Simultaneous Infection With Porcine Reproductive and Respiratory Syndrome and Influenza Viruses Abrogates Clinical Protection Induced by Live Attenuated Porcine Reproductive and Respiratory Syndrome Vaccination

Abstract

The porcine respiratory disease complex (PRDC) is responsible for significant economic losses in the pig industry worldwide. Porcine reproductive and respiratory syndrome virus (PRRSV) and swine influenza virus are major viral contributors to PRDC. Vaccines are cost-effective measures for controlling PRRS, however, their efficacy in the context of co-infections has been poorly investigated. In this study, we aimed to determine the effect of PRRSV-2 and swine influenza H3N2 virus co-infection on the efficacy of PRRSV modified live virus (MLV) vaccination, which is widely used in the field. Following simultaneous challenge with contemporary PRRSV-2 and H3N2 field isolates, we found that the protective effect of PRRS MLV vaccination on clinical disease and pathology was abrogated, although viral load was unaffected and antibody responses were enhanced. In contrast, co-infection in non-immunized animals reduced PRRSV-2 viremia and H3N2 virus load in the upper respiratory tract and potentiated T cell responses against both PRRSV-2 and H3N2 in the lung. Further analysis suggested that an upregulation of inhibitory cytokines gene expression in the lungs of vaccinated pigs may have influenced responses to H3N2 and PRRSV-2. These findings provide important insights into the effect of viral co-infections on PRRS vaccine efficacy that may help identify more effective vaccination strategies against PRDC in the field.

Keywords: co-infection; live attenuated vaccine; pig; porcine reproductive and respiratory syndrome virus; swine influenza A virus.

Figure 1

Clinical signs and lung lesions. (A) Pigs were vaccinated intramuscularly with Ingelvac PRRS^® MLV (Vac) or with PBS (Ctrl) or were untreated (naïve). Thirty-three days after the vaccination, pigs were challenged by intranasal inoculation with H3N2, PRRSV-2 or simultaneously with PRRSV-2/H3N2. Nasal swabs were daily taken after the challenge and pigs were culled 5 days later [38 days post-vaccination (dpv)]. Sera and PBMC were collected at 0, 20 and 38 dpv. Clinical signs and rectal temperature were monitored daily after the challenge. (B) Table indicating the number of pigs that developed fever after the challenge. (C-E) Lungs sections were scored for histopathological lesions (C); Morgan score), lesions with presence of influenza NP-positive cells (D); Iowa score) or lesions with presence of PRRSV N-positive cells (E); Salguero score). Each symbol represents an individual animal within the indicated group (n = 6 per group). The horizontal lines represent mean ± SD. Comparisons between 2 group were analyzed using Mann-Whitney test and asterisks indicate significant differences (*p < 0.05; **p < 0.01).

Figure 2

PRRSV-2 and H3N2 loads. Quantification of PRRSV-2 viral RNA in nasal swabs (A), BALF (B) and serum (C) were determined by qRT-PCR. H3N2 titers in nasal swabs (D) and BALF (E) were determined by plaque assay. The mean values (A, D) or individual values (B–E) for each group ± SD are indicated (n = 6 per group). P values were determined using Mann-Whitney test and asterisks indicate significant differences (*p < 0.05; **p < 0.01).

Figure 3

Antibody responses against PRRSV-2. (A) Detection of PRRSV N-specific Abs in serum at 0, 20 and 38 dpv was performed with a commercial ELISA test. The positive threshold is indicated with a horizontal dashed line. (B) PRRSV-specific Ab titers were measured in the sera of PRRS-immunized pigs at 0, 20 and 38 dpv with an in-house ELISA test. (C) Virus neutralizing Ab titers in the serum of PRRS-vaccinated pig at 0, 20 and 38 are shown. Sera from PRRSV-2 infected pigs from an unrelated study were used as positive controls (red symbols). Each pig serum is shown as a symbol within the indicated group (n = 6 per group) and the mean ± SD is represented. Starting dilution is indicated with dashed line. The comparison between the percentage positivity values or Ab titers measured at 20 and 38 dpv versus at 0 dpv for each vaccinated group were performed using the Wilcoxon test. Comparisons between groups were made using the Mann-Whitney test. Asterisks indicate significant differences (*p < 0.05; **p < 0.01). nd, not determined.

Figure 4

PRRSV-2-specific T cell responses in PBMC. PBMC isolated at 0, 20 and 38 dpv were restimulated in vitro for 18h with PRRSV-2 (MOI 0.1) or cultured with medium. Intracellular staining of IFN-γ, TNF, IL-2, was performed and frequencies of IFN-γ, TNF and IL-2 producing CD4⁺ (A) and CD8β⁺ (B) T cells were analyzed. The corrected frequencies values are shown (percentage of cytokine-producing cells subtracted with medium only). Data for individual pigs and the group mean ± SD are displayed (n = 5-6 per group). The Wilcoxon test was used to compare the T cell responses at day 0 and 20 dpv within the same group. Comparisons between 2 groups were performed using Mann-Whitney test. nd: not determined.

Figure 5

PRRSV-2-specific T cell responses in bronchoalveolar lavage. Cells isolated from BAL at 5 dpc were restimulated with PRRSV-2 (MOI 0.1) or cultured with medium. (A) Frequency of IFN-γ-, TNF- and IL-2-producing CD4⁺ and CD8β⁺ T cells are shown. (B) Frequency of IFN-γ-, TNF- and IL-17-producing CD2⁺ and CD2^- γδ T cells are represented. The corrected frequencies (percentage of cytokine-producing cells subtracted with medium only) of each individual pig and the group mean ± SD are displayed (n = 5-6 per group). Comparisons between groups were made using Mann-Whitney test. Asterisk indicates significant difference (*p < 0.05).

Figure 6

H3N2-specific T cell responses in bronchoalveolar lavage. Cells isolated from BALF at 5 dpc were restimulated with swIAV H3N2 (MOI 0.1) or cultured with medium. Cytokine secretion measured in CD4⁺ and CD8β⁺ (A), and CD2⁺ and CD2^- γδ (B) T cells are represented. Corrected frequencies of individual value and the mean ± SD are displayed (n = 5-6 per group). Comparisons between 2 groups were made using the Mann-Whitney test.

Figure 7

Gene expression in lung tissues. Total RNA was extracted from lung tissue collected at 5 dpc and the relative mRNA expression of IFN-α, IFN-γ, TNF, IL-12p40, IL-4, IL-6, IL-21, IL-10, TGF-β and CXCL-13 was assessed by qRT-PCR. Fold changes are shown over naïve group (dash line) after normalization with GAPDH and RPS24 genes. Individual pig values and the group mean ± SD are displayed (n = 5-6 per group). Comparisons were made using Kruskal-Wallis test and asterisks indicate significant differences (*p < 0.05).