Analysis of Immune Response of Chicken Primary Cells by Infection With Korean IBV Strain


 Infectious bronchitis virus (IBV), an avian coronavirus, is highly contagious, which develops acute pathogenesis in multiple organs. Frequent recombination of spike (S) glycoprotein leads to vaccine strategies insignificant. To understand IBV pathogenesis, we analyzed genetic distance of the Korean IBV isolate to different coronaviruses, including SARS-Cov2. For comprehensive information of the immune responses during IBV infection, we infected primary chicken embryonic kidney cells and performed transcriptome analysis. We observed functional pathways for innate immunity are associated and confirmed mRNA expressions of element genes that coordinate early immune responses. Immune profile of the host cell may assist vaccine development.


Introduction
Avian infectious bronchitis virus (IBV) is a momentous coronavirus that develops infectious bronchitis (IB) in the upper respiratory tract and spread to remote organs [1,2]. When it bears renal tropism, the infection induces interstitial nephritis and tubular pathology [3]. IBV is classi ed as gammacoronavirus while SARS-CoV-2 is classi ed as betacoronavirus. Swine coronavirus such as porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PdCV) are classi ed as alpha-and deltacoronaviruses, respectively [4]. The spike (S) glycoprotein is a key structure that mediates the coronavirus invasion into the host cell. Speci cally, it is a major inducer of host immune responses and determines host range and cell tropism [4][5][6]. The S glycoprotein is cleaved into S1 and S2 subunits and S1 protein facilitates viral attachment to the host cell receptor and the S2 protein mediates the fusion between the virus and cell membranes [5,7]. Once the virus arrives inside host cells, genetic modi cation and recombination of the S1 protein induces various viral subtypes. Consequently, the vaccine strategy is excluded by those genetic variations and results in poor cross protection [1,2,8].
We previously reported a genetic feature of a Korean IBV isolate (K047-12) that has kidney tropism [9], hence this study is designed to evaluate the genetic feature of the K047-12 strain and analyze the host immune responses during viral infection. First, we performed a phylogenetic sequence analysis of the S1 protein compared with other coronaviruses. We then investigated the genetic change in the chicken embryonic kidney (CEK) cells after acute infection with K047-12. Transcriptome analysis is a useful tool to anticipate unknown feature of immune systems [10] that we examined various pathways and genes for immune regulation. Our investigation in host immune responses by genetically modi ed coronaviruses is to broaden the understanding of pathogenesis during chicken coronavirus infection.
Moreover, comparing immune responses by coronavirus in animals to humans could contribute to prepare potential cross-species infection against modi ed viral strains.

Materials And Methods
The IBV K047-12 strain was isolated and propagated as reported previously [9]. Brie y, the virus was isolated from the cecal tonsil of a 6-week-old layer in 2012 from Pocheon city, Gyeonggi Province, Korea.
Then the virus was propagated in the SPF chicken embryos at embryo days (EDs) 9-11. The virus had been passaged three times in SPF embryonated eggs (Sungmin Farm, Korea).
The kidney tissue of SPF embryonated eggs at ED 18 were collected. Single cells from kidney (1×10 6 cells/mL) were obtained and cultured in DMEM supplemented with 10% FBS. Monolayers of primary kidney cells were prepared and split into K047-12 infected and uninfected control groups. After ve days of incubation, half of the medium was replaced with medium containing an IBV virus suspension for the infection group while the equal amount of fresh media was replaced in the control group. Cells were harvested after 48 hours post infection and stored in Tri reagent (Ambion, USA) for RNA extraction. The RNA from each group was sent to Macrogen Inc (Seoul, Korea) for RNA sequencing. The RNA was puri ed, fragmented, and reverse-transcripted before ampli cation by PCR for sequencing. The expression pro les for transcript were calculated as the fragments per kb of transcript per million mapped reads (FPKM). The ltered data was normalized by TMM normalization using the calcNormFactors method in the edgeR R library. The criteria for signi cant differentially expressed (DE) genes between two groups was as follows; l-fold change 1 ≥ 1.5, raw p value <0.05. The statistical signi cance of DE genes was determined using exactTest using edgeR. Gene ontology (GO) and functional annotation analysis for the differentially expressed genes was performed using gPro ler. The signi cant data were analyzed based on KEGG pathway enrichment. The transcriptome data for IBV strains (M41-CK, Beau-R, K047-12) were analyzed by hierarchical clustering (Euclidean distance and average linkage). The analysis was performed using Cluster 3.0 software and Java Treeview 1.2.0. The DE genes data for the M41-CK and Beau-R strains were acquired from a recent study [1].
Ten genes associated with immune responses were selected for validation at the mRNA level. The RNA was reverse-transcribed into cDNA using Accupower RT mix (Bioneer, Korea) according to the manufacturer's protocol. Quantitative PCR was conducted using a Maxima SYBR Green/Rox qPCR master mix kit (Thermo Fisher, USA). All reactions were set up in duplicate. The reaction conditions were 50°C for 2 min, 95°C for 10 min, and 40 ampli cation cycles consisting of 95°C for 15 s, 55°C (b-actin, Il1b, Il10, Il12a, Tlr4, Tlr7, Ccr2, Ccr7), or 50°C (Il6r, Il2rh, Il1rap) for 1 min. The melt curve stage was performed at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The threshold cycle (CT) values were collected. The gene expression was calculated using the formula 2 -ΔΔCT and mRNA levels were normalized against β-actin. Primers were made based on the published chicken genome sequences in NCBI. The primer sequences were sourced from published papers or designed by a primer designing tool (Primer 3 and BLAST, NCBI) and are listed in Table 1.

