Virus binding

SolHAs are of course an artificial system. As full length HAs form trimers and Fc-tagged HAs possibly only form dimers it was necessary to compare the artificial ver-sion to the in vivo version. Therefore binding tests with intact virions (with a virus

titer of 5x10⁵ ffu/ml) was performed under the same conditions as solHA binding was analysed. Virus incorporated HAs were stained with specific H7 and H9 antibodies (fig.4.15). The H9N2 strain used was the strain from which H9Fc is derived. As a con-trol for H7Fc a different H7N7 strain had to be used as H7Fc originates from a HPAI strain that has to be handled under BSL-3 conditions.

H7N7 bound weaker to all tested cells lines (MDCKII, Calu-3, A549 and CLEC213) than did the H9N2 virus. Unlike H7Fc, H7N7 did bind to A549 cells. This might be due to the presence of more HA trimers in each virion which could make it possible to give a detectable signal after binding to a scarcely expressed receptor that would not be detectable when a single solHA has bound.

Virus binding of NPTr and pCKC cells could not be performed as the polyclonal anti-H9N2 serum caused a high background staining. This problem could not be solved so far.

Nevertheless: binding of H7Fc and H9Fc is comparable to that of H7N7 and H9N2 virus in terms of intensity and distribution (see section 4.4).

5.3 Binding to respiratory tissue sections

Continuous cell lines have been immortalized and during this process expression of proteins and sialic acids changes. Thus they are only to a certain extent representative for the species and tissue they were isolated from. To analyse what tissues are suscep-tible for IAV binding and what cells within this tissues are targets for IAV attachment, primary material had to be used. Therefore, cryosections of chicken, turkey and pig trachea and pig lung were prepared and binding tests with solHAs performed.

Lectin staining of those tissues revealed an abundance of α2,3-linked sialic acids in chicken and turkey and of α2,6-linked sialic acids in the porcine respiratory tract. The respective other linkage type was also found but rather scarcely on the epithelium.

Es-pecially in the pig, MAAII staining was found with high intensities in e.g. basal cells or the submucosa. The presence of both sialic acid linkage types in the porcine respira-tory epithelium is consistent with other reports (Ito et al. [1998]) and results obtained by a independent study in our laboratory (Punyadarsaniya et al. [2011]) but other authors (Van Poucke et al. [2010], Nelli et al. [2010]) found no or only little MAAII staining in the porcine trachea and bronchus. The abundance of α2,3-linked sialic acids in the chicken trachea that was observed in this thesis does not match findings in other stud-ies, where alsoα2,6-linked sialic acids can be found in the chicken trachea (Yu [2010], Kuchipudi et al. [2009]). In these studies 4 or 8 week old SPF chicken were used which may explain the difference to our findings with embryonic tissues as sialic acid expres-sion changes during development. The presence of both sialic linkage types in turkey trachea sections with predominance of α2,3-linked sialic acids was also reported by others (Kimble et al. [2010], Costa et al. [2012]).

As expected, H7Fc and H9Fc both bound well to the respiratory epithelium of chicken and turkey as both solHAs were derived from strains isolated from chicken and both subtypes are known to infect chicken and turkey (Dong et al. [2011], A. S. Gambaryan et al. [2012]). H9Fc binding could also be observed in the submucosa. H9Fc also showed weak binding on the porcine respiratory tissue, especially in the trachea. H7Fc only bound very weakly to the porcine trachea but not in the lung. Binding tests with H7Fc and H9Fc clearly showed that there are receptors for these subtypes in the porcine trachea and for H9 in the lung as well despite the scarcity of α2,3-linked sialic acids as suggested by lectin staining. H9 viruses appear not only be able to bind to porcine respiratory epithelium but also can to replicate efficiently in pigs as H9N2 viruses circulate in Southeast China (Peiris et al. [2001], Lin et al. [2000]). Pun-yadarsaniya et al. [2011] showed that both H9N2 and H7N7 viruses can replicate in porcine precision cut lung slices though only to lower titers compared to porcine strains.

The discrepancy between infection of H7N7 and binding of H7Fc may be attributed to

the higher avidity of virus incorporated HAs compared to single dimers. Binding tests with a higher amount of H7Fc might show binding in the porcine lung.

H5Fc bound to chicken, turkey and swine trachea (fig.4.17). Strongest binding was seen in the turkey. As this strain was isolated from turkey and therefore the HA might be adapted to this epithelium. This adaptation to an epithelium expressing both sialic acid linkage types may include adaptation to sialoglycans that are present in both turkeys and pigs.

