Novel hepacivirus in Asian house shrew, China

Dear Editor,Hepatitis C virus (HCV) is a leading global cause of various liver diseases, including chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The genome of HCV is monopartite, single-stranded, positive RNA, about 10 kb in size.HCV is the prototype species of the Hepacivirus genus,which contains 14 species according to the update from the International Committee on Taxonomy of Viruses (Smith et al., 2016).

Identification and genetic characterization of bovine hepacivirus in China:A large scale epidemiological study

Bovine hepacivirus(BovHepV)is a novel virus that was recently discovered in Ghana and Germany in 2015.Until now,this virus has been identified in cattle population worldwide and is classified into subtypes A-G.To fully understand the epidemic situation and genetic characteristic of BovHepV in China,a total of 612 cattle serum samples were collected from 20 farms in seven provinces and municipality in China between 2018 and 2020 and were tested for the presence of BovHepV RNA via semi-nested PCR.The results demonstrated that 49(8.0%)samples were BovHepV RNA-positive.It is noted that BovHepV infection in yak was confirmed for the first time.BovHepV was detected in all the seven provinces,with the positive rate ranging from 3.1%to 13.3%,which indicates a wide geographical distribution pattern of BovHepV in China.Sequencing results revealed that 5'UTR of the 49 field BovHepV strains have a nucleotide similarity of 96.3%-100%between each other and 93.9%-100%with previously reported BovHepV strains.In addition,genetic analysis identified five critical nucleotide sites in 5'UTR to distinguish different subtypes,which was further verified by genomic sequencing and nucleotide similarity analysis.All the 49 Chinese field BovHepV strains were classified into subtype G and this subtype is only determined in cattle in China currently.This study will provide insights for us to better understand the epidemiology and genetic diversity of BovHepV.

Hepatitis C virus infection of human hepatoma cell line 7721 in vitro

AIM: To establish a cell culture system with long-term replication of hepatitis C virus in vitro. METHODS: Human hepatoma cell line 7721 was tested for its susceptibility to HCV by incubating with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various phases during the culturing periods. The presence of HCV RNA, the expression of HCV antigens in cells and/or supernatant were examined by RTPCR, in situ hybridization and immunohistochemistry respectively. RESULTS: The intracellular HCV RNA was first detected on d2 after infection and then could be intermittently detected in both cells and supernatant over a period of at least three months. The expression of HCV NS3,CP10 antigens could be observed in cells. The fresh cells could be infected by supernatant from cultured infected cells and the transmission of viral genome from HCV-infected 7721 cells to PBMCs was also observed. CONCLUSION: The hepatoma line 7721 is not only susceptible to HCV but also supports its long-term replication in vitro.

HCV-RNA positivity in peripheral blood mononuclear cells of patients with chronic HCV-infection: does it really mean viral replication?

AIM: To analyze the association of HCV-RNA with peripheral blood mononuclear cells (PBMC) and to answer the question whether HCV-RNA positivity in PBMC is due to viral replication. METHODS: HCV-RNA was monitored in serum and PBMC preparations from 15 patients with chronic HCV infection before, during and after an IFN-alpha therapy using a nested RT/PCR technique. In a second approach, PBMC from healthy donors were incubated in HCV positive plasma. RESULTS: In the IFN-alpha responding patients,HCV-RNA disappeared first from total RNA preparations of PBMC and then from serum. In contrast, in relapsing patients, HCV-RNA reappeared first in serum and then in PBMC. A quantitative analysis of the HCV-RNA concentration in serum was performed before and after transition from detectable to non detectable HCV-RNA in PBMC-RNA and vice versa. When HCV-RNA was detectable in PBMC preparations, the HCV concentration in serum was significantly higher than the serum HCV-RNA concentration when HCV-RNA in PBMC was not detectable. Furthermore, at no time during the observation period was HCV specific RNA observed in PBMC, if HCV-RNA in serum was under the detection limit. Incubation of PBMC from healthy donors with several dilutions of HCV positive plasma for two hours showed a concentration dependent PCR positivity for HCV-RNA in reisolated PBMC. CONCLUSION: The detectability of HCV-RNA in total RNA from PBMC seems to depend on the HCV concentration in serum. Contamination or passive adsorption by circulating virus could be the reason for detection of HCV-RNA in PBMC preparations of chronically infected patients.

