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Host Responses and Regulation by NFκB Signaling in the Liver and Liver Epithelial Cells Infected with A Novel Tick-borne Bunyavirus.

Sun Q, Jin C, Zhu L, Liang M, Li C, Cardona CJ, Li D, Xing Z - Sci Rep (2015)

Bottom Line: Viral nonstructural protein NSs was inhibitory to the induction of IFN-β, but interestingly, NFκB activation was enhanced in the presence of NSs.Therefore, NSs plays dual roles in the suppression of antiviral IFN-β induction as well as the promotion of proinflammatory responses.Our findings provide the first evidence for elucidating host responses and regulation in liver epithelial cells infected by an emerging bunyavirus.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology and Medical School, Nanjing University, Nanjing, China.

ABSTRACT
Infection in humans by severe fever with thrombocytopenia syndrome virus (SFTSV), a novel bunyavirus transmitted by ticks, is often associated with pronounced liver damage, especially in fatal cases. Little has been known, however, about how liver cells respond to SFTSV and how the response is regulated. In this study we report that proinflammatory cytokines were induced in liver tissues of C57/BL6 mice infected with SFTSV, which may cause tissue necrosis in mice. Human liver epithelial cells were susceptible to SFTSV and antiviral interferon (IFN) and IFN-inducible proteins were induced upon infection. We observed that infection of liver epithelial cells led to significant increases in proinflammatory cytokines and chemokines, including IL-6, RANTES, IP-10, and MIP-3a, which were regulated by NFκB signaling, and the activation of NFκB signaling during infection promoted viral replication in liver epithelial cells. Viral nonstructural protein NSs was inhibitory to the induction of IFN-β, but interestingly, NFκB activation was enhanced in the presence of NSs. Therefore, NSs plays dual roles in the suppression of antiviral IFN-β induction as well as the promotion of proinflammatory responses. Our findings provide the first evidence for elucidating host responses and regulation in liver epithelial cells infected by an emerging bunyavirus.

No MeSH data available.


Related in: MedlinePlus

Human liver epithelial cells (HepG2) were susceptible to SFTSV infection.HepG2 cells were mock-infected or infected with SFTSV at an m.o.i of 1. (A) Significant CPE was observed in infected cells (magnification 400×) at 48 and 72 hrs p.i. (B) Replicative curve of SFTSV in infected HepG2 cells. Culture media of the infected HepG2 cells were taken at various time points p.i., ten-fold serially diluted, and inoculated in Vero cells in 96-well plates. Infectious virus titers were determined after the cells were fixed and stained with an HRP-conjugated anti-viral NP antibody, followed by colorimetric development with TMB substrate. Infection foci were counted and TCID50 calculated based on the Reed and Muench method. (C) Quantitation of viral RNA in infected HepG2 cells. Total RNA was prepared from uninfected and infected cells at 6, 12, 24, 36, 48, and 72 hrs p.i. and viral RNA were qualified with real-time RT-PCR after reverse transcription. Specific primers for the S gene were used to quantify copy numbers of the S gene. The experiments were repeated at least three times and the data from one representative with two repeats were presented (* p < 0.05). (D) SFTSV antigens detected in infected HepG2 cells. Both infected and control cells were fixed at 10 or 20 hrs p.i. After permeablization with PFA, the cells were incubated with rabbit anti-NSs antibody at a dilution of 1:100, followed by staining with Alex Fluro488-conjugated secondary antibody.
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f2: Human liver epithelial cells (HepG2) were susceptible to SFTSV infection.HepG2 cells were mock-infected or infected with SFTSV at an m.o.i of 1. (A) Significant CPE was observed in infected cells (magnification 400×) at 48 and 72 hrs p.i. (B) Replicative curve of SFTSV in infected HepG2 cells. Culture media of the infected HepG2 cells were taken at various time points p.i., ten-fold serially diluted, and inoculated in Vero cells in 96-well plates. Infectious virus titers were determined after the cells were fixed and stained with an HRP-conjugated anti-viral NP antibody, followed by colorimetric development with TMB substrate. Infection foci were counted and TCID50 calculated based on the Reed and Muench method. (C) Quantitation of viral RNA in infected HepG2 cells. Total RNA was prepared from uninfected and infected cells at 6, 12, 24, 36, 48, and 72 hrs p.i. and viral RNA were qualified with real-time RT-PCR after reverse transcription. Specific primers for the S gene were used to quantify copy numbers of the S gene. The experiments were repeated at least three times and the data from one representative with two repeats were presented (* p < 0.05). (D) SFTSV antigens detected in infected HepG2 cells. Both infected and control cells were fixed at 10 or 20 hrs p.i. After permeablization with PFA, the cells were incubated with rabbit anti-NSs antibody at a dilution of 1:100, followed by staining with Alex Fluro488-conjugated secondary antibody.

