Microscopic Small Airway Abnormalities Identified in Early Idiopathic Pulmonary Fibrosis In Vivo Using Endobronchial Optical Coherence Tomography.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) affects subpleural lung, but is considered to spare small airways. Micro-CT studies demonstrated small airway reduction in end-stage IPF explanted lungs, raising questions about small airway involvement in early-stage disease. Endobronchial optical coherence tomography (EB-OCT) is a volumetric imaging modality that detects microscopic features from subpleural to proximal airways. We use EB-OCT to evaluate small airways in early IPF and control subjects in vivo. METHODS: EB-OCT was performed in 12 IPF and 5 control subjects (matched by age, sex, smoking-history, height, BMI). IPF subjects had early disease with mild restriction (FVC: 83.5% predicted), diagnosed per current guidelines and confirmed by surgical biopsy. EB-OCT volumetric imaging was acquired bronchoscopically in multiple, distinct, bilateral lung locations (total: 97 sites). IPF imaging sites were classified by severity into affected (all criteria for UIP present) and less affected (some but not all criteria for UIP present) sites. Bronchiole count and small airway stereology metrics were measured for each EB-OCT imaging site. RESULTS: Compared to control subjects (mean: 11.2 bronchioles/cm3; SD: 6.2), there was significant bronchiole reduction in IPF subjects (42% loss; mean: 6.5/cm3; SD: 3.4; p=0.0039), including in IPF affected (48% loss; mean: 5.8/cm3; SD: 2.8; p<0.00001) and IPF less affected (33% loss; mean: 7.5/cm3; SD: 4.1; p=0.024) sites. Stereology metrics showed IPF affected small airways were significantly larger and more distorted/irregular than in IPF less affected sites and control subjects. IPF less affected and control airways were statistically indistinguishable for all stereology parameters (p=0.36-1.0). CONCLUSION: EB-OCT demonstrated marked bronchiolar loss in early IPF (between 30 and 50%), even in areas minimally affected by disease, compared to matched controls. These findings support small airway disease as a feature of early IPF, providing novel insight into pathogenesis and potential therapeutic targets.

Balloon venoplasty for disdialysis syndrome due to pacemaker-related superior vena cava syndrome with chylothorax post-bacteraemia: A case report.

BACKGROUND: Although superior vena cava (SVC) syndrome has also been reported as a late complication of pacemaker (PM) implantation, acute onset of SVC syndrome caused by disdialysis syndrome in patients with PM implantation is very rare. There are no specific therapies or guidelines. CASE SUMMARY: A 96-year-old woman receiving dialysis was implanted with a PM due to sick sinus syndrome. She was referred to our facility for chest discomfort experienced during dialysis. Upon examination, unilateral pleural effusion on the right side was cloudy with a foul odour. The patient was diagnosed with pyothorax and treated with antibiotics. After the effusion was reduced, it gradually reaggravated and remained cloudy. In this case, SVC syndrome, which is generally considered a late complication after PM implantation, rapidly developed following the bacteraemia, resulting in impaired venous return, chylothorax, and disdialysis syndrome. After catheter intervention for SVC stenosis, the patient's symptoms promptly improved. The patient has been recurrence-free for a year. CONCLUSION: Acute SVC syndrome can cause dysdialysis in PM-implanted patients. Catheter intervention alone has improved this condition for a traceable period.

End-of-Life Care of Acute Myeloid Leukemia Compared with Aggressive lymphoma in Patients Who Are Eligible for Intensive Chemotherapy: An Observational Study in a Japanese Community Hospital.

Background: Patients with hematological malignancies (HMs) are reported to receive more aggressive care at the end of life (EOL) than patients with solid tumors. However, the reasons behind this occurrence are not fully understood. Objectives: To examine whether the care at EOL for HMs is mainly because of the disease characteristics or hematologists' attitudes and systems of care, we compared the EOL care of patients with acute myeloid leukemia (AML) and diffuse large B cell lymphoma (DLBCL). Design: We retrospectively analyzed the EOL care of patients with AML and DLBCL younger than 80 years who were receiving combination chemotherapy at a city hospital in Japan. Results: Fifty-nine patients with AML and 65 with DLBCL were included. Those with AML received chemotherapy more often within their last 30 days (48% vs. 19%, p < 0.001) and 14 days (37% vs. 1.5%, p < 0.001) of life, and consulted the palliative team less frequently (5.3% vs. 29%, p < 0.001). In the last 3 years, the mortality rate in hematological wards decreased from 74% to 29% in the DLBCL group, but only from 95% to 90% in the AML group. In multivariate analysis, AML (odds ratio [OR] 0.065) and death before 2018 (OR, 0.077) were significant factors associated with reduced referrals to specialized palliative teams. Conclusion: Patients with AML tend to have lesser access to specialized palliative care and fewer options for their place of death than those with DLBCL. Detailed EOL care plans are needed for these patients, considering the characteristics of the disease.

