Tools for the diagnosis of hepatitis C virus infection and hepatic fibrosis staging

Hepatitis C virus(HCV)infection represents a major public health issue.Hepatitis C can be cured bytherapy,but many infected individuals are unaware of their status.Effective HCV screening,fast diagnosis and characterization,and hepatic fibrosis staging are highly relevant for controlling transmission,treating infected patients and,consequently,avoiding end-stage liver disease.Exposure to HCV can be determined with high sensitivity and specificity with currently available third generation serology assays.Additionally,the use of point-of-care tests can increase HCV screening opportunities.However,active HCV infection must be confirmed by direct diagnosis methods.Additionally,HCV genotyping is required prior to starting any treatment.Increasingly,high-volume clinical laboratories use different types of automated platforms,which have simplified sample processing,reduced hands-on-time,minimized contamination risks and human error and ensured full traceability of results.Significant advances have also been made in the field of fibrosis stage assessment with the development of non-invasive methods,such as imaging techniques and serum-based tests.However,no single test is currently available that is able to completely replace liver biopsy.This review focuses on approved commercial tools used to diagnose HCV infection and the recommended hepatic fibrosis staging tests.

Hepatitis C virus: Virology, diagnosis and management ofantiviral therapy

Hepatitis C virus (HCV) infects approximately 170 million individuals worldwide. Prevention of HCV infection complications is based on antiviral therapy with the combination of pegylated interferon alfa and ribavirin. The use of serological and virological tests has become essential in the management of HCV infection in order to diagnose infection, guide treatment decisions and assess the virological response to antiviral therapy. Anti- HCV antibody testing and HCV RNA testing are used to diagnose acute and chronic hepatitis C. The HCV genotype should be systematically determined before treatment, as it determines the indication, the duration of treatment, the dose of ribavirin and the virological monitoring procedure. HCV RNA monitoring during therapy is used to tailor treatment duration in HCV genotype 1 infection, and molecular assays are used to assess the end-of-treatment and, most importantly the sustained virological response, i.e. the endpoint of therapy.

Potential mechanisms of hepatitis B virus induced liver injury

Chronic active hepatitis(CAH) is acknowledged as an imperative risk factor for the development of liver injury and hepatocellular carcinoma.The histological end points of CAH are chronic inflammation,fibrosis and cirrhosis which are coupled with increased DNA synthesis in cirrhotic vs healthy normal livers.The potential mechanism involved in CAH includes a combination of processes leading to liver cell necrosis,inflammation and cytokine production and liver scaring(fibrosis).The severity of liver damage is regulated by Hepatitis B virus genotypes and viral components.The viral and cellular factors that contribute to liver injury are discussed in this article.Liver injury caused by the viral infection affects many cellular processes such as cell signaling,apoptosis,transcription,DNA repair which in turn induce radical effects on cell survival,growth,transformation and maintenance.The consequence of such perturbations is resulted in the alteration of bile secretion,gluconeogenesis,glycolysis,detoxification and metabolism of carbohydrates,proteins,fat and balance of nutrients.The identification and elucidation of the molecular pathways perturbed by the viral proteins are important in order to design effective strategy to minimize and/or restore the hepatocytes injury.

Hepatitis B virus infection in Latin America:A genomic medicine approach

Hepatitis B virus(HBV)infection is the leading cause of severe chronic liver disease.This article provides a critical view of the importance of genomic medicine for the study of HBV infection and its clinical outcomes in Latin America.Three levels of evolutionary adaptation may correlate with the clinical outcomes of HBV infection.Infections in Latin America are predominantly of genotype H in Mexico and genotype F in Central and South America;these strains have historically circulated among the indigenous population.Both genotypes appear to be linked to a benign course of disease among the native and mestizo Mexicans and native South Americans.In contrast,genotypes F,A and D are common in acute and chronic infections among mestizos with Caucasian ancestry.Hepatocellular carcinoma is rare in Mexicans,but it has been associated with genotype F1b among Argentineans.This observation illustrates the significance of ascertaining the genetic and environmental factors involved in the development of HBV-related liver disease in Latin America,which contrast with those reported in other regions of the world.

