Infectious bronchitis corona virus establishes productive infection in avian macrophages interfering with selected antimicrobial functions

RESEARCH ARTICLE Infectious bronchitis corona virus establishes productive infection in avian macrophages interfering with selected antimicrobial functions Aruna Amarasinghe1, Mohamed Sarjoon Abdul-Cader1, Sadiya Nazir1, Upasama De Silva Senapathi1, Frank van der Meer1, Susan Catherine Cork1, Susantha Gomis2, Mohamed Faizal Abdul-Careem1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary, Health Research Innovation Center 2C53, Calgary, Alberta, Canada, 2 Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatoon, Canada * Abstract OPEN ACCESS Citation: Amarasinghe A, Abdul-Cader MS, Nazir S, De Silva Senapathi U, van der Meer F, Cork SC, et al. (2017) Infectious bronchitis corona virus establishes productive infection in avian macrophages interfering with selected antimicrobial functions. PLoS ONE 12(8): e0181801. https://doi.org/10.1371/journal. pone.0181801 Editor: Yongchang Cao, Sun Yat-Sen University, CHINA Received: April 20, 2017 Accepted: July 9, 2017 Published: August 1, 2017 Copyright: © 2017 Amarasinghe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All data is contained within the paper. Funding: SG received funding from Saskatchewan Agriculture Development Fund (grant number, 20140185; URL-https://www.saskatchewan.ca/ business/agriculture-natural-resources-andindustry/agribusiness-farmers-and-ranchers/ agricultural-research-programs/knowledgecreation/agriculture-development-fund) and MFAC Infectious bronchitis virus (IBV) causes respiratory disease leading to loss of egg and meat production in chickens. Although it is known that macrophage numbers are elevated in the respiratory tract of IBV infected chickens, the role played by macrophages in IBV infection, particularly as a target cell for viral replication, is unknown. In this study, first, we investigated the ability of IBV to establish productive replication in macrophages in lungs and trachea in vivo and in macrophage cell cultures in vitro using two pathogenic IBV strains. Using a double immunofluorescent technique, we observed that both IBV Massachusetts-type 41 (M41) and Connecticut A5968 (Conn A5968) strains replicate in avian macrophages at a low level in vivo. This in vivo observation was substantiated by demonstrating IBV antigens in macrophages following in vitro IBV infection. Further, IBV productive infection in macrophages was confirmed by demonstrating corona viral particles in macrophages and IBV ribonucleic acid (RNA) in culture supernatants. Evaluation of the functions of macrophages following infection of macrophages with IBV M41 and Conn A5968 strains revealed that the production of antimicrobial molecule, nitric oxide (NO) is inhibited. It was also noted that replication of IBV M41 and Conn A5968 strains in macrophages does not interfere with the induction of type 1 IFN activity by macrophages. In conclusion, both M41 and Con A5968 IBV strains infect macrophages in vivo and in vitro resulting productive replications. During the replication of IBV in macrophages, their ability to produce NO can be affected without affecting the ability to induce type 1 IFN activity. Further studies are warranted to uncover the significance of macrophage infection of IBV in the pathogenesis of IBV infection in chickens. PLOS ONE | https://doi.org/10.1371/journal.pone.0181801 August 1, 2017 1 / 21 Infectious bronchitis corona virus-avian macrophage interaction received funding from Natural Sciences and Engineering Research Council of Canada (grant number RT736458; URL-http://www.nserc-crsng. gc.ca/index_eng.asp). Competing interests: The authors have declared that no competing interests exist. Introduction Infectious bronchitis (IB) is primarily a respiratory disease of chickens but with potential to cause more widespread infection in the urinary and reproductive tracts in chicken leading to significant production losses in commercial broiler and layer flocks worldwide [1]. The causative infectious bronchitis virus (IBV) belongs to the family Coronaviridae. The disease is usually characterized by high morbidity and low mortality in mature birds, whereas in naive young birds (2–3 weeks of age), mortality up to 100% can be observed [1]. Being an RNA virus with the ability to mutate and recombine, IBV persist as numerous serotypes and strains. The control of IB relies on vaccination. Vaccines are available for commonly occurring serotypes and strains but they are not necessarily antigenically similar to the wild-type viral strains circulating in poultry barns. Although, these vaccine strains may provide some degree of protection for some related strains known as protectotypes [2], the commonly available vaccines may not elicit protective immune responses in a flock if the field strains are antigenically very different from the vaccine strains. Owing to this reason, vaccination against IBV is not currently considered to be a very effective control method and other biosecurity measures are necessary to prevent the introduction of IBV into poultry production facilities. IBV is known to replicate in the respiratory tract leading to changes in the muco-cilliary clearance mechanism, as such, expose the IBV infected birds to secondary bacterial infections. Additionally, IBV has tropisms for a variety of tissues. However, the mode of dissemination from the common route of entry, i.e. the respiratory route, to the rest of the body systems could potentially be due to the initial viremia [3]. Once disseminated, IBV infects epithelial cells of the reproductive and urinary systems, particularly the oviduct and kidney depending on the infecting strain [4]. Recently, it has been shown that a nephro-pathogenic strain of IBV (B1648) could replicate in peripheral blood monocytes leading to viremia [4]. The infection of circulating monocytes could potentially disseminate IBV to the urinary tract, liver and spleen [4]. Macrophages play roles in innate immune responses, as well as in mounting adaptive immune responses by functioning as antigen presenting cells, as such they are critical in protecting animals from microbial infections. Although it is known that macrophage numbers are elevated in the respiratory tract in response to IBV infection [5], the role played by macrophages in IBV infection, particularly if they serve as a target cell for viral replication is not known. Macrophages have been implicated to play in an important role in the pathogenesis of some animal and human viruses including Marek’s disease virus in birds [6], feline corona virus in cats [7], and human immunodeficiency virus (HIV) [8]. It was also (...truncated)