Molecular detection of Lotmaria passim in honeybees in Japan.

Crithidia mellificae (C. mellificae) and Lotmaria passim (L. passim) are trypanosomatids that infect Apis mellifera. We analyzed the prevalence of C. mellificae and L. passim in six regions of Japan from 2018 to 2019. The detection rate of C. mellificae was 0.0% in all regions, whereas L. passim was detected in 16.7%-66.7% of the honeybees. L. passim was detected at a significantly lower rate in the Cyugoku-Shikoku region than in other regions. Furthermore, phylogenetic analysis of the internal transcribed spacer 1 (ITS1) locus of related species was performed. All the samples in this study could be assigned to the L. passim clade. This study reveals that L. passim infection is predominantly prevalent in Japan. Further epidemiological surveys are needed to clarify the prevalence of C. mellificae infection in honeybees in Japan.

Mutant p53 Depletion by Novel Inhibitors for HSP40/J-Domain Proteins Derived from the Natural Compound Plumbagin.

Accumulation of missense mutant p53 (mutp53) in cancers promotes malignant progression. DNAJA1, a member of HSP40 (also known as J-domain proteins: JDPs), is shown to prevent misfolded or conformational mutp53 from proteasomal degradation. Given frequent addiction of cancers to oncogenic mutp53, depleting mutp53 by DNAJA1 inhibition is a promising approach for cancer therapy. However, there is no clinically available inhibitor for DNAJA1. Our in silico molecular docking study with a natural compound-derived small molecule library identified a plumbagin derivative, PLIHZ (plumbagin-isoniazid analog), as a potential compound binding to the J domain of DNAJA1. PLIHZ efficiently reduced the levels of DNAJA1 and several conformational mutp53 with minimal impact on DNA contact mutp53 and wild-type p53 (wtp53). An analog, called PLTFBH, which showed a similar activity to PLIHZ in reducing DNAJA1 and mutp53 levels, inhibited migration of cancer cells specifically carrying conformational mutp53, but not DNA contact mutp53, p53 null, and wtp53, which was attenuated by depletion of DNAJA1 or mutp53. Moreover, PLTFBH reduced levels of multiple other HSP40/JDPs with tyrosine 7 (Y7) and/or tyrosine 8 (Y8) but failed to deplete DNAJA1 mutants with alanine substitution of these amino acids. Our study suggests PLTFBH as a potential inhibitor for multiple HSP40/JDPs.

Reciprocal role of PLAP-1 in HIF-1α-mediated responses to hypoxia.

OBJECTIVE: To investigate the mutual regulation of hypoxia-inducible factor (HIF)-1α activity and periodontal ligament-associated protein-1 (PLAP-1) expression in human periodontal ligament cells (HPDLs). BACKGROUND: Cellular responses to hypoxia regulate various biological events (e.g., inflammation and tissue regeneration) through activation of HIF-1α. PLAP-1, an extracellular matrix protein preferentially expressed in the periodontal ligament, plays important roles in the functions of HPDLs. Although PLAP-1 expression has been demonstrated in hypoxic regions, the involvement of PLAP-1 in responses to hypoxia has not been revealed. METHODS: HPDLs were cultured under normoxic (20% O2 ) or hypoxic (1% O2 ) conditions with or without deferoxamine mesylate (chemical hypoxia inducer) or chetomin (HIF signaling inhibitor). Expression levels of PLAP-1 and HIF-1α were examined by real-time reverse transcription-polymerase chain reaction and western blot analysis. Luciferase reporter assays of HIF-1α activity were performed using 293T cells stably transfected with a hypoxia response element (HRE)-containing luciferase vector in the presence or absence of recombinant PLAP-1 or PLAP-1 gene transfection. RESULTS: Cultivation under hypoxic conditions elevated the gene and protein expression levels of PLAP-1 in HPDLs. Deferoxamine mesylate treatment also enhanced PLAP-1 expression in HPDLs. Hypoxia-induced PLAP-1 expression was significantly suppressed in the presence of chetomin. PLAP-1-suppressed HPDLs showed increased HIF-1α accumulation in the nucleus during culture under hypoxic conditions, but not in the presence of recombinant PLAP-1. In the presence of recombinant PLAP-1, hypoxia-induced HRE activity of 293T cells was significantly suppressed in a dose-dependent manner. Transfection of the PLAP-1 gene resulted in a significant reduction of HRE activity during culture under hypoxic conditions. CONCLUSION: PLAP-1 expression is upregulated under hypoxic conditions through HIF-1α activation. Moreover, hypoxia-induced PLAP-1 expression regulates HIF-1α signaling.

