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Comparative analysis of innate immune responses following infection of newborn calves with bovine rotavirus and bovine coronavirus

Radhey S. Kaushik

Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK S7N 5E3, Canada

Correspondence
Palok Aich
palok.aich{at}usask.ca

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Bovine rotavirus (BRV) and bovine coronavirus (BCV) are important causes of diarrhoea and death in newborn calves. Although these viruses belong to distinct viral classes, they both infect intestinal epithelial cells and induce similar clinical symptoms. Rotavirus usually causes an acute infection, but coronavirus infection can persist and reoccur in adults. Differences in viral structure and clinical outcome prompted us to postulate that innate mucosal immune responses would be markedly different following rotavirus and coronavirus infections. To address this hypothesis, gene expression following BRV and BCV infection was analysed in surgically prepared intestinal loops from 1-day-old colostrum-deprived calves. Gene expression was profiled at 18 h post-infection using bovine cDNA microarrays; the majority of differentially expressed significant genes were associated with the cell cycle and innate immune responses. A select group of these genes was validated by quantitative real-time PCR (qRT-PCR). The expression of genes associated with interferons (IFNs), cytokines and Toll-like receptors, which were not present on the microarray, was analysed further by qRT-PCR. Strong activation of TLR3, IL-6 and p65 was observed in BRV-infected host tissues, but not in tissues infected with BCV. Both viruses also downregulated IFN- and pro-inflammatory cytokine-associated pathways. In vitro studies confirmed that IFN inhibited viral replication. All of these results together suggested either that very early events of host responses at 18 h post-infection were being observed, or that both viruses have unique effective strategies to evade host immune responses.

Present address: Department of Biology and Microbiology and Veterinary Sciences, South Dakota State University, Brookings, SD 57007, USA.

A supplementary table showing primers used for the current study is available with the online version of this paper.

	INTRODUCTION

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Diarrhoea is a major cause of morbidity and mortality in young calves and results in greater financial loss to cattle producers than any other infectious disease (Busato et al., 1998; Durham et al., 1979; House, 1978). Bovine neonatal gastroenteritis is a multifactorial disease that can be caused by many pathogens, including bovine rotavirus (BRV) and bovine coronavirus (BCV), and these viruses can also be used as a model for understanding human rotavirus and coronavirus infections (Garcia et al., 2000). Worldwide, diarrhoea caused by rotavirus leads to approximately 6 % of all deaths in human children under the age of 5 years (Parashar et al., 2003). Coronavirus is also an important human pathogen causing enteritis, encephalitis and severe acute respiratory syndrome (SARS) (WHO, 2003).

BRV and BCV share properties such as stability at low pH, heat lability in the presence of proteolytic enzymes and transmission via the faecal–oral route. Both viruses can induce intestinal villous atrophy and diarrhoea, and co-infection may occur during natural infections (Acres et al., 1975; reviewed by Saif & Smith, 1985). Despite inducing similar clinical and physiological symptoms following infection, BRV [a non-enveloped, double-stranded RNA (dsRNA) virus] and BCV [an enveloped, (+) single-stranded RNA (ssRNA) virus] belong to different virus families and have very different physical characteristics (Bajolet & Chippaux-Hyppolite, 1998; Clark & McKendrick, 2004). Moreover, BRV infections are cleared more rapidly than BCV infections, which can persist longer and reoccur in adults (Crouch et al., 1985; Saif & Smith, 1985). These differences in viral structure and pathogenesis prompted us to hypothesize that innate mucosal immune responses would be significantly different following neonatal infections by these two enteric pathogens.

