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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 Mork 1 Martina Hesse 1 Sahar Abd El Rahman 2 SilkeRautenschlein 3 Georg Herrler 1 and Christine Winter 1 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 infec tious 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 geno types 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 reproduct ive 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 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 nephro pathogenic 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 Correspondence Christine Winter tiho hannover de 1 Institute of Virology University of Veterinary Medicine Hannover B nteweg 17 30559 Hannover Germany Full list of author information is available at the end of the article VETERINARY RESEARCH 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 recep tors 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 embryo nated 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 kid ney 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 segmentoftheoviductlongitudinallyandcuttingit 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 Eagle medium and Ham sF12 1 1 supplemented with 5 fetal bovine serum Biochrom Berlin Germany 1 chicken serum Sigma Aldrich Steinheim Germany 1 nonessential amino acids PAA Pasching Austria 1 Penicillin Streptomycin PAA Pasching Austria 0 01 Gentamycin PAA Pasching Austria 0 01 Amphothericine B Sigma Aldrich Germany filtered through 100 uni03BCmcellstrainer and seeded in tissue culture flasks 75 cm 2 After incuba tion for 2 h the cells were seeded in 6 well plates and incubated at 37 C and 5 CO 2 Preparation of oviduct explants The oviduct was aseptically collected from 16 to 18 weeks old premature SPF chicken After washing the oviduct with phosphate buffered saline PBS the different segments of the oviduct infundibulum magnum isthmus uterus were cut into approximately 5 mm thick rings and incubated at 37 C For cultivation we used the same medium mentioned above The next day the explants were used for infection studies with the strains QX and B1648 All experiments were performed in accordance with German animal welfare regulations Viability of oviduct explants In a live and dead staining LIVE DEAD Viability Cyto toxicity Kit Invitrogen Darmstadt Germany we tested the oviduct rings for viability Living cells are stained in green dead cells are stained in red Additionally we analyzed the oviduct rings for ciliary activity under the light microscope Preparations of cryosections of infected oviduct explants The explanted oviduct rings were infected 24 h post preparation with the IBV strains IBV QX and B1648 at a titer of 1 10 4 foci forming units FFU ring in duplicates in each of in total three experiments The infected rings were incubated for 1 h at 37 C on a shaker After 12 h 24 h and 48 h the rings were mounted on filter papers with tissue freezing medium Jung Heidelberg Germany and were subsequently frozen in liquid nitrogen and stored at 80 C until they were sectioned 10 uni03BCm with a cryostat Reichert Jung Nu loch Germany Immunofluorescence analysis The cryosections were fixed with 3 paraformaldehyde for 20 min and subsequently permeabilized with 0 2 Triton X 100 followed by three washing steps with PBS To detect infected cells a monoclonal antibody Ch IBV 48 4 Prionics Lelystad the Netherlands directed against the N protein of IBV in a dilution of 1 100 was used Bound antibodies were visualized by FITC labeled anti mouse antibodies Sigma Aldrich Steinheim Germany All antibodies were diluted in 1 bovine serum albumin Roth Karlsruhe Germany and incubated with the sections for 1 h at room temperature in a humid incuba tion chamber Fluorescence microscopy was performed with a Nikon Eclipse Ti Microscope Mork et al Veterinary Research 2014 45 67 Page 2 of 10 http www veterinaryresearch org content 45 1 67 Neuraminidase treatment of oviduct explants Explants of the oviduct had been prepared as described above and 24 h post preparation the explants were treated with each 300 mU neuraminidase from Clostridium perfringens Sigma Aldrich Steinheim Germany for 1 h at 37 C After careful washing of the explants they were infected with 1 10 4 FFU explant of IBV QX 24 hours post infection hpi the explants were snap frozen in liquid nitrogen and were used for cryosection preparation Lectin staining of oviduct epithelial cells To detect alpha 2 3 linked sialic acids we used the lectins Maackia amurensis agglutinin MAA in its two isoforms MAA I and MAA II Vector Laboratories USA The MAA I lectin was FITC labeled To detect alpha 2 6 linked sialic acids we used the lectin Sambucus nigra agglutinin SNA labeled with FITC Vector Laboratories USA Cryosections of the different