Antiviral effects of hepatitis B virus S gene-specific anti-gene locked nucleic acid in transgenic mice

AIM To assess the antiviral effects of hepatitis B virus(HBV) S gene-specific anti-gene locked nucleic acid(LNA) in transgenic mice.METHODS Thirty HBV transgenic mice were acclimatized to laboratory conditions and positive for serum HBV surface antigen(HBs Ag) and HBV DNA, were randomly divided into 5 groups(n = 7), including negative control(blank control, unrelated sequence control), positive control(lamivudine, anti-sense-LNA), and anti-gene-LNA experimental group. LNA was injected into transgenic mice by tail vein while lamivudine was administeredby gavage. Serum HBV DNA and HBs Ag levels were determined by fluorescence-based PCR and enzymelinked immune sorbent assay, respectively. HBV S gene expression amounts were assessed by reverse transcription polymerase chain reaction. Positive rates of HBsA g in liver cells were evaluated immunohistochemistry.RESULTS Average rate reductions of HBs Ag after treatment on the 3 rd, 5 th, and 7 th days were 32.34%, 45.96%, and 59.15%, respectively. The inhibitory effect of antigene-LNA on serum HBs Ag peaked on day 7, with statistically significant differences compared with pretreatment(0.96 ± 0.18 vs 2.35 ± 0.33, P < 0.05) and control values(P < 0.05 for all). Average reduction rates of HBV DNA on the 3 rd, 5 th, and 7 th days were 38.55%, 50.95%, and 62.26%, respectively. This inhibitory effect peaked on the 7 th day after treatment with anti-gene-LNA, with statistically significant differences compared with pre-treatment(4.17 ± 1.29 vs 11.05 ± 1.25, P < 0.05) and control values(P < 0.05 for all). The mR NA levels of the HBV S gene(P < 0.05 for all) and rates of HBsA g positive liver cells(P < 0.05 for all) were significantly reduced compared with the control groups. Liver and kidney function, and histology showed no abnormalities. CONCLUSION Anti-gene-LNA targeting the S gene of HBV displays strong inhibitory effects on HBV in transgenic mice, providing theoretical and experimental bases for gene therapy in HBV.

Epstein-Barr virus is related with 5-aminosalicylic acid, tonsillectomy, and CD19^+ cells in Crohn's disease

AIM: To study anti-Epstein-Barr virus(EBV) Ig G antibodies in Crohn′s disease in relation to treatment, immune cells, and prior tonsillectomy/appendectomy.METHODS: This study included 36 CD patients and 36 healthy individuals(controls), and evaluated different clinical scenarios(new patient, remission and active disease), previous mucosa-associated lymphoid tissue removal(tonsillectomy and appendectomy) and therapeutic regimens(5-aminosalicylic acid, azathioprine, anti-tumor necrosis factor, antibiotics, and corticosteroids). T and B cells subsets in peripheral blood were analyzed by flow cytometry(markers included: CD45, CD4, CD8, CD3, CD19, CD56, CD2, CD3, TCRαβ and TCRγδ) to relate with the levels of anti-EBV Ig G antibodies, determined by enzyme-linked immunosorbent assay.RESULTS: The lowest anti-EBV Ig G levels were observed in the group of patients that were not in a specific treatment(95.4 ± 53.9 U/m L vs 131.5 ± 46.2 U/m L, P = 0.038). The patients that were treated with 5-aminosalicylic acid showed the highest anti-EBV Ig G values(144.3 U/m L vs 102.6 U/m L, P = 0.045). CD19+ cells had the largest decrease in the group of CD patients that received treatment(138.6 vs 223.9; P = 0.022). The analysis of anti-EBV Ig G with respect to the presence or absence of tonsillectomy showed the highest values in the tonsillectomy group of CD patients(169.2 ± 20.7 U/m L vs 106.1 ± 50.3 U/m L, P = 0.002). However, in the group of healthy controls, no differences were seen between those who had been tonsillectomized and subjects who had not been operated on(134.0 ± 52.5 U/m L vs 127.7 ± 48.1 U/m L, P = 0.523).CONCLUSION: High anti-EBV Ig G levels in CD are associated with 5-aminosalicylic acid treatment, tonsillectomy, and decrease of CD19+ cells.

