Abstract
Porcine parainfluenza virus 1 (PPIV-1) is a recently characterized swine respirovirus. Previous experimental studies reported PPIV-1 replicates in the porcine respiratory tract causing minimal clinical disease or lesions. However, it is unknown if PPIV-1 co-infections with viral respiratory pathogens would cause respiratory disease consistent with natural infections reported in the field. The objective of this study was to evaluate if PPIV-1 increases the severity of influenza A virus respiratory disease in swine. Fifty conventional, five-week-old pigs were assigned to one of three challenge groups (n = 15) or a negative control group (n = 5). Pigs were challenged with a γ-cluster H1N2 influenza A virus in swine (IAV-S; A/Swine/North Carolina/00169/2006), PPIV-1 (USA/MN25890NS/2016), inoculum that contained equivalent titers of IAV-S and PPIV-1 (CO-IN), or negative control. Clinical scores representing respiratory disease and nasal swabs were collected daily and all pigs were necropsied five days post inoculation (DPI). The CO-IN group demonstrated a significantly lower percentage of pigs showing respiratory clinical signs relative to the IAV-S challenge group from 2 to 4 DPI. The IAV-S and CO-IN groups had significantly lower microscopic composite lesion scores in the upper respiratory tract compared to the PPIV-1 group although the IAV-S and CO-IN groups had significantly higher microscopic composite lung lesion scores. Collectively, PPIV-1 did not appear to influence severity of clinical disease, macroscopic lesions, or alter viral loads detected in nasal swabs or necropsy tissues when administered as a coinfection with IAV-S. Studies evaluating PPIV-1 coinfections with different strains of IAV-S, different respiratory pathogens or sequential exposure of PPIV-1 and IAV-S are warranted.
1. Introduction
Porcine parainfluenza virus 1 (PPIV-1) is a recently detected virus in swine in the family Paramyxoviridae and genus Respirovirus (Palinski et al., 2016, Wang et al., 2009). Related viruses in the same genus include bovine parainfluenza virus 3 (BPIV-3), human parainfluenza virus 1 (HPIV-1), human parainfluenza virus (HPIV-3), Sendai virus (SeV), and caprine parainfluenza virus 3 (CPIV-3) (Lau et al., 2013, Wang et al., 2009). Preliminary data suggest that PPIV-1 is widespread in United States (U.S.) swine (Park et al., 2019). However, veterinarians have reported PPIV-1 associated respiratory disease in the absence of other swine pathogens (Lower, 2018).
Experimental PPIV-1 challenge studies demonstrated high levels of viral nucleic acid found in the upper (URT) and lower respiratory tract (LRT) of swine (Welch et al., 2021). Additionally, high levels of viral antigen were observed in the respiratory epithelium of lung, trachea, and turbinate by PPIV-1 immunohistochemistry (IHC). The minimum infectious dose has not been experimentally determined. However, pigs indirectly exposed to PPIV-1 infected swine shed virus in nasal secretions one day post exposure, suggesting the virus may be highly contagious via aerosol transmission (Welch et al., 2021). Experimental PPIV-1 infection causes mild macroscopic lung lesions and clinical signs after challenge with approximately 105 50% tissue culture infectious dose per milliliter (TCID50/mL). However, it remains unclear if there is an interaction between PPIV-1 and other pathogens in the porcine respiratory disease complex (PRDC) that may influence the expression of clinical respiratory disease associated with PPIV-1 that is consistent with anecdotal reports from swine veterinarians.
In humans, parainfluenza viruses (PIV) cause minimal clinical disease in healthy individuals (Drews et al., 1997, Mehinagic et al., 2019). However, PIV infection in immunocompromised patients, such as hematopoietic transplant recipients and cancer patients, have been associated with poorer clinical outcomes and increased mortality (Lehners et al., 2016, Pochon and Voigt, 2018, Srinivasan et al., 2011). Parainfluenza-associated acute respiratory infections (ARIs) occur predominantly in young children and the elderly (Ruuskanen et al., 2011, Upadhyay et al., 2018). Seasonal peaks of parainfluenza-associated infections in humans occur annually during April-June and October-November closely following IAV trends (Liu et al., 2019). Clinical signs associated with intranasal, intratracheal, or aerosol exposure to parainfluenza has ranged from asymptomatic infections to severe pneumonia in bovine species (Dawson et al., 1965, Ellis, 2010, Frank and Marshall, 1971). Similar to infection in humans, seasonal patterns have also been observed in cattle, with 78% of viral respiratory infections detected in winter, excluding adenovirus and bovine viral diarrhea virus infections (Stott et al., 1980).
