- Email: [email protected]
Hemagglutinin and neuraminidase as determinants of influenza virus pathogenicity
International Congress Series 1219 (2001) 533 – 543
Hemagglutinin and neuraminidase as determinants of inf luenza virus pathogenicity Ralf Wagner a, Anke Feldmanna, Thorsten Wolff a, Stephan Pleschkab, Wolfgang Gartena, Hans-Dieter Klenka,* a
Institut fu¨r Virologie, Klinikum der Philipps-Universita¨t Marburg, Robert-Koch-Str. 17, 35037 Marburg, Germany b Institut fu¨r Virologie, Justus-Liebig-Universita¨t Giessen, Germany
Keywords: Pathogenicity; Fowl plague virus; Receptor binding specificity; Neurominidase activity
The pathogenicity of influenza viruses is determined by many of its other biological properties, such as efficiency of replication, tissue tropism, host range, spread of infection, as well as response to and modulation of host defense. All of these properties are controlled by the complex interplay of viral and host factors at virtually each stage in the life cycle of the virus. Activation of the hemagglutinin by host cell proteases has been shown in many studies to have a dramatic effect on pathogenesis, and the molecular details of proteolytic activation are well understood [16a]. We will concentrate here on recent studies in which we have analyzed the interplay of hemagglutinin (HA) and neuraminidase (NA) in receptor binding and release and some of the factors that determine spread of infection in the organism.
1. Balance of receptor binding and release regulates virus growth HA-mediated attachment of influenza viruses to sialic acid containing receptors on the host cell surface is the initial step of infection. Influenza HA contains at the tip a narrow crevice lined with highly conserved amino acids. By its ability to specifically bind sialic acids, this crevice has been identified as the receptor binding site [8,27,38]. The precise structure of this HA domain is known to be of crucial importance for the process of virus
*
Corresponding author. Tel.: +49-6421-286-6253; fax: +49-6421-286-8962. E-mail address: [email protected] (H.-D. Klenk).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 3 8 1 - 8
534
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
binding to its receptor. Accordingly, single amino acid substitutions in the binding pocket can result in altered receptor binding specificity and altered host range of the respective viruses [1,6,36]. Furthermore, employing vector-expressed FPV-HA, we could show that oligosaccharides flanking the binding site modulate receptor affinity [23]. To evaluate the impact of each individual N-glycan at the FPV-HA tip on the growth of intact viruses, we generated recombinant influenza viruses containing the oligosaccharide-deleted HA mutants [37] (Fig. 1). Our studies demonstrate that the glycans flanking the receptor binding pocket are potent regulators of virus growth in cell culture. The oligosaccharide attached to Asn 149 (absent in mutant G2) plays a dominant role in controlling virus spread while that attached to Asn 123 (absent in mutant G1) is less effective. Growth of viruses lacking both N-glycans was found to be reduced in cell culture due to a restricted release of progeny viruses from infected cells (Fig. 2). These findings on the growth of recombinant viruses are an important extension of our previous work investigating the receptor interaction of transiently expressed HA. There is now experimentally based evidence for a distinct regulatory function of individual N-
Fig. 1. Inset on the left: the head region of FPV-HA is shown on the top. N-linked oligosaccharides adjacent to the receptor binding pocket are indicated. Mutants G1 and G2 lack the glycosylation sites at Asn 123 and Asn 149, respectively. Both sites are absent in mutant G1,2. The arrow marks the entrance to the receptor binding pocket. Body of figure: recombinant viruses were generated by a RNA-polymerase I-based reverse genetics system. Two reassortants of strain A/WSN/33 were used as helper viruses to obtain two series of HA-mutant viruses only differing in NA.
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
535
Fig. 2. (a) Growth curves of recombinants in MDCK cells. Cell monolayers were infected at an MOI of 0.001 with recombinant viruses, and supernatants were monitored for HA titers at the time points indicated. The results obtained with viruses of the N2-series and of the N1-series are shown. (&) WT; (~) G1; (^) G2; (*) G1,2. (b) Comparison of specific NA-activities of WT/N1 and WT/N2 viruses. Different amounts of purified virus were incubated with 4-methylumbelliferyl N-acetylneuraminic acid for 20 min at 37 C. The reaction was stopped and NA activity was calculated by measuring the fluorescence of the liberated methylumbelliferone. The data are means of three experiments. They indicate that WT/N2 has a higher NA activity than WT/N1. (c) Release of recombinant viruses from MDCK cells. MDCK cells were infected at an MOI of 5 with recombinant viruses and incubated at 37 C overnight. One hour before virus harvest, VCNA was added to the culture media to one-half of the samples. Titers of progeny viruses released into the media were determined by plaque assay. Levels of virus release in the absence of VCNA are presented as percent values relative to the virus titers released after VCNA treatment (from Ref. [37]).
