ABSTRACT
The recently discovered influenza D virus (IDV) of the Orthomyxoviridae family has been detected in swine and ruminants with a worldwide distribution. Cattle are considered to be the primary host and reservoir, and previous studies suggested a tropism of IDV for the upper respiratory tract and a putative role in the bovine respiratory disease complex. This study aimed to characterize the pathogenicity of IDV in naive calves as well as the ability of this virus to transmit by air. Eight naive calves were infected by aerosol with a recent French isolate, D/bovine/France/5920/2014. Results show that IDV replicates not only in the upper respiratory tract but also in the lower respiratory tract (LRT), inducing moderate bronchopneumonia with restricted lesions of interstitial pneumonia. Inoculation was followed by IDV-specific IgG1 production as early as 10 days postchallenge and likely both Th1 and Th2 responses. Study of the innate immune response in the LRT of IDV-infected calves indicated the overexpression of pathogen recognition receptors and of chemokines CCL2, CCL3, and CCL4, but without overexpression of genes involved in the type I interferon pathway. Finally, virological examination of three aerosol-sentinel animals, housed 3 m apart from inoculated calves (and thus subject to infection by aerosol transmission), and IDV detection in air samples collected in different areas showed that IDV can be airborne transmitted and infect naive contact calves on short distances. This study suggests that IDV is a respiratory virus with moderate pathogenicity and probably a high level of transmission. It consequently can be considered predisposing to or a cofactor of respiratory disease.
IMPORTANCE Influenza D virus (IDV), a new genus of the Orthomyxoviridae family, has a broad geographical distribution and can infect several animal species. Cattle are so far considered the primary host for IDV, but the pathogenicity and the prevalence of this virus are still unclear. We demonstrated that under experimental conditions (in a controlled environment and in the absence of coinfecting pathogens), IDV is able to cause mild to moderate disease and targets both the upper and lower respiratory tracts. The virus can transmit by direct as well as aerosol contacts. While this study evidenced overexpression of pathogen recognition receptors and chemokines in the lower respiratory tract, IDV-specific IgG1 production as early as 10 days postchallenge, and likely both Th1 and Th2 responses, further studies are warranted to better understand the immune responses triggered by IDV and its role as part of the bovine respiratory disease complex.
INTRODUCTION
The bovine respiratory disease (BRD) complex is a significant economic and public health burden (1) that affects young bovines worldwide. BRD is triggered by the presence of one or several viruses and/or bacteria (2) that are favored by a set of predisposing factors such as altered state of the host immune system (3) and environmental factors (4). Respiratory viruses may induce the disease alone, as in the case of bovine respiratory syncytial virus (BRSV), or cause disease by initiating a cascade of subsequent events of immune suppression (3, 5), damaging the respiratory epithelium (6) and the airway clearance mechanisms (7) and altering the local biofilms (8–10) and normal commensal flora, thus leading to complex secondary bacterial invasion (8, 10, 11). Several viruses are thought to initiate and contribute to BRD, including BRSV, bovine parainfluenza type 3 virus (BPI3), bovine coronavirus (BCoV), bovine herpesvirus 1 (BoHV-1), and bovine viral diarrhea virus (BVDV). A new virus, now called influenza D virus (IDV), was identified in the United States in 2011, first in swine (12) and then in cattle (13) with respiratory disease. IDV is an enveloped single-stranded negative-sense RNA virus of the Orthomyxoviridae family. Like that of influenza C virus (ICV), the IDV genome is divided into seven segments, whereas the influenza A and B virus genomes are divided into eight segments (12, 13). The genetic identity on the whole genome between ICV and IDV only reaches 52%, and the two viruses do not seem able to reassort together (14). In addition, IDV was unable to cross-react with ICV-positive sera. Finally, a novel splicing strategy of IDV compared to ICV is also an important factor that led to the classification of the novel IDV in a new genus: the Deltainfluenzavirus genus of the Orthomyxoviridae family (13).
Since its discovery, both IDV and anti-IDV antibodies have been successfully detected in North and Central America, Europe, Asia, and Africa (12, 14–21). While IDV is thought to be circulating among several mammalian species (14), cattle are considered to be the primary host and reservoir. First, IDV was isolated on many occasions from bovine respiratory samples, and high seroprevalence and virus prevalence within bovine herds were recorded globally (12, 17, 21–23). Furthermore, the detection of IDV in the respiratory tract of cattle during BRD suggests that it could be a virus contributing to this major complex (23, 24). However, since IDV was also isolated from calves without BRD, its pathogenicity in cattle remains unclear. Two recent studies of IDV pathogenesis suggested that calves experimentally infected with strains isolated in the United States (24, 25) showed mild clinical signs of respiratory disease along with virus replication and lesions mainly restricted to the upper respiratory tract (URT). One study also clearly demonstrated that IDV is transmitted by direct close contact between calves (25).
The French IDV D/bovine/France/5920/2014 (IDV 5920) strain was recently isolated from the lower respiratory tract (LRT) of a calf suffering from BRD. Other classic respiratory viruses or bacteria were not detected (16), and this suggests that IDV 5920 may replicate in the LRT and induce clinical respiratory signs. Based on this virus, we developed a new calf model to (i) attempt to reproduce clinical signs in the LRT, (ii) study the viral replication, the viral tropism, and the pathology, (iii) characterize local and systemic immune responses, and (iv) investigate if this virus could be transmitted by airborne route over a short distance.
RESULTS
IDV induces moderate clinical signs and pathology in both the URT and LRT.After challenge and throughout the entire experiment (Fig. 1), no clinical signs (clinical score [CS] < 1; clinical cutoff of 1) were observed in calves in the control and aerosol sentinel groups, except in one control calf at days 12 and 13 (D12 and D13). This calf showed signs of apathy, loss of appetite, and difficulties in walking due to arthritis in one stifle, but no respiratory signs were observed. This animal recovered after antibiotic treatment. For the five calves inoculated with IDV 5920 and euthanized at the end of experimentation, clinical signs started at 4.6 ± 1.5 days, with a peak at day 8 ± 0.8 and a duration of CS of 6.4 ± 2.5 days. Calves showed mild (calves 7147 and 7161; sums of daily CS of 13.5 and 16) to moderate (calves 7142, 7165, and 7180; sums of daily CS of 33, 33.5, and 63) respiratory clinical signs. Mild clinical signs of respiratory disease included spontaneous coughing and slight tachypnea (35 to 40 breaths/min). Moderate respiratory signs, observed between D5 and D8, were characterized by repeated spontaneous coughing, abdominal dyspnea with increased respiratory rates (between 35 and 65 breaths/min), and abnormal lung sounds (wheezing) but without consequences on appetite or general state. No hyperthermia was detected in any of the infected animals, and all calves recovered by D12. The average CSs at D7, D8, and D9 were significantly higher in infected than in control animals (P < 0.0001 at D7 and D8 P > 0.05 at D9) (Fig. 2A). Moreover, the mean ACSs (mean of the individual area under daily clinical scores) were higher for the 5 direct-inoculated animals compared to controls (Fig. 2B), although no statistical significance could be demonstrated. Finally, one aerosol-sentinel calf (no. 7155; housed nearby and thus subject to aerosol transmission) showed mild clinical signs between D11 and D14, with individual clinical scores between 1.5 and 2.5.
