Kinetics of single and dual simultaneous infection of pigs with swine influenza A virus and porcine reproductive and respiratory syndrome virus

Abstract Background Simultaneous viral infections exhibit the phenomenon of viral interference, but understanding of the effect of one virus on another is limited. Objective Evaluate and compare clinical characteristics, immune and acute phase response, viral shedding and viral load in pigs singly and doubly inoculated with swine influenza A virus (swIAV) and porcine reproductive and respiratory syndrome virus (PRRSV). Animals Fifty‐four 7‐week‐old piglets. Methods Clinical status and gross lung lesions were scored. Titration of swIAV was carried out in Madin‐Darby canine kidney cells. The PRRSV RNA was quantified using a commercial qPCR kit. Antibodies were detected by hemagglutination inhibition assay and commercial ELISA. A lymphocyte proliferation assay was used to measure antigen‐specific T‐cell responses. Acute phase proteins were determined using ELISA. Results No differences were found between mean clinical scores, swIAV and PRRSV shedding, and magnitude of the humoral and T‐cell response between single‐inoculated and dual‐inoculated groups. Concentrations of C‐reactive protein and haptoglobin increased in PRRSV‐inoculated and coinoculated groups, whereas serum amyloid A concentration was increased in groups inoculated or coinoculated with swIAV. Mean swIAV TCID50 titers in the lungs did not differ significantly between coinoculated and swIAV single‐inoculated pigs. A significantly higher mean copy number of PRRSV was found in the lungs of PRRSV only‐inoculated pigs at 2 day postinoculation (DPI). From 4 DPI, no significant differences in PRRSV load were identified. Conclusions and Clinical Importance Coinfection of pigs with swIAV and PRRSV did not potentiate clinical signs, lung lesions, immune response, and replication of the viruses in the respiratory tract.

reported. [6][7][8] Moreover, PRRSV and swIAV, together and individually, frequently are primary or secondary agents responsible for porcine respiratory disease complex (PRDC). 9,10 Simultaneous viral infections can exhibit viral interference in which 1 virus blocks the growth of another virus. 11 Because mixed respiratory tract infections often are observed in animals, including pigs, the effect of the interaction of pathogens on the course of infection warrants further study. The impact of the intensity of coinfection on severity and clinical outcome still is unclear. Some studies determined that the clinical outcome of viral coinfections may be less, or at least not more, severe than infection by a single virus. 2,6,7,12,13 In contrast, other studies found that viral coinfection exacerbated the clinical course. 1,13 Contradictory consequences of viral coinfections also have been reported in the human medical literature. [14][15][16] Coinfections with swIAV and PRRSV are common in pig herds. [17][18][19] Both PRRSV and swIAV are responsible for PRDC, and some studies indicate the possibility of synergistic effects. 6,20 Because many PRRSV strains may have immunosuppressive potential, they may impact the immune response against other pathogens. 20,21 Previous studies showed various clinical outcomes with dual PRRSV and swIAV infection. 6,7,20 No significant changes in the clinical course of infection were found in a study in which piglets were infected with PRRSV and 1 week later infected with swIAV. 7 In contrast, another study reported more severe disease after dual infection compared to single PRRSV infection. 20 In yet another study of PRRSV and swIAV, variable clinical outcomes were observed in pigs coinfected with PRRSV and swIAV. 6 Regardless of the results of previous coinfection studies, our understanding of the effect of 1 virus on the other at both clinical and cellular levels still is limited. Thus, our objective was to assess and compare clinical characteristics, immune and acute phase response, viral shedding, and viral load between pigs singly and doubly inoculated with swIAV and PRRSV.

| Viruses
The swIAV used in our study, an avian-like H1N1

| Experimental design
Fifty-four 7-week-old conventional piglets from an influenza-and

| Clinical and pathological examination
Animals were examined daily from day 7 preinoculation until the end of the experiment at day 21 DPI or until euthanasia (at 2, 4, and 10 DPI). The pigs were observed and scored for respiratory signs as follows: respiratory rate: 0-normal, 0.33-slightly increased, 0.66-moderately increased, slight abdominal breathing, 1-clearly increased, distinct abdominal breathing; nasal discharge: 0-absent, 1 present; coughing: 0-absent, 1 present; sneezing: 0absent, 1 present, anorexia: 0-absent, 1 present. Rectal temperature was measured daily. Fever was recorded when rectal temperature reached or exceeded 40 C. When long-term fever (at least 3 days) was observed an additional point was added to the clinical score. Scores determined in each category were summated for a total clinical score for each individual pig (0-6). Nasal swabs were collected daily from all animals. Blood samples were collected at −7 days, day 0 (inoculation), and 1, 2, 3, 5, 7, 10, 14, and 21 DPI.
Three piglets of the inoculated and control groups were euthanized at 2, 4, and 10 DPI. The remaining inoculated pigs were euthanized and necropsied at 21 DPI.

