Characterization of Actinobacillus pleuropneumoniae antiviral effect against porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages

Le syndrome reproducteur et respiratoire porcin (SRRP) est la maladie infectieuse la plus économiquement importante de l’industrie porcine. Une étude récente a démontré que le surnageant de culture d’Actinobacillus pleuropneumoniae (App) inhibe l’infection du virus SRRP (VSRRP) in vitro dans des cellules de singe. L’objectif de cette étude est de démontrer l’effet antiviral d’App contre le VSRRP dans les cellules cibles du virus in vivo: les macrophages alvéolaires porcins (MAPs) et d’étudier les mécanismes spécifiques impliqués lors de l’inhibition virale. Les MAPs ont été traités avec App, avant et après l’infection avec le VSRRP. À différents temps post-infection, la réplication et la transcription du génome viral ont été quantifiées. L’expression des interférons (IFN) type I et II, ainsi que le profil protéomique en présence ou absence d’App ont été évalués. L’expression de certaines protéines a été confirmée par immunobuvardage et immunofluorescence (IF). Les résultats ont démontré que l’effet antiviral d’App n’est pas via l’induction des IFN type I et II. App inhibe l’infection virale dans MAPs avant la réplication et la transcription du génome viral, ce qui indique qu’App inhibe précocement le cycle réplicatif viral. Le profil protéomique a révélé qu’App augmentait l’expression de la cofiline, une protéine qui provoque la dépolymérisation de l’actine. De plus, ce phénomène de dépolymérisation a été confirmé par IF. Le traitement des MAPs avec la cytochalasin D (un composé qui provoque la fragmentation des microfilments) a démontré que comme pour App, cette drogue inhibe la réplication virale. Les résultats obtenus suggèrent que l’effet antiviral d’App est via l'activation de la cofiline et dépolymérisation de l’actine, affectant probablement l’endocytose du VSRRP.Porcine reproductive and respiratory syndrome (PRRS) is the most economically important infectious disease of swine production. A recent study has demonstrated that the culture supernatant of Actinobacilus pleuropneumoniae (App) inhibits PRRS virus (PRRSV) infection in vitro in a monkey cell line. Following this finding, the objective of this study was to demonstrate the antiviral effect of App in the primary target cells of PRRSV in vivo: porcine alveolar macrophages (PAM) and to elucidate how App inhibits PRRSV replication in PAM. Cells were treated with App before and after PRRSV infection. At different times postinfection, viral genome replication and transcription were measured in the presence of App. mRNA expression of type I and II interferon (IFN) and the proteomic profile of infected cells treated with App were evaluated. The expression of selected proteins was confirmed by immunofluorescence (IFA) and Western blot assays. Results showed that App antiviral effect against PRRSV is not via the induction of type I and II IFN expression. Moreover, it was observed that App inhibits PRRSV infection in PAM before its genome replication and transcription, indicating that App antiviral effect takes place early in PRRSV replication cycle. Proteomic results revealed that App increases cofilin, a protein that induces actin filaments depolymerisation in its active form. This depolymerisation phenomenon was further confirmed by IFA. Interestingly, a microfilament-disrupting compound (cytochalasin D) induced the same effect on PRRSV replication than App suggesting that App antiviral effect against PRRSV takes place via the activation of cofilin and thus actin depolymerisation, which probably affects PRRSV endocytosis

Actinobacillus pleuropneumoniae Possesses an Antiviral Activity against Porcine Reproductive and Respiratory Syndrome Virus

Pigs are often colonized by more than one bacterial and/or viral species during respiratory tract infections. This phenomenon is known as the porcine respiratory disease complex (PRDC). Actinobacillus pleuropneumoniae (App) and porcine reproductive and respiratory syndrome virus (PRRSV) are pathogens that are frequently involved in PRDC. The main objective of this project was to study the in vitro interactions between these two pathogens and the host cells in the context of mixed infections. To fulfill this objective, PRRSV permissive cell lines such as MARC-145, SJPL, and porcine alveolar macrophages (PAM) were used. A pre-infection with PRRSV was performed at 0.5 multiplicity of infection (MOI) followed by an infection with App at 10 MOI. Bacterial adherence and cell death were compared. Results showed that PRRSV preinfection did not affect bacterial adherence to the cells. PRRSV and App co-infection produced an additive cytotoxicity effect. Interestingly, a pre-infection of SJPL and PAM cells with App blocked completely PRRSV infection. Incubation of SJPL and PAM cells with an App cell-free culture supernatant is also sufficient to significantly block PRRSV infection. This antiviral activity is not due to LPS but rather by small molecular weight, heat-resistant App metabolites (,1 kDa). The antiviral activity was also observed in SJPL cells infected with swine influenza virus but to a much lower extent compared to PRRSV. More importantly, the PRRSV antiviral activity of App was also seen with PAM, the cells targeted by the virus in vivo during infection in pigs. The antiviral activity might be due, at least in part, to the production of interferon c. The use of in vitro experimental models to study viral and bacterial co-infections will lead to a better understanding of the interactions between pathogens and their host cells, and could allow the development of novel prophylactic and therapeutic tools

Actinobacillus pleuropneumoniae (App) culture supernatant antiviral effect against porcine reproductive and respiratory syndrome virus (PRRSV) occurs prior to the viral genome replication and transcription through actin depolymerization