A Retrospective Analysis of R-MPV Plus Response-adapted Whole-brain Radiotherapy for Elderly Patients with Primary Central Nervous System Lymphoma.

Objective Few reports have described the real-world outcomes of rituximab, methotrexate (MTX), procarbazine, and vincristine (R-MPV) plus response-adapted whole-brain radiotherapy (WBRT) for elderly patients with primary central nervous system lymphoma (PCNSL). We evaluated the outcome of this regimen. Methods We evaluated >60-year-old patients with newly diagnosed PCNSL who received R-MPV plus WBRT from January 2010 to December 2019 at Toyohashi Municipal Hospital. The patients' characteristics, regimen enforcement, response rate, survival, and toxicity were analyzed. Patients Ten patients were consecutively enrolled. Their median age was 69 years old, and 60% had a performance status of 3 or 4 before induction therapy. Results Seven patients achieved a complete response after induction, and all 10 patients achieved a complete response after consolidation. Seven received reduced-dose WBRT at 23.4 Gy, and 2 received WBRT at 45 Gy. The median follow-up was 44.4 months; the 3-year progression-free survival and overall survival rates were 60% and 80%, respectively; and the cumulative incidence of relapse was 40%. The incidence of symptomatic delayed neurotoxicity was 70%. Of the 7 patients who received reduced-dose WBRT, 4 (57%) developed delayed neurotoxicity, including 1 severely affected patient. Only one patient survived without relapse and delayed neurotoxicity. The ratio of patients who developed relapse or delayed neurotoxicity that impaired daily life was 33% and 100% in the MTX high- and low-intensity groups, respectively. Conclusion This regimen in elderly patients is unsatisfactory because of delayed neurotoxicity. We should consider maintaining an adequate MTX intensity, postponing or minimizing WBRT, and choosing high-dose consolidation therapy for select patients.

DNAJA1 promotes cancer metastasis through interaction with mutant p53.

Accumulation of mutant p53 (mutp53) is crucial for its oncogenic gain of function activity. DNAJA1, a member of J-domain containing proteins or heat shock protein 40, is shown to prevent unfolded mutp53 from proteasomal degradation. However, the biological function of DNAJA1 remains largely unknown. Here we show that DNAJA1 promotes tumor metastasis by accumulating unfolded mutp53. Levels of DNAJA1 in head and neck squamous cell carcinoma (HNSCC) tissues were higher than those in normal tissues. Knockdown of DNAJA1 in HNSCC cell lines carrying unfolded mutp53 significantly decreased the levels of mutp53, filopodia/lamellipodia formation, migratory potential, and active forms of CDC42/RAC1, which were not observed in HNSCC cells with DNA contact mutp53, wild-type p53, or p53 null. Such mutp53-dependent functions of DNAJA1 were supported by the observation that DNAJA1 selectively bound to unfolded mutp53. Moreover, DNAJA1 knockdown in HNSCC cells carrying unfolded mutp53 inhibited primary tumor growth and metastases to the lymph nodes and lungs. Our study suggests that DNAJA1 promotes HNSCC metastasis mainly in a manner dependent on mutp53 status, suggesting DNAJA1 as a potential therapeutic target for HNSCC harboring unfolded mutp53.

Transcriptional inhibition of feline immunodeficiency virus by alpha-amanitin.

Alpha-amanitin, one of the amatoxins in egg amanita, has a cyclic peptide structure, and was reported as having antiviral activity against several viruses. We investigated whether α-amanitin has antiviral activity against feline immunodeficiency virus (FIV). FL-4 cells persistently infected with FIV Petaluma were cultured with α-amanitin. Reverse transcriptase (RT) activity in the supernatant of FL-4 cells was significantly inhibited by α-amanitin. In addition, the production of FIV core protein in FL-4 cells was inhibited by α-amanitin when analyzed by western blotting. Furthermore, α-amanitin inhibited the transcription of FIV in real-time RT-PCR. These data suggested that α-amanitin showed anti-FIV activity by inhibiting the RNA transcription level.

[Lupus anticoagulant-hypoprothrombinemia syndrome associated with follicular lymphoma].

