viruses Article Molecular Biology and Pathological Process of an Infectious Bronchitis Virus with Enteric Tropism in Commercial Broilers Ana P. da Silva 1 , Ruediger Hauck 2 , Sabrina R. C. Nociti 3, Colin Kern 4, H. L. Shivaprasad 5, Huaijun Zhou 4 and Rodrigo A. Gallardo 1,* 1 Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis 1089 Veterinary Medicine Dr, 4008 VM3B, Davis, CA 95616, USA; [email protected] 2 Department of Pathobiology and Department of Poultry Science, Auburn University, 302J Poultry Science Building, 260 Lem Morrison Dr, Auburn, AL 36849, USA; [email protected] 3 Zootechnical Hygiene Laboratory, School of Animal Science and Food Engineering, University of Sao Paulo, 225 Duque de Caxias St, Pirassununga 13635-900, SP, Brazil; [email protected] 4 Department of Animal Science, College of Agriculture, University of California, Davis, One Shields Ave, Davis, CA 95616, USA; [email protected] (C.K.); [email protected] (H.Z.) 5 California Animal Health and Food Safety Laboratory System, Tulare Branch, 18760 Rd 112, Tulare, CA 93274, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-530-752-1078 Abstract: Infectious bronchitis virus (IBV) induces respiratory and urogenital disease in chickens. Although IBV replicates in the gastrointestinal tract, enteric lesions are uncommon. We have reported a case of runting-stunting syndrome in commercial broilers from which an IBV variant was isolated from the intestines. The isolate, CalEnt, demonstrated an enteric tissue tropism in chicken embryos and SPF chickens experimentally. Here, we determined the full genome of CalEnt and compared it Citation: da Silva, A.P.; Hauck, R.; to other IBV strains, in addition to comparing the pathobiology of CalEnt and M41 in commercial Nociti, S.R.C.; Kern, C.; Shivaprasad, broilers. Despite the high whole-genome identity to other IBV strains, CalEnt is rather unique in its H.L.; Zhou, H.; Gallardo, R.A. nucleotide composition. The S gene phylogenetic analyses showed great similarity between CalEnt Molecular Biology and Pathological Process of an Infectious Bronchitis and Cal 99. Clinically, vent staining was slightly more frequent in CalEnt-infected birds than those Virus with Enteric Tropism in challenged with M41. Furthermore, IBV IHC detection was more evident and the viral shedding in Commercial Broilers. Viruses 2021, 13, feces was overall higher with the CalEnt challenge compared with M41. Despite underlying intestinal 1477. https://doi.org/10.3390/ lesions caused by coccidiosis and salmonellosis vaccination, microscopic lesions in CalEnt-infected v13081477 chickens were more severe than in M41-infected chickens or controls, supporting the enteric tropism of CalEnt. Further studies in SPF chickens are needed to determine the pathogenesis of the virus, its Academic Editor: Faizal Careem molecular mechanisms for the enteric tropism, and its influence in intestinal health. Received: 29 June 2021 Keywords: IBV; infectious bronchitis; variants; whole-genome sequencing; enteric tropism; runting- Accepted: 24 July 2021 stunting syndrome Published: 28 July 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. Infectious bronchitis virus (IBV) belongs to the Gammacoronavirus genus and mainly causes respiratory and urogenital disease in chickens. Although IBV replicates and persists in the cecal tonsils and has been isolated from duodenum and jejunum, intestinal lesions are rare [1–3]. Genetic variation of the viral spike (S) gene may lead to an increased susceptibility to proteolytic activation and to an improved ability to bind cell receptors, Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. enhancing its infectivity. In addition, genomic changes modulate the virus entry and This article is an open access article influence tissue tropism, persistence, virulence, and host range [4]. This variability explains distributed under the terms and why coronaviruses genetically and antigenically similar to IBV can cause different clinical conditions of the Creative Commons outcomes. For example, the turkey coronavirus (TCoV) induces severe enteric disease by Attribution (CC BY) license (https:// the viral replication in enterocytes of the jejunum and ileum [5–7]. Molecular differences creativecommons.org/licenses/by/ between TCoV and IBV are mainly found in the S gene, in which homologies are as low as 4.0/). 