Differential expression of immune-related genes in the bursa of Fabricius of two inbred chicken lines following infection with very virulent infectious bursal disease virus

Comparative Immunology, Microbiology and Infectious Diseases 68 (2020) 101399

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Differential expression of immune-related genes in the bursa of Fabricius of two inbred chicken lines following infection with very virulent infectious bursal disease virus

T

Farhanah Mohd Isaa, Nagi Ahmed Al-Haja, Nurulfiza Mat Isaa,b, Aini Iderisa,c, Claire Powerse,f, Omobolanle Oladapoe, Venugopal Naire, Abdul Rahman Omara,d,* a

Laboratory of Vaccines and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Selangor, Malaysia Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Selangor, Malaysia d Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Selangor, Malaysia e Viral Oncogenesis Group, The Pirbright Institute, Pirbright, Woking, UK f The Jenner Institute, The Centre for Cellular and Molecular Physiology Roosevelt Drive, Oxford, United Kingdom b c

A R T I C LE I N FO

A B S T R A C T

Keywords: IBDV Very virulent Inbred lines Immune-related genes

Among different inbred chickens’ lines, we previously showed that lines P and N of Institute for Animal Health, Compton, UK are the most susceptible and the least affected lines, respectively, following infection with very virulent infectious bursal disease virus (vvIBDV). In this study, the differential expressions of 29 different immune-related genes were characterized. Although, birds from both lines succumbed to infection, line P showed greater bursal lesion scores and higher viral copy numbers compared to line N. Interestingly, line N showed greater down-regulation of B cell related genes (BLNK, TNFSF13B and CD72) compared to line P. While upregulation of T-cell related genes (CD86 and CTLA4) and Th1 associated cytokines (IFNG, IL2, IL12A and IL15) were documented in both lines, the expression levels of these genes were different in the two lines. Meanwhile, the expression of IFN-related genes IFNB, STAT1, and IRF10, but not IRF5, were up-regulated in both lines. The expression of pro-inflammatory cytokines (IL1B, IL6, IL18, and IL17) and chemokines (CXCLi2, CCL4, CCL5 and CCR5) were up-regulated in both lines with greater increase documented in line P compared to line N. Strikingly, the expression of IL12B was detected only in line P whilst the expression of IL15RA was detected only in line N. In conclusion, the bursal immunopathology of IBDV correlates more with expression of proinflammatory response related genes and does not related to expression of B-cell related genes.

1. Introduction Infectious bursal disease (IBD) is a highly contagious disease with great economic impact towards poultry industry worldwide. This disease caused by a small, non-enveloped double stranded RNA (dsRNA) virus known as infectious bursal disease virus (IBDV), a member of Birnaviridae family [1]. IBDV primarily targets IgM-bearing B-cells in the bursa of Fabricius, therefore leads to suppression of humoral immune response in the infected birds, creating immunocompromised birds that eventually have a higher risk towards secondary infections [2]. Other cells that are positive for IBDV infection and replication are

macrophages and dendritic cells. Meanwhile T cells that are refractory to IBDV infection, were reported to show an influx in the bursa following IBDV infection [3–5]. IBDV can be classified into several subtypes based on virus virulence, with very virulent (vv) strain caused high mortality rate in young chickens compared to the classical virulent strain, and layer-type chickens are more susceptible than broiler-type chicken following IBDV infection [6]. Differential expression of immune response genes provide important information on host pathogen interaction, including biological process involved during the infection. Studies on IBDV, showed that virus virulence and host genotype play an important role in stimulating

Abbreviations: IBD, infectious bursal disease; IBDV, infectious bursal disease virus; MHC, major histocompatibility complex; MD, Marek’s disease; HPAI, highly pathogenic avian influenza; vv, very virulent; p.i., post-infection; SPF, specific-pathogen-free; PBS, phosphate buffer saline; EID50, 50% embryo infectious dose; hpi, hour post infection; Ct, cycle threshold ⁎ Corresponding author at: Laboratory of Vaccines and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia. E-mail addresses: [email protected], [email protected] (A.R. Omar). https://doi.org/10.1016/j.cimid.2019.101399 Received 22 September 2019; Received in revised form 11 November 2019; Accepted 14 November 2019 0147-9571/ © 2019 Elsevier Ltd. All rights reserved.

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route. Meanwhile, each bird within control group for each line were mock-infected with sterile phosphate buffered saline (PBS). Bursa of Fabricius from three chickens of each line were harvested at 0 h postinfection (hpi) for the control groups, and at 24, 48 and 54 hpi for the infected groups. All the chickens were observed for any clinical signs, abnormalities and mortality during the trial.