(A) Experiment timeline and sampling. D0 is the day of inoculation. (B) Fourteen naive calves were allocated to two separated pens, one with 5 mock-infected calves (control group) and the second with 11 calves. In the latter pen, calves were divided into two groups separated by steel panels, 3 m apart. Eight calves (direct-inoculated group) were inoculated at day 0 (D0) with 107 TCID50 of IDV 5920, while the three others (aerosol-sentinel group) were not, in order to evaluate the aerosol transmission of IDV.
Mean clinical scores (A) and mean accumulated clinical scores (ACS, mean of the individual area under daily clinical scores using the trapezoid method) (GraphPad, La Jolla, CA) (B) of the five IDV direct-inoculated animals that were kept beyond day 8, all control uninfected calves, and all aerosol-sentinel calves.
Three direct-inoculated calves were euthanized at D8. At this time point, calf 6533 showed major respiratory clinical signs (CS of 6.5, starting at D4), while calves 6531 and 7144 were only mildly affected (CS of 2 starting at D6). These animals had macroscopic lung lesions characterized by patchy areas of atelectasis with a deep red texture. The lesions were restricted to the cranial right lobe and covered about 5% to 10% of the entire lung surface for two calves (no. 6531 and 6533) and less than 5% for calf no. 7144. No gross lesions were found in nasal cavities or the larynx or trachea. At D22 and D23 (end of the experiment), no macroscopic lesions were observed in any animal (including aerosol-sentinel calves), except in one direct-inoculated calf (no. 7165) which showed fibrinous and necrotic pleuropneumonia with several abscesses in the cranial lobes and one aerosol-sentinel (no. 7164) which showed limited lesions of BPI in the right cranial lobe. For calf 7165, bacteriology indicated the presence of Trueperella pyogenes, a bacterium typically involved in secondary respiratory infection of calves.
Histological examination was performed for all calves on olfactory bulb, nasal mucosa, trachea, mediastinal and tracheal lymph nodes, and lung tissue sections with gross lesions when present, collected from the right and left cranial, the intermediate, and the right and left caudal lobes. Microscopic lesions with light distribution (3 or fewer lesion foci per sample) were observed in the nasal mucosae and the right cranial lobes of 2 direct-inoculated euthanized at D8 (no. 6531 and 6533) and in the right cranial and accessory lobes of the lungs of two direct-inoculated euthanized at D23 (no. 7147 and 7165). No microscopic lesions were observed in the lung parenchyma from any other lobes. The lesions were typical of cases of subacute rhinitis (infiltration of the lamina propria by mononuclear cells in nasal epithelium) and subacute bronchointerstitial pneumonia (Fig. 3A) (neutrophils in bronchial lumens, neutrophilic and macrophagic alveolitis, and peribronchial and septal lymphoplasmocytic infiltration in the lung). At D8 (euthanasia), calf 6533 showed major respiratory clinical signs (CS of 6.5, starting at D4), while calf 6531 was only mildly affected (CS of 2). No microscopic lesions were observed in respiratory tissue of aerosol-sentinel calves.
(A) Subacute bronchointerstitial pneumonia with neutrophils in bronchial lumens, neutrophilic and macrophagic alveolitis and peribronchial and septal lymphoplasmocytic infiltration (left; magnification, ×200) and microscopic alveolar lesions at a higher magnification (right; magnification, ×400). (B) (Left) Immunostaining of left cranial lobe of calf no. 7165 inoculated with IDV showing cytoplasmic and nuclear labeling of the bronchiolar epithelium by the anti-IDV polyclonal antibody. (Right) Negative control of the same slide with the substitution of the IDV polyclonal serum by a rabbit normal serum (magnification, ×200).
IDV replicates in both URT and LRT.To assess the shedding of IDV in both URT and LRT, the presence of IDV RNA was monitored by reverse transcription-quantitative PCR (RT-qPCR) in deep nasal swabs (NS) between D0 and D22 and bronchoalveolar lavage specimens (BALs) on D0, D2, D7, D14, and D22. No virus was detected in control calves. IDV detection in NS started at D1 for four direct-inoculated calves and D2 for the other four. A peak of replication was observed at D4, with a mean titer of 5.6 × 108 (±3.9 standard deviations [SD]) IDV RNA copies/ml (Fig. 4A). The duration of excretion was 8.1 ± 1.9 days [interval of 5 to 12 days]. In addition, the IDV genome was detected in BALs of direct-inoculated calves with titers ranging between 5.1 × 103 and 1.4 × 105 RNA copies/ml in 3, 2, and 1 out of the 5 tested calves at D2, D7, and D14, respectively (Fig. 4B).
RT-qPCR IDV RNA loads in nasal secretions (A) and BALs (B) in the five IDV direct-inoculated animals that were kept beyond day 8 (blue), control animals (orange), and aerosol-sentinel calves (green). (C) IDV RNA loads were also detected in air samples collected over the course of the experiment in the direct-inoculated group area (infected area), the 3-m space between direct-inoculated and aerosol-sentinel calves (separated area), and the aerosol-sentinel area (contact area). Dashes represent negative results.
The presence of IDV in the lower respiratory tract was confirmed by analyses of IDV RNA in respiratory tissue samples collected from the three direct-inoculated calves euthanized at D8 (Table 1). Viral RNA was easily detected, with between 6.3 × 103 and 4 × 105 RNA copies/30 mg of tissue, in NS, trachea, cranial and caudal lobes of the lungs, and also in the mediastinal and tracheal lymph nodes. No virus could be detected in direct-inoculated calves euthanized at the end of the experiment on D23. In addition, samples from other collected tissues were all negative for IDV. Finally, to localize where IDV replicates, immunohistochemistry (IHC) was performed on lung tissue sections from two direct-inoculated calves euthanized at D8 (on cranial and intermediate lung lobes, which were positive by RT-qPCR) and on similar sections from control calves. IDV antigen deposition was detected in the epithelium of the bronchioles with a nuclear and cytoplasmic localization (Fig. 3B). All tissues from the control animals were IDV negative.