Parainfluenza virus infections commonly occur as coinfections with other pathogens in many species including dogs (Maboni et al., 2019), humans (Liu et al., 2019) and cattle (Ellis, 2010, Mehinagic et al., 2019). Detection of multiple respiratory viruses in human clinical samples is common, with some studies reporting the instance of coinfection at approximately 20–30% in infants (Zhong et al., 2019). Various mathematical approaches have attempted to model the effect of viral coinfection on biological systems although outcomes have often resulted in disparate stochastic predictions (Pinky et al., 2019). The importance of polymicrobial infections in swine respiratory disease has become more evident in recent years with advanced molecular diagnostic testing (Opriessnig et al., 2011). The objective of this study was to evaluate the potential role of PPIV-1 in the PRDC using a coinfection model with influenza A virus in swine (IAV-S) by comparing clinical signs, macroscopic and microscopic respiratory tract lesions and replication dynamics compared to the corresponding single infections.
2. Materials and methods
2.1. Virus and cell lines
A low passage PPIV-1 isolate USA/MN25890NS/2016, used in prior challenge studies (Welch et al., 2022a, Welch et al., 2022b, Welch et al., 2021), was propagated in swine testicular (ST) cells using a modified protocol (Park et al., 2019). The cells were maintained in Earles Minimum Essential Medium (MEM; Gibco™, Waltham, MA) supplemented with 10% fetal bovine serum (v/v) (FBS) (Atlas Biologicals, Fort Collins CO), 100 U penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine, 50 µg/mL gentamycin, (Gibco™, Waltham, MA) and 0.25 µg/mL amphotericin B (Sigma-Aldrich, St. Louis, MO). Post inoculation medium (PIM) consisted of MEM, antibiotics, and 1 µg/mL L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) treated trypsin. Pigs were challenged with a single PPIV-1 infection using 2 mL intratracheally and 1 mL intranasally split evenly between each nostril based on a back titration titer of 104.6 TCID50/mL. The titer was estimated using the method of Reed and Muench to determine 50% endpoint dilution (Reed and Muench, 1938).
A γ-cluster H1N2 IAV-S, isolate A/Swine/North Carolina/00169/2006, was provided at a titer of 106.1 50% egg infectious dose per mL (EID50/mL) by Boehringer Ingelheim Animal Health, Inc (BIAH). The IAV-S isolate was serially passaged and titrated in embryonated chicken eggs per established protocol (Zhang and Gauger, 2020). The inoculum was diluted to a concentration of 105.5 EID50/mL immediately prior to challenge with a single infection using the same volume and route as PPIV-1. The inoculum for the CO-IN group was prepared so that the viral titers of PPIV-1 and IAV-S were equal to the individually infected groups and administered using the same volume and route as the single inoculated groups.
2.2. Animal and study design
The animal study was approved by the BIAH institutional animal care and use committee with protocol number 100880. Fifty commercial, five-week-old pigs were purchased from a commercial farm screened for PPIV-1 antibody in the dams prior to selection of their pigs. Following arrival to the research facility, pigs were screened by reverse transcription real-time PCR (RT-rtPCR) or rtPCR and enzyme linked immunosorbent assay (ELISA) at the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) for agents commonly associated with PRDC including Mycoplasma hyopneumoniae (MHP), IAV-S, PPIV-1, Porcine circovirus 2 (PCV2) and Porcine reproductive and respiratory syndrome virus (PRRSV). Pigs were blocked by weight and litter prior to randomizing into three treatment groups of 15 pigs each and one negative control group of 5 pigs. Treatments consisted of PPIV-1 and IAV-S single infection groups, CO-IN group, and negative control (Neg Ctrl) groups. All pigs were challenged intranasally and intratracheally as described above using PPIV-1 or IAV-S as single virus infections or with an inoculum that contained equal volumes of PPIV-1 and IAV-S, and virus titers equivalent to the single infected pigs, representing the CO-IN challenge. Nasal swabs (NS) were collected daily from each nostril from 0 to 5 days post inoculation (DPI) and placed in 2 mL of MEM. Rectal temperatures were collected daily from 0 to 5 DPI and pigs designated with a fever had body temperatures ≥ 40.5 °C. Pigs were clinically evaluated daily from 0 to 5 DPI and scored for clinical signs associated with respiratory disease based on sneezing, respiratory rate, coughing, and lethargy (Table 1). The proportion of pigs demonstrating respiratory scores ≥ 1 and body temperatures ≥ 40.5 °C were computed for statistical analysis.
