Translational regulation of influenza virus mRNAs

cDNAs for genome RNAs of influenza virus A/PR/8/34 were cloned, and portions containing the ATG for initiation codon of translation were inserted into the 5 leader sequence of the chloramphenicol acetyltransferase (CAT) gene in a pSV2cat vector. When transfected cells were super-infected with influenza virus, the CAT activity was found to vary in a time-dependent fashion: A construct containing a cDNA segment for the nonstructural (NS) protein directed the highest activity during the early stage of infection, while a construct containing a cDNA segment for the neuraminidase (NA) directed the highest activity during the late stage of infection. This time-dependent variation in the CAT activity is in good agreement with that of the synthesis rate of respective viral proteins in infected cells. We propose that the translational efficiency of viral mRNA is subjected to temporal control following viral infection, although viral protein synthesis itself is regulated primarily at the level of mRNA synthesis.     

Detection of sequences in the structure of influenza virus proteins similar to vasoactive intestinal peptide

All-Union Influenza Research Institute, Ministry of Health of the USSR, Leningrad. Faculty of Biology, M. V. Lomonosov State University, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR I. P. Ashmarin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 4, pp. 371–373, April, 1991.     

禽流感病毒核蛋白单克隆抗体的制备及部分特性鉴定

目的：制备抗禽流感病毒（AIV）核蛋白（NP）的单克隆抗体（mAb）并进行特性鉴定。方法：分别用甲醛灭活的和TritonX-100裂解的AIV H9N2及AIVNP基因的原核表达产物免疫BALB／c小鼠。经细胞融合、间接ELISA筛选及克隆化，建立能稳定分泌抗AIV NP mAb的杂交瘤细胞株。mAb的效价采用间接ELISA测定，用交义反应试验及间接免疫荧光染色法检测mAb的特异性。结果：经细胞融合、筛选及克隆化，间接ELISA法测定，mAb共得到6株能稳定分泌抗禽流感病毒NPmAb的杂交瘤细胞株，分别命名为4F4、1C3、1G11、1C2、1D10及2E7。1G11、1D10腹水的ELISA效价最高，分别为2^-13和2^-14。交叉反应试验及间接免疫荧光染色检测表明，两株mAb的特异性良好。结论：其获得6株抗AIVNP的mAb，其中2株mAb1C11和1D10的效价最高，特异性良好，为AIV的研究及快速诊断方法的建立奠定了基础。     

Purification and enzymatic properties of an RNA polymerase-RNA complex from influenza virus

An RNA polymerase-viral RNA complex was purified from influenza A/PR/8 virions by combination of cesium trifluoroacetate centrifugation and phosphocellulose column chromatography. Surface proteins were removed from the detergent-treated virions by the centrifugation. Starting from the M protein-free ribonucleoprotein (RNP) fraction, an RNA polymerase-RNA complex lacking NP protein was isolated by repeated chromatography on phosphocellulose columns. The isolated RNA polymerase-RNA complex, which is composed of PB1, PB2, PA and vRNA, cleaved capped poly(A) endonucleolytically at 10-12 nucleotides from the 5' end and incorporated GMP into the 3' end of the resulting capped fragments. In the presence of all four ribonucleotide triphosphate substrates, the cleaved fragments were elongated to polynucleotides in the absence of exogenous vRNA. The RNA synthesis was primed not only by capped polynucleotides but also dinucleotide ApG. These results indicate that the purified RNA polymerase-RNA complex is as active in viral mRNAs synthesis as native RNP and that NP protein is not required for the catalytic function.     

Nucleotide sequence of the entire protein coding region of canine distemper virus polymerase-associated (P) protein mRNA

The entire coding region of the polymerase-associated (P) protein gene of canine distemper virus has been sequenced. A single cDNA clone which represents 98% of the mRNA encoding this protein was used to determine the nucleotide sequence. The sequence predicts a major protein of 507 amino acids and a molecular weight of 54 936. There is also a second, overlapping, open reading frame with a start signal 21 bases downstream of the first AUG which could code for a protein of 174 amino acids with a predicted molecular weight of 20 292. This arrangement of the genome for the P protein of canine distemper virus is exactly analogous to that published recently for the P gene of measles virus (Bellini, W.J. et al., 1985, J. Virol. 53, 908-919). When the sequences are aligned at the first AUG, considerable homology is seen at both the nucleotide and protein sequence level.     