Natural history of chronic hepatitis C in patients on hemodialysis: Case control study with 4-23 years of follow-up

AIM:Hepatitis C virus(HCV)infection is very common among end-stage kidney disease patients on hemodialysis,but its natural history is not known. METHODS:In this study,189 dialysis patients(case) positive for HCV antibodies who were followed up for more than 4 years were compared with twice as many sex/age matched controls with chronic hepatitis C who were diagnosed in the same month as the case and followed up for comparable periods.The longest follow-up was 23 years in dialysis cases. The disease activities were graded into'asymptomatic'if ALT was less than 40(35 in cases)IU/L,'low activities'if ALT was 40(35)-79 IU/L,and'high activities'if ALT was above 80 IU/L during the last or latest 4 year period. RESULTS:All 25 dialysis cases who were followed up for more than 15 years were asymptomatic and 15 of them were negative for HCV RNA.Of the 50 controls followed up for more than 15 years,34 had high activities,and none deared HCV RNA.There were 60 controls who were asymptomatic, but they were all positive for HCV RNA,while 22.3% of asymptomatic dialysis cases were RNA negative.No dialysis patients with chronic hepatitis C progressed to cirrhosis, whereas the disease progressed to cirrhosis in more than one quarter of the controls.These differences were highly significant(P<0.0001). CONCLUSION:Chronic hepatitic C among hemodialysis patients is mild in disease activity,and is not progressive, perhaps due to immunological abnormalities in these patients. Hepatic C virus is frequently cleared in asymptomatic dialysis patients during a long course.A possible mechanism for viral clearance is viral particle destruction on the surface of the dialyzer membrane.

HCV-specific cytokine induction in monocytes of patients with different outcomes of hepatitis C

AIM:Cytokine release by macrophages critically determines the type of immune response to an antigen.Therefore,we studied hepatitis C virus(HCV)-specific induction of interleukins-1β,-10,-12(IL-1β,IL-10,IL-12),and tumor necrosis factor-α(TNF-α)in monocytes. METHODS:Intracallular cytokine expression was studied by flow cytometry in 23 patients with chronic hepatitis C,14 anti-HCV seropositives without viremia and 11 controls after stimulation of peripheral blood mononuclear calls with recombinant core,NS3,NS4,NSSa and NSSb proteins. RESULTS:Patients with HCV viremia revealed greater spontaneous expression of IL-1β,TNF-α,and IL-10. Furthermore,greater than twofold higher IL-10 expression was induced by the HCV antigens in chronic hepatitis C than in the other two groups(P<0.05).In contrast,neither IL- 12 nor TNF-α was induced preferentially. CONCLUSION:In chronic hepatitis C antigen-specific cytokine induction in monocytes is apparently shifted towards predominant IL-10 induction-not counterbalanced by antiviral type 1 cytokines.This may contribute to persistent viral replication.

Effect of in vitro interferon-beta administration on hepatitis C virus in peripheral blood mononuclear cells as a predictive marker of clinical response to interferon treatment for chronic hepatitis C

