Chemical action of influenza virus on the red blood cell membrane. II. Action on membrane lipids and amino acids.

Haemagglutination by influenza virus strains PR8 and Lee as well as treatment of red blood cells by periodate produced numerous and manifold changes in the lipids and the amino acid composition of the erythrocytes. The changes induced by the influenza strains differed from each other and from those induced by periodate. Alterations in the membrane lipids observed after haemagglutination by heat treated strain PR8 and by its native form were identical.     

An immunoprecipitin study of the incidence of influenza A antibodies in animal sera in the Ottawa area.

A survey of over 600 'normal' sera from 14 animal species by immunoprecipitin tests in cellulose acetate using viron antigens revealed a high incidence of precipitating activity against a broad range of influenza A virus strains, particularly A2hHong Kong/1/68 and /PR8. However, serum treatments trypsin-heat-periodate, NaIO4, V. cholerae receptor-destroying enzyme (RDE), or kaolin eliminated most precipitating activity, which suggests that it was due to "non-specific" inhibitors of influenze viruses. A resistant minority could not be identified as inhibitor or antibody on this basis. Precipitation of the influenza A major type-specific antigen in virus-soluble antigens by human 7S gamma globulin antibody (IgG), demonstrated to be specific for influenza virus, was established as a reference reaction to identify similar immunoprecipitin reactions occurring between virus-soluble antigens and normal or immune sera. Complement fixation tests provided supplementary evidence for the presence of influenza A antibodies in these sera. Influenza A antibodies were found in only a few sera of six animal species: cat, dog, rabbit, goat, chipmunk, and sheep. Thus the animal species examined in the Ottawa area have not revealed an unequivocal reservoir for human influenza A viruses.     

Properties of the erythrocyte receptors for influenza C virus.

Properties of the receptor for influenza C virus were studied. Although the receptor for influenza C virus on chicken erythrocytes was destroyed by the homologous virion, neuraminidase activity could not be detected in any of the influenza C virus strains tested. The receptor activity of chicken erythrocytes for influenza C virus was diminished by formaldehyde treatment but not by periodate oxidation. There was a considerable variation in the pattern and the titer of hemagglutination of influenza C virus when human erythrocytes of different blood types were used; the virus agglutinated most type B erythrocytes but not type A erythrocytes. By using human type B erythrocytes, differences among strains of influenza C virus in the hemagglutinating activity were also demonstrated. These results showed that both the receptor for and the receptor-destroying activity of influenza C virus were completely different from those of influenza A or B virus and also that carbohydrates were not involved in the receptor for influenza C virus.     

Substituted Sialic Acid Prosthetic Groups as Determinants of Viral Hemagglutination

Inhibitors of hemagglutination by type A2 influenza virus and a recently isolated strain of type B influenza virus were separated by sucrose density gradient centrifugation and agarose gel filtration from horse serum. Using selected reagents, it was demonstrated that the active substituent on the horse serum inhibitor of A2 influenza virus was 4-O-acetyl-N-acetylneuraminic acid; however, the active substituent on the inhibitor of the influenza B virus was shown to be N-acetylneuraminic acid (NANA). Sodium metaperiodate treatment of a component of horse serum resulted in a 10 to 15-fold enhancement of inhibitory activity against the type B virus, whereas the A2 inhibitor was completely destroyed. Since this enhancement did not occur with influenza B viruses isolated prior to 1965, it was considered that this sensitivity to an oxidized NANA glycoside may have been a reflection of an antigenic change which occurred at that time. The use of different virus strains and selected chemical reagents to define the important sialic acid prosthetic groups active in inhibition was described.     

Peculiarities of influenza infection caused by highly and poorly immunogenic strains of influenza virus

The authors studied the peculiarities of the course of experimental influenza infection induced by the administration of highly and poorly immunogenic strains of influenza virus to mice. Influenza viruses with varying immunogenic activity were obtained from the vaccine strain A/Victoria/35/72/50 (H3N2) by immunoselection modelling the process of natural selection. The administration of strains with high and poor immunogenicity to mice of the F1 (CBA X C57B1) line led to the development of acute influenza infection accompanied by reproduction of viruses in the tissue of the lungs and other internal organs. The poorly immunogenic strain 5/II-Victoria, unlike the initial virus A/Victoria/35 and its highly immunogenic variant 2/I-Victoria, is able to circulate for a long time in the organism of the infected animal causing development of chronic inflammatory processes and stimulating the formation of neoplasms. Immunogenicity is thus one of the factors determining the character of the course of experimental influenza infection. A conclusion was drawn concerning the epidemiological and aetiological importance of viruses with reduced immunogenicity and their role in the evolution of influenza virus. It is presumed that reduced immunogenicity is one of the adaptation mechanisms of aggression permitting the population of influenza virus to escape control by specific humoral immunity.     

