Innate Host Response in Primary Human Hepatocytes with Hepatitis C Virus Infection

The interaction between hepatitis C virus (HCV) and innate antiviral defense systems in primary human hepatocytes is not well understood. The objective of this study is to examine how primary human hepatocytes response to HCV infection.An infectious HCV isolate JFH1 was used to infect isolated primary human hepatocytes. HCV RNA or NS5A protein in the cells was detected by real-time PCR or immunofluorescence staining respectively. Apoptosis was examined with flow cytometry. Mechanisms of HCV-induced IFN-β expression and apoptosis were determined.Primary human hepatocytes were susceptible to JFH1 virus and released infectious virus. IFN-α inhibited viral RNA replication in the cells. IFN-β and interferon-stimulated genes were induced in the cells during acute infection. HCV infection induced apoptosis of primary human hepatocytes through the TRAIL-mediated pathway. Silencing RIG-I expression in primary human hepatocytes inhibited IFN-β and TRAIL expression and blocked apoptosis of the cells, which facilitated viral RNA replication in the cells. Moreover, HCV NS34A protein inhibited viral induced IFN-β expression in primary human hepatocytes.Innate host response is intact in HCV-infected primary human hepatocytes. RIG-I plays a key role in the induction of IFN and TRAIL by viruses and apoptosis of primary human hepatocytes via activation of the TRAIL-mediated pathway. HCV NS34A protein appears to be capable of disrupting the innate antiviral host responses in primary human hepatocytes. Our study provides a novel mechanism by which primary human hepatocytes respond to natural HCV infection

Investigation of proton single-event transient in CMOS image sensor

With the increasingly widespread application of CMOS image sensors (CIS) in radiation environments, such as aerospace, their radiation effects have gained attention. This paper investigates single-event transient (SET) caused by the proton direct ionization on CIS, combining both experimental and simulation methods. The proton beam energy used in the experiment is 12 MeV, with a flux up to 3.5 × 108 p/(cm2 s). Due to the periodicity of the proton beam, the CIS output displays a phenomenon of alternating brightness and darkness. When the proton beam flux is low, numerous SET bright spots with different outputs are observed. To comprehensively analyze these experimental phenomena, a typical three-dimensional 4T pinned photodiode model is constructed in TCAD, and relevant SET simulation is carried out. The results indicate that incident position, incident time, and the number of incident protons significantly affect the output of SET-generated bright spots, which are key factors contributing to the different bright spots observed in the experiment

HCV induces apoptosis of PHH through TRAIL-mediated pathway.

<p>(<b>A</b>) HCV induces apoptosis of PHHs. Three different PHHs (Hu507, Hu606 and Hu831) from three donors were infected by JFH1 virus at MOI of 0.1. The cells were harvested at day 5 after infection and subjected to Annexin V analysis determined by Flow cytometry. The data represent the means of 3 different PHHs from 3 different donors. (<b>B</b>) Blocking viral entry by anti-CD81 antibody or suppression of HCV replication by IFN reduces apoptosis of PHHs. Three PHH (Hu507, Hu606 and Hu831) were treated with 100IU/mL or anti-CD81 two hours before viral infection. The cells were harvested at day 5 post-infection for Annexin V expression determined by Flow cytometry analysis. The data represent the means of 3 different PHHs from 3 different donors. (<b>C</b>) Kinetics of apoptosis in HCV-infected PHHs. Three different PHHs (Hu507, Hu606 and Hu831) from three donors were infected by JFH1 virus at MOI of 0.1. The cells were harvested every 24 hours and subjected to Annexin V analysis determined by Flow cytometry. The data represent the means of 3 different PHHs from 3 different donors. (<b>D</b>) HCV induces the expression of TRAIL, DR4 and DR5 in PHHs. Three different PHHs were infected by JFH1 virus and total RNA was purified from the cells at different time points. The expression of TRAIL, DR4 or DR5 was examined by real-time PCR analysis. The data were normalized with GAPDH and represent the means of 3 different PHHs (Hu507, Hu606 and Hu831) from 3 different donors. (<b>E</b>) TRAIL-specific siRNA knockdown HCV-induced TRAIL expression in PHH. TRAIL siRNA or control siRNA were delivered into PHH infected by HCV. The expression of TRAIL was examined using real-time PCR. The data were normalized with internal control GAPDH and represented the means of 3 different PHHs (Hu507, Hu606 and Hu831) from 3 different donors. The image showed the effectiveness of TRAIL-specific siRNA on knocking down TRAIL mRNA in HCV-infected PHHs, as determined by RT-PCR analysis using TRAIL-specific primer. (<b>F</b>) TRAIL knockdown with TRAIL-specific siRNA reduces HCV-induced apoptosis of PHHs. TRAIL siRNA or control siRNA was delivered into PHHs followed by HCV infection for 5 days. Apoptosis of PHHs was examined using flow cytometry. The data represent the means of 3 different PHHs (Hu507, Hu606 and Hu831) from 3 different donors.</p

