Evolutional analysis of human influenza A virus N2 neuraminidase genes based on the transition of the low-pH stability of sialidase activity

The 1957 and 1968 human pandemic influenza A virus strains as well as duck viruses possess sialidase activity under low-pH conditions, but human H3N2 strains isolated after 1968 do not possess such activity. We investigated the transition of avian (duck)-like low-pH stability of sialidase activities with the evolution of N2 neuraminidase (NA) genes in human influenza A virus strains. We found that the NA genes of H3N2 viruses isolated from 1971 to 1982 had evolved from the side branches of NA genes of H2N2 epidemic strains isolated in 1968 that were characterized by the low-pH-unstable sialidase activities, though the NA genes of the 1968 pandemic strains preserved the low-pH-stable sialidase. These findings suggest that the prototype of the H3N2 epidemic influenza strains isolated after 1968 probably acquired the NA gene from the H2N2 low-pH-unstable sialidase strain by second genetic reassortment in humans.     

Kinetic studies on the sialidase of three influenza B and three influenza A virus strains

Sialidase of influenza virus type A has been extensively studied through structural and kinetic approaches. However, sialidase of influenza virus type B has been less investigated. In this work, we have studied the activity and some properties (optimal pH, K_M, V_max, thermal stability) of sialidase in three influenza virus strains of type B (circulating in the period 1983–86) and also the activity and properties of sialidase from three virus strains of type A circulating at the same period of time.The results show that the activity and the V_max was always higher for sialidase of type A viruses relative to those values of type B. Differences were also found for optimal pH and, in some cases, for thermal stability of the sialidase between strains belonging to the influenza viruses type A and B. However, the behaviour for the sialidase in all strains was very similar towards two competitive inhibitors. Thus, it could be suggested that the evolution pattern of the sialidase of both types of influenza viruses determines some modifications which result in a higher efficiency for sialidase of some strains of influenza virus type A, but maintaining in the two types of viruses a similar behaviour towards competitive inhibitors.Dedicated to Professor Maurice Leclerc by one of us (J.A.C.) on the occasion of his retirement.     

Influenza A viruses lacking sialidase activity can undergo multiple cycles of replication in cell culture, eggs, or mice

Influenza A viruses possess both hemagglutinin (HA), which is responsible for binding to the terminal sialic acid of sialyloligosaccharides on the cell surface, and neuraminidase (NA), which contains sialidase activity that removes sialic acid from sialyloligosaccharides. Interplay between HA receptor-binding and NA receptor-destroying sialidase activity appears to be important for replication of the virus. Previous studies by others have shown that influenza A viruses lacking sialidase activity can undergo multiple cycles of replication if sialidase activity is provided exogenously. To investigate the sialidase requirement of influenza viruses further, we generated a series of sialidase-deficient mutants. Although their growth was less efficient than that of the parental NA-dependent virus, these viruses underwent multiple cycles of replication in cell culture, eggs, and mice. To understand the molecular basis of this viral growth adaptation in the absence of sialidase activity, we investigated changes in the HA receptor-binding affinity of the sialidase-deficient mutants. The results show that mutations around the HA receptor-binding pocket reduce the virus's affinity for cellular receptors, compensating for the loss of sialidase. Thus, sialidase activity is not absolutely required in the influenza A virus life cycle but appears to be necessary for efficient virus replication.     

Sialidase activity of influenza A virus in an endocytic pathway enhances viral replication

N2 neuraminidase (NA) genes of the 1957 and 1968 pandemic influenza virus strains possessed avian-like low-pH stability of sialidase activity, unlike most epidemic strains. We generated four reverse-genetics viruses from a genetic background of A/WSN/33 (H1N1) that included parental N2 NAs of 1968 pandemic (H3N2) and epidemic (H2N2) strains or their counterpart N2 NAs in which the low-pH stability of the sialidase activity was changed by substitutions of one or two amino acid residues. We found that the transfectant viruses bearing low-pH-stable sialidase (WSN/Stable-NAs) showed 25- to 80-times-greater ability to replicate in Madin-Darby canine kidney (MDCK) cells than did the transfectant viruses bearing low-pH-unstable sialidase (WSN/Unstable-NAs). Enzymatic activities of WSN/Stable-NAs were detected in endosomes of MDCK cells after 90 min of virus internalization by in situ fluorescent detection with 5-bromo-4-chloro-indole-3-yl-alpha-N-acetylneuraminic acid and Fast Red Violet LB. Inhibition of sialidase activity of WSN/Stable-NAs on the endocytic pathway by pretreatment with 4-guanidino-2,4-dideoxy-N-acetylneuraminic acid (zanamivir) resulted in a significant decrease in progeny viruses. In contrast, the enzymatic activities of WSN/Unstable-NAs, the replication of which had no effect on pretreatment with zanamivir, were undetectable in cells under the same conditions. Hemadsorption assays of transfectant-virus-infected cells revealed that the low-pH stability of the sialidase had no effect on the process of removal of sialic acid from hemagglutinin in the Golgi regions. Moreover, high titers of viruses were recovered from the lungs of mice infected with WSN/Stable-NAs on day 3 after intranasal inoculation, but WSN/Unstable-NAs were cleared from the lungs of the mice. These results indicate that sialidase activity in late endosome/lysosome traffic enhances influenza A virus replication.     