A 56-year-old woman was referred to our hospital with symptoms of swelling, purpura, and pain in her limbs. Prior to referral, bleeding in her limbs had spontaneously appeared and disappeared several times. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were prolonged, and the factor II level was 17%. The plasma-mixing test indicated lupus anticoagulant (LA), which was confirmed using aPTT-LA and dilute Russell's viper venom time (dRVVT). Therefore, she was diagnosed with lupus anticoagulant-hypoprothrombinemia syndrome (LAHPS). During screening for underlying disorders, chest computed tomography (CT) revealed a retrosternal mass. Biopsy was not performed because the administration of freshly frozen plasma failed to correct her coagulopathy. Prednisolone (PSL) treatment (1 mg/kg) was initiated, which normalized the coagulation tests. The retrosternal mass also disappeared. PSL was tapered without LAHPS recurrence; however, the follow-up CT revealed systemic lymphadenopathy. Follicular lymphoma was diagnosed using lymph-node biopsy. Considering the subsequent LAHPS recurrence, six cycles of bendamustine + rituximab were administered. Complete response with no LAHPS recurrence was observed at the time of drafting this report. LAHPS is rare and distinct from antiphospholipid syndrome because it can cause severe bleeding. Underlying disorders should be evaluated in cases of LAHPS.

Regulators of Oncogenic Mutant TP53 Gain of Function.

The tumor suppressor p53 (TP53) is the most frequently mutated human gene. Mutations in TP53 not only disrupt its tumor suppressor function, but also endow oncogenic gain-of-function (GOF) activities in a manner independent of wild-type TP53 (wtp53). Mutant TP53 (mutp53) GOF is mainly mediated by its binding with other tumor suppressive or oncogenic proteins. Increasing evidence indicates that stabilization of mutp53 is crucial for its GOF activity. However, little is known about factors that alter mutp53 stability and its oncogenic GOF activities. In this review article, we primarily summarize key regulators of mutp53 stability/activities, including genotoxic stress, post-translational modifications, ubiquitin ligases, and molecular chaperones, as well as a single nucleotide polymorphism (SNP) and dimer-forming mutations in mutp53.

Differential Distribution of Salmonella Serovars and Campylobacter spp. Isolates in Free-Living Crows and Broiler Chickens in Aomori, Japan.

Salmonella and Campylobacter cause foodborne enteritis mainly via the consumption of raw/undercooked contaminated poultry meat and products. Broiler flocks are primarily colonized with these bacteria; however, the underlying etiology remains unclear. The present study was conducted in order to obtain further information on the prevalence and genotypic distribution of Salmonella and Campylobacter in free-living crows and broiler flocks in a region for 2 years, thereby facilitating estimations of the potential risk of transmission of C. jejuni from crows to broiler flocks. Salmonella serovars Bredeney and Derby were isolated from 8 and 3 out of 123 captured crows, respectively, both of which are not common in broiler chickens. Campylobacter were isolated from all 89 crows tested and C. jejuni was prevalent (85 crows). Pulsed field gel electrophoresis showed broad diversity in the crow isolates of C. jejuni. However, 3 crow isolates and 2 broiler isolates showing similar banding patterns were assigned to different sequence types in multi-locus sequence typing. These results indicate that crows do not share Salmonella serovars with broilers, and harbor various genotypes of C. jejuni that differ from those of broiler flocks. Thus, our results indicate that crows are not a potential vector of these bacteria to broiler flocks in this region.

Drug screening to target nuclear orphan receptor NR4A2 for cancer therapeutics.

BACKGROUND: Our previous study suggested NR4A2, a subfamily member of orphan nuclear receptors, is essential for survival of human cancer cells such as mucoepidermoid carcinoma (MEC). METHODS: We conducted high throughput drug screening for NR4A2 inhibitors as a novel therapeutic modality. Positive screening was performed using a luciferase reporter vector containing NR4A2 binding sequence, and a CRE-reporter control vector was used to eliminate false positives. In vitro assays for positive hits were conducted. RESULTS: A total of 23 Food and Drug Administration (FDA) and 43 Life Science Library compounds were identified, including several epidermal growth factor inhibitors and Src inhibitors. Subsequent in vitro assays confirmed that identified compounds were preferentially active in NR4A2+ cancer cells. Several candidate compounds appeared to suppress NR4A2 via inhibition of p-ERK, whereas a novel compound KU0171309 may act as a more direct inhibitor. CONCLUSIONS: Further research should focus on homologue selectivity, in vivo activity, and definitively deciphering the mechanism of action of KU0171309.

Suppression of HBV replication by the expression of nickase- and nuclease dead-Cas9.

Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.

Host-derived apolipoproteins play comparable roles with viral secretory proteins Erns and NS1 in the infectious particle formation of Flaviviridae.

Amphipathic α-helices of exchangeable apolipoproteins have shown to play crucial roles in the formation of infectious hepatitis C virus (HCV) particles through the interaction with viral particles. Among the Flaviviridae members, pestivirus and flavivirus possess a viral structural protein Erns or a non-structural protein 1 (NS1) as secretory glycoproteins, respectively, while Hepacivirus including HCV has no secretory glycoprotein. In case of pestivirus replication, the C-terminal long amphipathic α-helices of Erns are important for anchoring to viral membrane. Here we show that host-derived apolipoproteins play functional roles similar to those of virally encoded Erns and NS1 in the formation of infectious particles. We examined whether Erns and NS1 could compensate for the role of apolipoproteins in particle formation of HCV in apolipoprotein B (ApoB) and ApoE double-knockout Huh7 (BE-KO), and non-hepatic 293T cells. We found that exogenous expression of either Erns or NS1 rescued infectious particle formation of HCV in the BE-KO and 293T cells. In addition, expression of apolipoproteins or NS1 partially rescued the production of infectious pestivirus particles in cells upon electroporation with an Erns-deleted non-infectious RNA. As with exchangeable apolipoproteins, the C-terminal amphipathic α-helices of Erns play the functional roles in the formation of infectious HCV or pestivirus particles. These results strongly suggest that the host- and virus-derived secretory glycoproteins have overlapping roles in the viral life cycle of Flaviviridae, especially in the maturation of infectious particles, while Erns and NS1 also participate in replication complex formation and viral entry, respectively. Considering the abundant hepatic expression and liver-specific propagation of these apolipoproteins, HCV might have evolved to utilize them in the formation of infectious particles through deletion of a secretory viral glycoprotein gene.

Effective chemomobilization with etoposide and cytarabine (EC regimen) in lymphoma patients: a single-center, retrospective, observational study.

Objective: Autologous stem cell transplantation is an important strategy for patients with relapsed or refractory lymphoma. Although various regimens for peripheral blood stem cell collection have been used, the optimal regimen has not yet been established. We aimed to evaluate the mobilization efficacy and safety of the regimen consisted of etoposide and cytarabine (EC regimen). Methods: We retrospectively analyzed the clinical data of 46 lymphoma patients who received peripheral blood stem cell mobilization with the EC regimen [etoposide (100 mg/m2/day, days 1-4) and cytarabine (100 mg/m2/day, days 1-4)] at Toyohashi municipal hospital from 2004 to 2013. Results: The median age of the patients was 55 years. The most common underlying diseases were diffuse large B-cell lymphoma (46%) and follicular lymphoma (26%). Three-quarters of patients were in their second complete or partial remission. The median total number of collected CD34+ cells was 10.6 × 106 kg-1. Forty-two patients (91%) yielded at least 2 × 106 kg-1 CD34+ cells within a median of 2 apheresis days, and 33 patients (72%) achieved it with only one apheresis. Successful mobilization was observed in five of six patients who failed to mobilize previously. Although febrile neutropenia occurred in 22 patients (48%), no fatal infection was observed. Conclusion: The EC regimen was highly effective in lymphoma patients, including patients who mobilized poorly with other regimens.

Characterization of miR-122-independent propagation of HCV.

miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5'UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients.

Quasispecies of Hepatitis C Virus Participate in Cell-Specific Infectivity.

It is well documented that a variety of viral quasispecies are found in the patients with chronic infection of hepatitis C virus (HCV). However, the significance of quasispecies in the specific infectivity to individual cell types remains unknown. In the present study, we analyzed the role of quasispecies of the genotype 2a clone, JFH1 (HCVcc), in specific infectivity to the hepatic cell lines, Huh7.5.1 and Hep3B. HCV RNA was electroporated into Huh7.5.1 cells and Hep3B/miR-122 cells expressing miR-122 at a high level. Then, we adapted the viruses to Huh7 and Hep3B/miR-122 cells by serial passages and termed the resulting viruses HCVcc/Huh7 and HCVcc/Hep3B, respectively. Interestingly, a higher viral load was obtained in the homologous combination of HCVcc/Huh7 in Huh7.5.1 cells or HCVcc/Hep3B in Hep3B/miR-122 cells compared with the heterologous combination. By using a reverse genetics system and deep sequence analysis, we identified several adaptive mutations involved in the high affinity for each cell line, suggesting that quasispecies of HCV participate in cell-specific infectivity.