IDV detection in postmortem samples of the three direct-inoculated calves euthanized at D8 and three aerosol-sentinel calves euthanized at D22 to D23 as determined by RT-qPCRa
IDV transmission may occur by direct or aerosol transmission.IDV was detected in nasal secretions from the three calves in the aerosol-sentinel group (separated by fences, 3 m apart from the infected group and without effluent contact), with shedding starting at least 10 days after that in the direct-inoculated calves, at D11, D17, and D21, respectively (Fig. 4A). For the calf starting shedding at D11 (no. 7155), IDV was detected in NS until D19. For the other two calves, IDV was still present in NS at the end of the experiment (D22 to 23). We also confirmed IDV in the tissues of the LRT of at least one aerosol-sentinel calf (no. 7164) at D22 and additionally in tonsils for one calf (no. 7164) and in the olfactory bulb for two calves (7163 and 7164 [Table 1]). Unfortunately, IHC results were negative for IDV in tonsils and olfactory bulbs of these calves. To investigate aerosol transmission, we performed virus detection in 24 air samples collected over the course of the experiment in the aerosol-sentinel calves’ area, the separating space, and the direct-inoculated calves’ area. The IDV genome was detected in air samples from the three locations at D3, D5, D7, D9, and D13 in a range of 5.2 to 6.1 log10 RNA copies/m3 of air (Fig. 4C), with a maximum between D5 and D7. IDV detection started at D3 in the direct-inoculated group area and D5 in the separating space and aerosol-sentinel group area. After D9, IDV was detected only in aerosols of the aerosol-sentinel group area. Comparing consensus sequences of genes coding for the hemagglutinin-esterase-fusion (HEF) protein in the inoculum virus and in NS of infected calves before and after the peak of virus replication (no. 7144 at D2 and no. 7180 at D8) did not reveal any nucleotide substitution. In contrast, the HEF sequence in NS of an aerosol-sentinel calf (no. 7155, at D14) had 3 substitutions compared to the inoculum virus: 2 silent (T729C and C1278T) and 1 nonsynonymous (T1988A, leading to an M663K substitution) mutation.
IDV infection induces innate immune responses in BALs characterized by mRNA overexpression of pathogen recognition receptors and proinflammatory cytokines.Since IDV was able to replicate in the LRT and no data were available on IDV innate local immunity in the natural host, we first analyzed BAL collected from direct-inoculated calves and controls, for the transcriptomic response of 46 molecules. These included pathogen recognition receptors (PRRs), cytokines, chemokines, and antiviral molecules involved in the interferon type I (IFN-I) response (Table 2). Four time points were looked at: D0 was considered as a reference before challenge, D2 corresponded to the initiation of IDV replication and innate immunity, D7 corresponded to the peak of clinical signs, and D14 corresponded to the recovery of direct-inoculated calves.
Comparative chemokine and cytokine mRNA expression (assessed by RT-qPCR Fluidigm assay) in BALs between direct-inoculated and control calves, expressed as fold changea
Four genes coding for interleukin 4 (IL-4), IL-5, IL-10, and MYD88 failed to pass the Fluidigm PCR quality check. Fold changes (mean calibrated normalized relative quantity [CNRQ] values of direct-inoculated calves on mean CNRQ values of control calves after normalization on D0) and significant statistical differences between the two groups and the way of the expression (over- or underexpressed) for direct-inoculated calves compared to controls are shown in Table 2 for each molecule and each day. The log transformed CNRQ data of Fluidigm qPCR are available as Data Set S1 in the supplemental material. The highest fold changes between direct-inoculated and control calves were found mainly after infection at D2 and, to a lesser extent, D7, for CCL2 (D2 and D7), CCL3 (D2), CXCL1 (D2), RIG-1 (D2 and D7), Toll-like receptor 7 (TLR7) (D2), SOCS1 (D2), SOCS3 (D2), IL-13 (D2), IL-1α (D2 and D7), and IL-8 (D2). When statistical analysis was applied, differences for IL-1α and IL-8 between the two groups were not significant despite high fold changes, due to high variability between animals. At D2, IDV infection induced a significant overexpression of PRR mRNA in direct-inoculated calves, mainly of TLR3, TLR7, TLR9, NOD2, and RIG-1 (Table 2). No significant differences were found for the IFN-α gene, genes involved in the type I interferon pathway (IRF1, IRF3, IRF7, and STAT2), or interferon antiviral-induced molecule ISG15. In contrast, SOCS1 and SOCS3 mRNAs were significantly overexpressed at D2 in direct-inoculated calves (P < 0.001). Concerning proinflammatory chemokine mRNAs, significant differences were observed mainly for CCL3, CCL4, and CX3CL1 at D2 and D14, CCL2 at D7 and D14, ITGAL (also named LFA-1A or CD11a) at D14, and CX3CR1 at all days. All the genes were overexpressed in direct-inoculated calves except for the ITGAL gene, which was significantly underexpressed (P < 0.01 at D14). In addition, IL-6 was significantly overexpressed at D2 (P < 0.01) and D14 (P < 0.01) for direct-inoculated calves, while no differences were observed between IDV and control groups for IL-1β, tumor necrosis factor alpha (TNF-α), TNF-β, and NF-κB. The cellular adaptive immune response was also characterized by an overexpression in the IDV group of IFN-γ at D2 and D14, IL-2 at D7, and IL-13 at D2, D7, and D14.
We then further analyzed protein expression of 15 bovine chemokines in BAL cells using a multiplex Luminex assay (Table 3; Data Set S2). When statistically analyzed using the same method as for mRNA expression, results of protein concentrations indicated in the direct-inoculated group significant overexpression of TNF-α at D2 and D7, of CXCL10, IL-1-R, and IL-10 at D7, and of IL-2 at D2 and D14 (Table 3). CCL3, IFN-γ, and IL-8 at D2 and D7 and CCL4 at D2 were also upregulated for IDV-inoculated calves but without significant differences, probably due to individual variability between calves. Protein concentrations were similar between the two groups for IL-4, IL-6, CCL2, IL-1α, IL-1β, and IL-17α. Identical results were obtained using median fluorescence intensity (MFI) or log transformed MFI as output (data not shown).
Comparative chemokine and cytokine concentrations in BAL cells between direct-inoculated calves and controls as determined by Luminex assaya
IDV induces both humoral and cell-mediated systemic immune responses.The three colostrum-deprived, mock-inoculated controls remained seronegative for IDV hemagglutination-inhibiting (HI) antibodies throughout the experiment, and no increase of HI antibody titers was detected in the serum of the two other mock-inoculated control calves that had been fed with IDV-positive colostrum (Fig. 5A). Two direct-inoculated calves had already seroconverted by D7 (no. 6131 and 7144), with HI titers of 1:10 (limit of detection [LOD], 1:10), and all five direct-inoculated calves that were kept beyond D8 had seroconverted by D15 (titers between 1:40 and 1:160 [Fig. 5A]). In addition, the aerosol-sentinel calf no. 7155 that started shedding IDV at D11 also developed a humoral response by D22, with an HI titer of 1:40. Sera from the five direct-inoculated calves that were kept beyond D8 were additionally analyzed by IDV-specific IgG1 and IgG2 enzyme-linked immunosorbent assays (ELISAs) (Fig. 5B). In agreement with the HI data, IDV-specific IgG1 seroconversions were detected in all direct-inoculated animals. While one calf had seroconverted by D10 (no. 7165), a slight response was observed below the LOD in sera of the remaining four calves by D10, and these had all seroconverted by D22 to D23. No IDV-specific IgG2 antibodies were detected throughout the study; however, a weak response below the LOD was observed in two direct-inoculated calves (no. 7142 and 7180) by D22 to D23, suggesting a low starting production of IgG2 in sera.
