Hepatitis C virus micro-elimination:Where do we stand?

Hepatitis C virus (HCV) elimination by 2030, using direct-acting antiviraltreatments, has been promoted by the World Health Organization. Thisachievement is not attainable, however, particularly after the 2020 pandemic ofthe coronavirus disease 2019. Consequently, the more realistic objective ofeliminating HCV from population segments for which targeted strategies ofprevention and treatment are easily attained has been promoted in Europe, as avalid alternative. The underlying idea is that micro-elimination will ultimatelylead to macro-elimination. The micro-elimination strategy may target differentspecific populations and at-risk groups. Different settings, including prisons andhospitals, have also been identified as micro-elimination scenarios. In addition,dedicated micro-elimination strategies have been designed that are tailored at thegeographical level according to HCV epidemiology and individual country’sincome. The main elements of a valid and successful micro-elimination project arereliable epidemiological data and active involvement of all the stakeholders.Community involvement represents another essential component for a successfulprogram.

Antibody to El peptide of hepatitis C virus genotype 4 inhibits virus binding and entry to HepG2 cells in vitro

AIM： To analyze the neutralizing activity of antibodies against E1 region of hepatitis C virus （HCV）. Specific polydonal antibody was raised via immunization of New Zealand rabbits with a synthetic peptide that had been derived from the E1 region of HCV and was shown to be highly conserved among HCV published genotypes. METHODS： Hyper-immune HCV E1 antibodies were incubated over night at 4 ℃ with serum samples positive for HCV RNA, with viral loads ranging from 615 to 3.2 million IU/mL. Treated sera were incubated with HepG2 cells for 90 min. Blocking of viral binding and entry into cells by anti E1 antibody were tested by means of RTPCR and flow cytometry. RESULTS： Direct immunostaining using FITC conjugated E1 antibody followed by Flow cytometric analysis showed reduced mean fluorescence intensity in samples pre-incubated with E1 antibody compared with untreated samples. Furthermore, 13 out of 18 positive sera （72%） showed complete inhibition of infectivity as detected by RT-PCR. CONCLUSION： In house produced E1 antibody, blocks binding and entry of HCV virion infection to target cells suggesting the involvement of this epitope in virus binding and entry. Isolation of these antibodies that block virus attachment to human cells are useful as therapeutic reagents.

Comparative Study on 4 EIA Kits for Screening Antibody to Hepatitis C Virus in Pooled Sera

Four enzyme immunoassay (EIA) test kits, 1 Canadian product and 3 Chinese products,were used in the comparative study. Each pool consisted of 5 sera, and the 5 single sera were tested as controls. The tests were carried out according to the instructions, keeping the same dilution of each serum in single and pool samples. It was found that with the Canadian kit,the positive and negative results of opled sera had no difference from that of the controls (P>0. 10). In the case of Chinese Yali and Kehua kits, the positive results of pooled sera showed no difference from the controls (P >0. 10), but the optical density (OD) of negative opls were increased (P < 0. 01 ), though quite distant from the cut-off values. In the case of Changzheng kit, the OD of opitive opls were significantly lower than those of the controls (P < 0. 05 ), and weak positive samples missed the detection. However this problem could be overcome by blocking the microwells beforehand. Our experiment demonstrate that not all EIA test kits are suitable for screening opls for antithey to hepatitis C virus, and that it is important to assess the sensitivity of the EIA kit to be used for this purpose.

Neutralizing antibodies in hepatitis C virus infection

Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays, the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses. In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodiesfor control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.