differential gene expression of those involved in host immune response including pro-inflammatory cytokines and chemokines, Th1-associated cytokines, stress-related and apoptosis-related genes [7,8]. Previous study has also emphasized on the early host immune response may provide resistance mechanism following virulent IBDV infection [9]. In addition to host genotype, chicken major histocompatibility complex (MHC) B haplotype found in B allele has a strong influence in disease resistance on different viral diseases such as Marek’s disease (MD) and highly pathogenic avian influenza (HPAI) [10,11]. In addition, B haplotype has been reported to be involved in the severity of thymus atrophy and antibody response in different inbred chicken lines during IBDV infection [12,13]. Selection and development of inbred chicken lines that have been maintained over several generations have provided valuable insights on gene function, such as viral receptor genes, immunoglobulin genes and MHC B haplotype on disease resistance [14]. Several inbred lines that were used to study avian virus infection include, the lines BrL and N, with formerly described as highly susceptible to vvIBDV infection [11]. In IBDV infection study by Smith et al. and Farhanah et al. on other inbred lines showed that the line 6 and line P birds were susceptible, meanwhile BrL and line N birds were highly resistant [9,15]. In addition, the association of host susceptibility on expression of immune response genes in the bursa was investigated, and it has been postulated that early immune response may have significant influence on chicken susceptibility via T cell function [16]. The complexity of immune response involving JAK-STAT and MAPK signaling pathway, as well as cytokine expression were also highlighted to be involved in modulating the disease severity in different inbred chickens [9,15]. Recently, we have characterized the transcriptome of bursa of Fabricius of six inbred chicken lines 6, 7, 15, N, 0 and P following infection with vvIBV strain UK661 at 3 days post-infection [15]. Although all the inbred lines succumbed to the infection, lines P and 6 were considered as the most susceptible lines, whilst line 15 and N were the least affected lines based on viral load and bursal lesion scoring at 3 days post infection. However, the expression profiles of the immunerelated genes at different days post-infection were not studied. In the present study, the host immune response through differential expression of selected immune-related genes in two inbred chicken lines with different MHC B haplotype namely line P (B19) and line N (B21) were characterized following UK661 vvIBDV infection.

2.2.2. Collection of bursal samples Each bursa samples were divided into two parts; one for pathological study in which the bursal tissue was fixed in 4% (v/v) paraformaldehyde, which later drained and replaced with phosphate buffered saline (PBS) (Sigma-Aldrich, UK), and the remaining bursal tissue were kept frozen in each individual vials for gene expression study. The bursal samples were air-couriered (World Courier, Malaysia) in dry ice. Upon arrival, bursal tissue kept in PBS was transferred to 4 °C chiller (NE Scientific, Malaysia), and the frozen bursal tissue was transferred to −80 °C freezer (Esco, Singapore). 2.2.3. Pathological scoring of bursal tissue Bursal section kept in PBS that was previously fixed in 4% paraformaldehyde from the control and infected samples collected at different time points were processed by embedding the tissues in paraffin blocks followed by tissue sectioning and then mounted them on glass slide followed by staining with hematoxylin and eosin according to the method described by Fischer et al. [18]. Bursal tissue was observed microscopically using FX100 light microscope (Olympus, Malaysia), and evaluated based on the severity of follicular damage, necrosis and lymphocytes depletion using a scoring system that was previously described by Sharma et al. [19]. 2.2.4. Bursal tissue RNA isolation and cDNA synthesis Total RNA from control and infected bursa were isolated using RNeasy Plus Mini Kit (Qiagen, UK) according to manufacturer’s guideline, and eluted in 40 μl RNase-free water. 1 μg of the isolated RNA was then reverse transcribed into cDNA using SensiFAST cDNA synthesis kit (Bioline, USA) in a total of 20 μl reaction mix. The cDNA synthesis reaction was performed in a thermal cycler (Biorad, USA) with following cycle profile: primer annealing at 25 °C for 10 min, reverse transcription at 42 °C for 15 min, inactivation at 85 °C for 5 min and finally hold or chill on ice at 4 °C. The synthesized cDNA were kept at −20 °C until further use.

2. Materials and methods 2.2.4.1. Viral load quantification. Viral load for each control and infected samples were measured based on absolute quantification. IBDV -specific primers were designed as follows: forward primer; 5′ATG CTC CAG ATG GGG TAC TTC-3′, and reverse primer; 5′-TTG GAC CCG GTG TTC ACG-3′ respectively, targeting VP4 region of IBDV [3]. The standard curve and melt curve for IBDV gene was generated through SensiFAST SYBR kit (Bioline, USA) using positive control cDNA obtained from bursa-infected vvIBDV at 10-fold serial dilution, ranging from 9 to 13 log10 viral copies number. A SYBR Green based real-time qPCR was performed in 3-steps cycling using a previously published method [3]. Standard curve generated was used to determine appropriate primer concentration and optimized concentration of the template, whilst melt curve generated was to verify the specificity of the amplification. All samples were assayed in triplicates, and the cycle threshold (Ct) values were recorded to quantify viral copy number based on log10 scale using the linear equation obtained from the standard curve.

2.1. IBDV strain Very virulent infectious bursal disease virus (vvIBDV) strain UK661 was isolated in the UK from 10-day-old broilers in 1989. The isolate was previously characterized as vvIBDV based on sequence analysis of VP2 (accession number NC_004178/9) [17]. 2.2. Chickens and IBDV infection Inbred specific-pathogen-free (SPF) chickens of lines P and N were obtained from unvaccinated flocks, supplied by Primary Production Unit of the Institute for Animal Health (Compton, United Kingdom). Details of these lines can be found at http://www.narf.ac.uk/chickens/. Line P with 30.2 % heterozygosity have B19 haplotype, meanwhile line N with 25.6% heterozygosity have B21 haplotype. These inbred lines have been maintained through over 20 generations by full sibling mating. Infected and age-matched control birds were housed in separate experimental animal rooms and supplied with both water and a vegetable-based feed ad libitum.