Enigmatic origin of hepatitis B virus:An ancient travelling companion or a recent encounter?

Hepatitis B virus(HBV)is the leading cause of liver disease and infects an estimated 240 million people worldwide.It is characterised by a high degree of genetic heterogeneity because of the use of a reverse transcriptase during viral replication.The ten genotypes(A-J)that have been described so far further segregate into a number of subgenotypes which have distinct ethno-geographic distribution.Genotypes A and D are ubiquitous and the most prevalent genotypes in Europe(mainly represented by subgenotypes D1-3 and A2);genotypes B and C are restricted to eastern Asia and Oceania;genotype E to central and western Africa;and genotypes H and F(classified into 4 subgenotypes)to Latin America and Alaska.This review summarises the data obtained by studying the global phylodynamics and phylogeography of HBV genotypes,particularly those concerning the origin and dispersion histories of genotypes A,D,E and F and their subgenotypes.The lack of any consensus concerning the HBV substitution rate and the conflicting data obtained using different calibration approaches make the time of origin and divergence of the various genotypes and subgenotypes largely uncertain.It is hypothesised that HBV evolutionary rates are time dependent,and that the changes depend on the main transmission routes of the genotypes and the dynamics of the infected populations.

Hepatitis B virus infection and the risk of hepatocellular carcinoma

Epidemiological studies have provided overwhelming evidence for a causal role of chronic hepatitis B virus(HBV) infection in the development of hepatocellular carcinoma(HCC).However,the pathogenesis of HBV infection and carcinogenesis of HBV-associated HCC are still elusive.This review will summarize the current knowledge on the mechanisms involved in HBV-related liver carcinogenesis.The role of HBV in tumor formation appears to be complex,and may involve both direct and indirect mechanisms.Integration of HBV DNA into the host genome occurs at early steps of clonal tumor expansion,and it has been shown to enhance the host chromosomal instability,leading to large inverted duplications,deletions and chromosomal translocations.It has been shown that the rate of chromosomal alterations is increased significantly in HBV-related tumors.Prolonged expression of the viral regulatory HBV x protein may contribute to regulating cellular transcription,protein degradation,proliferation,and apoptotic signaling pathways,and it plays a critical role in the development of hepatocellular carcinoma.

Determining hepatitis C virus genotype distribution among high-risk groups in Iran using real-time PCR

AIM: To assess hepatitis C virus (HCV) genotype patterns among high-risk Iranian groups, using real-time RT-PCR.

Hepatitis B virus genotypes in chronic liver disease patients from New Delhi,India

AIM: To study the Hepatitis B virus (HBV) genotypes and their effect on the progression and outcome in patients with chronic liver diseases from New Delhi, India. METHODS: Sera from 100 HBV-related chronic liver disease (CLDB) cases were tested for HBV genotype using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Type-specific primers-based PCR (TSP-PCR) targeting to the surface (S) gene encoding hepatitis B surface antigen. RESULTS: Only genotypes A and D were present and genotype D was dominant. Genotype D was present in all CLDB patient categories. The genotype distribution for the 100 patients with CLDB was as follows: genotype A, 16/100 (16%) (7/40- 17% chronic hepatitis B (CHB); 8/47, 17%, HBV-related cirrhosis (CRB); 1/13, 7.6%, HBV-related hepatocellular carcinoma (HCCB); genotype D- 84/100 (84%) (32/40- 80% CHB; 38/47- 81%, CRB; 11/13, 85%, HCCB); genotype A + D, 3/100 (3%) (1/40- 3% CHB; 1/47- 2%, CRB; 1/13, 7.6%, HCCB); C, 0; B, 0; E, 0; F, 0; G 0, H 0; (P < 0.01, genotype D vs A). CONCLUSION: Only HBV genotypes A and D were present in patients with CLDB from New Delhi, India. Compared with genotype D, genotype A patients had no significant clinical or biochemical differences (P > 0.05). Mixed infection with genotype A and D were seen in 3% of the cases. Genotype D was the dominant genotype prevalent in all patient categories.

