Co-infection of classic swine H1N1 influenza virus in pigs persistently infected with porcine rubulavirus

Abstract

Porcine rubulavirus (PorPV) and swine influenza virus infection causes respiratory disease in pigs. PorPV persistent infection could facilitate the establishment of secondary infections. The aim of this study was to analyse the pathogenicity of classic swine H1N1 influenza virus (swH1N1) in growing pigs persistently infected with porcine rubulavirus. Conventional six-week-old pigs were intranasally inoculated with PorPV, swH1N1, or PorPV/swH1N1. A mock-infected group was included. The co-infection with swH1N1 was at 44 days post-infection (DPI), right after clinical signs of PorPV infection had stopped. The pigs of the co-infection group presented an increase of clinical signs compared to the simple infection groups. In all infected groups, the most recurrent lung lesion was hyperplasia of the bronchiolar-associated lymphoid tissue and interstitial pneumonia. By means of immunohistochemical evaluation it was possible to demonstrate the presence of the two viral agents infecting simultaneously the bronchiolar epithelium. Viral excretion of PorPV in nasal and oral fluid was recorded at 28 and 52 DPI, respectively. PorPV persisted in several samples from respiratory tissues (RT), secondary lymphoid organs (SLO), and bronchoalveolar lavage fluid (BALF). For swH1N1, the viral excretion in nasal fluids was significantly higher in single-infected swH1N1 pigs than in the co-infected group. However, the co-infection group exhibited an increase in the presence of swH1N1 in RT, SLO, and BALF at two days after co-infection. In conclusion, the results obtained confirm an increase in the clinical signs of infection, and PorPV was observed to impact the spread of swH1N1 in analysed tissues in the early stage of co-infection, although viral shedding was not enhanced. In the present study, the interaction of swH1N1 infection is demonstrated in pigs persistently infected with PorPV.

Keywords: Classic swine H1N1 influenza virus; Co-infection; Pigs; Porcine rubulavirus; Respiratory disease.

Fig.1

Mean of the rectal temperatures (±SD) and clinical score in Mock/Mock (★), PorPV/Mock (●), PorPV/swH1N1 (○) and Mock/swH1N1 (▲) groups after simple inoculation with PorPV, swH1N1 and co-infection with both viruses. No significant differences between the observed groups (P > 0.05).

Fig. 2

Haematoxylin-eosin stained sections of lungs. (a) A Mock/Mock pig; (b) a pig co-infected with PorPV and swH1N1 at 52 DPI (PoRV/swH1N1 group); (c) a pig infected with PorPV at 52 DPI (PorPV/Mock group); (d) a pig infected with swH1N1 at 4 DPI (Mock/swH1N1 group); (e) a pig co-infected with PorPV and swH1N1 at 52 DPI, note the presence of multinucleated cells in the lumen of the alveoli (arrows); (f) a pig infected with PorPV at 52 DPI (PorPV/Mock group), the arrowhead indicates the presence of syncytia in the alveolar lumen. Immunohistochemistry of sections from the apical lung lobe. (g) Immunoreaction against HN of a lung section from a pig of group PorPV/swH1N1. (h) Immunoreaction against HA1 of a lung section from a pig of group PorPV/swH1N1. (i, j) Immunoreaction against HN of a lung section from a pig of group PorPV/Mock. (k, l) Immunoreaction against HA1 of a lung section from a pig of group Mock/swH1N1. The asterisk indicates the presence of positive immunoreaction. Magnification: ×100 (a-d), ×200 (i, k), ×400 (e, g, h, j and l) and ×640 (f).

Fig. 3

The individual and mean PorPV and swH1N1 viral load in nasal swabs in PorPV/Mock (●) and PorPV/swH1N1 (○) groups (A), and Mock/swH1N1 (▲) and PorPV/swH1N1 (○) groups (B), respectively. The dotted and solid lines indicate the mean of the viral quantification in each group.

Fig. 4

The individual and mean PorPV and swH1N1 viral load in oral swabs in PorPV/Mock (●) and PorPV/swH1N1 (○) groups (A), and Mock/swH1N1 (▲) and PorPV/swH1N1 (○) groups (B), respectively. The dotted and solid lines indicate the mean of the viral quantification in each group.