Antibody response in the five IDV direct-inoculated animals that were kept beyond D8 (blue) or all control (orange) calves assessed by IHA (A) and by specific ELISAs for IDV IgG1 (blue) or IgG2 (green) (B). The line in panel B represents the positive threshold of ELISAs.
In the search for IDV-specific cellular memory responses in peripheral blood, 22 days after challenge, we analyzed the proliferation of IDV-restimulated peripheral blood mononuclear cells (PBMC). The cells used for this purpose had been collected on D−1 and D22 from all five direct-inoculated animals that were kept beyond D8. To ignore any unspecific responses against the swine testis fibroblasts in which IDV was propagated, responses induced by uninfected cell lysate were substracted from those induced by IDV-infected cell lysate. IDV-specific responses were thus expressed as the difference between these conditions. Overall, the IDV-specific proliferation was significantly stronger at D22 than at D−1 (Fig. 6), after restimulation with both live and inactivated virus (P = 0.045 and P = 0.031, respectively, two-sided paired t test). The strongest proliferation was detected in calves 7142 and 7165 at D22, likewise after using both live and inactivated virus (Fig. 6).
IDV-specific lymphocyte proliferative responses of PBMC from all directly inoculated calves that were kept beyond D8. The cells were collected at D−1 and D22 and stimulated ex vivo with either heat-inactivated influenza D virus-infected or uninfected cell lysate (A) or untreated influenza D virus-infected or uninfected cell lysate (B). After 6 days of incubation, proliferative response was determined by corrected optical density (COD) of alamarBlue (Invitrogen, Sweden). Results are expressed as the mean COD of triplicate samples, with standard deviations indicated by upward deflecting lines.
We then compared cytokine production of IDV-restimulated PBMC from the 5 direct-inoculated calves before (D−1) and after infection (D22) to profile the immune response. Live virus induced a weaker proliferation or more cell death than inactivated virus but was nevertheless used for cytokine analyses to mimic the natural situation. Out of 15 cytokines analyzed by Luminex assay, only the IDV-specific CCL4 responses were significantly higher in PBMC collected on D22 than in those collected on D−1 (P = 0.025 [data not shown]). On the other hand, regardless of whether the calves from which cells were derived had previously been challenged, IDV infection of lymphocytes consequently induced IL-1α and IL-8, at statistically significantly greater levels than control antigen (P = 0.043 and P = 0.0003 for IL-1α and P = 0.005 and P = 0.0014 for IL-8 in cells from D−1 and D22, respectively [data not shown]).
DISCUSSION
Since its first detection in 2011, IDV has intrigued the scientific community: its pathogenesis, tropism, natural reservoir, route of transmission, and role in respiratory affections are still not well understood. Studies using next-generation sequencing on samples collected from young cattle with signs of respiratory disease suggested a positive relation between BRD and the presence of IDV in the URT (23, 26). On the other hand, when IDV was intranasally inoculated in calves, the virus and the microscopic lesions were detected mainly in the nasal cavities and trachea and only exceptionally in lungs (24, 25). In this study we confirmed a preferential tropism of IDV for the URT of cattle as also suggested in ferret, swine (12, 18), and guinea pig (27). However, in agreement with our previous field observations (16), we confirmed by virus detection and IHC in lung tissues a widespread infection of the LRT, which potentially makes the French IDV strain a stronger agent of bronchopneumonia. This is also suggested by the clinical signs typical of the LRT infection in two direct-inoculated calves, which was not the case in the two previous experimental infections performed with different IDV strains (24, 25). Differences may be related to the IDV strain, the method of inoculation, and/or the host. In this study, we used colostrum-deprived calves of dairy production, while Ferguson et al. (25) waited for the disappearance of maternal antibodies before inoculation. Consequently, infected calves were older and probably more mature immunologically. We also cannot exclude that colostrum from IDV-seropositive cows may have stimulated the priming of the cellular immune response in the study of Ferguson et al. (25). The French IDV strain used in this study and the D/bovine/Mississippi/C00046N/2014 virus used by Ferguson et al. are part of the D/swine/Oklahoma/2011 lineage (whole-genome sequences). No sequence data for the whole genome are to date available for the challenge strain, D/bovine/Kansas/162655/2012, used by Hause et al. (24). Therefore, no proper genetic comparison could be performed between the 3 inoculum stains of IDV, but we cannot rule out that differences in the genomes of the IDV challenge strain may have played a role in disease outcome and tissue tropism.
One interesting issue in this study is the similar pathogenicities observed after inoculation with the French IDV strain and natural infection in the aerosol-sentinel group. Indeed, the aerosol-sentinel calf no. 1755 starting nasal excretion at D11 showed identical clinical signs. The two other aerosol-sentinel calves remained healthy, but this could have been due to the short duration of the experiment, leading to their euthanasia before the onset of clinical signs (D4 to D6 of infection). While we cannot fully exclude lower IDV pathogenicity after natural infection, IDV tropism for the LRT was confirmed by virus RT-qPCR detection in respiratory samples of two aerosol-sentinel calves. Surprisingly, we were able to additionally detect IDV with high RNA loads in olfactory bulbs and tonsils in these aerosol-sentinel animals. We unfortunately could not confirm the IDV tropism for these tissues by IHC or virus isolation: additional necessary studies are needed to resolve this point. At this stage the IDV tropism seems to be mainly respiratory, as we could not detect IDV in tissues of other origins.
Despite the infection of the LRT, the clinical impact of IDV was moderate in this study, as direct-inoculated calves quickly recovered from the infection. The quick recovery of the calves correlates with the limited extension of gross and histopathologic lesions observed in the three animals euthanized at D8, indicating milder pathogenicity of IDV in the LRT than that of other respiratory viruses, such as BRSV. Besides the biological characteristics of IDV, mild pathogenicity may also be due to experimental conditions, including the virus, the dose, and the route of inoculation. We used a high quantity of virus to infect animals (107 50% tissue culture infective doses [TCID50] per calf) and a nebulization method which was previously shown to be very efficacious for BRSV experimental infection in calves (28). On the other hand, our IDV inoculum was produced on cells from human (HRT18G) and swine (ST) origins with 4 passages, which might have attenuated the virus. An inoculum produced by several passages in calves might be better suited since the virus may this way increase its fitness to the host and thereby raise its potential to replicate and induce clinical signs. Finally, the moderate IDV pathogenicity is in agreement with epidemiological findings. Seroprevalence of IDV in cattle is under investigation in France, and preliminary data indicate that over 50% of adult cattle may be seropositive (data not shown), suggesting that the virus may spread a lot without inducing clinical signs in a large part of the cattle population. In addition, IDV was detected only with low frequency in the LRT of calves suffering from severe BRD and frequently in association with other pathogens (16). All these points suggest that IDV is a respiratory virus that can replicate in both the URT and LRT and that can probably be considered as predisposing to or a cofactor of BPI, as already shown for BCoV and BPI3.