AIM:To test whether in vitro incubation of peripheral blood mononuclear cells (PBMC) with interferon (IFN) could efficiently decrease hepatitis C virus-RNA (HCV-RNA) amount and to analyze whether this effect was associated with clinical response to IFN.METHODS:Twenty-seven patients with histologically proven chronic hepatitis C were given intravenous administration of 6 million units (MU) IFN-β daily for 6 weeks followed by three times weekly for 20 weeks. PBMC collected before IFN therapy were incubated with IFN-β and HCV-RNA in PMBC was semi-quantitatively determined.RESULTS: Twenty-five patients completed IFN therapy.Eight patients (32%) had sustained loss of serum HCV-RNA with normal serum ALT levels after IFN therapy (complete responders).HCV-RNA in PBMC was detected in all patients,whereas it was not detected in PBMC from healthy subjects.In vitro administration of IFN-β decreased the amount of HCV-RNA in PMBC in 18 patients (72%). Eight of these patients obtained complete response. On the other hand,none of the patients whose HCV-RNA in PBMC did not decrease by IFN-β was complete responders. Multiple logistic regression analysis revealed that the decrease of HCV-RNA amount in PBMC by IFN-β was the only independent predictor for complete response (P<0.05).CONCLUSION:The effect of in vitro IFN-β on HCV in PBMC reflects clinical response and would be taken into account as a predictive marker of IFN therapy for chronic hepatitis C.

Assessment of correlation between serum titers of hepatitis c virus and severity of liver disease

AIM:The significance of hepatitis C virus (HCV) serum titers has been examined in several clinical situations. There is much evidence that patients with a lower viral load have better response rates to anti-viral therapy compared to those with higher levels.Moreover,a direct association has been observed between serum titers of HCV and transmission rates of the virus.The aim of the present study was to determine if there was any correlation between HCV viral load and the severity of liver disease. METHODS:Fifty patients with HCV infection were included in the study.These comprised of 34 subjects with a history of alcohol use and 16 non-alcoholics.Quantitative serum HCV RNA assay was carried out using the branched DNA (bDNA) technique.Linear regression analysis was performed between serum viral titers and liver tests.In addition,for the purpose of comparison,the subjects were divided into two groups:those with low viral liters (≤50 genome mEq/mL) and high titers (>50 mEq/mL). RESULTS:All subjects were men,with a mean±SD age of 47±7.8 years.The mean HCV RNA level in the blood was 76.3×10~5±109.1 genome equivalents/mL.There was no correlation between HCV RNA levels and age of the patients (r=0.181),and the history or amount (g/d) of alcohol consumption (r=0.07).Furthermore,no correlation was observed between serum HCV RNA levels and the severity of liver disease as judged by the values of serum albumin (r=0.175),bilirubin (r=0.217),ALT (r=0.06) and AST (r=0.004) levels.Similarly,no significant difference was observed between patients with low viral titers and high liters with respect to any of the parameters. CONCLUSION:Our results indicate that the severity of liver disease is independent of serum levels of hepatitis C virus.These findings are important since they have a direct impact on the current debate regarding the role of direct cytopathic effect of hepatitis C virus versus immune-mediated injury in the pathogenesis of HCV-related liver damage.

Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein

AIM: To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing. METHODS: HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively.Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products. RESULTS: E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively. Deglycosylation analysis showed that this difference was mainly due to different glycosylation. Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94. Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences. CONCLUSION: The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2. As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER. Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.

Identification of the epitopes on HCV core protein recognized by HLA-A2 restricted cytotoxic T lymphocytes

AIM: To identify hepatitis C virus(HCV) core protein epitopes recognized by HLA-A2 restricted cytotoxic T lymphocyte (CTL). METHODS: Utilizing the method of computer prediction followed by a 4h(51)Cr release assay confirmation. RESULTS: The results showed that peripheral blood mononuclear cells (PBMC) obtained from two HLA-A2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide &quot;ALAHGVRAL (core 150-158)&quot;. The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%, respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis. But blocking of CTL response with anti-CD8 mAb could abolish the lysis. CONCLUSION: The peptide (core 150-158) is the candidate epitope recognized by HLAA2 restricted CTL.

