Extracellular vesicles activate ATM-Chk2 signaling pathway through the intercellular transfer of mitochondrial DNA in HBV-infected human hepatocytes.

Hepatitis B virus (HBV) is a human hepatotropic pathogen causing hepatocellular carcinoma. We recently obtained HBV-susceptible immortalized human hepatocyte NKNT-3 by exogenously expressing NTCP and its derived cell clones, #28.3.8 and #28.3.25.13 exhibiting different levels of HBV susceptibility. In the present study, we showed that HBV infection activated the ATM-Chk2 signaling pathway in #28.3.25.13 cells but not in #28.3.8 cells. Both the cell culture supernatant and extracellular vesicles (EVs) derived from HBV-infected #28.3.25.13 cells also activated the ATM-Chk2 signaling pathway in naïve #28.3.25.13 cells. Interestingly, EVs derived from HBV-infected #28.3.25.13 cells included higher level of mitochondrial DNA (mtDNA) than those from HBV-infected #28.3.8 cells. Based on our results, we propose the novel model that EVs mediate the activation of ATM-Chk2 signaling pathway by the intercellular transfer of mtDNA in HBV-infected human hepatocyte.

Penetration factor and indoor deposition rate of elementary and particulate iodine in a Japanese house for assessing the effectiveness of sheltering for radiation exposures.

Sheltering is one of the countermeasures for protection against radiation exposures in nuclear accidents. The effectiveness of sheltering is often expressed by the reduction factor, that is the ratio of the indoor to the outdoor cumulative radioactivity concentrations or doses. The indoor concentration is mainly controlled by the air exchange rate, penetration factor, and indoor deposition rate. The penetration factor and indoor deposition rate depend on the surface and the materials and structure of windows and doors as it is these openings in the building envelope that control penetration. We investigated experimentally these parameters of I2and particles. The experiment was performed in two apartment houses, three single-family houses, and chambers. The obtained penetration factor ranged from 0.3 to 1 for particles of 0.3-1µm and 0.15-0.7 for I2depending on the air exchange rate. The indoor deposition rate for a house room ranged from 0.007 to 0.2 h-1for particles of 0.3-1µm and 0.2-1.5 h-1for I2.

Identification of ribavirin-responsive cis-elements for GPAM suppression in the GPAM genome.

Glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) is a rate-limiting enzyme catalyzing triglyceride synthesis. Recently, we demonstrated that the anti-viral drug ribavirin (RBV) reduces GPAM expression by downregulating CCAAT/enhancer-binding protein α (C/EBPα). However, the precise mechanisms of GPAM suppression have remained unclear. Here, we found that RBV suppressed GPAM expression by downregulating not only C/EBPα, but also sterol regulatory element-binding protein-1c (SREBP-1c). We also found that cis-elements regulated by C/EBPα and SREBP-1c functioned as distal and proximal enhancers, respectively, to express hepatocyte- and adipocytes-specific GPAM variants. These results imply that RBV disrupts formation of the enhancer machineries on the GPAM genome by downregulating both transcription factors. Our findings may contribute to the development of treatments for fatty liver diseases caused by aberrant triglyceride synthesis.

Study of multiple genetic variations caused by persistent hepatitis C virus replication in long-term cell culture.

The most characteristic feature of the hepatitis C virus (HCV) genome in patients with chronic hepatitis C is its remarkable variability and diversity. To better understand this feature, we performed genetic analysis of HCV replicons recovered from two human hepatoma HuH-7-derived cell lines after 1, 3, 5, 7, and 9 years in culture: The cell lines 50-1 and sO harbored HCV 1B-1 and O strain-derived HCV replicons established in 2002 and 2003, respectively. The results revealed that genetic variations in both replicons accumulated in a time-dependent manner at a constant rate despite the maintenance of moderate diversity (less than 1.8% difference) between the clones and that the mutation rate in the 50-1 and sO replicons was 2.5 and 2.9 × 10-3 base substitutions/site/year, respectively. We found that the genetic distance of both replicons increased from 7.9% to 10.5% after 9 years in culture. In addition, we observed that the guanine + cytosine (GC) content of both replicon RNAs increased in a time-dependent manner, as observed in our previous studies. Finally, we demonstrated that the high sensitivity of both replicons to direct-acting antivirals was maintained even after 9 years in culture. Our results suggest that long-term cultured HCV replicon-harboring cells are a useful model for understanding the variability and diversity of the HCV genome and the drug sensitivity of HCV in patients with chronic hepatitis C.

