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. 2016 Apr 15:6:24401.
doi: 10.1038/srep24401.

Matrine displayed antiviral activity in porcine alveolar macrophages co-infected by porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Na Sun  1 , Panpan Sun  1 , Haipeng Lv  1 , Yaogui Sun  1 , Jianhua Guo  2 , Zhirui Wang  3 , Tiantian Luo  4 , Shaoyu Wang  5 , Hongquan Li  1
Affiliations

Matrine displayed antiviral activity in porcine alveolar macrophages co-infected by porcine reproductive and respiratory syndrome virus and porcine circovirus type 2

Na Sun et al. Sci Rep. .

Abstract

The co-infection of porcine reproductive respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) is quite common in clinical settings and no effective treatment to the co-infection is available. In this study, we established the porcine alveolar macrophages (PAM) cells model co-infected with PRRSV/PCV2 with modification in vitro, and investigated the antiviral activity of Matrine on this cell model and further evaluated the effect of Matrine on virus-induced TLR3,4/NF-κB/TNF-α pathway. The results demonstrated PAM cells inoculated with PRRSV followed by PCV2 2 h later enhanced PRRSV and PCV2 replications. Matrine treatment suppressed both PRRSV and PCV2 infection at 12 h post infection. Furthermore, PRRSV/PCV2 co- infection induced IκBα degradation and phosphorylation as well as the translocation of NF-κB from the cytoplasm to the nucleus indicating that PRRSV/PCV2 co-infection induced NF-κB activation. Matrine treatment significantly down-regulated the expression of TLR3, TLR4 and TNF-α although it, to some extent, suppressed p-IκBα expression, suggesting that TLR3,4/NF-κB/TNF-α pathway play an important role of Matrine in combating PRRSV/PCV2 co-infection. It is concluded that Matrine possesses activity against PRRSV/PCV2 co-infection in vitro and suppression of the TLR3,4/NF-κB/TNF-α pathway as an important underlying molecular mechanism. These findings warrant Matrine to be further explored for its antiviral activity in clinical settings.