The NF‐κB inhibitor SC75741 efficiently blocks influenza virus propagation and confers a high barrier for development of viral resistance

Ongoing human infections with highly pathogenic avian H5N1 viruses and the emergence of the pandemic swine‐origin influenza viruses (IV) highlight the permanent threat elicited by these pathogens. Occurrence of resistant seasonal and pandemic strains against the currently licensed antiviral medications points to the urgent need for new and amply available anti‐influenza drugs. The recently identified virus‐supportive function of the cellular IKK/NF‐κB signalling pathway suggests this signalling module as a potential target for antiviral intervention. We characterized the NF‐κB inhibitor SC75741 as a broad and efficient blocker of IV replication in non‐toxic concentrations. The underlying molecular mechanism of SC75741 action involves impaired DNA binding of the NF‐κB subunit p65, resulting in reduced expression of cytokines, chemokines, and pro‐apoptotic factors, subsequent inhibition of caspase activation and block of caspase‐mediated nuclear export of viralribonucleoproteins. SC75741 reduces viral replication and H5N1‐induced IL‐6 and IP‐10 expression in the lung of infected mice. Besides its virustatic effect the drug suppresses virus‐induced overproduction of cytokines and chemokines, suggesting that it might prevent hypercytokinemia that is discussed to be an important pathogenicity determinant of highly pathogenic IV. Importantly the drug exhibits a high barrier for development of resistant virus variants. Thus, SC75741‐derived drugs may serve as broadly non‐toxic anti‐influenza agents.     

The DBA.2 mouse is susceptible to disease following infection with a broad, but limited, range of influenza A and B viruses

We assessed the relative susceptibilities to disease of the DBA.2 and C57BL/6 mouse models upon infection with a range of influenza A and B viruses. DBA.2 mice were more susceptible to disease upon inoculation with human H1N1 influenza A virus strains, several swine influenza viruses, and influenza B viruses but were not overtly susceptible to infection with human seasonal H3N2 strains. Hemagglutination inhibition and immunoglobulin isotype profiling indicated that DBA.2 and C57BL/6 mice generate comparable humoral responses upon equivalent 50% mouse lethal dose (MLD(50)) challenges with influenza virus. Our data demonstrate the utility of DBA.2 mice for the elucidation of influenza virus pathogenicity determinants and the testing of influenza vaccines.     

Prediction of selective inhibition of neuraminidase from various influenza virus strains by potential inhibitors

A universal model of inhibition of neuraminidases from various influenza virus strains by a particular inhibitor has been developed. It is based on known 3D structures for neuraminidases from three influenza virus strains (A/Tokyo/3/67, A/tern/Australia/G70C/75, B/Lee/40) and modeling of the 3D structure of neuraminidases from other strains (A/PR/8/34 and A/Aichi/2/68). Using docking and molecular dynamics, we have modeled 235 enzyme-ligand complexes for 185 compounds with known IC₅₀ values. Selection of final variants among three intermediate results obtained for each enzyme-ligand pair and calculation of independent variables for generation of linear regression equations were performed using MM-PBSA/MM-GBSA. This resulted in the set of equations for individual strains and the equations pooling all the data. Thus using this approach it is possible to predict inhibition for neuraminidase from each the considered strains by a particular inhibitor and to predict the range of its action on neuraminidases from various influenza virus strains.     

Human CD8+ and CD4+ T lymphocyte memory to influenza A viruses of swine and avian species.