The present study used immunohistological and functional genomic approaches to investigate the effects of BRV and BCV on bovine intestinal tissues at 18 h post-infection. We selected this time period based on an in vitro report with simian rotavirus in Caco-2 cells, which suggested a time course over 12 h to observe the innate immune response (Cuadras et al., 2002). An in vivo study in colostrum-deprived newborn calves with BRV also suggested induction of innate immune responses in less than 24 h (Schwers et al., 1983a, b; Vanden Broecke et al., 1984). In vitro analyses of rotavirus and coronavirus interaction with host cells are limited by the lytic activity of these viruses, and these studies cannot address potential indirect effects on uninfected crypt epithelial cells or leukocytes in the mucosal immune system. By developing a novel animal model, we were able to compare the direct and indirect effects of BRV and BCV infection on intestinal cells. Microarray analyses provide a global evaluation of host gene-expression patterns following BRV and BCV infection, and changes in gene expression were validated by quantitative real-time PCR (qRT-PCR) analyses.

We analysed transcriptional expression of genes by using bovine cDNA microarrays. We also studied expression of specific genes known to be key components of the innate immune response, such as interferon (IFN), pro-inflammatory cytokines and Toll-like receptor (TLR) families, which were not present on the microarray used. BRV, a dsRNA virus, is expected to be recognized by TLR3, whilst infection following ssRNA-containing BCV could activate TLR7/8-dependent pathways. This basic knowledge and a deeper understanding of innate immune responses following these two enteric viral infections would certainly help to develop effective intervention strategies. We report here a transcriptional analysis of innate mucosal immune responses at 18 h following BRV and BCV infection.

	METHODS

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Intestinal loop surgery.
An intestinal loop surgical technique developed in sheep (Gerdts et al., 2001) was adapted for use in newborn calves. Briefly, a 2–3 m section of jejunum was transected and isolated from the functional digestive tract without compromising the mesenteric attachment or altering intestinal contents. The continuity of the remaining digestive tract was re-established by end-to-end anastomoses and the isolated section of jejunum was closed at both ends by inversion sutures. This section was subdivided by using silk ligatures into 10–15 cm long segments, designated ‘loops’, and each loop was separated by 15–70 cm long interspaces. During the development of this model system, we observed that removal of ingesta and commensal flora markedly reduced the number of differentially expressed genes in the presence of coronavirus (Wilson et al., 2005). For this reason, ingesta and commensal flora were not removed prior to BRV or BCV infection studies.

Virus, viral titration and assays.
Field strain BCV 85-189 was a generous gift from Dr Gerald Woode, Texas A&M University, College Station, TX, USA. BRV 85 field isolate was obtained from a field sample from a calf with diarrhoea. The faecal sample was extracted with chloroform to remove any potential contaminating coronavirus. Capture ELISA analysis showed that this field isolate contained BRV (unpublished observations). This field isolate was used to challenge a newborn, gnotobiotic calf and its faecal material was used to infect three more newborn gnotobiotic calves. Faecal materials from these animals were pooled to create the BRV field-isolate stock 85-Rota.

Titration experiments were performed with each virus to identify the minimum infectious dose required to consistently infect mucosal epithelial cells (Fig. 1, inset). These titrations were performed over a 10-fold range (50–500 µl stock virus) using field isolate BRV (85-Rota, 35x10⁶ p.f.u. ml^–1) or field isolate BCV (85-189 Corona, 9x10⁶ p.f.u. ml^–1) diluted to a total volume of 5 ml with calcium- and magnesium-free PBS (Sigma-Aldrich) and injected into duplicate loops. Duplicate control loops were mock-infected with 5 ml PBS for at least four infected loops and two control loops per animal. Titration was done in three animals for each virus. Viral plaque assays were performed by using MA-104 cells as described previously (Aha & Sabara, 1990; Redmond et al., 1991) to confirm the titre of BRV and BCV in intestinal secretions of all loops.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Fluid accumulation upon viral infection. Mean volume of fluid accumulated in each loop for injection of PBS (as control) and different amounts (volume and viral titre) of BRV () and BCV () are shown. Inset: mean output viral titre per infected loop versus total input viral titre injected in loops. Lines were drawn connecting the points to show the trend in the change of viral titre or fluid-accumulated volume. Error bars on the data points represent SD.