Screening and identification of bioactive compounds from citrus against non-structural protein 3 protease of hepatitis C virus genotype 3a by fluorescence resonance energy transfer assay and mass spectrometry

BACKGROUND Hepatitis C virus genotype 3a(HCV G3a)is highly prevalent in Pakistan.Due to the elevated cost of available Food and Drug Administration-approved drugs against HCV,medicinal natural products of potent antiviral activity should be screened for the cost-effective treatment of the disease.Furthermore,from natural products,active compounds against vital HCV proteins like non-structural protein 3(NS3)protease could be identified to prevent viral proliferation in the host.AIM To develop cost-effective HCV genotype 3a NS3 protease inhibitors from citrus fruit extracts.METHODS Full-length NS3 without co-factor non-structural protein 4A(NS4A)and codon optimized NS3 protease in fusion with NS4A were expressed in Escherichia coli.The expressed protein was purified by metal ion affinity chromatography and gel filtration.Citrus fruit extracts were screened using fluorescence resonance energy transfer(FRET)assay against the protease and polyphenols were identified as potential inhibitors using electrospray ionization-mass spectrometry(MS)/MS technique.Among different polyphenols,highly potent compounds were screened using molecular modeling approaches and consequently the most active compound was further evaluated against HCV NS4A-NS3 protease domain using FRET assay.RESULTS NS4A fused with NS3 protease domain gene was overexpressed and the purified protein yield was high in comparison to the lower yield of the full-length NS3 protein.Furthermore,in enzyme kinetic studies,NS4A fused with NS3 protease proved to be functionally active compared to full-length NS3.So it was concluded that co-factor NS4A fusion is essential for the purification of functionally active protease.FRET assay was developed and validated by the half maximal inhibitory concentration(IC50)values of commercially available inhibitors.Screening of citrus fruit extracts against the native purified fused NS4A-NS3 protease domain showed that the grapefruit mesocarp extract exhibits the highest percentage inhibition 91%of protease activity.Among the compounds identified by LCMS analysis,hesperidin showed strong binding affinity with the protease catalytic triad having S-score value of-10.98.CONCLUSION Fused NS4A-NS3 protease is functionally more active,which is effectively inhibited by hesperidin from the grapefruit mesocarp extract with an IC50 value of 23.32μmol/L.

Effectiveness of entecavir in preventing hepatocellular carcinoma development is genotype-dependent in hepatitis B virus-associated liver cirrhosis

BACKGROUND The oral nucleos(t)ide analogue,entecavir(ETV)was demonstrated to reduce the rate of hepatocellular carcinoma(HCC)in patients with hepatitis B virus(HBV)-associated liver cirrhosis.However,the reduction of HCC differs in various regions of the world.AIM To investigate the reduction of HCC development due to ETV therapy by metaanalysis.METHODS We surveyed the differences in HCC development following ETV treatment based on published articles using PubMed(2004-2019).RESULTS The regions with the most marked reduction in HCC development due to ETV therapy were Spain(1.0%/year)and Canada(Southern part,1.3%/year),and the most ineffective areas were South Korea(3.6%-3.8%/year),China(3.3%/year),Taiwan(2.4%-3.1%/year),and Hong Kong(2.8%/year).Following ETV administration,the incidence of HCC in genotype D regions(1.89%±0.28%/year,mean±SE)was significantly lower than that in genotype C regions(2.91%±0.24%/year,P<0.01).With regard to the initial HBV-DNA level,in genotype C patients(average:5.61 Log10IU/mL)this was almost the same as that in genotype D patients(average:5.46 Log10IU/mL).Moreover,there was no association between the prevalence ratio of HBV and the incidence of HCC on ETV treatment.CONCLUSION The effectiveness of ETV in preventing HCC development in HBV-associated liver cirrhosis is genotype-dependent.