The local innate immune response induced by IDV in the LRT was also investigated. Our data suggest a high capacity of IDV to stimulate transcription of the membrane or cytosolic PRRs, such as TLR3, TLR7, TLR9, and RIG-1. In contrast, IFN-I and IFN-I pathway molecules such as IRF3, IRF7, and IFN-I-stimulated genes (ISG15) were not overexpressed in direct-inoculated calves compared to control animals. Phosphorylation and translocation of IRF3 and IRF7 were, however, not tested in this study. Furthermore, transcription of SOCS1 and SOCS3, which are cytokine-inducible negative regulators of cytokines and members of the STAT-induced STAT inhibitor (SSI) family, were overexpressed at the same time. These data allow us to further explore the ability of IDV to interfere with the IFN-I response at both the transcriptomic and protein levels as shown for IAV NS1 protein (29, 30). This also suggests that IDV is rather well adapted to cattle, and it would be interesting to investigate whether a similar response would be observed in pigs or other species. We also found overexpression of mRNAs of several chemokines along the course of infection, mainly observed for CCL2 (also named MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), and the tandem CX3CL1/CX3CR1. In agreement with this, CCL4 production was induced by IDV in PBMC in vitro and in larger amounts by PBMC collected after IDV challenge. The soluble form CX3CL1 is chemotactic for T cells and monocytes but not for neutrophils, while the membrane-bound form promotes adhesion of those leukocytes to endothelial cells. Both CCL3 and CCL4 act as a chemoattractant for monocytes, but also, in a lesser extent, neutrophils and natural killer (NK) cells, as well as antigen-specific T and B cells for CCL3 (31). Neutrophils might additionally have been stimulated by IL-8 production, as demonstrated in vitro after IDV infection of mononuclear cells and in vivo with a high but not significant fold change at D2 in BALs. These results may explain our histopathological observations in IDV direct-inoculated calves showing a peribronchial and septal lymphoplasmocytic infiltration in the lung, a moderate infiltration of neutrophils in the bronchial lumens, and a light neutrophilic and macrophagic alveolitis. The moderate infiltration of inflammatory cells is also correlated with a downregulation in IDV calves of ITGAL mRNA expression, which is involved in leukocyte adhesion and transmigration of leukocytes, including T cells and neutrophils (32). Taken together, these results also confirm well the interaction and replication of IDV in the lower respiratory tract. We do not know if overexpression of chemokines may contribute to limit the severity of the disease in our study. For IAV, both CCL3 and CCL4 contribute to promotion of recovery by inducing inflammatory responses (33), probably by inducing the synthesis and release of other proinflammatory cytokines such as IL-1, IL-6, and TNF-α from fibroblasts and macrophages (34, 35) and/or by skewing a Th1 protective response (36) when associated with its receptor, CCR5. We did not find IL-1β overexpression at either the mRNA or protein level in the lung, suggesting a moderate proinflammatory response and/or limited pathogenicity of IDV in the LRT. However, despite the fact that the expression levels of IL-1α and IL-1β were not significantly different, between groups we observed that IDV is able to induce high overexpression of IL-1α just after infection and IL-1α and IL-1β were induced by IDV in vitro. We also observed IL-6 overexpression at the transcription but not at the protein level, and inversely, TNF-α was significantly more abundant at D2 and D7 only at the protein level. These differences may be due to technical sensitivity or timing of sample collection, but overall, they suggest that an inflammatory response occurs. In addition, our results suggest a mixed local Th1/Th2 response, as both IFN-γ and IL-13 mRNA expressions were enhanced after IDV infection. Overexpressions of both IL-13 and IFN-γ were particularly found in the two direct-inoculated calves which showed a proliferative lymphocytic response. Unfortunately, we were not technically able to detect IL-4, IL-5, or IL-12 mRNA transcripts in BALs to more precisely define the cell-specific response. The systemic and local cell responses have to be investigated in more detail in the future.
Finally, the aerosol transmission of IDV was examined in this study. The airborne transmission of IAV was extensively studied, and it is now acknowledged that under certain conditions of temperature, humidity, and strain, particles of IAV could be transmitted by respiratory droplets at least over short distances (37). Our study demonstrated that the efficient transmission of IDV infection via aerosol to a seronegative calf can occur under experimental conditions and that IDV is an airborne transmitted pathogen. The first aerosol-sentinel calf began to shed virus actively at D11. Considering an incubation period of 1 to 3 days (direct-inoculated group), this calf may have been infected between D8 and D10, when IDV excretion in the direct-inoculated group remained high. For the two other aerosol-sentinel calves, we can estimate the infection period between D16 and D20, at the period of highest excretion of the first aerosol-sentinel calf. We can consequently hypothesize that the first aerosol-sentinel calf was infected by airborne droplets from the direct-inoculated group and that this calf contaminated the two other ones by direct contact. The aerosol transmission also correlates with progressive detection of the virus in air samples from D3 to D13 first in the infected zone and then in the intermediate and aerosol-sentinel areas. The relative quantities of IDV RNA in aerosols are relatively low (105 RNA copies/m3) but well in the range of the observed IAV RNA copies detected in swine barns (1.2 × 106 RNA copies/m3 [38] and 3 × 105 copies/m3 [39]). To date we do not know the minimum infectious dose for IDV in cattle. Since IDV RNA was found in the LRT of all the three aerosol-sentinel calves at D22, we can hypothesize an effective and probably high rate of IDV transmission between calves by natural transmission as also suggested by our epidemiological data. As we detected a nonsynonymous mutation in HEF in virus shed by an aerosol contact calf (no. 7155 at day 14), we could hypothesize that it may be an adaptation marker for aerosol transmission. The substitution (M663K) is, however, located at the very N-terminal end of the protein and not in the host receptor-binding site of the hemagglutinin as observed for the transmission substitutions in highly pathogenic influenza A viruses of the H5N1 subtype (40). There is a clear need for sequencing full genomes of IDV transmitted via the aerosol route, and in several animals to be able to draw hypotheses on molecular markers of transmission. In addition, it would be more informative to use deep sequencing to be able to detect minority variants and to assess the diversity of quasispecies in these samples. Finally, the capacity of IDV to be airborne transmitted, at least over short distances, and the high efficacy of transmission must also be considered in regard to the very high thermal and acid stability of the virus in comparison with that of IAV, IBV, or ICV (41) and potential high resistance of the virus outdoors. Further studies will be useful in order to explain the physical and chemical conditions and the strain-related differences in the aerosolization and transmission of IDV.