glycans located at the HA tip on the viral life cycle. By sequentially removing N-glycans from the vicinity of the HA receptor binding site, we have also delineated a novel approach to specifically generate influenza viruses with a gradual extension of attenuation in cell culture. By removing terminal sialic acid residues from oligosaccharide side chains of glycoconjugates, the viral neuraminidase (NA) acts as a receptor-destroying enzyme in influenza viruses [4,18]. When NA activity was blocked by either antibodies [5], inhibitors [12,24], or temperature-sensitive mutations [25], formation of large viral aggregates on the surface of infected cells was observed as was with virus lacking NA either partly [22] or completely [19]. Accordingly, viral NA is regarded an important factor for the release of
536
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
progeny virus from host cells promoting the efficient progression of an infection. In light of this, it was of special interest to examine how different NA subtypes affect the attenuated phenotype of the recombinant viruses lacking N-glycans at the HA tip. Several N1-NAs have a deletion in the stalk region that is most extensive with FPV-NA [14]. NA enzymatic activity has been reported to vary according to the length of the stalk region of the molecule with NA species containing a deletion in the stalk having a lower activity [2,9,20,21]. By choosing appropriate helper viruses, we generated recombinants in which the HA mutants were combined with either the WSN virus NA (N1 subtype) containing a stalk deletion or the Hong Kong virus NA (N2 subtype) that has no deletion. When assayed for neuraminidase activity, recombinant viruses carrying N2-NA exceeded those with N1-NA at least sixfold (Fig. 2). Thus, our set of recombinants was ideally suited to analyze in depth the impact of different NA activities on the growth of mutant influenza viruses specifically designed to show distinct receptor binding activities. Using this system, we were able to demonstrate that the growth behavior of HA mutant viruses is governed by the nature of the accompanying viral NA. Among the viruses with the highactivity N2-NA, growth restriction was observed only when the G1,2 mutant was present showing the highest receptor affinity, while recombinants containing G1 and G2 grew essentially like virus carrying wild type HA. Yet, the situation was different with viruses containing the low-activity N1-NA. Here, the growth of G1,2 mutant viruses was significantly impeded in cell culture due to a restricted release from host cells. This effect was less pronounced with G2 mutant viruses, but still evident. Obviously, unlike N2-NA, the lower-activity N1-NA is not capable to overcome the high affinity interaction of G1,2 and G2 HA with its receptor (Fig. 2). Hence, our data clearly point out that, for the establishment of productive infection, influenza viruses are strictly dependent on a highly balanced action of HA and NA. An increase in receptor binding affinity apparently needs to be accompanied by a concomitant increase in the receptor-destroying activity of the viral NA. Otherwise, the enhanced receptor binding is a serious disadvantage in the late stage of infection by preventing the release of progeny viruses from host cells. The need for such a match of HA and NA activities had so far only been deduced from studies analyzing natural virus isolates or laboratory generated reassortants [15,16,21,29]. Taken together, our work represents the first concise study of the functional interrelationship of distinct HA and NA species and provides experimental evidence for the strict requirement of a fine tuning of HA receptor binding and NA receptor destroying activity in order to allow for an efficient influenza virus propagation (Fig. 3). There is evidence that N-glycans flanking the receptor binding site not only modulate receptor affinity, but also control receptor specificity. Thus, H1 subtype influenza strains with an oligosaccharide in such a position have been shown to bind preferentially to a2,3-linked neuraminic acid, whereas mutants lacking this oligosaccharide had a preference for the a2,6-linkage [11,13]. Furthermore, it has been shown recently that glycans carrying neuraminic acid in a2,3 or a2,6 linkages gain access to the receptor binding pocket from opposite sides [8]. Sterical hindrance by a glycan adjacent to the receptor binding site may therefore be a determinant of receptor specificity. Finally, the number and structure of N-glycans neighbouring the receptor binding pocket have been suggested to determine host range and pathogenicity of influenza viruses [7,11,26]. In
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
537
Fig. 3. Regulation of virus binding and release by HA glycosylation and neuraminidase activity. Receptor affinity is controlled by the oligosaccharides adjacent to the receptor binding site on HA. The efficiency of release depends on the activity of NA.
view of these findings, it will now be interesting to employ our panel of recombinant viruses to elucidate the contributions of individual HA tip glycans to tissue tropism and host range.
2. Factors determining endotheliotropism of FPV Although cleavage of HA is an important determinant of spread of infection in the organism, it is not the only factor involved. This is illustrated by a recent study in which spread of FPV in the chick embryo was investigated [10]. We have found in this study that FPV shows strict endotheliotropism when infecting 11-day-old chick embryos (Fig. 4). Since hemorrhages and edema are major symptoms in FPV-infected chickens, it was not unexpected to see that the vasculature is an important target of infection. However, we were surprised when we could not detect viral replication in other cell types. Besides endothelia, myocytes and lymphatic tissues were found to be sites of virus replication, when hatched chickens were infected with other pathogenic H5 and H7 strains [17,34,35]. These differences in cell tropism may depend on the developmental stage of the host and on the virus strains used.
538
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
Fig. 4. Localization of FPV-infected cells in embryonic tissues by in situ hybridization. Brightfield photomicrographs showing autoradiograms with black grains representing bound HA-specific riboprobe in organs of the chick embryo. After in situ hybridization, slides were covered by photoemulsion, exposed for 2 days, developed, and counter-stained by hematoxilin-eosin. (a) Blood vessel (arrowheads indicating infected blood cells), (b) lung, (c) stomach, (d) heart, (e) liver, (f ) spleen. Magnification 75 (from Ref. [10]).
The results obtained with reassortants of FPV and virus N clearly indicate that endotheliotropism requires the presence of FPV-HA. This observation is in line with the concept that, because of its susceptibility to ubiquitous proteolytic activation, FPV-HA allows virus entry from the allantoic cavity into the highly vascularized mesenchymal layer of the chorioallantoic membrane, and thus, mediates hematogenic spread of infection. On the other hand, the restricted cleavability of virus N HA confines infection to the inner layer of the membrane and the allantoic cavity [28]. Therefore, reassortants containing virus N HA did not have access to endothelia when infected through the chorioallantoic route. Whereas cleavage activation of HA proved to be essential for targeting the virus to endothelia, it was not responsible for confining the infection to these cells. Furin and PC5/ PC6, the activating proteases of FPV-HA, were identified in all chicken tissues analyzed including endothelial cells. This observation indicates that the lack of spread of infection from endothelia to surrounding tissues cannot be attributed to the absence of activating proteases.