HCV NS34A inhibits IFN-β expression in viral-infected PHH.

<p>(<b>A</b>) The plasmid pTOPO-NS34A or control plasmid pTOPO was transfected into PHH, followed by HCV infection for 3 days. Total RNA was isolated and IFN-β level was detected using real-time PCR analysis. The expression level of IFN-β RNA was normalized with GAPDH. The data represent the means of 2 different PHHs (Hu507 and Hu593) from 2 different donors. (<b>B</b>) Overexpression of HCV NS34A in PHH. pTOPO or pTOPO-NS34A plasmid was transfected into PHHs respectively. Protein was isolated from the cells at day 2 after transfection and used for western blot analysis with anti- HCV NS34A antibody. (<b>C</b>) HCV NS34A inhibits the induction of IFN-β by NDV. PHH were transfected by plasmid pTOPO-NS34A or pTOPO, followed by NDV infection at MOI of 0.1. Total cellular RNA was isolated at 48 hours after infection and the level of IFN-β was determined with real-time RT-PCR analysis. The level of IFN-β RNA was normalized with GAPDH. The data represent the means of 3 different PHHs (Hu441, Hu507 and Hu593) from 3 different donors.</p

HCV infects primary human hepatocytes and induces IFN-β and ISGs in the cells.

<p>(<b>A</b>) Filtered supernatant from JFH1 RNA-transfected Huh7.5 cells was inoculated with naive Huh7.5 cells. Cells were immunostained with mouse monoclonal anti-NS5A antibody at day 5 after inoculation. DAPI was used for nuclear counterstaining. (<b>B</b>) Filtered supernatant from JFH1/GND RNA-transfected Huh7.5 cells was inoculated with naive Huh7.5 cells. Cells were stained at day 5 after inoculation as described in part A. (<b>C</b>) Filtered supernatant from JFH1 RNA-transfected Huh7.5 cells was inoculated with primary human hepatocytes Hu441. Cells were stained at day 5 after inoculation as described in part A. (<b>D</b>) Filtered supernatant from JFH1/GND RNA-transfected Huh7.5 cells was inoculated with primary human hepatocytes Hu441. At day 5 after inoculation, cells were stained as described in part A. (<b>E/F</b>) Naïve Huh7.5 cells were incubated for 3 days with filtered, conditioned media from Hu441 cells preinfection with supernatant from JFH1 RNA-transfected Huh7.5 cells (E) or Hu441 pre-inoculated with supernatant from JFH1/GND RNA-transfected Huh7.5 (F) and immunostained for NS5A expression. (<b>G</b>) Viral RNA kinetics determined by real-time PCR analysis in ten different PHH preparations and Huh7.5 cells infected by JFH1 virus. The HCV RNA in PHH was determined by quantitative real-time PCR analysis. The viral replication is represented by HCV genome equivalence (GE)/µg total RNA of HCV-infected PHH. The ten PHH preparations are primary human hepatocytes Hu401 (from 31-year-old female with liver mass in left lobe), Hu414 (from 65-year-old male with liver mass), Hu441 (from 65-year-old male with metastasis of a colic tumor), Hu583 (from 40-year-old female with liver adenoma), Hu0586 (from 69-year-old female with metastatic endometrial uterine cancer), Hu606 (from 44-year-old female with hemangioma), Hu749 (from 79-year-old female with metastatic ovarian), Hu750 (from 39-year-old female with hemangioma), Hu786 (from 70-year-old male with hepatocellular carcinoma), Hu831 (from 41-year-old male with