Recently, an avian influenza A virus (A/Hong Kong/156/97, H5N1) was isolated from a young child who had a fatal influenza illness. All eight RNA segments were of avian origin. The H5 hemagglutinin is not recognized by neutralizing Abs present in humans as a result of infection with the human H1, H2, or H3 subtypes of influenza A viruses. Subsequently, five other deaths and several more human infections in Hong Kong were associated with this avian-derived virus. We investigated whether influenza A-specific human CD8+ and CD4+ T lymphocytes would recognize epitopes on influenza A virus strains derived from swine or avian species, including the 1997 H5N1 Hong Kong virus strains. Our results demonstrate that adults living in an urban area of the U.S. possess influenza A cross-serotype reactive CD8+ and CD4+ CTL that recognize multiple epitopes on influenza A viruses of other species. Bulk culture cytotoxicity was demonstrated against avian and human influenza A viruses. Enzyme-linked immunospot assays detected precursor CTL specific for both human CTL epitopes and the corresponding A/HK/97 viral sequences. We hypothesize that these cross-reactive CTL might provide partial protection to humans against novel influenza A virus strains introduced into humans from other species.     

Distinct glycoprotein inhibitors of influenza A virus in different animal sera.

Normal horse and guinea pig sera contain the glycoprotein inhibitor alpha 2-macroglobulin, which inhibits the infectivity and hemagglutinating activity of influenza A viruses of the H2 and H3 subtypes. In the current study, the presence of inhibitors of influenza A virus in pig and rabbit sera was investigated. Variants of influenza virus type A/Los Angeles/2/87(H3N2) that were resistant to horse, pig, or rabbit serum were isolated. Analysis of the variant viruses with anti-hemagglutinin (HA) monoclonal antibodies revealed that antigenic changes occurred with the development of serum inhibitor resistance. Characterization of the inhibitors in pig and rabbit sera by using periodate and receptor-destroying enzyme demonstrated that carbohydrate is an important constituent of the active portion of both inhibitor molecules and that sialic acid is involved in the interaction of the inhibitors with influenza virus HA. Nucleotide sequence analysis of the HA molecule revealed that the serum-resistant variants each acquired a different set of amino acid alterations. The multiply resistant variants maintained the original amino acid changes and acquired additional changes. Sequence modifications in the HA involved the conserved amino acids within the receptor binding site (RBS) at position 137 and the second-shell RBS residues at positions 155 and 186. Amino acid changes also occurred within antigenic site A (position 145) and directly behind the receptor binding pocket (position 220). Amino acid alterations resulted in the acquisition of a potential glycosylation site at position 128 and the loss of potential glycosylation sites at positions 246 and 248. The localization of the amino acid changes in HA1 to the region of the RBS supports the concept of serum inhibitors as receptor analogs. The unique set of mutations acquired by the serum inhibitor-resistant variants strongly suggests that horse, pig, and rabbit sera each contain distinct glycoprotein inhibitors of influenza A virus.     

Vaccination with M2e-based multiple antigenic peptides: characterization of the B cell response and protection efficacy in inbred and outbred mice

Background

The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains.

Methodology/Principal Findings

Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs.

Conclusion/Significance

Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population.     

Andrographolide inhibits influenza A virus-induced inflammation in a murine model through NF-κB and JAK-STAT signaling pathway

Influenza viruses, the main cause of respiratory tract diseases, cause high morbidity and mortality in humans. Excessive inflammation in the lungs is proposed to be a hallmark for the severe influenza virus infection, especially influenza A virus infection. Strategies against inflammation induced by influenza A virus infection could be a potential anti-influenza therapy. Here, lethal dose of mouse-adapted H1N1 strain PR8A/PR/8/34 was inoculated C57BL/6 mice to detect the anti-influenza activity of andrographolide, the active component of traditional Chinese medicinal herb Andrographis paniculata, with or without influenza virus entry inhibitor CL-385319. Treatment was initiated on 4 days after infection. The survival rate, body weight, lung pathology, viral loads, cytokine expression were monitored in 14 days post inoculation. The combination group had the highest survival rate. Andrographolide treatment could increase the survival rate, diminish lung pathology, decrease the virus loads and the inflammatory cytokines expression induced by infection. Mechanism studies showed the NF-κB and JAK-STAT signaling pathway were involved in the activity of andrographolide. In conclusion, combination of virus entry inhibitor with immunomodulator might be a promising therapeutic approach for influenza.     

Efficacy of the New Neuraminidase Inhibitor CS-8958 against H5N1 Influenza Viruses

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 µg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants.     