Animals and infection model.
Six 1-day-old male Holstein calves were purchased and housed at the VIDO animal facility. The current infection study was completed in surgically prepared, non-sterile jejunal loops in 1-day-old calves as described previously (Aich et al., 2005). Calves were fed colostrum-free milk substitute to avoid acquisition of maternal antibodies. Two of four loops prepared from each animal (BRV, n=3; BCV, n=3) were injected with field isolates of either BRV or BCV diluted in 5 ml PBS, and the other two loops were injected with PBS (control). Tissue samples from duplicate loops (control and infected) were collected in either RNAlater (Ambion) or 10 % buffered formalin, or cryopreserved for gene-expression and histological studies. All experimental procedures were conducted in accordance with the Guide to the Care and Use of Experimental Animals, provided by the Canadian Council on Animal Care.

Sample collection.
Intestinal contents were collected from each intestinal loop at the time of tissue collection and the total volume of fluid/loop was recorded to determine total virus replication per intestinal loop. Formalin-fixed tissue samples were processed for histology, and stained with haematoxylin and eosin (H&E) to examine morphological changes in intestinal villi or immunohistochemical detection of virus by using protocols described by Ellis et al. (1995). These analyses were used to confirm a productive viral infection in challenged loops and the absence of viral infections in control loops.

Intestinal loop tissues were infected with equivalent infectious virus particles contained in the 500 µl BRV and 500 µl BCV stock for microarray analysis. Tissues were collected immediately following euthanasia, rinsed in PBS and stored in RNAlater (Ambion) for 24 h at room temperature before being frozen at –20 °C to preserve the RNA integrity.

Histology and immunohistochemistry.
Formalin-fixed tissue from infected and control loops was processed by the Prairie Diagnostic Centre Laboratory (Saskatoon, SK, Canada). Tissue sections were stained with H&E and examined under a light microscope for morphological changes in intestinal villi.

Indirect fluorescent antibody staining for BRV was performed with cryopreserved tissue as described previously (Ellis et al., 1995), with the exception that the primary antibody used was a mAb to group A rotavirus VP6 protein (clone RV 11-2 A; Rural Technologies). Binding of the mAb was detected by using fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Cappel Research Products). Immunohistochemical staining for BCV used a previously described immunohistochemical procedure, adapted for formalin-fixed tissues, and a robotic slide stainer (Brigati et al., 1988). BCV antigen was detected by using a polyclonal rabbit antiserum raised to purified BCV. Binding of the primary antibodies was detected by using biotinylated goat anti-rabbit IgG and horse anti-mouse IgG, respectively (Vector Laboratories Inc.), and an avidin–biotin complex immunoperoxidase method (Haines & Chelack, 1991). The slides were photographed by using an Axiovert 200 inverted light microscope (x40 magnification) and camera system (Carl Zeiss).

RNA-extraction protocol.
The protocol for RNA extraction was similar to that described previously (Aich et al., 2005). The RNA concentration was determined spectrophotometrically by measuring A₂₆₀ and purity was assessed by using an Agilent 2100 bioanalyser with RNA 6000 Nano kits (Agilent Technologies). The quality of total RNA was assessed by comparing the ratio of the area under the ribosomal peaks for 28S and 18S RNA (Gottwald et al., 2001). RNA with a ratio of >1.9 was considered of adequate quality and was used for the microarray analyses. Poly(A) mRNA from total RNA isolated from each tissue sample was converted into cDNA with oligo(dT) primers. RNA isolated from duplicate loops within each animal was pooled to provide a representative RNA population for each biological replicate.

