Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections,rt269L and rt269I

BACKGROUND The presence of two distinct hepatitis B virus(HBV)Pol RT polymorphisms,rt269L and rt269I,could contribute to the unique clinical or virological phenotype of HBV genotype C2.Therefore,a simple and sensitive method capable of identifying both types in chronic hepatitis B(CHB)patients infected with genotype C2 should be developed.AIM To develop a novel simple and sensitive locked nucleic acid(LNA)-real timepolymerase chain reaction(RT-PCR)method capable of identifying two rt269 types in CHB genotype C2 patients.METHODS We designed proper primer and probe sets for LNA-RT-PCR for the separation of rt269 types.Using synthesized DNAs of the wild type and variant forms,melting temperature analysis,detection sensitivity,and endpoint genotyping for LNA-RT-PCR were performed.The developed LNA-RT-PCR method was applied to a total of 94 CHB patients of genotype C2 for the identification of two rt269 polymorphisms,and these results were compared with those obtained by a direct sequencing protocol.RESULTS The LNA-RT-PCR method could identify two rt269L and rt269I polymorphisms of three genotypes,two rt269L types[‘L1’(WT)and‘L2’]and one rt269I type(‘I’)in single(63 samples,72.4%)or mixed forms(24 samples,27.6%)in 87(92.6%sensitivity)of 94 samples from Korean CHB patients.When the results were compared with those obtained by the direct sequencing protocol,the LNA-RT-PCR method showed the same results in all but one of 87 positive detected samples(98.9%specificity).CONCLUSION The newly developed LNA-RT-PCR method could identify two rt269 polymorphisms,rt269L and rt269I,in CHB patients with genotype C2 infections.This method could be effectively used for the understanding of disease progression in genotype C2 endemic areas.

T3098C and T53C Mutations of HBV Genotype C Is Associated with HBV Infection Progress

Objective To analyze the association between mutation（s） in preS region of HBV and hepatitis B disease progress in Chinese patients with genotype C chronic HBV infection. Methods Ninety-three patients with chronic genotype C HBV infection, including 24 asymptomatic carriers （ASC）, 26 patients with chronic hepatitis B （CHB）, 22 patients with liver cirrhosis （LC） and 21 HCC patients were investigated. Levels of HBV DNA, HBeAg, alanine aminotransferase （ALT）, asparate transaminase （AST） were measured. HBV preS region was analyzed by PCR direct sequencing. Results The prevalence of preS T3098C and T53C mutations ofgenotype C HBV was significantly higher in LC and HCC patients than ASC and CHB patients. The rate ofT3098C mutation in ASC, CHB, LC, and HCC patients were 0.00% （0/24）, 3.85% （1/26）, 9.09% （2/22）, and 30.77% （8/22）, respectively （P=0.0015）, while the rate of T53C mutation was I2.50% （3/24）, 3.85% （1/26）, 40.91% （9/22）, and 42.31% （11/26）, respectively （P=0.0012）. Conclusion The frequency of genotype C HBV preS T3098C and T53C mutations is associated with hepatitis B infection progression.

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.

鼠戊型肝炎病毒(Rocahepevirus ratti genotype C1)感染人类的研究进展

随着在中国香港、加拿大、西班牙和法国相继报道人感染鼠戊型肝炎病毒(Rocahepevirus ratti genotype C1;HEV-C1)病例,HEV-C1作为一种新发的人兽共患病毒,引起了全球的广泛关注。本研究对于HEV-C1感染人类相关的病原学特征、流行病学、基因组系统进化、动物和细胞模型以及病原学检测等进行系统综述,以期为HEV-C1的深入研究及疾病防控提供参考。

In vitro investigation of HBV clinical isolates from Chinese patients reveals that genotype C isolates possess higher infectivity than genotype B isolates

Hepatitis B virus(HBV)genotype B and C are two major genotypes that are prevalent in Asia and differ in natural history and disease progression.The impact of HBV genotypes on viral replication and protein expression has been explored by the transfection of hepatoma cells with replication-competent HBV DNA,which mimics the later stages of the viral life cycle.However,the influence of HBV genotypes on the early events of viral infection remains undetermined,mainly due to the difficulties in obtaining sufficient infectious viral particles for infection assays.Here,we report that a high-titer HBV inoculum can be generated from the transient transfection-based cell model after optimizing transfection conditions and modifying the HBV-expressing construct.By performing in vitro infection assays using transiently transfected derived viruses,we found that clinical genotype C isolates possessed higher infectivity than genotype B isolates.Moreover,we identified a naturally occurring mutation sL21S in small hepatitis B surface protein,which markedly decreased the infectivity of HBV genotype C isolates,but not that of genotype B isolates.In summary,using infectious viral particles provided by the optimized transient transfection-based cell model,we have been able to investigate a wide range of HBV variants on viral infectivity,which may contribute to our understanding of the reasons for different clinical outcomes in HBV infections and the development of therapeutic drugs targeting the early stages of HBV life cycle.

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.

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.

