the CAT gene (660 nt) or a fragment of the Mengo virus VP0 capsid gene (306 nt) under the control of a duplicated 3' promoter sequence. Despite slightly reduced NA...
Short interfering double-stranded RNAs (siRNAs) expressed under the control of an RNA polymerase I promoter system were used to target gene expression of influenza A and West Nile virus. Decreased RNA and protein expression was induced in a sequence-specific manner-reducing sequence complementarity from 21 to 17 nucleotides abrogated the siRNA effect. Reduced M 2 expression resulted in a decrease...
During influenza virus infection, transcription and replication of the viral RNA take place in the cell nucleus. Directly after entry in the nucleus the viral ribonucleoproteins (RNPs, the viral subunits containing vRNA, nucleoprotein and the viral polymerase) are tightly associated with the nuclear matrix. Here, we have analysed the binding of RNPs, M1 and NS2/NEP proteins to purified nucleosomes,...
In Japan, between the end of December 2003 and March 2004, four outbreaks of acute, highly transmissible and lethal disease occurred in birds in three prefectures separated by 150–450 km, involving three chicken farms and a group of chickens raised as pets. The cause of each outbreak was an H5N1 influenza A virus—the first highly pathogenic virus to be isolated from the outbreaks in Japan since 1925...
Three influenza viruses, A/Puerto Rico/8/34–A/England/939/69 clone 7a (H3N2), A/Fiji/15899/83 (H1N1), and A/Victoria/3/75 (H3N2), induce different levels of apoptosis in vitro at equal moi; Clone 7a > A/Victoria > A/Fiji. Previous studies have shown that several viral proteins from clone 7a and A/Fiji, including PB2, NA, NS1, M1, and M2, induce apoptosis when expressed individually fused to...
An H5N1 influenza A virus was isolated from duck meat processed for human consumption, imported to Japan from Shandong Province, China in 2003. This virus was antigenically different from other H5 viruses, including the Hong Kong H5N1 viruses isolated from humans in 1997 and 2003. Sequence analysis revealed that six genes (PB1, PA, HA, NA, M, and NS) of this virus showed > 97% nucleotide identity...
We generated recombinant A/WSN/33 influenza A viruses expressing a PB2 protein fused to a Flag epitope at the N- (Flag-PB2) or C-terminus (PB2-Flag), which replicated efficiently and proved to be stable upon serial passage in vitro on MDCK cells. Rescue of PB2-Flag viruses required that the 5′ end of the PB2 segment was kept identical to the wild-type beyond the 34 noncoding terminal nucleotides....
We generated novel recombinant influenza A viruses (vNA38) harboring dicistronic NA segments with an extended native 5′ terminal sequence of 70 nucleotides comprised of the last 42 nucleotides of the NA ORF and the 5′ noncoding region (5′ NCR). vNA38 viruses replicated stably and more efficiently than vNA35 viruses with a dicistronic NA segment comprised of the native 5′ NCR only, that we described...
We investigated the ability of a selection of human influenza A viruses, including recent clinical isolates, to induce IFN-β production in cultured cell lines. In contrast to the well-characterized laboratory strain A/PR/8/34, several, but not all, recent isolates of H3N2 viruses resulted in moderate IFN-β stimulation. Through the generation of recombinant viruses, we were able to show that this is...
Failure to isolate A/Fujian/411/2002 (H3N2) in embryonated chicken eggs resulted in its absence from the 2003/2004 vaccine. We analyzed the adaptation of this virus in eggs during serial passages in the amniotic then allantoic cavities. Amniotic passage allowed the virus to grow in the allantoic cavity. During adaptation, 6 amino acid substitutions occurred: 4 in HA (G186V, S219F, V226I, V309I) and...
The influenza virus RNA-dependent RNA polymerase interacts with the serine-5 phosphorylated carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). It was proposed that this interaction allows the viral RNA polymerase to gain access to host mRNA-derived capped RNA fragments required as primers for the initiation of viral mRNA synthesis. Here, we show, using a chromatin immunoprecipitation...
The role of serum components in enhancing virus neutralizing (VN) activity of influenza virus A/PR/8/34 hemagglutinin (HA)-specific MAbs in vitro was investigated. The degree of enhancement depended on the MAb's fine specificity and heavy chain isotype and on type of serum. Greatest enhancement (>100-fold) was seen with sera from immunodeficient mice that lacked serum immunoglobulin. At least two...
Effective vaccination strategies for infectious diseases take into account the induction, long-term maintenance and recall of memory T-cell populations. To understand the immunological cross-talk within the mucosal compartments, we compared intranasal to vaginal immunization and demonstrated that vaginal infection of BALB/c mice with influenza A virus provides protective mucosal immunity against both...
The transcription/replication activity of ribonucleoproteins derived from influenza A primary isolates of human (A/Paris/908/97) or avian origin (A/Mallard/Marquenterre/MZ237/83, A/Hong Kong/156/97) was compared upon reconstitution in mammalian or avian cells, using viral-like reporter RNAs synthesized under the control of the human and chicken RNA polymerase I promoters, respectively. In avian cells,...
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