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Figures

Figure 1
Figure 1. The chemical structure of Matrine.
Figure 2
Figure 2. Cytotoxicity of Matrine and Ribavirin on PAM cells.
PAM cells were incubated with different concentrations of Matrine and Ribavirin for 48 h. (a) the cell morphological changes by compound treatment. Typical vacuolization indicated by white arrows. (b) the cell viability rate was displayed “S” shape curve after treatment with Matrine or Ribavirin at different concentration, indicating the cytotoxicity of Matrine and Ribavirin on PAM cells was in a dose-dependent manner within the concentrations employed.
Figure 3
Figure 3
The detection of PRRSV N gene copies (a) and PCV2 CAP gene copies (b) respectively in PRRSV infected PAM cells, PCV2 infected PAM cells and PRRSV/PCV2 co-infected cells from 6 hpi to 96 hpi. The specific gene copies of PRRSV and PCV2 were determined by absolute quantitative real time PCR. Data were expressed as mean ± standard errors mean (SEM). Data with different letters (a–d) indicated that the group was significant different from other groups (p < 0.05).
Figure 4
Figure 4. PAM cells respectively infected with PRRSV, PCV2 and PRRSV/PCV2 from 6 hpi to 96 hpi.
Normal PAM cells displayed globular and clear cell boundary. At 96 hpi, in PRRSV alone group and PRRSV/PCV2 co-infection group, PAM cells presented various degree of morphological changes such as cell disruption, detachment of monolayer and aggregation (black arrows in Fig. 4). From 24 hpi to 72 hpi, the spindle cell formed in PCV2 alone infected PAM cells (white arrows in Fig. 4). Magnification:400×.
Figure 5
Figure 5. Detection of PCV2 CAP protein by IFA in PCV2 infected PAM cells.
After 12 and 72 hpi, cells were fixed and then treated with 0.1% triton-100 in PBS for 15 min. After that the cells were blocked by 3% BSA, then incubated with rabbit anti-CAP monoclonal antibody at 37 °C for 2 h. After washing, cells were stained with Alexa Fluor 488 conjugated affinipure goat anti-Rabbit IgG for 1 h. The cells were examined by a fluoresence microsope. No specific immunofluoresence in Normal cells (A). PCV2 CAP protein was detected in the cytoplasm of the infected PAM cells at 12 hpi (B). At 72 hpi, the cap protein was detected in the nucleus of the infected PAM cells (C).
Figure 6
Figure 6
Detection PRRSV N gene copies in PRRSV infected PAM cells and PRRSV/PCV2 co-infected cells from 6 hpi to 96 hpi (a). Detection of PCV2 CAP gene copies in PCV2 infected PAM cells and PRRSV/PCV2 co-infected cells from 6 hpi to 96 hpi (b). The specific gene copies of PRRSV and PCV2 were determined by absolute quantitative real time PCR. Data were expressed as mean ± standard errors mean (SEM). *means p < 0.05, **indicates p < 0.01, ***means p < 0.001.
Figure 7
Figure 7. Inhibition of PRRSV and PCV2 gene expression in PRRSV/PCV2 co-infection by Matrine.
Detection of PRRSV N gene (a) and protein (b and c) and of PCV2 CAP gene (d). Data were expressed as mean ± standard errors mean (SEM). Data with different letters (a,b,c,d,e) indicated that the group was significant different from other groups (p < 0.05).
Figure 8
Figure 8. Effect of Matrine on TLR3 and TLR4 expression.
PAM cells from all treatment groups were collected and extracted for RNA at 6, 12 and 24 hpi. The mRNA expression of TLR3 and TLR4 was detected using relative quantitative real time PCR. Data were expressed as mean ± standard errors mean (SEM). *indicates that the groups were significant different from cell control (p < 0.05). #means the compare between Matrine treatment group and virus control was significant different (p < 0.05), ##means p < 0.01, ###means p < 0.001.
Figure 9
Figure 9. PRRSV and PCV2 co-infection induced NF-κB activation while Matrine treatment barely block virus-induced NF-κB activation.
The expressions of IκBα, p-IκBα, cytoplasmic p65 and nuclear p65 were measured by western blot assay (a). Quantification of IκBα, p-IκBα, cytoplasmic p65 expression was normalized to GAPDH and the expression of nuclear p65 was normalized to TBP using Image Pro Plus software (b). *stands for the groups were significant different with cell control (p < 0.05), **means p < 0.01, ***means p < 0.001. #means the groups were significant different with virus control (p < 0.05), ##means p < 0.01, ###means p < 0.001.
Figure 10
Figure 10. Suppression of virus-induced TNF-α expression by Matrine. ELISA assay was used to measure the expression level of TNF-α at 6, 12 and 24 hpi.
Data were expressed as mean ± standard errors mean (SEM). *stands for the groups were significant different with cell control (p < 0.05). ##means the Matrine treatment group was significant different with virus control (p < 0.01).
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References

    1. Poljak Z. et al.. Spread of porcine circovirus associated disease (PCVAD) in Ontario (Canada) swine herds: Part 1. Exploratory spatial analysis. BMC Vet. Res. 6, 59 (2010). - PMC - PubMed
    1. Zhai S. L. et al.. Porcine circovirus type 2 in China: an update on and insights to its prevalaence and control. Virol. J. 11, 88 (2014). - PMC - PubMed
    1. Meng X. J. Porcine Circovirus Type 2 (PCV2): Pathogenesis and Interaction with the Immune System. Annu. Rev. Anim. Biosci. 1, 43–64 (2013). - PubMed
    1. Opriessnig T., Giménez-Lirola L. G. & Halbur P. G. Polymicrobial respiratory disease in pigs. Anim. Health Res. Rev. 2, 133–148 (2011). - PubMed
    1. Opriessnig T. & Halbur P. G. Concurrent infections are important for expression of porcine circovirus associated disease. Virus Res. 164, 20–32 (2012). - PMC - PubMed

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