Prospects for nucleic acid-based therapeutics against hepatitis C virus

In this review,we discuss recent advances in nucleic acid-based therapeutic technologies that target hepatitis C virus(HCV)infection.Because the HCV genome is present exclusively in RNA form during replication,various nucleic acid-based therapeutic approaches targeting the HCV genome,such as ribozymes,aptamers,siRNAs,and antisense oligonucleotides,have been suggested as potential tools against HCV.Nucleic acids are potentially immunogenic and typically require a delivery tool to be utilized as therapeutics.These limitations have hampered the clinical development of nucleic acid-based therapeutics.However,despite these limitations,nucleic acid-based therapeutics has clinical value due to their great specificity,easy and large-scale synthesis with chemical methods,and pharmaceutical flexibility.Moreover,nucleic acid therapeutics are expected to broaden the range of targetable molecules essential for the HCV replication cycle,and therefore they may prove to be more effective than existing therapeutics,such as interferon-αand ribavirin combination therapy.This review focuses on the current status and future prospects of ribozymes,aptamers,siRNAs,and antisense oligonucleotides as therapeutic reagents against HCV.

Construction of Three Nucleic Acid Vaccines of Swine Hepatitis E Virus ORF2 Ger\e Continuous Fragment

In order to develop swine hepatitis E （HE） genetically engineering vaccines, specific primers of genes LB1, LB2, LB3 of swine hepatitis E virus were designed and used for amplification, DNA amplieons generated by PCR assays were directly cloned into T-A plasmid and expressed using pEASY-M1 expression vector. Three recombinant eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3 were constructed. The eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2, and pEASY-LB3 were transfected into 293T cells, and three target genes were detected by real-time fluorescent quantitative RT-PCR. The results confirmed that three eukaryotic expression plasmids were transfected into 293Teells and target protein was expressed. Analysis by SDS-PAGE electrophoresis and Western-blot indicated that three target proteins were expressed in 293T cells transfected with eukaryotic expression plasmids of pEASY-LB1, pEASY-LB2 and pEASY-LB3. Antigenicity studies indicated good HEV responses. Therefore, three recombinant DNAs of HEV ORF2 nucleic acid vaccine candidates were ob- tained, which might lay the foundation for further studies in the future.

Consistency Analysis of Detection Results of Two Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kits

Objective: The objective of the study is to verify the clinical validity of the following kits with the comparative experimental analysis and evaluate whether their performance can meet the clinical requirements, i.e. Class III in vitro diagnostic reagent “Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kit (PCR-Fluorescence Probe Method)” of Daan Gene Co., Ltd. (Daan kit for short) and “Herpes Simplex Virus (HSV) Type II Nucleic Acid Detection Kit (Fluorescence PCR Method)” of Wuhan Biot Gene Co., Ltd. (Biot kit for short). Method: In the study process, the samples were divided into positive and negative groups according to the control test results, and the clinical application performance of Daan kit and Biot kit was evaluated by comparing their test results. Results: The results show that two kits indicate the same test results, i.e. 26 positive and 107 negative samples in a total of 133 male urethral discharge samples, and 32 positive and 238 negative samples in a total of 270 female cervical secretion samples. Conclusion: It can be concluded from the clinical test that Daan and Biot Herpes Simplex Virus (HSV) Type II Nuc- leic Acid Test Kits are reliable, accurate, safe, convenient for use, stable and high-value in the clinical application.