Characterization of the 1918 "Spanish" influenza virus matrix gene segment

The coding region of influenza A virus RNA segment 7 from the 1918 pandemic virus, consisting of the open reading frames of the two matrix genes M1 and M2, has been sequenced. While this segment is highly conserved among influenza virus strains, the 1918 sequence does not match any previously sequenced influenza virus strains. The 1918 sequence matches the consensus over the M1 RNA-binding domains and nuclear localization signal and the highly conserved transmembrane domain of M2. Amino acid changes that correlate with high yield and pathogenicity in animal models were not found in the 1918 strain. Phylogenetic analyses suggest that both genes were mammalian adapted and that the 1918 sequence is very similar to the common ancestor of all subsequent human and classical swine matrix segments. The 1918 sequence matches other mammalian strains at 4 amino acids in the extracellular domain of M2 that differ consistently between avian and mammalian strains, suggesting that the matrix segment may have been circulating in human strains for at least several years before 1918.     

Abnormal humoral immune response to influenza vaccination in pediatric type-1 human immunodeficiency virus infected patients receiving highly active antiretroviral therapy

Given that highly active antiretroviral therapy (HAART) has been demonstrated useful to restore immune competence in type-1 human immunodeficiency virus (HIV-1)-infected subjects, we evaluated the specific antibody response to influenza vaccine in a cohort of HIV-1-infected children on HAART so as to analyze the quality of this immune response in patients under antiretroviral therapy. Sixteen HIV-1-infected children and 10 HIV-1 seronegative controls were immunized with a commercially available trivalent inactivated influenza vaccine containing the strains A/H1N1, A/H3N2, and B. Serum hemagglutinin inhibition (HI) antibody titers were determined for the three viral strains at the time of vaccination and 1 month later. Immunization induced a significantly increased humoral response against the three influenza virus strains in controls, and only against A/H3N2 in HIV-1-infected children. The comparison of post-vaccination HI titers between HIV-1+ patients and HIV-1 negative controls showed significantly higher HI titers against the three strains in controls. In addition, post vaccination protective HI titers (defined as equal to or higher than 1:40) against the strains A/H3N2 and B were observed in a lower proportion of HIV-1+ children than in controls, while a similar proportion of individuals from each group achieved protective HI titers against the A/H1N1 strain. The CD4+ T cell count, CD4/CD8 T cells ratio, and serum viral load were not affected by influenza virus vaccination when pre- vs post-vaccination values were compared. These findings suggest that despite the fact that HAART is efficient in controlling HIV-1 replication and in increasing CD4+ T cell count in HIV-1-infected children, restoration of immune competence and response to cognate antigens remain incomplete, indicating that additional therapeutic strategies are required to achieve a full reconstitution of immune functions.     

Mast cell-induced lung injury in mice infected with H5N1 influenza virus

Although an important role for mast cells in several viral infections has been demonstrated, its role in the invasion of highly pathogenic H5N1 influenza virus is unknown. In the present study, we demonstrate that mast cells were activated significantly by H5N1 virus (A/chicken/Henan/1/2004) infection both in vivo and in vitro. Mast cells could possibly intensify the lung injury that results from H5N1 infection by releasing proinflammatory mediators, including histamine, tryptase, and gamma interferon (IFN-γ). Lung lesions and apoptosis induced by H5N1 infection were reduced dramatically by treatment with ketotifen, which is a mast cell degranulation inhibitor. A combination of ketotifen and the neuraminidase inhibitor oseltamivir protected 100% of the mice from death postinfection. In conclusion, our data suggest that mast cells play a crucial role in the early stages of H5N1 influenza virus infection and provide a new approach to combat highly pathogenic influenza virus infection.     

Molecular Mechanisms of Serum Resistance of Human Influenza H3N2 Virus and Their Involvement in Virus Adaptation in a New Host

