Showing papers on "Influenza A virus published in 1994"

Apoptosis: a mechanism of cell killing by influenza A and B viruses.

Abstract: In previous studies, we observed that the virulent avian influenza A virus A/Turkey/Ontario/7732/66 (Ty/Ont) induced severe lymphoid depletion in vivo and rapidly killed an avian lymphocyte cell line (RP9) in vitro. In examining the mechanism of cell killing by this virus, we found that Ty/Ont induced fragmentation of the RP9 cellular DNA into a 200-bp ladder and caused ultrastructural changes characteristic of apoptotic cell death by 5 h after infection. We next determined that the ability to induce apoptosis was not unique to Ty/Ont. In fact, a variety of influenza A viruses (avian, equine, swine, and human), as well as human influenza B viruses, induced DNA fragmentation in a permissive mammalian cell line, Madin-Darby canine kidney (MDCK), and this correlated with the development of a cytopathic effect during viral infection. Since the proto-oncogene bcl-2 is a known inhibitor of apoptosis, we transfected MDCK cells with the human bcl-2 gene; these stably transfected cells (MDCKbcl-2) did not undergo DNA fragmentation after virus infection. In addition, cytotoxicity assays at 48 to 72 h after virus infection showed a high level of cell viability for MDCKbcl-2 compared with a markedly lower level of viability for MDCK cells. These studies indicate that influenza A and B viruses induce apoptosis in cell cultures; thus, apoptosis may represent a general mechanism of cell death in hosts infected with influenza viruses.

Heterosubtypic immunity to influenza type A virus in mice. Effector mechanisms and their longevity.

Abstract: Immunity that cross-reacts between influenza type A viruses of distinct subtypes is called hetero(sub)typic (Het-I). We have studied Het-I by challenging PR8-immune mice with the heterosubtypic virus X31. Het-I did not prevent infection by X31 but, at its height, strongly aided in recovery. The nature of the effector mechanisms involved was investigated by simultaneous challenge with X31 and an immunologically unrelated influenza type B virus and by depleting individual lymphocyte subsets in PR8-immune mice before challenge. The study showed the following: 1) The effector mechanisms were intimately associated with immune recognition events. 2) In the nose, depletion of CD8+ or CD4+ T cells led to partial reduction of Het-I, and simultaneous depletion of both T cell subsets abrogated Het-I almost completely. This T cell-mediated immunity was short lived and had disappeared 4 to 5 mo after induction. 3) In trachea and lung, depletion of CD8+ T cells led to a partial reduction of Het-I, whereas depletion of CD4+ T cells was without significant effect. The CD8-mediated component appeared short lived, whereas the residual immunity (in CD4/8-depleted mice) was long lived and persisted past 7 mos after induction. 4) Depletion of NK cells did not significantly reduce the strength of Het-I in either nose or lung. In conclusion, the study shows that Het-I in this system is mediated by a complex combination of immune mechanisms that differ, in part, between upper and lower respiratory tract.

Potential for transmission of avian influenza viruses to pigs

Abstract: Pandemic strains of influenza A virus arise by genetic reassortment between avian and human viruses. Pigs have been suggested to generate such reassortants as intermediate hosts. In order for pigs to serve as ‘mixing vessels’ in genetic reassortment events, they must be susceptible to both human and avian influenza viruses. The ability of avian influenza viruses to replicate in pigs, however, has not been examined comprehensively. In this study, we assessed the growth potential of 42 strains of influenza virus in pigs. Of these, 38 were avian strains, including 27 with non-human-type haemagglutinins (HA; H4 to H13). At least one strain of each HA subtype replicated in the respiratory tract of pigs for 5 to 7 days to a level equivalent to that of swine and human viruses. These results indicate that avian influenza viruses with or without non-human-type HAs can be transmitted to pigs, thus raising the possibility of introduction of their genes into humans. Sera from pigs infected with avian viruses showed high titres of antibodies in ELISA and neutralization tests, but did not inhibit haemagglutination of homologous viruses, cautioning against the use of haemagglutination-inhibition tests to identify pigs infected with avian influenza viruses. Co-infection of pigs with a swine virus and with an avian virus unable to replicate in this animal generated reassortant viruses, whose polymerase and HA genes were entirely of avian origin, that could be passaged in pigs. This finding indicates that even avian viruses that do not replicate in pigs can contribute genes in the generation of reassortants.