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
539
In contrast, tissue-specific expression of virus receptors appears to be an important factor in restricting infection to endothelia. Using lectin binding assays we could detect a2,3-linked and a-2,6-linked neuraminic acid, which both proved to be able to serve as FPV receptors, only on epithelial cells and on cells of the reticulo-endothelial system. We could not detect, however, receptor determinants on other cells, such as myocytes, fibroblasts, and hepatocytes. Thus, it appears that cells lacking a measurable amount of neuraminic acid receptors cannot be infected by FPV and are therefore a barrier for the spread of infection. This concept is nicely supported by observations made in lung tissue. When this organ is infected via the hematogenic route as is the case in the embryo at day 11, the virus is retained in the endothelial cells of the capillary vessels. Since neuraminic acid is present in a2,6 linkage on these cells, it is clear that this type of neuraminic acid can serve as FPV receptor, although it appears that binding of avian strains is generally determined by the a2,3 linkage. The alveolar epithelia, although expressing virus receptor in large amounts, are not infected because virus access is prevented by the connective tissue lacking neuraminic acid. On the other hand, when embryos are infected through the airways, as can be done by inoculating virus 2 days before hatching into the now almost dry allantoic cavity, virus replication is readily detected in lung epithelia (data not shown). It has to be pointed out that expression of neuraminic acid in the chick embryo depends on tissue differentiation [3]. This may explain the absence of detectable amounts of neuraminic acid on fibroblasts in situ, whereas cultured fibroblasts readily express receptors as indicated by their ability to allow efficient virus replication. It has also to be assumed that the subendothelial connective tissue is not a very tight barrier in the chorioallantoic membrane where it allows penetration of the virus from the allantoic epithelium into the mesodermal endothelia. In fact, low amounts of virus budding from mesodermal fibroblasts have been observed, indicating that these cells may play a role in mediating spread of infection [28]. Whether the spread through the mesodermal layer is driven by the particularly high virus replication rates in the allantoic epithelium and the presence of neuraminic acid on mesodermal fibroblasts or by some other mechanism remains to be seen. Our data also show that the polarity of virus budding is another factor contributing to the confinement of infection to endothelial cells. Studies on Sendai virus in a mouse model have shown before that the sidedness of virus maturation has a distinct effect on spread of infection in the organism and on pathogenicity. Wild type Sendai virus released exclusively from the apical surface of lung epithelia is strictly pneumotropic, whereas the mutant F1-R which matures at the apical as well as the basolateral side causes pantropic infection [32,33]. It has long been known that FPV matures preferentially at the luminal side of endothelia [28], and the observations made here on virus budding and HA transport support this concept. The luminal budding polarity of FPV supports therefore the hematogenic spread of the virus and prevents at the same time infection of subendothelial cells. Taken together, our data indicate that endotheliotropism of FPV in the chick embryo is the result of an interplay of several factors determined by the virus and the host (Fig. 5). These include proteolytic activation of HA by ubiquitous proteases which is responsible for entry of the virus into the vascular system and at least two mechanisms contributing to the confinement of the virus to endothelia: the polarity of virus budding at the luminal side of endothelial cells and cell-specific differences in the expression of neuraminic acid
540
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
Fig. 5. The factors determining endotheliotropism of FPV. HA cleavage is responsible for virus entry into the vascular system. Polar budding and lack of receptors in adjacent tissues are responsible for restricting infection to endothelia.
receptors. Endotheliotropism without doubt plays an important role in the generalization of FPV infection and in the generation of typical symptoms of the disease, such as hemorrhages and edema. Systemic infection and severe vascular injury are also the central pathogenetic mechanisms of hemorrhagic fevers in primates caused by filoviruses and other agents, and there is evidence that at least some of these viruses replicate also in endothelia [30,31,39]. It will therefore be interesting to see if similar mechanisms as described here for FPV infection play also a role in the pathogenesis of hemorrhagic fevers in other species.
3. Conclusion The high variability of the influenza virus genome is reflected by a wide spectrum in host tropism, tissue specificity, and pathogenicity, ranging from local infection of the respiratory tract or the gut, as is the case with most mammalian strains and the apathogenic avian viruses, to systemic infection caused by fowl plague virus or other highly pathogenic avian strains. Pathogenicity is determined by the interaction of viral proteins with each other or with host factors. This concept is supported by many studies on the viral envelope proteins. Proteolytic activation of the hemagglutinin as a fusion protein has been known
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
541
for a long time to be an important determinant of pathogenicity. Receptor specificity is another hemagglutinin trait responsible for host range and tissue tropism. There is a close interdependence between hemagglutinin and neuraminidase in controlling binding and release of virus. The multifactorial character of tissue tropism and pathogenicity is illustrated by recent studies demonstrating that endotheliotropism of fowl plague virus in the chick embryo is determined, on one side, by the high cleavability of the hemagglutinin that mediates virus entry into the vascular system and, on the other hand, by restricted receptor expression and polar budding that prevent spread of infection into tissues surrounding endothelia.
Acknowledgements We are grateful to R. Rott and C. Scholtissek, Giessen, for helpful discussions and for providing influenza virus reassortants. The electron micrograph was made by B. Agricola. This study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 286 and KL 238/6-1) and from the Fonds der Chemischen Industrie.
References [1] S. Aytay, I.T. Schulze, Single amino acid substitutions in the hemagglutinin can alter the host range and receptor binding properties of H1 strains of influenza A virus, J. Virol. 65 (1991) 3022 – 3028. [2] M.R. Castrucci, Y. Kawaoka, Biologic importance of neuraminidase stalk length in influenza A virus, J. Virol. 67 (1993) 759 – 764. [3] P. Codogno, M. Aubery, Changes in cell-surface sialic acid content during chick embryo development,Mech. Ageing Dev. 23 (1983) 307 – 314. [4] P. Colman, Structure and function of the neuraminidase, in: K.G. Nicholson, R.G. Webster, A.J. Hay (Eds.), Textbook of Influenza, Blackwell, London, England, 1998, pp. 65 – 73. [5] R.W. Compans, N.J. Dimmock, H. Meier-Ewert, Effect of antibody to neuraminidase on the maturation and hemagglutinating activity of an influenza A2 virus, J. Virol. 4 (1969) 528 – 534. [6] R.J. Connor, Y. Kawaoka, R.G. Webster, J.C. Paulson, Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates, Virology 205 (1994) 17 – 23. [7] C.M. Deom, A.J. Caton, I.T. Schulze, Host cell-mediated selection of a mutant influenza A virus that has lost a complex oligosaccharide from the tip of the hemagglutinin, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 3771 – 3775. [8] M.B. Eisen, S. Sabesan, J.J. Skehel, D.C. Wiley, Binding of the influenza A virus to cell-surface receptors: structures of five hemagglutinin – sialyloligosaccharide complexes determined by X-ray crystallography, Virology 232 (1997) 19 – 31. [9] M.C. Els, G.M. Air, K.G. Murti, R.G. Webster, W.G. Laver, An 18-amino acid deletion in an influenza neuraminidase, Virology 142 (1985) 241 – 247. [10] A. Feldmann, M.K.-H. Scha¨fer, W. Garten, H.-D. Klenk, Targeted infection of endothelial cells by the avian influenza virus A/FPV/ROSTOCK/34 (H7N1) in chicken embryos, J. Virol. 74 (2000) 8018 – 8027. [11] A.S. Gambaryan, V.P. Marinina, A.B. Tuzikov, N.V. Bovin, I.A. Rudneva, B.V. Sinitsyn, A.A. Shilov, M.N. Matrosovich, Effects of host-dependent glycosylation of hemagglutinin on receptor-binding properties on H1N1 human influenza A virus grown in MDCK cells and in embryonated eggs, Virology 247 (1998) 170 – 177. [12] L.V. Gubareva, R. Bethell, G.J. Hart, K.G. Murti, C.R. Penn, R.G. Webster, Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en, J. Virol. 70 (1996) 1818 – 1827.
