Early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles

RESEARCH ARTICLE Early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles Cintia Hiromi Okino1¤*, Marcos Antônio Zanella Mores1, Iara Maria Trevisol1, Arlei Coldebella1, Hélio José Montassier2, Liana Brentano1 1 Embrapa Swine and Poultry, Concórdia, SC, Brazil, 2 Laboratory of Immunology and Virology (Imunovir), Department of Veterinary Pathology, Universidade Estadual Paulista—UNESP, Jaboticabal, SP, Brazil a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Okino CH, Mores MAZ, Trevisol IM, Coldebella A, Montassier HJ, Brentano L (2017) Early immune responses and development of pathogenesis of avian infectious bronchitis viruses with different virulence profiles. PLoS ONE 12(2): e0172275. doi:10.1371/journal.pone.0172275 Editor: Dong-Yan Jin, University of Hong Kong, HONG KONG Received: March 9, 2016 Accepted: February 1, 2017 Published: February 15, 2017 Copyright: © 2017 Okino 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 were inserted in the Results section of this manuscript, and some specific information could be found at Supporting Information. Funding: The whole study was supported by Brazilian Agricultural Research Corporation (Embrapa), a governamental institution os research from Brazil. The funder had role in study design, data collection and analysis, decision to publish and also on the preparation of the manuscript, as ¤ Current address: Embrapa Pecuária Sudeste, São Carlos, SP, Brazil * Abstract Avian infectious bronchitis virus (IBV) primarily replicates in epithelial cells of the upper respiratory tract of chickens, inducing both morphological and immune modulatory changes. However, the association between the local immune responses induced by IBV and the mechanisms of pathogenesis has not yet been completely elucidated. This study compared the expression profile of genes related to immune responses in tracheal samples after challenge with two Brazilian field isolates (A and B) of IBV from the same genotype, associating these responses with viral replication and with pathological changes in trachea and kidney. We detected a suppressive effect on the early activation of TLR7 pathway, followed by lower expression levels of inflammatory related genes induced by challenge with the IBV B isolate when compared to the challenge with to the IBV A isolate. Cell-mediated immune (CMI) related genes presented also lower levels of expression in tracheal samples from birds challenged with B isolate at 1dpi. Increased viral load and a higher percentage of birds with relevant lesions were observed in both tracheal and renal samples from chickens exposed to challenge with IBV B isolate. This differential pattern of early immune responses developed after challenge with IBV B isolate, related to the downregulation of TLR7, leading to insufficient pro-inflammatory response and lower CMI responses, seem to have an association with a most severe renal lesion and an enhanced capability of replication of this isolate in chicken. Introduction Avian infectious bronchitis virus (IBV) is a highly infectious causative agent of avian infectious bronchitis (IB), a disease of high economic impact, which affects poultry worldwide. IBV replicates primarily in tracheal epithelial cells, inducing several mucosal pathological changes, including ciliary loss, degeneration and necrosis of epithelial cells, glandular degeneration, inflammatory cell infiltration and epithelial hyperplasia [1,2]. After replication at primary site in tracheal mucosa, viraemia and IBV secondary replication are also found in other respiratory PLOS ONE | DOI:10.1371/journal.pone.0172275 February 15, 2017 1 / 17 Early immune response, pathogenesis and avian infectious bronchitis most of authors (CHO, MAZM, IMT, AC and LB) work for the funder. Competing interests: The authors have declared that no competing interests exist. tissues (nose, lungs and air sacs) and in many non-respiratory epithelial tissues (kidneys, testes, oviduct, and gastrointestinal tract). Nephritis is commonly observed, mainly in broilers, and depending on the pathotype of the IBV strain and on the bird age, it may cause high mortality, and microscopic lesions of degeneration and necrosis of the renal tubular cells and interstitial inflammatory cell infiltrate [3,4,5,6]. Prevention of IBV infection is currently achieved through vaccination, especially by attenuated viral vaccines, suggesting that local mucosal immunity is essential for induction of effective protection against disease [7,8,9]. In a previous study, we demonstrated that the dose of attenuated vaccine administered by local route is closely related to humoral and cellular immune responses at tracheal mucosal sites and their ability to confer effective protection against disease [8]. The continuous emergence of new IBV variants in several countries [3,10,11,12,13] are routinely pointed out as a cause of outbreaks in vaccinated flocks, leading to significant economic losses to the poultry industry [14]. Despite the great number of IBV strains and variants described over the last years [14], most of the studies have been limited to the classification and differentiation of IBV strains in genotypes, pathotypes, protectotypes, and/or serotypes, while, the specific immune mechanisms involved in pathogenesis of this virus remains poorly elucidated. A recent study has observed differential early immune response after infection with IBV when comparing IBV-susceptible and IBV-resistant chicken lines. Even before infection, specific genes, including genes related to innate immune response, were found to be differentially expressed in each chicken line. Despite these differences, viral loads were similar in tracheal samples of both chicken lines, indicating that IBV resistance might be associated with how the birds respond to the virus and not how they can prevent an initial infection [15]. The innate immune response is the first line of host defense against infections, and several immune and non-immune cells on mucosal surfaces are involved on recognition of pathogen associated molecular patterns (PAMP) of microorganisms. PAMPs are recognized through pattern recognition receptors (PRR), among which toll-like receptors (TLR) are an important group. During a RNA-virus infection, activation of innate immune responses depends on the interaction of endosome-associated (TLR3 and TLR7) and/or cytosol-located (RIG-I like receptors) PRRs. TLR3 and TLR7 interact with double-stranded (ds) RNA or single-strand (ss) RNA, respectively, and the subsequent activation pathways for TLR3 and TLR7 are mediated by TRIF (TIR-domain-containing adapter-inducing interf (...truncated)