Ribavirin-induced down-regulation of CCAAT/enhancer-binding protein α leads to suppression of lipogenesis.

Recently, we demonstrated that the anti-viral drug ribavirin (RBV) had the ability to suppress lipogenesis through down-regulation of retinoid X receptor α (RXRα) under the control of the intracellular GTP-level and AMP-activated protein kinase-related kinases, especially microtubule affinity regulating kinase 4 (MARK4). RXRα-overexpression attenuated but did not abolish lipogenesis suppression by RBV, implying that additional factor(s) were involved in this suppressive effect. In the present study, we found that the protein level, but not the mRNA level, of CCAAT/enhancer-binding protein α (C/EBPα) was down-regulated by RBV in hepatic cells. Treatment with proteasome inhibitor attenuated RBV-induced down-regulation of C/EBPα, suggesting that RBV promoted degradation of C/EBPα protein via the ubiquitin-proteasome pathway. Depletion of intracellular GTP through inosine monophosphate dehydrogenase inhibition by RBV led to down-regulation of C/EBPα. In contrast, down-regulation of C/EBPα by RBV was independent of RXRα and MARK4. Knockdown of C/EBPα reduced the intracellular neutral lipid levels and the expression of genes related to the triglyceride (TG) synthesis pathway, especially glycerol-3-phosphate acyltransferase, mitochondrial (GPAM), which encodes the first rate-limiting TG enzyme. Overexpression of C/EBPα yielded the opposite results. We also observed that RBV decreased GPAM expression. Moreover, overexpression of GPAM attenuated RBV-induced reduction in the intracellular neutral lipid levels. These data suggest that down-regulation of C/EBPα by RBV leads to the reduction in GPAM expression, which contributes to the suppression of lipogenesis. Our findings about the mechanism of RBV action in lipogenesis suppression will provide new insights for therapy against the active lipogenesis involved in hepatic steatosis and hepatocellular carcinomas.

Silent brain infarction after minimally invasive cardiac surgery with retrograde perfusion.

BACKGROUND AND AIM: There is no report on silent brain infarction (SBI) after minimally invasive cardiac surgery (MICS) with retrograde perfusion. Thus, the current study aimed to investigate the incidence of SBI after MICS using magnetic resonance imaging (MRI). METHODS: This study included 174 patients who underwent MICS with retrograde perfusion between July 2014 and July 2018. Preoperative computed tomography (CT) angiography was routinely performed and vascular pathology was evaluated for patient selection. Postoperative MRI was performed to investigate the occurrence of SBI. RESULTS: Out of the total 174 patients, 26 (14.9%) presented with SBI. A total of 61 SBI lesions were found in the 26 patients; of these, 34 (56%) SBI lesions were in the right hemisphere and 27 (44%) in the left hemisphere. SBIs were primarily observed in the posterior cerebral artery territory. Multivariate analysis revealed aortic stenosis to be the only risk factor of SBI. CONCLUSIONS: Retrograde perfusion via femoral cannulation may not increase the incidence of SBI in selected MICS patients based on preoperative CT findings.

A new hepatoma cell line exhibiting high susceptibility to hepatitis B virus infection.