Evidence for a protective role of pulmonary surfactant protein D (SP-D) against influenza A viruses.

Abstract: We tested the hypothesis that pulmonary surfactant-associated lectins--surfactant proteins A and D (SP-A, and -D)--contribute to initial protective mechanisms against influenza A viruses (IAVs). SP-D potently inhibited hemagglutination activity of several strains of IAV as well as causing viral aggregation. SP-D enhanced neutrophil binding of IAV and neutrophil respiratory burst responses to the virus. Neutrophil dysfunction resulting from IAV exposure was diminished when the virus was pre-incubated with SP-D. Each of these effects was mediated by the calcium-dependent carbohydrate-binding property of SP-D. Native SP-D preparations of both human and rat origin, as well as recombinant rat SP-D, had similar activity. SP-A also inhibited IAV hemagglutination activity. We have previously reported that related mammalian serum lectins (mannose-binding lectin [MBL] and conglutinin) have similar effects. SP-D was at least 10-fold more potent at causing hemagglutination inhibition than were SP-A or MBL. SP-D was shown to contribute to potent anti-IAV activity of human bronchoalveolar lavage fluid. These results suggest that SP-D--alone, and in conjunction with SP-A and phagocytic cells--constitutes an important component of the natural immune response to IAV infection within the respiratory tract.

Reverse genetics provides direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus.

Abstract: To obtain direct evidence for a relationship between hemagglutinin (HA) cleavability and the virulence of avian influenza A viruses, we generated a series of HA cleavage mutants from a virulent virus, A/turkey/Ontario/7732/66 (H5N9), by reverse genetics. A transfectant virus containing the wild-type HA with R-R-R-K-K-R at the cleavage site, which was readily cleaved by endogenous proteases in chicken embryo fibroblasts (CEF), was highly virulent in intramuscularly or intranasally/orally inoculated chickens. By contrast, a mutant containing the HA with an avirulent-like sequence (R-E-T-R) at the cleavage site, which was not cleaved by the proteases in CEF, was avirulent in chickens, indicating that a genetic alteration confined to the HA cleavage site can affect cleavability and virulence. Mutant viruses with HA cleavage site sequences of T-R-R-K-K-R or T-T-R-K-K-R were as virulent as viruses with the wild-type HA, whereas a mutant with a two-amino-acid deletion but retention of four consecutive basic residues (R-K-K-R) was as avirulent as a virus with the avirulent-type HA. Interestingly, although a mutant containing an HA with R-R-R-K-T-R, which has reduced cleavability in CEF, was as virulent as viruses with high HA cleavability when given intramuscularly, it was less virulent when given intranasally/orally. We conclude that the degree of HA cleavability in CEF predicts the virulence of avian influenza viruses.

Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses.

Abstract: Among the proprotein-processing subtilisin-related endoproteases, furin has been a leading candidate for the enzyme that activates the hemagglutinin (HA) of virulent avian influenza viruses. In the present study, we examined the cleavage activity of two other recently isolated ubiquitous subtilisin-related proteases, PACE4 and PC6, using wild-type HA of A/turkey/Ireland/1378/83 (H5N8) and a series of its mutant HAs. Vaccinia virus-expressed wild-type HA was not cleaved in human colon adenocarcinoma LoVo cells, which lack active furin. This processing defect was corrected by the expression of furin and PC6 but not of PACE4 and a control wild-type vaccinia virus. PC6 showed a sequence specificity similar to that with the endogenous proteases in cultured cells. When LoVo cells were infected with a virulent avian virus, A/turkey/Ontario/7732/66 (H5N9), only noninfectious virions were produced because of the lack of HA cleavage. However, when the cells were coinfected with vaccinia virus that expressed either furin or PC6, the avian virus underwent multiple cycles of replication, indicating that both furin and PC6 specifically cleave the virulent virus HA at the authentic site. These data suggest that PC6, as well as furin, can activate virulent avian influenza viruses in vivo, implying the presence of multiple HA cleavage enzymes in animals.

The long-term maintenance of cytotoxic T cell memory does not require persistence of antigen.