RNA labelling, hybridization and scanning protocol.
Resonance light scattering (RLS) microarray labelling and detection were performed following the manufacturer's instructions (Invitrogen). Microarray analysis was performed by using two technical replicates for each biological replicate. Isolated RNA was reverse-transcribed to cDNA by using a LabelStar Array kit (Qiagen) and cDNA was labelled with fluorescein-labelled dUTP (control) or biotin-labelled dUTP (sample) (both from Enzo Life Sciences). Animal-matched control (1 µg) (mock-infected loop) and sample (1 µg) (virus-infected loop) cDNAs were pooled and hybridized to Bovine 7800 series microarray slides (Pyxis Genomics) according to the instructions of the Genicon RLS two-colour kit (Invitrogen) for RLS detection. After hybridization for 24 h, silver-linked anti-fluorescein antibodies and gold-linked anti-biotin antibodies were incubated with the slides to create silver-labelled control cDNA and gold-labelled sample cDNA hybridized to the microarray. Microarrays were washed following the kit instructions, and the amount of reflected light from each metal particle was obtained by using a 16-bit CCD-based, white light illumination GSD-501 RLS Detection reader and ICS-501 image-capture software v. 2.2 (Invitrogen) (Bao et al., 2002).

Microarray data analysis.
Primary image analysis was performed by using ArrayVision (version 8.0, rev. 3.0; Imaging Research Inc.) for RLS images. Text files in the form of spreadsheets were generated from analysed data, consisting of spot-per-well identities, signal intensities for both channels (silver and gold) and background data. These data were background-corrected, normalized by the LOESS method from the Limma BioConductor package and subjected to Student's t-tests by using the ArrayPipe software (www.pathogenomics.ca/arraypipe/) (Hokamp et al., 2004). Microarray probe sequences were annotated by using ProbeLynx software (www.pathogenomics.ca/probelynx) (Roche et al., 2004). All genes were checked for false-discovery rate (at 30 % cut-off) by using significance analysis of microarrays (SAM) (Heller et al., 1997) and the genes that were expressed ±2-fold or more with respect to the control samples were considered as being differentially expressed and were selected for further analysis. These genes were annotated by using ArrayPipe (Hokamp et al., 2004; Roche et al., 2004) and classified based on Gene Ontology (GO).

Gene-validation and -expression studies using qRT-PCR.
We used qRT-PCR studies to determine the expression of selected genes. qRT-PCR using a SuperScript III Platinum two-step qRT-PCR kit with SYBR green (Invitrogen) was performed according to the manufacturer's instructions on a Bio-Rad iCycler, using previously described protocols (Aich et al., 2005; Wilson et al., 2005). Primers for sense and antisense strands of selected genes were designed by using Clone Manager 7.03 (Sci Ed Central) and Beacon Designer 2.1 from PREMIER Biosoft International (Bio-Rad). Primer information is provided in Supplementary Table S1, available in JGV Online. Primers were designed spanning an intron between exons wherever known and the specificity of PCR products was confirmed by gel electrophoresis and sequencing of PCR products. C_t (threshold cycle) values were normalized with respect to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) values and the error on each value was calculated from triplicate assays performed with cDNA from each biological replicate. GenBank accession numbers for genes studied are also listed in Supplementary Table S1 (available in JGV Online).

In vitro IFN-sensitivity assay.
Host cells [Madin–Darby bovine kidney (MDBK) or Georgia bovine kidney (GBK) cells] were seeded into a 24-well tissue-culture plate. Following cell attachment, the medium [minimal essential medium (MEM); Invitrogen] was removed from the plate, and 10-fold dilutions (from 10^–3 to 10^–9) of recombinant bovine IFN (IFN- or -; Genentech Inc.) were added to duplicate wells. The plate was incubated at 37 °C, 5 % CO₂. After 24 h, IFN was removed and an aliquot of BCV that gave 50 p.f.u. was added to each well and incubated for 1 h. The virus was then removed and the cell monolayer was washed once with MEM before overlaying with methylcellulose. Cultures were incubated for 48 h before staining with crystal violet (0.05 % in 80 % methanol) and plaques were counted. From the graph of plaque counts versus IFN dilutions, we determined the concentration of IFN that inhibited 50 % of viral plaques (IC₅₀). Vesicular stomatitis virus was used as a positive control for the IFN-sensitivity assay.

