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. 2021 Sep 6:8:722840.
doi: 10.3389/fvets.2021.722840. eCollection 2021.

Antiviral Effectivity of Favipiravir Against Peste Des Petits Ruminants Virus Is Mediated by the JAK/STAT and PI3K/AKT Pathways

Weifeng Zhang  1 , Hualong Deng  1 , Yanfen Liu  1 , Shaohong Chen  2 , You Liu  2 , Yuntao Zhao  2
Affiliations

Antiviral Effectivity of Favipiravir Against Peste Des Petits Ruminants Virus Is Mediated by the JAK/STAT and PI3K/AKT Pathways

Weifeng Zhang et al. Front Vet Sci. .

Abstract

Peste des petits ruminants virus (PPRV), belonging to the genus Morbillivirus in the family Paramyxoviridae, causes severe infectious disease in small ruminants and has been rapidly spreading in many parts of Africa, the Middle East, and Asia. Although vaccination is considered to be an effective means of controlling PPR, the heat-sensitive nature of the vaccines against PPRV greatly limits their application in areas with a hot climate. In the present study, we investigated the anti-PPRV effects of favipiravir and sought to identify the underlying mechanisms in vitro using the Vero cell line. MTT assays, Western blotting, indirect immunofluorescence assays, virus plaque formation assays, and qRT-PCR were used to assess the effects of favipiravir on the life cycle of PPRV and the expression of RNA-dependent RNA polymerase (RdRp). Additionally, the expression levels of JAK1, STAT1, phosphorylated (p)-STAT1, PI3K, AKT, and p-AKT, as well as those of signaling molecules acting downstream of the JAK/STAT and PI3K/AKT signaling pathways, were determined by Western blotting and qRT-PCR. The results indicated that, in PPRV-infected, favipiravir-treated Vero cells, the attachment, invasion, replication, and release of PPRV were significantly inhibited, as was the expression of RdRp, when compared with that in untreated PPRV-infected cells. Furthermore, in favipiravir-treated cells, the expression of JAK1 and STAT1 was downregulated, whereas that of p-STAT1 was significantly upregulated. Similarly, the expression levels of PKR, IRF9, ISG54, and MxA proteins that are associated with innate antiviral activity in host cells were also markedly increased. Moreover, with favipiravir treatment, the expression of PI3K and p-AKT and the p-AKT/AKT ratio were significantly decreased, whereas the expression of AKT was noticeably upregulated. The expression of GSK3, NF-κB p65, p-NF-κB p65, and BAD was also increased with favipiravir treatment, while the expression of CREB, p-CREB, p-GSK3, and Bcl-2 was slightly decreased. In addition, all the p-GSK3/GSK3, p-CREB/CREB, p-NF-κB/NF-κB, and p-BAD/BAD ratios were significantly reduced in favipiravir-treated cells. These results implied that the antiviral effectivity of favipiravir against PPRV is mediated by the JAK/STAT and PI3K/AKT pathways and that favipiravir has potential for use as an effective antiviral agent against PPRV.