Hepatitis B virus (HBV) infection, which increases the risk of cirrhosis and hepatocellular carcinoma and requires lifelong treatment, has become a major global health problem. However, host factors essential to the HBV life cycle are still unclear, and the development of new drugs is needed. Cells derived from the human hepatoma cell line HepG2 and engineered to overexpress sodium taurocholate cotransporting polypeptide (NTCP: a receptor for HBV), termed HepG2/NTCP cells, are widely used as the cell-based HBV infection and replication systems for HBV research. We recently found that human hepatoma cell line Li23-derived cells overexpressing NTCP (A8 cells subcloned from Li23 cells), whose gene expression profile was distinct from that of HepG2/NTCP cells, were also sensitive to HBV infection. However, the HBV susceptibility of A8 cells was around 1/100 that of HepG2/NTCP cells. Since we considered that plural cell assay systems will be needed for the objective evaluation of anti-HBV reagents, as we previously demonstrated in hepatitis C virus research, we here attempted to develop a new Li23 cell-derived assay system equivalent to that using HepG2/NTCP cells. By repeated subcloning of A8 cells, we successfully established a new cell line (A8.15.78.10) exhibiting high HBV susceptibility equal to that of HepG2/NTCP cells. Characterization of A8.15.78.10 cells revealed that the increase of HBV susceptibility was correlated with increases in the protein and glycosylation levels of NTCP, and with decreased expression of STING, a factor contributing to innate immunity. Finally, we performed a comparative evaluation of HBV entry inhibitors (cyclosporin A and rosiglitazone) by an HBV/secNL reporter assay using A8.15.78.10 cells or HepG2/NTCP cells. The results confirmed that cyclosporin A exhibited anti-HBV activity in both cell lines, as previously reported. However, we found that rosiglitazone did not show the anti-HBV activity in A8.15.78.10 cells, although it worked in HepG2/NTCP cells as previously reported. This suggested that the difference in anti-HBV activity between cyclosporin A and rosiglitazone was due to the different types of cells used for the assay. In conclusion, plural assay systems using different types of cells are required for the objective and impartial evaluation of anti-HBV reagents.

Daunorubicin, a topoisomerase II poison, suppresses viral production of hepatitis B virus by inducing cGAS-dependent innate immune response.

Hepatitis B virus (HBV) causes hepatic diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. These diseases are closely associated with persistent HBV infection. To prevent the progression of hepatic diseases, it is thus important to suppress persistent HBV infection. Daunorubicin (DNR), a topoisomerase II (Top II) poison, is a clinically used anticancer agent with a wide spectrum of activity against malignancies. DNR was recently reported to cause DNA damage-dependent interferon (IFN)-ß induction through exogenous cyclic GMP-AMP synthetase (cGAS) and subsequently to suppress Ebola virus replication. In the present study, we demonstrated that DNR caused the inhibition of cell proliferation, but not cell death, through the DNA damage response in immortalized human hepatocyte NKNT-3/NTCP cells. Interestingly, DNR triggered the endogenous cGAS-dependent innate immune response and subsequently suppressed viral production of HBV in NKNT-3/NTCP cells. Top II poisons may be anti-HBV drug candidates.

Dual effects of the Nrf2 inhibitor for inhibition of hepatitis C virus and hepatic cancer cells.

BACKGROUND: We previously showed that knockdown of nuclear factor E2-related factor 2 (Nrf2) resulted in suppression of hepatitis C virus (HCV) infection. In this study, whether brusatol, an Nrf2 inhibitor, has dual anti-HCV and anticancer effects was explored. METHODS: The anti-HCV effect of brusatol was investigated by analyzing HCV RNA and proteins in a hepatic cell line persistently-infected with HCV, HPI cells, and by analyzing HCV replication in a replicon-replicating hepatic cell line, OR6 cells. Then, dual anti-HCV and anticancer effects of brusatol and enhancement of the effects by the combination of brusatol with anticancer drugs including sorafenib, which has been reported to have the dual effects, were then investigated. RESULTS: Brusatol suppressed the persistent HCV infection at both the RNA and protein levels in association with a reduction in Nrf2 protein in the HPI cells. Analysis of the OR6 cells treated with brusatol indicated that brusatol inhibited HCV persistence by inhibiting HCV replication. Combination of brusatol with an anticancer drug not only enhanced the anticancer effect but also, in the case of the combination with sorafenib, strongly suppressed HCV infection. CONCLUSIONS: Brusatol has dual anti-HCV and anticancer effects and can enhance the comparable effects of sorafenib. There is therefore the potential for combination therapy of brusatol and sorafenib for HCV-related hepatocellular carcinoma.