Distribution and changes in hepatitis C virus genotype in China from 2010 to 2020

BACKGROUND Hepatitis C virus(HCV)causes a large number of infections worldwide.New infections seem to be increasing according to a report of the World Health Organization in 2015.Although direct-acting antivirals are quite effective for most genotypes of the HCV,some genotypes fail to respond.Therefore,the trend of genotype distribution is vital to better control the development of this infection.AIM To analyze the distribution and trends of the HCV genotype before and after the emergence of direct-acting antivirals in China.METHODS We searched all literature published in five electronic databases-China National Knowledge Infrastructure,Wan Fang Data,VIP Chinese Journal Database,Chinese Biomedical Literature Service System,and PubMed-from January 1,2010 to December 31,2020.The search strategy combined medical subject headings and free-text terms,including“hepatitis C virus”or“HCV”and“genotype”or“subtype”and“China”or“Chinese”.Additional relevant articles were searched by manual selection.Data were extracted to build a database.All of the data were totaled according to regions,periods,routes of transmission,and sexes.The percentages in various stratifications were calculated.RESULTS There were 76110 samples from 30 provinces included in the study.Genotype 1(G1)accounted for 58.2%of cases nationwide,followed by G2,G6,G3b,G3a,unclassified and mixed infections(17.5%,7.8%,6.4%,4.9%,1.8%,and 1.2%,respectively).The constitution of genotype varied among different regions,with G6 and G3b being more common in the south and southwest,respectively(28.1%,15.4%).The past ten years have witnessed a decrease in G1 and G2 and an increase in G3 and G6 in almost all regions.The drug-use population had the most abundant genotypes,with G6 ranking first(33.3%),followed by G1 and G3b(23.4%,18.5%).CONCLUSION G3 and G6 pose a new challenge for HCV infection.This study revealed the distribution of HCV genotypes in China over the past 10 years,providing information for HCV management strategies.

Hepatitis C virus in human B lymphocytes transformed by Epstein-Barr virus in vitro by in situ reverse transcriptase-polymerase chain reaction

AIM: To study persistence and replication of hepatitis C virus (HCV) in patients' peripheral blood mononuclear cells (PBMC) cultured in vitro. METHODS: Epstein Barr virus (EBV) was used to transform the hepatitis C virus from a HCV positive patient to permanent lymphoblastoid cell lines (LCL). Positive and negative HCV RNA strands of the cultured cells and growth media were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) each month. Core and NS5 proteins of HCV were further tested using immunohistochemical SP method and in situ RT-PCR. RESULTS: HCV RNA positive strands were consistently detected the cultured cells for one year. The negative-strand RNA in LCL cells and the positive-strand RNA in supernatants were observed intermittently. Immunohistochemical results medicated expression of HCV NS3 and C proteins in LCL cytoplasm mostly. The positive signal of PCR product was dark blue and mainly localized to the LCL cytoplasm. The RT-PCR signal was eliminated by overnight RNase digestion but not DNase digestion. CONCLUSION: HCV may exist and remain functional in a cultured cell line for a long period.

SEN virus does not affect treatment response in hepatitis C virus coinfected patients but SEN virus response depends on SEN virus DNA concentration

AIM: To clarify the effect of SEN virus (SENV) infection on a combination therapy including interferon alfa (IFN-α) or pegylated-IFN with ribavirin in patients with chronic hepatitis and the effect of a combination therapy on SENV.METHODS: SENV DNA was determined by polymerase chain reaction in serum samples from 95 patients with chronic hepatitis C. Quantitative analysis was done for SENV H DNA.RESULTS: Twenty-one (22%) of 95 patients were positive for SENV DNA. There was no difference in clinical and biochemical parameters between patients with HCV infection alone and coinfected patients. The sustained response rate for HCV clearance after combination therapy did not differ between patients with SENV (52%) and without SENV(50%, n.s.). SENV DNA was undetectable in 76% of the initially SENV positive patients at the end of follow-up. SENV H response to combination therapy was significantly correlated with SENV DNA level (P=-0.05).CONCLUSION: SENV infection had no influence on the HCV sustained response rate to the combination therapy.Response rate of SENV to the combination therapy depends on SENV DNA level.