Table 1. Respiratory clinical signs were evaluated in all experimental groups using four parameters that were scored based on presence and level of severity observed from 0 to 5 days post inoculation.
| Score | Sneezing | Respiratory Rate | Coughing | Lethargy |
|---|---|---|---|---|
| 0 | Absent | Normal | Absent | Normal |
| 1 | Mild: mild sneeze observed | Mild: increase in respiratory rate | Mild: mild cough observed | Depressed: slight decrease in movement and attitude |
| 2 | Moderate: notable increased sustained sneezing | Moderate: dyspnea and notable increase in respiratory rate | Moderate: notable increase in sustained coughing | Lethargic: requires aggressive physical stimulation before pig will rise |
| 3 | No score | Severe: dyspnea and exaggerated abdominal breathing | Severe: loud barking cough | Recumbent: laying down, unable to rise when provoked with physical stimulus |
2.3. Animal necropsy
All pigs were humanely euthanized by electrocution at 5 DPI immediately prior to necropsy and consistent with established American Veterinary Medical Association guidelines. Lungs were examined grossly for the presence of macroscopic lesions by a veterinary pathologist and given a score estimating the percent of each affected lobe with a weighted score representing the entire affected lung as previously described (Halbur et al., 1995). Fresh samples collected at necropsy consisted of serum, tracheal swabs (TS), bronchoalveolar lavage fluid (BALF), lung homogenate (LH), and nasal turbinates (NT). Formalin fixed (10%) samples consisted of lung, trachea, and NT.
2.4. Microscopic evaluation
Tissues were fixed in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA) for 48 h and embedded in paraffin blocks (FFPE) per ISU VDL protocol. The FFPE blocks were cut into 4 µm thick sections before staining with hematoxylin and eosin (H&E) and PPIV-1 and IAV-S specific immunohistochemistry (IHC) per ISU VDL protocol.
Microscopic lung, trachea, and NT lesions and the PPIV-1 and IAV-S IHC signals were scored by a veterinary pathologist blinded to treatment groups as previously described (Gauger et al., 2014, Welch et al., 2021). The microscopic lung lesion scores were computed into an LRT composite score (0−22) for statistical analysis. Proximal and distal trachea inflammation scores, trachea epithelial necrosis scores, and right and left NT epithelial necrosis scores were combined for an URT composite score (0−24) for statistical analysis. The following lesions were included in the composite microscopic lung score: interstitial pneumonia (0−4), peribronchiolar cuffing (0−4), airway epithelial necrosis (0−4), suppurative bronchiolitis (0−4), epithelial microabscesses (0−3), and alveolar edema (0−3). Proximal and distal trachea as well as right and left NT sections were scored separately. The composite URT score consisted of proximal (0−4) and distal (0−4) tracheitis, proximal (0−4) and distal (0−4) trachea epithelial necrosis, and necrosis of the right (0−4) and left (0−4) NT epithelium.
2.5. Nucleic acid extraction and RT-qPCR assay
All sample processing and RT-rtPCR or reverse transcription real-time quantitative PCR (RT-qPCR) assays were conducted as previously described by ISU-VDL protocols (Park et al., 2019, Welch et al., 2021). Briefly, nucleic acid extraction was conducted via a 5XAmbion® MagMAX™− 96 Viral RNA Kit and automated Kingfisher 96® magnetic particle processor (ThermoFisher™ Scientific, Waltham MA) using the high-volume protocol per manufacturer instructions. Detection of PPIV-1 viral RNA was conducted using the Ambion® Path-ID™ RT-qPCR kit (Life Technologies, Carlsbad CA) with a 7500 Fast Real-time PCR System (Applied biosystems®, Foster City CA). The PCR reaction was run to 40 cycles. Any PPIV-1 sample with fluorescence signal below a threshold of 0.2 at the end of the run was considered negative; the baseline was automatically calculated by ABI software using the “auto-baseline” setting. IAV-S detection was performed with the VetMax™ Gold SIV Detection Kit (ThermoFisher™ Scientific, Waltham MA) per manufacturer instructions. Amplification curves were analyzed using commercial software. Genomic copies per mL (GC/mL) were determined based on standard curves from serial dilutions of in-vitro transcribed RNA quantities conducted at the ISU-VDL targeting the N gene of PPIV-1 (Park et al., 2019) and the M gene of IAV-S.