Impact of hepatitis B virus infection on hepatic metabolic signaling pathway

A growing body of epidemiologic research has demonstrated that metabolic derangement exists in patients with hepatitis B virus(HBV) infection, indicating that there are clinical associations between HBV infection and host metabolism. In order to understand the complex interplay between HBV and hepatic metabolism in greater depth, we systematically reviewed these alterations in different metabolic signaling pathways due to HBV infection. HBV infection interfered with most aspects of hepatic metabolic responses, including glucose, lipid, nucleic acid, bile acid and vitamin metabolism. Glucose and lipid metabolism is a particular focus due to the significant promotion of gluconeogenesis, glucose aerobic oxidation, the pentose phosphate pathway, fatty acid synthesis or oxidation, phospholipid and cholesterol biosynthesis affected by HBV. These altered metabolic pathways are involved in the pathological process of not only hepatitis B, but also metabolic disorders, increasing the occurrence of complications, such as hepatocellular carcinoma and liver steatosis. Thus, a clearer understanding of the hepatic metabolic pathways affected by HBV and its pathogenesis is necessary to develop more novel therapeutic strategies targeting viral eradication.

Evidence-based consensus on the diagnosis, prevention and management of hepatitis C virus disease

Hepatitis C virus(HCV) is a potent human pathogen and is one of the main causes of chronic hepatitis round the world. The present review describes the evidencebased consensus on the diagnosis, prevention and management of HCV disease. Various techniques, for the detection of anti-HCV immunoglobulin G immunoassays, detection of HCV RNA by identifying virus-specific molecules nucleic acid testings, recognition of core antigen for diagnosis of HCV, quantitative antigenassay, have been used to detect HCV RNA and core antigen. Advanced technologies such as nanoparticlebased diagnostic assays, loop-mediated isothermal amplification and aptamers and Ortho trak-C assay have also come to the front that provides best detection results with greater ease and specificity for detection of HCV. It is of immense importance to prevent this infection especially among the sexual partners, injecting drug users, mother-to-infant transmission of HCV, household contact, healthcare workers and people who get tattoos and piercing on their skin. Management of this infection is intended to eradicate it out of the body of patients. Management includes examining the treatment(efficacy and protection), assessment of hepatic condition before commencing therapy, controlling the parameters upon which dual and triple therapies work, monitoring the body after treatment and adjusting the co-factors. Examining the treatment in some special groups of people(HIV/HCV co-infected, hemodialysis patients, renal transplanted patients).

Current testing strategies for hepatitis C virus infection in blood donors and the way forward

Screening tests for blood donations are based upon sensitivity, cost-effectiveness and their suitability for high-throughput testing. Enzyme immunoassay (EIAs) for hepatitis C virus (HCV) antibodies were the initial screening tests introduced. The &#x0201d;first generation&#x0201c; antibody EIAs detected seroconversion after unduly long infectious window period. Improved HCV antibody assays still had an infectious window period around 66 d. HCV core antigen EIAs shortened the window period considerably, but high costs did not lead to widespread acceptance. A fourth-generation HCV antigen and antibody assay (combination EIA) is more convenient as two infectious markers of HCV are detected in the same assay. Molecular testing for HCV-RNA utilizing nucleic acid amplification technology (NAT) is the most sensitive assay and shortens the window period to only 4 d. Implementation of NAT in many developed countries around the world has resulted in dramatic reductions in transfusion transmissible HCV and relative risk is now &#x0003c; 1 per million donations. However, HCV serology still continues to be retained as some donations are serology positive but NAT negative. In resource constrained countries HCV screening is highly variable, depending upon infrastructure, trained manpower and financial resource. Rapid tests which do not require instrumentation and are simple to perform are used in many small and remotely located blood centres. The sensitivity as compared to EIAs is less and wherever feasible HCV antibody EIAs are most frequently used screening assays. Efforts have been made to implement combined antigen-antibody assays and even NAT in some of these countries.

Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis

Hepatitis C virus(HCV)infection represents a significant health problem and represents a heavy load on some countries like Egypt in which about 20%of the total population are infected.Initial infection is usually asymptomatic and result in chronic hepatitis that give rise to complications including cirrhosis and hepatocellular carcinoma.The management of HCV infection should not only be focus on therapy,but also to screen carrier individuals in order to prevent transmission.In the present,molecular detection and quantification of HCV genome by real time polymerase chain reaction(PCR)represent the gold standard in HCV diagnosis and plays a crucial role in the management of therapeutic regimens.However,real time PCR is a complicated approach and of limited distribution.On the other hand,isothermal DNA amplification techniques have been developed and offer molecular diagnosis of infectious dieses at point-of-care.In this review we discuss recombinase polymerase amplification technique and illustrate its diagnostic value over both PCR and other isothermal amplification techniques.

Hepatitis C virus:Virology,diagnosis and treatment

More than twenty years of study has provided a better understanding of hepatitis C virus(HCV) life cycle,including the general properties of viral RNA and proteins. This effort facilitates the development of sensitive diagnostic tools and effective antiviraltreatments. At present,serologic screening test is recommended to perform on individuals in the high risk groups and nucleic acid tests are recommended to confirm the active HCV infections. Quantization and genotyping of HCV RNAs are important to determine the optimal duration of anti-viral therapy and predict the likelihood of response. In the early 2000 s,pegylated interferon plus ribavirin became the standard antiHCV treatment. However,this therapy is not ideal. To 2014,boceprevir,telaprevir,simeprevir,sofosbuvir and Harvoni are approved by Food and Drug Administration for the treat of HCV infections. It is likely that the new all-oral,interferon-free,pan-genotyping anti-HCV therapy will be available within the next few years. Majority of HCV infections will be cured by these antiviral treatments. However,not all patients are expected to be cured due to viral resistance and the high cost of antiviral treatments. Thus,an efficient prophylactic vaccine will be the next challenge in the fight against HCV infection.

A brief review of novel nucleic acid test biosensors and their application prospects for salmonids viral diseases detection

Viral diseases represent one of the major threats for salmonids aquaculture.Early detection and identification of viral pathogens is the main prerequisite prior to undertaking effective prevention and control measures.Rapid,sensitive,efficient and portable detection method is highly essential for fish viral diseases detection.Biosensor strategies are highly prevalent and fulfill the expanding demands of on-site detection with fast response,cost-effectiveness,high sensitivity,and selectivity.With the development of material science,the nucleic acid biosensors fabricated by semiconductor have shown great potential in rapid and early detection or screening for diseases at salmonids fisheries.This paper reviews the current detection development of salmonids viral diseases.The present limitations and challenges of salmonids virus diseases surveillance and early detection are presented.Novel nucleic acid semiconductor biosensors are briefly reviewed.The perspective and potential application of biosensors in the on-site detection of salmonids diseases are discussed.

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

Molecular detection of SARS-CoV-2 being challenged by virus variation and asymptomatic infection

Coronavirus disease 2019(COVID-19)has been a pandemic for more than a year.With the expanding second wave of the pandemic in winter,the continuous evolution of SARS-CoV-2 has brought new issues,including the significance of virus mutations in infection and the detection of asymptomatic infection.In this review,we first introduced several major SARS-CoV-2 mutations since the COVID-19 outbreak and then mentioned the widely used molecular detection techniques to diagnose COVID-19,primarily focusing on their strengths and limitations.We further discussed the effects of viral genetic variation and asymptomatic infection on the molecular detection of SARS-CoV-2 infection.The review finally summarized useful insights into the molecular diagnosis of COVID-19 under the special situation being challenged by virus mutation and asymptomatic infection.