hepatocellular carcinoma). (<b>H</b>) IFN inhibited HCV RNA replication in PHH in a dose-dependent manner. The data represent the relative RNA levels after 2 days of IFN-α treatment. These results are representative of observations made with PHH cultures from three different donors (PHHs are Hu750, Hu786 and Hu787 from 48-year-old female with metastasis of a colic tumor). (<b>I</b>) Anti-CD81 antibody blocked HCV infection in PHH. Primary human hepatocytes were pretreated with anti-CD81 antibody for 2 hours before viral inoculation. Viral RNA was analyzed by real-time PCR at day 3 post-infection. Data are means from three independent assays. These results are representative of observations made with PHH cultures from three different donors (PHH are Hu750, Hu786 and Hu787). (<b>J/K</b>) Kinetics of IFN-β and G1P3 in viral infected PHH. Three different PHH preparations (Hu583, Hu586 and Hu786) from three donors were infected with JFH1 virus at MOI of 0.1. Cells were harvested for total RNA extraction at different time points. The kinetics of induction of IFN-β (J) and G1P3 (K) was analyzed by real-time PCR assay. The data were normalized with internal control glyceraldehyde-3- phosphate dehydrogenase (GAPDH) and represent the means of 3 different PHHs from 3 different donors.</p

Silencing RIG-I expression in PHH inhibits the expression of IFN-β and reduces apoptosis of PHH in response to HCV infection.

<p>(<b>A</b>) RIG-I knockdown with RIG-I-specific siRNA inhibits IFN-β expression in PHH. RIG-I siRNA or control siRNA was delivered into PHH. The expression of IFN-β was examined using real-time PCR. The data were normalized with internal control GAPDH and represented the means of 3 different PHH (Hu507, Hu606 and Hu831) from 3 different donors. The image showed the effectiveness of RIG-I-specific siRNA on knocking down RIG-I mRNA in viral infected PHHs, as determined by RT-PCR analysis using RIG-I-specific primer. (<b>B</b>) RIG-I regulates TRAIL expression in HCV-infected PHH. PHHs were transfected with control siRNA or RIG-I-specific siRNA for 24 hours, followed by viral infection. The cells were harvested for total cellular RNA extraction and the expression of TRAIL in the cells was examined using real-time RT-PCR. (<b>C</b>) RIG-I knockdown with RIG-I-specific siRNA reduces HCV-induced apoptosis of PHH. RIG-I siRNA or control siRNA was delivered into PHH followed by HCV infection for 5 days. Apoptosis of PHH was examined using flow cytometry. The data represent the means of 3 different PHHs (Hu507, Hu606 and Hu831) from 3 different donors. (<b>D</b>) Relative HCV RNA level in PHH transfected with RIG-I siRNA. PHH were transfected with RIG-I or control siRNA, followed by viral infection for 72 hours. Total RNA was isolated and HCV RNA was detected using real-time PCR. The data was normalized with GAPDH and represented the means of 3 different PHHs (Hu507, Hu606 and Hu831) from 3 different donors. (<b>E</b>) Immunostaining of HCV NS5A protein in PHH with RIG-I downregulation. PHHs were transfected with RIG-I or control siRNA, followed by viral infection for 72 hours. The cells were immunostained with mouse monoclonal anti-NS5A antibody. DAPI was used for nuclear counterstaining.</p