2.2.4.2. Preparation of standard curve for gene primers. Standard curves were generated for 31 genes namely IFNG, IFNB, IL1B, IL2, IL6, IL12A, IL12B, IL15, IL15RA, IL17, IL18, TLR3, CXCLi2, CCL4, CCL5, CCR5, HSPA5, HSPB1, CTLA4, IGJ, BLNK, CD72, CD86, IRF5, IRF10, NOS2, STAT1, TNFSF13B, CASP3, GAPDH and ACTB using positive control cDNA obtained from bursa-infected vvIBDV in a series of 10-fold

2.2.1. IBDV challenge procedures Each bird within the infected group for each line were infected with 105.43 EID50 of UK661 vvIBDV strain at 6-weeks old via the intra-nasal 2

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dilution ranging from 1000 ηg to 0.01 ηg using TaqMan Fast Advance Master Mix (Applied Biosystems, USA). Each gene was run in triplicate with following cycle profile: one cycle at 50 °C for 2 min and, at 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min on CFX96 real-time system (Bio-rad, USA). The data were expressed in cycle threshold (Ct) values to plot a standard curve in which used to determine PCR amplification efficiency, linear dynamic range of reaction and coefficient of determination (R2) as described by Bustin et al. [20].

(p < 0.05). Although line P showed slightly higher bursal lesion compared to line N at 48 and 54 hpi, they are not statistically significant at p < 0.05 (Fig. 1). 3.3. Standard curves generation for gene primers for real-time qPCR analysis Standard curves were generated for 29 target genes, 2 reference genes, and also for VP4 region of IBDV for quantification and determination of expression of immune-related genes and viral copy number, respectively. A linear relationship was observed in target genes, reference genes and IBDV-specific primer over five log10 dilutions. Genes with high PCR efficiency (R2: 90% - 110%) [22] were used in the high-throughput real-time qPCR reaction. The PCR efficiency, correlation of determination (R2) and equation of standard curve for each genes are presented in Table 1.

2.2.5. High-throughput real-time qPCR analysis and data processing A total of 29 target and 2 housekeeping 20X TaqMan® Gene Expression Assay (Applied Biosystems, USA) were pooled together at 2 μl each in DNA Suspension buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to make a final concentration of 0.2X, and amplified using PreAmp Master Mix (Fluidigm Corporation, USA) according to manufacturer’s protocol. The samples and 10X TaqMan gene expression assay (Applied Biosystems, USA) were prepared according to manufacturer’s protocol. Gene expression assay was run on 96.96 dynamic arrays integrated fluidic circuit (IFC) chip (Fluidigm Corporation, USA) according to manufacturer’s guideline. Each sample was prepared in triplicate while each assay was prepared in duplicate. The IFC chip was placed in Biomark™ HD System (Fluidigm Corporation, USA) for qPCR reaction. The data generated by this platform was analyzed with Fluidigm RealTime PCR Analysis Software (Fluidigm Corporation, USA) using the Linear (Derivative) Baseline Correction and the Auto (Global) Ct Threshold Method for analysis setting. Gene expression data was obtained from the fold-change value calculated from infected to control expression ratio using -ΔΔCT method in which expression value was normalized against two reference genes; GAPDH and ACTB. Gene expression of target genes was reported as log2 (fold-change) [21]. For gene with undetected expression in the uninfected control group, log2 fold-change cannot be calculated, and therefore expression of this gene was expressed in Ct value.

3.4. Detection of viral copy number in the bursa of Fabricius following vvIBDV infection The UK661 vvIBDV was not detected in the bursa of both uninfected control lines P and N at 0 hpi, however, this virus was detected in both lines as early as 24 h following the infection with values of 8.29 log10 and 8.13 log10 copies number, respectively. The copy number of the virus was detected higher in line P and significantly increased by 1.4fold with peak expression at 48 hpi (p < 0.05). The viral copy in line N also significantly increased at 48 hpi compared to 24 hpi (p < 0.05). The viral copy number of vvIBDV was significantly higher in line P compared to line N at 48 and 54 hpi (p < 0.05) (Table 2). 3.5. Gene expression and KEGG pathway analysis High-throughput gene expression analysis using microfluidic dynamic arrays was conducted to quantify mRNA expression levels of various immune-related genes in the bursa of Fabricius tissue of inbred lines P and N following infection with UK661 vvIBDV strain at 24, 48 and 54 hpi compared to uninfected control groups at 0 hpi. Gene expression of the target genes were reported as log2 (fold-change) with log2 (fold-change) > 2 considered as differentially up-regulated, meanwhile log2 (fold-change) < -2 considered as differentially downregulated. The target genes included in this study can be classified as Bcell related genes : BLNK, IGJ, TNFSF13B and CD72, T-cell related genes : CD86 and CTLA4, Th1 associated cytokines: IFNG, IL12A, IL12B, IL2, IL15 and IL15RA, pro-inflammatory cytokines and chemokines: IL1B, IL6, IL18, IL17, CXCLi2, CCL4, CCL5 and CCR5, IFN related genes: IFNB, STAT1, IRF5, and IRF10, stress-related genes: HSPA5 and HSPB1, and other immune-related genes including TLR3, NOS2, and CASP3.