Gene therapeutic approaches to inhibit hepatitis B virus replication

Acute and chronic hepatitis B virus(HBV) infections remain to present a major global health problem. The infection can be associated with acute symptomatic or asymptomatic hepatitis which can cause chronic inflammation of the liver and over years this can lead to cirrhosis and the development of hepatocellularcarcinomas. Currently available therapeutics for chronically infected individuals aim at reducing viral replication and to slow down or stop the progression of the disease. Therefore, novel treatment options are needed to efficiently combat and eradicate this disease. Here we provide a state of the art overview of gene therapeutic approaches to inhibit HBV replication. We discuss non-viral and viral approaches which were explored to deliver therapeutic nucleic acids aiming at reducing HBV replication. Types of delivered therapeutic nucleic acids which were studied since many years include antisense oligodeoxynucleotides and antisense RNA, ribozymes and DNAzymes, RNA interference, and external guide sequences. More recently designer nucleases gained increased attention and were exploited to destroy the HBV genome. In addition we mention other strategies to reduce HBV replication based on delivery of DNA encoding dominant negative mutants and DNA vaccination. In combination with available cell culture and animal models for HBV infection, in vitro and in vivo studies can be performed to test efficacy of gene therapeutic approaches. Recent progress but also challenges will be specified and future perspectives will be discussed. This is an exciting time to explore such approaches because recent successes of gene therapeutic strategies in the clinic to treat genetic diseases raise hope to find alternative treatment options for patients chronically infected with HBV.

PLASMID VACCINE ENCODING HN GENE FROM NEWCASTLE DISEASE VIRUS HAS MARKED ANTITUMORAL EFFECT IN VITRO

Objective: To explore the antitumor effects of hemaagglutinin-neuraminase gene (HN gene) from Newcastle disease virus. Methods: Plasmid vaccine of pIRHN was constructed and transfected into HeLa cells. The expression of HN was analyzed by Western blot analysis, and the mode of cell death was detected by fluorescence microscope, gel electrophoresis and TUNEL assay and the expression of p53 and bcl-2 was also analyzed in transfected Hela cells. The effect of pIRHN on sialic acid contents in the Hela cell was examined. Results: pIRHN nucleic acid vaccines could be expressed in eukaryotic cell. pIRHN could induce apoptosis after HeLa cells were transfected. The effect of antitumor responses of pIRHN was correlated with the contents of sialic acid in tumor cells, and there was no prominent evidence for the relatedness of the antitumor effect with the expression of p53 and bcl-2. Conclusion: pIRHN may become a new antitumor biological agent.

CONSECUTIVE IMMUNIZATION WITH RECOMBINANT FOWLPOX VIRUS AND PLASMID DNA FOR ENHANCING CELLULAR AND HUMORAL IMMUNITY

Objective: To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD 4 + T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.

Transfusion-transmitted virus co-infection in other types of hepatitis and its genotypes

OBJECTIVE: To identify the influence of transfusion transmitted virus (TTV) co-infection in other virus infected patients and its genotypes. METHODS: A conservative sequence of ORFl in the TTV genome was selected as primers and TTV DNA was measured in students and other hepatitis patients by using microplate nucleic acid hybridization and ELISA. The results were statistically analyzed. Nucleotide sequence of divergence >50% was used as color probe for distinguishing TTV genotypesⅠorⅡ. RESULTS: TTV DNA was detected in the sera from 2 (3.3%) of 60 students, 2 (14.3%) of 14 patients with non A-non E hepatitis, 6 (12%) of 50 patients with chronic hepatitis B, and 4 (16%) of 25 patients with liver cirrhosis, respectively. Statistical difference was observed between the patient group and the student group (P<0.05), but no significant difference in age, gender, serum ALT levels and TBiL between TTV DNA positive and negative patients (P>0.05). TTV genotype Type Ⅰ was by far the most frequent viral genotype (66.7%), followed by type Ⅱ (25%), and mixed infection (8.3%). CONCLUSIONS: These results suggest that the routes of TTV infection may be similar to those of HBV and HCV, and concurrent infection with HBV, HCV are common. TTV co-infection could not affect the clinical features of patients with liver diseases and the pathological process. TTV is not a main causative factor for patients with non A-non E hepatitis. Further study is needed to clarify the role of TTV in patients with non A-non E hepatitis.