An O-glycoside of sialic acid derivative that inhibits both hemagglutinin and sialidase activities of influenza viruses

The compound Neu5Ac3alphaF-DSPE (4), in which the C-3 position was modified with an axial fluorine atom, inhibited the catalytic hydrolysis of influenza virus sialidase and the binding activity of hemagglutinin. The inhibitory activities to sialidases were independent of virus isolates examined. With the positive results obtained for inhibition of hemagglutination and hemolysis induced by A/Aichi/2/68 virus, the inhibitory effect of Neu5Ac3alphaF-DSPE (4) against MDCK cells was examined, and it was found that 4 inhibits the viral infection with IC50 value of 5.6 microM based on the cytopathic effects. The experimental results indicate that compound 4 not only inhibits the attachment of virus to the cell surface receptor but also disturbs the release of the progeny viruses from infected cells by inhibiting both hemagglutinin and sialidase of the influenza viruses. The study suggested that the compound is a new class of bifunctional drug candidates for the future chemotherapy of influenza.     

Duck and human pandemic influenza A viruses retain sialidase activity under low pH conditions

The majority of influenza A viruses isolated from wild birds, but not humans, can replicate in the duck intestinal tract. Here we demonstrate that all duck isolates tested universally retain sialidase activities under low pH conditions independent of their neuraminidase (NA) subtypes. In contrast, the sialidase activities of most isolates from humans and pigs practically disappear below pH 4.5, with the exception of four human pandemic viruses isolated in 1957 and 1968. Sequence comparisons among duck, human, and swine N2 NA subtypes indicate that amino acids at positions 153, 253, 307, 329, 344, 347, 356, 368, 390, and 431 may be associated with the low pH stability of duck and human pandemic N2 NAs. This finding suggests that the low pH stability of duck influenza A virus NA may be a critical factor for replication in the intestinal tract through the digestive tract of ducks, and that the properties of NAs are important for understanding the epidemiology of the influenza virus.     

Inhibition of sialidases from viral,bacterial and mammalian sources by analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid modified at the C-4 position

The inhibition of sialidase activity from influenza viruses A and B, parainfluenza 2 virus,Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, and sheep liver by a range of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified at the C-4 position has been studied. All substitutions tested resulted in a decrease in the degree of inhibition of the bacterial and mammalian sialidases. For sialidases from influenza viruses A and B, on the other hand, most of the substitutions tested either had no significant effect on binding or, in the case of the basic amino and guanidino substituents, resulted in significantly stronger inhibition. The results for parainfluenza 2 virus sialidase were mostly intermediate, in that inhibition was neither significantly increased nor decreased by most of the modifications. We conclude that only the influenza A and B sialidase active sites possess acid groups correctly positioned to participate in charge-charge interactions in the region of C-4 of bound substrate, and that the C-4 binding pockets of the bacterial and mammalian sialidases examined are considerably smaller than is observed for either the influenza virus or parainfluenza virus sialidases.This paper is dedicated to the memory of Professor Dr E. Zbiral.     

New ganglioside analogs that inhibit influenza virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac alpha(2-S-6)Glc beta(1-1)Ceramide (1) and the GM3 analog Neu5Ac alpha(2-S-6)Gal beta(1-4)Glc beta(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent Ki value of 2.8 x 10(-6) and 1.5 x 10(-5) M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac alpha(2-S-6)Gal beta(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (Ki = 1.0 x 10(-4) M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were non-hydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases from Clostridium perfringens and Arthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.     