Flow cytometric detection of hepatitis C virus antigens in infected peripheral blood leukocytes: Binding and entry

AIM： We designed two synthetic-core-specific peptides core 1 （C1） and core 2 （C2）, and an E1-specific peptide （El）. We produced specific polyclonal antibodies against these peptides and used the antibodies for detection of HCV antigens on surface and within infected peripheral blood leukocytes. METHODS： Peripheral blood from a healthy individual who tested negative for HCV RNA was incubated with HCV type 4 infected serum for i h and 24 h at 37 ℃. Cells were stained by direct and indirect immunofluorescence and measured by flow cytometry. RESULTS： After 1 h of incubation, antibodies against C1, C2, and El detected HCV antigens on the surface of 27%, 26% and 73% of monocytes respectively, while 10%, 5% and 9% of lymphocytes were positive with anti-C1, anti-C2 and anti-E1 respectively. Only 1-3% of granulocytes showed positive staining with anti-C1, anti-C2 and anti E1 antibodies. After 24 h of incubation, we found no surface staining with anti-C1, anti-C2 or anti-E1. Direct immunostaining using anti-C2 could not detect intracellular HCV antigens, after 1 h of incubation with the virus, while after 24 h of incubation, 28% of infected cells showed positive staining. Only plus strand RNA was detectable intracellularly as early as 1 h after incubation, and remained detectable throughout 48 h post-infection. Interestingly, minus RNA strand could not be detected after 1 h, but became strongly detectable intracellularly after 24 h post-infection. CONCLUSION： Monocytes and lymphocytes are the preferred target cells for HCV infection in peripheral blood leukocytes. Our specific anti-core and anti-E1 antibodies are valuable reagents for demonstration of HCV cell cycle. Also, HCV is capable of infecting and replicating in peripheral blood mononuclear cells as confirmed by detection of minus strand HCV RNA as well as intracellular staining of core HCV antigen.

Spontaneous elimination of hepatitis C virus infection: A retrospective study on demographic, clinical, and serological correlates

To find correlates to spontaneous clearance of hepatitis C virus （HCV） infection, this study compared individuals with self-limited and chronic infection with regard to clinical, demographic, and serological pa- rameters. METHODS： Sixty-seven anti-HCV positive and repeatedly HCV RNA negative individuals were considered to have resolved HCV infection spontaneously. To determine the viral genotype these patients had been infected with HCV serotyping was performed. For comparison reasons, 62 consecutive patients with chronic hepatitis C were enrolled. Cases and controls were compared stratified for age and sex. RESULTS： Retrospective analysis showed （1） a lower humoral reactivity to HCV in patients with self-limited compared to chronic HCV-infection and （2） that younger age, history of iv drug use, and acute/post-acute hepatitis A or B co-infections, but not viral genotypes, are independent correlates for spontaneous HCV clearance. CONCLUSION： The stronger humoral reactivity to HCV in patients with persistent infections and in those with a history of iv drug use is supposed to be due to continuous or repeated contact（s） to the antigen. Metachronous hepatitis A or hepatitis B infections might favor HCV clearance.

Virus-host Interactions during Hepatitis C Virus Entry-Implications for Pathogenesis and Novel Treatment Approaches

Hepatitis C virus (HCV) is a member of the Flaviviridae family and causes acute and chronic hepatitis. Chronic HCV infection may result in severe liver damage including liver cirrhosis and hepatocellular carcinoma. The liver is the primary target organ of HCV, and the hepatocyte is its primary target cell. Attachment of the virus to the cell surface followed by viral entry is the first step in a cascade of interactions between the virus and the target cell that is required for successful entry into the cell and initiation of infection. This step is an important determinant of tissue tropism and pathogenesis; it thus represents a major target for antiviral host cell responses, such as antibody-mediated virus neutralization. Following the development of novel cell culture models for HCV infection our understanding of the HCV entry process and mechanisms of virus neutralization has been markedly advanced. In this review we summarize recent developments in the molecular biology of viral entry and its impact on pathogenesis of HCV infection, development of novel preventive and therapeutic antiviral strategies.

