Female and castrated male, 5- to 6-month-old, crossbred (Angus X Hereford) calves (n = 30) were purchased from a single commercial herd. Calves were identified as seronegative for BHV-1 by screening with a recombinant, truncated glycoprotein D antibody capture ELISA (31). Calves were weaned, transported to the research facility, and adapted for 2 weeks to a diet of free choice hay and 0.5 kg oats/day. During the adaptation period, calves were housed as a single group and there was no contact with other cattle. The average weight of calves was 232 kg (range = 174 and 242 kg), and the animals were housed in a single pen throughout the study. All experimental procedures were conducted according to the Guide to the Care and Use of Experimental Animals, provided by the Canadian Council on Animal Care and the experimental protocols were approved by the University of Saskatchewan Animal Care Committee (Protocol #19940211 and 19940218). The animals used for tissue collection were the same animals used to analyze virus shedding, IFN gene expression, and the expression of IFN-stimulated genes in a previous study (4). In our previous study, we demonstrated that all infected animals shed greater than 10⁵ infectious virus particles/ml of nasal secretion at the time tissues were collected between days 3 and 7 pi (4). BHV-1 infection of nasal turbinate mucosal epithelium was also confirmed by immunohistochemical (IHC) staining (4).

Six (6) of the 30 calves served as uninfected controls and tissue samples were collected from the control animals immediately prior to challenging the remaining 24 calves with BHV-1. This ensured there was no potential exposure of control calves to BHV-1. The 24 infected calves were aerosol challenged with BHV-1 isolate 108 (5 × 10⁷ pfu/animal) on experimental day 0 as previously described (32). A clinical veterinarian, blinded to treatment group, examined calves daily and recorded body weight and temperature. For tissue collection, cohorts of six calves were randomly selected and euthanized with an intravenous injection of Euthanyl (240 mg/ml; Bimeda-MTC, Canada) on days 3, 5 7, and 10 pi. Tissue samples were collected within 15–20 min after euthanasia, and three replicate samples were immediately placed in RNAlater or 10% buffered formalin. Tissue samples from nasal turbinates were collected 10–12 cm from the external nares of all calves (Figure S1 in Supplementary Material). A 4–5 mm² piece of nasal turbinate was placed on the surface of a 4–5 mm² and 1-mm thick slice of fresh liver tissue with the epithelial surface of the turbinate supported by the liver. This protected the mucosal epithelium from damage during cryosectioning. Tissue blocks were snap-frozen in liquid nitrogen and stored at −80°C until tissues were used for cryosectioning and IHC staining. Formalin-fixed tissues were used for histology and IHC detection of BHV-1 proteins. Tissues fixed with RNAlater were stored at −80°C until RNA was extracted for qRT-PCR analysis of bovine chemokine gene expression.

The mAbs used for IHC and immunofluorescence (IF) and relevant isotype controls are listed in Table 1, which provides details on mAb specificity and the concentration of each mAb used for staining.

Three 4–5 mm² nasal turbinate tissue samples were collected from each animal and tissue blocks were randomly selected for cryosectioning. Tissue blocks were sectioned at −20°C, and 5-µm tissue sections were cut using a cryostat (DAMON/IEC Division, Microtome 3398) and mounted on Superfrost Plus slides (Fisherbrand#12550215, ON, Canada). Serial tissue sections were selected for IHC staining on the basis of optimum tissue morphology. Tissue sections were fixed in chilled 100% acetone for 8 min and air-dried for 20 min prior to storage at 4°C. Indirect-immunolabelling was performed as described previously (33) to visualize the location and distribution of IFN-α and IFN-γ in tissue sections and to perform morphometric analyses of the frequency of CD335⁺, CD3⁺, CD8⁺, and CD4⁺ cells in nasal turbinates before infection (day 0) and on day 3, 5, 7, and 10 post-BHV-1 infection. Biotin-conjugated goat-anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA #BA-9200) and the Vectastain Elite Avidin-Biotin complex Kit (Vector Laboratories, Burlingame, CA, USA #PK4000) were used to visualize bound mAbs. BHV-1 infection occurs primarily within mucosal epithelial cells of the nasal turbinate (4). Thus, boundaries set for morphometric analysis of individual lymphocyte populations included the mucosal epithelium and the underlying lamina propria region. Five contiguous microscopic fields (total area = 0.196 mm²) were analyzed within each tissue section, and cells with visible staining were manually counted using an Olympus CX31 microscope (Olympus, Center Valley, PA, USA) with a 40× objective. The first field counted was located at the upper left corner of the cryosection and four contiguous fields were then counted. The top margin of each field was defined by the external surface of the mucosal epithelium and included the underlying lamina propria. An Olympus BX51 microscope and DP70 digital camera system (Olympus, Center Valley, PA, USA) were used to capture images of IHC-stained cryosections.