542
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
[13] I. Gu¨nther, B. Glatthaar, G. Doller, W. Garten, A H1 hemagglutinin of a human influenza A virus with a carbohydrate-modulated receptor binding site and an unusual cleavage site, Virus Res. 27 (1993) 147 – 160. [14] J. Hausmann, E. Kretzschmar, W. Garten, H.D. Klenk, Biosynthesis, intracellular transport and enzymatic activity of an avian influenza A virus neuraminidase: role of unpaired cysteines and individual oligosaccharides, J. Gen. Virol. 78 (1997) 3233 – 3245. [15] N.V. Kaverin, A.S. Gambaryan, N.V. Bovin, I.A. Rudneva, A.A. Shilov, O.M. Khodova, N.L. Varich, B.V. Sinitsin, N.V. Makarova, E.A. Kropotkina, Post reassortment changes in influenza A virus hemagglutinin restoring HA-NA functional match, Virology 244 (1998) 315 – 321. [16] (a) H.D. Klenk, W. Garten, Activation cleevage of viral spike proteins. In: E. Wimmer (Ed.), Cold Spring Harbor Laboratory Press, 1994, pp. 241 – 280; (b) N.V. Kaverin, H.D. Klenk, Strain-specific differences in the effect of influenza A virus neuraminidase on vector-expressed hemagglutinin, Arch. Virol. 144 (1999) 781 – 786. [17] Y. Kobayashi, T. Horimoto, Y. Kawaoka, D. Alexander, C. Itakura, Pathological studies of chickens experimentally infected with two highly pathogenic avian influenza viruses, Avian Pathol. 25 (1996) 285 – 304. [18] R.A. Lamb, R.M. Krug, Orthomyxoviridae: The viruses and their replication, in: B.N. Fields, D.M. Knipe, P.M. Howley (Eds.), Fields Virology, Lippincott-Raven Publishers, Philadelphia, USA, 1996, pp. 1353 – 1395. [19] C. Liu, M.C. Eichelberger, R.W. Compans, G.M. Air, Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding, J. Virol. 69 (1995) 1099 – 1106. [20] G. Luo, J. Chung, P. Palese, Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions, Virus Res. 29 (1993) 321. [21] M. Matrosovich, N. Zhou, Y. Kawaoka, R. Webster, The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties, J. Virol. 73 (1999) 1146 – 1155. [22] L.J. Mitnaul, M.R. Castrucci, K.G. Murti, Y. Kawaoka, The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication, J. Virol. 70 (1996) 873 – 879. [23] M. Ohuchi, R. Ohuchi, A. Feldmann, H.D. Klenk, Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety, J. Virol. 71 (1997) 8377 – 8384. [24] P. Palese, R.W. Compans, Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydroN-trifluoroacetylneuraminic acid (FANA): mechanism of action, J. Gen. Virol. 33 (1976) 159 – 163. [25] P. Palese, K. Tobita, M. Ueda, R.W. Compans, Characterization of temperature sensitive influenza virus mutants defective in neuraminidase, Virology 61 (1974) 397 – 410. [26] M.L. Perdue, J.W. Latimer, J.M. Crawford, A novel carbohydrate addition site on the hemagglutinin protein of a highly pathogenic H7 subtype avian influenza virus, Virology 213 (1995) 276 – 281. [27] G.N. Rogers, J.C. Paulson, R.S. Daniels, J.J. Skehel, I.A. Wilson, D.C. Wiley, Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity, Nature 304 (1983) 76 – 78. [28] R. Rott, M. Reinacher, M. Orlich, H.D. Klenk, Cleavability of hemagglutinin determines spread of avian influenza viruses in the chorioallantoic membrane of chicken embryo, Arch. Virol. 65 (1980) 123 – 133. [29] I.A. Rudneva, E.I. Sklyanskaya, O.S. Barulina, S.S. Yamnikova, V.P. Kovaleva, I.V. Tsvetkova, N.V. Kaverin, Phenotypic expression of HA-NA combinations in human-avian influenza A virus reassortants, Arch. Virol. 141 (1996) 1091 – 1099. [30] E.J. Ryabchikova, L.V. Kolesnikova, S.V. Netesov, Animal pathology of filoviral infections, Curr. Top. Microbiol. Immunol. 235 (1999) 145 – 173. [31] H.J. Schnittler, F. Mahner, D. Drenckhahn, H.-D. Klenk, H. Feldmann, Replication of Marburg virus in human endothelial cells: a possible mechanism for the development of viral hemorrhagic disease, J. Clin. Invest. 91 (1993) 1301 – 1309. [32] M. Tashiro, J.T. Seto, S. Choosakul, M. Yamakawa, H.D. Klenk, R. Rott, Budding site of Sendai virus in polarized epithelial cells is one of the determinants for tropism and pathogenicity in mice, Virology 187 (1992) 413 – 422. [33] M. Tashiro, M. Yamakawa, K. Tobita, J.T. Seto, H.D. Klenk, R. Rott, Altered budding site of a pantropic mutant of Sendai virus F1-R, in polarized epithelial cells, J. Virol. 64 (1990) 4672 – 4677.
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
543
[34] H. van Campen, B.C. Easterday, V.S. Hinshaw, Destruction of lymphocytes by a virulent avian influenza A virus, J. Gen. Virol. 70 (1989) 467 – 472. [35] H. van Campen, B.C. Easterday, V.S. Hinshaw, Virulent avian influenza A viruses: their effect on avian lymphocytes and macrophages in vivo and in vitro, J. Gen. Virol. 70 (1989) 2887 – 2895. [36] A. Vines, K. Wells, M. Matrosovich, M.R. Castrucci, T. Ito, Y. Kawaoka, The role of influenza A virus hemagglutinin residues 226 and 228 in receptor specificity and host range restriction, J. Virol. 72 (1998) 7626 – 7631. [37] R. Wagner, T. Wolff, A. Herwig, S. Pleschka, H.-D. Klenk, Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth — a study by reverse genetics, J. Virol. 74 (2000) 6316 – 6323. [38] W. Weis, J.H. Brown, S. Cusack, J.C. Paulson, J.J. Skehel, D.C. Wiley, Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid, Nature 333 (1988) 426 – 431. [39] S.R. Zaki, C.S. Goldsmith, Pathologic features of filovirus infections in humans, Curr. Top. Microbiol. Immunol. 235 (1999) 97 – 116.
