Genomic analysis of matrix gene and antigenic studies of its gene product (M1) of a swine influenza virus (H1N1) causing chronic respiratory disease in pigs.

The nucleotide sequence of gene coding for the matrix protein (M1 and M2) of swine influenza (H1N1) virus, A/Sw/Quebec/5393/91 (SwQc91), associated with chronic respiratory disease in pigs, was determined. The deduced amino acid (aa) sequence was compared with the other North American swine strains including the A/Sw/Quebec/192/81 (SwQc81) strain associated with the chronic and acute respiratory disease in pigs. Separate analysis of the M1 and M2 gene products showed different evolutions. M1 had 2 aas changes among 252 aas and these were at positions 4 and 205. The mutation rate was 0.08%, aa changes per residue per year, and its homology with other strains was 99.2%. The M2 protein (97 aas) was relatively more variable than M1 with 5 substitutions. Differences observed were at positions 4, 16, 21, 54 and 95. The mutation rate was 0.51% and its homology with other strains was 94.8%. The M1 gene was cloned in the procaryotic plasmid pET21a and the recombinant plasmid was expressed in Escherichia coli under pre-determined optimal conditions. The recombinant M1 protein (RM1P) (approximately 28 kDa) comigrated as a single band on SDS-PAGE. RM1P was antigenic and reacted with polyclonal sera and 5 monoclonal antibodies (MAbs) spanning 4 epitopes including the membrane binding site and the transcription inhibition activity site. RM1P was immunogenic. The mouse anti-RM1P ELISA antibodies reacted with the purified viral M1 protein and the whole virus.

Influenza virus hemagglutinin stimulates the protein kinase C activity of human polymorphonuclear leucocytes.

Human polymorphonuclear leukocytes (PMNL) incubated with influenza virus, A/USSR/90/77 (H1N1) or its hemagglutinin produced a significant increase in their PKC activity when compared with untreated PMNL. The activated kinase translocated from cytosol to cellular membrane. The calcium-dependent enzyme activity was inhibited by a specific inhibitor suggesting that alpha and/or beta isoforms of PKC were involved.

Genomic study of hemagglutinins of swine influenza (H1N1) viruses associated with acute and chronic respiratory diseases in pigs.

The nucleotide sequences of HA1 domain of hemagglutinin of clinical H1N1 influenza viruses, isolated during recent outbreaks of respiratory problems in pig farms of Quebec, was determined. The viruses A/Sw/Quebec/3291/90 (SwQc3291) and A/Sw/Quebec/1747/90 (SwQc1747), associated with chronic respiratory disease, showed close similarity for their deduced aa sequences. When compared with the published data of A/Sw/Quebec/5393/91 (SwQc91), the variations observed included Cb and Ca antigenic sites in SwQc3291 and Sb and Ca sites in SwQc1747 isolates. These variants were antigenically related to SwQc91 virus associated with chronic respiratory disease, but differed from the more classical A/Sw/Quebec/192/81 (SwQc81) strain. In contrast, A/Sw/Quebec/1192/86 (SwQc1192) isolate, associated with acute respiratory influenza, showed maximum number of differences including Ca, Cb, Sa and Sb antigenic sites. The latter, as well as the SwQc81 strain, were antigenically distinct from SwQc91 virus on the basis of its cross-reactivity to MAbs directed against the HA glycoprotein. Estimation of genetic distances and phylogenic tree analysis showed that SwQc1747 and SwQc3291 were closely related, but these viruses along with SwQc1192 were considerably divergent from SwQc91 virus.

Superoxide anion production in influenza protein-activated NADPH oxidase of human polymorphonuclear leukocytes.

There are conflicting reports regarding superoxide anion (O2-) production by human polymorphonuclear leukocytes (PMNL) that have been activated by influenza virus. In the present study, the output of O2- was determined by measuring superoxide dismutase-inhibitable cytochrome c reduction. Incubation of PMNL with purified influenza matrix (M) protein, neuraminidase (NA), or hemagglutinin (HA) enhanced the production of O2-: 4.93 nmol of O2-/4 x 10(5) cells/15 min was produced with M protein, 5.20 with NA, and 6.89 with HA. These values were significantly higher (P < .05) than that for untreated PMNL (1.51). Both nonglycosylated and glycosylated proteins had the potential to generate O2- in human PMNL. Neither the hemagglutinating activity of HA nor the enzymatic activity of NA were necessary for viral protein activation of PMNL.

