Showing papers on "Influenza A virus published in 1991"

Clearance of Influenza Virus Respiratory Infection in Mice Lacking Class I Major Histocompatibility Complex-restricted CD8 + T Cells

Abstract: Transgenic mice homozygous for a beta 2-microglobulin (beta 2-m) gene disruption and normal mice that had been treated with a CD8-specific mAb were infected intranasally with an H3N2 influenza A virus. Both groups of CD8T cell-deficient mice eliminated the virus from the infected respiratory tract. Potent CTL activity was detected in lung lavage populations taken from mice with intact CD8+ T cell function, with minimal levels of cytotoxicity being found for inflammatory cells obtained from the antibody-treated and beta 2-m mutant mice. We therefore conclude that cells infected with an influenza A virus can be cleared from the respiratory tract of mice lacking both functional class I major histocompatibility complex (MHC) glycoproteins and class I MHC-restricted, CD8+ effector T cells.

Positive Darwinian evolution in human influenza A viruses.

Abstract: We earlier suggested that type A human influenza virus genes undergo positive Darwinian selection through immune surveillance. This requires more favorable amino acid replacements fixed in antigenic sites among the surviving lineages than among the extinct lineages. We now show that viral hemagglutinins fix proportionately more amino acid replacements in antigenic sites in the trunk of the evolutionary tree (survivors) than in the branches (nonsurvivors), demonstrating that type A human influenza virus is undergoing positive Darwinian evolution. The hemagglutinin gene is evolving 3 times faster than the nonstructural gene and the average age of the sampled nonsurvivors is only 1.6 years, so that extinction is not only common but rapid.

Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins.

Abstract: Phylogenetic analysis of 42 membrane protein (M) genes of influenza A viruses from a variety of hosts and geographic locations showed that these genes have evolved into at least four major host-related lineages: (i) A/Equine/prague/56, which has the most divergent M gene; (ii) a lineage containing only H13 gull viruses; (iii) a lineage containing both human and classical swine viruses; and (iv) an avian lineage subdivided into North American avian viruses (including recent equine viruses) and Old World avian viruses (including avianlike swine strains). The M gene evolutionary tree differs from those published for other influenza virus genes (e.g., PB1, PB2, PA, and NP) but shows the most similarity to the NP gene phylogeny. Separate analyses of the M1 and M2 genes and their products revealed very different patterns of evolution. Compared with other influenza virus genes (e.g., PB2 and NP), the M1 and M2 genes are evolving relatively slowly, especially the M1 gene. The M1 and M2 gene products, which are encoded in different but partially overlapping reading frames, revealed that the M1 protein is evolving very slowly in all lineages, whereas the M2 protein shows significant evolution in human and swine lineages but virtually none in avian lineages. The evolutionary rates of the M1 proteins were much lower than those of M2 proteins and other internal proteins of influenza viruses (e.g., PB2 and NP), while M2 proteins showed less rapid evolution compared with other surface proteins (e.g., H3HA). Our results also indicate that for influenza A viruses, the evolution of one protein of a bicistronic gene can affect the evolution of the other protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Extensive conservation of alpha-chain and beta-chain of the human t-cell antigen receptor recognizing hla-a2 and influenza-a matrix peptide

Abstract: The major histocompatibility complex class I molecule HLA-A2.1 presents the influenza A virus matrix peptide 57-68 to cytotoxic T lymphocytes in all individuals with this common HLA type and is among the most thoroughly studied immune responses in humans. We have studied the T-cell receptor (TCR) heterogeneity of T cells specific for HLA-A2 and influenza A matrix peptide using the polymerase chain reaction. The usage of V-alpha and V-beta-sequences seen on these T cells is remarkably conserved as are certain junctional sequences associated with alpha and beta-chains. Furthermore, two unrelated HLA-A2 individuals have a similar pattern of TCR usage, implying that this is a predominant response in HLA-A2 populations. Analysis in one individual showed that the conserved TCR V-alpha and V-beta-genes are minor members of the peripheral blood TCR repertoire. The sequences provide important information on the TCR necessary for the final structural analysis of this ternary complex.

Evolution of influenza A virus nucleoprotein genes: implications for the origins of H1N1 human and classical swine viruses.