Hepatitis C virus may infect extrahepatic tissues in patients with hepatitis C

AIM To explore the status of extrahepatichepatitis C virus(HCV)infection and replicationin hepatitis C patients,and its potentialimplication in HCV infection and pathogenicity.METHODS By reverse-transcriptase poly-merase chain reaction(RT-PCR),in situhybridization(ISH)and immunohistochemistry,HCV RNA,HCV replicative intermediate(minus-strand of HCV RNA),and HCV antigens weredetected in 38 autopsy extrahepatic tissuespecimens(including 9 kidneys,9 hearts,9pancreas,5 intestines,2 adrenal glands,2spleens,1 lymph node,and 1 gallbladder)from 9hepatitis C patients,respectively;and thestatus of HCV replication in extrahepatic tissueswas studied.RESULTS By RT-PCR,all 9 patients werepositive for HCV RNA in kidney,heart,pancreas,and intestine,but only 6(66.7%)patients were positive for HCV replicativeintermediate.HCV RNA and HCV antigens weredetected in kidney,heart,pancreas,intestine,adrenal gland,lymph node,and gallbladder in 5(55.6%)and 6(66.7%)patients by ISH andimmunohistochemistry,respectively.HCV RNA and HCV antigens were not detected in theseextrahepatic organs in 3(33.3%)patients,although their livers were positive for HCV.HCVreplicative intermediate detected by RT-PCR wasconsistent with HCV RNA and HCV antigensdetected by ISH and immunohistochemistry(Kappa=0.42-0.75).HCV RNA and HCVantigens were detected in myocardial cells,epithelial cells of intestinal gladular,interstitialcells of kidney,epithelial cells of tubules andglomerulus,pancreas acinar cells and epithelialcells of pancreatic duct,epithelial cells ofmucous membrane sinus of gallbladder,cortexand medulla cells in adrenal gland,andmononuclear cells in lymph node.HCV RNA wasalso detected in bile duct epithelial cells,sinusoidal cells,and mononuclear cells in livertissues by ISH.CONCLUSION HCV can infect extrahepatictissues,and many various tissue cells maysupport HCV replication;extrahepatic HCVinfection and replication may be of'concomitantstate'in most of patients with hepatitis C.Theinfected extrahepatic tissues might act as areservoir for HCV,and play a role in both HCVpersistence and reactivation of infection.HCVas an etiologic agent replicating and expressingviral proteins in extrahepatic tissues itselfcontributes to extrahepatic syndromeassociated.HCV infection in a few patients withchronic HCV infection.

Exploring the hepatitis C virus genome using single molecule realtime sequencing

Single molecular real-time(SMRT)sequencing,also called third-generation sequencing,is a novel sequencing technique capable of generating extremely long contiguous sequence reads.While conventional short-read sequencing cannot evaluate the linkage of nucleotide substitutions distant from one another,SMRT sequencing can directly demonstrate linkage of nucleotide changes over a span of more than 20 kbp,and thus can be applied to directly examine the haplotypes of viruses or bacteria whose genome structures are changing in real time.In addition,an error correction method(circular consensus sequencing)has been established and repeated sequencing of a single-molecule DNA template can result in extremely high accuracy.The advantages of long read sequencing enable accurate determination of the haplotypes of individual viral clones.SMRT sequencing has been applied in various studies of viral genomes including determination of the full-length contiguous genome sequence of hepatitis C virus(HCV),targeted deep sequencing of the HCV NS5A gene,and assessment of heterogeneity among viral populations.Recently,the emergence of multi-drug resistant HCV viruses has become a significant clinical issue and has been also demonstrated using SMRT sequencing.In this review,we introduce the novel third-generation PacBio RSII/Sequel systems,compare them with conventional next-generation sequencers,and summarize previous studies in which SMRT sequencing technology has been applied for HCV genome analysis.We also refer to another long-read sequencing platform,nanopore sequencing technology,and discuss the advantages,limitations and future perspectives in using these thirdgeneration sequencers for HCV genome analysis.