Sulfatide is required for efficient replication of influenza A virus

Sulfatide is abundantly expressed in various mammalian organs, including the intestines and trachea, in which influenza A viruses (IAVs) replicate. However, the function of sulfatide in IAV infection remains unknown. Sulfatide is synthesized by two transferases, ceramide galactosyltransferase (CGT) and cerebroside sulfotransferase (CST), and is degraded by arylsulfatase A (ASA). In this study, we demonstrated that sulfatide enhanced IAV replication through efficient translocation of the newly synthesized IAV nucleoprotein (NP) from the nucleus to the cytoplasm, by using genetically produced cells in which sulfatide expression was down-regulated by RNA interference against CST mRNA or overexpression of the ASA gene and in which sulfatide expression was up-regulated by overexpression of both the CST and CGT genes. Treatment of IAV-infected cells with an antisulfatide monoclonal antibody (MAb) or an anti-hemagglutinin (HA) MAb, which blocks the binding of IAV and sulfatide, resulted in a significant reduction in IAV replication and accumulation of the viral NP in the nucleus. Furthermore, antisulfatide MAb protected mice against lethal challenge with pathogenic influenza A/WSN/33 (H1N1) virus. These results indicate that association of sulfatide with HA delivered to the cell surface induces translocation of the newly synthesized IAV ribonucleoprotein complexes from the nucleus to the cytoplasm. Our findings provide new insights into IAV replication and suggest new therapeutic strategies.     

The low-pH stability discovered in neuraminidase of 1918 pandemic influenza A virus enhances virus replication

The "Spanish" pandemic influenza A virus, which killed more than 20 million worldwide in 1918-19, is one of the serious pathogens in recorded history. Characterization of the 1918 pandemic virus reconstructed by reverse genetics showed that PB1, hemagglutinin (HA), and neuraminidase (NA) genes contributed to the viral replication and virulence of the 1918 pandemic influenza virus. However, the function of the NA gene has remained unknown. Here we show that the avian-like low-pH stability of sialidase activity discovered in the 1918 pandemic virus NA contributes to the viral replication efficiency. We found that deletion of Thr at position 435 or deletion of Gly at position 455 in the 1918 pandemic virus NA was related to the low-pH stability of the sialidase activity in the 1918 pandemic virus NA by comparison with the sequences of other human N1 NAs and sialidase activity of chimeric constructs. Both amino acids were located in or near the amino acid resides that were important for stabilization of the native tetramer structure in a low-pH condition like the N2 NAs of pandemic viruses that emerged in 1957 and 1968. Two reverse-genetic viruses were generated from a genetic background of A/WSN/33 (H1N1) that included low-pH-unstable N1 NA from A/USSR/92/77 (H1N1) and its counterpart N1 NA in which sialidase activity was converted to a low-pH-stable property by a deletion and substitutions of two amino acid residues at position 435 and 455 related to the low-pH stability of the sialidase activity in 1918 NA. The mutant virus that included "Spanish Flu"-like low-pH-stable NA showed remarkable replication in comparison with the mutant virus that included low-pH-unstable N1 NA. Our results suggest that the avian-like low-pH stability of sialidase activity in the 1918 pandemic virus NA contributes to the viral replication efficiency.     

Synthesis and anti-influenza evaluation of orally active bicyclic ether derivatives related to zanamivir

We synthesized bicyclic ether sialidase inhibitors such as tetrahydro-furan-2-yl, tetrahydro-pyran-2-yl, and oxepan-2-yl derivatives related to zanamivir. These compounds substituted by diol at the C-3' and C-4' positions resulted in the retention of low nanomolar inhibitory activities against not only influenza A virus sialidase but also influenza A virus in cell culture. Compound 11a in particular showed comparable efficacy in vivo relative to that of oseltamivir phosphate.     

The hemagglutinins of the human influenza viruses A and B recognize different receptor microdomains

A cryptically I-active sialylglycoprotein (glycoprotein 2) isolated from bovine erythrocyte membranes as Sendai virus receptor (Suzuki, Y., Suzuki, T. and Matsumoto, M. (1983) J. Biochem. 93, 1621-1633) contains N-glycolylneuraminic acid (NeuGc) as its predominate sialic acid and exhibits poor receptor activity for a variety of influenza viruses. Enzymatic modification of asialoglycoprotein-2 to contain N-acetylneuraminic acid (NeuAc) in the NeuAc alpha 2-3Gal and NeuAc alpha 2-6Gal sequences using specific sialyltransferase resulted in the appearance of receptor activity toward human influenza viruses A and B. The biological responsiveness chicken erythrocytes treated with sialidase and then reconstituted with derivatized glycoprotein 2 showed considerable recovery to influenza virus hemagglutinin-mediated agglutination, low-pH fusion and hemolysis. Specific hemagglutination inhibition activity of derivatized glycoprotein 2 was 5-16-times higher than that of human glycophorin. A/PR/8/34 (H1N1) virus preferentially recognized derivatized glycoprotein 2 containing NeuAc alpha 2-3Gal sequence over that containing NeuAc alpha 2-6Gal while the specificity of A/Aichi/2/68 (H3N2) for the sialyl linkages was reversed. B/Lee virus recognized both sequences almost equally. The biological responsiveness to the viruses of the erythrocytes labeled with the derivatized glycoprotein 2 containing NeuGc was considerably lower than that of derivatized glycoprotein 2 containing NeuAc. The results demonstrate that the hemagglutinins of human isolates of influenza viruses A and B differ in the recognition of microdomains (NeuAc, NeuGc) of the receptors for binding and fusion activities in viral penetration and the sequence to which sialic acid (SA) is attached (SA alpha 2-3Gal, SA alpha 2-6Gal). Inner I-active neolacto-series type II sugar chains may be important in revealing the receptor activity toward the hemagglutinin of both human influenza viruses A and B.     