| Lung score
Gross lung lesions were used to assign a lung score (LS) as described previously. 23 Each lung lobe was assigned a number reflecting an approximate volume percentage of the entire lung represented by that lobe. Ten possible points (5 for dorsal, 5 for ventral) were assigned each to the right anterior lobe, right middle lobe, anterior part of the left anterior lobe, and caudal part of the left anterior lobe.
The accessory lobe was assigned 5 points, and 27.5 points (15 for dorsal and 12.5 for ventral) were assigned to each of the right and left caudal lobes for a total of 100 points. The evaluation based on this procedure resulted in a LS that corresponded to the percentage of the lung affected by pneumonia.

| Serological tests
All sera were examined using a hemagglutination inhibition (HI) assay against challenge SwH1N1 strain and ELISA (VetExpert PRRS Ab The ELISA assays for PRRSV-specific antibodies were conducted according to manufacturer's recommendations.

| Lymphocyte proliferation assay
The T-cell proliferation assay to measure SwH1N1 and PRRSV-specific T-cell responses of pigs was performed at 0, 7, 14 and 21 DPI, as described previously. 27 Briefly, peripheral blood mononuclear cells (PBMC) were isolated from blood samples by centrifugation on Kildare, Ireland). The quantity of the protein was calculated based on the standard curve for each protein using FindGraph software.

| Pathogen shedding
The AUC value for SwH1N1 and PRRSV shedding, which was obtained by plotting SwH1N1 titers or PRRSV genomic copies of pigs sampled from day 0 to the last day when the virus was shed (TCID 50 titer or copy number/mL below the detection limit) vs each time point, did not differ significantly between single inoculated (both viruses) and coinoculated pigs (P ≥ .05). The dynamics of shedding of both pathogens (mean ± SD) during study period are presented in Figure 3A (PRRSV) and B (swIAV).

| PRRSV in serum
The AUC for PRRSV viremia, which was obtained by plotting genomic copy number against each sampling point, did not differ significantly between pigs single-inoculated and pigs coinoculated (P ≥ .05). The dynamics of PRRSV viremia (mean ± SD) during the study period are presented in Figure 4.

| Humoral immune response
The humoral response after inoculations of pigs with SwH1N1, PRRSV, or both is presented in Figure 5A,   Figure 6A,B.

| Acute phase proteins
In the control pigs, serum concentrations of all investigated acute phase proteins were stable during the study period and did not differ significantly from concentrations observed at −7 DPI (Figure 7A-D).
The serum concentration of CRP increased significantly only in pigs inoculated with PRRSV and in coinoculated pigs ( Figure 7A) as compared to controls (P < .05). In piglets in the coinoculated group, the mean serum concentration of CRP was significantly increased from 2 DPI until the end of the study (as compared to day 0 concentration and to control animals).
In the PRRSV group, different kinetics of serum CRP concentration were noted. The mean serum concentrations of this protein was significantly higher at 4, 10, and 14 DPI as compared to control animals (P < .05).
The serum concentration of Hp increased significantly in pigs single or coinoculated with PRRSV as compared to control animals ( Figure 7B). The dynamics of serum Hp concentration were similar in both groups inoculated with PRRSV. In piglets from the PRRS + swIAV and PRRSV groups, mean serum concentrations of Hp were significantly increased from 4 to 14 DPI (as compared to day 0 serum concentrations and to control animals).
The serum concentrations of SAA were significantly increased from 2 to 4 DPI in groups inoculated with SIV and PRRSV + swIAV as compared to control pigs (P < .05). No significant differences were found between control and PRRSV as well as swIAV and PRRSV + swIAV groups (P ≥ .05). Starting from 5 DPI the serum concentrations of SAA in inoculated groups did not differ significantly from those of control animals (P ≥ .05; Figure 7C).
The serum concentration of Pig-MAP remained unchanged as compared to its preinoculation concentration (P ≥ .05) in pigs inoculated with SwH1N1 and in control animals ( Figure 7D). In piglets from the PRRSV and swIAV + PRRSV groups, significant increases were observed from 3 DPI. The serum concentration of Pig-MAP remained increased in both groups until 7 DPI as compared to the day 0 concentration and to concentrations in control pigs (P < .05). No differences were found between pigs singly and coinoculated with PRRSV (P ≥ .05). F I G U R E 9 Swine influenza A virus (swIAV) titers, A and the mean copy number of porcine reproductive and respiratory syndrome virus (PRRSV), B, (mean ± SD) in the lung at 2, 4, 10, and 21 days after single or coinoculation of pigs. The dashed line represents the detection limit. a, significant differences between PRRSV and PRRS + swIAV group (P < .05) (P < .05). At 10 DPI, the mean LS in the coinoculated pigs was higher than in the PRRSV and control groups (P < .05). Mean LS differed significantly at 21 DPI between pigs singly or coinoculated with PRRSV and controls (P < .05), as well as compared to swIAV single-inoculated pigs (P < .05). The mean LS observed in all experimental groups are presented in Figure 8.
The RNA of swIAV was detected in all samples taken from the right lungs of pigs inoculated with swIAV at 2 and 4 DPI. At 10 and 21 DPI, no swIAV was detected in any of the groups. The mean swIAV TCID 50 titers did not differ significantly between pigs singly-inoculated and coinoculated with swIAV (P ≥ .05). In contrast, significant differences were found between mean copy number of PRRSV in lungs taken from pigs in the PRRSV and PRRSV + swIAV groups (P < .05) at 2 DPI, which was significantly higher in PRRSV only-inoculated pigs. At 4, 10 and 21 DPI, no significant differences in lung PRRSV load were observed (P ≥ .05). The PRRSV was detected in samples taken from right lung samples of all PRRSV singly and coinoculated pigs from 2 to 21 DPI. Virus loads in the lungs at 2, 4, 10, and 21 DPI are presented in Figure 9A,B.