Hemagglutinin and neuraminidase as determinants of inf luenza virus pathogenicity Ralf Wagner a, Anke Feldmanna, Thorsten Wolff a, Stephan Pleschkab, Wolfgang Gartena, Hans-Dieter Klenka,* a
Institut fu¨r Virologie, Klinikum der Philipps-Universita¨t Marburg, Robert-Koch-Str. 17, 35037 Marburg, Germany b Institut fu¨r Virologie, Justus-Liebig-Universita¨t Giessen, Germany
Keywords: Pathogenicity; Fowl plague virus; Receptor binding specificity; Neurominidase activity
The pathogenicity of influenza viruses is determined by many of its other biological properties, such as efficiency of replication, tissue tropism, host range, spread of infection, as well as response to and modulation of host defense. All of these properties are controlled by the complex interplay of viral and host factors at virtually each stage in the life cycle of the virus. Activation of the hemagglutinin by host cell proteases has been shown in many studies to have a dramatic effect on pathogenesis, and the molecular details of proteolytic activation are well understood [16a]. We will concentrate here on recent studies in which we have analyzed the interplay of hemagglutinin (HA) and neuraminidase (NA) in receptor binding and release and some of the factors that determine spread of infection in the organism.
1. Balance of receptor binding and release regulates virus growth HA-mediated attachment of influenza viruses to sialic acid containing receptors on the host cell surface is the initial step of infection. Influenza HA contains at the tip a narrow crevice lined with highly conserved amino acids. By its ability to specifically bind sialic acids, this crevice has been identified as the receptor binding site [8,27,38]. The precise structure of this HA domain is known to be of crucial importance for the process of virus
*
Corresponding author. Tel.: +49-6421-286-6253; fax: +49-6421-286-8962. E-mail address: [email protected] (H.-D. Klenk).
0531-5131/01/$ – see front matter D 2001 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 1 ) 0 0 3 8 1 - 8
534
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
binding to its receptor. Accordingly, single amino acid substitutions in the binding pocket can result in altered receptor binding specificity and altered host range of the respective viruses [1,6,36]. Furthermore, employing vector-expressed FPV-HA, we could show that oligosaccharides flanking the binding site modulate receptor affinity [23]. To evaluate the impact of each individual N-glycan at the FPV-HA tip on the growth of intact viruses, we generated recombinant influenza viruses containing the oligosaccharide-deleted HA mutants [37] (Fig. 1). Our studies demonstrate that the glycans flanking the receptor binding pocket are potent regulators of virus growth in cell culture. The oligosaccharide attached to Asn 149 (absent in mutant G2) plays a dominant role in controlling virus spread while that attached to Asn 123 (absent in mutant G1) is less effective. Growth of viruses lacking both N-glycans was found to be reduced in cell culture due to a restricted release of progeny viruses from infected cells (Fig. 2). These findings on the growth of recombinant viruses are an important extension of our previous work investigating the receptor interaction of transiently expressed HA. There is now experimentally based evidence for a distinct regulatory function of individual N-
Fig. 1. Inset on the left: the head region of FPV-HA is shown on the top. N-linked oligosaccharides adjacent to the receptor binding pocket are indicated. Mutants G1 and G2 lack the glycosylation sites at Asn 123 and Asn 149, respectively. Both sites are absent in mutant G1,2. The arrow marks the entrance to the receptor binding pocket. Body of figure: recombinant viruses were generated by a RNA-polymerase I-based reverse genetics system. Two reassortants of strain A/WSN/33 were used as helper viruses to obtain two series of HA-mutant viruses only differing in NA.
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
535
Fig. 2. (a) Growth curves of recombinants in MDCK cells. Cell monolayers were infected at an MOI of 0.001 with recombinant viruses, and supernatants were monitored for HA titers at the time points indicated. The results obtained with viruses of the N2-series and of the N1-series are shown. (&) WT; (~) G1; (^) G2; (*) G1,2. (b) Comparison of specific NA-activities of WT/N1 and WT/N2 viruses. Different amounts of purified virus were incubated with 4-methylumbelliferyl N-acetylneuraminic acid for 20 min at 37 C. The reaction was stopped and NA activity was calculated by measuring the fluorescence of the liberated methylumbelliferone. The data are means of three experiments. They indicate that WT/N2 has a higher NA activity than WT/N1. (c) Release of recombinant viruses from MDCK cells. MDCK cells were infected at an MOI of 5 with recombinant viruses and incubated at 37 C overnight. One hour before virus harvest, VCNA was added to the culture media to one-half of the samples. Titers of progeny viruses released into the media were determined by plaque assay. Levels of virus release in the absence of VCNA are presented as percent values relative to the virus titers released after VCNA treatment (from Ref. [37]).