2.6. Statistical analysis
Data comparisons between experimental groups were performed in the open-source statistics program R (version 3.6.3). IAV-S and PPIV-1 GC/mL in NS and necropsy samples were compared using a linear mixed model in the “lme4″ package (version 1.1–23). Individual pairwise comparisons of estimated mixed marginal means were conducted with the package “emmeans” (version 1.4.4). A Kruskal Wallace rank sum test and post-hoc Mann-Whitney U tests were used to compare severity of macroscopic lesions, microscopic lesion scores, and IHC scores. Overall and pairwise differences in the percentage of pigs showing respiratory clinical signs were analyzed with a Fishers exact test of association.
3. Results
3.1. Pre-Challenge microbiology demonstrated pigs were negative for extraneous respiratory viruses and bacteria
Based on negative PCR results, the pre-challenge screening demonstrated no evidence of PRRSV or PCV2 viremia or IAV-S, PPIV-1 or MHP nasal shedding. All pigs were free of PPIV-1, IAV-S, PRRSV and MHP antibodies pre-screen. However, 54 pigs tested positive for PCV2 antibody by ELISA.
3.2. Co-infected and PPIV-1 challenged groups demonstrated less clinical respiratory disease compared to the IAV-S challenged group
Pigs challenged with IAV-S demonstrated a significantly higher proportion of animals with a clinical respiratory disease score ≥ 1 compared to those challenged only with PPIV-1 or the CO-IN group (Fig. 1 A). Clinical signs observed in the IAV-S group were consistent with previous reports of influenza respiratory disease in swine (Van Reeth and Vincent, 2019). There were 8/15–11/15 pigs that developed respiratory clinical signs in the IAV-S group on 2–4 DPI. Few pigs in the CO-IN group (1/15–2/15) showed respiratory clinical signs during the same time (Fig. 1 A).
Fig. 1. (A) Composite respiratory scores representing sneezing, respiratory rate, coughing and lethargy were used to calculate the percentage of pigs with clinical signs. A significantly higher (*, p < 0.05) percentage of pigs in the IAV-S only group showed respiratory clinical signs from day 2–4 relative to the other groups. (B) Febrile animals (≥ 40.5 °C) as a percentage of group. On 1 DPI, 7/15 and 5/15 of the IAV-S and CO-IN animals, respectively, had fevers greater than 40.5ºC. Fever was not observed in the PPIV-1 or Neg Ctrl groups. Error bars represent the standard error of the proportion (SEp) calculated as
.
No pigs had body temperatures ≥ 40.5 ºC prior to challenge regardless of the experimental group (Fig. 1B). At 1 DPI, 7/15 IAV-S and 5/15 CO-IN pigs had fevers in contrast to 0/15 and 0/5 detected in the PPIV-1 and Neg Ctrl pigs, respectively. Body temperatures remained lower than 40.5 °C from 14/15 pigs, or more, in all groups from 2 to 5 DPI and is consistent with rectal temperatures of nursery-age pigs under thermoneutral conditions (Soerensen and Pedersen, 2015).
3.3. Co-infection reduced PPIV-1 replication in the lower respiratory tract in contrast to IAV-S replication that was similar to the co-infected group
Pigs challenged with IAV-S or PPIV-1, regardless of CO-IN or single inoculation, demonstrated high levels of virus replication detected in NS, LH, TS, and NT consistent with prior challenge studies (Khatri et al., 2010, Welch et al., 2021). Coinfection did not appear to affect replication kinetics of either virus relative to their respective PPIV-1 (Fig. 2A) and IAV-S (Fig. 2B) challenge groups in NS.
Fig. 2. (A) Viral loads in NS at 3 and 5 DPI detected by PPIV-1 specific RT-qPCR. No significant differences were observed between the PPIV-1 and CO-IN groups on 3 and 5 DPI. (B) Viral loads in NS at 3 and 5 DPI detected by IAV-S specific RT-qPCR. No differences were observed between IAV-S and CO-IN groups at 3 and 5 DPI.
Necropsy tissues representing the LRT (BALF and LH) demonstrated that quantities of PPIV-1 RNA detected in the CO-IN group were significantly lower compared to the PPIV-1 group suggesting coinfection may have reduced PPIV-1 replication (Fig. 3 A). In contrast, necropsy tissues from the URT (NT and TS) demonstrated PPIV-1 RNA genomic copies were similar between the CO-IN and PPIV-1 groups in spite of the lower trend demonstrated in the CO-IN group. In contrast, the quantities of IAV-S RNA were similar in the CO-IN and IAV-S groups regardless of LRT or URT tissue samples (Fig. 3B).
