Differences in the tissue tropism to chicken oviduct epithelial cells between avian coronavirus IBV strains QX and B1648 are not related to the sialic acid binding properties of their spike proteins

Mork et al. Veterinary Research 2014, 45:67 http://www.veterinaryresearch.org/content/45/1/67 RESEARCH VETERINARY RESEARCH Open Access Differences in the tissue tropism to chicken oviduct epithelial cells between avian coronavirus IBV strains QX and B1648 are not related to the sialic acid binding properties of their spike proteins Ann-Kathrin Mork1, Martina Hesse1, Sahar Abd El Rahman2, Silke Rautenschlein3, Georg Herrler1 and Christine Winter1* Abstract The avian coronavirus (AvCoV) infectious bronchitis virus (IBV) is a major poultry pathogen. A characteristic feature of IBV is the occurrence of many different strains belonging to different serotypes, which makes a complete control of the disease by vaccinations a challenging task. Reasons for differences in the tissue tropism and pathogenicity between IBV strains, e.g. a predilection for the kidneys or the oviduct are still an open question. Strains of the QX genotype have been major pathogens in poultry flocks in Asia, Europe and other parts of the world. They are the cause of severe problems with kidney disease and reproductive tract disorders. We analysed infectivity and binding properties of the QX strain and compared them with those of the nephropathogenic strain B1648. As most IBV strains do not infect permanent cell lines and show infection only in primary chicken cells of the target organs, we developed a culture system for chicken oviduct explants. The epithelial cells of the oviduct showed a high susceptibility to infection by the QX strain and were almost resistant to infection by the nephropathogenic B1648 strain. Binding tests with isolated primary oviduct epithelial cells and soluble S1 proteins revealed that S1 proteins of two IBV strains bound with the same efficiency to oviduct epithelial cells. This attachment was sialic acid dependent, indicating that the sugar binding property of IBV spike proteins is not the limiting factor for differences in infection efficiency for the oviduct of the corresponding viruses. Introduction Coronaviruses are pathogens of birds and mammals including humans. The avian coronavirus (AvCoV) infectious bronchitis virus (IBV) as a representative of the Gamma-coronavirus genus infects mainly chickens and other galliforme birds. Within the species AvCoV, there are many strains belonging to different serotypes, genotypes and/or different pathotypes. Some of these strains cause only respiratory disease whereas other strains can spread to other organs like the kidneys and the reproductive tract (reviewed in [1]). The clinical manifestations of IBV in the kidney and the oviduct are of high economic importance in the poultry business. When the kidneys of young broilers are affected, mortality rates may be as high as 60% [2]. An infection of the reproductive tract * Correspondence: 1 Institute of Virology, University of Veterinary Medicine Hannover, Bünteweg 17, 30559 Hannover, Germany Full list of author information is available at the end of the article may have severe implications comprising a drop in egg production, bad egg quality and the occurrence of so-called false layers. Viruses of the QX genotype have been related to problems in layer flocks causing cystic oviducts and inducing false layers [3,4]. The chicken oviduct is a large organ, where secretion of egg white and the egg shell development takes place. It is divided into four different functional parts, including the infundibulum, the magnum, the isthmus and the uterus. In all four parts, a sheet of epithelial cells forms the outer cell layer facing the lumen of the oviduct. Whether epithelial cells of the different segments of the oviduct differ in their susceptibility to infection by IBV or whether IBV strains differ in the ability to infect oviduct epithelial cells is not known. Recent publications discuss a high nephropathogenic potential of the QX strain [5], indicating that this strain has a broad tissue tropism in the bird, which may- at least in part- explain the high pathogenicity. The © 2014 Mork et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Mork et al. Veterinary Research 2014, 45:67 http://www.veterinaryresearch.org/content/45/1/67 B1648 strain has a predilection for the kidneys and has been shown to reproducibly induce kidney disease [6]. An involvement of the reproductive organs in a B1648 infection has not been described. It has been reported that differences in the organ and cell tropism and thus differences in the pathogenicity of IBV strains may be associated with differences in the binding properties of their spike proteins [7,8]. As the binding to susceptible host cells is the first important step in a virus life cycle, this would partly explain why some strains are able to spread to kidneys and/or to the oviduct of chickens. For several coronaviruses, the receptors have been identified. For IBV no cellular protein is known to function as a cellular receptor, but alpha 2,3-linked sialic acids serve as receptor determinants for this virus [9]. The importance of sialic acids for the infection of chicken host cells was shown for both, the QX strain and the B1648 strain [10,11]. We analysed in this study whether the differences in the ability of the two viruses to infect oviduct epithelial cells were related to differences in binding properties of their spike proteins. Materials and methods Viruses Virus stocks of the IBV strains QX and B1648 were obtained by propagation in specific pathogen free embryonated chicken eggs (VALO SPF, Cuxhaven, Germany). The allantoic fluid was harvested, clarified by low–speed centrifugation and stored at −80 °C. The viral titer was determined by titration in primary chicken embryo kidney cells. The IBV strain QX was kindly provided by Hans Christian Philipp (Lohmann Tierzucht, Cuxhaven, Germany). The IBV strain B1648 was kindly provided by Dave Cavanagh (Institute for Animal Health, Compton, UK). S1 sequences can be found in Additional file 1. Cells Primary chicken embryo kidney cells were prepared from 20 day old SPF chicken embryos as described previously [9]. Epithelial cells of the chicken oviduct from 16–19 weeks old chickens were isolated by opening the magnum segment of the oviduct longitudinally and cutting it into small pieces. After incubation with 0.4 mg protease from Streptomyces griseus, Type XIV (Sigma-Aldrich, Steinheim, Germany)/mL medium for 3 h, the remaining tissue was removed and the cells in the supernatant were pelleted by centrifugation. The pellet was resuspended with Dulbecco’s modified (...truncated)