Genetic variation in swine influenza virus A isolate associated with proliferative and necrotizing pneumonia in pigs.

A new antigenic variant of H1N1 swine influenza virus A (Sw/QC/5393/91 [QC/91]) has been found to be associated with porcine proliferative and necrotizing pneumonia. Analysis of its genomic RNA by T1 oligonucleotide mapping revealed that considerable genomic divergence exists between QC/91 and the swine influenza viruses currently circulating in North American swine herds. Analysis of the nucleotide sequence of the HA1 region of the hemagglutinin RNA of QC/91, in comparison with those of most common H1N1 human and swine influenza A viruses, showed the presence of multiple point mutations. Two amino acid substitutions appeared to be located in antigenic sites Sb and Ca. This correlates with antigenic variations demonstrated between A/NJ/8/76, A/Sw/WI/49/76, and Québec isolate A/Sw/QC/5393/91 of swine influenza virus A. Another mutation was responsible for the loss of a glycosylation site, which may have also affected the antigenicity. The other mutations seem to have been accumulated progressively over time. This significant constancy in the fixation of mutations with time suggests that genetic diversity of these viruses may best be interpreted as the result of drifts in the population of circulating swine influenza viruses in Québec.

Administration of inactivated and detergent-treated influenza virus to mice before virus challenge reduces mortality.

Formalin-inactivated virus (FIV) and the detergent-treated virus (DTV) preparations were tested for their ability to enhance the resistance of mice to experimental influenza infection. FIV (100 micrograms) was administered intravenously to mice. After 24 hr, animals were challenged with 5 LD50 dose intranasally. FIV-treated and non-treated (control) mice had 10% and 100% mortality, respectively. Similar results were obtained with the DTV (40 micrograms) preparation. The pulmonary virus titer of FIV-treated mice was lower when compared with the control. Mechanisms other than acquired immunity may have conferred the early resistance to virus infection in mice.

Modulation of murine macrophage responses stimulated with influenza glycoproteins.

Previously it was reported that influenza virus stimulated, nonspecific resistance was largely due to its glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The enhancement of natural killer cell activity was the intrinsic property of NA and HA. In the present study, the stimulatory effect of these glycoproteins on the murine peritoneal macrophages was studied. Electrophoretically purified glycoproteins, NA and HA, of influenza virus A/USSR/90/77 (H1N1) were administered intraperitoneally to C3H/HeN mice, with or without stearyl tyrosine (ST). Macrophages were isolated and were restimulated with phorbol myristate acetate. H2O2 secretion was determined by horseradish peroxidase dependent oxidation of phenol red assay. HA enhanced H2O2 secretion only in the presence of ST (60 nmol.mg-1.h-1), whereas NA alone stimulated H2O2 secretion (83 nmol.mg-1.h-1), by 6-fold over control (13 nmol.mg-1.h-1), and this stimulation was further increased (136 nmol.mg-1.h-1) in the presence of ST. Interleukin 1 (IL-1) activity was determined by using D10.G4.1 cells. There was a little stimulation of IL-1 activity (less than 1 U/mL) of macrophages isolated from HA-primed of HA+ST-primed mice restimulated with HA. On the other hand, IL-1 activity of macrophages isolated from NA-primed mice restimulated with NA significantly increased (102 U/mL) over control (less than 1 U/mL), and an additional 2-fold increase (231 U/mL) resulted when macrophages from NA+ST-primed mice were used. Tumor necrosis factor (TNF) activity was examined by using L929 cells. Negligible TNF activity was observed in macrophages isolated from either HA-primed or HA+ST-primed mice restimulated with HA.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of separated protein bands in unstained electrophoresed polyacrylamide gradient slab gel.

Polyacrylamide gel electrophoresis has become the most widely used separation method in biological science. Once electrophoresis is complete the protein bands must be localized prior to excision. A zig-zag gel cutter is described which cuts a strip of gel from the side of a slab gradient gel or a gel of uniform concentration in peaks and valleys. The location of the protein of interest is determined by counting the number of peaks on the stained side strip. The portion of the unstained gel corresponding to the same count (number of valleys) is cut to recover the protein of interest from the main gel for further manipulations.