Results
Using the GenBank database, we analyzed the genetic differences of the Korean IBV isolate (K047-12) to other coronaviruses. As expected, the sequence of the Korean IBV isolate was most similar to standard IBV strains. Of interest, IBV strains showed highest similarity to SARS-CoV-2, and bat coronavirus. On the other hand, alpha-and deltacoronavirus were less correlated in terms of their S1 protein sequences (Fig.  1A). This result identi es the adjacent genetic distance between IBV and SARS-Cov-2 and potential cross antigenicity between human and chicken. Because the K047-12 strain is adapted to renal damage, we infected the virus on a primary chicken kidney cell and performed RNA sequencing to comprehensively investigate genetic regulation. Among 17,026 total genes, we identi ed 1,084 DE genes that showed greater than 1.5-fold change. Within those, we recognized 787 upregulated genes and 297 downregulated genes by volcano plot (Fig. 1B).
We further classi ed the DE genes using the GO enrichment analysis and signi cant changes were detected in biological process and molecular function. In the biological process, K047-12 infection signi cantly changed about 150 genes that regulate leukocyte activation, in ammatory response, and cytokine-mediated signaling pathway. Arrangement by molecular function revealed that pathways for cytokines and receptors were highly expressed by IBV infection (Fig. 2A). In addition, we examined functional classi cation by KEGG pathway analysis. In accordance with the GO enrichment analysis, the top-ranked functions were closely correlated with major immune responses, including metabolic pathway, cytokine-cytokine receptor interaction, and the toll-like receptor (TLR) signaling pathway (Fig. 2B). The functional classi cation demonstrates that the K047-12 infection in primary renal cells essentially activates the immune system.
To identify the precise action of the K047-12 strain in a host immune system, we clustered and compared the gene expression among IBV strains. From a previous publication, we retrieved the raw data for gene expression from primary renal cells that were infected with M41-CK or Beau-R [1]. We then clustered genes by functions for immune response, cytokine responses, and cell activation. As shown in Fig.1A, the S1 genetic distance of M41-CK was closer to Beau-R than K047-12. However, K047-12-activating genes were more analogous to M41-CK than the Beau-R strain (Fig. 3A). This con icting coherence suggests that the immune system is not completely coded by S1 sequence during infection. During acute infection, TLR activation turns on intracellular signals to stimulate effector cytokines and their receptors followed by cytotoxic T cell generation which is critical for protection, but an exacerbated cytokine secretion is detrimental to the host [11,12]. Based on functional classi cation, we determined highly regulated genes that are associated with innate immunity (Table 2). To verify the transcriptome analysis, we designed primers for ten genes and con rmed their expression at the mRNA level. By K047-12 infection, genetic levels of il1b, il10, il1rap, and il2rg for in ammatory signaling, tlr4 and tlr7 for RNA virus-speci c PRRs, and ccr2 and ccr7 for lymphocyte migration were signi cantly increased ( Figure 3B). Nevertheless a few gene expressions are not signi cantly changed but showed increasing trend overall. This result validates kidney cells is a source of critical immune regulators during early stage of IBV infection.