Monoclonal antibodies:Principles and applications of immmunodiagnosis and immunotherapy for hepatitis C virus

Hepatitis C virus(HCV) is a major health problem worldwide. Early detection of the infection will help better management of the infected cases. The monoclonal antibodies(m Ab) of mice are predominantly used for the immunodiagnosis of several viral,bacterial,and parasitic antigens. Serological detection of HCV antigens and antibodies provide simple and rapid methods of detection but lack sensitivity specially in the window phase between the infection and antibody development. Human mA b are used in the immunotherapy of several blood malignancies,such as lymphoma and leukemia,as well as for autoimmune diseases. In this review article,we will discuss methods of mouse and human monoclonal antibody production. We will demonstrate the role of mouse mA b in the detection of HCV antigens as rapid and sensitive immunodiagnostic assays for the detection of HCV,which is a major health problem throughout the world,particularly in Egypt. We will discuss the value of HCV-neutralizing antibodies and their roles in the immunotherapy of HCV infections and in HCV vaccine development. We will also discuss the different mechanisms by which the virus escape the effect of neutralizing mA b. Finally,we will discuss available and new trends to produce antibodies,such as egg yolk-based antibodies(Ig Y),production in transgenic plants,and the synthetic antibody mimics approach.

Immune mechanisms of vaccine induced protection against chronic hepatitis C virus infection in chimpanzees

Hepatitis C virus(HCV) infection is characterized by a high propensity for development of life-long viral persistence. An estimated 170 million people suffer from chronic hepatitis caused by HCV. Currently,there is no approved prophylactic HCV vaccine available.With the near disappearance of the most relevant animal model for HCV,the chimpanzee,we review the progression that has been made regarding prophylactic vaccine development against HCV. We describe the results of the individual vaccine evaluation experiments in chimpanzees,in relation to what has been observed in humans. The results of the different studies indicate that partial protection against infection can be achieved,but a clear correlate of protection has thus far not yet been defined.

Studies on hepatitis C virus and prevalence of its infection in China

HCV infection is one of the most important health problems in China. In this project starting from 1989, the authors carried out a survey on the prevalence of HCV infection in eastern areas of China, collected large amounts of blood samples from individuals of high risk groups from Shanghai and the Provinces of Jiangsu,Hebei, Shandong and Hunan, constructed a random-primed Chinese HCV cDNA λgt11 library, developed diagnostic systems for the detection of anti-HCV, HCV genomic RNA as well as for HCV genotyping,and investigated the possible relationship between HCV infection and the development of hepatocellular carcinoma (HCC). According to our epidemiological data, about 2%-5% of the population was estimated to be infected by HCV in this country. The high anti-HCV positive rate (4. 1% - 65. 5% ) in blood donors indicated that HCV infection was a principal cause of post-transfusion hepatitis. DNA sequencing data of the clones obtained by immunoscreening or PCR method from the cDNA library showed that HCV genotype Ⅱ was the major strain in China. Some of the antigenic epitopes identified from HCV C and NSS regions-derived clones were chosen for the development of anti-HCV EIA kit. The kit showed good agreement with that from UBI, with the total coincidence of 99. 1 % in 736 specimens,and seemed to be more adaptive in the detection of Chinese hepatitis C patients. It was interesting that HCV RNA was detectable in 33. 3% liver tissue specimens of HCC patients. This rate is much higher than that of anti-HCV (10. 5%) in serum specimens of these patients. By in situ hybridization and peroxidase-antiperoxidase (PAP) detection, 26. 0% and 28. 8% of HCC liver specimens were found positive for HCV RNA and antigens. Our findings demonstrated that HCV infection was also a high risk factor of HCC in China.

Murine antibody against E2 can capture hepatitis C virus in vitro

Objective To find neutralizing antibody candidates against hepatitis C virus (HCV) infection.Methods We constructed two eukaryotic expression vectors which contained the E1 and E2 gene of HCV, and detected their expression in mammalian cells with transient expression. BALB/c mice were given subculaneous injections of constructed vectors combined with the IL-2 gene intraepidermally and evaluated for induced humoral immune responses by enzyme-linked immunosorbent assay (ELISA). We used an antibody-virus interaction assay to analyze the interaction of the antisera and HCV viral particles in vitro.Results Anti E1 and anti-E2 antisera were obtained from immunized mice. The serum of mice immunized with the E2 gene immunoprecipitated the HCV isolate in source serum and reacted with the isolates unrelated to the original one.Conclusions Anti-E2 antibody in induced mice can cross-reactively capture HCV particles, highlighting the possibility of generating broadly reactive anti-E2 antibodies.