	RESULTS

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BRV and BCV infections in intestinal loops
Intestinal loops within each animal were injected with either PBS or varying doses of virus; the results showed that 500 µl stock virus (35x10⁶ p.f.u. ml^–1 for BRV and 9x10⁶ p.f.u. ml^–1 for BCV) was sufficient to induce consistent fluid accumulation (Fig. 1) and visible histological changes in the intestinal villi (Fig. 2). The mean volume of fluid recovered from uninfected loops was 3–5 ml and the mean volume of fluid recovered from infected loops was 23 and 18 ml for BRV and BCV, respectively. Viral titre at 18 h post-infection revealed a 2-log increase in BRV (4.75x10⁸ p.f.u. per loop) and BCV (4.0x10⁸ p.f.u. per loop) titres in infected loops. The inset to Fig. 1 shows the viral titres following 18 h infection. We noticed that, despite a 3-fold difference in input viral particles, the output viral titre and fluid accumulation were similar (Fig. 1). No viral titre was obtained in control or adjacent uninfected loops.

View larger version (146K): [in this window] [in a new window]   Fig. 2. Histology of jejunal mucosa in control and BRV- and BCV-infected loops. Tissues were collected 18 h post-infection, fixed and stained with H&E. (a) Control loop and (b) BRV-infected loop were collected from the same animal. (c) Control loop and (d) BCV-infected loop were collected from the same animal. Bars, 100 µm.

View larger version (115K): [in this window] [in a new window]   Fig. 3. Immunohistochemical staining to detect viral protein in mucosal epithelium. Intestinal loops were injected with PBS (control), BRV or BCV. Tissues were collected 18 h post-infection. Tissues from BCV-infected animals were formalin-fixed and tissues from BRV-infected animals were cryopreserved. Immunofluorescent staining was used to detect BRV antigen in control (a) and BRV-infected (b) loops collected from the same animal. Immunoperoxidase staining was used to detect BCV antigen in control (c) and BCV-infected (d) loops collected from the same animal. Bars, 100 µm.

We therefore used qRT-PCR analysis to confirm the gene-expression trend observed from microarray analysis and extended this analysis to include a set of cell cycle-regulatory genes that were not present on the microarray (Fig. 4). The results in Fig. 4 show the expression patterns of key cell cycle-regulatory genes, such as cyclins A1, B2, E1 and E2; cell-dependent cycle (Cdc) 2, 20, 26; and A8 (Fig. 4a); cyclin-dependent kinases (Cdk) 2, 4 and 6; and kinase inhibitors (Cdkn) 1A, 1C and 2D (Fig. 4b). These analyses revealed upregulation of the majority of these genes. Genes that were not upregulated include cyclin E1, Cdc20, CdcA8 and Cdk2 following BRV infection and Cdc20, CdcA8, Cdk2 and TP53 following BCV infection.

View larger version (17K): [in this window] [in a new window]   Fig. 4. qRT-PCR fold changes for cell cycle-associated genes. Fold-change values from qRT-PCR analyses of genes belong to (a) cell division cycles and cyclins and (b) cyclin-dependent kinases and inhibitors following BRV (empty bars) and BCV (filled bars) infection are shown. Error bars represent SD, calculated based on data from three animals.

To validate the hypothesis of disparate innate immune signalling following BRV and BCV infections, we selected genes that could be grouped broadly as IFN-response or -regulatory genes (IFN1@, IFNAA, IFNb, IFNg, IFNGR2, IP-10/CXCL10, OAS1, IRF1, IRF3 and ISGF3G), pro-inflammatory genes (IL-1A, IL-1B, IL-6, IL-10, IL-15, CEBPB, p65/RelA, NF-B1, NF-BAP and TNF-) and TLR and associated genes (TLR3, TLR7, TLR8, TLR9, MyD88, TBK1, TRAF6, JAK2, JAK3, DUSP1, p38MAPK, TRAF2 and TRAF3). These genes were selected based on their known involvement in responses to viral infections (Bickel et al., 1990; Biondillo et al., 1994; Frangogiannis et al., 2000) and to provide a more detailed analysis of innate immune responses following viral infections.