Mentions: To determine how liver cells develop pathogenicity in response to SFTSV, we inoculated human liver epithelial cultures using the human hepatocellular carcinoma cell lines, HepG2 and MHCC-LM3, with SFTSV at an m.o.i. of 1. Typical cytopathic effects (CPEs) appeared at 48 hr and the cell monolayers deteriorated 72 hr p.i. in HepG2 cells (Fig. 2A), demonstrating that SFTSV is able to infect and replicate in human liver epithelial cells. Similar results with cell death were observed with MHCC-LM3 cells (data not shown). We also examined the viral replicative curves as exhibited in Fig. 2B, indicating that SFTSV replicates efficiently in HepG2 with an infectious dose at m.o.i. of either 1 or 10, reaching the same level of about 105.5TCID50/ml in culture media at 72 hr p.i. To further confirm efficient viral replication and infection, we extracted total RNA from the cells at various time points p.i. and measured viral S gene copy numbers by realtime RT-PCR. As early as 12 hr p.i., we detected the viral S gene copies at a level of 5.2 × 102, and copy numbers increased to 2.1 × 104 and 5.5 × 104 at 24 and 48 hr p.i., respectively (Fig. 2C).


Host Responses and Regulation by NFκB Signaling in the Liver and Liver Epithelial Cells Infected with A Novel Tick-borne Bunyavirus.

Sun Q, Jin C, Zhu L, Liang M, Li C, Cardona CJ, Li D, Xing Z - Sci Rep (2015)

Human liver epithelial cells (HepG2) were susceptible to SFTSV infection.HepG2 cells were mock-infected or infected with SFTSV at an m.o.i of 1. (A) Significant CPE was observed in infected cells (magnification 400×) at 48 and 72 hrs p.i. (B) Replicative curve of SFTSV in infected HepG2 cells. Culture media of the infected HepG2 cells were taken at various time points p.i., ten-fold serially diluted, and inoculated in Vero cells in 96-well plates. Infectious virus titers were determined after the cells were fixed and stained with an HRP-conjugated anti-viral NP antibody, followed by colorimetric development with TMB substrate. Infection foci were counted and TCID50 calculated based on the Reed and Muench method. (C) Quantitation of viral RNA in infected HepG2 cells. Total RNA was prepared from uninfected and infected cells at 6, 12, 24, 36, 48, and 72 hrs p.i. and viral RNA were qualified with real-time RT-PCR after reverse transcription. Specific primers for the S gene were used to quantify copy numbers of the S gene. The experiments were repeated at least three times and the data from one representative with two repeats were presented (* p < 0.05). (D) SFTSV antigens detected in infected HepG2 cells. Both infected and control cells were fixed at 10 or 20 hrs p.i. After permeablization with PFA, the cells were incubated with rabbit anti-NSs antibody at a dilution of 1:100, followed by staining with Alex Fluro488-conjugated secondary antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4488873&req=5

