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Proc Natl Acad Sci U S A. 2009 January 6; 106(1): 286–291. Published online 2008 December 30. doi: 10.1073/pnas.0811052106. | PMCID: PMC2629220 |
Copyright © 2008 by The National Academy of Sciences of the USA Microbiology Changes in H5N1 influenza virus hemagglutinin receptor binding domain affect systemic spread Hui-Ling Yen,a Jerry R. Aldridge,a Adrianus C. M. Boon,a Natalia A. Ilyushina,a Rachelle Salomon,a Diane J. Hulse-Post,a Henju Marjuki,a John Franks,a David A. Boltz,a Dorothy Bush,b Aleksandr S. Lipatov,a Richard J. Webby,a Jerold E. Rehg,b and Robert G. Webstera1 aDivision of Virology, Department of Infectious Diseases and bDepartment of Pathology, St. Jude Children's Research Hospital, Memphis, TN 38105 Received August 14, 2008. The HA of influenza virus is a receptor-binding and fusion protein that is required to initiate infection. The HA receptor-binding domain determines the species of sialyl receptors recognized by influenza viruses. Here, we demonstrate that changes in the HA receptor-binding domain alter the ability of the H5N1 virus to spread systemically in mice. The A/Vietnam/1203/04 (VN1203) and A/Hong Kong/213/03 (HK213) viruses are consistently lethal to domestic chickens but differ in their pathogenicity to mammals. Insertion of the VN1203 HA and neuraminidase (NA) genes into recombinant HK213 virus expanded its tissue tropism and increased its lethality in mice; conversely, insertion of HK213 HA and NA genes into recombinant VN1203 virus decreased its systemic spread and lethality. The VN1203 and HK213 HAs differ by 10 aa, and HK213 HA has shown greater binding affinity for synthetic α2,6-linked sialyl receptor. Introduction of an S227N change and removal of N-linked glycosylation at residue 158 increased the α2,6-binding affinity of VN1203 HA. Recombinant VN1203 virus carrying the S227N change alone or with the residue-158 glycosylation site removed showed reduced lethality and systemic spread in mice but not in domestic chickens. Wild-type VN1203 virus exhibited the greatest efficiency in systemic spread after intramuscular inoculation and in infection of mouse bone marrow-derived dendritic cells and conventional pulmonary dendritic cells. These results show that VN1203 HA glycoprotein confers pathogenicity by facilitating systemic spread in mice; they also suggest that a minor change in receptor binding domain may modulate the virulence of H5N1 viruses. Keywords: dendritic cell, H5N1, pathogenesis, mice The HA glycoprotein of influenza A virus is a type I transmembrane protein that binds to sialic acid residues on target-cell glycoproteins or glycolipids ( 1). The receptor-binding domain is located at the HA membrane distal end, where the 130-loop, 220-loop, 190-helix, and other conserved residues (Y98, W153, H183, Y195) form the receptor-binding site ( 1, 2). HA is a host-range determinant, because human and avian influenza virus HAs preferentially recognize α2,6- and α2,3-linked sialyl receptors, respectively ( 3, 4). In addition to the terminal sialic-acid linkages, internal linkages, as well as fucosylation, sulfation, and sialylation at the inner oligosaccharides, also determine receptor-binding affinity and specificity ( 2, 5). Despite their preferential recognition of α2,3-linked sialyl receptors, highly pathogenic H5N1 avian influenza viruses are able to infect humans and cause severe disease. Glycan-array profiling has shown differences between seasonal human influenza virus and H5N1 viruses ( 2, 6), but current techniques are at an early stage to map the glycans in tissues, and it is not known whether differences in glycan recognition affect influenza virus pathogenicity. The highly pathogenic H5N1 viruses are consistently lethal to domestic chickens but differ in their pathogenicity to small mammals, including mice and ferrets ( 7– 9). Although H5N1 virulence in mammals is known to be a polygenic trait ( 10– 15), H5N1 viruses that are highly lethal to mice or ferrets commonly spread systemically after intranasal inoculation ( 8, 9). Furthermore, H5N1 virus or RNA has been isolated from human specimens