IPS-1 is essential for type III IFN production by hepatocytes and dendritic cells in response to hepatitis C virus infection.

Hepatitis C virus (HCV) is a major cause of liver disease. The innate immune system is essential for controlling HCV replication, and HCV is recognized by RIG-I and TLR3, which evoke innate immune responses through IPS-1 and TICAM-1 adaptor molecules, respectively. IL-28B is a type III IFN, and genetic polymorphisms upstream of its gene are strongly associated with the efficacy of polyethylene glycol-IFN and ribavirin therapy. As seen with type I IFNs, type III IFNs induce antiviral responses to HCV. Recent studies established the essential role of TLR3-TICAM-1 pathway in type III IFN production in response to HCV infection. Contrary to previous studies, we revealed an essential role of IPS-1 in type III IFN production in response to HCV. First, using IPS-1 knockout mice, we revealed that IPS-1 was essential for type III IFN production by mouse hepatocytes and CD8(+) dendritic cells (DCs) in response to cytoplasmic HCV RNA. Second, we demonstrated that type III IFN induced RIG-I but not TLR3 expression in CD8(+) DCs and augmented type III IFN production in response to cytoplasmic HCV RNA. Moreover, we showed that type III IFN induced cytoplasmic antiviral protein expression in DCs and hepatocytes but failed to promote DC-mediated NK cell activation or cross-priming. Our study indicated that IPS-1-dependent pathway plays a crucial role in type III IFN production by CD8(+) DCs and hepatocytes in response to HCV, leading to cytoplasmic antiviral protein expressions.

Rab13 Is Involved in the Entry Step of Hepatitis C Virus Infection.

Membrane transport probably participates in the lifecycle of hepatitis C virus (HCV). Rab proteins are essential host factors for HCV RNA replication, but these proteins' roles in other steps of the HCV lifecycle are not clear. The tight junction (TJ) plays a key role in HCV infection. Rab13 regulates the endocytic recycling of the TJ-associated proteins. Here we investigated whether Rab13 is involved in the HCV entry step. We used HuH-7-derived RSc cells and Li23-derived D7 cells. To evaluate the effect of Rab13 in HCV infection, we transfected the cells with siRNA targeting Rab13 before HCV infection. The down-regulation of Rab13 inhibited HCV infection. The D7 cells had showed a greater inhibitory effect against HCV infection compared to that in the RSc cells by Rab13 knockdown. Next, to evaluate the effect of Rab13 after infection, we inoculated the cells with HCV before transfection of the siRNA. The down-regulation of Rab13 did not show any effects after HCV infection. We further examined whether Rab13 would influence HCV RNA replication by using HCV replicon-harboring cells. The results revealed that Rab13 did not affect the step of HCV RNA replication. These results suggest that Rab13 plays an important role in the step of HCV entry.

Molecular Mechanism Underlying the Suppression of CPB2 Expression Caused by Persistent Hepatitis C Virus RNA Replication.

The mechanisms of hepatitis C virus (HCV)-associated hepatocarcinogenesis and disease progression are unclear. We previously observed that the expression level of carboxypeptidase B2 (CPB2) gene was remarkably suppressed by persistent HCV RNA replication in human hepatoma cell line Li23- derived cells. The results of the present study demonstrated that the CPB2 expression in patients with chronic hepatitis C was inversely correlated with several risk factors of hepatic fibrosis or steatosis, although ectopic CPB2 expression did not suppress the expression of fibrogenic or lipogenic genes. The suppressed CPB2 expression was restored by treatment with 5-azacytidine. To clarify the mechanism underlying this phenomenon, we analyzed the CPB2 promoter, and the results revealed that (1) hepatocyte nuclear factor 1 (HNF1), especially HNF1α, was essential for the CPB2 promoter, and (2) CPB2 promoter was not methylated by persistent HCV RNA replication. The expression levels of HNF1α and HNF1ß were also not changed by persistent HCV RNA replication. These results suggest the existence of 5-azacytidine-inducible or -reducible unknown factor(s) that can control the CPB2 expression. To evaluate this idea we performed a microarray analysis, and several gene candidates corresponding to the suggested factor(s) were identified.

