In vitro study on role of σB protein in avian reovirus pathogenesis

Abstract

Avian reoviruses, members of Orthoreovirus genus was known to cause diseases like tenosynovitis, runting-stunting syndrome in chickens. Among eight structural proteins, the proteins of S-class are mainly associated with viral arthritis but the significance of σB protein in arthritis is not established till date. In this infection pathological condition together with infection of joints often leads to arthritis because joints consists of cartilage which forms lubricating surface between two bones, and has limited metabolic, replicative and repair capacity. To establish the role of σB protein in arthritis, an in-vitro microarray study was conducted consisting four groups viz. virus infected and control; pDsRed-Express-N1-σB and empty pDs-Red transfected, CEF cells. With cut-off value as FC ≥2, p value <0.05, 6709 and 4026 numbers of DEGs in virus and σB, respectively were identified. The Ingenuity Pathway Analysis gave an idea about the involvement of σB protein in "osteoarthritis pathway", which was activated with z-score with 3.151. The pathway "Role of IL-17A in arthritis pathway" was also enriched with -log (p-value) 1.64. Among total 122 genes involved in osteoarthritis pathway, 28 upregulated and 11 downregulated DEGs were common to both virus and σB treated cells. Moreover, 14 upregulated and 7 downregulated were unique in σB transfected cells. Using qRT-PCR for IL-1B, BMP2, SMAD1, SPP1 genes, the microarray data was validated. We concluded that during ARV infection σB protein, if not fully partially leads to molecular alteration of various genes of host orchestrating the different molecular pattern in joints, leading to tenosynovitis syndrome.

Keywords: S3 gene; arthritis; avian reovirus; microarray; σB protein.

Figure 1. Schematic representation of workflow of microarray experiments for delineating osteoarthritic pathways in ARV and σB treated CEF cells

Figure 2. Cloning and expression of recombinant σB protein

(a) PCR amplification of ARV σB gene. Lane M: 1 Kb plus DNA ladder, Lane 1: NTC, Lane 2: σB 1104 bp PCR product; (b) Lane M: 1 Kb plus DNA ladder, Lane 1: NTC, Lane 2-3: colony PCR yielding σB 1104bp PCR product; Lane 4: Plasmid construct pDsRed-Express-N1-σB; (c) Western blot analysis using hyperimmune sera rose against recombinant σB protein in SPF chicken. Lane 1: 24 h, Lane 2: E. coli expressed σB protein, Lane 3: 48 h, Lane 4: 72 h post pDsRed-Express-N1-σB plasmid transfection CEF cell lystae showing expressed eukaryotic σB protein.

Figure 3. Activated canonical pathways in ARV infected and pDsRed-Express-N1-σB transfected groups

(a) Depicts the activated canonical pathways based on their z-score ≥2 analysed through IPA, (b) Venn diagram of differentially expressed genes (DEGs). All DEGs are clustered into four comparison groups represented by four ellipses. The sum of all the figures in one ellipse represents the number of DEGs in one comparison group (e.g., ARV infected vs. σB transfected). The overlapping parts of different ellipses represent the number of DEGs in common from those comparison groups. Diagram shows the upregulated and downregulated unique and common genes related to osteoarthritis pathway of both ARV infected and σB transfected CEF cells.

Figure 4

Delineated mechanism of osteoarthritis changes in response to σB protein (a) Pathway contains the differentially expressed genes related to ARV infected cells showing their mode of action and relation with each other in arthritis activation. (b) Predicted mechanism of arthritis induced by σB protein by NFkB pathway and the up and down regulation of closely related genes of NFkB network. Red colour indicates the upregulation of particular gene and green colour indicates the downregulation of particular gene. Blue line indicates leads to inhibition and orange line indicates lead to activation and orange line indicates “finding inconsistent with state of downstream molecule”.

Figure 4

Delineated mechanism of osteoarthritis changes in response to σB protein (a) Pathway contains the differentially expressed genes related to ARV infected cells showing their mode of action and relation with each other in arthritis activation. (b) Predicted mechanism of arthritis induced by σB protein by NFkB pathway and the up and down regulation of closely related genes of NFkB network. Red colour indicates the upregulation of particular gene and green colour indicates the downregulation of particular gene. Blue line indicates leads to inhibition and orange line indicates lead to activation and orange line indicates “finding inconsistent with state of downstream molecule”.

Figure 5

Interconnected network of differentially expressed osteoarthritis related genes (a) DEGs of ARV infected CEF cells compared to whole DEGs. Each circle indicating the node or member genes of the network are related to the hub i.e. with higher diameter than other arranged according to their degree; (b) DEGs of σB transfected CEF cells compared to whole DEGs. Each circle indicating the node or member genes of the network are related to the hub i.e. with higher diameter than other arranged according to their degree.

Figure 6

The Y-axis of the bar graph indicates the log2 fold changes of respective gene in ARV infected (a) and σB transfected (b) CEF cells. cDNA derived from CEF cells at 48hr of infection/transfection was compared to respective control by qRT-PCR and microarray.