outside the respiratory tract, including blood, cerebrospinal fluid, intestine, feces, placenta, and a fetus ( 16– 20). The mechanism of systemic spread of H5N1 viruses in mammals has not been established, although detection of virus in blood ( 18, 21) or nerves ( 22) suggests possible routes. We and others have found that A/Vietnam/1203/04 (VN1203) virus, whose HA recognizes synthetic α2,3-linked sialyl receptor, is highly pathogenic in the ferret and mouse models, whereas A/Hong Kong/213/03 (HK213) virus, whose HA recognizes both α2,3-linked and α2,6-linked synthetic sialyl receptors, is less lethal ( 8, 9, 23, 24). Surface glycoproteins have also been reported to play a role in the pathogenicity of H5N1 viruses isolated in Hong Kong in 1997 ( 11, 25), H7N7 virus isolated in the Netherlands ( 26), and the H1N1 1918 pandemic virus ( 27). We hypothesized that changes in the HA receptor binding domain might modulate the virulence of H5N1 viruses in mammals by affecting the spread of virus from the respiratory tract to other tissues. VN1203 HA and Neuraminidase Increase the Virulence of HK213 in Mice. The VN1203 virus spreads systemically in inoculated mice and ferrets whereas the HK213 virus is detected largely in their respiratory tracts ( 8, 9, 23, 24). To determine whether the HA and neuraminidase (NA) of VN1203 contribute to its systemic spread, we first investigated the tissue tropism of HK213 virus carrying VN1203 HA or VN1203 HA and NA. HK213, HK213xVN1203 HA, and HK213xVN1203 HANA recombinant viruses grew to comparable titers in vitro ( Table S1), and one 50% mouse lethal dose (MLD 50) in BALB/cJ mice was 4,295, 413.3, and 41.8 pfu, respectively. Therefore, the lethality of HK213 virus was increased one log by inserting VN1203 virus HA and two logs by inserting VN1203 HA and NA. Furthermore, ≈40% of mice inoculated with HK213xVN1203 HA or HK213xVN1203 HANA virus, but none of the mice inoculated with HK213, developed neurological signs (hind limb paralysis or convulsion). The lung titers of HK213 and HK213xVN1203 HANA viruses differed on day 1 ( P = 0.0174, Tukey's test) but were comparable on days 3, 5, and 7 after inoculation ( P = 0.0802, 2-way ANOVA) ( A). HK213xVN1203 HA and HK213xVN1203 HANA viruses were detected in brain or blood, suggesting that infection was not contained in the mouse lungs ( A). The results suggest that the HA glycoproteins of VN1203 contribute to viral spread from the respiratory tract. Determinants of HA Affinity for α2,6-Linked Sialyl Receptor. We assess possible functional differences between the HK213 and VN1203 HA glycoproteins. The two HA glycoproteins were shown to differ in their affinity for synthetic α2,3-linked 3′-sialyllactose (p3′SL) and α2,6-linked 6′-sialyllactose (p6′SL) receptors ( 24), although a recent glycan array study found no significant difference between their recognized glycan species ( 28). Ten amino acid differences between the VN1203 and HK213 HA glycoproteins were identified ( Table S2); differences within or near the receptor binding site included one change among the conserved residues in the 220-loop (S227N in HK213), one difference in the 190-helix (R193 in HK213 and K193 in VN1203), and two differences (residues 158–160: NST for VN1203, NNA for HK213) that resulted in a potential N-linked glycosylation site at residue 158 of VN1203 HA. Another human H5N1 isolate (A/Turkey/65–596/06), which exhibits dual binding affinity for p3′SL and p6′SL ( 24), also shows the S227N change and de-glycosylation at residue 158. To investigate the effect of these differences on receptor binding affinity and specificity, we generated recombinant VN1203, VN1203-HA S227N, and VN1203-HA S227N+158GlyΔ viruses (de-glycosylation by introducing S159N and T160A mutations). The binding affinity of the recombinant viruses to synthetic sialyl receptor substrates was measured ( Table 1). The S227N change alone did not significantly increase binding affinity for p6′SL, consistent with previous report ( 2). VN1203-HA S227N also exhibited reduced binding to fetuin (containing α2,3- and α2,6-linked sialyl receptors) and to 3′-sialyl ( N-acetyllactosamine) (p3′SLN). The