H3N2 human influenza viruses that are resistant to horse, pig, or rabbit serum possess unique amino acid mutations in their hemagglutinin (HA) protein. To determine the molecular mechanisms of this resistance, we characterized the receptor-binding properties of these mutants by measuring their affinity for total serum protein inhibitors and for soluble receptor analogs. Pig serum-resistant variants displayed a markedly decreased affinity for total pig serum sialylglycoproteins (which contain predominantly 2-6 linkage between sialic acid and galactose residues) and for the sialyloligosaccharide 6′-sialyl(N-acetyllactosamine). These properties correlated with the substitution 186S→I in HA1. The major inhibitory activity in rabbit serum was found to be a β inhibitor with characteristics of mannose-binding lectins. Rabbit serum-resistant variants exhibited decreased sensitivity to this inhibitor due to the loss of a glycosylation sequon at positions 246 to 248 of the HA. In addition to a somewhat reduced affinity for 6′-sialyl(N-acetyllactosamine)-containing receptors, horse serum-resistant variants lost the ability to bind the viral neuraminidase-resistant 4-O-acetylated sialic acid moieties of equine α₂-macroglobulin because of the mutation 145N→K/D in their HA1. These results indicate that influenza viruses become resistant to serum inhibitors because their affinity for these inhibitors is reduced. To determine whether natural inhibitors play a role in viral evolution during interspecies transmission, we compared the receptor-binding properties of H3N8 avian and equine viruses, including two strains isolated during the 1989 to 1990 equine influenza outbreak, which was caused by an avian virus in China. Avian strains bound 4-O-acetylated sialic acid residues of equine α₂-macroglobulin, whereas equine strains did not. The earliest avian-like isolate from a horse influenza outbreak bound to this sialic acid with an affinity similar to that of avian viruses; a later isolate, however, displayed binding properties more similar to those of classical equine strains. These data suggest that the neuraminidase-resistant sialylglycoconjugates present in horses exert selective pressure on the receptor-binding properties of avian virus HA after its introduction into this host.Influenza A viruses possess two envelope glycoproteins:hemagglutinin (HA) and neuraminidase (NA). HA binds to cell surface sialylglycoconjugates and mediates virus attachment to target cells (19, 30). NA cleaves the α-glycosidic linkage between sialic acid and an adjacent sugar residue, facilitating elution of virus progeny from infected cells and preventing self-aggregation of the virus (1, 13). Natural sialylglycoconjugates are structurally diverse (37, 40), and the preferential recognition of distinct sialyloligosaccharides by HA and NA correlates with the host species from which the viruses are isolated (reviewed in references 19, 30, and 38; see also references 4, 6, 7, 11, and 28).The receptor-binding activity of influenza viruses can be inhibited by certain molecules present in the sera and fluid secretions of animals (see references 14 and 21 for reviews). These inhibitors are classified as α, β, and γ types based on their thermal stability, virus-neutralizing activity, and sensitivity to inactivation by NA and periodate treatments. The β inhibitors are thermolabile mannose-binding lectins that interact with the oligosaccharide moieties on viral glycoproteins. They neutralize virus by steric hindrance of HA and by activation of the complement-dependent pathway (2, 3). By contrast, the α and γ inhibitors are heat-stable sialylated glycoproteins that mimic the structure of the cellular receptors of influenza viruses and competitively block the receptor-binding sites of HA. Influenza viruses are neutralized by γ inhibitors but not by α inhibitors, which are considered to be sensitive to viral NA. However, the distinction between α and γ inhibitors is strain dependent and rather arbitrary, as described by Gottschalk et al. (14). Although inhibitors in serum or other body fluids are believed to influence the selection of influenza virus receptor variants in natural hosts, no direct experimental support for this hypothesis has been presented.A potent γ inhibitor of H2 and H3 human influenza viruses, equine α₂-macroglobulin (EM), contains a Neu4,5Ac₂2-6Gal moiety that is insensitive to viral NA and thus resists inactivation by this enzyme (16, 24, 31). Cultivation of human H3 influenza viruses in the presence of horse serum results in the selection of variants that have a decreased affinity for the Neu5Ac2-6Gal-specific receptors due to a single amino acid substitution (226L→Q) in their HA (32, 33). One of these mutants (X31/HS strain) does not bind the Neu4,5Ac₂ (4-O-acetylated sialic acid) species (25). Therefore, there are at least two mechanisms by which a virus can become resistant to the horse serum inhibitor: a change in the recognition of the type of Sia-Gal linkage, and a change in the recognition of the 4-O-acetylated sialic acid. The relative contributions of these mechanisms to the resistant phenotype are yet to be defined.We have previously shown that horse, pig, and rabbit sera all contain distinct heat-resistant inhibitors of the H3N2 human influenza virus A/Los Angeles/2/87 (LA/87), because variants resistant to these sera possess unique mutations in their HA receptor-binding regions (34). The major inhibitor in pig serum was later identified as α₂-macroglobulin that contains predominantly 2-6 linkage between sialic acid and galactose (35). Gimsa et al. (12) recently showed that pig serum-resistant human and swine strains exhibit decreased affinity for human erythrocytes that had been modified to contain terminal Neu5Ac2-6Gal residues. However, the nature of the rabbit serum inhibitor and the mechanisms of influenza virus resistance to each serum inhibitor remain unknown.To understand the molecular mechanisms by which influenza viruses become resistant to horse, pig, and rabbit serum inhibitors, we compared the receptor-binding characteristics of LA/87 and its serum-resistant variants and analyzed these data in relation to the known amino acid substitutions in the HA of the mutants. We then analyzed the receptor-binding properties of viruses isolated during an equine influenza outbreak that was caused by an avian virus, in order to evaluate the influence of natural inhibitors on the evolution of virus in a new host.     