Abstract: A phylogenetic analysis of 52 published and 37 new nucleoprotein (NP) gene sequences addressed the evolution and origin of human and swine influenza A viruses. H1N1 human and classical swine viruses (i.e., those related to Swine/Iowa/15/30) share a single common ancestor, which was estimated to have occurred in 1912 to 1913. From this common ancestor, human and classical swine virus NP genes have evolved at similar rates that are higher than in avian virus NP genes (3.31 to 3.41 versus 1.90 nucleotide changes per year). At the protein level, human virus NPs have evolved twice as fast as classical swine virus NPs (0.66 versus 0.34 amino acid change per year). Despite evidence of frequent interspecies transmission of human and classical swine viruses, our analysis indicates that these viruses have evolved independently since well before the first isolates in the early 1930s. Although our analysis cannot reveal the original host, the ancestor virus was avianlike, showing only five amino acid differences from the root of the avian virus NP lineage. The common pattern of relationship and origin for the NP and other genes of H1N1 human and classical swine viruses suggests that the common ancestor was an avian virus and not a reassortant derived from previous human or swine influenza A viruses. The new avianlike H1N1 swine viruses in Europe may provide a model for the evolution of newly introduced avian viruses into the swine host reservoir. The NPs of these viruses are evolving more rapidly than those of human or classical swine viruses (4.50 nucleotide changes and 0.74 amino acid change per year), and when these rates are applied to pre-1930s human and classical swine virus NPs, the predicted date of a common ancestor is 1918 rather than 1912 to 1913. Thus, our NP phylogeny is consistent with historical records and the proposal that a short time before 1918, a new H1N1 avianlike virus entered human or swine hosts (O. T. Gorman, R. O. Donis, Y. Kawaoka, and R. G. Webster, J. Virol. 64:4893-4902, 1990). This virus provided the ancestors of all known human influenza A virus genes, except for HA, NA, and PB1, which have since been reassorted from avian viruses. We propose that during 1918 a virulent strain of this new avianlike virus caused a severe human influenza pandemic and that the pandemic virus was introduced into North American swine populations, constituting the origin of classical swine virus.

Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules.

Abstract: Mice that were intranasally immunized with different influenza A virus hemagglutinins (HA), derived from PR8 (H1N1), A/Yamagata (H1N1) or A/Fukuoka (H3N2) virus, together with cholera toxin B subunit as an adjuvant, were examined for protection against PR8 infection; PR8 HA and A/Yamagata HA immunization conferred complete protection, while A/Fukuoka HA immunization failed to confer protection. In parallel with protection, PR8 HA-, A/Yamagata HA-, and A/Fukuoka HA-immunized mice produced a high, a moderate and a low level of PR8 HA-reactive IgA in the respiratory tract, respectively. These IgA antibodies were not only higher in content in the nasal secretions, but also more cross-reactive than IgG. The purified IgA antibodies from respiratory tract washings of PR8 HA-immunized mice, which contained the HA-specific IgA corresponding to the amount detected in the nasal wash, were able to protect mice from PR8 challenge when transferred to the respiratory tract of naive mice. The transfer of IgA from A/Yamagata HA-immunized mice also afforded cross-protection against PR8 infection, whereas the IgA from A/Fukuoka HA-immunized mice failed to provide protection. The ability of transferred IgA to prevent viral infection was dependent on the amount of HA-reactive IgA remaining in the respiratory tract of the host at the time of infection. These experiments directly demonstrate that IgA antibodies to influenza A virus HA by themselves play a pivotal role in defence not only against homologous virus infection, but also against heterologous drift virus infection at the respiratory mucosa, the portal of entry for the viruses.

An influenza A virus containing influenza B virus 5' and 3' noncoding regions on the neuraminidase gene is attenuated in mice.

Abstract: Influenza A and B viruses have not been shown to form reassortants. It had been assumed that the lack of genotypic mixing between influenza virus types reflected differences in polymerase and packaging specificity. In this study, we show that an influenza A virus polymerase transcribes and replicates a chloramphenicol acetyltransferase (CAT) gene flanked by the nontranslated sequences of an influenza B virus gene. Although the transcription level of this CAT gene was several times lower than that of a CAT gene flanked by the homologous nontranslated sequences of an influenza A virus, we proceeded to construct a chimeric type A/B influenza virus. Using recombinant DNA techniques, a chimeric neuraminidase gene was introduced into the genome of influenza A/WSN/33 virus. The hybrid influenza A/B virus gene contained the coding region of the A/WSN neuraminidase and the 3' and 5' nontranslated sequences of the nonstructural gene of influenza B/Lee virus. The resulting chimeric virus formed plaques in Madin-Darby bovine kidney cells but replicated more slowly and achieved lower titers than wild-type influenza A/WSN/33 virus. The chimeric virus was attenuated for mice as indicated by a 400-fold increase in its LD50. Interestingly, the virus was greatly restricted in replication in the upper respiratory tract and partially restricted in the lungs. Animals infected with the transfectant virus were highly resistant to influenza virus challenge. It appears that this chimeric virus has many of the properties desirable for a live attenuated virus vaccine.