Evaluation of an attenuated vaccine candidate based on the genotype C of bovine parainfluenza virus type 3 in albino guinea pigs

Bovine parainfluenza virus type 3（BPIV3） is considered as one of the most important respiratory tract pathogens of both young and adult cattle, and widespread among cattle in the world. BPIV3 was first reported in China in 2008 and four strains of BPIV3 were isolated from Shandong Province, known as genotype C（BPIV3c）. Pathogen investigations had shown that BPIV3 c infection was very common among cattle in China. To date, BPIV3 can be classified into genotypes A, B and C based on genetic and phylogenetic analysis. Serological survey also demonstrates that BPIV3 infection is widespread in China, however, there is still no available vaccine for BPIV3 prevention in China nowadays. In the present study, the BPIV3 c strain SD0835 was continuously passaged on Madin-Darby bovine kidney（MDBK） cells for hundreds of times, and the pathogenicity of passage 209 was reduced in guinea pigs. The passage 209 of BPIV3 c strain SD0835 was used as a live vaccine candidate to immunize the guinea pigs. The vaccination results revealed that two vaccinations could induce excellent serum neutralizing antibody responses as well as proliferation of T lymphocytes. The vaccinated guinea pigs were well protected against challenge with a low passage of BPIV3 c strain SD0835. Additionally, the percentages of CD4~＋ and CD8~＋ T cell subsets of animals in vaccinated group increased after immunization; T cell subsets on day 2 after challenge in both groups decreased, and the decline of CD4~＋ and CD8~＋ T cell subsets levels of four guinea pigs in vaccinated group was relatively moderate, comparing with that of the control group. These data support further testing of the attenuated virus as an effective candidate vaccine.

Hepatocellular carcinoma in African Blacks: Recent progress in etiology and pathogenesis

Occult hepatitis B virus (HBV) infection was shown to be present in 75% of Black Africans with hepatocellular carcinoma (HCC) in whom the tumor was hitherto not thought to be caused by chronic HBV infection. The association between chronic HBV infection and the development of the tumor is thus even closer than was originally thought. HBV viral load was found to be significantly higher in patients with HCC than in Black African controls. As in other populations, HBV e antigen-positive patients with hepatocellular carcinoma had significantly higher viral loads than patients negative for this antigen. The significance of this finding is discussed. The risk for HCC development with genotype A of HBV, the predominant genotype in African isolates, has not been investigated. Genotype A was shown to be 4.5 times more likely than other genotypes to cause HCC in Black Africans, and tumours occurred at a significantly younger age. Increasing numbers of patients with human immunodeficiency virus (HIV) and HBV co-infection are being reported to develop HCC. A preliminary case/control comparison supports the belief that HIV co-infection enhances the hepatocarcinogenic potential of HBV. A study from The Gambia provides the first evidence that dietary exposure to afltoxin B1 may cause cirrhosis and thatthis may play a contributory role in the pathogenesis of aflatoxin-induced HCC. An animal model has provided experimental support for the clinical evidence that dietary iron overload in the African is directly hepatocarcinogenic, in addition to causing the tumor indirectly through the development of cirrhosis.

Extended-therapy duration for chronic hepatitis C,genotype 1:The long and the short of it

With pegylated interferon and ribavirin, more than half of all chronically-infected hepatitis C patients can achieve a sustained virologic response; however, patients with genotype 1 infections and those with other poor prognostic factors have relatively inferior treatment response rates. Since new therapies are still years away from approval, it is incumbent upon providers to maximize the therapeutic efficacy of today＇s treatment. The later the virus is undetectable in serum during treatment, the less likely it will be eradicated. Patients with a delayed or slow virologic response to therapy （at least a 2-1og10 decrease in baseline hepatitis C RNA yet detectable viremia at 12 wk of therapy and undetectable virus 12 wk subsequently） may, therefore, benefit from an extended therapy course beyond one of standard duration. Although higher rates of treatment discontinuation may plague this approach, 72 wk of treatment for genotype 1-infected slow-responders may improve response rates and diminish relapse rates relative to those of 48 wk. Based on data from both viral kinetic and clinical studies, therapy prolongation in slow responders may be a reasonable strategy to improve response rates in these treatment-refractory patients.