Fig. 3. (A) Viral loads detected by PPIV-1 RT-qPCR in necropsy samples on 5 DPI. The data are expressed as the mean log10 genomic copies per milliliter (GC/mL) of the sample homogenate. Significantly lower PPIV-1 nucleic acid was detected in the LRT of the CO-IN group relative to the PPIV-1 only group in BALF and LH. Connecting lines indicate significant differences between PPIV-1 and CO-IN groups. (B) Viral loads detected by IAV-S RT-qPCR in necropsy samples on 5 DPI. The data are expressed as the mean log10 genomic copies per milliliter (GC/mL) of the sample homogenate. No differences were observed between the CO-IN and IAV-S groups in BALF, LH, TS, or NT.
3.4. Co-infected and IAV-S challenged groups demonstrated higher percentage of macroscopic pneumonia compared to the PPIV-1 challenged group
Macroscopic lung lesions were consistent with cranioventral consolidation typical of influenza infection in the IAV-S and CO-IN groups (data not shown). However, macroscopic lung lesions were minimal or not observed in the PPIV-1 and Neg Ctrl pigs. The percent pneumonia was significantly higher in the IAV-S and CO-IN groups compared to the PPIV-1 and Neg Ctrl groups (Fig. 4A). No significant difference in macroscopic pneumonia lesions were observed between the CO-IN and IAV-S groups or between the PPIV-1 and Neg Ctrl groups (Fig. 4A).
Fig. 4. Median percent macroscopic lung consolidation, composite microscopic LRT and URT lesion scores and composite IAV-S and PPIV-1 IHC scores by treatment group at 5 DPI. A) Median, 25th and 75th quartile percent macroscopic lung consolidation based on weighted averages of individual lung lobes. IAV-S and CO-IN groups had significantly increased median percent lung consolidation relative to the PPIV-1 and Neg Ctrl groups. B) Median, 25th and 75th quartile microscopic composite LRT lesion score. Both the IAV-S and CO-IN groups had significantly increased microscopic LRT lesion scores relative to the PPIV-1 and Neg Ctrl groups. C) Median, 25th and 75th quartile microscopic composite URT score. Significantly higher scores were observed in the URT of the PPIV-1 group relative to all other treatment groups. D and E) Median, 25th and 75th quartile LRT composite IHC score for IAV-S and PPIV-1, respectively. F) Median, 25th and 75th quartile URT composite PPIV IHC score. Significantly increased signal was observed in the PPIV-1 and CO-IN groups relative to the Neg Ctrl group. No IAV IHC signal was observed in the upper respiratory tract of the IAV-S or CO-IN groups (data not shown). Brackets with p-values indicate significant differences in ranked percentages or scores were based on Kruskal-Wallace and post-hoc Mann-Whitney U tests.
3.5. Co-infected and IAV-S lower respiratory tract microscopic lesions were similar and significantly higher compared to the PPIV-1 challenged group
Microscopic lesions in the Neg Ctrl group were not observed in lung (Fig. 5A), trachea (Fig. 5E) or NT (data not shown). The PPIV-1 challenged pigs demonstrated minimal to mild bronchiolar epithelial proliferation and mild peribronchiolar lymphocytic cuffing with occasional interstitial pneumonia (Fig. 5B). In contrast, microscopic lung lesions in the IAV-S (Fig. 5C) and CO-IN (Fig. 5D) groups consisted of marked bronchiolar epithelial necrosis, peribronchiolar lymphocytic cuffing, interstitial pneumonia, and suppurative bronchiolitis. Median composite microscopic lung lesion scores representing the LRT were significantly increased in the IAV-S and CO-IN groups compared to the PPIV-1 and Neg Ctrl groups (Fig. 4B).