Abstract: I have used the transfer of primed lymphocytes into syngeneic irradiated recipients to investigate whether the persistence of antigen is required in the long-term maintenance of cytolytic T cell memory to influenza virus. Animals were immunized with influenza virus (A/WSN) and used 17 wk later as either donors for T cells or as lethally irradiated recipients. Naive age-matched mice served as controls. At intervals of 4, 8, 16, and 25 wk after T cell transfer, experimental and control groups were immunized with a heterologous virus (A/JAP) and splenocytes tested for lytic activity to influenza virus 3 and 6 d after immunization. Lytic activity 3 d after infection (a property exclusive to a memory cytotoxic T cell response) (Effros, R. B., J. Bennink, and P. C. Doherty. 1978. Cell. Immunol. 36:345.; and Hill, A. B., R. V. Blanden, C. R. Parrish, and A. Mullbacher. 1992. Immunol. Cell Biol. 70:259), was only observed by primed and naive irradiated recipients reconstituted with memory T cells. No day 3 responses were observed when naive T cells were transferred into irradiated primed or unprimed recipients. These observations demonstrate that cytolytic T cell memory to influenza virus is long lived in the absence of antigen.

Influenza A virus infections among hospitalized adult bone marrow transplant recipients.

Abstract: Although influenza virus continues to cause annual epidemics of respiratory diseases, surprisingly little is known about the frequency and clinical course of influenza among adult patients with cancer. During the 1991-92 influenza epidemic in Houston, Texas, we followed all adult BMT recipients hospitalized at M.D. Anderson Cancer Center. None of these 68 patients had received prophylaxis for influenza. Influenza virus type A was isolated from 8 (29%) of 28 BMT recipients with an acute respiratory illness. Five of these infections were acquired in the hospital. All 8 patients presented with an upper respiratory tract illness. In 6 patients, the infection was complicated by pneumonia. The frequency of influenza was similar among autologous (5 of 18) and allogeneic (3 of 10) BMT recipients. The risk of developing pneumonia was not related to the type of transplant or to the engraftment status. All patients received broad-spectrum antibiotics. The 2 patients who did not develop pneumonia also received amantadine. The mortality with pneumonia was 17%. During community outbreaks, influenza is a frequent cause of acute respiratory illness among hospitalized adult BMT recipients and is frequently complicated by pneumonia. Studies are needed to define the optimal means of preventing and treating influenza in BMT recipients.

Efficacy of inactivated vaccine in preventing antigenically drifted influenza type A and well-matched type B.

Abstract: Objective. —To evaluate the efficacy of currently used inactivated influenza vaccine during a severe epidemic caused by antigenically drifted influenza type A(H3N2) and well-matched type B viruses during the 1992-1993 season. Design. —Prospective nonrandomized controlled trial. Setting. —An urban general hospital pediatric asthma clinic in Japan. Participants. —A total of 137 children with moderate to severe asthma (mean age, 7.0 years; range, 2 to 14 years). Interventions. —Eighty-five children received trivalent split-antigen vaccine containing A/Beijing/352/89 (H3N2) and B/Bangkok/163/90 (B/Panama/45/90—like strain). Fifty-two were unvaccinated. Main Outcome Measures. —Protection against infection was determined using hemagglutination inhibition test and virus isolation. Clinical efficacy was estimated based on febrile episodes with antibody rise or virus isolation. Results. —Although marked antigenic drift in hemagglutinin was demonstrated in the epidemic virus (A/Beijing/32/92—like strain), the protection against influenza type A(H3N2) infection was 67.5% (P Conclusions. —Our data suggest that current inactivated vaccine is highly effective for protection against influenza type A(H3N2) virus infection regardless of antigenic drift. In contrast, the protective efficacy obtained by vaccination may not be sufficient against influenza type B virus infection, and especially in young children, it does not offer protection. (JAMA. 1994;272:1122-1126)

Influenza virus M2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport.

Abstract: The influenza A/fowl plague virus/Rostock/34 hemagglutinin (HA), which is cleaved intracellularly and has a high pH threshold (pH 5.9) for undergoing its conformational change to the low-pH form, was expressed from cDNA in CV-1 and HeLa T4 cells in the absence of other influenza virus proteins. It was found, by biochemical assays, that the majority of the HA molecules were in a form indistinguishable from the low-pH form of HA. The acidotropic agent, ammonium chloride, stabilized the accumulation of HA in its native form. Coexpression of HA and the homotypic influenza virus M2 protein, which has ion channel activity, stabilized the accumulation of HA in its pH neutral (native) form, and the M2 protein ion channel blocker, amantadine, prevented the rescue of HA in its native form. These data provide direct evidence that the influenza virus M2 protein ion channel activity can affect the status of the conformational form of cleaved HA during intracellular transport.