Class A scavenger receptor 1 (MSR1) restricts hepatitis C virus replication by mediating toll-like receptor 3 recognition of viral RNAs produced in neighboring cells.

Persistent infections with hepatitis C virus (HCV) may result in life-threatening liver disease, including cirrhosis and cancer, and impose an important burden on human health. Understanding how the virus is capable of achieving persistence in the majority of those infected is thus an important goal. Although HCV has evolved multiple mechanisms to disrupt and block cellular signaling pathways involved in the induction of interferon (IFN) responses, IFN-stimulated gene (ISG) expression is typically prominent in the HCV-infected liver. Here, we show that Toll-like receptor 3 (TLR3) expressed within uninfected hepatocytes is capable of sensing infection in adjacent cells, initiating a local antiviral response that partially restricts HCV replication. We demonstrate that this is dependent upon the expression of class A scavenger receptor type 1 (MSR1). MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. RNAi-mediated knockdown of MSR1 expression blocks TLR3 sensing of HCV in infected hepatocyte cultures, leading to increased cellular permissiveness to virus infection. Exogenous expression of Myc-MSR1 restores TLR3 signaling in MSR1-depleted cells with subsequent induction of an antiviral state. A series of conserved basic residues within the carboxy-terminus of the collagen superfamily domain of MSR1 are required for binding and transport of dsRNA, and likely facilitate acidification-dependent release of dsRNA at the site of TLR3 expression in the endosome. Our findings reveal MSR1 to be a critical component of a TLR3-mediated pattern recognition receptor response that exerts an antiviral state in both infected and uninfected hepatocytes, thereby limiting the impact of HCV proteins that disrupt IFN signaling in infected cells and restricting the spread of HCV within the liver.

Hepatitis C virus core protein suppresses mitophagy by interacting with parkin in the context of mitochondrial depolarization.

Hepatitis C virus (HCV) causes mitochondrial injury and oxidative stress, and impaired mitochondria are selectively eliminated through autophagy-dependent degradation (mitophagy). We investigated whether HCV affects mitophagy in terms of mitochondrial quality control. The effect of HCV on mitophagy was examined using HCV-Japanese fulminant hepatitis-1-infected cells and the uncoupling reagent carbonyl cyanide m-chlorophenylhydrazone as a mitophagy inducer. In addition, liver cells from transgenic mice expressing the HCV polyprotein and human hepatocyte chimeric mice were examined for mitophagy. Translocation of the E3 ubiquitin ligase Parkin to the mitochondria was impaired without a reduction of pentaerythritol tetranitrate-induced kinase 1 activity in the presence of HCV infection both in vitro and in vivo. Coimmunoprecipitation assays revealed that Parkin associated with the HCV core protein. Furthermore, a Yeast Two-Hybrid assay identified a specific interaction between the HCV core protein and an N-terminal Parkin fragment. Silencing Parkin suppressed HCV core protein expression, suggesting a functional role for the interaction between the HCV core protein and Parkin in HCV propagation. The suppressed Parkin translocation to the mitochondria inhibited mitochondrial ubiquitination, decreased the number of mitochondria sequestered in isolation membranes, and reduced autophagic degradation activity. Through a direct interaction with Parkin, the HCV core protein suppressed mitophagy by inhibiting Parkin translocation to the mitochondria. This inhibition may amplify and sustain HCV-induced mitochondrial injury.

Annexin A1 negatively regulates viral RNA replication of hepatitis C virus.