Keywords: antiviral activity; favipiravir; peste des petits ruminants virus; ruminants; signal pathway.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Determination of the maximal noncytotoxic concentration of favipiravir. (A) The viability of Vero cells treated with serial dilutions of favipiravir for 48 h from a starting concentration of 100 μg/ml. (B) The viability of Vero cells treated with 100 TCID50 peste des petits ruminants virus (PPRV) and favipiravir at maximal non-toxic concentration for 48 h (*p < 0.05, ***p < 0.001 vs. the cell control).
Figure 2
Figure 2
The anti-peste des petits ruminants virus (anti-PPRV) effect of favipiravir in Vero cells. (A,B) Vero cells were treated with PPRV at 100 TCID50 and favipiravir at 50 μg/ml for 48 h, following which the expression of the structural proteins H, F, and N was analyzed by Western blotting (***p < 0.001 vs. the virus-only control). (C) Vero cells were treated with favipiravir and PPRV simultaneously for 48 h, and then the relative mRNA expression levels of the H and N genes were measured by qRT-PCR (***p < 0.001 vs. the virus-only control). (D) The anti-PPRV activity of favipiravir was detected by immunofluorescence assay (IFA) in PPRV-infected Vero cells after 48 h. PPRV is in green; nuclei are stained blue (DAPI). Scale bars = 50 μm.
Figure 3
Figure 3
The effect of favipiravir on peste des petits ruminants virus (PPRV) attachment to host cells. Vero cells were pre-incubated with favipiravir at 50 μg/ml or an equal volume of medium at 37°C for 1 h and then infected with PPRV at 4°C for 2 h. The cells were washed five times with cold phosphate-buffered saline (PBS) and collected for qPCR and immunofluorescence assay (IFA) detection (**p < 0.01, ***p < 0.001 vs. the virus-only control). (A) The mRNA expression of the H and N genes. (B) PPRV distribution in the cytoplasm was detected by indirect IFA. Scale bars = 100 μm.
Figure 4
Figure 4
Vero cells were infected with peste des petits ruminants virus (PPRV) at 100 TCID50/ml at 4°C for 1 h, washed with cold phosphate-buffered saline (PBS) to remove unattached viruses, and then treated with favipiravir at 50 μg/ml in maintenance medium supplemented with 2% fetal calf serum (FCS) for 1 h at 37°C. The cells were then washed again with PBS to remove any extracellular viruses, and maintenance medium was added. The plates were incubated at 37°C, and the expression of the viral structural proteins H, F, and N was analyzed by Western blotting at (A) 36, (B) 48, and (C) 60 h post-invasion (**p < 0.01, ***p < 0.001 vs. the virus-only control).
Figure 5
Figure 5
The effect of favipiravir on the release of progeny peste des petits ruminants virus (PPRV) particles from host cells. (A,B) Vero cells were infected with 100 TCID50 PPRV at 37°C for 2 h and washed five times with cold phosphate-buffered saline (PBS) to remove unattached viruses, followed by supplementation with maintenance medium. The cells were incubated at 37°C for 48 h and washed five times with cold PBS. Finally, the cells were incubated with maintenance medium containing favipiravir at 50 μg/ml for 2 h. The supernatants were collected, and the virus titers were determined by plaque formation assay (**p < 0.01 vs. the virus-only control).
Figure 6
Figure 6
The effect of favipiravir on the expression levels of proteins and mRNAs related to the JAK/STAT signaling pathway (A–I). Vero cells were simultaneously treated with favipiravir and infected with peste des petits ruminants virus (PPRV), and protein expression levels were measured 48 h after infection. The detection of JAK1 (B), STAT1 (C), and p-STAT1 (D) expression levels by Western blotting. The relative mRNA levels of PKR (E), IRF9 (F), ISG20 (G), ISG54 (H), and MxA (I) were detected by qRT-PCR (&p < 0.05, &&p < 0.01, &&&p < 0.001 vs. the virus-only control; *p < 0.05, **p < 0.01, ***p < 0.001 vs. the cell control).
Figure 7
Figure 7
The effects of favipiravir on the expression levels of key proteins related to the PI3K/AKT signaling pathway. Vero cells were simultaneously treated with favipiravir and infected with peste des petits ruminants virus (PPRV); and the expression levels of PI3K, p-AKT, AKT, p-GSK3, GSK3, p-CREB, CREB, p-NF-κB p65, NF-κB p65, p-BAD, BAD, and Bcl-2 (A–H) were detected by Western blotting 48 h after infection (&&&p < 0.001 vs. the virus-only control; **p < 0.01, ***p < 0.001 vs. the cell control).
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References

    1. Wernike K, Eschbaumer M, Breithaupt A, Maltzan J, Wiesner H, Beer M, et al. . Experimental infection of sheep and goats with a recent isolate of peste des petits ruminants virus from Kurdistan. Vet Microbiol. (2014) 172:140–5. 10.1016/j.vetmic.2014.05.010 - DOI - PubMed
    1. Kamel M, El-Sayed A. Toward peste des petits virus (PPRV) eradication: Diagnostic approaches, novel vaccines, and control strategies. Virus Res. (2019) 274:197774–85. 10.1016/j.virusres.2019.197774 - DOI - PubMed
    1. Pope RA, Parida S, Bailey D, Brownli, Table e J, Barrett T, et al. . Early events following experimental infection with Peste-Des-Petits ruminants virus suggest immune cell targeting. PLoS One. (2013) 8:e55830–45. 10.1371/journal.pone.0055830 - DOI - PMC - PubMed
    1. Altan E, Parida S, Mahapatra M, Turan N, Yilmaz H. Molecular characterization of Peste des petits ruminants viruses in the Marmara Region of Turkey. Transbound Emerg Dis. (2019) 66:865–72. 10.1111/tbed.13095 - DOI - PMC - PubMed
    1. Rojas JM, Moreno H, Valcárcel F, Peña L, Sevilla N, Martín V. Vaccination with recombinant adenoviruses expressing the peste des petits ruminants virus F or H proteins overcomes viral immunosuppression and induces protective immunity against PPRV challenge in sheep. PLoS One. (2014) 9:e101226–32. 10.1371/journal.pone.0101226 - DOI - PMC - PubMed