Expression of a foreign protein by influenza A virus.

Abstract: In this report we describe the rescue of a transfectant influenza A virus which stably expresses a heterologous protein, bacterial chloramphenicol acetyltransferase (CAT) The foreign sequences encoding CAT are expressed as part of an essential influenza virus segment, that coding for the neuraminidase (NA) protein The novel way by which this was achieved involved inserting in frame the 16-amino-acid self-cleaving 2A protease of foot-and-mouth disease virus between the CAT and the NA coding sequences The resultant gene produces a polyprotein which is proteolytically cleaved to release both CAT and NA The intramolecular cleavage occurs at the C terminus of the 2A sequence between a glycine-proline dipeptide motif such that the released NA protein has an additional N-terminal proline residue The transfectant virus is stable upon passage in tissue culture CAT activity is expressed at high levels in cell culture supernatants and in the allantoic fluid of infected eggs Since the chimeric segment must maintain the heterologous reading frame to retain viability, the virus stability is dependent upon concomitant synthesis of the heterologous protein This design may be particularly appropriate for utilization of influenza virus as a mammalian expression vector

Antigenicity and immunogenicity of equine influenza vaccines containing a Carbomer adjuvant

Abstract: Equine influenza vaccines containing inactivated whole virus and Carbomer adjuvant stimulated higher levels and longer lasting antibody to haemagglutinin in ponies than vaccines of equivalent antigenic content containing aluminium phosphate adjuvants. Five months after the third dose of vaccine containing Carbomer adjuvant, ponies were protected against clinical disease induced by an aerosol of virulent influenza virus (A/equine/Newmarket/79, H3N8). In contrast ponies which received vaccine containing aluminium phosphate adjuvant were susceptible to infection and disease. There was an inverse correlation between prechallenge levels of antibody detected by single radial haemolysis (SRH) and duration of virus excretion, pyrexia and coughing. All ponies with antibody levels equivalent to SRH zones of > or = 154 mm2 were protected against infection and all those with levels > or = 85 mm2 were protected from disease.

Protection against the mouse-adapted A/FM/1/47 strain of influenza A virus in mice by a monoclonal antibody with cross-neutralizing activity among H1 and H2 strains.

Abstract: The monoclonal antibody designated C179 was found to neutralize all of the H1 and H2 strains of influenza A virus studied (Y Okuno, Y Isegawa, F Sasao, and S Ueda, J Virol 67:2552-2558, 1993) In the present study, the ability of C179 to protect mice from the lethal effect of the A/FM/1/47 (H1N1) strain was examined When the mice were injected intraperitoneally with 100 micrograms of C179 per mouse a day before the virus challenge (20 x 10(3) focus-forming units per mouse), all of the mice survived Moreover, significantly higher survival rates were observed in mice receiving 1,000 micrograms of C179 per mouse 2 days after the virus challenge than in those receiving phosphate-buffered saline alone These results indicate that C179 is effective not only for prevention but also for treatment of mice infected with H1 and H2 strains The possibility that C179 can be used for passive immunization in humans is discussed

Fine structure of influenza A virus observed by electron cryo-microscopy.

Abstract: A rapidly frozen vitrified aqueous suspension of influenza A virus was observed by high resolution electron cryomicroscopy The influenza particles were grouped into small (diameter < 150 nm) spherical particles with well organized interiors, large spherical ones with less internal organization, and filamentous ones Envelopes of most of the large virus particles were phospholipid bilayers, and the chromatography fraction containing these large particles was largely devoid of viral activity The envelopes of most of the filamentous and small spherical virus particles, on the other hand, gave a strange contrast which could be ascribed to a combination of a thin outer lipid monolayer and a 72 nm thick protein-containing inner layer These latter particles represented most of the viral activity in the preparation Densitometric traces of the near in-focus images confirmed these structural differences Some viral envelope structures apparently intermediate between these two distinct types of membrane were also detected A structural model of intact biologically active influenza virus particles was formulated from these results, together with computer simulations

Comparison of rapid detection methods for influenza A virus and their value in health-care management of institutionalized geriatric patients.