2.3. Statistical analysis All quantitative data were analyzed using SPSS version 21 (IBM Corporation, Armonk, NY). The results from this study were reported as mean ± standard error of mean (SEM). Data were evaluated for statistical significant differences using a one-way ANOVA analysis followed by Duncan post hoc test. Data with p value < 0.05 were considered as significantly different from the control or between two different inbred lines in the same experiment. 3. Results 3.1. Results of vvIBDV infection challenge test in inbred lines

3.5.1. Expression of B-cell related genes, T-cell related genes and Th1 associated cytokines In general, line N (B21 haplotype) showed greater down-regulation of B cell-related genes compared to line P following vvIBDV infection (p < 0.05). Down-regulation of B cell related genes namely BLNK, TNFSF13B and CD72 were detected starting at 48 hpi in line N. However, expression of these genes were not differentially expressed in line P (B19 haplotype). Greatest down-regulation was documented for CD72 in line N at log2 fold-change of -4.24. Differential down-regulation of B-cell related gene in line P was only documented for CD72 at 54 hpi (Fig. 2). Although expression of B-cell related genes were down-regulated in the infected lines, we documented an up-regulation of T-cell related genes such as CD86 and CTLA4, and Th1 associated cytokines such as IFNG, IL12B, and IL2. Initially, CD86 was down-regulated in line P at 24 hpi, but this gene was then up-regulated at 48 hpi (Table S1). Other genes such as IL2, CTLA4 and IFNG showed a significant up-regulation

There was no clinical signs and mortality recorded in control groups for lines P and N throughout the course of experiment. In the infected groups, the clinical signs started to develop at 48 hpi, with all birds from the infected line N showed clinical signs. Meanwhile in line P, the birds showed clinical signs later on that day. By 54 hpi, however, all infected birds required humane euthanisation as they reached the clinical end points. 3.2. Histopathological changes of the bursa of Fabricius following vvIBDV infection As expected, uninfected control chickens of both lines P and N had no bursal lesion at 0 hpi. At 24 hpi, line P showed greater bursal lesion compared to line N, however the bursal score was not statistically significant at p < 0.05 (Fig. 1). Nevertheless, at 48 hpi onwards, both lines had significantly severe depletion of lymphoid cells and degeneration of bursal follicles compared to bursal score at 24 hpi 3

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Fig. 1. Histopathological changes of the bursa of Fabricius of line P and N birds following UK661 vvIBDV infection. Different letters signify: (nf) normal follicles, (mdn) mild degeneration and necrosis of lymphoid cells, (fcvi) follicular cyst, vacuolation, and infiltration of fibroblast, (isi) interstitial space infiltrated with inflammatory cells, (dcm) degeneration of cortex and medulla and (vm) vacuolation in the medulla. Bursal lesion was scored between 0–5 based on lesion severity. Scale bar; magnification of 200X = 50 μm.

hand, was not detected in line N (Table S1). In addition, expression for IL12A was also documented, however its expression could not be measured based on relative expression log2 fold-change, as it could not be detected in the uninfected sample in both lines P and N. For this reason, Ct value was used to quantify IL12A level, with high Ct value implies low quantity of IL12A, while low Ct

in line P compared to line N (p < 0.05). Furthermore, the peak expression of CD86, CTLA4, and IFNG were detected in line P at 48 hpi except for IL2 which peak at 54 hpi (Table S1). The expression of CTLA4 however, was not differentially expressed in line N at all time points (Table S1) Meanwhile, expression of IL12B that was detected in line P, with differential up-regulation at 48 and54 hpi, on the other

4

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Table 1 Standard curve information generated from real-time qPCR for target genes, references genes and IBDV-specific primers. Genes Target genes: IL12A IL12B IFNB IFNG IL1B IL2 IL6 IL15 IL15RA IL18 IL17 CCL4 CCL5 CCR5 CXCLi2 TLR3 CTLA4 STAT1 TNFSF13B NOS2 CASP3 IGJ BLNK CD86 CD72 IRF5 IRF10 HSPA5 HSPB1 Reference genes: GAPDH ACTB IBDV-specific primer (VP4)

Assay ID/ Accession No.

PCR efficiencies (%)

Correlation of determination (R2)

Standard curve equation

Gg03349762_m1 Gg03349681_m1 Gg03344129_s1 Gg03348618_m1 Gg03347155_g1 Gg03346865_m1 Gg03337980_m1 Gg03337642_m1 Gg03315880_m1 Gg03337832_g1 Gg03365522_m1 Gg03338617_m1 Gg03360169_m1 Gg03360184_s1 GG03349360_m1 Gg03359006_m1 Gg03359552_m1 Gg03343393_m1 Gg03347037_m1 Gg03347740_m1 Gg03338677_m1 Gg03346952_m1 Gg03339304_m1 Gg03357700_m1 Gg03348391_g1 Gg03354005_m1 Gg03337559_g1 Gg03340218_m1 Gg03348779_m1

108.8 102.9 103.4 100.4 93.8 91.1 97.8 97.5 98.2 98.1 109.7 93.6 99.0 100.4 96.2 101.2 100.1 98.4 96.0 92.0 94.7 94.0 98.1 102.6 97.5 100.9 98.0 102.0 98.6

0.994 0.978 0.985 0.995 0.997 0.997 0.997 0.999 0.999 0.994 0.995 0.996 0.995 0.993 0.997 0.994 0.996 0.999 0.999 0.998 0.999 0.999 0.997 0.997 0.999 0.997 0.992 0.998 0.998

y y y y y y y y y y y y y y y y y y y y y y y y y y y y y

Gg03346984_g1 Gg03370230_s1 AY520910

98.3 90.7 90.6

1.000 0.999 0.997

y = -3.37x + 40.71 y = -3.57x + 41.62 y = - 3.57x + 60.05

= = = = = = = = = = = = = = = = = = = = = = = = = = = = =

-3.13x -3.25x -3.24x -3.31x -3.48x -3.56x -3.38x -3.38x -3.37x -3.37x -3.11x -3.49x -3.35x -3.31x -3.42x -3.30x -3.32x -3.36x -3.42x -3.53x -3.46x -3.48x -3.37x -3.26x -3.38x -3.30x -3.37x -3.27x -3.35x

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + +

42.45 51.38 38.55 46.06 45.89 52.53 50.00 45.64 46.86 46.56 49.61 46.30 50.36 46.41 43.27 44.63 49.99 38.41 45.57 41.91 44.50 44.07 46.09 45.01 43.88 45.86 46.22 38.85 41.69

The assay ID for reference and target genes were provided by manufacturer (Applied Biosystems, USA).

