Specific activation of 2'-5'oligoadenylate synthetase gene promoter by hepatitis C virus-core protein:A potential for developing hepatitis C virus targeting gene therapy

AIM： TO examine whether 2＇-5＇oligoadenylate synthetase （OAS） gene promoter can be specifically activated by hepatitis C virus （HCV）-core protein. METHODS： Human embryo hepatic cell line L02 was transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence was amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luci plasmid was transiently transfected into L02/core cells and luciferase activity was assayed. I^ESULTS： L02/core cell line stably expressing HCV- core protein was established. The pGL3-OAS-Luci construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells. CONCLUSION： HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.

Expression of co-stimulatory molecules B7-2 and PD-L1 on peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection

Objective： To explore the roles of the expression of the co-stimulatory molecule, B7-2, and the co-inhibitory molecule, PD-L1, on peripheral blood mononuclear cells in the mechanism of immunotolerance in chronic hepatitis B virus infection. Methods： Thirty HBV infected patients in the immunoreactive phase and 20 patients in the immunotolerant phase were enrolled in the study, while 20 healthy volunteers were used as controls. RT- PCR and real-time PCR methods were used to detect the expression levels of B7-2 and PD-L1 mRNA in peripheral blood mononuclear cells in chronic HBV infected patients. Results： The B7-2 expression in irnrnunoreactive and immunotolerant patients was significantly lower than that in the controls （P all 〈 0.01 ）; B7-2 expression in immunoreactive patients was significantly lower than in immunotolerant patients （P 〈 0.01）. PD-L1 expression in irnmunoreactive patients and immunotolerant patients was significantly higher than that in normal controls （P all 〈 0.01）. The PD-L1/BT-2 ratios in immunoreactive and immunotolerant patients were significantly higher than that of the healthy controls （P all 〈 0.01）; the PD-L1/ B7-2 ratio was significantly higher in the immunoreactive patients than in the immunotolerant patients （P 〈 0.01）. Conclusion： In chronic HBV infection, changes in the expression of co-stimulatory and co-inhibitory molecules imply a protective adjustment against the patient＇ s immune response that may result in increased immunotolerance and persistent HBV infection.

A retrospective investigation on the phenotype and stability of the E-protein gene in Japanese Encephalitis (JE) virus strain SA14-14-2 used live-attenuated JE vaccine

To detect retrospectively the phenotype and stability of the E-protein gene in Japanese Encephalitis （JE） virus strain SA14-14-2 used in the live-attenuated JE vaccine prepears, the viral titer was titrated by plaque formation in BHK-21 cell cultures, and the neuro-virulence of viruses was assayed in mice with body weight of 12-14 g by intracerebral inoculation. Meanwhile, the total RNA of virus gene was extracted and amplified by RT-PCR with the designed primers, and then it was purified and cloned to the expression vector pGEM-T. The recombinant plasmid was purified and sequenced. It was found that the loss of viral titer of vaccines stored in -20℃ for longer than 10 years was less than 0.5 Lg PFU/ml. No mice inoculated intracerebrally showed signs of illness or even death. The size of plagues of the vaccine virus remained to be small, and the E genes of primary virus seed SA14-14-2 and the vaccines prepared at different years （1987-2001） were unchanged, in- cluding the 8 critical amino acid sites which were different from the parent wild virus strain SA14 and the related neuro-virulence. These results indicate that the genotypic and biological characteristics of the attenuated JE virus strain SA14-14-2 and its vaccines sion noted. prepared are quite stable without any reversion noted.

Study on pharmacodynamics of recombinant interferon α-2b suppository

Objective To investigate the antiviral activity of recombinant interferonα-2b suppository(IFNα-2b)in vivo and in vitro.Methods The cytopathic-effect inhibition assay was applied in this study to investigate the antiviral activity of this drug as well as yingtelong and axiluowei as positive control.The guinea pig model of vaginitis and skin infection caused by HSV-2 infection were established,treated with IFNα-2b suppository at dosages of 60000、180000、540000 IU,using IFNα-2b injection 180000 IU·kg-1 as controls.Score the pathological changes of appearance and skin,the virus activities of vaginal secretion and tissue sections of viginae were assayed after treatment.Results The TD50 of IFN α-2b and yingtelong for Vero cells was(>100)μg·mL-1 and(>100000)IU·mL-1,respectively.The IC50 of IFN α-2b and yingtelong and axiluowei for Herpes virus type 1 was(0.29±0.08)μg·mL-1 and(185.0±28.8)IU·mL-1 and(0.19±0.03)μg·mL-1,respectively.The mean scores for vaginal and skin lesion of the treated groups were lower than those of untreated group.Among these concentrations,the IFNα-2b suppository of 540000 IU·kg-1 group.Showed highest anti-viral activity.The virus activity in vaginal secretion of treated group was lower than that of untreated group too(P<0.01 or P<0.05).Tissue sections of viginae after treatment with IFNα-2b suppository showed significantly therapeutical effects on the degrees of vaginal lesion.At the same dosage,The anti-HSV activity of IFNα-2b suppository was also compared with IFNα-2b injection,the results showed that the activity of suppository of 540000 IU·kg-1 group was similar to that of the injection.Conclusions The IFNα-2b suppository has anti-viruses function both in vivo and in vitro.