Abstract: Respiratory specimens from 160 geriatric patients with suspected influenza illness were used to evaluate the abilities of two enzyme immunoassays (EIAs; Directigen FLU-A [Becton Dickinson Microbiology Systems, Cockeysville, Md] and Prima EIA [Baxter/Bartels Diagnostics, Inc, Issaquah, Wash]) and direct immunofluorescence testing (immunofluorescence assay [IFA]) to identify influenza A virus In comparison with culture isolation, the sensitivities and specificities of the IFA, Directigen FLU-A, and Prima EIA were 925 and 972%, 868 and 991%, and 925 and 981%, respectively In contrast to EIA, IFA was labor intensive and required a high degree of technical expertise, and the results of IFA were difficult to interpret These factors may preclude the use of IFA for testing large numbers of specimens A retrospective epidemiologic survey of influenza infection was done in six geriatric institutions which had used either rapid and culture testing or culture alone Preventable cases of influenza A virus infection ranged from 9 to 38% of all cases in facilities which used culture testing only and which had not instituted amantadine prophylaxis The use of direct specimen testing is recommended as an adjunct to culture isolation for the identification of influenza A virus Use of a combination of these methods permits the timely administration of appropriate antiviral therapy and infection control measures, while it also permits the antigenic surveillance of circulating influenza strains, which is necessary for present vaccine efficacy evaluations and the creation of future effective vaccine formulations

Sequence specificity of furin, a proprotein-processing endoprotease, for the hemagglutinin of a virulent avian influenza virus.

Abstract: The virulence of avian influenza viruses correlates with the sensitivity of their hemagglutinin (HA) to cellular proteases. Furin, a proprotein-processing subtilisin-related endoprotease, is a leading candidate for the enzyme that cleaves the HA of virulent avian viruses. We therefore compared the specificity of furin with those of proteases in a variety of cultured cells and in a rat Golgi fraction, using the HA cleavage mutants of a virulent avian influenza virus, A/Turkey/Ireland/1378/85 (H5N8). The results indicated similar sequence specificities among the endoproteases when purified furin was used. In experiments with the vaccinia virus expression system, overexpressed furin cleaved mutant HAs that were not recognized by the endogenous proteases, resulting in an apparent broader specificity of furin. These findings authenticate the proposed role of furin as an HA-activating protease in vivo and caution against the use of expression vectors to study protease sequence specificity.

Statistical analysis of nucleotide sequences of the hemagglutinin gene of human influenza A viruses.

Abstract: To examine whether positive selection operates on the hemagglutinin 1 (HA1) gene of human influenza A viruses (H1 subtype), 21 nucleotide sequences of the HA1 gene were statistically analyzed. The nucleotide sequences were divided into antigenic and nonantigenic sites. The nucleotide diversities for antigenic and nonantigenic sites of the HA1 gene were computed at synonymous and nonsynonymous sites separately. For nonantigenic sites, the nucleotide diversities were larger at synonymous sites than at nonsynonymous sites. This is consistent with the neutral theory of molecular evolution. For antigenic sites, however, the nucleotide diversities at nonsynonymous sites were larger than those at synonymous sites. These results suggest that positive selection operates on antigenic sites of the HA1 gene of human influenza A viruses (H1 subtype).

Characterization of the polyadenylation signal of influenza virus RNA.

Abstract: It has been shown that a stretch of uridines (U's) near the 5' end of the virion RNA of influenza A virus is the polyadenylation site for viral mRNA synthesis In addition, the RNA duplex made up the 3' and 5' terminal sequences adjacent to the U stretch is also involved in polyadenylation We have further characterized the polyadenylation signal of influenza virus RNA with a ribonucleoprotein transfection system We found that the optimal length of the U stretch is 5 to 7 uridine residues We also showed that the upstream sequence at the 5' end is not involved in polyadenylation and that the optimal distance between the 5' end and the U stretch is 16 nucleotides The combination of these features defines the polyadenylation site and differentiates this signal from other U stretches scattered throughout the genomes of influenza viruses

An influenza A (H1N1) virus, closely related to swine influenza virus, responsible for a fatal case of human influenza.