Fig. 5 shows fold-change values determined from qRT-PCR data for selected genes mentioned in the preceding section. Fold-change values for the IFN group of genes (Fig. 5a) showed that IRF3, most type I and II IFNs and IFN-inducible genes such as IP10 and OAS1 were either unchanged or downregulated following both viral infections. IFNGR2 was upregulated following BCV infection, but downregulated following BRV infection, and IRF1 was upregulated following BRV infection and downregulated or unchanged following BCV infection. ISGF3G was upregulated following both viral infections. To determine the IFN sensitivity of these viruses, we performed in vitro plaque-inhibition assays to quantify the IFN- and IFN- sensitivity of BRV and BCV. We observed that plaque inhibition by recombinant bovine IFN was 0.8 ng ml^–1 for IFN- and 8x10^–3 ng ml^–1 for IFN- for both viruses.

View larger version (23K): [in this window] [in a new window]   Fig. 5. qRT-PCR fold changes for innate immune genes. Fold-change values for genes representative of (a) IFN-response, (b) pro-inflammatory, (c) TLR and (d) related signalling pathways as expressed in bovine intestinal loop tissues 18 h post-BRV and -BCV infection. The relative fold-change values of these genes from qRT-PCR results for BRV-infected (empty bars) and BCV-infected (filled bars) intestinal ‘loop’ tissues (with respect to uninfected-control tissue) are plotted as the mean of three animals, with 1 SD of data calculated from three biological replicates.

qRT-PCR results showed that TLR3 was upregulated markedly following BRV infection and that TLR9 was upregulated modestly following both viral infections (Fig. 5c). All other genes analysed within the TLR pathways except TLR8 and TRAF6, as shown in Fig. 5(c), were downregulated by 18 h post-infection with both viruses. We also tested the transcriptional expression profile of other genes that are important intermediates in various innate immune responses, such as JAK2, JAK3, TRAF2 and TRAF3, as well as mitogen-activated kinases such as DUSP1, p38 and GTPase regulatory gene ARHA. These analyses revealed a number of marked differences in gene expression following these two infections. We observed a >1000-fold increase in TLR3 expression and 10-fold increased expression of IL-6 and IL-6-dependent/binding gene CEBPB relative to control loops in BRV-infected intestinal tissue. A 10-fold decrease in expression of IL-6 and a 5-fold decrease in CEBPB expression were observed following BCV infection.

A direct comparison of normalized fold-change values between viruses revealed other biologically relevant differences in host responses (Table 3). The comparison revealed that expression of TLR3, TLR9, IFN-, IL-6, IL-10, IL-1B and p65 was greater following BRV rather than BCV infection. In contrast, IFNGR2, p38 and p50 expression was lower in BRV than in BCV infection.

	DISCUSSION

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Expression profile of cell-cycle genes in BRV- and BCV-infected tissues
The functional genomic analysis indicated that both viruses led to increased expression of genes involved in cellular proliferation. To evaluate whether BRV and BCV infections promoted the coordinate expression of genes whose products play roles throughout the cell cycle, qRT-PCR analyses were performed. Data from Boshuizen et al. (2003a) and the present study (Fig. 4) indicated that viral infection had an indirect effect on the number of cells leaving G₀. These data do not address, however, whether the cells completed the cell cycle or whether they were arrested at one or more phases of the cell cycle. BRV and BCV infection may promote disparate rates of crypt cell regeneration and replenishment of epithelial villi, which may explain why BCV-infected calves have delayed resolution of clinical symptoms compared with BRV-infected animals (Boshuizen et al., 2003b). Global gene-expression analyses indicated that BRV and BCV infection of bovine intestinal tissues promoted differential expression of a number of genes whose products play a role in cell-cycle progression (Fig. 4). Expression of some genes, such as Cdc20 and CdcA8, was downregulated following both infections, cyclin E1 and cdk2D were downregulated after BRV infection and TP53 was repressed following BCV infection. The downregulation of these genes may be because of other cell cycle-regulatory processes or may reflect the kinetics of gene expression associated with the single-time-point data.