glycans located at the HA tip on the viral life cycle. By sequentially removing N-glycans from the vicinity of the HA receptor binding site, we have also delineated a novel approach to specifically generate influenza viruses with a gradual extension of attenuation in cell culture. By removing terminal sialic acid residues from oligosaccharide side chains of glycoconjugates, the viral neuraminidase (NA) acts as a receptor-destroying enzyme in influenza viruses [4,18]. When NA activity was blocked by either antibodies [5], inhibitors [12,24], or temperature-sensitive mutations [25], formation of large viral aggregates on the surface of infected cells was observed as was with virus lacking NA either partly [22] or completely [19]. Accordingly, viral NA is regarded an important factor for the release of
536
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
progeny virus from host cells promoting the efficient progression of an infection. In light of this, it was of special interest to examine how different NA subtypes affect the attenuated phenotype of the recombinant viruses lacking N-glycans at the HA tip. Several N1-NAs have a deletion in the stalk region that is most extensive with FPV-NA [14]. NA enzymatic activity has been reported to vary according to the length of the stalk region of the molecule with NA species containing a deletion in the stalk having a lower activity [2,9,20,21]. By choosing appropriate helper viruses, we generated recombinants in which the HA mutants were combined with either the WSN virus NA (N1 subtype) containing a stalk deletion or the Hong Kong virus NA (N2 subtype) that has no deletion. When assayed for neuraminidase activity, recombinant viruses carrying N2-NA exceeded those with N1-NA at least sixfold (Fig. 2). Thus, our set of recombinants was ideally suited to analyze in depth the impact of different NA activities on the growth of mutant influenza viruses specifically designed to show distinct receptor binding activities. Using this system, we were able to demonstrate that the growth behavior of HA mutant viruses is governed by the nature of the accompanying viral NA. Among the viruses with the highactivity N2-NA, growth restriction was observed only when the G1,2 mutant was present showing the highest receptor affinity, while recombinants containing G1 and G2 grew essentially like virus carrying wild type HA. Yet, the situation was different with viruses containing the low-activity N1-NA. Here, the growth of G1,2 mutant viruses was significantly impeded in cell culture due to a restricted release from host cells. This effect was less pronounced with G2 mutant viruses, but still evident. Obviously, unlike N2-NA, the lower-activity N1-NA is not capable to overcome the high affinity interaction of G1,2 and G2 HA with its receptor (Fig. 2). Hence, our data clearly point out that, for the establishment of productive infection, influenza viruses are strictly dependent on a highly balanced action of HA and NA. An increase in receptor binding affinity apparently needs to be accompanied by a concomitant increase in the receptor-destroying activity of the viral NA. Otherwise, the enhanced receptor binding is a serious disadvantage in the late stage of infection by preventing the release of progeny viruses from host cells. The need for such a match of HA and NA activities had so far only been deduced from studies analyzing natural virus isolates or laboratory generated reassortants [15,16,21,29]. Taken together, our work represents the first concise study of the functional interrelationship of distinct HA and NA species and provides experimental evidence for the strict requirement of a fine tuning of HA receptor binding and NA receptor destroying activity in order to allow for an efficient influenza virus propagation (Fig. 3). There is evidence that N-glycans flanking the receptor binding site not only modulate receptor affinity, but also control receptor specificity. Thus, H1 subtype influenza strains with an oligosaccharide in such a position have been shown to bind preferentially to a2,3-linked neuraminic acid, whereas mutants lacking this oligosaccharide had a preference for the a2,6-linkage [11,13]. Furthermore, it has been shown recently that glycans carrying neuraminic acid in a2,3 or a2,6 linkages gain access to the receptor binding pocket from opposite sides [8]. Sterical hindrance by a glycan adjacent to the receptor binding site may therefore be a determinant of receptor specificity. Finally, the number and structure of N-glycans neighbouring the receptor binding pocket have been suggested to determine host range and pathogenicity of influenza viruses [7,11,26]. In
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
537
Fig. 3. Regulation of virus binding and release by HA glycosylation and neuraminidase activity. Receptor affinity is controlled by the oligosaccharides adjacent to the receptor binding site on HA. The efficiency of release depends on the activity of NA.
view of these findings, it will now be interesting to employ our panel of recombinant viruses to elucidate the contributions of individual HA tip glycans to tissue tropism and host range.
2. Factors determining endotheliotropism of FPV Although cleavage of HA is an important determinant of spread of infection in the organism, it is not the only factor involved. This is illustrated by a recent study in which spread of FPV in the chick embryo was investigated [10]. We have found in this study that FPV shows strict endotheliotropism when infecting 11-day-old chick embryos (Fig. 4). Since hemorrhages and edema are major symptoms in FPV-infected chickens, it was not unexpected to see that the vasculature is an important target of infection. However, we were surprised when we could not detect viral replication in other cell types. Besides endothelia, myocytes and lymphatic tissues were found to be sites of virus replication, when hatched chickens were infected with other pathogenic H5 and H7 strains [17,34,35]. These differences in cell tropism may depend on the developmental stage of the host and on the virus strains used.
538
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
Fig. 4. Localization of FPV-infected cells in embryonic tissues by in situ hybridization. Brightfield photomicrographs showing autoradiograms with black grains representing bound HA-specific riboprobe in organs of the chick embryo. After in situ hybridization, slides were covered by photoemulsion, exposed for 2 days, developed, and counter-stained by hematoxilin-eosin. (a) Blood vessel (arrowheads indicating infected blood cells), (b) lung, (c) stomach, (d) heart, (e) liver, (f ) spleen. Magnification 75 (from Ref. [10]).
The results obtained with reassortants of FPV and virus N clearly indicate that endotheliotropism requires the presence of FPV-HA. This observation is in line with the concept that, because of its susceptibility to ubiquitous proteolytic activation, FPV-HA allows virus entry from the allantoic cavity into the highly vascularized mesenchymal layer of the chorioallantoic membrane, and thus, mediates hematogenic spread of infection. On the other hand, the restricted cleavability of virus N HA confines infection to the inner layer of the membrane and the allantoic cavity [28]. Therefore, reassortants containing virus N HA did not have access to endothelia when infected through the chorioallantoic route. Whereas cleavage activation of HA proved to be essential for targeting the virus to endothelia, it was not responsible for confining the infection to these cells. Furin and PC5/ PC6, the activating proteases of FPV-HA, were identified in all chicken tissues analyzed including endothelial cells. This observation indicates that the lack of spread of infection from endothelia to surrounding tissues cannot be attributed to the absence of activating proteases.
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
539
In contrast, tissue-specific expression of virus receptors appears to be an important factor in restricting infection to endothelia. Using lectin binding assays we could detect a2,3-linked and a-2,6-linked neuraminic acid, which both proved to be able to serve as FPV receptors, only on epithelial cells and on cells of the reticulo-endothelial system. We could not detect, however, receptor determinants on other cells, such as myocytes, fibroblasts, and hepatocytes. Thus, it appears that cells lacking a measurable amount of neuraminic acid receptors cannot be infected by FPV and are therefore a barrier for the spread of infection. This concept is nicely supported by observations made in lung tissue. When this organ is infected via the hematogenic route as is the case in the embryo at day 11, the virus is retained in the endothelial cells of the capillary vessels. Since neuraminic acid is present in a2,6 linkage on these cells, it is clear that this type of neuraminic acid can serve as FPV receptor, although it appears that binding of avian strains is generally determined by the a2,3 linkage. The alveolar epithelia, although expressing virus receptor in large amounts, are not infected because virus access is prevented by the connective tissue lacking neuraminic acid. On the other hand, when embryos are infected through the airways, as can be done by inoculating virus 2 days before hatching into the now almost dry allantoic cavity, virus replication is readily detected in lung epithelia (data not shown). It has to be pointed out that expression of neuraminic acid in the chick embryo depends on tissue differentiation [3]. This may explain the absence of detectable amounts of neuraminic acid on fibroblasts in situ, whereas cultured fibroblasts readily express receptors as indicated by their ability to allow efficient virus replication. It has also to be assumed that the subendothelial connective tissue is not a very tight barrier in the chorioallantoic membrane where it allows penetration of the virus from the allantoic epithelium into the mesodermal endothelia. In fact, low amounts of virus budding from mesodermal fibroblasts have been observed, indicating that these cells may play a role in mediating spread of infection [28]. Whether the spread through the mesodermal layer is driven by the particularly high virus replication rates in the allantoic epithelium and the presence of neuraminic acid on mesodermal fibroblasts or by some other mechanism remains to be seen. Our data also show that the polarity of virus budding is another factor contributing to the confinement of infection to endothelial cells. Studies on Sendai virus in a mouse model have shown before that the sidedness of virus maturation has a distinct effect on spread of infection in the organism and on pathogenicity. Wild type Sendai virus released exclusively from the apical surface of lung epithelia is strictly pneumotropic, whereas the mutant F1-R which matures at the apical as well as the basolateral side causes pantropic infection [32,33]. It has long been known that FPV matures preferentially at the luminal side of endothelia [28], and the observations made here on virus budding and HA transport support this concept. The luminal budding polarity of FPV supports therefore the hematogenic spread of the virus and prevents at the same time infection of subendothelial cells. Taken together, our data indicate that endotheliotropism of FPV in the chick embryo is the result of an interplay of several factors determined by the virus and the host (Fig. 5). These include proteolytic activation of HA by ubiquitous proteases which is responsible for entry of the virus into the vascular system and at least two mechanisms contributing to the confinement of the virus to endothelia: the polarity of virus budding at the luminal side of endothelial cells and cell-specific differences in the expression of neuraminic acid
540
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
Fig. 5. The factors determining endotheliotropism of FPV. HA cleavage is responsible for virus entry into the vascular system. Polar budding and lack of receptors in adjacent tissues are responsible for restricting infection to endothelia.