Persistent infection with hepatitis C virus (HCV) often causes chronic hepatitis, and then shows a high rate of progression to liver cirrhosis and hepatocellular carcinoma. To clarify the mechanism of the persistent HCV infection is considered to be important for the discovery of new target(s) for the development of anti-HCV strategies. In the present study, we found that the expression level of annexin A1 (ANXA1) in human hepatoma cell line Li23-derived D7 cells was remarkably lower than that in parental Li23 cells, whereas the susceptibility of D7 cells to HCV infection was much higher than that of Li23 cells. Therefore, we hypothesized that ANXA1 negatively regulates persistent HCV infection through the inhibition of viral RNA replication. The results revealed that HCV production was significantly inhibited without a concomitant reduction in the amount of lipid droplets in the D7 cells stably expressing exogenous ANXA1. Further, we demonstrated that ANXA1 negatively regulated the step of viral RNA replication rather than that of viral entry in human hepatocytes. These results suggest that ANXA1 would be a novel target for the development of anti-HCV strategies.

[Recognition mechanism of hepatitis C viral infection].

In the viral reproduction, hepatitis C virus(HCV) produces double-stranded RNA (dsRNA) as a replication intermediate. RIG-I(retinoic acid inducible protein I) recognizes the intracellular HCV dsRNA as a "non self" molecule, and triggers the induction of interferon (IFN)-ß and then numerous IFN-stimulated genes(ISGs). On the other hand, one of toll-like receptors, TLR3 also recognizes the extracellular HCV dsRNA, and subsequently triggers the induction of IFN-ß and ISGs. We recently reported class A scavenger receptor (MSR1) was required for TLR3-mediated recognition of the extracellular HCV dsRNA. In this review, we summarize current knowledge about RIG-I- and TLR3/MSR1-mediated recognition mechanisms of HCV infection.

Alternative endocytosis pathway for productive entry of hepatitis C virus.

Previous studies have shown that hepatitis C virus (HCV) enters human hepatic cells through interaction with a series of cellular receptors, followed by clathrin-mediated, pH-dependent endocytosis. Here, we investigated the mechanisms of HCV entry into multiple HCV-permissive human hepatocyte-derived cells using trans-complemented HCV particles (HCVtcp). Knockdown of CD81 and claudin-1, or treatment with bafilomycin A1, reduced infection in Huh-7 and Huh7.5.1 cells, suggesting that HCV entered both cell types via receptor-mediated, pH-dependent endocytosis. Interestingly, knockdown of the clathrin heavy chain or dynamin-2 (Dyn2), as well as expression of the dominant-negative form of Dyn2, reduced infection of Huh-7 cells with HCVtcp, whereas infectious entry of HCVtcp into Huh7.5.1 cells was not impaired. Infection of Huh7.5.1 cells with culture-derived HCV (HCVcc) via a clathrin-independent pathway was also observed. Knockdown of caveolin-1, ADP-ribosylation factor 6 (Arf6), flotillin, p21-activated kinase 1 (PAK1) and the PAK1 effector C-terminal binding protein 1 of E1A had no inhibitory effects on HCVtcp infection into Huh7.5.1 cells, thus suggesting that the infectious entry pathway of HCV into Huh7.5.1 cells was not caveolae-mediated, or Arf6- and flotillin-mediated endocytosis and macropinocytosis, but rather may have occurred via an undefined endocytic pathway. Further analysis revealed that HCV entry was clathrin- and dynamin-dependent in ORL8c and HepCD81/miR122 cells, but productive entry of HCV was clathrin- and dynamin-independent in Hep3B/miR122 cells. Collectively, these data indicated that HCV entered different target cells through different entry routes.

Anti-HCV activity of the Chinese medicinal fungus Cordyceps militaris.