IL18, and IL17 were generally up-regulated in both lines as early as 48 hpi, with greater up-regulation documented in line P compared to line N (Fig. 2a and b). The expression of IL1B and IL17 were initially downregulated in line P and line N at 24 hpi, respectively, however their expression peaking up at 48 hpi. Expression of IL18 however, was not differentially expressed in line N at all time points. Overall, IL6 has highest up-regulation in both lines compared to other pro-inflammatory cytokines peaking at 48 hpi. At 54 hpi, the expression IL1B and IL6 decreased but remain differentially expressed in both lines. On the other hand, expression of IL18 was not differentially expressed in line P at 54 hpi (Fig. 2a). Also, the expressions of these pro-inflammatory cytokines significantly different between line P and line N at 48 hpi (p < 0.05). Up-regulation of pro-inflammatory cytokines were accompanied by up-regulation of pro-inflammatory chemokines namely CXCLi2, CCL4, CCL5 and associated receptor CCR5. In general, expression of CXCLi2, CCL4, CCL5 and CCR5 were up-regulated in both lines as early as 48 hpi, with higher expression documented in line P (Fig. 2a). Initially, the expressions of the pro-inflammatory chemokines were not differentially expressed in both lines at 24 hpi. Expression of CXCLi2, CCL4 and CCR5 were significantly different between line P and line N at 48 hpi (p < 0.05), and later during infection, the expression of these genes was slightly decreased in both lines at 54 hpi but remain differentially expressed (Fig. 2a and 2b). Overall, CCL4 has the highest expression value followed by CXCLi2 and CCL5 in both lines.

Table 2 Quantification of vvIBDV copy number in the bursa of Fabricius by real-time qPCR. Time (hours)

Uninfected 24 48 54

vvIBDV copy number (log10) in inbred chicken line P

N

ND* 8.29 ± 0.00a 11.40 ± 0.32b 11.14 ± 0.04b

ND* 8.13 ± 0.29a 10.19 ± 0.45c 10.11 ± 0.34c

The vvIBDV viral copy number was obtained from cycle threshold (Ct) using real-time qPCR platform. Using an equation y = m(x)+ c, the Ct value was then calculated to obtain a viral copy number based on log10 transformation. At 0 hpi, the uninfected bursa has no detection of vvIBDV. The data were presented as mean value of log10 (viral copy number) ± SEM taken from three birds, each with three technical replicates. abc Values with different superscripts differ significantly using Duncan’s test at p < 0.05. *ND: not detected.

value implies high quantity of IL12A. This gene was not detected in the uninfected samples with Ct value set to a maximum value of 35. IL12A was only detected in infected lines P and N as early as 48 hpi with Ct value of 22.55 and 25.30, respectively. Expression of IL12A was statistically significantly between line P and line N at 48 hpi and 54 hpi with p < 0.05 (Fig. 2c). Additionally, Th1 associated cytokine IL15 was differentially expressed only in line N at 54 hpi with log2 foldchange -2.75. On the other hand, the receptor for IL15, IL15RA was only detected in line N with peak up-regulation at 48 hpi at log2 foldchange 5.37 (Fig. 2b).

3.5.3. Expression of IFN-related and stress related genes The expressions of interferon (IFN) related genes including IFNB, STAT1, IRF5, and IRF10 were measured following vvIBDV infection at selected time points. In general, IFNB, STAT1, and IRF10 were upregulated in both line P and N as early as 48 hpi, meanwhile and the

3.5.2. Expression of pro-inflammatory cytokines and chemokines The expression of pro-inflammatory cytokines such as IL1B, IL6, 5

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Fig. 2. Quantification of immune-related genes expression by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis for up- and downregulated genes following vvIBDV infection at different time points. Greatest down-regulation was documented for CD72 in line N at log2 fold-change of -4.24. Differential down-regulation of B-cell related gene in line P was only documented for CD72 at 54 hpi. Immune-related genes of the bursa of Fabricius tissue of inbred (a) lines P and (b) line N following infection with UK661 vvIBDV strain at 24, 48 and 54 hpi compared to uninfected control groups at 0 hpi and the data were quantified based on log2 fold-change by comparing infected samples to non-infected samples, and normalized to the expression level of reference genes. (c) The expression of Th1 cytokines gene IL12A following vvIBDV infection at different time points. Expression of IL12A was statistically significant between line P and line N at 48 hpi and 54 hpi with p < 0.05 and IL12A was only detected in infected lines P and N as early as 48 hpi with Ct value of 22.55 and 25.30, respectively, (corrected 40-CT ± the standard errors). *abc Groups labelled with different superscripts differ significantly at p < 0.05.