33%, validating the divergent tissue tropism of these closely related viruses [8]. Viruses 2021, 13, 1477. https://doi.org/10.3390/v13081477 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 1477 2 of 12 An enterotropic IBV strain has been described for causing intestinal lesions, mainly in the rectum, consisting of the desquamation of epithelial cells on the villi tips and congestion of the intestinal submucosa [9,10]. This virus was originally isolated from the intestines of chickens presenting with respiratory signs [11]. In another study, an IBV strain isolated from broilers with enteric and respiratory diseases was compared to an isolate from broilers that prompted only respiratory disease. Both isolates induced similar pathology after experimental infection; both viruses were detected in the intestines but their infection did not cause enteric lesions [12]. IBV was also one of several pathogens isolated from the intestines of commercial broilers presenting with runting-stunting syndrome. Infection with these IBV isolates alone or combined with other microorganisms induced a reduced body weight in experimentally infected chickens [13]. Previously, we have described the detection and isolation of an IBV variant from the intestines of brown broiler chickens showing runting-stunting signs. Interestingly, the virus was not retrieved from the kidneys or respiratory tract. The enteric isolate was named CalEnt and was isolated from the intestines of chicken embryos, but not from the chorioallantoic membrane. Additionally, CalEnt induced runting-stunting syndrome-like lesions after experimental oculonasal infection of specific-pathogen-free (SPF) chickens. The S1 gene sequence of CalEnt showed nucleic acid sequence identities of 93.8% to IBV California 99 (Cal 99) and of 85.7% to IBV Arkansas DPI (Ark DPI) [14]. The aim of the present investigation was to determine the full genome of CalEnt and compare its genomic identity to other IBV strains, as well as to compare the pathobiology of CalEnt to that of the respiratory strain M41 after a controlled infection of commercial broilers. 2. Materials and Methods 2.1. Viruses IBV CalEnt and M41 strains were used in experimental challenges. Both IBV strains were grown and titrated in embryonated chicken SPF eggs (Charles River, CT, USA) using 4 standard procedures [15,16]. The viral dose of IBV CalEnt was 2 × 10 EID50 and the dose 5 of IBV M41 was 2 × 10 EID50 in a 200-µL inoculum. The challenge was performed either oculonasally or via crop gavage. 2.2. Whole Genome Sequencing Intestines from embryos infected with CalEnt were homogenized with PBS and RNA was extracted from a volume of 100 µL using TRIzol (ThermoFisher, Waltham, MA, USA). DNA depletion was performed using the Turbo DNA-Free Kit (ThermoFisher, Waltham, MA, USA) followed by rRNA depletion using the Terminator 50-Phosphate-Dependent Exonuclease (Epicentre Biotechnologies, Madison, WI, USA) as per the manufacturer’s instructions. The DNase reaction was ceased by adding EDTA at a concentration of 5 mmol/L. The RNA was purified using the QIAamp Viral RNA Mini Kit (QIAgen, Valencia, CA, USA) without the addition of the carrier RNA, and eluted to a final volume of 30 µL. The RNA quality was evaluated using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). cDNA libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). Libraries were sequenced using the Illumina at 100 bp paired end. Quality control of the reads, adaptor trimming, merging of paired end reads, and assembly were performed using Geneious Prime 2021.1.1. The sequences were assembled using IBV Cal99 as a reference (accession number AY414485). A second round of reference-based assembly was performed using the consensus sequence obtained in the first round as a reference. For accuracy verification, de novo assembly was performed using the SPAdes assembler. The two assemblies shared 100% nucleotide identity. The consensus was uploaded to GenBank (accession number MW556742). The obtained consensus sequence was aligned with several IBV variants and TCoV using the MAFFT plugin [17] in Geneious Prime 2020.1.1. Nucleotide sequences were used to calculate homologies and for phylogenetic analyses using the maximum likelihood method based on the GTRGAMMAI model with 1000 bootstraps in Geneious Prime Viruses 2021, 13, 1477 3 of 12 with the RaxML plugin [18]. Homology matrices were calculated, and phylogeny was performed in whole genomes, full S gene sequences, and 751 bp fragments of the S gene bearing the S1 hypervariable region (nucleotide positions 20,368 to 21,119 of GenBank accession number MW556742). The S gene of CalEnt was submitted to the basic local alignment search tool (BLAST, blast.ncbi.nlm.nih.gov, accessed on 20 July 2021) and the whole S gene from sequences showing the highest homologies was aligned using the MAFFT plugin [17] in Geneious Prime 2020.1.1. The alignment was uploaded in SimPlot,