Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs

Influenza viruses are responsible for acute febrile respiratory disease. When deaths occur, definitive diagnosis requires viral isolation because no characteristic viral inclusions are seen. We examined the distribution of influenza A virus in tissues from 8 patients with fatal infection using 2 immunohistochemical assays (monoclonal antibodies to nucleoprotein [NP] and hemagglutinin [HA]) and 2 in situ hybridization (ISH) assays (digoxigenin-labeled probes that hybridized to HA and NP genes). Five patients had prominent bronchitis; by immunohistochemical assay, influenza A staining was present focally in the epithelium of larger bronchi (intact and detached necrotic cells) and in rare interstitial cells. The anti-NP antibody stained primarily cell nuclei, and the anti-HA antibody stained mainly the cytoplasm. In 4 of these cases, nucleic acids (ISH) were identified in the same areas. Three patients had lymphohistiocytic alveolitis and showed no immunohistochemical or ISH staining. Both techniques were useful for detection of influenza virus antigens and nucleic acids in formalin-fixed paraffin-embedded tissues and can enable further understanding of fatal influenza A virus infections in humans.     

A new influenza A virus infection in turkeys

Summary In transmission experiments, the influenza A virus isolate turkey/ Ontario 7732/66 caused an acute disease in chickens and turkeys, but was apathogenic to ducks, geese and pigeons. After an incubation period from two to eight days, turkeys and chickens became rapidly depressed and died usually within the following four days. Other clinical signs were variable for the two gallinaceous species, such as exudative head swellings and gangrenous comb lesions in chickens, and diarrhea in turkeys. Infection by even minimal virus doses was fatal in turkeys, whereas chickens sometimes recovered from the disease or remained unaffected by the infection. Serial passage of the virus in chicken embryos accentuated this difference in species susceptibility still more. The infection spread easily by close contact among turkeys, but less among chickens.The signs and course of the disease by virus 7732 are compared to those described for classical fowl plague, and it is concluded that these two avian influenza virus infections cannot be differentiated by clinical criteria.     

Correlation of influenza A virus nucleoprotein genes with host species

The RNAs coding for the nucleoproteins of a panel of influenza isolates from human and nonhuman hosts were compared by RNA-RNA hybridization to determine the extent of genetic diversity of this protein and to determine if related nucleoproteins (NP) are consistenly found in viruses from certain hosts. Five nucleoprotein groups were defined. Group 1 contains nearly all of the avian influenza viruses, group 2 includes only certain viruses isolated from gulls, group 3 includes all recent equine influenza strains, group 4 contains only equine/Prague/1/56, and group 5 contains all human and swine influenza isolates. The maintenance of specific nucleoproteins in viruses from certain species suggests that these proteins have evolved functionally significant differences that favor their replication in a specific host.     

Evidence for antigenic variation in influenza A nucleoprotein.

Nucleoprotein (NP) antigens isolated from sodium sarcosyl detergent-disrupted influenza A virus particles by cellulose acetate electrophoresis were used to prepare specific immune sera. Antigenic analysis of the nucleoproteins by immuno-double-diffusion and antibody absorption tests revealed antigenic differences in the nucleoprotein antigens of human A/PR8/34 (HON1) virus and viruses of the Hong Kong (H3N2) subtype. The nucleoprotein antigens of avian A/duck/Ukraine/1/63 (Hav7Neg2) and A/swine/Iowa/15/30 (Hsw1Nl) viruses resembled more closely the NP of A/PR8/34 virus than the NP of human H3N2 strains. Antigenic analysis of recombinant strains prepared from A/PR8/34 and H3N2 parent viruses indicated that the parental origin of the NP antigens could be clearly identified. Identification of the nucleoproteins of parental and recombinant influenza A viruses by migration rate analysis of NP polypeptides and RNA genes by electrophoresis on polyacrylamide gels gave results which correlated with the antigenic characterization of their NP antigens. The findings suggested that antigenic “drift” occurs in the nucleoproteins of human influenza A viruses.     