S227N change and de-glycosylation at residue 158 significantly increased the binding affinity of VN1203 HA for p6′SL (see Table 1) without reducing its binding affinity for p3′SL. The importance of glycosylation at residue 158 was recently demonstrated ( 28). The majority (99%) of the clade 2.2 H5N1 viruses lack the glycosylation site at residue 158 ( 28) and retain binding specificity for α2,3-linked sialyl receptors. Thus, the absence of glycosylation at residue 158 alone is not sufficient to confer preferential binding to p6′SL. Our results suggest that the increased binding affinity of HK213 for p6′SL was mediated mainly by the S227N change and the absence of N-linked glycosylation at residue 158. | Table 1.Affinity of the recombinant H5N1 influenza viruses to sialyl substrates |
Introduced HA Mutations Do Not Affect Viral Growth In Vitro or Membrane Fusion pH. We assessed whether the introduced HA changes affected growth of recombinant VN1203 viruses in vitro. VN1203xHK213 HANA virus was generated for comparison. The recombinant viruses grew comparably in Madin-Darby canine kidney (MDCK) cells but formed different plaque sizes (see Table S1). At a multiplicity of infection (MOI) = 0.0001 pfu/cell, multistep growth curves showed comparable titers of VN1203, VN1203-HA S227N, and VN1203-HA S227N+158GlyΔ viruses in MDCK cells ( Fig. S1). We also evaluated whether the introduced HA mutations would affect membrane fusion activity of the VN1203 recombinant viruses by determining the pH of syncytium formation in Vero cells. Vero cells infected with VN1203, VN1203-HA S227N, and VN1203-HA S227N+158GlyΔ viruses formed syncytia at or below pH 5.8, 5.9, and 5.8, respectively. Syncytium formation was observed at or below pH 5.7 for the VN1203xHK213 HANA virus. The results suggest that the introduced HA mutations did not significantly impair viral fitness in vitro. Changes in the HA Receptor-Binding Motif Affect Virulence in Mice but Not in Chickens. Despite their comparable growth in vitro, the four recombinant viruses differed in their lethality to BALB/cJ mice. VN1203 virus was most lethal (1 MLD50 = 0.6 pfu), followed by VN1203-HAS227N+158GlyΔ (8.6 pfu), VN1203xHK213HANA (74.8 pfu), and VN1203-HAS227N (85.4 pfu) viruses. The ≈100-fold reduction in the lethality of VN1203 virus carrying HK213 HA and NA confirmed that VN1203 HA and NA surface glycoproteins are virulence factors. We observed neurological signs in ≈40% of VN1203-inoculated and 20% of VN1203-HA S227N+158GlyΔ-inoculated mice, but none in VN1203-HA S227N or VN1203xHK213 HANA-inoculated mice. Viral replication in mouse lungs was comparable at days 1, 3, and 5 ( B), but the day-7 lung titer of VN1203xHK213 HANA was significantly lower than those of the VN1203 or VN1203-HA S227N+158GlyΔ viruses ( P = 0.0043, Tukey's test) ( P = 0.0113, 2-way ANOVA). Although all viruses were detectable in the brain (including olfactory bulbs) at day 7 after inoculation, only VN1203 and VN1203-HA S227N+158GlyΔ replicated to high titers ( B). Detection of VN1203xHK213 HANA virus in mouse brain on day 7 in the absence of neurological signs may be attributable to virus replication in the olfactory bulbs after intranasal inoculation. Alternatively, the efficient transcription and replication activity of VN1203 polymerase complex ( 13) may facilitate the spread of VN1203xHK213 HANA viruses by a route other than blood (viremia was detected only in mice inoculated with VN1203 virus) ( B). To test whether VN1203 virus possessed the greatest ability to spread systemically after respiratory tract infection, we monitored virus replication kinetics after intramuscular (i.m.) inoculation with 100 pfu. All viruses were detectable at the inoculation site (thigh muscle and popliteal lymph node) until day 3, and VN1203 virus remained detectable until day 7 (A). In addition, virus was detected in the inguinal lymph nodes of mice inoculated with VN1203 or VN1203-HAS227N+158GlyΔ virus (B). Virus was detected in the blood only in mice inoculated with VN1203 (C). VN1203 virus was detected in the lungs as early as day 3 and in the brain as early as day 5 after inoculation ( D and