f2: Human liver epithelial cells (HepG2) were susceptible to SFTSV infection.HepG2 cells were mock-infected or infected with SFTSV at an m.o.i of 1. (A) Significant CPE was observed in infected cells (magnification 400×) at 48 and 72 hrs p.i. (B) Replicative curve of SFTSV in infected HepG2 cells. Culture media of the infected HepG2 cells were taken at various time points p.i., ten-fold serially diluted, and inoculated in Vero cells in 96-well plates. Infectious virus titers were determined after the cells were fixed and stained with an HRP-conjugated anti-viral NP antibody, followed by colorimetric development with TMB substrate. Infection foci were counted and TCID50 calculated based on the Reed and Muench method. (C) Quantitation of viral RNA in infected HepG2 cells. Total RNA was prepared from uninfected and infected cells at 6, 12, 24, 36, 48, and 72 hrs p.i. and viral RNA were qualified with real-time RT-PCR after reverse transcription. Specific primers for the S gene were used to quantify copy numbers of the S gene. The experiments were repeated at least three times and the data from one representative with two repeats were presented (* p < 0.05). (D) SFTSV antigens detected in infected HepG2 cells. Both infected and control cells were fixed at 10 or 20 hrs p.i. After permeablization with PFA, the cells were incubated with rabbit anti-NSs antibody at a dilution of 1:100, followed by staining with Alex Fluro488-conjugated secondary antibody.
Mentions: To determine how liver cells develop pathogenicity in response to SFTSV, we inoculated human liver epithelial cultures using the human hepatocellular carcinoma cell lines, HepG2 and MHCC-LM3, with SFTSV at an m.o.i. of 1. Typical cytopathic effects (CPEs) appeared at 48 hr and the cell monolayers deteriorated 72 hr p.i. in HepG2 cells (Fig. 2A), demonstrating that SFTSV is able to infect and replicate in human liver epithelial cells. Similar results with cell death were observed with MHCC-LM3 cells (data not shown). We also examined the viral replicative curves as exhibited in Fig. 2B, indicating that SFTSV replicates efficiently in HepG2 with an infectious dose at m.o.i. of either 1 or 10, reaching the same level of about 105.5TCID50/ml in culture media at 72 hr p.i. To further confirm efficient viral replication and infection, we extracted total RNA from the cells at various time points p.i. and measured viral S gene copy numbers by realtime RT-PCR. As early as 12 hr p.i., we detected the viral S gene copies at a level of 5.2 × 102, and copy numbers increased to 2.1 × 104 and 5.5 × 104 at 24 and 48 hr p.i., respectively (Fig. 2C).

Bottom Line: Viral nonstructural protein NSs was inhibitory to the induction of IFN-β, but interestingly, NFκB activation was enhanced in the presence of NSs.Therefore, NSs plays dual roles in the suppression of antiviral IFN-β induction as well as the promotion of proinflammatory responses.Our findings provide the first evidence for elucidating host responses and regulation in liver epithelial cells infected by an emerging bunyavirus.

View Article: PubMed Central - PubMed

Affiliation: The State Key Laboratory of Pharmaceutical Biotechnology and Medical School, Nanjing University, Nanjing, China.

ABSTRACT
Infection in humans by severe fever with thrombocytopenia syndrome virus (SFTSV), a novel bunyavirus transmitted by ticks, is often associated with pronounced liver damage, especially in fatal cases. Little has been known, however, about how liver cells respond to SFTSV and how the response is regulated. In this study we report that proinflammatory cytokines were induced in liver tissues of C57/BL6 mice infected with SFTSV, which may cause tissue necrosis in mice. Human liver epithelial cells were susceptible to SFTSV and antiviral interferon (IFN) and IFN-inducible proteins were induced upon infection. We observed that infection of liver epithelial cells led to significant increases in proinflammatory cytokines and chemokines, including IL-6, RANTES, IP-10, and MIP-3a, which were regulated by NFκB signaling, and the activation of NFκB signaling during infection promoted viral replication in liver epithelial cells. Viral nonstructural protein NSs was inhibitory to the induction of IFN-β, but interestingly, NFκB activation was enhanced in the presence of NSs. Therefore, NSs plays dual roles in the suppression of antiviral IFN-β induction as well as the promotion of proinflammatory responses. Our findings provide the first evidence for elucidating host responses and regulation in liver epithelial cells infected by an emerging bunyavirus.

No MeSH data available.


Related in: MedlinePlus