Targeted infection of endothelial cells by avian influenza virus A/FPV/Rostock/34 (H7N1) in chicken embryos

Abstract

The tissue tropism and spread of infection of the highly pathogenic avian influenza virus A/FPV/Rostock/34 (H7N1) (FPV) were analyzed in 11-day-old chicken embryos. As shown by in situ hybridization, the virus caused generalized infection that was strictly confined to endothelial cells in all organs. Studies with reassortants of FPV and the apathogenic avian strain A/chick/Germany/N/49 (H10N7) revealed that endotheliotropism was linked to FPV hemagglutinin (HA). To further analyze the factors determining endotheliotropism, the HA-activating protease furin was cloned from chicken tissue. Ubiquitous expression of furin and other proprotein convertases in the chick embryo indicated that proteolytic activation of HA was not responsible for restriction of infection to the endothelium. To determine the expression of virus receptors in embryonic tissues, histochemical analysis of alpha2,3- and alpha2,6-linked neuraminic acid was carried out by lectin-binding assays. These receptors were found on endothelial cells and on several epithelial cells, but not on tissues surrounding endothelia. Finally, we analyzed the polarity of virus maturation in endothelial cells. Studies on cultured human endothelial cells employing confocal laser scanning microscopy revealed that HA is specifically targeted to the apical surface of these cells, and electron microscopy of embryonic tissues showed that virus maturation occurs also at the luminar side. Taken together, these observations indicate that endotheliotropism of FPV in the chicken embryo is determined, on one hand, by the high cleavability of HA, which mediates virus entry into the vascular system, and, on the other hand, by restricted receptor expression and polar budding, which prevent spread of infection into tissues surrounding endothelia.

FIG. 1

Spread of FPV and virus N in the chick embryo. Embryonated eggs were infected with 10³ PFU via the allantoic route. At 17 h (FPV) or 40 h (N) p.i., cryosections were prepared that were subjected to autoradiography after in situ hybridization with [³⁵S]UTP-labeled riboprobes directed against mRNA of H7 HA (a) and H10 HA (c). Sections of uninfected chick embryos were used as controls (b and d). Exposure time for autoradiography was 8 h.

FIG. 2

Localization of FPV-infected cells in embryonic tissues by in situ hybridization. Bright-field 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 counterstained by hematoxylin-eosin. (a) Blood vessel (arrowheads indicate infected blood cells); (b) lung; (c) stomach; (d) heart; (e) liver; (f) spleen. Magnification, ×57.

FIG. 3

Identification of endothelia by in situ hybridization using VEGF receptor 2 as a marker. Dark-field microscopy showing autoradiographic silver grains representing bound VEGF receptor 2-specific mRNA in lung and spleen of chick embryos. After in situ hybridization with a radioactive probe directed against mRNA of VEGF receptor 2, the slides were covered by photoemulsion, exposed for 30 days, and developed. Magnification, ×71.

FIG. 4

Cleavage of HA by chicken furin. LoVo cells (35) were infected with VV:gfur and VV:HAwt (22), each at an MOI of 10. Virus inocula were replaced by DMEM without FCS 1 h after infection. At 4 h after infection, LoVo cells (diameter of the culture dishes, 35 mm) were starved for methionine for 1 h and then labeled with 100 μCi of [³⁵S]methionine (1,000 Ci/mmol; Amersham, Braunschweig, Germany) for 3 h in 0.5 ml of methionine-free MEM. The medium was replaced by MEM containing nonradioactive methionine, and incubation was continued for an additional hour. Cells were lysed in radioimmunoprecipitation buffer. After immunoprecipitation with anti-FPV or anti-hfur rabbit serum (final dilution, 1:500) and protein A-Sepharose CL-4B (Sigma, Deisenhofen, Germany), proteins were analyzed by SDS–10% PAGE under reducing conditions. Sizes are shown in kilodaltons.

FIG. 5

Localization and identification of FPV and proprotein convertases in feather germs by in situ hybridization. For the analysis of proprotein convertases, cryosections of uninfected chicken embryos were incubated with [³⁵S]UTP-labeled riboprobes directed against mRNA of the proteases. Slides were covered by photoemulsion, exposed for 21 days, developed, and inspected by dark-field microscopy. For analysis of FPV-infected cells, cryosections prepared at 17 h p.i. were subjected to in situ hybridization with an HA-specific probe, covered with photoemulsion, exposed for 2 days, developed, stained with hematoxylin-eosin, and inspected by bright-field microscopy. Magnification, ×75.

FIG. 6

Replication of FPV in HUVEC. (A) Confluent cell layer of HUVEC was infected with egg-grown FPV at an MOI of 1. Release of virus was measured by hemagglutinating activity in the supernatant. (B) Autoradiography of [³⁵S]methionine-labeled proteins of virus particles purified from the supernatant of infected HUVEC. At 20 h p.i., virus in the cell supernatant was centrifuged through a 20% sucrose cushion, and purified virus was disrupted in sample buffer containing 2% 2-mercaptoethanol and separated on a 10% polyacrylamide gel followed by autoradiography. Sizes are shown in kilodaltons.

FIG. 7

Histochemical analysis of NeuAc-α2,3-Gal and NeuA-cα2,6-Gal in the lung and the liver of chicken embryos by lectin binding. Cryosections of uninfected chicken embryos were incubated with MAA, specific for NeuAc-α2,3-Gal, or SNA, specific for NeuAc-α2,6-Gal, as described in Materials and Methods. Bound lectins were identified using the DIG-glycan differentiation kit (Boehringer). Magnifications: ×75x and ×150 (liver). Symbols in lung sections indicate endothelial cells (solid arrowheads), mesenchymal cells (stars), alveolar and epithelia (open arrowheads). Symbols in liver sections indicate endothelial cells (solid arrowheads), Kupffer cells (open arrowheads), hepatocytes (asterisks), and sinusoids (arrows).

FIG. 8

Electron micrograph of an FPV-infected endothelial cell from a heart capillary. At 18 h p.i., the heart of an infected chicken embryo was prepared for transmission electron microscopy. (Top) Numerous virus particles bud from the luminal side of the endothelial cell. (Bottom) The section boxed in the top panel at higher magnification.

FIG. 9

Polarized expression of HA at the luminal side of FPV-infected HUVEC. (A) HUVEC were grown on Transwell filters. At 4 h p.i. with FPV, they were analyzed by confocal laser scanning microscopy using an HA-specific monoclonal antibody (HA1-A11H7) for immunofluorescence labeling. Magnification, ×630. (B) Distribution of viral glycoproteins on the apical (a) and basolateral (b) surfaces of HUVEC infected with either FPV or VSV. Surface proteins were labeled by domain-specific biotinylation and immunoprecipitated using the monoclonal antibody against FPV HA or a polyclonal antiserum against VSV (for details, see Materials and Methods).