Productive replication of nephropathogenic infectious bronchitis virus in peripheral blood monocytic cells, a strategy for viral dissemination and kidney infection in chickens

Reddy et al. Vet Res (2016) 47:70 DOI 10.1186/s13567-016-0354-9 Open Access RESEARCH ARTICLE Productive replication of nephropathogenic infectious bronchitis virus in peripheral blood monocytic cells, a strategy for viral dissemination and kidney infection in chickens Vishwanatha R. A. P. Reddy*, Ivan Trus, Lowiese M. B. Desmarets, Yewei Li, Sebastiaan Theuns and Hans J. Nauwynck Abstract In the present study, the replication kinetics of nephropathogenic (B1648) and respiratory (Massachusetts-M41) IBV strains were compared in vitro in respiratory mucosa explants and blood monocytes (KUL01+ cells), and in vivo in chickens to understand why some IBV strains have a kidney tropism. B1648 was replicating somewhat better than M41 in the epithelium of the respiratory mucosa explants and used more KUL01+ cells to penetrate the deeper layers of the respiratory tract. B1648 was productively replicating in KUL01+ monocytic cells in contrast with M41. In B1648 inoculated animals, 102.7–6.8 viral RNA copies/100 mg were detected in tracheal secretions at 2, 4, 6, 8, 10 and 12 days post inoculation (dpi), 102.4–4.5 viral RNA copies/mL in plasma at 2, 4, 6, 8, 10 and 12 dpi and 101.8–4.4 viral RNA copies/106 mononuclear cells in blood at 2, 4, 6 and 8 dpi. In M41 inoculated animals, 102.6–7.0 viral RNA copies/100 mg were detected in tracheal secretions at 2, 4, 6, 8 and 10 dpi, but viral RNA was not demonstrated in plasma and mononuclear cells (except in one chicken at 6 dpi). Infectious virus was detected only in plasma and mononuclear cells of the B1648 group. At euthanasia (12 dpi), viral RNA and antigen positive cells were detected in lungs, liver, spleen and kidneys of only the B1648 group and in tracheas of both the B1648 and M41 group. In conclusion, only B1648 can easily disseminate to internal organs via a cell-free and -associated viremia with KUL01+ cells as important carrier cells. Introduction Avian infectious bronchitis virus (IBV) causes mild to acute respiratory disease in chickens, characterized by coughing, sneezing, tracheal rales and dyspnea [1]. IBV belongs to the order of the Nidovirales, family Coronaviridae, subfamily Coronavirinae and genus Gammacoronavirus [2]. Worldwide, IBV causes huge economic losses in both broilers and layers. IBV has a tropism not only for the epithelium of the respiratory tract but also for the epithelium of kidneys, oviduct, gastrointestinal tract *Correspondence: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium (oesophagus, proventriculus, duodenum, jejunum, bursa of Fabricius, caecal tonsils, rectum and cloaca) and testes [3, 4]. IBV is clinically associated with poor performance of birds, reduced egg production and quality, as well as increased predisposition to other secondary bacterial infection [5]. IBV is highly contagious. Currently, multiple serotypes of IBV exist, and new variants emerge due to frequent point mutations and recombination events in the viral genome [4]. Vaccination failure is very common against IBV due to poor or no cross-protection between different IBV serotypes. The first IBV was isolated from birds showing respiratory problems in the United States in 1931 [6]. In the early 1950s, the well-known respiratory Massachusetts type of IBV (Mass) was isolated in the United States. In © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Reddy et al. Vet Res (2016) 47:70 subsequent years, Mass-type (prototype: M41) strains have been identified worldwide, and many variants emerged. Some IBV strains were called nephropathogenic because the initial respiratory infection was followed by severe kidney infection. Important clinical signs of nephropathogenic IBV strains include increased water consumption, low body weight gain, watery droppings and significant mortality. Necropsy of birds that died during a nephropathogenic infection reveals enlarged and pale kidneys with urates in the collecting tubules [7]. In the 1960s, the first nephropathogenic IBV strains were reported in the US and Australia, and later worldwide. In the last 15 years, nephropathogenic IBV strains have been emerging as most prevalent IBV strains in commercial poultry [8–12]. The B1648 strain is a Belgian reference nephropathogenic IBV serotype, that was responsible for large outbreaks of kidney disease in broiler farms in Belgium, The Netherlands and Northern France, and was first isolated in 1984 [7, 13–15]. In September 2012, a novel coronavirus emerged in humans, designated Middle East respiratory syndrome coronavirus (MERS-CoV). MERS-CoV has a higher mortality rate (>35%) than another well-known coronavirus, the severe acute respiratory syndrome coronavirus (SARS-CoV) (9.6%). The MERS-CoV infected patients usually end up with a severe pneumonia complicated with kidney failure. The severity of MERS-CoV infections in humans, caused by its extra-pulmonary infection of kidneys have prompted us to question why this virus has a strong tropism for the kidneys. The same question has been raised for the kidney tropism of certain IBV strains, for the past 25 years [7, 13–15]. Hence, in the present study, we aimed to explore the tissue tropism characteristics of IBV nephropathogenic (B1648) and respiratory (M41) strains in chickens. To this end, replication kinetics of IBV B1648 and M41 were evaluated in vitro in tracheal mucosa explants and blood monocytes by a reproducible quantitative analysis system using confocal microscopy [16–18]. A new 5′ RT-qPCR was validated and used for comparing in vivo the viral replication kinetics in the respiratory tract and dissemination in blood of IBV B1648 and M41 [19]. Elucidating the tissue tropism mechanisms of B1648 and M41 is important to plan better prevention strategies for emerging highly nephropathogenic IBV infections. Materials and methods IBV B1648 and M41 replication characteristics in tracheal mucosa explants and peripheral blood monocytes Viruses The virulent nephropathogenic IBV B1648 and the respiratory prototype M41 were used in this study. B1648 is a Belgian field isolate obtained in 1984 and described Page 2 of 19 previously [13, 15, 20]. M41 with unknown passage history was obtained from the avian pathology laboratory, Ghent University [21]. Virus was propagated in specific pathogen free (SPF) 10 days old embryonated chicken eggs. (...truncated)