| DISCUSSION
The viruses PRRSV and swIAV, alone or in combination, are 2 important pathogens among viruses contributing to porcine respiratory infections. 5,9,10 The PRRSV predisposes pigs to coinfection by other respiratory viruses, because of destruction of pulmonary tissues. 18,21,28,29 Additionally, immunosuppression induced by PRRSV may enhance the severity of other respiratory viral coinfections 20,21 and decrease the efficacy of immunization, including vaccination against swIAV. 13 Previous studies indicated that pigs infected with PRRSV were more likely to be coinfected with swIAV and developed 11%-50% higher LS. Moreover, PRRSV-infected pigs at the ages of 9 and 16 weeks were 15.57 and 5.75 times more prone to swIAV coinfection. 4  PRRSV and coinfected with swIAV 3 days later. 20 Another study found significant enhancement of clinical respiratory lesions and PRRSV-related interstitial pneumonia in pigs simultaneously infected with swIAV and PRRSV at 7 weeks of age. 13 In another study, in which piglets were infected intranasally with PRRSV, followed 1 week later with H3N2 swIAV strain, no influence on the clinical course of influenza infection was detected. 7 Another study observed variable clinical outcomes, depending on both the time interval between infections and the health status of pigs used in the study. 6 Another factor increasing variability among different studies may be the inherent virulence of viral strains used, especially PRRSV. One groups of investigators infected pigs with PRRSV-2, considered to be more virulent than PRRSV-1 used in other studies. 13 Another study determined that swIAV replication was slightly affected by prior infection with PRRSV, and viral excretion in the PRRSV-swIAV group was delayed by 2 days, not only with regard to presence of the virus, but also with respect to the peak amount. 20 We did not observe any delay or decrease in swIAV shedding in the coin- Our results also show that coinfection with local PRRSV and swIAV strains did not affect the intensity and kinetics of the acute phase and immune responses. The results of a previous study identified that the acute phase response was markedly different between strains in terms of intensity and duration, 38 but Hp was the most sensitive biomarker for PRRSV infection. In addition, Hp and CRP discriminated between infected and control pigs. This finding is in agreement with our results, because the significant increase in Hp and CRP was noted only in groups inoculated or coinoculated with PRRSV. In addition, significant differences in Hp between PRRSV and PRRSV + swIAV groups and the swIAV group were observed from 4 to 14 DPI. No significant differences in magnitude of PBMC proliferation and humoral response against both pathogens were noted between respective singly or coinoculated groups (P ≥ .05). In contrast, another study observed that PRRSV increased swIAV-specific lymphocyte proliferation in PBMCs collected 4 weeks after coinfection, but the PRRSV-2 strain was used. 13 Few effects on the innate immune response after coinfection with swIAV and PRRSV also were observed in a previous study conducted on conventional pigs. 39 The investigators concluded that coinfection with PRRSV and swIAV has additive effects only on the mRNA expression of interleukin (IL) 6 and IL-10,

CONFLICT OF INTEREST DECLARATION
Authors declare no conflict of interest.

OFF-LABEL ANTIMICROBIAL DECLARATION
Authors declare no off-label use of antimicrobials.

HUMAN ETHICS APPROVAL DECLARATION
Authors declare human ethics approval was not needed for this study.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.