Functional expression of influenza A viral nucleoprotein in cells transformed with cloned DNA

Simian cells permissive for influenza A virus infection were stably transformed with a full-length cloned influenza A nucleoprotein gene under the control of an inducible metallothionein promoter and linked to a dihydrofolate reductase gene to facilitate cell selection. Transformed cells synthesized a virus-specific nucleoprotein which was indistinguishable from the nucleoprotein synthesized in virus-infected cells with respect to molecular weight and intracellular localization. It was estimated that transformed cells produced only 1% of the amount of nucleoprotein synthesized in simian cells infected with influenza A virus. Nonetheless, when transformed cells were infected with influenza virus mutants which synthesized temperature-sensitive nucleoprotein, protein expressed by the cloned gene was able to complement the synthesis of plus-strand and minus-strand viral RNA for one mutant and only plus-strand synthesis for another mutant. This indicated that the influenza A nucleoprotein expressed in the transformed cells exhibited functional activity.     

甲型流感病毒核蛋白基因的克隆表达及纯化

目的　将甲型流感病毒核蛋白(NP)基因克隆到原核表达载体进行可溶性融合表达,制备纯化的病毒核蛋白,为制备甲型流感病毒单克隆抗体提供材料。方法　提取甲型流感病毒RNA ,设计引物,RT PCR扩增NP基因,利用基因工程的手段,将甲型流感病毒的NP基因在大肠埃希菌中进行融合表达,并将表达产物进行亲和层析。结果　成功构建了甲型流感病毒NP基因原核表达载体,经亲和层析制备了较高纯度的目的表达产物。结论　通过合理控制发酵时间、生长温度和诱导物浓度,制备了较为理想的可溶性甲型流感病毒核蛋白。     

Associated seroconversions to respiratory viruses in volunteers with experimental influenza infection.

Serological examinations of 573 volunteers with mild experimental influenza infection and 86 volunteers of a control group hospitalized in a special clinic revealed a significant rise in the titre of antibodies (seroconversion) not only to influenza A or B viruses used for the experimental infection but in 23.3 to 29.8% of cases also to other respiratory viruses. Based on a number of arguments, associated seroconversions are interpreted as due to mixed or sequential infections of different aetiology.     

Immunogenic properties of ISCOM prepared with influenza virus nucleoprotein

Summary After covalent attachment of bacterial lipopolysaccharide to the nucleoprotein of influenza A virus, this water-soluble antigen could be incorporated firmly into ISCOM. This potent immunostimulating complex induced the production of high antibody titers in mice and could partially protect the animals from a lethal challenge infection. After immunization with ISCOM preparations NP-specific cytotoxic T cell activity could not be demonstrated.     

Immunohistochemical demonstration of Chlamydial antigens in association with prostatitis

The occurrence of Chlamydia trachomatis in association with histologically proven prostatitis was investigated. We used the immunoperoxidase technique with a monoclonal antibody against Chlamydia and evaluated formalin-fixed, paraffin-embedded sections from 16 cases of histologically proven prostatitis. Chlamydial antigens were detected within the prostate glands in 5 (31%) of these 16 cases. In contrast, none of 19 cases of prostatic hyperplasia without significant inflammation used as a control group showed staining for Chlamydia in the prostatic glands (P less than 0.03). Chlamydial antigens were, however, detected in the urothelium of the prostatic urethra in one of these control cases. Staining for Chlamydia occurred focally in atrophic glands and was associated with a predominantly chronic nonspecific inflammation. No cytoplasmic inclusions or other specific morphologic features of chlamydial infection could be identified on routine hematoxylin-eosin- or Giemsa-stained tissue sections from these specimens. Our results demonstrate that Chlamydia can infect the prostatic glands, and that its presence is statistically associated with marked nonspecific inflammation. We suggest that C. trachomatis may have an etiologic role in nonbacterial prostatitis.     

Antibody-binding epitope differences in the nucleoprotein of avian and mammalian influenza A viruses

Abstract Influenza virus nucleoprotein (NP) binds to the viral genome RNA and forms the internal ribonucleoprotein complex of the virus particle. Avian and human influenza virus NP have characteristic differences at several amino acid positions. It is not known whether any of these differences can be recognized by antibodies. In the present study five monoclonal antibodies (MAbs) were produced against NP of A/Duck/Novosibirsk/56/05 (H5N1) influenza virus. Two MAbs discerned human and avian influenza strains on ELISA testing. The NP expressed in a prokaryotic system was used for the analysis of site-specific mutants carrying amino acid substitutions in the relevant positions. Amino acid residues in positions 100 and 101 were shown to be recognized by the MAbs. The residue in position 100 is host-specific, and its recognition by the MAb 2E6 may be useful for the differentiation of human and avian viruses. The data are discussed in view of the effects of amino acid substitutions in influenza virus NP affecting both host range and antibody-binding specificity.     