Stimulation of tumor necrosis factor secretion by purified influenza virus neuraminidase.

We showed that purified neuraminidase (NA) of influenza virus, but not hemagglutinin (HA), possessed the potential to increase in vitro and in vivo the interleukin 1 (IL 1) activity of mouse peritoneal macrophages. In this study, we report the effect of NA and HA on the secretion of tumor necrosis factor (TNF) activity by murine peritoneal macrophages. TNF being a cytokine sharing many related and overlapping biological functions with IL 1. The two glycoproteins of the strain A/USSR/90/77 (H1N1) were purified electrophoretically and were tested in vitro at doses ranging from 0.5 to 5.0 micrograms using the adherent peritoneal macrophages of C3H/HeN mice elicited with thioglycolate. The TNF activity of culture supernatants, collected 24 hr after stimulation with viral protein, was evaluated by the standard cytolytic assay using L929 and WEHI.164 cells. No increase of the TNF activity was observed at 0.5 micrograms of NA (4.8 Units (U)/ml in the L929 assay and 20.4 U/ml in the WEHI assay) but further increase of NA to 1.0 microgram had a significant effect on the TNF activity (39.7 and 88.8 U/ml, respectively). Higher concentrations of NA (2.0 and 5.0 micrograms) did not improve the TNF activity. The addition of a rabbit anti-TNF-alpha serum to the assay system reduced the lysis of L929 cells by 85%, suggesting that the observed activity was due to TNF. In parallel, the enhancement of IL 1 activity due to NA was reverified using D10.G4.1 cells instead of the C3H/HeJ thymocytes assay used previously. NA augmented the IL 1 activity up to 1.0 micrograms (25.8 U/ml). The addition of monoclonal anti-IL 1 antibodies (100 neutralizing units) to the supernatants reduced the incorporation of [3H]-thymidine by 90 to 95%, suggesting that the observed activity was due to IL 1. Comparative results of NA and HA showed that only NA stimulated the TNF and IL 1 activities of murine macrophages.

Human influenza virus neuraminidase, but not hemagglutinin, induces murine macrophage interleukin 1 in vivo and in vitro.

Since our previous studies showed that the viral glycoproteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA), are involved in stimulating the human and mouse natural killer (NK) cell activities, it was considered appropriate to study the effect of these glycoproteins on macrophages, another component of nonspecific resistance mechanisms. The glycoproteins HA and NA, of the strain A/USSR/90/77 (H1N1), were purified electrophoretically and were tested in vitro using the adherent peritoneal macrophages of C3H/HeN mice elicited with thioglycolate. IL-1 activity of culture supernatants, collected 24 h after the addition of viral proteins, was evaluated by the standard IL-1 assay using C3H/HeJ thymocytes. Viral NA, but not HA, induced a significant increase of IL-1 activity (p less than 0.05) at dilutions ranging from 1:2 (about thirty-fold augmentation) to 1:256 (nine-fold augmentation) compared to control. The in vivo data showed that intraperitoneal administration of either glycoprotein (2 micrograms) alone did not increase the IL-1 activity; but a six-fold increase (p less than 0.05) of IL-1 activity was observed when the adherent macrophages prepared from NA-primed mice (24 h and 96 h postinoculation) were restimulated in vitro with NA. Similar experiments carried out with HA showed no increase in the IL-1 activity. These and other results suggest that influenza virus NA is superior to HA in stimulating some components of the nonspecific resistance mechanisms.

Purified glycoproteins of influenza virus stimulate cell-mediated cytotoxicity in vivo.