Dual-color staining of tissue sections was performed using fluorochrome-conjugated mAbs that reacted with bovine CD3, CD8, and CD335. The mAbs (Table 1) were conjugated with fluorochromes using Molecular Probes^® Antibody Labeling Kit (Thermofisher Scientific, Carlsbad, CA, USA), following the manufacturer’s instructions. The anti-bovine CD3 mAb was conjugated with Alexa Fluor^® 594. The anti-bovine CD8 mAb was conjugated with Alexa Fluor^® 488 and Alexa Fluor^® 594, so it could be used in appropriate combinations with the fluorochrome-conjugated CD3 and CD335 mAbs. IgG1 isotype control mAb (Table 1) was conjugated with Alexa Fluor^® 594 to be used for confirmation of staining specificity of the three other fluorochrome-conjugated antibodies. Direct fluorochrome labeling was necessary since the three mAbs are all IgG1 isotype (Table 1) and mAb reactivity after fluorochrome conjugation was confirmed by flow cytometric analysis of labeled blood mononuclear cells. Serial nasal turbinate cryosections were cut and fixed as described previously and stored at 4°C overnight. Acetone-fixed sections were brought to room temperature, air-dried for 30 min, and then rehydrated with phosphate buffered saline (PBS). The PBS solution was changed three times at 5 min intervals before slides were blocked by incubating with 10% normal goal serum for 3 h, followed by a 5 min wash with PBS. Tissue sections were then flooded with 200 µl of PBS containing 5% normal goat serum and one of the following combinations of fluorochrome-conjugated mAbs: CD3 and CD335; CD335 and CD8; or CD3 and CD8. Cryosections were incubated in the dark at room temperature for 2.5 h before washing twice for 5 min with PBS that was mixed with a magnetic stirrer. Tissue sections were counterstained by flooding with 200 µl 4′,6-diamidino-2-phenylindole (1 µg/ml methanol) (Life Technologies, Camarillo, CA, USA) for 10 min at room temperature to visualize cell nuclei and then washed for 5 min with PBS. Tissue sections were cover slipped with Cytoseal 60 mounting media (Thermofisher Scientific, Carlsbad, CA, USA) and stored overnight at 4°C before fluorescent imaging was completed. Confocal images were generated using a Leica TCS SP8 scanning confocal microscope (Leica Microsystems, Wetzlar, Germany), equipped with a 63× oil immersion objective and utilizing the 405 nm (UV) and 561 nm laser lines. Confocal images from a minimum of 15 microscopic fields were captured per sample, and a minimum of 100 CD335⁺, CD3⁺, and CD8⁺ cells were counted per tissue section. Tissue sections were analyzed for nasal turbinate samples collected from three animals on day 5 pi to determine the frequency of CD335⁺CD3⁺, CD335⁺CD8⁺, and CD3⁺CD8⁺ cells.

RNAlater^® fixed nasal turbinate samples were homogenized as previously described (34) and total RNA was extracted using the RNeasy mini Kit (Qiagen Inc., Ontario, CA, USA) following the manufacturer’s protocol. RNA quality and total RNA were determined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Samples with an RNA integrity number of eight or higher were used to prepare cDNA.

Total RNA (1 µg) was reverse-transcribed using qScript™ cDNA SuperMix (Quanta Biosciences™ Beverly, MA, USA), following the manufacturer’s protocol. cDNA samples were diluted in DNAse/RNAse free water for use as template in qRT-PCR reactions.

Primer pairs for chemokine transcripts (Table 2) were designed using Clone Manager 9.0 program (Sci-Ed Software). Primer specificity for individual chemokine genes was confirmed through generation of a single peak in the melting curve and detecting a single amplicon following gel electrophoresis of PCR products. PCR products were also cloned in the PCR 2.1 vector using the TA cloning kit (Life Technologies, Camarillo, CA, USA). Cloned products were sequenced with a CEQ 200XL DNA Analysis system (Beckman Coulter) to confirm product identity and size. Primer amplification efficiency (Table 2) was determined using standard curves as described previously (35).