As we analysed the gene-expression profile of mixed cell populations from intestinal loops, we must be careful not to overinterpret our results. We cannot link gene-expression changes directly to a single cell type within the tissues (i.e. crypt cells). However, tissue samples were obtained 18 h post-infection for both pathogens, and infection with either pathogen produced characteristic villous atrophy and showed similar cell cycle-related gene expression. Therefore, qRT-PCR analysis confirmed that a number of genes encoding proteins that promote cell proliferation were upregulated in tissues infected with BRV and BCV compared with medium controls, indicating increased cellular proliferation in bovine intestinal tissues following enteric viral infections.

Expression of innate immune genes in BRV- and BCV-infected tissues
Innate immune responses at mucosal surfaces following viral infections remain poorly defined. For the present investigation, we selected a group of immune-response genes that are commonly activated following various viral infections and that can be considered as innate immune genes. These two important enteric pathogens cause very similar clinical disease and microarray analysis identified numerous innate immune genes that were common to both infections. There were also unique patterns of gene expression following BRV and BCV infection. However, due to the limited number of innate immune genes on the microarray used, we also selected a number of genes known to be involved in innate immune responses following viral infection for further qRT-PCR analyses.

qRT-PCR analysis revealed important differences in innate immune gene expression following BRV and BCV infection. Specifically, it appeared that BRV activated TLR3, NF-B p65 and the pro-inflammatory gene IL-6 at 18 h post-infection. In contrast, BCV did not induce a detectable pro-inflammatory response, despite the activation of p50. The current study, however, does not suggest any plausible difference in the activation of pathways. It was shown in a recent study that, despite transcriptional activation of TLR3 by dsRNA, innate epithelial responses could not be inhibited by blocking the activation of TLR3. An alternate non-TLR pathway via activation of protein kinase R could play an important role in innate mucosal immunity following rotavirus infection (Vijay-Kumar et al., 2005). It is also important to note that IRF1, which is required to activate type I IFN responses, was activated following BRV infection, and IFNGR2, a downstream receptor for IFN-, was activated following BCV infection. All other IFN-regulatory and -stimulatory genes evaluated in this study, except for ISGF3G, were either downregulated or unchanged following both viral infections. This may indicate either that both viruses suppress the IFN response or that this response was not activated at 18 h post-infection. The results from in vitro IFN-sensitivity assays support the conclusion that both viruses may have evolved mechanism(s) to inhibit the IFN response. Susceptibility of BCV to IFN was tested in bovine cells. Our results for the susceptibility of BRV to IFN are comparable to results reported by Dagenais et al. (1981).

Although IFN and pro-inflammatory responses in host cells are commonly observed following viral infection, we did not detect significant expression changes for these genes following BRV infection, except for CEBPB, IL-6 and p65 (Fig. 5). A previous study with rhesus rotavirus-infected human intestinal Caco-2 cells reported activation of various type I IFN responses at 16 h post-infection (Cuadras et al., 2002). The apparent differences in IFN responses between this in vitro study and the current in vivo study may be due to either differences in the viral strains or an increased sensitivity of detection in vitro with a homogeneous cell population. We observed activation of p50 by BCV and p65 by BRV, which together are responsible for activation of the NF-B transcription factor, but their activation mechanisms are distinct (Fujita et al., 1992). Our results also suggest that BRV and BCV control activation of these two transcription factors differently. Whilst p65 and p50 are two important subunits of the transcription factor NF-B, in our study it was not clear what this differential response following BRV and BCV infection might mean. Activation of p65 has also been implicated in cellular proliferation through an active dimeric p65, cdc2 and cyclin B (CcnB) complex (Meikrantz et al., 1990). In our study, cdc2 and CcnB2 were upregulated following both viral infections, but p65 was only activated following BRV infection. This might suggest that cellular proliferation and cell division were more active following BRV infection than BCV infection and that increased cell proliferation may be consistent with rapid intestinal recovery following BRV infection.