Previously, we reported that purified surface influenza viral glycoproteins can induce cell-mediated cytotoxicity (CMC) in vitro. Both neuraminidase (NA) and hemagglutinin (HA) were equally good stimulators, on an equimolar basis. In order to broaden the scope of these observations, we examined whether these glycoproteins stimulate natural killer (NK) activity in vivo. Biologically active preparations of glycoproteins NA and HA were purified from virus A/USSR/90/77 (H1N1) and recombinant virus A/USSR/92/77 (H1) x A/Prague/1/56 (N7), respectively. The studies were carried out using the optimal doses of NA and HA. In a 4-hour NK assay, using NK-sensitive YAC-1 cells as targets, both viral glycoproteins stimulated the NK activity of splenocytes of BALB/c and C3H mice. This stimulation was independent of the route of administration (intravenous or intraperitoneal) of the antigen. The observed NK activity was viral antigen-specific and could be modulated to levels comparable to those observed with the standard stimulator, polyinosinic acid-polycytidylic acid, by the use of an appropriate synthetic adjuvant, stearyl tyrosinate. Direct and indirect evidences suggest that the enhanced CMC is due to NK cells. These observations imply that enhancement of NK activity is the intrinsic property of influenza NA and HA.

Human influenza viral neuraminidases augment cell-mediated cytotoxicity in vitro.

Previously, we reported that influenza virus-induced cell-mediated cytotoxicity (CMC) was largely due to its glycoproteins, hemagglutinin and neuraminidase (NA). These observations were based on the use of a single influenza virus strain, the A/Port Chalmers/3/73 (H3N2), and these were considered insufficient to generalize that all human influenza virus NAs augment CMC. Therefore, antigenically different NAs of human influenza strains were used to study whether (a) all NAs possess the potential to stimulate NK activity and (b) does the enzymatic activity of NA play a role in the CMC stimulation. Biologically active preparations of N1 subtype NA (A/USSR/90/77 (H1N1) and N2 subtype NAs (A/Aichi/2/68 (H3N2) and A/Port Chalmers) were evaluated for NK activity stimulation in an overnight radiolabeled chromium-release assay consisting of human peripheral blood lymphocytes and K562 target cells. The level of CMC stimulation was the same at equivalent protein concentrations with all the NAs tested. The addition of homologous NA-monospecific antibody almost completely reduced the CMC stimulation, while the addition of homosubtypic antibody reduced the CMC by 56-75%. However, in the presence of heterosubtypic monospecific antibody, NA-augmented CMC was reduced by 27-47% in most experiments. The results suggest that the CMC stimulation site is probably the same in all NAs tested. This putative site is thermo-resistant and is independent of the conformational change of the NA molecule. Furthermore, it is distinct from the enzymatic and probably from the antigenic sites.

Characterization of influenza virus neuraminidase with hemagglutinin activity and its comparison with that of viral neuraminidase.

The neuraminidase associated with the bifunctional protein, hemagglutinin-neuraminidase, of influenza virus has been characterized. The enzyme has a pH optimum of 4.5, does not require Ca2+ and is inactivated (98%) by incubation at 50 degrees C. The enzyme has a Km of 2.00 X 10(-3) M and 0.06 X 10(-3) M with the substrates 2-(3-methoxyphenyl)-N-acetylneuraminic acid and fetuin, respectively. The Ki is 400 X 10(-6) with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid. The incorporation of labeled cysteine, valine and leucine in the hemagglutinin-neuraminidase protein is different from that of viral neuraminidase. A comparison of the properties of the neuraminidase associated with protein hemagglutinin-neuraminidase with that of viral neuraminidase or sialidase showed that the former is biochemically different and an antigenically distinct enzyme. The unique feature of the new enzyme is that it has the hemagglutinin activity as well. The two biological activities could not be separated from each other in all systems used. Apparently, protein hemagglutinin-neuraminidase is genetically transferable and it is detectable in a laboratory recombinant virus E-2971 (H3 Aichi X N7). These results suggest that protein hemagglutinin-neuraminidase is a unique surface protein of the influenza virus A/Aichi/2/68 (H3N2).

Influenza viral glycoproteins induced cell-mediated cytotoxicity by an interferon-independent mechanism.

Human natural killer (NK) cells exposed to the influenza surface antigen neuraminidase (NA) show high cytotoxic activity, as evaluated using chromium-labeled K562 target cells in a standard overnight cytotoxicity assay. The role of interferon (IFN) in the stimulation of NK cells was examined by using three separate approaches. The use of appropriate antibodies to check IFN- and NA-specific cell-mediated cytotoxicity (CMC) stimulation showed that antibodies to IFN (-alpha, -gamma) did not alter NA-induced CMC, and vice versa. The treatment of NK cells with actinomycin D, before or after stimulation with IFN and NA revealed that only IFN-induced CMC was inhibited (50 to 100%). However, NK cells that were stimulated with NA before their exposure to actinomycin D became susceptible to stimulation by IFN. The interaction kinetics between IFN and NA demonstrated the presence of two mechanisms of CMC stimulation. Taken together, the results clearly show that stimulation of CMC by a viral component is effected through an IFN-independent pathway, and that this mechanism is probably followed by IFN under certain conditions.