receptors. Endotheliotropism without doubt plays an important role in the generalization of FPV infection and in the generation of typical symptoms of the disease, such as hemorrhages and edema. Systemic infection and severe vascular injury are also the central pathogenetic mechanisms of hemorrhagic fevers in primates caused by filoviruses and other agents, and there is evidence that at least some of these viruses replicate also in endothelia [30,31,39]. It will therefore be interesting to see if similar mechanisms as described here for FPV infection play also a role in the pathogenesis of hemorrhagic fevers in other species.
3. Conclusion The high variability of the influenza virus genome is reflected by a wide spectrum in host tropism, tissue specificity, and pathogenicity, ranging from local infection of the respiratory tract or the gut, as is the case with most mammalian strains and the apathogenic avian viruses, to systemic infection caused by fowl plague virus or other highly pathogenic avian strains. Pathogenicity is determined by the interaction of viral proteins with each other or with host factors. This concept is supported by many studies on the viral envelope proteins. Proteolytic activation of the hemagglutinin as a fusion protein has been known
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
541
for a long time to be an important determinant of pathogenicity. Receptor specificity is another hemagglutinin trait responsible for host range and tissue tropism. There is a close interdependence between hemagglutinin and neuraminidase in controlling binding and release of virus. The multifactorial character of tissue tropism and pathogenicity is illustrated by recent studies demonstrating that endotheliotropism of fowl plague virus in the chick embryo is determined, on one side, by the high cleavability of the hemagglutinin that mediates virus entry into the vascular system and, on the other hand, by restricted receptor expression and polar budding that prevent spread of infection into tissues surrounding endothelia.
Acknowledgements We are grateful to R. Rott and C. Scholtissek, Giessen, for helpful discussions and for providing influenza virus reassortants. The electron micrograph was made by B. Agricola. This study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 286 and KL 238/6-1) and from the Fonds der Chemischen Industrie.
References [1] S. Aytay, I.T. Schulze, Single amino acid substitutions in the hemagglutinin can alter the host range and receptor binding properties of H1 strains of influenza A virus, J. Virol. 65 (1991) 3022 – 3028. [2] M.R. Castrucci, Y. Kawaoka, Biologic importance of neuraminidase stalk length in influenza A virus, J. Virol. 67 (1993) 759 – 764. [3] P. Codogno, M. Aubery, Changes in cell-surface sialic acid content during chick embryo development,Mech. Ageing Dev. 23 (1983) 307 – 314. [4] P. Colman, Structure and function of the neuraminidase, in: K.G. Nicholson, R.G. Webster, A.J. Hay (Eds.), Textbook of Influenza, Blackwell, London, England, 1998, pp. 65 – 73. [5] R.W. Compans, N.J. Dimmock, H. Meier-Ewert, Effect of antibody to neuraminidase on the maturation and hemagglutinating activity of an influenza A2 virus, J. Virol. 4 (1969) 528 – 534. [6] R.J. Connor, Y. Kawaoka, R.G. Webster, J.C. Paulson, Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates, Virology 205 (1994) 17 – 23. [7] C.M. Deom, A.J. Caton, I.T. Schulze, Host cell-mediated selection of a mutant influenza A virus that has lost a complex oligosaccharide from the tip of the hemagglutinin, Proc. Natl. Acad. Sci. U. S. A. 83 (1986) 3771 – 3775. [8] M.B. Eisen, S. Sabesan, J.J. Skehel, D.C. Wiley, Binding of the influenza A virus to cell-surface receptors: structures of five hemagglutinin – sialyloligosaccharide complexes determined by X-ray crystallography, Virology 232 (1997) 19 – 31. [9] M.C. Els, G.M. Air, K.G. Murti, R.G. Webster, W.G. Laver, An 18-amino acid deletion in an influenza neuraminidase, Virology 142 (1985) 241 – 247. [10] A. Feldmann, M.K.-H. Scha¨fer, W. Garten, H.-D. Klenk, Targeted infection of endothelial cells by the avian influenza virus A/FPV/ROSTOCK/34 (H7N1) in chicken embryos, J. Virol. 74 (2000) 8018 – 8027. [11] A.S. Gambaryan, V.P. Marinina, A.B. Tuzikov, N.V. Bovin, I.A. Rudneva, B.V. Sinitsyn, A.A. Shilov, M.N. Matrosovich, Effects of host-dependent glycosylation of hemagglutinin on receptor-binding properties on H1N1 human influenza A virus grown in MDCK cells and in embryonated eggs, Virology 247 (1998) 170 – 177. [12] L.V. Gubareva, R. Bethell, G.J. Hart, K.G. Murti, C.R. Penn, R.G. Webster, Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en, J. Virol. 70 (1996) 1818 – 1827.