Effects of anti-HPV bioprotein dressing combined with interferon α-2b therapy on the malignant molecule expression in patients with CINⅢ complicated by high-risk HPV positive

Objective: To study the effects of anti-HPV bioprotein dressing combined with interferon α-2b therapy on the malignant molecule expression in patients with cervical intraepithelial neoplasia (CIN) Ⅲ complicated by high-risk HPV positive. Methods: Patients who were diagnosed with CINⅢ and high-risk HPV positive and underwent conization in the 3201 Hospital Affiliated to Xi'an Jiaotong University between June 2014 and February 2017 were selected and randomly divided into the observation group who received preoperative anti-HPV bioprotein dressing combined with interferon α-2b therapy and the control group who received no special treatment. CIN lesion was collected to determine the expression of pro-proliferation molecules, pro-apoptosis molecules and epithelial-mesenchymal transition molecules. Results: Rsf1, Piwil2, TOPK, p38MAPK, ERK, Snail, Twist, N-cadherin and Vimentin mRNA expression in cervical intraepithelial neoplasia lesions of observation group were greatly lower than those of control group whereas LRIG3, SARI, IEX-1, FHIT and E-cadherin mRNA expression were greatly higher than those of control group. Conclusion: Anti-HPV bioprotein dressing combined with interferon α-2b therapy can inhibit the proliferation and invasive growth of tumor cells in patients with CINⅢ complicated by high-risk HPV positive.

Identification of African swine fever virus MGF505-2R as a potent inhibitor of innate immunity in vitro

African swine fever(ASF)is etiologically an acute,highly contagious and hemorrhagic disease caused by African swine fever virus(ASFV).Due to its genetic variation and phenotypic diversity,until now,no efficient commercial vaccines or therapeutic options are available.The ASFV genome contains a conserved middle region and two flexible ends that code for five multigene families(MGFs),while the biological functions of the MGFs are not fully characterized.Here,ASFV MGF505-2R-deficient mutant ASFV-Δ2R was constructed based on a highly virulent genotype II field isolate ASFV CN/GS/2018 currently circulating in China.Transcriptomic profiling demonstrated that ASFV-Δ2R was capable of inducing a larger number of differentially expressed genes(DEGs)compared with ASFV CN/GS/2018.Hierarchical clustering of up-regulated DEGs revealed that ASFV-Δ2R induced the most dramatic expression of interferon-related genes and inflammatory and innate immune genes,as further validated by RT-qPCR.The GO and KEGG pathway analysis identified significantly enriched pathways involved in pathogen recognition and innate antiviral immunity.Conversely,pharmacological activation of those antiviral immune responses by exogenous cytokines,including type I/II IFNs,TNF-αand IL-1β,exerted combinatory effects and synergized in antiviral capacity against ASFV replication.Collectively,MGF505-2R is a newly identified inhibitor of innate immunity potentially implicated in immune evasion.

Microalbuminuria in hepatitis C-genotype 4:Effect of pegylated interferon and ribavirin

AIM:To study the relation between hepatitis C virus (HCV) genotype 4 and microalbuminuria and renal impairment in relation to hepatic histology, and viremia in the absence of cryoglobulinemia, and to examine the effect of treatment on microalbuminuria.METHODS: Three hundred subjects, including 233 HCV genotype-4 infected patients, were tested for cryoglobulinemia, microalbuminuria, albumin creatinine ratio (ACR), urea, creatinine, and estimated glomerular filtration rate (eGFR). The parameters were measured again in the HCV patients after 48 wk of treatment with pegylated interferon and ribavirin.RESULTS: Significantly higher levels of microalbumin- uria were detected in HCV-positive patients compared to HCV-negative controls (median 9.5 vs 5.9, respectively, Kruskal-Wallis P=0.017). Log microalbuminuria was significantly correlated with hepatic inflammation (r=0.13, P=0.036) and f ibrosis (r=0.12, P=0.061), but not with viral load (r=-0.03, P=0.610), or alanine transaminase (r=-0.03, P=0.617). Diabetes mellitus neither significantly moderated (χ2=0.13, P=0.720), nor mediated (Sobel test P=0.49) the HCV effect. HCV status was signifi cantly associated with log microalbu-minuria (χ2=4.97, P=0.026), adjusting for age, gender, diabetes, cryoglobulinemia, urea and creatinine. A positive HCV status was not significantly associated with low eGFR (<60 mL/min every 1.73 m2) [odds ratio (OR): 0.5, 95% confidence interval (CI):0.2-1.4], nor with high ACR (OR: 1.7, 95% CI:0.7-4.1). End-of-treatment response (ETR) was achieved in 51.9% of patients. Individuals with ETR had significantly lower microalbuminuria post-treatment (χ2=8.19, P=0.004).CONCLUSION: HCV affected the development of microalbuminuria independent of diabetes or cryoglobulinemia. Combination therapy of pegylated interferon-ribavirin had a positive effect in reducing microalbuminuria.