Gene expression data for individual samples was normalized relative to β-actin, which was confirmed to be stable over multiple time points and not affected by BHV-1 infection in nasal turbinates (data not shown). We also analyzed the expression of GAPDH and RPS9 reference genes. The expression of the three reference genes did not differ significantly when comparing samples collected at different time points following BHV-1 infection (data not shown) but β-actin transcription displayed the least variability among animals at each time point. The β-actin gene also had the lowest quantification cycle (Cq) value when comparing among the three reference genes and was therefore selected as the best reference gene for detection of genes with high transcriptional levels. qRT-PCR was performed using the PerfeCTa^® SYBR^® Green FastMix for iQ (Quanta BioScience, Beverly, MA, USA). Briefly, 9 µl of Perfecta SYBR green master mix (2×), 3 µl of the primer pair at 3.3 µM, and 3 µl cDNA template were added to a 15 µl final volume. The reaction was performed in BioRad iCycler iQ PCR detection system using the following program: 1 cycle at 95°C for 30 s, 45 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. After cycling, the temperature was increased starting from 56°C at a rate of 1°C every 10 s to build a melting curve (40 times). Amplification data obtained for individual genes was expressed as Cq and was subtracted from the Cq of the β-actin reference gene to obtain ΔCq (ΔCq = Cq gene − Cq β-actin).

All statistical analyses in were performed using GraphPad software (Version 7.0a, La Jolla, CA, USA). A one-way ANOVA with Holm–Sidak’s multiple comparisons test was used to compare the frequency of leukocyte subpopulations in nasal turbinates before BHV-1 infection (day 0) with each day sampled pi. A Mann–Whitney test was used to analyze changes in the expression level of individual chemokine genes in nasal turbinates following BHV-1 infection when compared to preinfection (day 0) levels.

Previous studies confirmed IFN-α and -γ gene expression in nasal turbinates and the concentration of IFN in nasal secretions increased following a primary BHV-1 infection (4, 32). IHC staining of nasal turbinate cryosections with mAbs specific for bovine IFN-α and -γ was performed to determine if the production of these cytokines could be localized to specific cells within the tissue. IHC staining revealed, however, a diffuse staining pattern with the most intense staining associated primarily with mucosal epithelial cells (Figures 1C,D). Diffuse staining was occasionally observed throughout the lamina propria but there was no consistent pattern and staining could not be localized to individual cells. Isotype-matched mAbs were used to confirm the specificity of the staining observed at the mucosal surface and in the lamina propria (data not shown). Due to the diffuse and inconsistent staining pattern observed for IFN in tissue sections, a qualitative analysis was used to determine the frequency of positive staining in nasal turbinates collected from animals before and after BHV-1 infection. Some of the nasal turbinate samples collected from uninfected animals were positive for IFN-α (3/6 animals; 50%) and IFN-γ (4/6 animals; 60%) (Figures 1A,B). IHC staining of day 5 tissue sections revealed more intense IFN-α staining of the epithelial cell layer in all animals but only 80% (5/6 animals) of the tissue samples were positively stained for IFN-γ. The staining reaction was darkest at the epithelial surface and generally less intense in the lamina propria (Figures 1C,D). Positive staining for type I IFN on day 0 (Figure 1E) may reflect environmental exposure to other pathogens or commensal microbes that maintain a basal level of innate immune activation in mucosal epithelium of the URT. IFN-α is a potent activator of NK cells, which may explain the concurrent expression of IFN-γ (Figure 1F) if these innate immune cells are present in the tissue. Therefore, we investigated the types of lymphocytes present in nasal turbinates before and after BHV-1 infection.

We previously demonstrated that bovine IFN-γ had limited direct antiviral activity, despite high levels of IFN-γ production during a primary BHV-1 infection (4). Thus, the role IFN-γ may play in clearing a primary BHV-1 respiratory infection is not known. One hypothesis is that increased IFN-γ production may reflect increased recruitment of either NK cells or T-cells to the site of viral infection and subsequent activation of these cells by Type I IFNs. Nasal turbinates are a site of BHV-1 infection and replication, and therefore, to test this hypothesis, nasal turbinate tissue was collected from BHV-1 infected calves. IHC was used to analyze the phenotype and frequency of innate and adaptive lymphocytes present in the tissue.