We also checked expression of the TNF gene, which encodes a multifunctional pro-inflammatory cytokine. This cytokine is secreted mainly by macrophages and is involved in the regulation of a wide spectrum of biological processes, including cell proliferation, differentiation, apoptosis, lipid metabolism and coagulation. qRT-PCR studies showed that this gene was repressed by both viruses (Fig. 5b). We also observed repression of genes such as IFNGR2 and p38MAPK following BRV infection compared with BCV infection. The detailed effects of these genes are not evident from this single-time-point study.

The results of the current investigation, when compared with in vitro gene-expression profiles for other pathogens, revealed that BRV, a dsRNA virus, and BCV, which replicates via intermediary dsRNA, induced distinct innate immune responses in vivo. IL-15 was upregulated following influenza virus infection of dendritic cells and IL-15 has also been shown to play an important role in many other viral (herpes simplex virus, Epstein–Barr virus, respiratory syncytial virus, reovirus and Sendai virus) infections through activation of NK cells (Fawaz et al., 1999). In the present report, IL-15 was unchanged following both infections. This comparison suggests again that, whilst in vitro studies are important for understanding the response of specific cell types following viral infection, the actual host response in vivo may be markedly different, due to possible cellular interactions or the release of either local or systemic soluble factors. The list of activated genes indicates clearly that BRV induces TLR-mediated responses, which further activate downstream pro-inflammatory genes such as IFN-, IL-6, IL-10, IL-1B and p65. Collectively, these differences in innate immune responses indicate that these two viruses have developed very different strategies to evade IFN and pro-inflammatory responses. Activation of pro-inflammatory genes at 18 h post-BRV infection may be consistent with the rapid clearance of this viral infection. The absence of this response following BCV infection may result in a more prolonged infection.

In summary, the present functional genomic analyses revealed that, in terms of cell-cycle regulation, the host response at 18 h following BRV and BCV infection was similar. However, innate mucosal immune responses to BRV and BCV were markedly different at 18 h post-infection. Downregulation of IFN responses following both viral infections may suggest a similar immune-evasion strategy. The innate immune genes that were actively upregulated, however, suggest clearly that BRV and BCV have evolved different mechanisms to activate the innate immune response. This proposed differential regulation of innate mucosal immune responses will require further verification through time-course analyses of viral infection and protein-based studies. Understanding how these physically divergent viruses induce similar symptoms, but different innate mucosal immune responses, might also identify unique strategies by which these pathogens evade host defences. A comparative analysis of host responses following infection by viruses that belong to different families, but cause similar syndromes, can be very important in understanding the host innate immunity and for developing successful intervention strategies.

	ACKNOWLEDGEMENTS

 
Funding was provided by Genome Canada, Pyxis Genomics (Canada) and Inimex Pharmaceuticals Inc. (Canada) to Genome Prairie (Canada). H. L. W. was supported by a post-doctoral fellowship from HSURC (now SHRF, Saskatchewan, SK, Canada). A. A. P. is the holder of an NSERC Senior Industrial Research Chair and L. A. B. is a recipient of the Canada Research Chair in Vaccinology. This manuscript is published with permission of the Director of VIDO as article number 378. The authors would like to thank the animal-care staff at VIDO for their handling and care of the animals and Ashley Begg, Wayne Connor and Elaine Van Moorlehem for their technical support in histology, qRT-PCR and viral assays, respectively.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
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Received 18 January 2007; accepted 1 June 2007.

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