Prevalence of HCV Antibody and its Associated Factors: A Study from Sentinel Hospitals in China

Objective The prevalence and related factors of serum anti-HCV in different regions and hospitals have not been studied extensively in China. We used routine screening data to determine the prevalence of HCV antibody in hospital patients, evaluate the epidemic trend of hepatitis C and formulate screening strategies.Methods Patient information and HCV antibody testing results were collected from January 2017 to December 2019 in 77 HCV sentinel hospitals in China. Univariate and multivariate logistic regression was used to determine the characteristics and associations.Results HCV antibody prevalence rates were distinct among patients in different departments, with a range of 0.33%–6.93%. Patients who were admitted to the liver disease-related departments(aOR =10.76;95% CI, 10.27–11.28), Internal Medicine(aOR = 2.87;95% CI, 2.75–3.00), and Department of Surgery(aOR = 1.95;95% CI, 1.87–2.04), were more likely to be tested for HCV antibody positive. HCV antibody prevalence was associated with patients aged 45 years and older(aOR = 2.74;95% CI,2.69–2.80), testing in infetious disease hospitals(aOR = 2.33;95% CI, 2.26–2.40) and secondary hospitals(aOR = 1.72;95% CI, 1.69–1.75). Patients in sentinel hospitals of the Northeast(aOR = 12.75;95% CI,12.40–13.11), the Central(aOR = 1.65;95% CI, 1.61–1.70), and the West(aOR = 1.78;95% CI, 1.73–1.83)China had higher HCV prevalence than those who were in the Eastern coastal area. Conclusion Those who were over 45 years old and saw doctors for liver diseases, and invasive diagnosis and treatment should be referred to HCV antibody testing.

Autoantibody profiles in autoimmune hepatitis and chronic hepatitis C identifies similarities in patients with severe disease

To determine how the auto-antibodies (Abs) profiles overlap in chronic hepatitis C infection (CHC) and autoimmune hepatitis (AIH) and correlate to liver disease.METHODSLevels of antinuclear Ab, smooth muscle antibody (SMA) and liver/kidney microsomal-1 (LKM-1) Ab and markers of liver damage were determined in the sera of 50 patients with CHC infection, 20 AIH patients and 20 healthy controls using enzyme linked immunosorbent assay and other immune assays.RESULTSWe found that AIH patients had more severe liver disease as determined by elevation of total IgG, alkaline phosphatase, total serum bilirubin and serum transaminases and significantly higher prevalence of the three non-organ-specific autoantibodies (auto-Abs) than CHC patients. Antinuclear Ab, SMA and LKM-1 Ab were also present in 36% of CHC patients and related to disease severity. CHC cases positive for auto-Abs were directly comparable to AIH in respect of most markers of liver damage and total IgG. These cases had longer disease duration compared with auto-Ab negative cases, but there was no difference in gender, age or viral load. KLM-1+ Ab CHC cases showed best overlap with AIH.CONCLUSIONAuto-Ab levels in CHC may be important markers of disease severity and positive cases have a disease similar to AIH. Auto-Abs might have a pathogenic role as indicated by elevated markers of liver damage. Future studies will unravel any novel associations between these two diseases, whether genetic or other.

Replication priority of hepatitis C virus genotype 2a in a Chinese cohort

HCV genotypes have been documented in clinical practice.The aim of this study was to determine the replication priority of different HCV genotypes in a Chinese HCV positive cohort.Serum samples from 491 apparently healthy Chinese blood donors testing positive for HCV antibodies and naive to antiviral drug therapy were tested.Genotyping analysis showed that genotypes 1b and 2a were predominant and accounted for 77.6%of the HCV infections.Among the genotype groups,individuals infected with genotype 2a had an HCV RNA viral load(108 copies/mL)about 200-fold(lg,2.3)greater than those infected with other genotypes(10^(4)–10^(5)copies/mL)indicating a replication priority of genotype 2a.However,there was no correlation between HCV genotype and antibody response suggesting that the amplification advantage of genotype 2a results from a favorable interaction with the host cellular environment.In conclusion,HCV genotypes 1b and 2a are the predominant genotypes in China and genotype 2a possesses a significant replication priority compared with the other genotypes.This suggests the existence of host cellular factors that may act as drug-targets for entirely clearing HCV infection in the future.