oviduct parts were incubated with the indicated lectins for 1 h at room temperature MAA II was used after preincubation of sections with an Avidin Biotin Blocking kit from Vector Laboratories USA Detection of bound MAA II lectins was carried out with streptavidin labeled with Cy3 Sigma Aldrich Preparation of soluble spike proteins cDNA of the S1 subunits was generated by RT PCR of the indicated strains and cloned into the vector pCG1 Fc to obtain fusion proteins consisting of the S1 gene part AA 1 536 for B1648 and AA 1 524 for QX fused to the human IgG Fc domain For the production of soluble spike proteins BHK 21 cells were transfected using poly ethylenimine Polysciences Eppelheim Germany with the plasmids following the instructions of the manufac turer Supernatants containing the soluble proteins were harvested 24 h and 48 h post transfection To concentrate the proteins in the supernatant Amicon Ultra 15 Centrifuge Filter devices were used Aliquots of the soluble proteins were stored at 20 C Binding assay on cryosections of the oviduct To analyze binding of the soluble spike proteins of the strains QX and B1648 we incubated the soluble proteins on cryosections of the oviduct at 4 C for 1 h To test if binding is sialic acid dependent we incubated the cryo sections with 300 mU neuraminidase from Clostridium perfringens Sigma Aldrich Steinheim Germany for 1 h at 37 C before the incubation with the soluble proteins Binding was detected with a secondary anti human Fc Cy 3 labeled antibody in a concentration of 1 750 Sigma Aldrich Steinheim Germany Binding of virus particles Allantoic fluid containing 3 10 5 FFU of either QX or B1648 and 3 mL allantoic fluid of uninfected SPF chicken eggs was ultracentrifuged through a 25 sucrose cushion at 100 000 g for 2 h at 4 C The pelleted virus particles were resuspended in PBS and applied to cryosections of the isthmus part of the chicken oviduct for 1 h at room temperature After three washing steps a monoclonal anti body directed against the spike protein Prionics Lelystad the Netherlands and followed by a FITC labeled second ary antibody was used to detect bound virus particles Flow cytometric analysis of isolated oviduct epithelial cells with bound spike proteins To quantify the binding of soluble S1 proteins to iso lated chicken oviduct epithelial cells the isolated cells were seeded in 6 well plates Greiner Frickenhausen Germany The next day the cells were washed with PBS and detached using 400 uni03BCL trypsin and the cells were subsequently resuspended in PBS containing 1 0 bovine serum albumin After pelleting by centrifugation at 1000 rpm for 5 min the cells were incubated with the solubleS1proteinsfor1hat4 Cinanoverhead shaker After 3 washing steps with PBS containing 1 0 BSA and pelleting of the cells by centrifugation at 1000 rpm for 5 min after every washing step the cells were incubated with an anti human PE conjugated secondary antibody in a concentration of 1 200 Beckman Coulter Marseille France for 1 h at 4 C in an overhead shaker After three more washing steps with PBS containing 1 0 BSA the cells were directly subjected to flow cytometric analysis on a Beckman Coulter Epics XL flow cytometer and analyzed using EXPO32 analysis software Results Epithelial cells of the oviduct are highly susceptible to the QX strain Explant cultures of premature oviducts were prepared and analysed for their viability with a live dead staining kit The majority of the epithelial cells were alive for more than 72 h data not shown An advantage of the preparation of this oviduct explants is that the epithelial cells are in the same arrangement as in vivo The oviduct explants of the different segments infundibulum mag num isthmus and uterus were infected 24 hours post preparation with 10 4 FFU of either of two IBV strains QX and B1648 At 12 24 and 48 hpi the organ explants were frozen in liquid nitrogen and cryosections were prepared and stained for IBV antigen We observed clear differences between the IBV strains The epithelial cells of all segments were highly susceptible to infection with the QX strain which led to a high antigen detection rate and an increasing cytopathogenic effect over the three analysed time points Infection with the B1648 strain was only detected in the infundibulum at very low rates Figure 1 and Table 1 Mork et al Veterinary Research 2014 45 67 Page 3 of 10 http www veterinaryresearch org content 45 1 67 Figure 1 Infection of oviduct explants with IBV strains The infundibulum magnum isthmus and uterus were infected with IBV QX and IBV B1648 Rings were fixed at 12 hpi The nuclei were stained with DAPI blue The infection was detected with a monoclonal antibody against the N protein and a FITC labeled secondary antibody green Scale bar 50 uni03BCm Table 1 