Recovery of drug-resistant influenza A virus during therapeutic use of rimantadine.

Abstract: The therapeutic activity of rimantadine and its relationship to the shedding of drug-resistant influenza A virus were assessed in two randomized, double-blind, placebo-controlled trials involving patients with laboratory-documented influenza A virus (H3N2 subtype) illness of 2 days' duration or less. In a family-based study, rimantadine treatment for 10 days (24 children and adults) was associated with significant decreases in the number of days to a 50% reduction in symptoms (mean difference, 2.5 days), days of fever (1.6 days), and days of restricted activity (1.5 days) compared with the results obtained with placebo-treated patients (32 children and adults). Drug-resistant virus was recovered from eight (33%) of the rimantadine recipients on day 5. No differences in patient demographics or illness severity at the time of enrollment in the study were apparent between those who shed resistant virus and those who did not. Illness resolution tended to be slower in those who shed resistant virus compared with that in those who did not. In a study of adults treated for 5 days (six treated with rimantadine, six treated with placebo), resistant virus was recovered in three rimantadine recipients by day 3 of treatment. The results indicate that drug-resistant influenza A virus (H3N2) can be recovered from rimantadine-treated children and adults as early as 2 days after starting treatment, but that rimantadine retains a net therapeutic benefit compared with that of placebo.

NS2 protein of influenza virus is found in purified virus and phosphorylated in infected cells

Abstract: Purified viral preparations of influenza A virus were examined for the presence of NS2 protein hitherto considered as a viral nonstructural protein that is present only in infected cells. Analysis of purified virus by radioimmunoprecipitation with monospecific antisera to NS2 revealed its presence in the virus particle suggesting that it is a viral structural protein. NS2 protein was also shown to be phosphorylated in infected cells in this study. This brings the number of influenza virus phosphoproteins to three which include NP, NS1, and NS2. These observations raise important questions about the role of NS2 in the replication of influenza virus.

Differential inhibitory effects of sulfated polysaccharides and polymers on the replication of various myxoviruses and retroviruses, depending on the composition of the target amino acid sequences of the viral envelope glycoproteins.

Abstract: Sulfated polysaccharides (i.e., dextran sulfate) and sulfated polymers (i.e., sulfated polyvinylalcohol and sulfated copolymers of acrylic acid with vinylalcohol) were found to be potent and selective inhibitors of the replication of respiratory syncytial virus (RSV) and influenza virus type A (influenza A virus) but not of other myxoviruses (parainfluenza 3, measles, and influenza B viruses). The compounds were also inhibitory to human immunodeficiency virus type 1 (HIV-1) and HIV-2 and simian immunodeficiency virus but not simian AIDS-related virus. The mode of antiviral action of the sulfated polysaccharides and polymers can be attributed to an inhibition of virus binding to the cells (HIV-1), inhibition of virus-cell fusion (influenza A virus), or inhibition of both virus-cell binding and fusion (RSV). The fact that the sulfated polysaccharides and polymers are inhibitory to some myxoviruses and retroviruses but not to others seems to depend on the composition of the amino acid sequences of the viral envelope glycoproteins that are involved in virus-cell binding and fusion. All myxoviruses and retroviruses that are sensitive to the sulfated polysaccharides and polymers share a tripeptide segment (Phe-Leu-Gly). This tripeptide segment may be involved either directly (as a target sequence) or indirectly in the inhibitory effects of the compounds on virus-cell binding and fusion.