FoxJ1 inhibits African swine fever virus replication and viral S273R protein decreases the expression of FoxJ1 to impair its antiviral effect

African swine fever(ASF)is a highly pathogenic swine infectious disease that affects domestic pigs and wild boar,which is caused by the African swine fever virus(ASFV).ASF has caused huge economic losses to the pig industry and seriously threatens global food security and livestock health.To date,there is no safe and effective commercial vaccine against ASF.Unveiling the underlying mechanisms of ASFV-host interplay is critical for developing effective vaccines and drugs against ASFV.In the present study,RNA-sequencing,RT-qPCR and Western blotting analysis revealed that the transcriptional and protein levels of the host factor FoxJ1 were significantly down-regulated in primary porcine alveolar macrophages(PAMs)infected by ASFV.RT-qPCR analysis showed that overexpression of FoxJ1 upregulated the transcription of type I interferon and interferon stimulating genes(ISGs)induced by poly(dA:dT).FoxJ1 revealed a function to positively regulate innate immune response,therefore,suppressing the replication of ASFV.In addition,Western blotting analysis indicated that FoxJ1 degraded ASFV MGF505-2R and E165R proteins through autophagy pathway.Meanwhile,RT-qPCR and Western blotting analysis showed that ASFV S273R inhibited the expression of FoxJ1.Altogether,we determined that FoxJ1 plays an antiviral role against ASFV replication,and ASFV protein impairs FoxJ1-mediated antiviral effect by degradation of FoxJ1.Our findings provide new insights into the antiviral function of FoxJ1,which might help design antiviral drugs or vaccines against ASFV infection.

Screening of Fungi from Chinese Medical Plants for Anti-Human Immunodeficiency Virus Type 1 Activity

In order to Isolate anti-human Immunodeflclency virus （HIV） agents from natural products, 97 ethanollc extracts of 90 fungi were tested for their Inhibitory activity on HIV-1. Most of the extracts tested were relatively non-toxic to human lymphocytic MT-4 cells, but extracts of some fungi exhibited potent antl-HIV activity In an in vitro 3-（4, 5-dlmethyl-2 thlazoyl）-2,5-dlphenyl-2H-tetrazollum bromide assay with a selectivity Index greater than 3. Most fungi were Isolated from Dendrobium sp. and Taxus sp.

Decreases in Both the Seroprevalence of Serum Antibodies and Seroprotection against Japanese Encephalitis Virus among Vaccinated Children

The incidence of Japanese encephalitis(JE)has significantly decreased in China due to JE vaccines.In this study,we investigated the post-JE vaccination seroprevalence and protection provided by vaccinated sera against Japanese encephalitis virus(JEV)to elucidate the persistence and waning of antibodies to JEV among JE-SA14-14-2-vaccinated children.A total of 300 serum samples were collected from vaccinated children aged 3-10 years in Zhaotong,Yunnan,China.The seroprevalence of anti-JEV antibodies was determined by enzyme-linked immune sorbent assay and plaque reduction neutralization test.The highest seropositivity of 82%was observed in vaccinated children during the first 0.5-1.5 years after booster vaccination.Then,the seropositivity began to decline and remained lower than the original level observed in the 0.5-1.5-year group.An association was found between the waning of seroprevalence and elapsed time of the post-booster vaccination.Similarly,the neutralizing antibody(nAb)titres gradually decreased over time,and the levels showed a positive correlation with the protective efficacy in mice.This finding suggests that nAbs play an important role in the antiviral process and that the nAb titre is an adequately credible parameter for evaluating the protective efficacy induced by the JE vaccine.Our results provide data that clarify the persistence and waning of antibodies to JEV,which may help elucidate the pathogenesis of JE.

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宿主细胞蛋白HSPA2与TGEV Nsp2的相互作用及其对病毒复制的影响

为了探究猪传染性胃肠炎病毒(Transmissible gastroenteritis virus,TGEV)非结构蛋白2(Nsp2)与宿主细胞热休克蛋白70-2(HSPA2)的相互作用,以及HSPA2表达对TGEV复制的影响,试验利用RT-PCR方法获得猪源HSPA2基因并构建其真核表达载体pCMV-Myc-HSPA2,通过Western-blot和间接免疫荧光试验检测真核表达载体pCMV-Myc-HSPA2在IPEC-J2细胞中的表达情况。利用免疫共沉淀(Co-IP)和共定位试验进一步验证TGEV Nsp2和宿主细胞蛋白HSPA2之间是否存在相互作用,利用基因过表达技术在IPEC-J2细胞中过表达HSPA2后接种TGEV,分别测定接种后36,48小时时病毒TCID50。结果表明:真核表达载体pCMV-Myc-HSPA2能够在IPEC-J2细胞中成功表达,HSPA2与Nsp2之间存在相互作用,并且HSPA2表达量升高后TGEV的TCID50略有升高。说明宿主细胞蛋白HSPA2与TGEV Nsp2之间存在相互作用,且HSPA2的表达对TGEV复制具有一定促进作用。