Infection of chicken oviduct explants with the IBV strains QX and B1648 IBV QX 12 hpi IBV B1648 12 hpi IBV QX 24 hpi IBV B1648 24 hpi IBV QX 48 hpi IBV B1648 48 hpi Infundibulum Magnum Isthmus Uterus Infection score no infection detectable infection of 1 10 cells infection of 11 25 cells infection of 26 and more cells per cryo section Mork et al Veterinary Research 2014 45 67 Page 4 of 10 http www veterinaryresearch org content 45 1 67 Infection of oviduct epithelial cells with QX is sialic acid dependent To analyse the importance of sialic acids for the infection of oviduct epithelial cells by IBV explant cultures of the oviduct were mock treated or treated with neuraminidase to release sialic acids from the cell surfaces Desialylated and control samples were infected with 10 4 FFU of the QX strain At 12 hpi the explants were frozen and cryosections were prepared and stained for the presence of IBV antigen After neuraminidase treatment of the explants hardly any infected cells were detectable whereas in the mock treated explants the majority of the epithelial cells in all sections of the oviduct were infected Figure 2 This result indicates that the infection of oviduct epithelial cells by the QX strain requires the presence of sialic acids on the cell surface Lectin staining revealed expression of alpha 2 3 linked sialic acids in all parts of the chicken oviduct To analyze if the receptor determinant of IBV alpha 2 3 linked sialic acid 9 11 is expressed in all parts of the oviduct or whether variations in the expression of this sugar may explain the observed differences in the sus ceptibility of the cells to IBV infections we performed lectin staining of cryosections of oviduct explants We applied two isoforms of the lectin Maackia Amurensis agglutinin MAAI and MAAII to detect alpha 2 3 linked sialic acid as well as Sambucas nigra agglutinin SNA to detect alpha 2 6 linked sialic acids We ob served that MAAII bound in all parts of the oviduct in dicating expression of alpha 2 3 linked sialic acids on the majority of epithelial cells whereas the isoform MAAI bound only in the magnum and uterus A staining with SNA revealed that alpha 2 6 linked sialic acids were present on the epithelial cells of infundibulum magnum and isthmus and absent on cells of the uterus Figure 3 and Table 2 Soluble S1 proteins of QX and B1648 bind both to oviduct epithelial cells and in a sialic acid dependent manner To find out whether the different parts of the oviduct differ in the presence of binding sites for IBV binding tests were performed with S1 protein connected to the Fc portion of human IgG Figure 4 The S1 subunit is sufficient for attachment to host cells Cryosections of the different segments of the oviduct were incubated with the S1 Fc proteins Binding of S1 Fc was detected on all four parts of the oviduct but staining was most intense in the isthmus whereas in the uterus fluorescent signals were restricted to single cells This variation was observed irrespective of the source of the S1 protein i e S1 Fc from strain QX showed a similar binding profile compared to the S1 from B1648 Binding of both proteins was dependent on the presence of sialic acid on the surface of the oviduct epithelium because detection of fluorescent signals was largely abolished after neuraminid ase treatment Figure 5 To analyse whether the observed binding of the soluble S1 proteins is comparable to that of trimerized spike proteins of virus particles we performed a binding test with virions on oviduct epithelial cells Both QX and B1648 viruses could bind to epithelial cells indicating that the soluble spike constructs have similar bindingpropertiesastheviruses Figure6 No differences of the binding intensity of S1 proteins to oviduct epithelial cells To get quantitative data on the binding properties of the two spike proteins FACS analysis was performed with primary isolated oviduct epithelial cells that retained their differentiated status which could be estimated by analysing the ciliary activity of the isolated epithelial cells under the light microscope The isolated oviduct epithelial cells were incubated with the soluble spike proteins and analysed by flow cytometric analysis for the percentage of cells with bound spike proteins No significant differences in the binding efficiency between S1 spike proteins of QX and B1648 was observed Figure 7 Discussion Analysis of IBV infections under in vitro conditions is difficult Most strains do not replicate in permanent cell cultures We could demonstrate in former studies that primary cell culture systems of the respiratory tract are suitable tools to investigate and compare different IBV strains 11 12 As IBV infection of the reproductive tract is one major economic problem in laying flocks we developed