Comparison of Heterotypic Protection against Influenza A/Taiwan/86 (H1N1) by Attenuated and Inactivated Vaccines to A/Chile/83-like Viruses

Abstract: Children (n = 192) aged 3-19 years from 98 families completed this double-blind, placebo-controlled study comparing the efficacy of a bivalent attenuated (CR) vaccine with trivalent inactivated (TI) vaccine. Both vaccines contained A/Chile/83 (H1N1)-like antigens. After vaccination the geometric mean titer to A/Taiwan/86 (H1N1) was 1:36 in the CR group, 1:92 in the TI group, and 1:5 in the placebo group. During the influenza A/Taiwan/86 (H1N1) epidemic, 21.4% of CR recipients, 16.7% of TI recipients, and 43.9% of placebo recipients were infected with influenza A/Taiwan. TI vaccine provided better heterotypic protection than did CR vaccine for children aged 10-18 years (infection rate, 0 vs. 24%, respectively; P less than .025); in contrast, in the younger children (3-9 years), CR vaccine tended to be more protective (19% vs. 26% for TI).

Amantadine selection of a mutant influenza virus containing an acid-stable hemagglutinin glycoprotein: evidence for virus-specific regulation of the pH of glycoprotein transport vesicles

Abstract: Mutants of influenza Rostock virus (H7N1 subtype) were selected for resistance to amantadine hydrochloride at concentrations of the antiviral drug known to affect the function of the virus M2 transmembrane protein. Sequence analysis revealed that three mutants had no changes in M2 but contained a lysine to isoleucine substitution in the hemagglutinin (HA) membrane glycoprotein at position 58 of HA2. The mutant viruses were found to fuse membranes at a pH value 0.7 lower than wild type and to exhibit changes in the conformation of their HAs specifically at the lower pH. The homologous lysine to isoleucine substitution was introduced by site-specific mutagenesis into the HA of X-31 influenza virus (H3 subtype), which was expressed by using vaccinia virus recombinants. The expressed HA also mediated membrane fusion and changed in conformation at a pH value 0.7 lower than wild type. These results indicate that increased acid stability of the HA obviates the consequences of the inhibition of M2 function by amantadine and provide further evidence for the role of M2 in regulating the pH of vesicles involved in glycoprotein transport to the cell surface.

Influenza A virus infection of macrophages. Enhanced tumor necrosis factor-alpha (TNF-alpha) gene expression and lipopolysaccharide-triggered TNF-alpha release.

Abstract: We have previously shown that infection of macrophages by influenza A virus is capable of priming for a high TNF-alpha production in response to LPS. The present study was designed to examine in more detail TNF-alpha gene expression and TNF-alpha protein release of virus-infected, murine PU5-1.8 macrophages in the presence or absence of low and by itself rather inefficient concentrations of LPS (10 ng/ml). Although influenza A virus infection alone induced a massive TNF-alpha mRNA accumulation, translation into the bioactive TNF-alpha protein was low as intra- and extracellularly determined by bioassay, specific ELISA and Western blot. However, when LPS was added simultaneously or up to 4 h after infection, a high TNF-alpha production was initiated. The virus-induced TNF-alpha mRNA accumulation appeared to be due to both transcriptional and post-transcriptional changes: an enhanced TNF-alpha gene transcription as determined by nuclear run-on transcription assay and a markedly prolonged half-life of TNF-alpha mRNA as shown in actinomycin D-treated macrophages. These findings imply that influenza A virus may 1) either directly or indirectly stimulate TNF-alpha gene transcription activators or may interfere with labile transcription repressor proteins and 2) may stabilize TNF-alpha mRNA by delaying its degradation. Both mechanisms, taken together, prime influenza A virus-infected macrophages for a high TNF-alpha release in response to LPS which, as clinical cases show, may adversely affect patients with combined influenza A virus and bacterial infections.

Influences of Dietary Restriction on Immunity to Influenza in Aged Mice

Abstract: Our previous studies of the immune response of aged mice inoculated with influenza A virus revealed age-related decreases in antigen-specific cytotoxic T-lymphocyte function (CTL), T-cell proliferation, IL-2 production, antigen presentation, and antibody production. Because dietary restriction (DR) of rodents has been shown to extend maximum life span, delay the onset of tumors, and improve many immunologic parameters in aged animals, we tested the effect of such a regimen on the immune response to the influenza virus. We report that DR significantly inhibited the age-related decline in antigen presentation and T-cell proliferation. It also reduced the decline in antibody production to the virus. This is the first demonstration of improved immunity to an actual infectious agent resulting from DR. The improvement appears to be on a number of levels and to reflect more than one operative mechanism.