P compared to line N at 48 hpi and 54 hpi (Fig. 2a and b). The expression of stress related genes such as HSPB1 and HSPA5 were also measured following vvIBDV infection. In general, expression of HSPB1 was up-regulated in line P and line N at 48 and 54 hpi, Line P showed higher up-regulation of this gene (p < 0.05). On the other hand, expression of HSPA5 was not differentially expressed in both lines (Fig. 2a and b).

expression of IRF5 was not differentially expressed in both lines. Initially, the expression of IFNB, STAT1 and IRF10 were not differentially expressed in line P at 24 hpi. Expression of STAT1 and IRF10 were also not differentially expressed in line N at 24 hpi. However, the expression of IFNB was slightly up-regulated at 24 hpi (Fig. 2a and 2b). The expression of STAT1 and IFNB were significantly different between line P and N at 48 hpi and 54 hpi (p < 0.05), but expression of IRF10 was only significantly different in both lines at 54 hpi (p < 0.05). In addition to that, expression of IFNB was up-regulated by ∼2-fold in line 6

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and medulla. In agreement with previous study, severity of bursal lesion was not associated with differences in the B haplotype [12]. Furthermore, severity of bursal lesion also was not dependent on amount of viral load, but instead the magnitude of T-cell response in the bursa that is influenced by strain virulence [26]. The immunosuppressive effects caused by IBDV were directed towards depletion of B lymphocytes and the down-regulation of B cellspecific genes such as BLNK, IGJ, and VPREB3 [8,27]. Down-regulation of B cell-related genes was also recorded in the current study including the expression of BLNK, TNFSF13B and CD72 with peak down-regulation observed in line N at 48 hpi. However, expression of B cell-related genes was not directly associated with viral copy number or changes in bursal lesion, since these genes were not differentially expressed in line P that has higher viral copy number. Interestingly, line N which seems to have lower bursal lesion scoring compared to line P showed higher down-regulation of B cell-related genes. Further analysis was done to study innate and adaptive immunity in lines P and N following vvIBDV infection. Innate immunity plays an important role as an early defense mechanism against viral infection in the avian host through expression of various immune mediators such as cytokines and chemokines [28]. Type-1 IFN such as IFNB is a critical gene with antiviral activity, as this gene has been previously documented to be expressed in HD11 cell line and also in vivo study of chicken infected with very virulent strain [29,30]. In this study, expression of IFNB was up-regulated as early as 48 hpi, with line P showed significantly higher expression of this gene compared to line N (p < 0.05). Further to that, expression of TLR3 has been previously reported to be up-regulated in the classical IBDV infected chicken at day 3 post-inoculation, but down-regulated in variant IBDV infected chicken, portraying that expression of TLR3 is modulated by different strains of IBDV [31]. However, in the current study, TLR3 was only upregulated in line P at 48 hpi suggesting the influence of genetic background of the host in modulating the expression of this gene during IBDV infection. In addition to that, macrophage activation through secretion of iNOS and pro-inflammatory cytokines such as IL1B, IL6 and IL18 were of importance during IBDV infection as these genes may promote cellular destruction [4]. In this study, expression of NOS2 was up-regulated as early as 48 hpi, with line P showed significantly higher expression of this gene compared to line N (p < 0.05). Expression of proinflammatory cytokines IL1B, IL6, IL18 and IL17 were also higher in line P at 48 hpi compared to line N. Expression of STAT1 and IRF10 which are important in IFN signaling pathway were also up-regulated, with significantly higher expression of STAT1 in line P at 48 and 54 hpi, and IRF10 at 54 hpi compared to line N (p < 0.05). Therefore, inbred line P significantly induced expression of pro-inflammatory cytokines, as well as IFN-related genes following vvIBDV infection. In addition, several inflammatory chemokines have been identified following IBDV infection through up-regulation of CXCLi2 and CCL4 at 2 dpi following vvIBDV and virulent IBDV infection, respectively [9,24,32]. In the current study, CXCLi2 and CCL4 were up-regulated in both lines at 48 hpi with an additional chemokine CCL5. Expression of

3.5.4. Expression of other Immune-related genes Additionally, genes that involve in immune response mechanisms such as TLR3, NOS2, and CASP3 were also measured following vvIBDV infection (Fig. 2a and 2b). The expression of TLR3 was reported not differentially expressed in both lines at 24 and 54 hpi, while the expression of NOS2 was up-regulated in both lines at 48 and 54 hpi with line P showed greater up-regulation of the gene (p < 0.05). Expression of CASP3, which is an apoptotic gene was down-regulated in both lines with line N showed greater down-regulation of the gene (Fig. 2a and b). In general, vvIBDV infection caused an up-regulation of immunerelated genes of those involve as Th1-associated cytokines, pro-inflammatory cytokines and chemokines, IFN-related genes, and stressrelated genes, and up-regulation of immune-related genes mostly take place at 48 hpi (Fig. 2). Meanwhile, genes that are considered as B-cell related genes were downregulated (Fig. 2; detailed information of gene expression value is available in Table S1). The highly expressed genes in this study were IL6 and IL2 in both infected lines P and N (Fig. 2). Pathway enrichment analysis on this set of genes showed that the genes were mostly and significantly involved in cytokine-cytokine receptor interaction and toll-like receptor signaling pathway. Genes that were differentially expressed were also significantly involved in virus or bacterial infection such as herpes simplex, influenza and salmonella (p < 0.05; Table 3).