Fig. 5. Microscopic lung and trachea lesions at 200x magnification, H&E stain and immunohistochemistry for IAV-S and PPIV-1. A) Neg Ctrl, lung, no microscopic lesions. B) PPIV-1, lung, mild bronchiolar epithelial proliferation. C) IAV-S, lung, marked bronchiolar epithelial necrosis, peribronchiolar lymphocytic cuffing, and interstitial pneumonia. D) CO-IN, lung, marked bronchiolar epithelial necrosis, peribronchiolar lymphocytic cuffing, and interstitial pneumonia. E) Neg Ctrl, trachea, no microscopic lesions. F) PPIV-1, trachea, moderate epithelium necrosis with macrophages and lymphocytes in the lamina propria. G) IAV-S, trachea, minimal epithelium attenuation. H) CO-IN, trachea, mild to moderate epithelial attenuation and mild submucosal inflammation. I) Neg ctrl, lung, PPIV-1 IHC signals not observed. J) PPIV-1, lung, abundant PPIV-1 IHC signal in the bronchiolar epithelium. K) IAV-S, lung, PPIVl-1 signal was not detected. L) CO-IN, lung, mild PPIV-1 IHC signal in the bronchiolar epithelium. M) Neg Ctrl, trachea, PPIV-1 IHC signal not observed. N) PPIV-1, trachea, abundant PPIV-1 IHC signal observed in tracheal epithelium. O) IAV-S, trachea, PPIV-1 IHC signal not observed. P) CO-IN, trachea, abundant PPIV-1 IHC signal observed in tracheal epithelium. Q) Neg Ctrl, lung, IAV-S IHC signal not observed. R) PPIV-1, lung, IAV-S IHC signal not observed. S) IAV-S, lung, abundant IAV-S IHC signal observed in bronchiolar epithelium. T) CO-IN, lung, moderate to abundant IAV-S IHC signal observed in bronchiolar epithelium. Neg Ctrl: Negative control; PPIV-1: Porcine parainfluenza 1; IAV-S: influenza A virus in swine; CO-IN: coinfected group.
3.6. PPIV-1 upper respiratory tract microscopic lesions were significantly higher compared to the co-infected and IAV-S challenged groups
Microscopic lesions in the trachea representing the URT consisted of moderate trachea epithelial necrosis and mild tracheitis in the PPIV-1 group (Fig. 5F). However, trachea lesions were minimal to mild in the IAV-S (Fig. 5G) and CO-IN (Fig. 5H) groups. Mild to moderate nasal turbinate epithelial necrosis was observed regardless of PPIV-1, IAV-S, or CO-IN challenge groups (data not shown). Median composite microscopic lesion scores from the URT were significantly higher in the PPIV-1 group compared to IAV-S, CO-IN, and Neg Ctrl groups (Fig. 4C).
3.7. PPIV-1 or IAV-S immunohistochemistry signal was detected in the lower respiratory tract in co-infected or single infected groups
There was no PPIV-1 or IAV-S IHC signal observed in sections of lung, trachea, or NT (NT data not shown) from the Neg Ctrl group (Fig. 5I & M PPIV-1 IHC; Fig. 5Q IAV-S IHC). Moderate PPIV-1 IHC signal was detected in sections of bronchiolar epithelium in the PPIV-1 (Fig. 5J) and CO-IN (Fig. 5L) challenged pigs but was absent in the IAV-S (Fig. 5K) challenge group. In contrast, abundant PPIV-1 IHC signal was observed in the trachea and NT in both PPIV-1 (Fig. 5N) and CO-IN (Fig. 5P) groups (NT PPIV-1 IHC not shown) but was not observed in trachea from the IAV-S (Fig. 5O) group. No IAV-S IHC signal was observed in the bronchiolar epithelium of the PPIV-1 group (Fig. 5R). However, abundant IAV-S IHC signal was observed in the bronchiolar epithelium of the IAV-S group (Fig. 5S) and CO-IN group (Fig. 5T). In addition, IAV-S IHC signal was absent in sections of trachea and NT regardless of PPIV-1, IAV-S, or CO-IN groups (data not shown).
3.8. PPIV-1 immunohistochemistry signal was significantly higher in the lower respiratory tract compared to the co-infected group but was similar in the upper respiratory tract
Median IAV-S IHC scores in the lung were significantly higher in the IAV-S and CO-IN groups compared to the Neg Ctrl pigs (Fig. 4D) although IAV-S IHC scores were similar in the IAV-S and CO-IN groups. In contrast, median PPIV-1 IHC scores in the lung were significantly higher in the PPIV-1 challenged pigs compared to the CO-IN and Neg Ctrl (Fig. 4E). In addition, significantly higher PPIV-1 IHC scores were reported in tissues collected from the URT (Fig. 4F) in the PPIV-1 and CO-IN pigs compared to the Neg Ctrl although no difference was detected between the PPIV-1 and CO-IN groups. IAV-S IHC signal was not observed in sections from the URT regardless of the group (data not shown).