Efficacy and safety of telaprevir- and simeprevir-based triple therapies for older patients with chronic hepatitis C

AIMTo evaluate and compare the efficacy and safety of telaprevir (TVR)-and simeprevir (SMV)-based triple therapies in elderly patients, specifically patients aged 66 years or older. METHODSThe present study enrolled 112 and 76 Japanese patients with chronic hepatitis C virus genotype 1b infection who were treated with a 12-wk TVR-based or SMV-based triple therapy, respectively, followed by a dual therapy that included pegylated interferon α and ribavirin (RBV) for 12 wk. The patients were categorized into two groups according to age as follows: A younger group of patients aged ≤ 65 years old and an older group of patients aged > 65 years old. Among the patients treated with TVR-based triple therapy, 34 patients were included in the older group. The median ages were 56 years (range: 28-65 years) in the younger group and 69 years (range: 66-81 years) in the older group. Among the patients treated with SMV-based triple therapy, 39 patients were included in the older group. The median ages were 59 years (range: 36-65 years) in the younger group and 71 years (range: 66-86 years) in the older group. The clinical, biochemical and virological data were analyzed before and during treatment. RESULTSAmong the patients treated with the TVR-based triple therapy, no significant difference in the sustained virological response (SVR) was found between the younger (80.8%) and older (88.2%) groups. The SVR rates for patients with the interleukin 28B (IL28B) (rs8099917) TG/GG-genotypes (73.9% and 60.0% in the younger and older groups, respectively) were significantly lower than for patients with the IL28B TT-genotype (86.3% and 92.9%, respectively). The cumulative exposure to RBV for the entire 24-wk treatment period (as a percentage of the target dose) was significantly higher in the younger group than in the older group (91.7% vs 66.7%, respectively, P vs 81.9%, respectively). A multivariate analysis identified the TT-genotype of IL28B (OR = 8.160; 95%CI: 1.593-41.804, P = 0.012) and the adherence of RBV (> 60%) (OR = 11.052; 95%CI: 1.160-105.273, P = 0.037) as independent factors associated with the SVR. Adverse events resulted in discontinuation of the treatment in 11.3% and 14.7% of the younger and older groups, respectively. Among the patients treated with the SMV-based triple therapy, no significant difference in the SVR rare was found between the younger (81.1%) and older (82.1%) groups. The SVR rates for patients with the IL28B TG/GG-genotypes (77.8% and 64.7% in the younger and older groups, respectively) were significantly lower than for patients with the IL28B TT-genotype (88.2% and 100%, respectively). A multivariate analysis identified the TT-genotype of IL28B as an independent factor associated with the SVR (OR = 9.677; 95%CI: 1.114-84.087, P = 0.040). Adverse events resulted in discontinuation of the treatment in 7.0% and 14.3% of patients in the younger and older groups, respectively. CONCLUSIONBoth TVR- and SMV-based triple therapies can be successfully used to treat patients aged 66 years or older with genotype 1b chronic hepatitis C. Genotyping of the IL28B indicates a potential to achieve SVR in these difficult-to-treat elderly patients.

Impact of IL28B gene polymorphisms rs8099917 and rs12980275 on response to pegylated interferon-α/ribavirin therapy in chronic hepatitis C genotype 4 patients

Host genetic factors may predict the outcome and treatment response in hepatitis C virus（HCV）infection.One of these factors is the single nucleotide polymorphisms of the interleukin 28B（IL28B）gene.We sought to evaluate the outcome of pegylated interferon and ribavirin therapy in association with IL-28B rs8099917 and rsl2980275 in patients infected with HCV genotype 4.A total of 180 patients with chronic hepatitis C were selected from Egyptians who have received combined therapy with pegylated interferon and ribavirin for 6 months and their response was evaluated after follow-up at 0,6,12,24 and 48 weeks from the beginning of the therapy.Blood samples were collected from responders and non-responders.Genomic DNA was extracted from whole blood and genotyping was carried out by polymerase chain reaction and restriction fragment length polymorphism（PCR-RFLP）.Our results showed that TT genotype of rs8099917 was associated with higher sustained viral response（SVR）rates and G allele represented a risk factor for failure of response（OR=3.7,CI=1.8：7.64）while rs12980275 was not significantly associated with SVR in genotype 4 Egyptian patients.The determination of 1L-28B SNPs may be useful in enhancing correct prediction of SVR achievement in treating this group of genotype 4 patients.

Genotyping of Hepatitis C Virus in Patients with Hepatocellular Carcinoma.