Concentration and purification of influenza virus from allantoic fluid.

A simple procedure which enables the concentration and purification of influenza virus, using an angular rotor, is described. Virus is concentrated over a sucrose step gradient. The same gradients are reused and volumes up to 4 liters are concentrated in 1 day. The concentrated virus is further purified by a simplified density-gradient technique. No host cell protein is detectable in the final product. The technique offers a broad application potential for concentrating and purifying other viruses.

In vitro enhancement of human natural cell-mediated cytotoxicity by purified influenza virus glycoproteins.

The role of the glycoproteins of influenza virus, hemagglutinin (HA), and neuraminidase (NA) in the in vitro stimulation of natural cell-mediated cytotoxicity (NCMC) or natural killer activity of human peripheral blood lymphocytes was evaluated with radiolabeled K562 cells as target cells in an overnight chromium release assay. Three different approaches were used. (i) Purified viral proteins were obtained by extraction with Nonidet P-40, separation on a sucrose gradient, and further purification by affinity chromatography. Ficoll-Hypaque-purified peripheral blood lymphocytes exposed to HA or NA individually or to a mixture of both significantly increased NCMC (32 to 50%). (ii) Treatment of HA and NA with their respective homologous antisera or F(ab')2 antibody abrogated the stimulation of NCMC by these glycoproteins. (iii) Virions treated with proteolytic enzymes resulted in viral cores lacking either HA or NA or both activities. Compared to whole virions, viral cores devoid of HA activity only induced a 50% increase in NCMC, whereas viral cores lacking HA activity and with traces of NA activity stimulated only 10% of the NCMC. These results suggest that influenza virus-induced cell-mediated cytotoxicity is largely due to its glycoproteins.

The complete neuraminidase of influenza A/PR8/34 (H0N1) is not detectable in its recombinant virus.

The neuraminidase located on the influenza virus recombinant A/Eq (Heq1) x A/PR8 (N1) was relatively a poor antigen (NI titre less than 10) as compared with the enzyme present on the parent virus A/PR8/34 (H0N1) (NI titre 160). This difference in the antigenic behavior of the neuraminidase of A/PR8/34 (H0N1) virus was explored. Results obtained showed that rabbit anti-A/PR8/34 (H0N1) serum contained two distinct types of antineuraminidase antibody and only one type of antineuraminidase antibody was precipitable by the neuraminidase located on the recombinant virus. Evidence suggests that the parent enzyme(s) is not detectable in its entirety on its recombinant virus.

The presence of two neuraminidases in an influenza virus.

The wild type influenza strain A/Aichi/2/68 (H3N2), when disrupted with SDS and electrophoresed on cellulose acetate paper, yielded two separate neuraminidases, NA(H+) and NA(H-). These enzymes after extraction were biologically active and possessed different specific activities. Enzyme NA(H+) possessed neuraminidase as well as hemagglutinin activities whereas enzyme NA(H-) demonstrated only neuraminidase activity. Similar results were obtained when the Aichi strain was treated with Tween-ether and the two enzymes were separated by affinity chromatography. Techniques used failed to separate the hemagglutinin activity from neuraminidase NA(H+). These results suggest that the dual activity present in enzyme NA(H+) may be characteristic of this protein. Both enzymes are antigenically different and are apparently present as distinct entities in the Aichi strain. Experiments showed that only enzyme NA(H-) of the Aichi strain was incorporated into the hybrid X-32 virus during genetic recombination.

The isolation and properties of a ribonuclease associated with influenza virus.

A ribonuclease activity associated with influenza virus has been purified to homogeneity. This preparation is free of DNAase, phosphodiesterase, and phosphatase activities. The purified enzyme has a pH optima of 7.0 at 37 degrees C, and moves as a single band on sodium dodecyl sulfate - polyacrylamide gel with an estimated molecular weight of 84 000.