PURPOSE: Recently, the strong antiviral activity of an Actinobacillus pleuropneumoniae (App) culture supernatant against porcine reproductive and respiratory syndrome virus (PRRSV) was discovered. Following this finding, the objective of the present study was to understand how the App culture supernatant inhibits PRRSV replication in its natural targeted host cells, i.e. porcine alveolar macrophages (PAMs). METHODOLOGY: Several assays were conducted with App culture supernatant-treated PRRSV-infected cell lines, such as PAM, St-Jude porcine lung and MARC-145 cells. RT-qPCR assays were used to determine the expression levels of type I and II IFN mRNAs, viral genomic (gRNA) and sub-genomic RNAs (sgRNAs). Proteomic, Western blot and immunofluorescence assays were conducted to determine the involvement of actin filaments in the App culture supernatant antiviral effect.Results/Key findings. Type I and II IFN mRNA expressions were not upregulated by the App culture supernatant. Time courses of gRNA and sgRNA expression levels demonstrated that the App culture supernatant inhibits PRRSV infection before the first viral transcription cycle. Western blot experiments confirmed an increase in the expression of cofilin (actin cytoskeleton dynamics regulator) and immunofluorescence also demonstrated a significant decrease of actin filaments in App culture supernatant-treated PRRSV-infected PAM cells. App culture supernatant antiviral activity was also demonstrated against other PRRSV strains of genotypes I and II. CONCLUSION: App culture supernatant antiviral effect against PRRSV takes place early during PRRSV infection. Results suggest that App culture supernatant antiviral effect may take place via the activation of cofilin, which induces actin depolymerization and subsequently, probably affects PRRSV endocytosis. Other experiments are needed to fully validate this latest hypothesis

Antiviral activity of <i>AppΔapxIΔapxIIC</i> supernatant against several animal DNA and RNA viruses in SJPL infected cells.

<p>All experiences were performed at least 2 times.</p><p>Statistical <i>P</i> value compared to <i>AppΔapxIΔapxIIC</i> untreated cells: <i>P</i> = 0.15.</p><p>Statistically significative compared to <i>AppΔapxIΔapxIIC</i> untreated cells: *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p><p>Statistically significative compared to other viruses: <i>^aP</i><0.01.</p

Bacterial adherence over time of <i>Appwt</i> or <i>AppΔapxIΔapxIIC</i> in PRRSV co-infected SJPL and MARC-145 cells.

<p>SJPL (A) and MARC-145 (B) cells were infected with or without PRRSV at an MOI of 0.5 during 72 hours, and then cells were co-infected with <i>Appwt</i> or <i>AppΔapxIΔapxIIC</i> at an MOI of 10. Bacterial adherence was measured in CFU per well after 1, 2 and 3 hours post bacterial infection as described in Auger <i>et al</i>., 2009 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098434#pone.0098434-Auger1" target="_blank">[20]</a>. Values are presented as ± Standard Deviation (SD). No statistical significance was obtained following two-away ANOVA analysis. All experiments were repeated 3 times.</p

<i>AppΔapxIΔapxIIC</i> cell culture supernatant <1 kDa fraction antiviral activity against PRRSV.

<p>Detection of the N viral protein in PRRSV infected SJPL cells by immunofluorescence. SJPL cells were infected with 0.5 MOI of PRRSV for 4 hours then incubated with DMEM culture medium alone (DMEM) (A) or either a DMEM culture medium fraction of <1 kDa (DMEM <1 kDa) (B) or a <i>AppΔapxIΔapxIIC</i> cell culture supernatant <1 kDa fraction (<i>App</i><1 kDa) (C) added to complete SJPL culture medium for 48 hours. White scale bar represents 200 µm. Pictures were taken at 100X magnification.</p

Cytotoxicity over time of <i>Appwt</i> or <i>AppΔapxIΔapxIIC</i> in PRRSV co-infected SJPL and MARC-145 cells.

<p>SJPL (A and B) and MARC-145 cells (C and D) were infected with or without PRRSV at an MOI of 0.5 during 72 hours, and then cells were co-infected with <i>App</i> (for 1 or 2 hours) (A and C, respectively) or with <i>AppΔapxIΔapxIIC</i> (for 4, 5 and 6 hours) (B and D, respectively) at an MOI of 10. Cytotoxicity was measured in % using lactate dehydrogenase (LDH) CytoTox assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098434#pone.0098434-Auger1" target="_blank">[20]</a>. Values are presented as ± Standard Deviation (SD). Two-away ANOVA analysis was used to obtain statistical data. *<i>P</i><0.05. All experiments were performed 3 times.</p

<i>AppΔapxIΔapxIIC</i> cell culture supernatant and PRRSV effects on mRNA quantification of type I (IFNα, IFNβ) and type II (IFNγ) interferons.

<p>qRT-PCR results expressed in relative expression (ΔΔCT) for IFNα (A), IFNβ (B) and IFNγ (C) in SJPL cells. The cells were mock infected or infected with 0.5 MOI of PRRSV for 4 hours then treated without or with <i>AppΔapxICΔapxIIC</i> cell culture supernatant for 48 hours. Poly (I:C) and LPS were used as positive controls. Data labeled with superscripts of different letters indicates that these sets of data are statistically different (P<0.05).</p