4. Discussion
Diagnosis of PPIV-1 clinical respiratory disease is challenging as the virus has been detected in both symptomatic and asymptomatic herds based on submissions to the ISU VDL and reports from swine veterinarians (Lower, 2018). A prior report supporting the endemic nature of PPIV-1 indicated approximately 43% of 842 respiratory specimens submitted to the ISU VDL were PPIV-1 RT-rtPCR positive by retrospective surveillance, demonstrating the virus is frequently detected in U.S. swine (Park et al., 2019). However, previous challenge studies conducted with the same PPIV-1 isolate (USA/MN25890NS/2016) used in this report did not show significant clinical signs or macroscopic lung lesions (Welch et al., 2022a, Welch et al., 2021). Therefore, it remained unclear if coinfections with other agents in the PRDC, such as IAV-S, were necessary to replicate clinical disease observed in conventionally raised swine reported in the field.
The results of this study demonstrated a PPIV-1 and IAV-S coinfection administered simultaneously to conventional nursery pigs did not appear to exacerbate clinical signs compared to their respective PPIV-1 or IAV-S individually challenged groups. In fact, the CO-IN and PPIV-1 groups demonstrated a significantly lower percentage of pigs with clinical signs associated with respiratory disease relative to the IAV-S challenged pigs from 2 to 4 DPI. This result was unexpected considering at minimum, similar clinical signs would have been observed in the CO-IN and IAV-S groups. The data in this report suggests a PPIV-1 and IAV-S simultaneous coinfection did not impact the severity of clinical respiratory disease compared to a single virus challenge under experimental conditions. Parainfluenza viruses are commonly associated with coinfections in pediatric human patients with reports as high as 77% of cases, most commonly with respiratory syncytial virus and rhinovirus (Martin et al., 2013). However, prior studies have demonstrated similar results where viral coinfections did not impact or increase respiratory clinical signs and risk of hospitalization relative to individual infections in humans (Martin et al., 2012). In contrast, other studies found that children less than 5 years of age with viral coinfections were more likely to require hospitalization relative to those with individual infections (Goka et al., 2015). These studies often do not reliably identify the time of infection relative to onset of clinical signs. Furthermore, the pathogenesis of simultaneous coinfections and manifestation of clinical disease may present differently if viruses were challenged sequentially at different times. Therefore, study limitations and methodology may play a role explaining conflicting results between different studies. Models were developed that investigated distinct secondary coinfections in mice between SeV and IAV that demonstrated increased morbidity when mice were initially challenged with SeV and subsequently challenged with IAV suggesting the timing of the initial and subsequent coinfections may impact manifestation of clinical disease. In addition, clinical respiratory scores are subjectively evaluated at a single timepoint during the day that may also impact outcomes or create variability in the data. Further studies are necessary to evaluate if the sequence of PPIV-1 and IAV-S coinfections may impact the magnitude of clinical disease in swine.
The current study found significantly higher macroscopic pneumonia and microscopic lung lesion scores in the CO-IN and IAV-S groups relative to the PPIV-1 and Neg Ctrl pigs. These data suggest that lung lesions observed in the CO-IN group may be attributed to IAV-S and not PPIV-1 considering IAV-S and CO-IN pathology in the LRT were similar in appearance and median lesion scores were not significantly different. In contrast, the CO-IN and PPIV-1 groups had significantly increased URT median composite lesion scores and PPIV-1 IHC signal relative to the IAV-S and Neg Ctrl groups. Parainfluenza viruses have been reported to preferentially replicate in the respiratory epithelium of the nasopharynx regardless of the species, which is consistent with the higher levels of PPIV-1 RNA detected in the URT in the current and previous experimental challenge studies compared to virus levels in the lower respiratory tract (Ellis, 2010, Henrickson, 2003, Welch et al., 2021).
The γ-cluster H1N2 preferential replication detected in LRT tissue, that was demonstrated in this study, may be attributed to several factors. Studies have shown by viral IHC that isolates representing different H1 genetic clusters will vary in their level of binding affinity to the respiratory epithelium. However, prior reports have shown that γ-cluster H1 viruses efficiently replicated in both the URT and LRT (Detmer et al., 2012). Sialic acid receptor distribution throughout the respiratory tract could play a role in isolate-specific virus distribution. As in humans, the α2,3- and α2,6- sialic acid receptor distribution in swine is different between the URT and LRT (Rajao et al., 2019). Two PPIV-1 isolates have been characterized that have shown preferential replication in the URT, namely the nasopharynx, oropharynx, and trachea (Welch et al., 2020, Welch et al., 2021). The difference in virus tropism or location of virus attachment found between these IAV-S and PPIV-1 isolates may suggest the difference in replication levels between the URT and LRT described in this study.