Abstract: In July 1991, an influenza A virus, designated A/Maryland/12/91 (A/MD), was isolated from the bronchial secretions of a 27-year-old animal caretaker. He had been admitted to the hospital with bilateral pneumonia and died of acute respiratory distress syndrome 13 days later. Antigenic analyses with postinfection ferret antisera and monoclonal antibodies to recent H1 swine hemagglutinins indicated that the hemagglutinin of this virus was antigenically related to, but distinguishable from, those of other influenza A (H1N1) viruses currently circulating in swine. Oligonucleotide mapping of total viral RNAs revealed differences between A/MD and other contemporary swine viruses. However, partial sequencing of each RNA segment of A/MD demonstrated that all segments were related to those of currently circulating swine viruses. Sequence analysis of the entire hemagglutinin, nucleoprotein, and matrix genes of A/MD revealed a high level of identity with other contemporary swine viruses. Our studies on A/MD emphasize that H1N1 viruses in pigs obviously continue to cross species barriers and infect humans.

The influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity.

Abstract: The influenza A virus hemagglutinin (HA) glycoprotein contains a cytoplasmic tail which consists of 10-11 amino acids, of which five residues re conserved in all subtypes of influenza A virus. As the cytoplasmic tail is not needed for intracellular transport to the plasma membrane, it has become virtually dogma that the role of the cytoplasmic tail is in forming protein-protein interactions necessary for creating an infectious budding virus. To investigate the role of the HA cytoplasmic tail in virus replication, reverse genetics was used to obtain an influenza virus that lacked an HA cytoplasmic tail. The rescued virus contained the HA of subtype A/Udorn/72 in a helper virus (subtype A/WSN/33) background. Biochemical analysis indicated that only the introduced tail- HA was incorporated into virions and these particles lacked a detectable fragment of the helper virus HA. The tail- HA rescued virus assembled and replicated almost as efficiently as virions containing wild-type HA, suggesting that the cytoplasmic tail is not essential for the virus assembly process. Nonetheless, a revertant virus was isolated, suggesting that possession of a cytoplasmic tail does confer an advantage.

Mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation.

Abstract: The carboxy terminus of the hemagglutinin (HA) of influenza A viruses contains three cysteine residues which are highly conserved among HA subtypes. It has previously been shown for the H2, H3, and H7 subtypes of HA that these cysteine residues are modified by the covalent attachment of palmitic acid. In order to study the role of the acylated cysteines in the formation of infectious influenza viruses, we introduced mutations into the HA of influenza A/WSN/33 virus (H1 subtype) by reverse-genetics techniques. We found that the cysteine at position 563 of the cytoplasmic tail is required for infectious-particle formation. The cysteine at position 560 can be changed to alanine or tyrosine to yield virus strains that are attenuated in cell cultures. The change from cysteine at position 553 to serine or alanine does not significantly alter the phenotype of the virus. The requirement for a cysteine at position 563 suggests a functional role for palmitylation of the cytoplasmic tail. This interpretation is further supported by experiments in which two or more of the cysteine residues were mutated, eliminating potential palmitylation sites. None of these double or triple mutations resulted in infectious virus. Selection of revertants of the attenuated cysteine-to-tyrosine mutant (mutation at position 560) always resulted in reversion to cysteine rather than to other amino acids. Although our data indicate a biological role for the conserved cysteine residues in the cytoplasmic tail of the HA of influenza viruses, we cannot exclude the possibility that structural constraints in the cytoplasmic tail of the HA--rather than altered palmitylation--are the determining factors for infectious-particle formation.

Use of a mammalian internal ribosomal entry site element for expression of a foreign protein by a transfectant influenza virus.

Abstract: Influenza A viruses arenegative-strand RNA viruses which havea segmented genome witha coding capacity for10 polypeptides. Thevirus genome iscomposed ofeight different RNA segmentswhichare tightly associated withtheviral nucleoprotein andpolymerases inribonucleoprotein (RNP) complexes (14). Because ofthenoninfectious natureofthe genomicRNAs,influenza viruses were until recently not amenable tostandard techniques forgenetic manipulation. Thedevelopment ofa new transfection methodology, based on theinvitro reconstitution ofRNPsusing synthetic RNAs,has now madeitpossible toconstruct influenza viruses containing plasmid-derived RNA segments(5,19;fora review,see reference 8).Thissystemallows thestudy ofcis-acting sequencesintheviral RNAsaswell asthefunctional analysis of theviral proteins. Inaddition, this methodology hasbeenused togenerate recombinant influenza virus vectorsexpressing foreign sequences. A foreign geneflanked bythe3'and5'noncoding sequences ofinfluenza virus RNA segments canbeamplified, expressed, andpackaged into virus particles after RNP transfection into influenza virus-infected cells. However, therecombinant (nonessential) geneislost after several viral passages.Thegeneration ofstable influenza virus vectors expressing foreign amino acid sequenceswas achieved byinserting theforeign sequence into an essential viral gene.Thehemagglutinin (HA)and neuraminidase (NA)arespike-like proteins on theinfluenza virusenvelope; theyare alsoexpressed on thesurface of influenza virus-infected cells. Forthese reasons,theyare attractive proteins forthepresentation offoreign B-cell and T-cell epitopes. Transfectant influenza viruseswhichcontain