E); VN1203-HAS227N+158GlyΔ virus was detected in lungs but not in brain, and the VN1203-HAS227N and VN1203xHK213HANA viruses were not detected in lungs or brain. All mice inoculated with VN1203 (n = 17) died 7–8 days after inoculation; 4 of 17 mice inoculated with VN1203-HAS227N+158GlyΔ and 1 of 7 mice inoculated with VN1203xHK213HANA were euthanized because of hind-limb paralysis, but mice inoculated with VN1203-HAS227N (n = 17) showed no clinical signs (F). At an i.m. dose of 105 pfu, all VN1203-inoculated mice (n = 8) died 7 to 8 days after inoculation; 7 of 8 VN1203-HAS227N+158GlyΔ-inoculated mice, 3 of 8 VN1203xHK213HANA-inoculated mice, and 1 of 8 VN1203-HAS227N-inoculated mice were euthanized because of hind-limb paralysis. All mice that survived the 105 pfu i.m. inoculation were protected from homologous intranasal rechallenge with 103 pfu. The HA and NA genes of virus isolated from two mouse lungs 7 days after i.m. inoculation with 100 pfu of VN1203-HAS227N+158GlyΔ virus were analyzed; the introduced HA mutations were retained and no additional NA mutations were identified. Because VN1203-HAS227N virus showed significantly reduced lethality in mice, we also tested its lethality in domestic chickens. The i.v. pathogenicity indexes of the VN1203, VN1203-HAS227N, and VN1203-HAS227N+158GlyΔ viruses were 3.00, 2.95, and 2.98, respectively. All chickens died within 48 h after inoculation. Overall, our results demonstrated that the VN1203 recombinant viruses with different receptor recognition differed in their ability to spread systemically in mice but remained universally lethal to domestic chickens. Target Cells of the H5N1 Viruses. Systemic spread requires both target cell binding and systemic trafficking. We first compared the cell types bound by the surface glycoproteins of HK213 and VN1203 viruses by immunohistochemistry (IHC) with FITC-labeled viruses and normal mouse tissues ( 29). To perform IHC in a biosafety level 2 laboratory, we used recombinant A/Puerto Rico/8/34 (PR8) viruses carrying the HA (with the basic amino acid motif removed) and NA glycoproteins of HK213 or VN1203 (designated PR8xHK213 HANA and PR8xVN1203 HANA). The overall patterns of attachment of the PR8xHK213 HANA and PR8xVN1203 HANA viruses in the mouse respiratory tract (moderately positive in trachea, bronchi, bronchioles, and alveoli) (see Table S3) were similar to those of A/Vietnam/1194/04 (H5N1) virus ( 29). Viral attachment was observed outside the respiratory tract in endothelial cells of the brain, biliary epithelial cells of the liver, and ciliated ependymal cells lining the brain ventricles ( Table S3 and Fig. S2). The similar attachment patterns of the PR8xHK213 HANA and PR8xVN1203 HANA viruses suggest that the HK213 and VN1203 viruses have similar affinity for these target cells; therefore, the different systemic spread of these viruses is likely to reflect their ability to reach these target cells. Infection of Mouse Monocytes In Vitro and In Vivo. Because systemic spread depends on systemic trafficking and target-cell binding, we next investigated the ability of the H5N1 viruses to infect mouse dendritic cells (DCs). DCs appeared to be good candidates for systemic trafficking vehicles because they are professional antigen-presenting cells that are widely distributed in tissues and can migrate to draining lymph nodes for antigen presentation. The detection of VN1203 virus in the popliteal lymph node and blood after i.m. inoculation supports the role of DCs in facilitating systemic spread. Furthermore, recent studies have shown that DCs are susceptible to H5N1 infection and can potentially disseminate the virus in vivo ( 30, 31). We first infected differentiated mouse bone marrow-derived DCs (BMDCs) in vitro with recombinant VN1203, VN1203-HAS227N, VN1203-HAS227N+158GlyΔ, and VN1203xHK213HANA viruses and with PR8 (H1N1) virus at MOIs of 0.2, 2, or 10 pfu/cell. After 15 h, cells were analyzed by flow cytometry for CD11b (monocyte marker), CD11c (DC marker), and influenza nucleoprotein (NP). At an MOI of 0.2 or 2.0, the percentage of CD11c+NP+ cells was greater (59.4% and 83.6%, respectively) in BMDCs infected with