Acute hepatitis C in a chronically HIV-infected patient:Evolution of different viral genomic regions

AIM: To analyze the molecular evolution of different viral genomic regions of HCV in an acute HCV infected patient chronically infected with HIV through a 42-month follow-up.METHODS: Serum samples of a chronically HIV infected patient that seroconverted to anti HCV antibodies were sequenced, from the event of superinfection through a period of 17 months and in a late sample (42nd month). Hypervariable genomic regions of HIV (V3 loop of the gp120) and HCV (HVR-1 on the E2 glycoprotein gene) were studied. In order to analyze genomic regions involved in different biological functions and with the cellular immune response, HCV core and NS5A were also chosen to be sequenced. Amplification of the different regions was done by RT-PCR and directly sequenced. Confirmation of sequences was done on reamplified material. Nucleotide sequences of the different time points were aligned with CLUSTAL W 1.5, and the corresponding amino acid ones were deduced.RESULTS: Hypervariable genomic regions of both viruses (HVR1 and gp120 V3 loop) presented several nonsynonymous changes but, while in the gp120 V3 loop mutations were detected in the sample obtained right after HCV superinfection and maintained throughout, they occurred following a sequential and cumulative pattern in the HVR1. In the NS5A region of HCV, two amino acid changes were detected during the follow-up period, whereas the core region presented several amino acid replacements, once the HCV chronic infection had been established.CONCLUSION: During the HIV-HCV superinfection, each genomic region analyzed shows a different evolutionary pattem.Most of the nucleotide substitutions observed are nonsynonymous and clustered in previously described epitopes,thus suggesting an immune-driven evolutionary process.

Is laparoscopy an advantage in the diagnosis of cirrhosis in chronic hepatitis C virus infection?

AIM:To evaluate the potential of laparoscopy in the diagnosis of cirrhosis and outcome of interferon treatment in HCV-infected patients. METHODS:In this retrospective study,diagnostic laparoscopy with laparoscopic liver biopsy was performed in 72 consecutive patients with chronic HCV infection.The presence or absence of drrhosis was analyzed macroscopically by laparoscopy and microscopically by liver biopsy specimens.Clinical and laboratory data and outcome of interferon-alfa treatment were compared between cirrhotic and noncirrhotic patients. RESULTS:Laparoscopically,cirrhosis was seen in 29.2 % (21/72)and non-cirrhosis in 70.8 %(51/72)of patients. Cirrhotic patients were significantly older with a significant longer duration of HCV infection than noncirrhotic patients. Laboratory parameters(AST,y-GT,y-globulin fraction)were measured significantly higher as well as significantly lower (prothrombin index,platelet count)in cirrhotic patients than in non-cirrhotic patients.Histologically,cirrhosis was confirmed in 11.1%(8/72)and non cirrhosis in 88.9 %(64/72).Patients with macroscopically confirmed cirrhosis(n=21)showed histologically cirrhosis in 38.2 %(8/21)and histologically non- cirrhosis in 61.9 %(13/21).In contrast,patients with macroscopically non-cirrhosis(n=51)showed histologically non cirrhosis in all cases(51/51).Thirty-nine of 72 patients were treated with interferon-alfa,resulting in 35.9 %(14/39) patients with sustained response and 64.1%(25/39)with non response.Non-responders showed significantly more macroscopically cirrhosis than sustained responders.In contrast,there were no significant histological differences between non-responders and sustained responders. CONCLUSION:Diagnostic laparoscopy is more accurate than liver biopsy in recognizing cirrhosis in patients with chronic HCV infection.Liver biopsy is the best way to assess inflammatory grade and fibrotic stage.The invasive marker for staging,prognosis and management,and treatment outcome of chronic HCV-infected patients need further research and dinical thals.Laparoscopy should be performed for recognition of drrhosis if this parameter is found to be of prognostic and therapeutic relevance in patients with chronic HCV infection.

Tricistronic hepatitis C virus subgenomic replicon expressing double transgenes

AIM: To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.

黄病毒科的黄病毒和肝炎病毒研究进展

黄病毒科的黄病毒和肝炎病毒可引起许多严重的人类疾病。该文概述了这两类病毒的体外细胞培养系统、病毒蛋白的抗原结构与功能、病毒毒力的分子基础。