MATERIALS AND METHODS
Cells and virus.The influenza D strain D/bovine/France/5920/2014 was isolated from a lung fragment of a calf dead from BRD in 2014 in France (16). Lung extract was propagated on human rectal tumor 18G (ATCC CRL-11663) cells for 5 days in the presence of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK) trypsin (1 μg/ml; Thermo Fisher Scientific, MA) in Opti-MEM reduced serum medium (Thermo Fisher Scientific) supplemented with penicillin (500 μg/ml)-streptomycin (500 U/ml) (Life Technologies, Carlsbad, CA), ciprofloxacin (10 μg/ml; Sigma-Aldrich, MO), amphotericin B (2.5 μg/ml; Sigma-Aldrich), and BM-cyclin (15 μg/ml; Sigma-Aldrich). For the second, third, and fourth passages, the supernatant was propagated on swine testis (ST) cells for 5 days in the presence of Opti-MEM medium supplemented with the same products and the progeny supernatant was filtered. The virus solution was verified free from other major respiratory pathogens by qPCR using the LSI VetMAX Screening pack—Ruminant Respiratory Pathogens real-time PCR kit (Life Technologies) and titrated using the 50% tissue culture infective dose (TCID50) as previously described (25), using the Spearman-Kärber titration method.
Ethics statement.Animal experimentation was performed in biosafety level 3 facilities at the Research Platform for Infectious Disease (PFIE, National Institute for Agronomic Research [INRA], Nouzilly, France) in accordance with accepted human standards of animal care (European Community council 2010/63/EU) and under the national ethical agreement (number APAFIS#6204-20 1 60725 14353285 v5; French Ministry of Agriculture, Ethics Committee no. 019).
Calves and experimental design.Fourteen Normand and Holstein calves, born in a bovine herpesvirus 1 (BoHV-1)- and bovine viral diarrhea virus (BVDV)-free experimental station of INRA, were colostrum deprived (42) and transferred to PFIE between 3 and 7 days of age. The calves were then fed with commercial milk and reared for 2 to 6 weeks before inoculation. To reach the number of animals necessary for the statistical significance of some of the tests, two additional calves with maternal antibodies against IDV were added in the control group. Before challenge, all calves tested negative for IDV, BRSV, BCoV, BPI3, Mycobacterium bovis, Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica (LSI VetMAX Screening pack qPCR—Ruminant Respiratory Pathogens; Life Technologies, Carlsbad, CA). Absence of BVDV was confirmed by negative detection of the NS3 antigen (Serelisa BVDV-BD; Synbiotics, Lyon, France). In addition, the 14 colostrum-deprived calves also tested negative for anti-IDV antibodies using HI assay.
Two separated pens were used; 5 mock-infected calves (control group; 3 deprived and 2 IDV antibody-positive calves) were placed in the first pen, whereas 11 colostrum-deprived calves, 8 of which were to be infected, were placed in the second pen. In the latter pen, calves were divided into two groups separated by steel panels, 3 m apart. Eight calves were inoculated (direct-inoculated group), while the three others (aerosol-sentinel group) were not, in order to evaluate the aerosol transmission of IDV. The two groups did not share any equipment, food, water, space, or fomites. The rule consisted of handling the three aerosol-sentinel contact calves first, and then personal protective equipment was completely changed before manipulating direct-inoculated calves. The eight direct-inoculated calves were infected at day 0 (D0) with 107 TCID50 (10 ml in Opti-MEM) of D/bovine/France/5920/2014 by aerosol inhalation using a compressor connected to a mask covering the nostrils and mouth as previously described (28). Calves of the control group received ST cell supernatant (10 ml in Opti-MEM). The aerosol-sentinel calves were not inoculated.
Clinical examination.Calves were examined by the same investigator twice a day from 3 days before infection (D−3) to D22 to D23 for body temperature, nasal discharge, coughing, decreased appetite, general state, abnormal breathing, respiratory rate, and abnormal lung sounds. Clinical scores were assessed for each calf as previously described (42). Briefly scores were assessed as follows: scores for rectal temperatures (T) were 0 (T < 39°C), 1 (39.1°C < T < 40°C), 2 (40.1°C < T < 41°C) or 3 (T > 41°C); scores for respiratory rates (RR) per minute were 0 (RR < 30), 1 (31 < RR < 40), 2 (41 < RR < 60), 3 (61 < RR < 80), or 4 (RR > 80); a score of 0 (normal), 1 (mild), or 2 (severe) was assigned for nasal discharge, coughing, or the general state, respectively, while dyspnea (absent, weak, moderate, or high) and appetite (drop in milk consumption of 0%, <30%, 30 to 60%, or >60%) were scored from 0 to 3. Daily individual accumulated scores were calculated for each calf by adding the score of every parameter. In addition, mean accumulated clinical scores (ACSs) were calculated for each group as the mean of the individual area under daily clinical scores using the trapezoid method (GraphPad, La Jolla, CA).
Gross lesions, histopathology, and immunohistochemistry.Euthanasia and necropsy were carried out at D8 for 3 direct-inoculated calves and at D22–D23 for the remaining 13 calves. Euthanasia was achieved by intravenous injection of 25 ml of pentobarbital sodium injection (Dolethal; Vetoquinol, France; 182.2 mg of pentobarbital per kg of body weight) before complete exsanguination. Extracted lungs were photographed, examined for gross lesions, and then lavaged with saline isotonic fluid. Tissue samples were then collected for histopathology and virology, including cranial right and left, intermediate, caudal right and left lobes of the lungs, the nasal and tracheal mucosa, tonsils, lymph nodes (mediastinal, tracheobronchial, and mesenteric), kidney, spleen, liver, and intestine (duodenum, jejunum, and colon). Every tissue and organ was divided in two parts, one placed in 10% buffered formalin for histology and immunohistology and one stored at −80°C for RNA isolation and subsequent RT-qPCR.
For histopathology, fixed tissue samples were paraffin wax embedded, sectioned at 3 to 5 μm, and stained with hematoxylin and eosin. A pathologist described and scored the severity of microscopic lesions in slides of the lungs and respiratory lymph nodes as either light (3 or fewer small lesion foci in one section), moderate (>3 small lesion foci per section), or marked (diffuse lesions in the section).
To confirm the presence of IDV in the respiratory tract tissues, immunohistochemical staining was performed on paraffin-embedded sections of lungs with a polyclonal rabbit anti-influenza D virus antibody obtained by multiple immunizations of rabbits. Slides were retrieved in pronase antigen retrieval solution (Dako, USA) at 0.05% and left for 10 min at 37°C. They were then incubated with the rabbit antiserum at a 1/100 dilution overnight at 4°C. The staining was revealed with a polymer conjugated with horseradish peroxidase (HRP) (polymer-HRP dual EnVision; Dako, USA) and the diaminobenzidine chromogen of the HRP (Diagomics, France). A rabbit normal serum was used as a negative control.