VN1203 than in BMDCs infected with VN1203-HAS227N (9.8% and 63.8%, respectively), VN1203-HAS227N+158GlyΔ (11.9% and 68%, respectively), VN1203xHK213HANA (12.1% and 64.8%, respectively), or PR8 (4.4% and 18.1%, respectively) viruses (A). At MOI = 10, the percentage of CD11c+NP+ cells was similar in all cells infected with the H5N1 recombinants (69.2%–78.7%) but was lower in those infected with PR8 virus (45.1%). To differentiate active viral replication from antigen uptake, we analyzed BDMCs incubated for 1 h or 16 h with live or heat-inactivated (60°C for 15 min) viruses at MOI = 0.2 or 2 pfu/ cell. After 1-h incubation at MOI = 0.2, the percentage of CD11c+NP+ cells was similar in BMDCs incubated with heat-inactivated (0.4–0.8%) and live (0.3–0.7%) viruses. However, after 16-h incubation, the percentage of CD11c+NP+ cells was significantly different in BMDCs incubated with heat-inactivated (0.3–1.2%) and live (19.3–53.2%) viruses. The percentage of CD11c+NP+ cells was highest in BMDCs infected with VN1203 (53.2%), followed by VN1203xHK213HANA (34%), VN1203-HAS227N+158GlyΔ (30.7%), VN1203-HAS227N (20.8%), and PR8 (19.3%). Similarly, after 1-h incubation at MOI = 2.0, the percentage of CD11c+NP+ cells was comparable in BMDCs incubated with heat-inactivated (0.4–3.1%) and live (0.5–6.6%) viruses. The percentage of live CD11c+NP+ cells remained low at 0.2 to 0.3% in BMDCs incubated with heat-inactivated viruses for 16 h; however, the percentage of CD11c+NP+ cells was 68.4%, 64.2%, 58.6%, 58.5%, and 49.8% in BMDCs incubated with live VN1203, VN1203-HAS227N+158GlyΔ, VN1203-HAS227N, VN1203xHK213HANA, and PR8 viruses, respectively. Overall, these results suggest that all 5 viruses tested were able to replicate (produce NP protein) in the infected BMDCs, and that VN1203 virus infects and replicates most efficiently in these cells. We next analyzed the mononuclear infiltrate in mouse lungs 5 days after intranasal inoculation of 100 pfu of the recombinant H5N1 viruses (n = 5 per group). All 4 viruses had been shown to replicate to comparable titers in mouse lungs on day 5 (see D). In total lung homogenate, the mean percentage of NP+ cells was higher in mice inoculated with VN1203 [43.8%±10.0% (SD)] than in mice inoculated with VN1203-HAS227N (37.3 ± 6.6%), VN1203-HAS227N+158GlyΔ (27.0 ± 5.7%), or VN1203xHK213HANA (27.7 ± 4.4%) (P = 0.0039, 1-way ANOVA). Tukey's test showed a significantly higher percentage of NP+ cells after VN1203 inoculation than after VN1203-HAS227N+158GlyΔ (P < 0.01) or VN1203xHK213HANA (P < 0.05) inoculation. The mean percentage of CD11b+NP+ cells was also higher, although not significantly so, after inoculation with VN1203 (54.9 ± 14.0%) vs. VN1203-HAS227N (46.3 ± 8.9%), VN1203-HAS227N+158GlyΔ (39.2 ± 10.0%), or VN1203xHK213HANA (39.5 ± 7.7%) virus (P = 0.0959, 1-way ANOVA). The percentage of CD11c+NP+ cells did not differ and overall >99% of the conventional DC population (CD11chiCD11bint) was NP+. However, the percentage of conventional DCs in total lung homogenate was lowest in VN1203-inoculated (4.5 ± 1.2%) and VN1203-HAS227N-inoculated (4.5 ± 0.8%) mice, followed by VN1203-HAS227N+158GlyΔ-inoculated (7.7 ± 2.2%) and VN1203xHK213HANA-inoculated (6.1 ± 1.5%) mice (P = 0.0108, 1-way ANOVA). In Tukey's test, these percentages differed significantly between mice inoculated with VN1203 or VN1203-HAS227N vs. VN1203xHK213HANA virus (P < 0.05). In a repeated experiment, the number of conventional DCs in the mouse lung 5 days after inoculation was calculated. The mean number of conventional DCs was lowest with VN1203-inoculated mice (84,337.6), followed by VN1203-HAS227N-inoculated (110,697.2), VN1203-HAS227N+158GlyΔ-inoculated (149,171.2), and VN1203xHK213HANA-inoculated (169,913.3) mice. We further differentiated the conventional DC population into NP int and NP hi based on the level of cellular NP expression ( B). As DCs mature, antigen internalization is down-regulated ( 32). Therefore, a high level of NP expression (NP hi) is indicative of cells in which virus is replicating, whereas an intermediate level of NP expression (NP int) is indicative of antigen internalization. The percentage