Lymphoid populations present in nasal turbinates were first analyzed by staining for T-cells (CD3) and both conventional NK cells and non-conventional T-cells, which express CD335. Tissue sections were also stained for CD4 and CD8 which are markers for subsets of α/βTcR T-cells (36) and are also expressed on bovine NK cells and non-conventional T-cells (12). Morphometric analysis of tissue sections revealed that both T-cells (CD3⁺) and either conventional NK cells or non-conventional T-cells (CD335⁺) were present at low levels in nasal turbinates prior to BHV-1 infection (Figure 2). Cells staining for CD4 and CD8 were also present at low levels in the lamina propria prior to viral infection (Figures 2B,C and 3C; CD4 staining not shown). The number of CD335⁺, CD8⁺, and CD3⁺ cells present in the intraepithelial and lamina propria compartments increased significantly (P < 0.05) on day 5 pi (Figures 2C and 3) when compared to the number of cells present prior to infection (Figure 2). The increased number of cells present in nasal turbinates on day 5 then declined to preinfection levels on days 7 and 10 pi. In contrast, there was a significant (P < 0.05) decrease in the number of CD4⁺ cells on days 3, 7, and 10 pi (Figure 2). Cells staining for CD3 were the most abundant on day 5 pi, exceeding 100 cells per field (0.196 mm²) examined. The high frequency of CD3 cells on day 5 pi could not be accounted for by adding the total number of CD4⁺ and CD8⁺ cells, suggesting that other CD3 co-expressing cell populations may be recruited to the nasal turbinates following viral infection. γδTCR T-cells are a major T-cell subpopulation present at mucosal surfaces of ruminants (37) but the mAbs evaluated for bovine γδTCR staining reacted strongly with the mucosal epithelium. This non-specific staining precluded an accurate enumeration of all known T-cell subpopulations present in nasal turbinates following BHV-1 infection. IHC staining for IgA plasma cells also revealed consistently low numbers both before and after BHV-1 infection (data not shown).

The CD335 mAb binds to the NKp46 receptor on innate immune cells and is involved in the activation of NK cells and target cell lysis (9).The NKp46 receptor is, however, expressed on a variety of lymphocytes, which includes both conventional NK cells (CD335⁺CD3⁻) and non-conventional T-cells (CD335⁺CD3⁺) that play key roles in host immune defenses and inflammation (12). Therefore, we further investigated the phenotype of the CD335 cells recruited to nasal turbinates during BHV-1 infection to determine if this might account for the large increase in CD3⁺ cells. A double staining protocol was optimized to determine if CD335⁺ cells co-expressed CD8 (Figures 4A–C) or CD3 (Figures 4D–F). On average, 77.5% of CD335⁺ cells present in nasal turbinates on day 5 pi co-expressed CD3 while 22.5% of the CD335⁺ cells expressed markers typical for conventional NK cells (CD335⁺CD3⁻) (Figure 4G). Further, 89.7% of CD335⁺ cells present on day 5 pi co-expressed CD8 (Figure 4G). Conversely, 78.5% of CD3⁺ cells present in the nasal turbinates on day 5 pi co-expressed CD335 while 21.8% of the CD3⁺ cells co-expressed the markers of conventional T-cells (CD3⁺CD335⁻) (Figure 4I). Furthermore, co-staining for CD8 and CD3 revealed 86.6% of CD8⁺cells recruited to the nasal turbinates on day 5 pi co-expressed CD3 (data not shown). Double staining for CD8 and CD3 also indicated that only 73.4% of CD3 cells co-expressed CD8, which verified that other CD3-expressing cell populations were recruited to the nasal turbinates following BHV-1 infection. The specificity of the fluorescent signal observed when dual-staining cells was confirmed by the absence of detectable fluorescent signal in the mucosal epithelium and lamina propria when tissue sections were staining with flourochrome-conjugated isotype control mAbs (Figure S2 in Supplementary Material). Thus, co-expression analysis revealed that the predominant population of lymphoid cells recruited to nasal turbinates on day 5 pi were characterized by co-expression of CD335 and CD3 and the majority of these non-conventional T-cells also co-expressed CD8.

Human and mouse NKT cells express many different chemokine receptors that are involved in the recruitment and localization of NKT cells at sites of inflammation (20). Chemokine receptor expression has not been characterized for bovine NKT cells but the production of IFN-γ by human NKT has been shown to provide a positive feedback mechanism that enhances further NKT cell recruitment through induction of chemokines (38) Peak IFN-γ production following BHV-1 infection occurs on day 5 pi and this coincides with maximum recruitment of non-conventional T-cells (Figure 2). Therefore, we designed and validated qRT-PCR primers for known bovine chemokine genes (Table 2) to investigate whether BHV-1 infection altered the expression of chemokines that may be involved in recruitment of non-conventional T-cells. Among the 10 bovine chemokine genes analyzed, transcript levels for CCL4 (Figure 5A; P < 0.05), CCL5 (Figure 5B; P < 0.05), and CXCL9 (Figure 5C; P < 0.01) were significantly upregulated on day 7 pi. Thus, there did not appear to be a close temporal association between initial non-conventional T-cell recruitment to nasal turbinates and the onset of tissue expression of chemokine genes known to be involved in NKT cell recruitment.