Antigenic and genetic variation in influenza A (H1N1) virus isolates recovered from a persistently infected immunodeficient child.

Abstract: Antigenic and genetic variations have been analyzed in eight consecutive isolates recovered from a child with severe combined immunodeficiency syndrome persistently infected with naturally acquired type A (H1N1) influenza virus over a 10-month period. Hemagglutination inhibition reactions and T1 oligonucleotide fingerprinting demonstrated that these viruses were related to strains causing outbreaks in the United States at that time (1983 to 1984) but that antigenic and genetic differences between consecutive isolates could be detected. This variation between isolates was examined further by sequencing the RNAs encoding the HA1 region of the hemagglutinin (HA) and the nucleoprotein (NP) in five of the consecutive isolates. Multiple point mutations were detected in both genes, and a deletion of one amino acid was detected in the HA. Depending on the isolates compared, 5.8 x 10(-3) to 17 x 10(-3) substitutions per nucleotide site per year were detected in the RNAs encoding the HA1, and 3.5 x 10(-3) to 24 x 10(-3) substitutions per nucleotide site per year were detected in the NP gene. Fifty-four percent of the base changes in the HA1 and 73% in the NP led to amino acid substitutions. A progressive accumulation of mutations over time was not observed, suggesting that the genetic diversity of these viruses may best be interpreted as the result of shifts in the population equilibrium (quasi-species) of replicating variant genomes.

Age distribution of patients with medically-attended illnesses caused by sequential variants of influenza A/H1N1 : comparison to age-specific infection rates, 1978-1989

Abstract: Since influenza A/H1N1 viruses reappeared during the 1977-1978 season, this subtype has contributed 27% of 6,609 documented influenza infections of persons with acute respiratory disease presenting to clinics serving as surveillance sites of the Influenza Research Center in Houston for the 12-year period ending June 1989. Wide differences in the distribution of H1N1 viruses occurred by age group: more than 50% of H1N1 infections were detected among persons aged 10-34 years, compared with 28% for influenza A/H3N2 and 35% for influenza B. Over age 35 years, the contribution of H1N1 viruses dropped to only 4%, compared with 20% and 16% for influenza A/H3N2 and influenza B, respectively. When birth dates of persons with positive cultures were examined, it was found that most of the H1N1-positive persons were born after 1950. Concurrently, longitudinal studies of families and other adults under intensive surveillance for infection, including cultures of all respiratory illnesses and tests for serum antibody rise over the respiratory disease season, revealed appreciable infection rates for adults born before 1950. Furthermore, the annual peak of hospitalization of older persons with pneumonia and other acute respiratory illnesses was significantly correlated with the peak of H1N1 virus activity in 1978-1979, a year when H1N1 viruses were the only influenza viruses prevalent. These observations indicate that many persons infected with influenza A/H1N1 viruses that circulated from 1946 through 1953 have immunity which has persisted for more than 25 years but this immunity is not complete. Reinfection that may result in serious illness in older vulnerable adults does occur but with lower frequency than with influenza A/H3N2 infection. Currently prevalent H1N1 variants are antigenically different from those that circulated in the 1950s; however, older adults readily acquire immunity to these new variants--perhaps as a result of immunologic priming that occurred in childhood.

Guillain-Barré Syndrome and Influenza Vaccination in the US Army, 1980–1988

Abstract: An increased incidence of Guillain-Barre syndrome (polyradiculoneuritis) occurred in individuals who received the A/New Jersey (swine) influenza vaccine in 1976-1977. A retrospective study encompassing the years 1980-1988 was conducted to determine if the US Army's mass influenza vaccination program has been associated with an increased incidence of Guillain-Barre syndrome in active duty soldiers during the study years. No temporally related increase in Guillain-Barre syndrome was found during the study years.

Bronchial Hyperresponsiveness in Normal Subjects during Attenuated Influenza Virus Infection

Abstract: Fourteen healthy male subjects with hemagglutination-inhibition antibody titers of 1:8 or less to homologous influenza A virus were studied. Six subjects received live, attenuated influenza virus by nasal drops and by aerosol. Although infection occurred in these six subjects, with the development of 4-fold or greater increases in hem agglutination-inhibit ion antibody titers, they remained asymptomatic. Eight subjects received placebo via the same route, and did not develop symptoms and showed no increase in antibody titer. Prior to administration of virus or placebo, histamine diphosphate aerosol increased airway resistance only slightly, and there was no difference between the virus and placebo groups. Two days after inoculation, bronchomotor responses in the placebo group were unchanged (p > 0.05), but in the virus-infected group, bronchomotor responses were significantly greater than in the preinfected state (p < 0.01). Isoproterenol hydro-chloride reversed and prevented the increase in airway resist...