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

AIM To prepare hybridoma cell lines which secrete anti HCV recombinant NS3 and NS5 proteins′ monoclonal antibodies, and to evaluate their usage in the study of the distribution of HCV NS3 and NS5 antigen in liver tissues. METHODS The hybridoma cell lines were raised using the spleen cells of BALB/C mouse immunized with recombinant NS3 and NS5 proteins according to the conventional protocols. The antibody secreting cells were screened using solid phase ELISA and cloned by limited dilution method. In order to determine the specificity of these hybridoma cell lines, the culture supernatant of these cells was western blot assayed with expression and nonexpresion E. coli and ELISA with other antigens, including HCV core and NS3 and HBsAg. Immunohistochemistry of 51 cases paraffin embedded liver tissues was performed to determine the distribution of HCV NS5 antigen as well as NS3 antigen in liver tissues. RESULTS Eight hybridoma cell lines secreting monoclonal antibodies against HCV NS3 and NS5 proteins were raised. They are named 2B6, 2F3, 3D8, 3D9, 8B2, 6F11, 4C6 and 7D9. Among them only 2B6 against NS3 protein can react with the polypipetides of C7 that is another recombinant polypipetides of NS3 gene. Others have no reaction with HCV core and HBsAg of HBV, and there is no cross reaction between NS3Ag and anti NS5Ag McAb and between NS5Ag and anti NS3 McAb. The immunohistochemistry results indicate that no HCV antigen was detected in the specimens of HBV infection in 20 cases. In 31 HCV infected specimens the positive rate of NS3Ag and NS5Ag are 51 6% (16/31) and 54 9% (17/31), respectively. There were six pure HCV infected specimens in these 31 specimens and half of them were HCV NS3Ag and NS5Ag positive. In the co infection of HBV and HCV group the positive rate of NS3Ag and NS5Ag were 52% (13/25) and 56% (14/25), respectively, almost the same with that of pure HCV infected group. The positive rates of HCV antigens were 70 6% (12/17) and 76 5% (13/17) in CAC patients. CONCLUSION The monoclonal antibodies we prepared are specific to the recombinant HCV NS3 and NS5 proteins and can be used in the clinical immunohistochemistry diagnosis.

Evaluate the prevalence and trend of hepatitis B and hepatitis C in Nahavand: west of Iran, 2013–2017

Viral hepatitis is a global threat to public health and one of the leading causes of death worldwide.Often,acute viral cases in children and adults are associated with viral hepatitis A,B,C,D and E,or co-infection with two types of hepatitis.Infection with these viruses is a global health problem and continuous efforts are in place to identify infected people through targeted screening,preventing new infections through vaccination,monitoring and treating people at risk for complications of all types of hepatitis.The aim of this study was to determine the evaluate the prevalence and trends of hepatitis B and C infection in the Nahavand city during 5 consecutive years(2013–2017).The total number of patients with hepatitis B and C was 141 persons from March 2013 to March 2017,of these,101 had hepatitis B,and 40 had hepatitis C.The prevalence of hepatitis B and C was higher in men than women.The percentage frequency hepatitis B in the city in the last five years was 0.05 percent.11 cases(10.89%)pregnant women and Six cases(5.9%)receiving blood(blood transfusions)in Hepatitis B was observed.the prevalence of hepatitis C was 0.2%at the end of 2017.The study on the cause of hepatitis C in Nahavand has shown that 21(52.5%)of the total of 40 people were infected with addiction.The interesting point in this report is that according to reports from viral hepatitis testing questionnaires,24 of 101 people with type B hepatitis have 23.7%of people with a history of complete vaccination of hepatitis B and one person(0.9%)had incomplete vaccination.A significant relationship was found between the level of education and the prevalence of hepatitis(P=0.005).