Nucleotide sequences of the neuraminidase genes of influenza A/Leningrad/134/57 (H2N2) virus and two of its live,attenuated, cold-adapted variants

The nucleotide sequences of the neuraminidase (NA) genes of the A/Leningrad/134/57 (H2N2) wild-type (Len/wt) virus as well as two of its live attenuated, cold-adapted (ca) variants, A/Leningrad/134/17/57 (Len/17) and A/Leningrad/134/47/57 (Len/47), were determined. In comparison with Len/wt, one nucleotide change (C-225 to A) was found in the NA gene of Len/17. This change codes for a Thr-to-Asn substitution at position 69 of NA. The NA gene of the more attenuated Len/47 ca virus has one silent (T-814 to C) and two coding nucleotide substitutions, C-78 to T (Ala-20 to Val) and C-225 to A (Thr-69 to Asn). These sequence data were used to design a PCR-restriction technique to determine the origin of the NA gene in candidate live, attenuated vaccine reassortants made by reassorting these ca strains with current field viruses.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers L37329-L37331.     

Antigenic variations of human influenza virus in Shiraz,Iran

Purpose: Influenza virus is a major cause of human respiratory infections and responsible for pandemics and regional outbreaks around the world. This investigation aims to determine the prevalent influenza genotypes during 2005-2007 outbreaks in Shiraz, the capital city of Fars province, southern Iran and compare the results obtained with those of previous study. Materials and Method: Of the 300 pharyngeal swabs collected from influenza patients, 26 were found to be positive by culture and hemagglutination (HA) assays. Typing and subtyping of the isolates carried out by using multiplex RT-PCR and phylogenetic analysis performed on isolated HA genes using neighbour-joining method. Result: Out of 26 positive isolates 12 and 14 were H1N1 and H3N2 respectively. The phylogenetic and amino acid sequence analyses of our H1N1 isolates showed 99-100% genetic resemblance to A/NewCaledonia/20/99 (H1N1) vaccine strain. Most of the Iranian H3N2 isolates varied form A/California/7/2004 vaccine strain in 20 amino acids of which positions 189,226 and 227 were located in antigenic sites of HA1 molecule. These substitutions were not observed in any of the H3N2 subtypes from the same region reported previously. Conclusion: The H3N2 subtype strains prevalent during the 2005/7 influenza outbreak in southern Iran demonstrated a drastic antigenic variation and differed from A/California/7/2004 vaccine strain. The H1N1 subtypes showed a notable resemblance to A/NewCaledonia/20/99 vaccine strain and therefore were predicted to be capable of conferring sufficient immunity against H1N1 subtypes.     

T cell recognition of the posttranslationally cleaved intersubunit region of influenza virus hemagglutinin

The influenza virus hemagglutinin is synthesized as a single polypeptide chain, but upon maturation it will posttranslationally be modified by a host cell related trypsin-like enzyme. The enzymatic cleavage attacks the so-called intersubunit region of the molecule giving rise to covalently linked HA1 and HA2 subunits. An I-E^drestricted T cell epitope was identified in the highly conserved intact intersubunit region of the influenza virus hemagglutinin. T cell recognition of a 25-mer synthetic peptide comprising the intact intersubunit region does not require further processing and the elimination of the intervening Arg residue coupling the fusion peptide to the C-terminal segment of HA1 does not abolish the T cell activating capacity. The fine specificity pattern of a T cell hybridoma similar to that of the polyclonal T cell response demonstrates that a single T cell receptor is able to recognize peptides of different sizes representing not only the uncleaved but also the cleaved form of this hemagglutinin region. Based on specificity studies the epitope was localized to the C-terminal 11 amino acids of the HA1 subunit. The cross-reactivity of peptide-primed T cells with influenza virus infected antigen-presenting cells shows that fragments comprising the identified epitope of the intersubunit region can be generated as a result of natural processing of the hemagglutinin molecule. As antigen-presenting cells are lacking the enzyme which is responsible for the posttranslational modification of newly synthesized hemagglutinin molecules, the role of immature viral proteins in immune recognition is discussed.     