Primary pulmonary cytotoxic T lymphocytes induced by immunization with a vaccinia virus recombinant expressing influenza A virus nucleoprotein peptide do not protect mice against challenge.

Abstract: The nucleoprotein (NP) of influenza A virus is the dominant antigen recognized by influenza virus-specific cytotoxic T lymphocytes (CTLs), and adoptive transfer of NP-specific CTLs protects mice from influenza A virus infection. BALB/c mouse cells (H-2d) recognize a single Kd-restricted CTL epitope of NP consisting of amino acids 147 to 155. In the present study, mice were immunized with various vaccinia virus recombinant viruses to examine the effect of the induction of primary pulmonary CTLs on resistance to challenge with influenza A/Puerto Rico/8/34 virus. The minigene ESNP(147-155)-VAC construct, composed of a signal sequence from the adenovirus E3/19K glycoprotein (designated ES) and expressing the 9-amino-acid NP natural determinant (amino acids 147 to 155) preceded by an alanine residue, a similar minigene NP(Met 147-155)-VAC lacking ES, and a full-length NP-VAC recombinant of influenza virus were analyzed. The two minigene NP-VAC recombinants induced a greater primary pulmonary CTL response than the full-length NP-VAC recombinant. However, NP-specific CTLs induced by immunization with ESNP(147-155)-VAC did not decrease peak virus titer or accelerate clearance of virus in the lungs of mice challenged intranasally with A/PR/8/34. Furthermore, NP-specific CTLs induced by immunization did not protect mice challenged intranasally with a lethal dose of A/PR/8/34. Sequence analysis of the NP CTL epitope of A/PR/8/34 challenge virus obtained from lungs after 8 days of replication in ESNP(147-155)-VAC-immunized mice showed identity with that of the input virus, demonstrating that an escape mutant had not emerged during replication in vivo. Thus, in contrast to adoptively transferred CTLs, pulmonary NP-specific CTLs induced by recombinant vaccinia virus immunization do not have protective in vivo antiviral activity against influenza virus infection.

Reconstitution of the influenza virus M2 ion channel in lipid bilayers

Abstract: M2, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M2 baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25-500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25-90 pS). (3) The channels were selective for cations; tNa = 0.75 when 150 mM NaCl bathed both sides of the membrane. (4) Amantadine reduced the probablity of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs approximately Na > Li. The pH activation, amantadine block and ion selectivity of the M2 protein ion channel in bilayers are consistent with those observed on expression of the M2 protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M2 protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.

Administration of anti-IFN-gamma antibody to beta 2-microglobulin-deficient mice delays influenza virus clearance but does not switch the response to a T helper cell 2 phenotype.

Abstract: Treatment of mice that were homozygous for a beta 2-microglobulin gene disruption with a mAb that was specific for IFN-gamma delayed clearance of an influenza A virus from the respiratory tract for at least 3 days, whereas administration of an anti-IL-4 mAb had no effect. However, all mice survived and eventually cleared the virus. The anti-IFN-gamma significantly decreased both the level of class II MHC glycoprotein expression and the numbers of CD4+ lymphocytes in the inflammatory populations recovered by bronchoalveolar lavage of the pneumonic lung, whereas the total cell counts remained the same. These differences were not apparent for the regional mediastinal lymph nodes, although the frequency of lymph node B cells producing virus-specific Ab of the IgG2a subclass was greatly reduced. However, neither the anti-IFN-gamma nor anti-IL-4 treatments drastically altered the cytokine production profiles detected for freshly isolated lymphocytes by the using single cell ELISPOT assay or by ELISA of culture supernatants after in vitro restimulation with virus. Thus, neutralization of secreted IFN-gamma during the course of an influenza-specific response in beta 2-microglobulin-deficient mice that lack CD8+ T cells delays virus clearance and modifies the character of the host response, but does not cause the CD4+ subset to switch to a Th2 cytokine profile.