Synthesis of potent pyrrolidine influenza neuraminidase inhibitors

The synthesis of several pyrrolidine inhibitor analogs is described that possess nanomolar in vitro potencies against the neuraminidase enzymes expressed by the B/Memphis/3/89 and A/N1/PR/8/34 influenza strains.     

Involvement of the mannose receptor in infection of macrophages by influenza virus

Influenza viruses A/PR/8/34 (PR8; H1N1), A/Aichi/68 X-31 (HKx31; H3N2), and A/Beijing/89 X-109 (BJx109; H3N2) show marked differences in their ability to infect murine macrophages, including resident alveolar and peritoneal macrophages as well as the macrophage-derived cell line J774. The hierarchy in infectivity of the viruses (PR8 < HKx31 < BJx109) resembles that of their reactivity with mannose-binding lectins of the collectin family. Since the macrophage mannose receptor recognizes the same spectrum of monosaccharides as the collectins do, we investigated the possible involvement of this receptor in infection of macrophages by influenza virus. In competitive binding studies, the binding of (125)I-labeled mannosylated bovine serum albumin to macrophages was inhibited by the purified hemagglutinin and neuraminidase (HANA) glycoproteins of influenza virus but not by HANA that had been treated with periodate to oxidize its oligosaccharide side chains. The inhibitory activity of HANA from the three strains of virus differed markedly and correlated with the infectivity of each virus for macrophages. Infection of macrophages, but not MDCK cells, by influenza virus was inhibited by yeast mannan. A variant line of J774 cells, J774E, which expresses elevated levels of the mannose receptor, was more readily infected than J774, and the sensitivity of J774E cells to infection was greatly reduced by culture in the presence of D-mannose, which down-modulated mannose receptor expression. Together, the data implicate the mannose receptor as a major endocytic receptor in the infectious entry of influenza virus, and perhaps other enveloped viruses, into murine macrophages.     

Comparative analyses of pandemic H1N1 and seasonal H1N1, H3N2, and influenza B infections depict distinct clinical pictures in ferrets

Influenza A and B infections are a worldwide health concern to both humans and animals. High genetic evolution rates of the influenza virus allow the constant emergence of new strains and cause illness variation. Since human influenza infections are often complicated by secondary factors such as age and underlying medical conditions, strain or subtype specific clinical features are difficult to assess. Here we infected ferrets with 13 currently circulating influenza strains (including strains of pandemic 2009 H1N1 [H1N1pdm] and seasonal A/H1N1, A/H3N2, and B viruses). The clinical parameters were measured daily for 14 days in stable environmental conditions to compare clinical characteristics. We found that H1N1pdm strains had a more severe physiological impact than all season strains where pandemic A/California/07/2009 was the most clinically pathogenic pandemic strain. The most serious illness among seasonal A/H1N1 and A/H3N2 groups was caused by A/Solomon Islands/03/2006 and A/Perth/16/2009, respectively. Among the 13 studied strains, B/Hubei-Wujiagang/158/2009 presented the mildest clinical symptoms. We have also discovered that disease severity (by clinical illness and histopathology) correlated with influenza specific antibody response but not viral replication in the upper respiratory tract. H1N1pdm induced the highest and most rapid antibody response followed by seasonal A/H3N2, seasonal A/H1N1 and seasonal influenza B (with B/Hubei-Wujiagang/158/2009 inducing the weakest response). Our study is the first to compare the clinical features of multiple circulating influenza strains in ferrets. These findings will help to characterize the clinical pictures of specific influenza strains as well as give insights into the development and administration of appropriate influenza therapeutics.