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although the sustained virologic response rate in the treatment of genotype 1 using new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has been improved by more than 70%, several severe side effects such as skin rash/ageusia and advanced anemia have become a problem. Under these circumstances, a new type of anti-HCV oral drug with few side effects is needed. Our recently developed HCV drug assay systems, including the HuH-7 cell line-derived OR6 and AH1R, and the Li23 cell line-derived ORL8 and ORL11, allow genome-length HCV RNAs (several strains of genotype 1b) encoding renilla luciferase to replicate efficiently. Using these systems as anti-HCV candidates, we have identified numerous existing medicines that can be used against HCV with few side effects, such as statins and teprenon. To obtain additional anti-HCV candidates, we evaluated a number of oral health supplements, and found that the capsule but not the liquid form of Cordyceps militaris (CM) (Ascomycotinanorth, North Chinese caterpillar fungus), which is used as a Chinese herbal medicine, exhibited moderate anti-HCV activity. In combination with interferon-α or ribavirin, CM exhibited an additive inhibitory effect. Among the main components of CM, cordycepin, but not ergosterol, contributed to the anti-HCV activity of CM. In consideration of all these results, we suggest that CM would be useful as an oral anti-HCV agent in combination with interferon-α and/or ribavirin.

Sensitive and specific PCR systems for detection of both Chinese and Japanese severe fever with thrombocytopenia syndrome virus strains and prediction of patient survival based on viral load.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease with a high case fatality risk and is caused by the SFTS virus (SFTSV). A retrospective study conducted after the first identification of an SFTS patient in Japan revealed that SFTS is endemic to the region, and the virus exists indigenously in Japan. Since the nucleotide sequence of Japanese SFTSV strains contains considerable differences compared with that of Chinese strains, there is an urgent need to establish a sensitive and specific method capable of detecting the Chinese and Japanese strains of SFTSV. A conventional one-step reverse transcription-PCR (RT-PCR) (cvPCR) method and a quantitative one-step RT-PCR (qPCR) method were developed to detect the SFTSV genome. Both cvPCR and qPCR detected a Chinese SFTSV strain. Forty-one of 108 Japanese patients suspected of having SFTS showed a positive reaction by cvPCR. The results from the samples of 108 Japanese patients determined by the qPCR method were in almost complete agreement with those determined by cvPCR. The analyses of the viral copy number level in the patient blood samples at the acute phase determined by qPCR in association with the patient outcome confirmed that the SFTSV RNA load in the blood of the nonsurviving patients was significantly higher than that of the surviving patients. Therefore, the cvPCR and qPCR methods developed in this study can provide a powerful means for diagnosing SFTS. In addition, the detection of the SFTSV genome level by qPCR in the blood of the patients at the acute phase may serve as an indicator to predict the outcome of SFTS.

Adenosine kinase is a key determinant for the anti-HCV activity of ribavirin.

UNLABELLED: Ribavirin (RBV) is often used in conjunction with interferon-based therapy for patients with chronic hepatitis C. There is a drastic difference in the anti-hepatitis C virus (HCV) activity of RBV between the HuH-7-derived assay system, OR6, possessing the RBV-resistant phenotype (50% effective concentration [EC50 ]: >100 µM) and the recently discovered Li23-derived assay system, ORL8, possessing the RBV-sensitive phenotype (EC50 : 8 µM; clinically achievable concentration). This is because the anti-HCV activity of RBV was mediated by the inhibition of inosine monophosphate dehydrogenase in RBV-sensitive ORL8 cells harboring HCV RNA. By means of comparative analyses using RBV-resistant OR6 cells and RBV-sensitive ORL8 cells, we tried to identify host factor(s) determining the anti-HCV activity of RBV. We found that the expression of adenosine kinase (ADK) in ORL8 cells was significantly higher than that in RBV-resistant OR6 cells harboring HCV RNA. Ectopic ADK expression in OR6 cells converted them from an RBV-resistant to an RBV-sensitive phenotype, and inhibition of ADK abolished the activity of RBV. We showed that the differential ADK expression between ORL8 and OR6 cells was not the result of genetic polymorphisms in the ADK gene promoter region and was not mediated by a microRNA control mechanism. We found that the 5' untranslated region (UTR) of ADK messenger RNA in ORL8 cells was longer than that in OR6 cells, and that only a long 5' UTR possessed internal ribosome entry site (IRES) activity. Finally, we demonstrated that the long 5' UTR functioned as an IRES in primary human hepatocytes. CONCLUSION: These results indicate that ADK acts as a determinant for the activity of RBV and provide new insight into the molecular mechanism underlying differential drug sensitivity.