Sample collection.Nasal swabs (both nostrils) were collected daily from D0 to D22 in 1 ml of phosphate-buffered saline (PBS) and directly stored at −80°C. Bronchoalveolar lavage specimens (BALs) were obtained from 5 control calves and 5 direct-inoculated calves at D0, D2, D6 or D7, D14, and D22 to D23 by endoscopy (Olympus CF-EL; Shinjuku, Tokyo, Japan) under general anesthesia (10 mg/kg of ketamine and 10 mg/kg of diazepam) as previously described (42). During the process, airway abnormalities (inflammation and fibrinous content) were recorded. For each BAL, 100 ml of a sterile isotonic sodium chloride solution was injected into the same location of the right lobe. BALs after euthanasia (D22 to D23 and D8 for the 3 other direct-inoculated calves) were obtained by direct extraction and lavages of the lungs with 250 ml of sterile isotonic saline solution as previously described (43). Within an hour postcollection, crude BAL, BAL supernatant, and cells (obtained after centrifugation of 15 ml of BAL at 2500 rpm for 15 min) were aliquoted and stored at −80°C.
Air sampling was carried out at D1, D2, D4, D6, D8, D10, D12, D14, D16, and D18 postinfection in the same locations every time: starting in the center of the contact calf area, then the center of the separating space, and finally the center of the infected calf area. Air samples were collected using the Coriolis MICRO (Bertin Instruments, Montigny-le-Bretonneux, France) air sampler according to the manufacturer’s procedures. The collector was placed 1 m above ground, and after every collection, the collector was thoroughly rinsed and disinfected. Briefly, 10 ml of PBS supplemented with 0.01% Tween 20 (Sigma-Aldrich, MO) was added to the collector reservoir, and the Coriolis collector was run for 15 min, during which 4.5 m3 of ambient air was captured and mixed in the PBS-Tween solution. Immediately after collection, aliquots of 140 μl were stored at −80°C for further RNA extraction and IDV genome detection by RT-qPCR.
Virus quantification.IDV detection and quantification were performed by RT-qPCR in nasal swabs, BALs, and air samples. Viral RNA was extracted from liquid samples using the QIAamp cador PAthogen extraction kit (Qiagen, Hilden, Germany) on the Qiagen Qiacube robot platform. Tissues were extracted manually using Qiagen QiaAmp and RNA blood (Macherey-Nagel, Düren, Germany).
The IDV genome was analyzed by an RT-qPCR targeting a 135-bp fragment of polymerase basic 1 (PB1) segment gene as previously described (12) in the LightCycler 96 real-time PCR system (Roche, Basel, Switzerland) using the QuantiNova Probe PCR kit (Qiagen, Hilden, Germany). In order to quantify the IDV genome in samples, the blunt-ended PCR product of D/bovine/5920/2014 was inserted in the plasmid pSC-amp/kan of the StrataClone blunt PCR cloning kit (Agilent, Santa Clara, CA). Amplified plasmids were linearized with EcoRV restriction enzyme before in vitro transcription according to the instructions of the manufacturer (MEGAscript kit; Thermo Fisher Scientific). Purified RNA was then quantified by UV light absorbance (A260) using a CLARIOstar reader (BMG Labtech, Ortenberg, Germany), stored at −80°C, and used to make a standard curve for PB1 gene quantification. IDV loads in NS and BALS were expressed as the daily mean. In addition, individual accumulated viral shedding (AVS) in nasal secretions was calculated for each calf as the area under the curve of IDV titers using the trapezoid method (GraphPad Prism 7.0; La Jolla, USA) as previously described (28). To compare the inoculum virus, D/bovine/France/5920/2014, and virus from NS from infected calves, HEF genes were amplified and sequenced as previously described (16).
Host humoral response.Antibody response was first tested by HI at D0, D3, D6, D10, D15, and D22 to D23 as described previously (44). The sera were all treated with receptor-destroying enzyme (RDE) (Denka Seiken, Japan) and hemadsorbed on packed horse red blood cells (RBC). Four hemagglutination units of D/bovine/France/5920/2014 and 1% horse RBC were used for HI assays. The IDV antibody response was also assessed by two ELISAs for detection of IDV IgG1 and IgG2 at D0, D10, and D22 to D23. For antigen preparation, IDV strain D/bovine/France/5920/2014 was propagated on swine testis fibroblasts (ATCC CRL-1746). Infected and uninfected cells were frozen on day 4 postinoculation and then thawed and pelleted by centrifugation at 1,000 × g and 21°C for 10 min. The cell pellets were resuspended in H2O containing 0.5% Triton X (Sigma) and centrifuged as described above, and the supernatants were used as ELISA antigen and control antigen, respectively. For analysis of IDV-specific IgG1 and IgG2 antibodies, 96-well ELISA plates (PolySorp; Nunc, Denmark) were coated during 18 h at 4°C with IDV antigen or control antigen in 0.05 M sodium carbonate-bicarbonate buffer, pH 9.6. The wells were thereafter blocked for 1 h at 25°C with 0.05% PBS-Tween prior to addition of (i) 1:50 diluted sera in 0.05% PBS-Tween, (ii) monoclonal mouse anti-bovine IgG1 antibodies (MCA627; Bio-Rad; clone K37 2G6) or mouse anti-bovine IgG2 antibodies (MCA626; Bio-Rad; clone K192 4F10), (iii) monoclonal rat anti-mouse IgG1 antibodies conjugated with HRP (MCA336P; Bio-Rad; clone LO-MG1-2), (iv) tetramethylbenzidine (TMB) substrate, and (v) H2O2. Samples and antibodies were incubated for 1 h at 37°C prior to three washes with 0.05% PBS-Tween solution. Corrected OD (COD) values were calculated by subtracting absorbance values at 450 nm of wells containing control antigen from wells containing IDV antigen. Data were expressed as percentage of the COD of positive control sera.
Host cell-mediated immune response.The cell-mediated immune response was tested by IDV-specific lymphocyte proliferation assays. Peripheral blood mononuclear cells (PBMC) were obtained through centrifugation of heparinized blood diluted 1:1 in PBS, at 1,100 × g and 21°C for 45 min, over Ficoll-Paque PLUS (density, 1.077 g/ml; GE Healthcare). The cells were washed three times with PBS and by centrifugation (once at 500 × g and 21°C for 10 min and twice at 200 × g and 21°C for 10 min) and were frozen at −80°C until analysis. Cells from five IDV direct-inoculated calves, collected on the day before challenge (D−1) and D22, were thawed and immediately stimulated ex vivo, in triplicate with either (i) heat-inactivated IDV-infected or uninfected cell lysate or (ii) unheated IDV-infected or uninfected cell lysate. The strain D/bovine/France/5920/2014 propagated in swine testis fibroblasts was used for this purpose. After 6 days of incubation at 37°C, alamarBlue reagent (Invitrogen, Sweden) was added according to the manufacturer’s instructions. Absorbance was measured by spectrophotometry at 570 nm and 595 nm after another 24 h of incubation at 37°C. Corrected OD values were calculated between IDV and control antigen-stimulated PBMC, as described previously (28).