4. Discussion IBDV infection generally associated with induction of pro-inflammatory cytokines, influx of T cells and macrophages into the bursa, and also depletion of B cells following classical, variant and very virulent IBDV infection. Previously, we have showed that inbred chickens of different MHC (B) haplotypes exhibit different susceptible spectrum toward IBDV based on bursal lesion scoring and viral load, with line P (B19 haplotype) is more susceptible compared to line N (B21 haplotype) following infection with vvIBDV strain UK661 [15]. However, the dynamics in the expression of innate immune related genes in the bursa of these inbred lines were not well studied. Based on real-time qPCR, the viral copy number of UK661 vvIBDV was detected in the bursa of Fabricius of infected line P and N as early as 24 hpi at log10 8.0 which later increased by ∼1.4-fold at 48 hpi and 54 hpi in both lines. In a study using SPF chickens infected with different strains, namely UPM0081 and DK01, IBDV positive bursa were detected during the course of infection at day 1 and day 2, meanwhile infection with less virulent strain such as vaccine strain D78, the viruses had a delayed detection at day 4 [23,24]. This finding also suggested rapid replication of the very virulent strain of IBDV as has been suggested [25]. The association of viral copy number and changes in a histopathological lesion of the bursa of Fabricius was evaluated, with line P showed greater bursal lesion compared to line N but the differences were not significant at different time points (p > 0.05, Fig.1). However, there was a major change in bursal lesion at 48 hpi as the bursa had severe atrophy of bursa follicles and degeneration of both cortex Table 3 KEGG pathway analysis of differentially expressed genes. KEGG pathway

No. of Genes

P-Value

Genes

gga04060:Cytokine-cytokine receptor interaction gga04620:Toll-like receptor signaling pathway gga05168:Herpes simplex infection gga05164:Influenza A gga04672:Intestinal immune network for IgA production gga04630:Jak-STAT signaling pathway gga04623:Cytosolic DNA-sensing pathway gga05132:Salmonella infection gga04621:NOD-like receptor signaling pathway

13 10 10 9 6 8 6 6 4

1.29E-12 5.23E-11 6.43E-09 6.63E-08 2.46E-07 6.41E-07 9.25E-07 1.16E-05 9.17E-04

IFNB, IL6, TNFSF13B, CCR5, IL18, IFNG, IL1B, IL15RA, IL15, IL12B, CCL5, CCL4, IL2 IFNB, IL6, CD86, IRF5, IL1B, TLR3, IL12B, CCL5, STAT1, CCL4 IFNB, CASP3, IL6, IFNG, IL1B, TLR3, IL15, IL12B, CCL5, STAT1 IFNB, IL6, IL18, IFNG, IL1B, TLR3, IL12B, CCL5, STAT1 IL6, CD86, TNFSF13B, IL15RA, IL15, IL2 IFNB, IL6, IFNG, IL15RA, IL15, IL12B, STAT1, IL2 IFNB, IL6, IL18, IL1B, CCL5, CCL4 IL6, IL18, IFNG, IL1B, NOS2, CCL4 IL6, IL18, IL1B, CCL5

7

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regulation of B-cell related genes compared to line P. It seems that the bursal immunopathology of IBDV correlates with the expression of proinflammatory related mediators and not with the expression of Bcell genes.

CCL5 (RANTES) has been extensively studied in a murine model. In a chicken model, expression of CCL5 was found to be up-regulated in the H9N2-infected macrophages [33]. In this study, CCL5 expression was successfully documented and found to be up-regulated following vvIBDV infection in both lines P and N, however expression of this gene was not significantly different among both lines. The potential role of CCL5 in modulating IBDV infection was also supported through upregulation of its corresponding receptor CCR5 in both lines. Study on Sendai virus-infected mice demonstrated that CCL5/CCR5 interaction inhibits virus-induced apoptosis and promote macrophage survival [34]. A virus-induced apoptotic gene CASP3 was found to be downregulated in both infected lines P and N following vvIBDV infection, which is in agreement with a previous study using classical IBDV strain F52/70 [8]. An in vitro study on influenza virus infection documented that an efficient virus replication requires caspase 3 activation [35]. The association of down-regulation of CASP3 with IBDV-induced apoptosis and IBDV-replication is of interest for future study. Previous study showed that infection of H5N1, Newcastle disease virus and IBDV in chickens induced stress response genes namely HSP60, HSP70 and HSP47 [8,36]. In current study, we documented an up-regulation in the expression of HSPB1 (HSP27) in both lines, which is a downstream target of p38 in MAPK pathway [37], and this gene was significantly higher in line P compared to line N (p < 0.05). Expression of HSPB1 was previously associated with viral infection [38], and play an important role in the expression of pro-inflammatory cytokines such as IL6 and CXCLi2 [39]. Despite non-differential expression of HSPA5 (GRP78) in both infected lines, this gene showed a striking difference in expression pattern with an up-regulated pattern in line P and downregulated pattern in line N. Therefore, B haplotype play an important role in inducing stress response, with B19 haplotype developed enhanced stress response compared to B21 haplotype. This study also analyzed the involvement of cell-mediated immune response of the infected host. Previous study reported that Th1 and Th2 responses were dependent on virus virulence [40]. In present study, line P showed enhanced expression of Th1-associated cytokines such as IL12A, IL12B, IFNG, and IL2, compared to line N. Expression of IL12B, however, was undetected in line N at 48 hpi and 54 hpi. Expression of IL12B was previously reported to be highly regulated as it was mainly produced by activated macrophages meanwhile expression of IL12A was detected ubiquitously in many cell types [41] might explain the undetected expression of IL12B in line N. Apart from that, expression of IL15 was found to be down-regulated in line N at 54 hpi, but not differentially expressed in line P. Study using another strain of vvIBDV also reported the non-differential expression of IL15 in SPF-infected chicken [24]. However, it is important to note that the complementary receptor for IL15, IL15RA was only detected in line P. A study in murine model documented that over-expression of IL15RA enhanced IL15mediated T cell survival in mice splenocytes, and signaling of IL15/ IL15RA was not limited to T-cell function but also other cell populations including NK cells, dendritic cells, B-cells and macrophages [42,43]. This study suggests that CTLA4 and CD86 are crucial in modulating T-cell activation [44] following vvIBDV infection. We documented that line P showed significantly higher expression of CD86 and CTLA4 at 48 hpi compared to line N (p < 0.05). CTLA4 is a protein receptor expressed on T-cells with inhibitory function may bind to CD86 expressed on antigen presenting cells (APC), and expression of these genes were found to be up-regulated in chicken lung infected with H9N2 [45,46]. Therefore, expression of CTLA4 and CD86 may as well associated in inbred chicken lines with different B haplotype.

