Comparative features of infections of two Massachusetts (Mass) infectious bronchitis virus (IBV) variants isolated from Western Canadian layer flocks

Amarasinghe et al. BMC Veterinary Research https://doi.org/10.1186/s12917-018-1720-9 (2018) 14:391 RESEARCH ARTICLE Open Access Comparative features of infections of two Massachusetts (Mass) infectious bronchitis virus (IBV) variants isolated from Western Canadian layer flocks Aruna Amarasinghe1, Upasama De Silva Senapathi1, Mohamed Sarjoon Abdul-Cader1, Shelly Popowich2, Frank Marshall3, Susan C. Cork1, Frank van der Meer1, Susantha Gomis2 and Mohamed Faizal Abdul-Careem1* Abstract Background: Infectious bronchitis virus (IBV) is one of the leading causes of mortality and morbidity in chickens. There are numerous serotypes and variants, which do not confer cross protection resulting in failure of currently used IBV vaccines. Although variant IBV isolates with major genetic differences have been subjected to comparative studies, it is unknown whether minor genetic differences in IBV variants within a serotype are different in terms of pathogenesis and eliciting host responses. Two Massachusetts (Mass) variant IBV isolates recovered from commercial layer flocks in the Western Canadian provinces of Alberta (AB) and Saskatchewan (SK) were compared genetically and evaluated for their pathogenicity, tissue distribution and ability to recruit and replicate in macrophages. Results: Although whole genome sequencing of these two Mass IBV isolates showed low similarity with the M41 vaccinal strain, they had an identical nucleotide sequence at open reading frames (ORFs) 3a, 3b, envelop (E), matrix (M), 5a and 5b. The rest of the ORFs of these 2 IBV isolates showed 99.9% nucleotide similarity. However, upon experimental infection, we found that the IBV isolate originating from AB was different to the one that originated in SK due to higher tracheal lesion scores and lower lung viral replication and lower genome loads in cecal tonsils. Nevertheless, both IBV isolates elicited host responses characterized by significant macrophage recruitment to the respiratory tract and there was evidence that both IBV isolates replicated within tracheal and lung macrophages. Conclusions: Overall, this study shows that Mass variant IBV isolates, although possessing minor genetic variations, can lead to significant differences in pathogenicity in young chickens. Further studies are required to investigate the pathogenicity of these two Mass variant IBV isolates in laying hens. Keywords: Infectious bronchitis virus, Whole genome sequencing, Tissue distribution, Pathogenicity, Macrophage response Background Infectious bronchitis virus (IBV) belongs to the family Coronaviridae. Traditionally, IBV is considered to be a host-specific respiratory pathogen in chickens and IBV initially replicates at the route of entry, the tracheal mucosa [1, 2]. However, identification of new variants and/ or serotypes of IBV have shown a wide variation of tissue tropism including urinary [3–10], gastrointestinal * Correspondence: 1 Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary, Health Research Innovation Center 2C53, 3330 Hospital Drive NW, Calgary, AB T2N 4N1, Canada Full list of author information is available at the end of the article [6, 9, 11, 12], oviduct [9, 13] and bursa of Fabricius [11, 14]. IBV is known to replicate in the reproductive tract epithelium in layers leading to reduced egg production and shell defective eggs [15, 16]. False layer syndrome, which is associated with cystic oviduct formation occurs with IBV infection in early life [17, 18]. IBV can also replicate in the testes of cockerels [19]. An array of serotypes and strains of IBV infect chickens throughout the world [2]. Genetic events such as insertion(s) and deletions [20, 21], point mutations [22], and recombination [23–27] contribute to genomic variations of IBV [28]. The spike 1 (S1) gene is highly © The Author(s). 2018 Open Access 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. Amarasinghe et al. BMC Veterinary Research (2018) 14:391 variable among IBV strains and it encodes epitopes, which bind to neutralizing antibodies [29]. A change in the amino acid sequence as small as 2 to 3% in the S1 subunit can result in changes in the antigenicity of the virus [30]. Based on these changes of the S1 protein, numerous IBV strains have been characterized throughout the world [1]. Therefore, either the partial [31–33] or the full-length [34–39] of the S1 glycoprotein gene has been used in the molecular characterization of IBV isolates. However, using whole genome sequencing it has been observed that genes other than S1 may play a role in the pathogenicity of IBV infection [40, 41]. Currently, there are no IBV reference full genome sequences available for Canadian IBV isolates but partial [33] or complete [37] S1 sequences are available. Previously, comparative studies have been conducted to elucidate differential pathogenicity and host responses between variant IBV isolates with larger genome variability such as nephropathogenic and Massachusetts (Mass) IBV isolates recovered from various geographical areas [18, 42–46]. Since it is well known that very minor changes in the genome of viruses [47] including IBV [48, 49] can lead to difference in pathogenicity, comparative studies involving variant IBV isolates are required. A wide array of IBV variants are affecting commercial broiler, layer and breeder flocks in Canada [33, 37, 50, 51]. For example, Mass type IBV variants are impacting the commercial egg production in Western Canada [50]. This study showed that Mass type IBV infection of 27-week old layers at peak production, lead to loss of egg production for about 2 weeks followed by production of shell less, small and defective eggs for another 2 days before the egg production bounced back to normal production. The duration of the loss of marketable eggs appears to be 16 days. In an 8000 bird layer flock of Western Canada, we observed 47.6% drop in egg production for 10 days and we isolated a Mass type variant IBV from this flock. Based on Egg Farmers of Alberta’s price of $2.15/dozen of large eggs, the loss for 10 days is calculated to be $6823 for this particular outbreak and the loss will be higher if the infection persists beyond 10 days or reoccur. In addition to this direct loss of egg production, egg grading stations need to outsource supply of eggs during these production drops and also some of the affected birds may succumb to secondary bacterial infections (...truncated)