Transcriptomic analyses in BALs.BALs at D0, D2, D7, and D10 were analyzed for calf transcriptomic response of 46 bovine genes involved in the inflammatory response and innate immune response using the high-throughput microfluidic qPCR platform BioMark (Fluidigm, CA) technology. Briefly, cDNAs were generated from 300 ng of clean total RNA using random hexamer primers and the Revertaid reverse transcriptase (Thermo Fisher). Primer pairs were designed using Primer3 software based on the relevant bovine mRNA sequences and synthesized commercially (Eurogentec). They were previously validated for RT-qPCR (Roche; LightCycler480 system) using negative and positive samples. Two genes encoding IL-4 and IL-5 did not pass the validation and were ruled out. Genes and primers are listed in Data Set S3. Preamplification of cDNA was carried out with a pool of forward and reverse primers (0.2 μM) on an ABI thermocycler with an initial step of activation (95°C for 10 min) followed by 14 cycles of two steps (95°C for 15 s and 60°C for 4 min). Preamplification products were then treated with exonuclease I (40 IU) and diluted 5 times in Tris-EDTA (TE). Standards were prepared by sequential 2-fold dilutions of a pool of all samples (2 μl from each). qPCR of preamplification products were then run on the BioMark qPCR with a cycling program as follows: 50°C for 2 min for the amplification phase and 10 min at 95°C for activation of the hot-start enzyme, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 1 min, and elongation at 72°C for 20 s. Melting-curve analysis was performed after completion of the qPCR collecting fluorescence intensities between 60 and 95°C. Once the quality test passed, the data of the run were exported to Biogazelle qBase+ software (Biogazelle, Gent, Belgium). Relative expressions for each day were calculated by threshold cycle (ΔΔCT) analysis after normalization (GeNorm analysis) on the two most stable bovine housekeeping genes (hprt and ywha7) on a list of 6 genes previously mentioned in the literature (gapdh, hprt, rpl19, rpl26, sdha, and ywha7). Results were expressed as log transformed calibrated normalized relative quantities (CNRQ) values. For each day (D2, D6, and D14) and each molecule, fold changes were expressed as mean CNRQ values of direct-inoculated calves on mean CNRQ values of control calves after normalization on D0 using Biogazelle qBase+ software.
Protein quantification in BALs and PBMC supernatant using bioluminoassay.Protein extraction on cell extracts of BALs was done after two washings with ice-cold PBS. Just before use, the cell pellet was resuspended for 15 min on ice in NP-40 cell lysis buffer (Thermo Fisher; catalog no. FNN0021) containing 1 mM phenylmethylsulfonyl fluoride (PMSF; stock 0.3 M in dimethyl sulfoxide [DMSO]) and protease inhibitor cocktail (Sigma; catalog no. P-2714). The lysate was centrifuged at 14,000 rpm for 10 min at 2 to 8°C and stored at −20°C after total protein concentration determination. Protein extraction on (24-h-) stimulated lymphocyte supernatants was done after cell centrifugation at 14,000 rpm for 10 min at 4°C to remove any cells or cellular debris. Clarified medium was aliquoted and stored at −80°C for analysis. Just before analysis, thawed samples were clarified by centrifugation at 14,000 rpm for 10 min at 4°C in a refrigerated microcentrifuge to prevent clogging of the Luminex probe and/or filter plate. Protein quantification was performed by Luminex technology using the Millipex MAX magnetic bead panel coupled with the Luminex xMAP platform (SPRCUS706; EMD Millipore, Burlington, MA) according to the manufacturer’s instructions. Fifteen bovine proteins were quantified: IL-1α, IL-1β, IL-1RA, IL-6, IL-4, IL-17α, IL-2, IFN-γ, IL-8, IP-10, IL-10, CCL2, CCL3, CCL4, and TNF-α. Mean fluorescence or protein concentration (according to a reference range for each target) was analyzed as described below.
Statistical analysis.Statistical analysis for clinical and virological examinations were performed using GraphPad (La Jolla, CA). Logarithmic transformation was applied to fulfill the conditions of variances in homogeneity and normality when necessary (qPCR data). Data were expressed as arithmetic means ± standard errors of the means (SEM) or standard deviations (SD). A two-way analysis of variance (ANOVA) with repeated measures (three-factor splitplot ANOVA) was used to analyze the clinical and qPCR results. When effects of the day and treatment factors were significant among interactions, a Bonferroni test between contrasts was used to compare the treatments on each day postchallenge. P values have been indicated in the text; in figures, levels of significance are indicated as follows: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. A one-way ANOVA was used to compare the AVS and ACS. When the effect of the treatment factor was significant, a Newman-Keuls test was used to compare the treatment effects at each time point. A t test (Mann-Whitney test) was also run for these parameters.
Statistical analysis of Fluidigm transcriptomic results was carried out on the log transformed calibrated normalized relative quantities (CNRQ) values of mRNA expression by comparing the slopes from D−1 to Dx between infected and control calves. We used a linear mixed model with random effect for group, considering interactions between time and status (infected or control) and fit by maximum likelihood t tests using Satterthwaite approximations to degrees of freedom [formula: Y ∼ status × time + (1 | calves)]. This model takes into account the heterogeneity of cytokine RNA data at D0, when calves were not infected, and the different predictions of evolution between infected and control groups when interaction (ANOVA type III) is significant. The cytokine and proliferation data from in vitro PBMC stimulations were analyzed by using two-sided paired t tests in Minitab 18 statistical software.
ACKNOWLEDGMENTS
We thank Gilles Foucras and Didier Raboisson for their valuable scientific support, Angelique Teillaud, Sarah Walachowski, and Cecile Caubet for precious technical assistance, Olivier Boulesteix and Edouard Guitton for their work in the animal facility, and Maxime Fontanié for his help with the Qiagen Qiacube robot. We also thank the GeT-PlaGe platform (Toulouse) for the Fluidigm experiments.
This work was funded by the French National Agency for Research (ANR), project ANR-15-CE35-0005 FLUD. E. Salem is supported by a Ph.D. scholarship from the Lebanese University.
The SLU authors would like to thank the Cells for Life Platform, partly funded by the Infrastructure Committee at SLU, Sweden, for providing facilities and equipment, and the staff at the Clinical Science laboratory at SLU for their technical support.
FOOTNOTES
- Received 16 October 2018.
- Accepted 3 January 2019.
- Accepted manuscript posted online 23 January 2019.
Supplemental material for this article may be found at https://doi.org/10.1128/JVI.01853-18.
- Copyright © 2019 American Society for Microbiology.