Computational biology approach to uncover hepatitis C virus helicase operation

Hepatitis C virus(HCV)helicase is a molecular motor that splits nucleic acid duplex structures during viral replication,therefore representing a promising target for antiviral treatment.Hence,a detailed understanding of the mechanism by which it operates would facilitate the development of efficient drug-assisted therapies aiming to inhibit helicase activity.Despite extensive investigations performed in the past,a thorough understanding of the activity of this important protein was lacking since the underlying internal conformational motions could not be resolved.Here we review investigations that have been previously performed by us for HCV helicase.Using methods of structure-based computational modelling it became possible to follow entire operation cycles of this motor protein in structurally resolved simulations and uncover the mechanism by which it moves along the nucleic acid and accomplishes strand separation.We also discuss observations from that study in the light of recent experimental studies that confirm our findings.

Immune Responses to Varicella-Zoster Virus Glycoprotein E Formulated with Poly(Lactic-co-Glycolic Acid) Nanoparticles and Nucleic Acid Adjuvants in Mice

The subunit herpes zoster vaccine Shingrix is superior to attenuated vaccine Zostavax in both safety and efficacy,yet its unlyophilizable liposome delivery system and the limited supply of naturally sourced immunological adjuvant QS-21 still need to be improved.Based on poly(lactic-co-glycolic acid)(PLGA)delivery systems that are stable during the lyophilization and rehydration process and using a double-emulsion(w/o/w)solvent evaporation method,we designed a series of nanoparticles with varicella-zoster virus antigen glycoprotein E(VZV-g E)as an antigen and nucleic acids including polyinosinic-polycytidylic acid(Poly I:C)and phosphodiester Cp G oligodeoxynucleotide(Cp G ODN),encapsulated as immune stimulators.While cationic lipids(DOTAP)have more potential than neutral lipids(DOPC)for activating g E-specific cell-mediated immunity(CMI)in immunized mice,especially when g E is encapsulated in and presented on the surface of nanoparticles,PLGA particles without lipids have the greatest potential to induce not only the highest g Especific Ig G titers but also the strongest g E-specific CMI responses,including the highest proportions of interferon-c(IFNc)-and interleukin-2(IL-2)-producing CD4?/CD8?T cells according to a flow cytometry assay and the greatest numbers of IFN-c-and IL-2-producing splenocytes according to an enzyme-linked immunospot(ELISPOT)assay.These results showed that immune-stimulating nucleic acids together with the PLGA delivery system showed promise as a safe and economical varicella and zoster vaccine candidate.

HBV、HCV、HIV血筛多中心研究免疫学灰区的核酸检测分析与临床特征研究

目的分析化学发光灰区标本的临床特征及核酸检测对化学发光灰区标本结果判断的指导性意义。方法收集2021年7月—12月全国不同地区的5家综合医院入院患者术前/输血前血源性传播疾病样本检测结果,对化学发光灰区检测结果的标本进行核酸检测结果及临床特征分析。结果5723例样本中总计检出HBV免疫灰区样本28例(占比0.49%),HCV灰区样本20例(占比0.35%)。经核酸检测验证,28例HBV灰区样本中,15例HBV样本核酸检测为阳性(占比53.5%),其HBcAb也均为阳性;13例HBV样本核酸检测为阴性(占比46.5%),其中HBcAb阳性4例。HBV与HCV免疫检测灰区在临床各个科室均有发现,出现HBV灰区样本最多的前三科室为骨科、妇科、泌尿科,灰区样本核酸验证假阳性最多的临床科室为妇科与骨科。HCV灰区样本最多的前三科室为泌尿、肾内、外科,且均为假阳性。HBV灰区样本患者临床诊断结果有35.7%(10/28)为肿瘤类疾病,HCV灰区样本患者临床诊断结果有40%(8/20)为肿瘤类疾病。结论化学发光法容易造成假阳性结果,应注意复检验证,且设置灰区并非必要。灰区样本可见于多个临床科室,具有一定的临床分布特征。核酸检测可以提高检测灵敏度并且更大限度保证结果的准确性,能够验证免疫检测灰区。