Discussion
Complete prevention of IB by vaccination has not been very successful due to the rise of variant virus strains. The respiratory tract is the primary organ affected by IBV invasion, but the genetic modi cation of the S1 protein expands the range of infection to remote tissues [13][14][15]. Earlier IBV studies focused on viral activity rather than the host-pathogen interaction. Recently we isolated a novel IBV strain with kidney tropism and reported its genetic characteristics by comparing IBV isolates from different regions [9].
Current study, we examined the genetic correlation of the K047-12 strain with coronaviruses from different host species. Interestingly, IBV showed a relative closeness to betacoronavirus such as SARS-CoV2 and bat coronavirus. Considering the repeated emergence of new variants amid the coronavirus pandemic (COVID-19), we speculate on the appearance of a zoonotic coronavirus through IBV similar to avian in uenza virus (AIV). Hence, we asked how host cells react with IBV and regulate innate immune response. Infection by kidney-adapted IBV is detrimental to chickens and develops a systemic pathology. As the rst line of defense, innate immunity is activated within two days after antigenic stimulation. At this initial stage of IBV infection, cytokines and chemokines are produced and co-stimulatory pathways are activated [3,16,17]. Then we speculated the transcriptome analysis of infected kidney tissue by an IBV strain with renal tropism might be helpful to understand the pathogenesis of IB. For precise genetic information for innate immunity during kidney infection, we infected a Korean IBV isolate (K047-12) on primary kidney cells for 48 hours and analyzed genes in immune functions. We rst detected expression of 1,084 genes that were signi cantly changed, while 787 genes were enhanced but 297 genes were suppressed. Assuming upregulated genes are linked to initial immune responses, functional signi cance by GO enrichment and the KEGG pathway were examined. Functions for innate immunity regulation, such as leukocyte activation, in ammatory response, and cytokine-mediated signaling pathway, are notably activated by K047-12. The study that infected the M41-CK strain also observed comparable gene regulation. For example, rsad2, snx10, stat1, tnip2, il6, il8l1 and dll1 genes that are associated with cytokine pathway were enhanced by kidney-adapted IBV [1]. Despite of the genetic variance of the S1 gene, IBVs might utilize common pathways for host protection against IBV. However, it is debatable whether certain genetic modi cations signi cantly contribute to the pathogenicity and host immune reaction. Although the modi cation of a few amino acids is enough to affect the infectivity [18,19], additional studies are necessary to understand precise immune regulation. Moreover, our examination suggests that the relationship between the S1 protein variation and host response is not always correlated. In a phylogenetic tree analysis for the S1 protein sequence, the genetic distances between K047-12 to M41-CK strain and K047-12 to the Beau-R strain were not different (Fig.1A). According to the immunological classi cation, however, K047-12 was closer to M41-CK than the Beau-R strain (Fig.3A). This observation emphasizes the advantage of transcriptome analysis in host cells over the viral examination. Identi cation of major cytokine regulation during acute IBV infection opens paths to gure out following adaptive immunity that manipulate systemic in ammation and we pinpointed substantial genes for innate immunity. In the M41-CK strain infection, il6 and il1b were activated in lung tissue when an IBV stain for kidney tropism accelerated immune modulators, such as il6, il10ra, il17ra, ccl4, and ccl17 [3,20,21]. Among the top twenty list of highly regulated genes, we also found overlapping genes including il1b, il10, il12a, il1rap, il2rg, il6r, tlr4m tl47, ccr2, and ccr7. We then further validated the mRNA expression of those genes using real-time PCR and identi ed il1b and il10 as signature genes. Produced by mesangial cell, IL-10 is a critical cytokine for pathophysiology in renal disorders. Nonetheless of its anti-in ammatory action, IL-10 is upregulated during nephritis where the bloom of in ammatory lymphocytes to be controlled and regulatory T cells are required [22,23]. Along with IL-6, IL-1β is also secreted from renal mesangial cells and mediates in ammation. These are key activators for Th17 cell generation that develops numerous renal disorders [24,25] Detecting PAMPs, TLR activation signals for proin ammatory cytokine production [26]. For instance, TLR3 and TLR7 are critical mediators for IBV recognition and initiation of innate immune response [20]. TLR3 and TLR4 are also activated in SARS coronavirus infection [27,28]. Considering TLR4 and TLR7 promotion by K047-12, we speculate that IBV might share in common with betacoronavirus in the regulation of PRR recognition. The knowledge from rodent and human immunology in regulation of the chemokine receptors is frequently applied in chicken study. CCR2 receives the CCL2 signal and activates monocyte migration for viral clearance thus its de ciency is fatal during infection [29]. CCR7 is activated by CCL19 and CCL21, and a study examined its critical role for T migration in chickens [30]. In addition, human patients with SARS coronavirus infections were activated by CCR2 and CCR7 expression [31,32]. We also observed ccr2 and ccr7 activation by K047-12 which suggests that the avian immune system activates comparable chemokine receptors to mouse and human and directs immune cell migration and host protection.
A genetic modi cation endows IBV for various tissue tropisms, which makes it di cult to save animals from the endemic outbreak. IBV strains with renal tropism are lethal because they also attack both urogenital organs and systemic organs [33]. Therefore, to understand the immune responses of target organs against viral infection, we applied the transcriptome analysis of primary renal cells. In speci c, we found the genetic pro le in immunologic function for pathogen recognition, cytokine regulation, and signals is closely associated with innate immune responses during viral infection. In this regard, this study can be utilized to identify unknown immune pathways that avian viruses prefer to utilize. Therefore, our results could expand the knowledge in avian immunology that is applied for vaccine strategies and prevention of new variant viruses. Tables   Table 1. Primer information for qRT-PCR