The data presented in this study indicated the CO-IN pigs had lower levels of PPIV-1 RNA and antigen expression in the LRT compared to the PPIV-1 group suggesting the coinfection with IAV-S may have negatively impacted PPIV-1 replication in the LRT (Figs. 3A, 4E). However, IAV-S and PPIV-1 coinfection did not appear to negatively impact PPIV-1 replication in the URT (Figs. 3A, 4F). Surveillance of hospitalized children with acute respiratory infections showed HPIV-1 viral shedding decreased in the presence of viral coinfection. However, this was not observed in coinfections involving HPIV-3 or IAV (Martin et al., 2012). Mathematical models of respiratory infections in human hospitalized patients predicted that the virus replicating more rapidly will have a competitive advantage and therefore often dominates in a coinfection (Pinky and Dobrovolny, 2016). Replication of slower growing viruses may be suppressed in the presence of viruses with a shorter life-cycle. In contrast, alternative models suggest slower replicating viruses can also stochastically outcompete a faster growing virus provided the initial growth rates are similar in some situations. These respiratory models are often criticized for ignoring the impact of the immune response of the host (Pinky and Dobrovolny, 2016, Pinky et al., 2019).
Limitations of the current study include the use of a specific H1N2 IAV-S subtype in the co-infection model and administering the co-infected challenge material at equal titers and at the same time. In particular, utilizing different IAV-S in the co-infection may have produced different clinical outcomes that could be influenced by subtype, phylogenetic clade, or strain of the virus. The IAV-S currently circulating in U.S. swine are genetically diverse and include H1N1, H1N2 and H3N2 subtypes representing eleven different phylogenetic clades including the gamma H1N2 used in this study (Anderson et al., 2021). This suggests different strains of IAV-S co-infected with PPIV-1 may impact the presence of clinical respiratory disease that was lacking under the conditions of the current study. Additional PPIV-1 coinfection studies are warranted to further evaluate the impact of PPIV-1 replication with different strains of IAV-S or other PRDC pathogens.
5. Conclusion
Collectively, the results of this study did not demonstrate more severe clinical respiratory disease in the CO-IN pigs compared to the IAV-S or PPIV-1 challenged groups based on the conditions of this study. In addition, replication in the LRT and URT in CO-IN pigs was either similar to, or lower than, the pigs challenged with only IAV-S or PPIV-1. However, there were some differences demonstrated in the gross and microscopic lesions and IHC scores between CO-IN and the IAV-S or PPIV-1 pigs although clinical relevance was insignificant. Although this study was the first to evaluate the role of PPIV-1 coinfection in pigs, the scope of the study is limited in several ways. It is unknown how PPIV-1 contributes to coinfection with different strains of IAV-S or with other respiratory or systemic viruses and bacteria. In humans, HPIV with bacterial superinfection was associated with increased clinical disease scores, perinatal intensive care unit admissions, and increased disease severity while failing to show a correlation between disease severity and viral coinfections. However, the authors also acknowledged that there is conflicting evidence depending on the study and agents investigated (Cebey-López et al., 2016). Future research is needed to investigate the interaction of PPIV-1 with common porcine bacterial respiratory pathogens involved in the PRDC to evaluate PPIV-1 potential involvement in clinical disease that was not observed in this study using a simultaneous IAV-S and PPIV-1 coinfection.
Ethics Approval
All animal challenge studies were approved by the Institutional Animal Care and Use Committee, protocol 100880, at Boehringer Ingelheim Animal Health, Inc.
Funding statement
This work was supported by funding provided by Boehringer Ingelheim Animal Health, Inc. Duluth, Georgia, USA and were involved in study design, data collection data analysis, data interpretation, and editing the report.
Declaration of Competing Interest
The authors declare the following financial interests/personal relationships which may be considered as potential competing interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr. Abby Patterson is an employee of Boehringer Ingelheim Animal Health, Inc. who sponsored the study. Involvement in this research was academic and did not involve other competing interests.
Acknowledgements
Facilities and laboratory equipment were provided by Boehringer Ingelheim Animal Health and the Iowa State University Veterinary Diagnostic Laboratory. Special thanks to the staff at Boehringer Ingelheim Animal Health for assisting with study logistics and daily animal care.
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