Forty-two patients with hepatocellular carcinoma（HCC）were examined for hepatitis C virus（HCV）RNA in liver tissues by reverse transcription-polymerase chain reaction（RT PCR）.Typing of HCV liver samples of 18 patientswas dependent on the amplication of NSregion gy PCR using type-specific primers.

Molecular epidemiology of respiratory syncytial virus

Objective To determine the epidemiologic pattern of subgroups A and B and genotypes of respiratory syncytial virus (RSV) during two noncontinuous epidemics during 1990-1991 and 1997-1998 in Beijing Methods Nasopharyngeal secretion (NPS) samples of RSV positive or RSV isolates tested by indirect immunofluorescence (IIF) assay were classified into subgroups A and B Isolates of RSV were divided into at least six different lineages, designated NP1 NP6, by restriction mapping of the N gene Np1, 3 and 6 were given by subgroup B isolates, while NP2, 4 and 5 were given by subgroup A isolates Strains of subgroup A were further subdivided into six lineages SHL1 SHL6 on the basis of the SH gene sequence SH lineages were closely related to each other and to NP1 NP6 Strains of SHL1, 3 and 4 were closely related and belonged to NP2, SHL2 and 6 to NP4, and SHL5 to NP5 Results Of 145 RSV NPS samples from the 1997-1998 epidemic, 83 (57 2%) were of subgroup B RSV positive, 62 (42 8%) of subgroup A RSV positive The rate of occurrence of subgroup A to B strains was about 1∶1 3 Two of 10 isolates during the epidemic were subgroup A strains, whereas 8 were subgroup B strains The rate of occurrence of subgroup A to B strains was 1∶4 Eight subgroup A strains of 10 isolates from the 1990-1991 epidemic were dominant; the proportion of subgroup A to B strains was 4∶1 With 10 RSV isolates in 1997-1998, all 2 subgroup A strains gave N gene fragment restriction pattern NP4, and fell into SH lineage SHL2, whereas 8 subgroup B strains all belonged to NP3 All 8 subgroup A isolates from the 1990-1991 epidemic gave pattern NP4, and fell into SHL2, while 2 subgroup B strains all belonged to NP3 The classification of subgroups A and B deduced from NP patterns corresponded to the definition of these subgroups by monoclonal antibodies Conclusions These observations confirm that subgroups A and B or multiple lineages of RSV co circulated in Beijing, but different genome types predominated each year Moreover, very similar viruses were isolated up to more than 5 years ago, indicating that despite apparent diversity of the subgroup A strains, the separate lineages might be relatively stable

An improved reverse genetics system for Newcastle disease virus genotype Ⅶ

Dear Editor, Newcastle disease virus （NDV）, also known as avian paramyxovirus serotype 1 （APMV-1）, is a member of the genus Avulavirus within the family Paramyxoviridae, or- der Mononegavirales （Miller et al., 2010）. Although all isolated NDV strains belong to a single serotype, epi- demiological studies have revealed that the genotype VII strain is currently the most prevalent circulating geno- type worldwide and is associated with many of the most recent outbreaks in China since 1997 （Liu et al., 2007）.

VIS Atlas:A Database of Virus Integration Sites in Human Genome from NGS Data to Explore Integration Patterns

Integration of oncogenic DNA viruses into the human genome is a key step in most virusinduced carcinogenesis.Here,we constructed a virus integration site(VIS)Atlas database,an extensive collection of integration breakpoints for three most prevalent oncoviruses,human papillomavirus,hepatitis B virus,and Epstein-Barr virus based on the next-generation sequencing(NGS)data,literature,and experimental data.There are 63,179 breakpoints and 47,411 junctional sequences with full annotations deposited in the VIS Atlas database,comprising 47 virus genotypes and 17 disease types.The VIS Atlas database provides(1)a genome browser for NGS breakpoint quality check,visualization of VISs,and the local genomic context;(2)a novel platform to discover integration patterns;and(3)a statistics interface for a comprehensive investigation of genotypespecific integration features.Data collected in the VIS Atlas aid to provide insights into virus pathogenic mechanisms and the development of novel antitumor drugs.