Location and architecture of an antibody-binding site of influenza A virus nucleoprotein

Amino acid positions recognized by monoclonal antibodies (MAbs) in the influenza A virus nucleoprotein (NP) have been reported. As these residues were scattered in the three-dimensional (3D) structure of NP, no patterns of the architecture of antibody-binding sites could be inferred. Here, we used site-specific mutagenesis and ELISA to screen the amino acids surrounding position 470 recognized by the MAb 3/1 as a linear epitope. Ten amino acid residues involved in the reaction of NP with the MAb 3/1 and the MAb 469/6 were identified. Our data are the first to outline a compact site recognized by MAbs in the 3D structure of the influenza virus NP.     

Inhibition of influenza A virus replication by influenza B virus nucleoprotein: an insight into interference between influenza A and B viruses

Given that co-infection of cells with equivalent titers of influenza A and B viruses (FluA and FluB) has been shown to result in suppression of FluA growth, it is possible that FluB-specific proteins might hinder FluA polymerase activity and replication. We addressed this possibility by individually determining the effect of each gene of FluB on the FluA polymerase assay and found that the nucleoprotein of FluB (NP_FluB) inhibits polymerase activity of FluA in a dose-dependent manner. Mutational analyses of NP_FluB suggest that functional NP_FluB is necessary for this inhibition. Slower growth of FluA was also observed in MDCK cells stably expressing NP_FluB. Further analysis of NP_FluB indicated that it does not affect nuclear import of NP_FluA. Taken together, these findings suggest a novel role of NP_FluB in inhibiting replication of FluA, providing more insights into the mechanism of interference between FluA and FluB and the lack of reassortants between them.     

Differential processing of influenza nucleoprotein in human and mouse cells

To investigate how early events in antigen processing affect the repertoire of peptides presented by MHC class I molecules, we compared the presentation of the influenza A nucleoprotein epitope 265 – 273 by HLA-A3 class I molecules in human and mouse cells. Mouse cells that express HLA-A3 failed to present the NP265 – 273 peptide when contained within the full-length nucleoprotein, to HLA-A3-restricted human cytotoxic T lymphocytes. However, when the epitope was generated directly in the cytosol using a recombinant vaccinia virus that expressed the nonamer peptide, mouse cells were recognized by HLA-A3-restricted CTL. Poor transport of the peptide by mouse TAP was not responsible for the defect as co-infection of mouse cells with recombinant vaccinia viruses encoding the full-length nucleoprotein and the human TAP1 and TAP2 peptide transporter complex failed to restore presentation. These results therefore demonstrate a differential processing of the influenza nucleoprotein in mouse and human cells. This polymorphism influences the repertoire of peptides presented by MHC class I molecules at the cell surface.     

Sequence variation in the influenza A virus nucleoprotein associated with escape from cytotoxic T lymphocytes

CD8+ cytotoxic T lymphocytes (CTLs) contribute to the control of viral infections by recognizing peptides of viral proteins presented by MHC class I molecules on infected cells. Some viruses have developed strategies to evade recognition by CTL. One of these strategies involves antigenic variation in CTL epitopes as described for viruses chronically infecting their host like EBV, HIV, HBV and HCV. Here we show three examples of variation in CTL epitopes in the influenza virus nucleoprotein (NP) associated with escape from CTL immunity. The first two involve a mutation at position 384 of the NP, which is the anchor residue of a HLA-B*2705-restricted epitope NP383-391 (SRYWAIRTR) and the HLA-B*08-restricted epitope NP380-388 (ELRSRYWAI). It was shown that these mutations have arisen in the 1993/1994 season and that these mutant variants completely replaced the virus strains containing the wild-type epitopes. Furthermore, T cell recognition was completely abrogated by the R384G mutation. A third example of variation in an influenza virus CTL epitope was found in a newly identified HLA-B*3501-restricted CTL epitope. This immunodominant epitope exhibited extensive amino acid sequence variation and the variants emerged in a chronological order. Again CTL specific for older variants failed to recognize more recent strains of influenza A virus, indicating an escape from CTL immunity. Thus, in addition to the introduction of mutations in the surface glycoproteins like the hemagglutinin, allowing escape from antibody-mediated immunity, there is now evidence that influenza viruses can escape in a similar way from CTL-mediated immunity.     

