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.

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.

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.

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,order Mononegavirales(Miller et al.,2010).Although all isolated NDV strains belong to a single serotype,epidemiological studies have revealed that the genotype

Investigation of the genotype distribution of hepatitis C virus among Turkish population in Turkey and various European countries

Hepatitis C virus （HCV） infection is a global health problem. HCV is one of the leading causes for acute and chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The prevalence of HCV infection varies from 0. 1% to 0. 2% in the West, to 3% in Mediterranean countries and up to 6% in tropical areas. The prevalence in Turkey is about 1.8%. HCV is classified into six different genotypes （genotypes 1 -6 ）, each consisting of different subtypes. HCV genotypes and their subtypes coexist in various geographic locations but show different prevalence levels.