Novel Pandemic Influenza A(H1N1) Viruses Are Potently Inhibited by DAS181, a Sialidase Fusion Protein

Background

The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses.

Methods and Findings

The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus.

Conclusions

The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.     

Synthesis and anti-influenza virus activity of 4-guanidino-7-substituted Neu5Ac2en derivatives

Substitution of 7-OH by small hydrophobic groups on zanamivir resulted in the retaining of low nanomolar inhibitory activities against not only influenza A virus sialidase but also influenza A virus in cell culture. These compounds were prepared by treatment of the corresponding 7-substituted sialic acids derived from 4-modified N-acetyl-D-mannosamine (ManNAc) using enzyme-catalyzed aldol condensation.     

Adaptation of influenza A viruses to cells expressing low levels of sialic acid leads to loss of neuraminidase activity

Influenza A viruses possess two virion surface proteins, hemagglutinin (HA) and neuraminidase (NA). The HA binds to sialyloligosaccharide viral receptors, while the NA removes sialic acids from the host cell and viral sialyloligosaccarides. Alterations of the HA occur during adaptation of influenza viruses to new host species, as in the 1957 and 1968 influenza pandemics. To gain a better understanding of the contributions of the HA and possibly the NA to this process, we generated cell lines expressing reduced levels of the influenza virus receptor determinant, sialic acid, by selecting Madin-Darby canine kidney cells resistant to a lectin specific for sialic acid linked to galactose by alpha(2-3) or alpha(2-6) linkages. One of these cell lines had less than 1/10 as much N-acetylneuraminic acid as its parent cell line. When serially passaged in this cell line, human H3N2 viruses lost sialidase activity due to a large internal deletion in the NA gene, without alteration of the HA gene. These findings indicate that NA mutations can contribute to the adaptation of influenza A virus to new host environments and hence may play a role in the transmission of virus across species.     

Human trachea primary epithelial cells express both sialyl(α2-3)Gal receptor for human parainfluenza virus type 1 and avian influenza viruses, and sialyl(α2-6)Gal receptor for human influenza viruses

We reported previously that the dominant receptors of influenza A and B viruses, and human and murine respiroviruses, were sialylglycoproteins and gangliosides containing monosialo-lactosamine type I-and II-residues, such as sialic acid-α2-3(6)-Galβ1-3(4)-GlcNAcβ1-. In addition, the Siaα2-3Gal linkage was predominantly recognized by avian and horse influenza viruses, and human parainfluenza virus type 1 (hPIV-1), whereas the Siaα2-6Gal linkage was mainly recognized by human influenza viruses (Paulson JC in “The Receptors' [Conn M Ed] 2, 131–219 (1985); Suzuki Y, Prog Lipid Res 33, 429–57 (1994); Ito T, J Virol 73, 6743–51 (2000); Suzuki Y, J Virol 74, 11825–31 (2000); Suzuki T, J. Virol 75, 4604–4613 (2001); Suzuki Y, Biol. Pharm. Bull. 28, 399–408 (2005)). To clarify the distribution of influenza virus receptors on the human bronchial epithelium cell surface, we investigated a primary culture of normal human bronchial epithelial (NHBE) cells using two types of lectin (MAA and SNA), which recognize sialyl linkages (α2-3 and α2-6), using fluorescence-activated cell-sorting analysis. The results showed that both α2-3- and α2-6-linked Sias were expressed on the surface of primary human bronchial epithelial cells. The cells infected by hPIV-1 bound to MAA, confirming that cells targeted by hPIV-1 have α2-3-linked oligosaccharides. We also compared the ability of hPIV-1 and human influenza A virus to infect primary human bronchial epithelial cells pre-treated with Siaα2-3Gal-specific sialidase from Salmonella typhimurium. No difference was observed in the number of sialidase pre-treated and non-treated cells infected with human influenza A virus, which binds to Siaα2-6Gal-linked oligosaccharides. By contrast, the number of cells infected with hPIV-1 decreased significantly upon sialidase treatment. Thus, cultured NHBE cells showed both α2-3-linked Sias recognized by hPIV-1 and avian influenza virus receptors, and α2-6-linked Sias recognized by human influenza virus receptors.     