Immunohistochemical demonstration of chromogranin in endometrial carcinomas with argyrophil cells

Forty-five endometrial carcinomas, 36 of which contained argyrophil cells and nine of which were nonargyrophilic by the Grimelius method, were examined immunohistochemically for chromogranin. Chromogranin immunoreactivity was present in 19 of the 36 tumors with argyrophil cells (53 per cent) and in none of the nine tumors lacking these cells. All six of the tumors that contained argyrophil cells resembling enterochromaffin cells were chromogranin-positive, with the staining corresponding to the argyrophilia. In contrast, only 13 of the 30 tumors in which argyrophilia was present in the apical region or throughout the cytoplasm of the cells showed chromogranin immunoreactivity. In seven of these tumors, an excellent correlation existed between the distribution of argyrophilia and chromogranin positivity, but in the other six tumors argyrophilia was more pronounced than chromogranin immunoreactivity. Adjacent to one tumor, unusual cells in which argyrophil granules were packed predominantly in the basal portion of the cytoplasm were encountered in a focus of atypical hyperplasia; these cells were also chromogranin-positive. The present observations suggest that endometrial carcinoma cells with diffuse or apical chromogranin immunoreactivity may represent an early stage in the development of cells resembling those of the enterochromaffin type.     

Immunohistochemical demonstration of migration inhibitory factor (MIF) in experimental allergic contact dermatitis.

The kinetics of appearance of MIF+ cells was investigated in experimental contact dermatitis using a monoclonal antibody (7D10) against murine MIF which was reacted with cryostat sections of tissues and detected by the indirect immunoperoxidase test. Four groups of BALB/c mice were investigated: (1) sensitized with 2,4-dinitrofluorobenzene (DNFB); (2) unsensitized controls; (3) tolerized; (4) unsensitized. A challenge dose of DNFB was applied to the ear of animals of groups 1-3 and of croton oil to those of group 4. Three phases could be distinguished in group 1: (a) an initial vascular and exudative reaction; (b) an early cellular phase; and (c) a late cellular phase. At zero time rarely any T lymphocytes (Lyt 1+; Lyt 2+) were seen in all four groups. Within less than 30 min venous endothelial cells became strongly MIF+. This was followed by an influx of monocytes/macrophages reaching a maximum of 72 h in group 1 and a slight peak at 12 h in groups 2 and 3. At 16-24 h in all groups the endothelial reaction weakened while many 7D10+ macrophages appeared in group 1. By double-labelling it was shown that lymphocytes were 7D10-. The influx of lymphocytes, part of which carried the T cell receptor, began at 12 h, reaching a maximum at 72 h in group 1. In groups 2 and 3 only a weak lymphocytic infiltrate developed which declined at 24 h. Group 4 developed an inflammatory reaction after the initial phase with similar kinetics as in group 1. The data suggest that an immune inflammatory reaction is preceded by a nonspecific reaction of the vascular endothelium and the mononuclear phagocytic system and that MIF is playing a central role in these events.     

Mutants and revertants of an avian influenza A virus with temperature-sensitive defects in the nucleoprotein and PB2

ts19 is a temperature-sensitive (ts) mutant of the influenza A fowl plague virus with a defect in the nucleoprotein (NP). In ts19-infected chicken embryo cells all viral components are synthesized in normal yields at the nonpermissive temperature, but infectious virus is not formed. Under these conditions the migration of the NP and M of ts19 from the cell nucleus to the cytoplasm is affected. This ts defect is due to a single amino acid replacement (R162K) in a completely conserved region of the NP. Another mutant with a different defect in the NP is ts81. After infection with ts81 at 40 degrees no vRNA is being synthesized. By backcross of a revertant derived from ts81 many isolates with a ts defect in the PB2 protein were obtained. This ts defect seems to extragenically suppress the ts defect in the NP gene and to be dominant in a wild-type background.