of conventional DCs expressing NP hi was higher in VN1203-inoculated mice than in mice inoculated with VN1203-HA S227N, VN1203-HA S227N+158GlyΔ, or VN1203xHK213 HANA ( P = 0.001, 1-way ANOVA) ( C); the percentage of conventional DCs expressing NP int was lowest in VN1203-inoculated mice ( P = 0.001, 1-way ANOVA) ( D). Overall, the total lung homogenate of VN1203-inoculated mice showed the lowest percentage of conventional DCs but the highest percentage of conventional DCs expressing NP hi (indicating active viral replication). The low proportion of DCs may therefore reflect the death of VN1203-infected cells. These results support the potential mediation of systemic spread by VN1203-infected DCs. Effect of Surface Glycoproteins on Cytokine/Chemokine Production. The pathogenicity of H5N1 viruses is reportedly associated with high levels of proinflammatory cytokines ( 14, 18). There has been evidence suggesting that viral glycoproteins may interact with pattern recognition receptors to induce inflammatory cytokine production ( 33). To determine whether changes in surface glycoproteins affect the level of proinflammatory cytokine production, we assayed IFN-α, IL-6, TNF-α, MCP-1 (CCL2), MIP-1α (CCL3), and IFN-γ in mouse lung homogenate on days 3 and 6 after inoculation with 1,000 pfu of recombinant HK213, HK213x VN1203 HANA, VN1203, or VN1203xHK213 HANA virus ( Fig. S3). The pulmonary levels of MCP-1 and TNF-α differed significantly on days 3 and 6 between mice inoculated with HK213 vs. HK213xVN1203 HANA viruses and between mice inoculated with VN1203 vs. VN1203xHK213 HANA viruses). Therefore, VN1203 HA and NA glycoproteins specifically induce greater production of these cytokines. Virus containing the VN1203 internal genes (VN1203 and VN1203xHK213 HANA) induced higher levels of IFN-α on day 3 and higher levels of TNF-α and MCP-1 on days 3 and 6 than did HK213 and HK213xVN1203 HANA viruses. On day 6, VN1203-inoculated mice had a significantly higher pulmonary level of IL-6 and lower level of MIP-1α and IFN-γ than did other mice. The low MIP-1α level on day 6 suggested the possibility of reduced trafficking of CD8 + T cells into the lungs. Poor IFN-γ induction indicates an inefficient cell-mediated immune response, and is consistent with the comparatively low percentage of conventional DCs observed in the lungs of VN1203-inoculated mice. Influenza virus surface glycoproteins are known to contribute to pathogenicity ( 11, 25– 27). Here, we showed that changes in the HA receptor-binding domain affect the ability of highly pathogenic H5N1 virus to spread systemically, thereby modifying its pathogenicity in a mouse model. The differing ability of VN1203, VN1203-HA S227N, VN1203-HA S227N+158Gly, and VN1203xHK213 HANA viruses to infect BMDC in vitro and conventional DCs in mouse lungs in vivo suggests that the efficient systemic spread of VN1203 may be mediated by the interaction of its HA glycoprotein with specific cellular receptors in the monocyte population. Detection of VN1203 virus in the inguinal lymph node and in blood from the retro-orbital plexus after i.m. inoculation further supports the possibility of viral spread via infected monocytes. A change from α2,3-linked to α2,6-linked sialyl receptor recognition has been observed repeatedly during the evolution of pandemic influenza viruses (i.e., the 1918, 1957, and 1968 pandemics) ( 5, 34). As a consequence, human influenza viruses recognize predominantly α2,6-linked sialyl receptors ( 6). Whereas this change may facilitate efficient human-to-human transmission, reduced affinity for a spectrum of α2,3-linked sialyl receptors may affect pathogenicity. H5N1 viruses continue to pose a serious pandemic threat, but it is not clear whether they would follow the evolutionary path of previous pandemic influenza viruses by gradually acquiring changes in their receptor recognition pattern. In the present study, the differences we observed between VN1203, VN1203-HA S227N, VN1203-HA S227N+158Gly, and VN1203xHK213 HANA in systemic spread cannot be simply explained by the differential usage of α2,3- vs. α2,6-linked sialyl receptors. The introduced HA mutations