Comparison of enzyme-linked immunosorbent assay, indirect immunofluorescence assay, and virus isolation for detection of respiratory viruses in nasopharyngeal secretions.

Abstract: Nasopharyngeal secretions obtained from 94 children with acute respiratory illness were examined for the presence of respiratory syncytial virus (RSV), adenovirus, and influenza virus type A by virus culturing (virus isolation technique [VIT]), immunofluorescence assay (IFA), and enzyme-linked immunosorbent assay (ELISA). Similar results were obtained in at least two tests for RSV, influenza virus type A, and adenovirus in 92 (97.9%), 88 (93.6%), and 88 (93.6%) cases, respectively. Both rapid virus detection methods showed good specificity for the diagnosis of these virus infections (greater than or equal to 90.7%) and were more sensitive than was VIT for RSV detection. In a more accurate statistical analysis, the indexes of agreement between VIT and ELISA were substantial for RSV (kappa = 0.69; zeta = 5.5; P less than 0.0001), influenza virus type A (kappa = 0.67; zeta = 5.3; P less than 0.0001), and adenovirus (kappa = 0.71; zeta = 6.0; P less than 0.0001), while it was almost perfect for RSV when ELISA was compared with IFA (kappa = 0.88; zeta = 5.7; P less than 0.0001). Although the observed agreement was good in the comparison of these two tests for these three viruses (89%0, the indexes of agreement were moderate in the comparison of IFA and VIT for RSV (K = 0.55; Z = 2.0; P

Imaging in influenza A encephalitis.

Abstract: Two cases of influenza A encephalitis seen during an outbreak of influenza types A/England/427/88 (H3N2) and A/Taiwan/1/86 (H1N1) in December 1989 are described. In both children the encephalitis developed within three days of the respiratory symptoms and both became comatose within 48 hours. Virological studies showed that the patients had had a recent influenza A infection. Symmetrical localised hypodense lesions within the thalami and pons were demonstrated in both cases on computed tomography of the brain and striking findings in the pons in one case on magnetic resonance imaging. Influenza A encephalitis is not easy to recognise clinically and serological confirmation can only be made after 10 days. Imaging may provide evidence in the acute stage to support a diagnosis of influenza encephalitis during influenza outbreaks.

Virulence of Rimantadine-Resistant Human Influenza A (H3N2) Viruses in Ferrets

Abstract: The influence of rimantadine-resistance mutations on the virulence of human H3N2 viruses in ferrets was examined. The similarities in virulence of the drug-resistant mutants with single amino acid substitutions at three different locations, 27, 30, and 31, within the M2 sequence and their corresponding sensitive wild-type isolates contrasted with differences in virulence between the three pairs of viruses. These data provide further evidence that rimantadine-resistant viruses that emerge during treatment of patients with the drug are unaltered both in their growth characteristics and virulence.

The chronicle of influenza epidemics.

Abstract: Epidemics that were probably influenza have been reported throughout recorded history. There were 13 fairly severe epidemics during the 18th century and 12 during the 19th century. Probably 8 of these 25 were influenza pandemic. In the 20th century there have been 4 pandemic (1918/19, 1957/58, 1968/69 and 1977) due to the emergence of new subtypes of influenza A virus. The great pandemic of 1918/19 caused an estimated 20 million deaths. Between pandemics usually there have been epidemics of varying severity at intervals of one to three years and a trickle of sporadic cases every winter. The morbidity and mortality rates have varied greatly from epidemic to epidemic and from place to place during the same epidemic. Generally the morbidity has been lowest in people over 60 years of age, but, except for 1918/19, the mortality has been predominantly in old people. The epidemic behaviour of influenza has been so erratic and difficult to understand that there are still a few scientists who consider that extraterrestrial influences operate. These views are not taken seriously by most virologists but there are puzzling aspects of influenza that are not yet understood.