Variability of tropism and replicative capacity of two naturally occurring influenza A H9N2 viruses in cell cultures from different tissues

Studies carried out on cell permissivity are of great interest to understand virus replication and pathogenicity. We described the results of a comparative analysis of replication efficiency of two naturally occurring influenza A H9N2 variants isolated from poultry and wild birds, differing by only two substitutions Q226L and T384N, in the receptor-binding site of haemagglutinin and the 380 loop region of NA proteins, respectively. Considering the overall growth of both viruses, lung cultures ensured the most efficient growth of TUN12L226N384 strain with titres up to 10⁹ TCID₅₀/ml whereas small intestine culture was highly susceptible to the TUN51Q226T384 virus reaching a titre of 10⁶ TCID50/ml. The lowest replication was shown in liver cells. The addition of trypsin was essential for the replication of either virus in primary fibroblasts, but it had a marginal positive effect on virus replication in the four other culture types with maximum titres of 10⁸ TCID₅₀/ml. This means that in chicken, the proteolytic activation of the H9N2 viruses with the cleavage motif RSSR may be mediated by other endoproteases than trypsin. Further investigations should concentrate on the production of the appropriate set of viruses by a reverse genetics approach and the examination of cellular protease expression in chicken tissues. This would lead to a more complete understanding of the tropism of low-pathogenic Influenza A viruses.     

Detection and identification of influenza virus antigens by nylon-coupled enzyme-linked immunoassay

A direct solid-phase enzyme-linked immunoassay for rapid detection and typing of influenza virus was developed utilizing antibodies immobilized by covalent linkage to nylon beads. Covalent linkage of antibody to nylon was accomplished by treatment of partially hydrolyzed nylon with glutaraldehyde. For comparison to conventional enzyme-linked immunosorbent assays (ELISA), IgG fractions were adsorbed to polystyrene beads. Influenza type-specific immunoglobulins coupled to nylon beads were used in an enzyme-linked immunoassay to identify influenza A/USSR/77(H1N1), and A/Texas/75 (H3N2). In titrations of viral antigen, antibody coupled to nylon beads detected 1.9 × 10⁴ plaqueforming units (PFU) per assay, whereas 2.2 × 10⁵ PFU were required in assays utilizing antibody adsorbed to polystyrene beads. Use of fluorogenic or radioactive substrates for alkaline phosphataselabeled antibodies increased the sensitivity for virus detection 10-fold with this enzyme, but were only slightly more sensitive than chromogenic substrates with peroxidase-labeled antibody.     

Epitope specific T‐cell responses against influenza A in a healthy population

Pre‐existing human CD4⁺ and CD8⁺ T‐cell‐mediated immunity may be a useful correlate of protection against severe influenza disease. Identification and evaluation of common epitopes recognized by T cells with broad cross‐reactivity is therefore important to guide universal influenza vaccine development, and to monitor immunological preparedness against pandemics. We have retrieved an optimal combination of MHC class I and class II restricted epitopes from the Immune Epitope Database ( www.iedb.org ), by defining a fitness score function depending on prevalence, sequence conservancy and HLA super‐type coverage. Optimized libraries of CD4⁺ and CD8⁺ T‐cell epitopes were selected from influenza antigens commonly present in seasonal and pandemic influenza strains from 1934 to 2009. These epitope pools were used to characterize human T‐cell responses in healthy donors using interferon‐γ ELISPOT assays. Upon stimulation, significant CD4⁺ and CD8⁺ T‐cell responses were induced, primarily recognizing epitopes from the conserved viral core proteins. Furthermore, the CD4⁺ and CD8⁺ T cells were phenotypically characterized regarding functionality, cytotoxic potential and memory phenotype using flow cytometry. Optimized sets of T‐cell peptide epitopes may be a useful tool to monitor the efficacy of clinical trials, the immune status of a population to predict immunological preparedness against pandemics, as well as being candidates for universal influenza vaccines.     

荧光定量PCR在筛查甲型流感病毒中的诊断价值

目的:探讨荧光定量PCR技术在筛查甲型流感病毒中的诊断价值。方法:用荧光定量RT-PCR方法检测150例疑似流感患者咽拭子中的甲型流感病毒RNA,用XT-1800全自动血液分析仪检测150例疑似流感患者EDTA抗凝全血中的白细胞计数,用胶体金法检测54例甲型流感病毒RNA阳性患者的甲型流感病毒核心蛋白。结果:在150例患者中,甲型流感病毒RNA检测阳性者有54例,阳性率为36%。54例甲型流感病毒RNA检测阳性者采用胶体金法检测甲型流感病毒核心蛋白,结果均为阴性。甲型流感病毒RNA检测阳性者白细胞计数为(6.81±2.12)×109/L,阴性者为(6.64±3.13)×109/L,白细胞计数在甲型流感患者和非甲型流感患者中无统计学差异(P0.05)。结论:荧光定量RT-PCR在检测甲型流感病毒RNA中有较好的阳性检出率,其敏感性和特异性明显优于胶体金方法和白细胞计数,能快速有效筛选出甲型流感患者,防止疫情爆发流行。     