did not affect the pH for syncytium formation or reduce viral lethality in domestic chickens, but other mechanisms that may explain our differing observations in vivo cannot be excluded. For example, the introduced HA mutations may affect HA susceptibility to protease cleavage in mammalian cells or influence viral transport in the endocytic pathways. However, the location of these HA mutations and the fact that they affect binding affinity for p3′SL and p6′SL leads us to speculate that they affected binding affinity for or recognition of fine glycan structures that cannot be fully characterized by current methods. The glycan array profiles of VN1203 and VN1203-HA S227N viruses ( 2) indicate that both viruses bind to the carbohydrate determinant sialyl lewis x [Neu5Acα2–3Galβ1–4(Fucα1–3)GlcNAc], but only VN1203 binds to sialyl lewis a [Neu5Acα2–3Galβ1–3(Fucα1–4)GlcNAc] ( 2). Both sialyl lewis x and sialyl lewis a are important in leukocyte rolling and cancer cell metastasis ( 35, 36). Hypercytokinemia and high viral load are associated with fatal human H5N1 infection ( 18). We observed that the most lethal virus studied, VN1203, induced an elevated proinflammatory cytokine response in mouse lungs (TNF-α, MCP-1, and MIP-1 α on day 3 after inoculation; IL-6 and MCP-1 on day 6). Infection of monocytes with VN1203 virus may lead to apoptosis of these cells and increased level of proinflammatory cytokine response in mouse lungs. The observation that VN1203 surface glycoproteins specifically induced higher level of TNF-α and MCP-1 suggest that HA and NA may affect viral infectivity in monocytes. Furthermore, infection of the conventional pulmonary DC population would impede DC antigen presentation to T cells and might convey inhibitory signals to T cells ( 37). Lymphopenia has been reported in humans infected with H5N1 virus ( 38) and in mice inoculated with highly pathogenic H5N1 virus ( 21). The internal genes of VN1203 induced higher levels of IFN-α on day 3 and of TNF-α and MCP-1 on days 3 and 6 after inoculation. The polymerase complex of VN1203 is reported to confer virulence in mammals by enhancing replication and transcription efficiency ( 13). In our studies, VN1203 polymerase complex showed significantly greater transcription and replication efficiency than HK213 virus in human lung epithelial A549 cells but not in quail fibroblast QT6 cells (Yen et al., unpublished data). In this study, the VN1203 recombinant viruses replicated more efficiently in mouse lungs (range, 4.8–6.5 log 10TCID 50/ gram tissue) than did HK213 recombinant viruses (range, 3.5–4.8 log 10TCID 50/ gram tissue) on day 1 after inoculation with 100 pfu of virus, regardless of the surface glycoproteins they carried. Therefore, polymerase activity may affect viral replication efficiency in mouse lungs and further modulate the cytokine response. Influenza pathogenicity is a polygenic trait ( 10– 15). Although we show here that the HA glycoprotein can facilitate systemic spread, the role of NA glycoprotein should not be overlooked. Further studies are also needed to elucidate whether VN1203 HA glycoprotein facilitates systemic spread through efficient interaction with specific receptors on monocytes. Recent studies have identified that DC-SIGN, a member of the C-type lectin family, may interact with the HA of H5N1 viruses by facilitating capture and attachment of the virus particles to DCs ( 31, 39). DC-SIGN is known to mediate the HIV-1 infection of T cells ( 40); it also reportedly interacts with the glycoproteins of Ebola virus, hepatitis C virus, dengue virus, measles viruses, and SARS coronavirus ( 37). In our preliminary experiment, recombinant human DC-SIGN, DC-SIGNR, and Langerin did not neutralize VN1203 infection of MDCK cells (data not shown), suggesting that these C-type lectins may not served as the major receptors for entry ( 39). In addition to C-type lectins, interaction between viral glycoproteins and Toll-like receptors 2 (TLR2) and TLR4 have been reported ( 41), and interaction between UV-inactivated H5N1 virus and TLR4 has been recently shown to induce acute lung injury in mice ( 42). Experiments are being planned