In elderly persons live attenuated influenza A virus vaccines do not offer an advantage over inactivated virus vaccine in inducing serum or secretory antibodies or local immunologic memory.

Abstract: In a double-blind, randomized trial, 102 healthy elderly subjects were inoculated with one of four preparations: (i) intranasal bivalent live attenuated influenza vaccine containing cold-adapted A/Kawasaki/86 (H1N1) and cold-adapted A/Bethesda/85 (H3N2) viruses; (ii) parenteral trivalent inactivated subvirion vaccine containing A/Taiwan/86 (H1N1), A/Leningrad/86 (H3N2), and B/Ann Arbor/86 antigens; (iii) both vaccines; or (iv) placebo. To determine whether local or systemic immunization augmented mucosal immunologic memory, all volunteers were challenged intranasally 12 weeks later with the inactivated virus vaccine. We used a hemagglutination inhibition assay to measure antibodies in sera and a kinetic enzyme-linked immunosorbent assay to measure immunoglobulin G (IgG) and IgA antibodies in sera and nasal washes, respectively. In comparison with the live virus vaccine, the inactivated virus vaccine elicited higher and more frequent rises of serum antibodies, while nasal wash antibody responses were similar. The vaccine combination induced serum and local antibodies slightly more often than the inactivated vaccine alone did. Coadministration of live influenza A virus vaccine did not alter the serum antibody response to the influenza B virus component of the inactivated vaccine. The anamnestic nasal antibody response elicited by intranasal inactivated virus challenge did not differ in the live, inactivated, or combined vaccine groups from that observed in the placebo group not previously immunized. These results suggest that in elderly persons cold-adapted influenza A virus vaccines offer little advantage over inactivated virus vaccines in terms of inducing serum or secretory antibody or local immunological memory. Studies are needed to determine whether both vaccines in combination are more efficacious than inactivated vaccine alone in people in this age group.

Inhibition of influenza A virus hemagglutinin and induction of interferon by synthetic sialylated glycoconjugates.

Abstract: Multivalent forms of neoglycoproteins and polyacrylamides containing sialic acid were prepared and shown to be potent inhibitors of influenza A virus (H3N2) hemagglutinin with chick red blood cells...

The effect of restraint stress on the kinetics, magnitude, and isotype of the humoral immune response to influenza virus infection.

Abstract: The stress of physical restraint has been shown to modulate the cellular immune response during a viral infection. We have studied the effects of stress on the humoral immune response during infection by influenza virus. Restraint stress altered the kinetics of the antibody response; seroconversion in the IgG and IgA isotypes was delayed in virus-infected C57BL/6 mice subjected to repeated cycles of physical restraint. However, the magnitude and isotype of the mature antibody response were unaffected during the plateau phase; no significant differences were observed between restrained/infected and nonrestrained/infected mice. Thus, the time during infection at which the antibody response was measured was a significant variable in the study of stress-induced alterations of the host's response to a replicating viral antigen. While restraint stress did not significantly affect the magnitude or class of the humoral response, it did alter the kinetics of response.

Single amino acid substitutions in the hemagglutinin can alter the host range and receptor binding properties of H1 strains of influenza A virus.

Abstract: We have previously characterized an influenza A (H1N1) virus which has host-dependent growth and receptor binding properties and have shown that a mutation which removes an oligosaccharide from the tip of the hemagglutinin (HA) by changing Asn-129 to Asp permits this virus to grow to high titer in MDBK cells, (C. M. Deom, A. J. Caton, and I. T. Schulze, Proc. Natl. Acad. Sci. USA 83:3771-3775, 1986). We have now isolated monoclonal antibodies specific for the mutant HA and have used escape mutants to identify alterations in HA sequence which reduce virus yields from MDBK cells without reducing those from chicken embryo fibroblasts. Two types of escape mutants which grow equally well in chicken embryo fibroblasts were obtained. Those with the parent phenotype contain Asn at residue 129 and are glycosylated at that site. Those with the mutant phenotype are unchanged at residue 129 but have a Gly to Glu substitution at residue 158, which is close to residue 129 on the HA1 subunit. Binding assays with neoglycoproteins containing N-acetylneuraminic acid in either alpha 2,3 or alpha 2,6 linkage to galactose showed that the MDBK-synthesized oligosaccharides at Asn-129 reduce binding to both of these receptors, leaving the HA's preference for alpha 2,6 linkages unchanged. Glu at residue 158 greatly reduces binding to both receptors without reducing virus yields from MDBK cells. We conclude that changes in the receptor binding properties of the HA can result either from direct alteration of the HA protein by host cell glycosylation or from mutations in the HA gene and that these changes generate heterogeneity that can contribute to the survival of influenza A virus populations in nature.

