3.4.1 Propagation of virus in embryonated chicken eggs
LPAI viruses were propagated in chicken eggs. For this purpose 10 day-old embry-onated chicken eggs were inoculated with 100 µl virus suspension. Every day the embryos were screened for viability. Dead eggs were stored at 4◦C. After three days the remaining eggs were incubated at 4◦C for at least 2 h. Afterwards the eggs were opened at the side of the air cell and the allantoic fluid was harvested. Aliquots were stored at -80◦C.
3.4.2 Immunoplaque assay
MDCKII cells were seeded in 96 well plates on day prior to infection. The next day the 90 - 100% confluent cells were washed and infected with serial 10-fold dilutions of each virus, 50 µl per well including an uninfected control. The cells were incubated under gentle shaking for 1h at 37◦C. Afterwards the inoculum was removed and the cells covered with methylcellulose containing acetylated trypsin (1:1000). The cells were further incubated for 48h and then fixed using 3% PFA.
The cells were permeabilized with 0.2% Triton X-100 and then incubated with α-NP antibody, 25 µl per well. After 1h at RT the cells were washed and incubated with the secondary antibodyα-mouse-Cy3 for 1h at RT in the dark. The cells were washed again with PBS and water, foci of infected cells were counted under a fluorescence microscope. The viral titer was calculated in ffu/ml.
Titer= mean of foci count
dilution factor×0.05 (3.3)
3.4.3 Virus binding assay
Binding of virus particles was visualized by immunofluorescence microscopy (see sec-tion 3.3.4 for details). Cells seeded on coverslips were fixed, quenched and washed.
Then they were incubated with a virus suspension for 1 h at 4◦C (dilution, application and removal of the virus was performed underneath a laminar flow at BSL 2 conditions).
Afterwards the coverslips were washed and virus binding visualized with antibodies:
H7N7 virus was detected using mouse anti-H7 and anti-mouse-FITC as secondary antibody; for H9N2 a polyclonal anti-H9N2 serum was used with anti-rabbit-FITC as secondary antibody. After 45min at 4◦C cells were embedded in DAPI-mowiol. Binding was analysed by confocal laser scanning microscopy.
4.1 Expression and purification of soluble HAs
The soluble hemagglutinins analysed in this study were generated as follows:
Soluble H7Fc was cloned by Dr. Maren Bohm (Bohm [2010]) and initially tested in my Master Thesis (Sauer 2009). Soluble Fc and his-tagged constructs of the hu-man swine-origin H1 and the 1918 H1 (H1_2009Fc and H1_2009This; H1_1918Fc and H1_1918This) were created and tested by Dr. Meike Erdt (Erdt [2012]).
The full length H1 (porcine), H5 (avian), H9 (avian) and H17 (bat) HAs were modified by PCR with specific primers to delete the transmembrane anchor and cytosolic do-main and to add PacI restriction sites to the sequences. PCR products were restriction digested and ligated into pCGFc and pCGT6his vectors as described in the methods section (3.2). Although all constructs indicated in section 2.6 were cloned, not all of them are yet fully analysed with regard of their binding properties. The solHAs that were used in this work are indicated in table 4.1.
Plasmids of all solHA constructs were transfected into 293T cells. Due to the lack of a membrane anchor, the HAs were secreted into the cell culture supernatant which was collected after 48 h and 72 h post transfection and cleared by centrifugation. After purification and concentration by FPLC the quality of the solHAs was determined by control Western blot and control binding tests (section 3.3.1).
Maximum expression of solHAs was observed in medium containing 3% FCS with peak
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Table 4.1:List of soluble HAs used in this work(* indicates the HAs cloned by Maren Bohm and ** indicates HAs cloned by Meike Erdt)
Name Vector Primer No. Restriction Enzymes bp / aa
H7Fc* pCGFc / PacI / XbaI 2346/782
H7T6his pCGT6his 16, 17 PacI / XbaI 1722/574
H9Fc pCGFc 8, 9 PacI 2325/775
H9T6his pCGT6his 8, 9 PacI 1701/567
H5Fc pCGFc 1, 2 PacI 2367/789
H1_2009Fc** pCGFc / PacI 2352/784
H1_1918Fc** pCGFc / PacI 2331/777
H1_WFc pCGFc 18, 19 PacI 2367/789
H17Fc pCGTFc 30, 31 PacI 2364/788
values at 48 h post-transfection. Transfection and purification protocols for FPLC have been established during this thesis together with Dr. Katarina Shawhan.
In fig.4.1, a Western blot of all Fc-tagged solHAs is shown. In all solHA prepara-tions a high molecular weight band above 300 kDa is visible which may represent an oligomeric form. A lower band at around 250 kDa is the strongest and may be a highly glycosylated dimeric HAFc protein as the average predicted molecular weight for ungly-cosylated Fc-tagged HAs lies around 86 to 90 kDa with 4 to 11 potential N-glycosylation sites. The negative control FcATG appears under non-reducing conditions as a 50 kDa band of the dimeric form.
Under reducing conditions, the oligomers are dissociated into monomers. In this case, H7Fc is mainly observed as a band at around 60 kDa which represents the HA2 sub-unit carrying the Fc-tag (fig. 4.2). Under these conditions, the second subsub-unit, HA1, is not detected because immunostaining of the WB was directed against the Fc-tag. In Coomassie-stained gels, both HA1 and HA2-Fc are visible only in a broad band, be-cause both have about the same size (not shown). H9Fc is not cleaved by endogenous proteases of the cells and appears only as a higher band at around 100 kDa, which represents the uncleaved precursor HA0. FcATG is also separated into monomers by DTT treatment (fig.4.2).
His-tagged versions of solHAs were cloned for all subtypes. A Western blot of H7T6his
and H9T6his stained with mouse-anti-his antibodies and peroxidase -labeled anti-mouse-IgG antibodies is shown in fig. 4.3. When analysed under non-reducing conditions, again a high molecular weight band appears at the top of the blot. H7T6his forms bands with an estimated size of around 200 kDa and about 110 kDa respectively. H9T6his shows as several bands in the range of 240 to 180 kDa that may represent proteins differing in extent of glycosylation. A very small band at 80 kDa is also visible.
Figure 4.1: Western blot of Fc-tagged solHA proteins and FcATG under non-reducing conditions. The blot was immunostained with anti-human-IgG-PO.
Figure 4.2: Western blot of H9Fc, H7Fc and FcATG under reducing conditions. The blot was immunostained with anti-human-IgG-PO
Figure 4.3: Western blot of H7T6his and H9T6his. The blot was immunostained with mouse anti-his and anti-mouse-PO.