The two human subtypes in this study also bound to the turkey trachea rather strong, but probably not to chicken respiratory epithelium. Infection of turkeys with pandemic 2009 H1N1 viruses has been reported and local outbreaks in Chile occurred in 2010 (Mathieu et al. [2010]). Infection was asymptomatic when turkeys were experimentally infected via intranasal or intraocular routes (Russell et al. [2009]). Intrauterine and intracloacal infection however led to diarrhea and decrease in egg production (Pantin-Jackwood et al. [2010]). In the turkey respiratory tract, 2009 H1N1 viruses apparently are able to attach and maybe to infect cells but are unable to establish efficient repli-cation. Pandemic 2009 H1N1 and 1918 H1N1 viruses are not pathogenic in chicken (Babiuk et al. [2010]) and as shown here, the respective HAs do not bind to the embry-onic chicken trachea.

Human solHAs also bound to the swine trachea. In the concentration used in this thesis, H1_2009Fc did not bind to the swine lung but H1_1918Fc did bind to single cells. Thus although both H1_2009Fc and H1_1918Fc share the same amino acids in their RBS they behave differently in the porcine lung. There appears to be a sufficient number of receptors for H1_1918Fc to be present but not for H1_2009Fc to show a clear binding. Experimental infection of pigs with 1918 H1N1 virus only results in mild disease with fever and mild respiratory symptoms (Weingartl et al. [2009]). Differences between H1_2009Fc and H1_1918Fc binding to the porcine lung may again be ex-plained by amino acid substitutions outside the RBS as described previously in section

5.2 and de Vries et al. [2011] that results in changed receptor binding properties and efficiencies. Binding studies with a higher concentration of solHAs might reveal binding of H1_2009Fc to swine lung sections.

The only porcine H1 did hardly bind to avian respiratory tissue of either species al-though the other H1 subtypes were able to bind in turkey sections. H1_WFc pos-sesses a RBS amino acid composition resulting in α2,6-linkage specificity. Although turkey trachea shows SNA staining, the α2,6-linked sialoglycans might not be suit-able as receptors for this subtype or are only recognized with low affinity. Although the porcine respiratory epithelium presents a lot ofα2,6-linked sialic acids, H1_1918Fc and H1_WFc only bound to single cells in the porcine lung. Thus, among the expressed α2,6-linked sialic acids that are stained by SNA only few are able to serve as receptors, or the receptors can in this case not all be detected by SNA. Yet, studies with H1_WFc have to be repeated to confirm these results.

Another aspect of H1_WFc binding is the adaptation to Neu5Gc. This could be one of the reasons why H1_WFc did not bind to turkey and chicken trachea as this modifica-tion of neuraminic acid is abundant in swine but not in birds and humans. Adaptamodifica-tion of H1_W to pigs may have resulted in adaptation to Neu5Gc terminated sugars. For some equine H7 strains it is known that adaptation to Neu5Gc occurs (A. S. Gambaryan et al. [2012]) and Neu5Gc binding is important for successful replication in ducks (Ito &

Kawaoka [2000]).

For all six subtypes the porcine trachea did show binding of solHAs. So at least con-cerning receptor binding, the porcine trachea is susceptible for IAV infection with dif-ferent subtypes and therefore could serve as the famous mixing vessel for reassort-ment of viruses or as intermediate host allowing the adaptation to different receptors.

This does not seem necessary for attachment as binding is already possible but fur-ther adaptation may increase infectivity or transmissibility and fur-therefore increase the zoonotic threat as successful replication in a host is not only a matter of attachment.

The same holds true for turkey trachea as both avian and human strains were able to bind here.

Future analyses have to show to what kind of cells solHAs bind. Especially the combi-nation H1_1918Fc, H1_WFc and porcine lung, where only single cells showed binding, is of great interest concerning the question if a specific cell type harbors the receptors for these HA subtypes (e.g. ciliated or mucus producing cells). On most other tissues binding of solHAs is mostly distributed over the epithelial surface. Use of lower con-centrations might reveal differences e.g. areas or cells that the HAs bind stronger to.

It would also be interesting to extend the binding studies to the lung of adult turkey and chicken or to perform these tests on human tissue samples to investigate binding ability of different HA subtypes in these tissues. Receptor binding however is only one essential step in a successful infection. Other viral components like the polymerase or NS1 protein and interaction with host factors are important for establishing infection in a cell. High binding affinity to a cell or a tissue does not necessarily mean that a virus can successfully replicate in this cell and changes outside the RBS of the HA might be important for transmission to new hosts (Baigent & McCauley [2003]).