Funding information The study was supported by the Institute of Bioscience, Higher Institution Centre of Excellence (IBS HICoE) grant no. 6,369,101 from the Ministry of Higher Education, Government of Malaysia. This work was also carried out as part of the UK’s Biotechnology and Biological Sciences Research Council (BBSRC) funded projects ‘Developing Rapid Responses to Emerging Virus Infections of Poultry (DRREVIP)’ via grant BB/K002465/1, and Avian Viral Diseases Programme via grant BB/ J004448/1. Ethics statement This study was carried out according to the guidance and regulations of the UK Home Office under the provisions of the project license Number 30/3169 issued by the Secretary of the State, Her Majesty’s Government of the United Kingdom. The experiments were carried out by animal handling staff and scientists holding personal licenses. The collected animal tissues were couriered to Institute of Biosciences, Universiti Putra Malaysia according to accepted import permit and health certificate, and received frozen in dry ice. Declaration of Competing Interest The authors declare that there are no conflicts of interest. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.cimid.2019.101399. References [1] P. Dobos, B.J. Hill, R. Hallett, D.T. Kells, H. Becht, D. Teninges, Biophysical and biochemical characterization of five animal viruses with bisegmented doublestranded RNA genomes, J. Virol. 32 (1979) 593–605. [2] Y.M. Saif, Immunosuppression induced by infectious bursal disease virus, Vet. Immunol. Immunopathol. 30 (1991) 45–50, https://doi.org/10.1016/01652427(91)90007-Y. [3] A.R. Yasmin, S.K. Yeap, M. Hair-Bejo, A.R. Omar, Characterization of chicken splenic-derived dendritic cells following vaccine and very virulent strains of infectious bursal disease virus infection, Avian Dis. 60 (2016) 739–751, https://doi. org/10.1637/11275-091315-Reg.1. [4] M. Khatri, J.M. Palmquist, R.M. Cha, J.M. Sharma, Infection and activation of bursal macrophages by virulent infectious bursal disease virus, Virus Res. 113 (2005) 44–50, https://doi.org/10.1016/j.virusres.2005.04.014. [5] I.J. Kim, S.K. You, H. Kim, H.Y. Yeh, J.M. Sharma, Characteristics of bursal T lymphocytes induced by infectious bursal disease virus, J. Virol. 74 (2000) 8884–8892 . (accessed November 14, 2016) http://www.ncbi.nlm.nih.gov/ pubmed/10982331. [6] H. Scanavini Neto, N.M.K. Ito, C.I. Miyaji, E.D.A. Lima, S. Okayabayashi, A.R.A. Correa, G.C. Eleuterio, M.A. Zuanaze, Infectious bursal disease virus: case report and experimental studies in vaccinated and unvaccinated SPF chickens and commercial broiler chicks, Rev. Bras. Cienc. Avic. 61 (2004) 41–54. [7] I. Eldaghayes, L. Rothwell, A. Williams, D. Withers, S. Balu, F. Davison, P. Kaiser, Infectious bursal disease virus: strains that differ in virulence differentially modulate the innate immune response to infection in the chicken bursa, Viral Immunol. 19 (2006) 83–91, https://doi.org/10.1089/vim.2006.19.83. [8] T. Ruby, C. Whittaker, D.R. Withers, M.K. Chelbi-Alix, V. Morin, A. Oudin, J.R. Young, R. Zoorob, Transcriptional profiling reveals a possible role for the timing of the inflammatory response in determining susceptibility to a viral infection, J. Virol. 80 (2006) 9207–9216, https://doi.org/10.1128/JVI.00929-06. [9] J. Smith, J.-R. Sadeyen, C. Butter, P. Kaiser, D.W. Burt, Analysis of the early immune response to infection by infectious bursal disease virus in chickens differing in their resistance to the disease, J. Virol. 89 (2015), https://doi.org/10.1128/JVI. 02828-14 2469–82. [10] H.D. Hunt, S. Jadhao, D.E. Swayne, Major histocompatibility complex and background genes in chickens influence susceptibility to high pathogenicity avian influenza virus, Avian Dis. 54 (2010) 572–575, https://doi.org/10.1637/8888-

5. Conclusions Line P which is more susceptible to vvIBDV showed greater expressions of proinflammatory mediators compared to line N while, line N which is the least affected by vvIBDV showed greater down8

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