Development of rHA1-ELISA for specific and sensitive detection of H9 subtype influenza virus

A recombinant antigen-based single serum dilution ELISA was developed for simultaneous detection and subtyping of influenza viruses. Recombinant baculovirus encoding the hemagglutinin (HA₁ subunit) of H9N2 virus was generated. To evaluate the rHA1-ELISA, microplates were coated with purified HA1 protein and tested with reference control sera. Subsequently, 92 field sera collected from chickens suspected to be infected with H9N2 AIV were employed to test the efficacy of the rHA1-ELISA. The sera were tested simultaneously by HI and a commercial AIV ELISA kit. The rHA1-ELISA appeared to be highly specific and sensitive for direct detection of H9N2 antibodies in serum samples.     

用8质粒病毒拯救系统产生冷适应减毒的重组A型人流感病毒的研究

目的 利用流感病毒8质粒病毒拯救系统,产生冷适应减毒的重组A型人流感病毒,建立以冷适应流感病毒株为拯救骨架的反向遗传学技术平台.方法 以冷适应、温度敏感、减毒的A/Ann Arbor/6/60(H2N2)流感病毒株作为拯救病毒的骨架,人工合成了该病毒株的6个内部基因片段,即PB2、PB1、PA、NP、M和NS,同时引入5个氨基酸突变作为标签.6个基因片段通过与改造后的转录载体pAD3000连接,构建6个基因的拯救载体,经测序获得序列准确的拯救质粒:pMDV-A-PB2、pMDV-A-PB1、pMDV-A-PA、pMDV-A-NP、pMDV-A-M、pMDV-A-NS.结果 6质粒与PR8的表面基因HA和NA进行"6+2"组合的病毒拯救,8个重组质粒共转染COS-1细胞,成功拯救出了具有血凝活性的冷适应减毒的重组A型人流感病毒,命名为rMDV-A.鸡胚尿囊液中重组病毒的血凝(HA)效价为1∶2 9～1∶2 10.结论 构建的A/Ann Arbor/6/60的6个内部基因的病毒骨架拯救系统,为深入研究冷适应减毒人流感病毒的基因功能和新型疫苗研发提供了试验材料.     

Nucleotide sequence of the avian influenza virus A/fowl plague/Rostock/34 segment 1 encoding the PB2 polypeptide

The nucleotide sequence of segment 1 of fowl plague virus (A/FPV/Rostock/34) was determined from a DNA copy inserted at the Pst I site of pBR322. Segment 1 is 2341 nucleotides long and encodes a highly basic protein of 759 amino acid residues (PB2). This protein forms part of the viral polymerase complex and is involved in the recognition of cap structures and endonucleolytic cleavage of eukaryotic mRNAs. Comparisons made between segment 1 of fowl plague virus and three human strains of influenza virus show approximately 85% nucleotide sequence homology and 94% homology between amino acid sequences.     

In vivo selection of a TCR Vbeta repertoire directed against an immunodominant influenza virus CTL epitope

Little is known about the mechanisms governing TCR repertoire selection in response to foreign antigens. Here, we evaluate the molecular features of the murine C57BL/6 (B6) TCR Vbeta repertoire directed at the NP(366-374) immunodominant epitope of the influenza virus nucleoprotein. Common or 'public' beta chains are shared among individuals following either primary or secondary infection. Importantly, repertoire diversity decreases substantially after a second viral exposure due to enrichment of TCRs sharing Vbeta CDR3 loops of identical length and highly related amino acid sequences. TCRs from these secondary T cell populations possess greater overall avidity for the NP(366-374)/D(b) complex compared to those from the primary repertoire. Thus, expansion of CD8(+) T cells expressing a favored germline Vbeta gene segment in the primary response and further selection for CDR3beta loops during the secondary response, contribute to optimization of immune recognition against certain viral epitopes.