to further characterize the interaction between HA and DCs. Taken together, our results show that changes in the HA receptor binding domain can alter the ability of H5N1 virus to spread systemically in a mouse model. Our results also suggest that H5N1 infected DCs may serve as vehicles for systemic spread. Viruses. H5N1 isolates were obtained through the World Health Organization Global Influenza Surveillance Network. The 8 gene segments of HK213 virus were amplified by RT-PCR and cloned into plasmid vector pHW2000 ( 43). The plasmids encoding the 8 genes of VN1203 virus have been described ( 13). Amino acid changes were introduced by using the QuikChange site-directed mutagenesis kit (Stratagene). Recombinant HK213 and VN1203 viruses were generated by cotransfecting 293T cells (TransIT-LT1, Mirus) with the appropriate 8 plasmids ( 43), and virus stocks were prepared in embryonated chicken eggs. The full genome sequences of the recombinant viruses were verified. Experiments with H5N1 viruses were conducted in a United States Department of Agriculture-approved biosafety level 3+ containment facility. Binding of Virus to Sialic Acid-Containing Substrates. Virus binding affinity to HRP-conjugated fetuin was tested in a direct solid-phase assay with the immobilized virus ( 44). Virus binding affinity to the synthetic sialyl receptors p3′SL and p6′SL (provided by Dr. Nicolai V. Bovin, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow) was tested by competitive assay based on inhibition of binding to HRP-labeled fetuin ( 45). The dissociation constants ( Kdiss) of virus-receptor analog complexes were calculated as the means of 4 individual experiments. Animal Experiments. The MLD50 was determined by intranasally inoculating groups of 6-week-old female BALB/cJ mice (Jackson Laboratories) with 10-fold serial dilutions of virus in 50 μl of PBS under isoflurane anesthesia. Mice were inoculated i.m. with virus diluted in 100 μl of PBS with 26-gauge needles. Mouse blood and organs were collected and stored at −70°C before titration in MDCK cells. Clinical signs were monitored daily. Mice that developed neurological signs or lost >25% of their original weight were euthanized. The i.v. pathogenicity index was determined in groups of 10 8-week-old chickens by i.v. injection with 0.1 ml of 10-fold dilutions of virus-containing allantoic fluid. Deaths and disease signs were recorded daily for 10 days. All animal studies were conducted under applicable laws and guidelines and were approved by the St. Jude Children's Research Hospital Animal Care and Use Committee. In Vitro Infection of Bone Marrow-Derived DCs. BMDC were generated as described ( 47, 48) by culturing bone marrow cells harvested from the femurs and tibias of C57BL/6 mice in the presence of GM-CSF (200 units/ml) (Peprotech). Half of the medium was replaced every 2 days, and nonadherent cells were collected on day 8. Harvested cells were resuspended (2.5 × 10 6 cells per reaction) in RPMI1640 containing live or heat-inactivated influenza viruses at different MOIs and incubated at 37°C for 1 h. Cells were washed twice with HBSS, plated in 6-well plates, and incubated at 37°C before flow-cytometry analysis. Flow Cytometry. Mouse lung monocyte populations were analyzed after intranasal inoculation of 100 pfu of recombinant VN1203 viruses. Single cell suspensions were prepared by passing lung tissue through a 100-μm nylon mesh (BD). For surface staining, isolated cells (1 × 106) were stained with mAbs to CD11b (monocyte marker) (M1/70; BD) and CD11c (DC marker) (N418; eBiosciences). For intracellular staining, cells were fixed and permeabilized (Cytofix/Cytoperm kit, BD), then stained with a FITC-labeled mAb specific for influenza NP (Argene). The experiments were repeated independently and the representative result is reported. All flow-cytometry data were collected on a BD FACSCalibur (BD) and analyzed by FlowJo software (Tree Star, Inc.). For additional information, see SI Text. We thank the World Health Organization Global Influenza Surveillance Network for providing the H5N1 viruses; Nicolai V. 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