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 HAactivating 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 ␣2,3-and ␣2,6-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.

Spread of Infection and Lymphocyte Depletion in Mice Depends on Polymerase of Influenza Virus

“…Using another HPAIV of subtype H7, we have previously shown that endotheliotropism plays a crucial role in systemic infection of the chicken embryo. 22,31 In the present study, there was no evidence for infection of endothelia in any of the analyzed organs. Thus, endotheliotropism can be ruled out as a mechanism promoting systemic spread of SC35M infection in mice.…”

“…22 Linearization of this plasmid with HindIII and subsequent T7 RNA polymerase transcription produced an antisense RNA probe suited to detect NP-specific RNA. Control RNA probes were obtained from the vector containing the dual-promoter plasmid of coxsackievirus B3 (pCVB3-R1).…”

Receptor-Binding Profiles of H7 Subtype Influenza Viruses in Different Host Species

“…Given that both selectins and their ligands are evolutionary conserved molecules, one can predict expression of Su-SLe x on avian endothelial cells. Interestingly, three previous reports described a strong HA-dependent tropism of H5 and H7 poultry influenza viruses to endothelial cells of chicken embryo cells (13) and to cultured human pulmonary microvascular endothelial cells (45,69). Thus, enhanced binding of poultry influenza viruses to Su-SLe x and other sulfated and fucosylated sialyloligosaccharide sequences could have emerged as an adaptive change required for viral endotheliotropism in birds, a wellknown feature of highly pathogenic poultry influenza viruses with H5 and H7 HA (28).…”

Influenza A Virus Infections in Swine

“…2 When prominent, this leakage corresponds to extremely high cytokine levels in the tissues (cytokine storm). 162 Although some studies have described receptors for influenza virus on the surface of human endothelial cells 170 and virus infection of avian 28,65 and human endothelial cells, 175 these studies suggest the endothelial cell damage is due to cytokines. Influenza virus infection of endothelial cells in swine lungs has not been documented.…”