Influenza A and the virus associated haemophagocytic syndrome: cluster of three cases in children with acute leukaemia.

Abstract: At the height of the United Kingdom influenza A epidemic in December 1989, three children receiving treatment for non-T cell acute leukaemia developed pancytopenia with concomitant influenza A infection. Bone marrow histology showed prominent marrow erythrophagocytosis by morphologically mature histiocytes, consistent with the picture of virus associated haemophagocytic syndrome (VAHS). In two cases there was an initial spontaneous recovery, though recurrence of VAHS developed in one case in association with a different viral infection (cytomegalovirus) following autologous bone marrow transplantation. The third child died from cardiorespiratory failure secondary to infection with influenza A and Klebsiella pneumoniae sepsis. It is suggested that influenza A should be added to the list of infective causative agents.

Identification of eight determinants in the hemagglutinin molecule of influenza virus A/PR/8/34 (H1N1) which are recognized by class II-restricted T cells from BALB/c mice.

Abstract: Eight nonoverlapping regions of the hemagglutinin (HA) molecule of influenza virus A/PR/8/34 (PR8), which serve as recognition sites for class II-restricted T cells (TH) from BALB/c mice, have been identified in the form of 10- to 15-amino-acid-long synthetic peptides. These TH determinants are located between residues 110 to 313 of the HA1 polypeptide. From a total of 36 HA-specific TH clones and limiting-dilution cultures of independent clonal origins, 33 (90%) responded to stimulation with one of these peptides. The residual three TH clones appeared to recognize a single additional determinant on the HA1 polypeptide which could not be isolated, however, in the form of a stimulatory peptide. None of the motifs that have been proposed to typify TH determinants were displayed by more than half of these recognition sites. Most unexpected was the finding that none of the TH determinants was located in the ectodomain of the HA2 polypeptide that makes up roughly one-third of the HA molecule. Possible reasons for the preferential recognition of HA1 as opposed to HA2 by TH are discussed.

The M2 protein of influenza A virus is acylated.

Abstract: The M2 protein of influenza A virus, a 97 amino acid integral membrane protein expressed on the surface of infected cells, is covalently modified with long chain fatty acids. The fatty acid bond is sensitive to treatment with neutral hydroxylamine and mercaptoethanol, which indicates a labile thioester type linkage. Thinlayer chromatographic fatty acid analysis of [3H]myristic and [3H]palmitic acid-labelled M2 protein shows that palmitic acid is the predominant fatty acid linked to this polypeptide. Palmitoylation of M2 occurs post-translationally and causes an upward shift in the SDS-PAGE mobility of the protein.

Human influenza virus hemagglutinin with high sensitivity to proteolytic activation.

Abstract: To examine the prerequisites for cleavage activation of the hemagglutinin of human influenza viruses, a cDNA clone obtained from strain A/Port Chalmers/1/73 (serotype H3) was subjected to site-directed mutagenesis and expressed in CV-1 cells by using a simian virus 40 vector. The number of basic residues at the cleavage site, which consists of a single arginine with wild-type hemagglutinin, was increased by inserting two, three, or four additional arginines. Like wild-type hemagglutinin, mutants with up to three additional arginines were not cleaved in CV-1 cells, but insertion of four arginines resulted in activation. When the oligosaccharide at asparagine 22 of the HA1 subunit of the hemagglutinin was removed by site-directed mutagenesis of the respective glycosylation site, only three inserted arginines were required to obtain cleavage. Mutants containing a series of four basic residues were also generated by substituting arginine for uncharged amino acids immediately preceding the cleavage site. The observation that these mutants were not cleaved, even when the carbohydrate at asparagine 22 of HA1 was absent, underscores the fact that the basic peptide had to be generated by insertion to obtain cleavage. The data show that the hemagglutinin of a human influenza virus can acquire high cleavability, a property known to be an important determinant for the pathogenicity of avian influenza viruses. Factors important for cleavability are the number of basic residues at the cleavage site, the oligosaccharide at asparagine 22, and the length of the carboxy terminus of HA1.