Influenza B virus requires BM2 protein for replication

The BM2 protein of influenza B virus functions as an ion channel, which is suggested to be important for virus uncoating in endosomes of virus-infected cells. Because direct support for this function is lacking, whether BM2 plays an essential role in the viral life cycle remains unknown. We therefore attempted to generate BM2 knockout viruses by reverse genetics. Mutant viruses possessing M segments with the mutated initiation codon of BM2 protein at the stop-start pentanucleotide were viable and still expressed BM2. The introduction of multiple stop codons and a one-nucleotide deletion downstream of the stop-start pentanucleotide, in addition to disablement of the BM2 initiation codon, failed to generate viable mutant viruses, but the mutant M segments still expressed proteins that reacted with the BM2 peptide antiserum. To completely abolish BM2 expression, we generated a mutant M gene whose BM2 open reading frame was deleted. Although this mutant was not able to replicate in normal MDCK cells, it did replicate in a cell line that we established which constitutively expresses BM2. Furthermore, a virus possessing the mutant M gene lacking the BM2 open reading frame and a mutant NA gene containing the BM2 open reading frame instead of the NA open reading frame underwent multiple cycles of replication in MDCK cells, with exogenous sialidase used to supplement the deleted viral sialidase activity. These findings demonstrate that the BM2 protein is essential for influenza B virus replication.     

Inhibition of mouse liver sialidase by plant flavonoids

Flavonoids (103 species) were tested for inhibitory activity against mouse liver sialidase using sodium p-nitrophenyl-N-acetyl-alpha-D-neuraminate (PNP-NeuAc) as substrate. Isoscutellarein-8-O-glucuronide from the leaf of Scutellaria baicalensis showed most potent activity (IC50, 40 microM), and this flavone appeared to be a non-competitive inhibitor of the enzyme. This flavone inhibited the lysosomal solubilized sialidase against PNP-NeuAc and sialyllactose effectively, but not microsomal enzyme against gangliosides and colominic acid, whereas, negligible or weak inhibitory activities were observed for influenza virus sialidase, beta-galactosidase, alpha-mannosidase, and alpha-glucosidase tested. These results indicate that this flavone may be useful to elucidate the function of the lysosomal solubilized sialidase.     

Relationship between mitogenic activity of influenza viruses and the receptor-binding specificity of their hemagglutinin molecules

The relationship between the mitogenic activity of influenza type A viruses for murine B lymphocytes and the receptor-binding specificity of their hemagglutinin was examined. Receptor-binding specificity was determined by the ability of the virus to agglutinate erythrocytes that had been sialidase treated and then enzymatically resialylated to contain sialyloligosaccharides with defined sequences. Distinct differences in receptor-binding specificity were observed between strongly and weakly mitogenic viruses of the H3 subtype, with strong mitogenic activity correlating with the ability of the virus to recognize the sequence N-glycolylneuraminic acid alpha 2,6 galactose (NeuGc alpha 2,6Gal). Viruses isolated early in the evolution of the H3 subtype (from 1968 to 1971) are relatively weak mitogens and recognize the sequence N-acetylneuraminic acid alpha 2,6 galactose (NeuAc alpha 2,6Gal) but not NeuGc alpha 2,6Gal. H3 viruses isolated since 1972 are strongly mitogenic, and these viruses recognize both NeuGc alpha 2,6Gal and NeuAc alpha 2,6Gal. The amino acid substitution of Tyr for Thr at residue 155 of HA1 may be critical to this change in receptor-binding specificity and mitogenic activity of the later H3 viruses. Horse serum-resistant variants of H3 viruses, which bind preferentially to the sequence NeuAc alpha 2,3Gal, are poorly mitogenic. Differences were also observed between the receptor-binding specificity of the strongly mitogenic H3 viruses and viruses of the H2 and H6 subtypes, the mitogenic activity of which is limited to strains of mice that express the class II major histocompatibility complex glycoprotein I-E. The results indicate that the receptor-binding specificity of the hemagglutinin plays a critical role in determining the mitogenic activity of influenza viruses.