542
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
[13] I. Gu¨nther, B. Glatthaar, G. Doller, W. Garten, A H1 hemagglutinin of a human influenza A virus with a carbohydrate-modulated receptor binding site and an unusual cleavage site, Virus Res. 27 (1993) 147 – 160. [14] J. Hausmann, E. Kretzschmar, W. Garten, H.D. Klenk, Biosynthesis, intracellular transport and enzymatic activity of an avian influenza A virus neuraminidase: role of unpaired cysteines and individual oligosaccharides, J. Gen. Virol. 78 (1997) 3233 – 3245. [15] N.V. Kaverin, A.S. Gambaryan, N.V. Bovin, I.A. Rudneva, A.A. Shilov, O.M. Khodova, N.L. Varich, B.V. Sinitsin, N.V. Makarova, E.A. Kropotkina, Post reassortment changes in influenza A virus hemagglutinin restoring HA-NA functional match, Virology 244 (1998) 315 – 321. [16] (a) H.D. Klenk, W. Garten, Activation cleevage of viral spike proteins. In: E. Wimmer (Ed.), Cold Spring Harbor Laboratory Press, 1994, pp. 241 – 280; (b) N.V. Kaverin, H.D. Klenk, Strain-specific differences in the effect of influenza A virus neuraminidase on vector-expressed hemagglutinin, Arch. Virol. 144 (1999) 781 – 786. [17] Y. Kobayashi, T. Horimoto, Y. Kawaoka, D. Alexander, C. Itakura, Pathological studies of chickens experimentally infected with two highly pathogenic avian influenza viruses, Avian Pathol. 25 (1996) 285 – 304. [18] R.A. Lamb, R.M. Krug, Orthomyxoviridae: The viruses and their replication, in: B.N. Fields, D.M. Knipe, P.M. Howley (Eds.), Fields Virology, Lippincott-Raven Publishers, Philadelphia, USA, 1996, pp. 1353 – 1395. [19] C. Liu, M.C. Eichelberger, R.W. Compans, G.M. Air, Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding, J. Virol. 69 (1995) 1099 – 1106. [20] G. Luo, J. Chung, P. Palese, Alterations of the stalk of the influenza virus neuraminidase: deletions and insertions, Virus Res. 29 (1993) 321. [21] M. Matrosovich, N. Zhou, Y. Kawaoka, R. Webster, The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties, J. Virol. 73 (1999) 1146 – 1155. [22] L.J. Mitnaul, M.R. Castrucci, K.G. Murti, Y. Kawaoka, The cytoplasmic tail of influenza A virus neuraminidase (NA) affects NA incorporation into virions, virion morphology, and virulence in mice but is not essential for virus replication, J. Virol. 70 (1996) 873 – 879. [23] M. Ohuchi, R. Ohuchi, A. Feldmann, H.D. Klenk, Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety, J. Virol. 71 (1997) 8377 – 8384. [24] P. Palese, R.W. Compans, Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydroN-trifluoroacetylneuraminic acid (FANA): mechanism of action, J. Gen. Virol. 33 (1976) 159 – 163. [25] P. Palese, K. Tobita, M. Ueda, R.W. Compans, Characterization of temperature sensitive influenza virus mutants defective in neuraminidase, Virology 61 (1974) 397 – 410. [26] M.L. Perdue, J.W. Latimer, J.M. Crawford, A novel carbohydrate addition site on the hemagglutinin protein of a highly pathogenic H7 subtype avian influenza virus, Virology 213 (1995) 276 – 281. [27] G.N. Rogers, J.C. Paulson, R.S. Daniels, J.J. Skehel, I.A. Wilson, D.C. Wiley, Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity, Nature 304 (1983) 76 – 78. [28] R. Rott, M. Reinacher, M. Orlich, H.D. Klenk, Cleavability of hemagglutinin determines spread of avian influenza viruses in the chorioallantoic membrane of chicken embryo, Arch. Virol. 65 (1980) 123 – 133. [29] I.A. Rudneva, E.I. Sklyanskaya, O.S. Barulina, S.S. Yamnikova, V.P. Kovaleva, I.V. Tsvetkova, N.V. Kaverin, Phenotypic expression of HA-NA combinations in human-avian influenza A virus reassortants, Arch. Virol. 141 (1996) 1091 – 1099. [30] E.J. Ryabchikova, L.V. Kolesnikova, S.V. Netesov, Animal pathology of filoviral infections, Curr. Top. Microbiol. Immunol. 235 (1999) 145 – 173. [31] H.J. Schnittler, F. Mahner, D. Drenckhahn, H.-D. Klenk, H. Feldmann, Replication of Marburg virus in human endothelial cells: a possible mechanism for the development of viral hemorrhagic disease, J. Clin. Invest. 91 (1993) 1301 – 1309. [32] M. Tashiro, J.T. Seto, S. Choosakul, M. Yamakawa, H.D. Klenk, R. Rott, Budding site of Sendai virus in polarized epithelial cells is one of the determinants for tropism and pathogenicity in mice, Virology 187 (1992) 413 – 422. [33] M. Tashiro, M. Yamakawa, K. Tobita, J.T. Seto, H.D. Klenk, R. Rott, Altered budding site of a pantropic mutant of Sendai virus F1-R, in polarized epithelial cells, J. Virol. 64 (1990) 4672 – 4677.
R. Wagner et al. / International Congress Series 1219 (2001) 533–543
543
[34] H. van Campen, B.C. Easterday, V.S. Hinshaw, Destruction of lymphocytes by a virulent avian influenza A virus, J. Gen. Virol. 70 (1989) 467 – 472. [35] H. van Campen, B.C. Easterday, V.S. Hinshaw, Virulent avian influenza A viruses: their effect on avian lymphocytes and macrophages in vivo and in vitro, J. Gen. Virol. 70 (1989) 2887 – 2895. [36] A. Vines, K. Wells, M. Matrosovich, M.R. Castrucci, T. Ito, Y. Kawaoka, The role of influenza A virus hemagglutinin residues 226 and 228 in receptor specificity and host range restriction, J. Virol. 72 (1998) 7626 – 7631. [37] R. Wagner, T. Wolff, A. Herwig, S. Pleschka, H.-D. Klenk, Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth — a study by reverse genetics, J. Virol. 74 (2000) 6316 – 6323. [38] W. Weis, J.H. Brown, S. Cusack, J.C. Paulson, J.J. Skehel, D.C. Wiley, Structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid, Nature 333 (1988) 426 – 431. [39] S.R. Zaki, C.S. Goldsmith, Pathologic features of filovirus infections in humans, Curr. Top. Microbiol. Immunol. 235 (1999) 97 – 116.