Remission of hepatotoxicity in chronic pulmonary aspergillosis patients after lowering trough concentration of voriconazole

BACKGROUND Chronic pulmonary aspergillosis(CPA)is a rare syndrome that is often accompanied by gradual lung tissue destruction.Voriconazole is usually employed as the first-line agent for CPA treatment.However,some patients can develop hepatotoxicity and often were forced to stop voriconazole treatment.AIM To record the improving trend of liver function and the therapeutic effects in patients after lowering the trough concentration of voriconazole.METHODS This study retrospectively analyzed 12 adult CPA patients who developed hepatotoxicity during the voriconazole treatment.In these patients,the oral dose was reduced to 3/4 or 1/2 of the standard dose(4 mg/kg,twice daily),and the lower limit of voriconazole trough concentration was maintained more than 0.5μg/m L.The trend of remission of liver toxicity after drug reduction in 12 patients was recorded.During the same period,25 patients who received standard doses served as the control group.Data from the two groups were collected and analyzed for different parameters such as demographic characteristics,underlying pulmonary disorders,laboratory tests,and therapeutic effect.The differences between the two groups were statistically compared.RESULTS Hepatotoxicity occurred in 12 patients within 28-65 d after oral voriconazole treatment.Hepatotoxicity was mainly manifested by the significantly increased level of gamma-glutamyltransferase and a slight increase of alanine aminotransferase and aspartate aminotransferase.The oral dose of voriconazole was reduced to approximately 3 mg/kg in seven patients and approximately 2 mg/kg in five patients.The average trough concentrations for the 12 patients before and after voriconazole oral dose reduction were 3.17±1.47μg/m L(1.5-6.0μg/m L)and 1.70±0.78μg/m L(0.6-3.3μg/m L),respectively(P=0.02).After lowering the trough concentrations,the hepatotoxicity was alleviated in all the patients.However,gamma-glutamyltransferase levels declined slowly.After 4 mo of treatment,7 of the 12 patients were successfully treated in the low trough concentrations group(41.7%).Similarly,8 of the 25 patients in the standard treatment dose group(32.0%)were effectively treated.There was no statistical difference between the groups(P=0.72).CONCLUSION Reducing the lower limit of the voriconazole trough concentration to 0.5μg/m L can alleviate the hepatotoxicity and maintained certain clinical efficacy in CPA patients;however,patients should be closely monitored.

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.

Vitamin A supplementation downregulates ADH1C and ALDH1A1 mRNA expression in weaned beef calves

Our previous studies demonstrated that oral vitamin A supplementation during late-stage pregnancy and the neonatal stage enhances birth weight,growth performance,and mRNA expression related to muscle and preadipocyte development in beef cattle.The alcohol dehydrogenase 1C(ADH1C)c.-64T>C genotype also correlated with vitamin A concentration in beef production.This study aimed to investigate the effects of vitamin A supplementation on the muscle development and vitamin A metabolism in weaned beef calves with different ADH1C genotypes.Twenty male calves(90 d of age;initial BW:89.03 kg[SD 8.60])were stratified according to ADH1C genotype and vitamin A treatment(duration:3 months)and randomly assigned to 4 groups with a 22 factorial arrangement.Vitamin A treatments included the following:control(10,000 IU/kg of as-fed,a.TT type;b.TC type);treatment(40,000 IU/kg of as-fed,c.TT type;and d.TC type).Parameters including BW,FI,blood,longissimus dorsi muscle,and liver status during the experimental period were analyzed using the generalized linear model(GLM)procedure and Tukey's test by SAS 9.4 program.Serum vitamin A was significantly increased(P<0.05)in the vitamin A treatment group at 4 and 6 months of age.TT type calves showed higher serum vitamin A concentration(P<0.05)than the TC type calves.Serum triglyceride and non-esterified fatty acid(NEFA)levels increased(P<0.05)in the treatment group compared with the control at 6 months of age.However,BW,ADG and FI showed no differences between the groups.In addition,mRNA expression in longissimus dorsi muscle revealed upregulation of paired box 7(PAX7)(P<0.05)after the vitamin A treatment period based on biopsy results.Both ADH1C and aldehyde dehydrogenase(ALDH)1A1 mRNA expression was downregulated(P<0.01)by vitamin A supplementation.The TC type of ADH1C showed higher mRNA expression than the TT type.However,no effect was observed on adipogenic mRNA expression(preadipocyte factor-1[PREF-1],peroxisome proliferator-activated receptor gamma[PPARg],fatty acid binding protein 4[FABP4])in all groups.Our findings suggest that weaned calves treated with vitamin A may promote the storage of satellite cells by elevating PAX7 gene expression in the muscle.The TC type calves may show increased capacity for vitamin A metabolism,which can be used in genetically customizing feed management to maximize beef production in the calves.

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.

Generation of a Double KO Mouse by Simultaneous Targeting of the Neighboring Genes Tmem176a and Tmem176b Using CRISPR/Cas9:Key Steps from Design to Genotyping

The CRISPR/Cas9 system has been tailored to a revolutionary genetic tool because of its remarkable simplicity and efficacy.While complex genome editing in the mouse since the 1990 s has been dominated by the use of embryonic stem（ES） cells,CRISPR/Cas9 now offers a versatile and fast approach to precisely modify virtually any DNA regions directly in mouse zygotes.Yet,this relative simplicity does not preclude a conscientious preparatory work that is often neglected when initiating a project.Here,we describe the key steps leading to successful generation of a double knockout（KO） mouse by simultaneously targeting two homolog genes,Tmem176 a and Tmem176 b,which are located in the same genomic locus.Additionally,we show that similar efficiency can be obtained in a mixed genetic background or directly in the C57BL/6 inbred strain.Thus,presented as a detailed case study that should be helpful to the non-specialists,we focus on the genotyping strategy to anticipate the various possibilities.