对两株NSP3区插入外源基因的重组轮状病毒遗传稳定性的研究

目的分析2株插入和表达外源基因的重组轮状病毒rLLR/NSP3-NLuc和rLLR/NSP3-CoV2/RBD在MA104细胞上连续传代增殖的遗传稳定性。方法将2株重组轮状病毒滴度测定后, 按照MOI0.01传至P2代, 随后按1∶100稀释并活化上一代病毒裂解液, 连续18代(P20)感染MA104细胞, 用间接免疫荧光法测定P1, P5, P10, P15, P20代细胞裂解液病毒滴度, 进行RT-PCR鉴定以及dsRNA-PAGE银染实验, 并检测rLLR/NSP3-NLuc的荧光素酶活性。结果重组轮状病毒rLLR/NSP3-NLuc在20代内均未发现插入片段丢失, 重组病毒滴度为3.85～5.16×106 FFU/ml, 各代次均有较强的荧光素酶信号。重组轮状病毒rLLR/NSP3-CoV2/RBD在第6代出现了插入片段的丢失, 该重组病毒前5代的感染性滴度为:2.6～3.36×106 FFU/ml。结论 NSP3基因末端插入582 bp NLuc的重组轮状病毒具有很好的遗传稳定性, 而NSP3基因末端插入885 bp RBD的重组轮状病毒仅在前5代稳定, 提示重组轮状病毒NS...     

发热伴血小板减少综合征病毒热稳定性和灭活条件初步研究

目的 阐明新发危害严重的发热伴血小板减少综合征病毒的稳定性和理化灭活条件.方法 评估细胞培养制备的SFTS病毒在不同温度下的热稳定性,对紫外线、酸性环境、常用消毒剂、有机溶剂敏感性.处理后病毒感染Vero细胞,用间接免疫荧光法确定病毒复制,并采用基于病毒核蛋白的双抗体夹心ELISA方法滴定病毒与无相应处理的对照组,比较分析各种理化条件对病毒感染性的影响.结果 SFTS病毒在37℃能够存活较短时间,感染性下降较快,在4℃能保持相对稳定,1周内感染性无明显下降.对热敏感,60℃30 min能够完全灭活病毒.对紫外线敏感,185 μW/cm2紫外线照射30 min可灭活病毒.对乙醚、氯仿等有机溶剂,β-丙内酯、甲醛和常用有机氯消毒剂敏感,在合适的浓度下可在较短时间内有效灭活病毒,400 mg/L的有效氯灭活病毒需室温放置10 min以上,在pH3.0条件对病毒活力有损害,但不能完全灭活病毒.结论 研究结果初步客观的评价了SFTSV热稳定性和灭活条件,为科学研究和疾病控制中样本采集、病毒灭活、安全防护等工作提供了科学依据.     

肾综合征出血热纯化疫苗候选毒株在Vero细胞上的适应传代及其免疫原性研究

目的 将肾综合征出血热纯化疫苗候选毒株适应于Vero细胞，并对其抗原性和免疫原性进行研究。方法 将汉滩病毒H8207株和汉城病毒Y86013株在Vero细胞上进行连续终末稀释传代，采用间接免疫荧光法、酶联免疫吸附试验和空斑减少中和试验，研究传代后毒株的繁殖特性、病毒滴度、抗原量及免疫原性。结果 经过5次终末稀释传代，两株病毒在Vero细胞上均显示出良好的适应性，从第六代开始病毒滴度稳定在7．0Lg TCID50/m1以上，第八代两毒株抗原量均已达到1：64，H8207株抗原量继续升高，最高时达1：256。用两株病毒不同培养代次上清液制备的单价原液灭活疫苗免疫家兔二针，免疫血清对同型毒株的中和效价均达到1：10。结论 两株病毒已适应于Vero细胞，且具有病毒滴度高和免疫原性良好的特性，适合用作Vero细胞肾综合征出血热灭活纯化疫苗的候选毒株。     

创伤弧菌紫外线抗性观察

目的:观察创伤弧菌对紫外线(UV)的抵抗力,确定紫外线有效灭杀创伤弧菌的时间,为创伤弧菌消毒提供实验依据.方法:创伤弧菌ATCC27562、大肠杆菌ATCC25922悬液及平板在强度值达90μW/cm2的紫外线照射不同时间后,进行培养并定量测照射前后的活菌浓度,统计杀菌率,绘制照射前后灭活曲线(t-△lg).结果:创伤弧菌在强度值达90 μW/cm2的紫外线照射下,15 min后定性培养为阴性,定量检测1 min平均灭活对数值LIV悬液法为4.37±0.13、平板法为5.10±0.19,平均杀菌率悬液法为(99.99±0.01)%、平板法为(99.99±0.05)%,照射15min后无菌存活.大肠杆菌在照射1 min后达到消毒水平,10min后达到无菌状态.结论:创伤弧菌经强度值达90 μW/cm2的紫外线照射1 min达到消毒水平(LIV≥4.00),照射15 min创伤弧菌能被迅速有效灭杀.     

适宜标记的HFRS病毒组特异性McAb的建立与应用

本文用陕西省从黑线姬鼠肺分离的HFRS病毒81-14A株,感染BHK细胞,收集感染细胞。经紫外线灭活后,病毒抗原通过粗提,免疫BALB/c小鼠,通过细胞融合,建立了适宜于标记的HFRS病毒组特异性3-1C_4 McAb。荧光素标记McAb效价为1:5120,标记后一年半效价下降一个滴度,与26株从不同地区分离的HFRS病毒株作直接免疫荧光反应,均产生较     

猪小肠抗菌肽杀菌机理研究

抗菌肽(Antibacterial peptide)是一类新记述的膜活性多肽,广泛存在于生物界,具广谱的抗微生物和细胞毒活性。作者由猪小肠上段组织在酸性(pH2.5)条件下提取抗菌肽,提取率为湿重组织的0.19％。在pH4.0和pH8.6条件下电泳,从正极向负极迁移,证明为强碱性阳离子多肽。耐热,在100℃水浴30分钟未破坏杀菌活性。测试11种13株细菌、真菌、病毒及原虫的活性,结果以大肠杆菌最敏感,当该多肽为80μg/ml,细菌2×10~5CFU/ml,经37℃,3h,大肠杆菌100％被灭活;对其他革兰氏阳性、阴性、球菌、杆菌、白色念珠菌亦有一定灭活作用。对高度耐药的金葡菌和伤寒杆菌(耐11种抗生素株)也有灭活作用。     

β-丙内酯对流感病毒鸡胚高产母本株灭活条件的研究

目的 流感病毒鸡胚高产母本株A/PR8/34株和X-157株灭活条件的研究.方法 应用改良后的β-丙内酯灭活法对流感病毒鸡胚高产母本株进行灭活.经试验证明,0.5‰β-丙内酯在37℃水浴,pH值范围在7.4-8.0之间条件下作用2h.补加0.5‰β-丙内酯,pH值为9,温度为4℃条件下过夜,能彻底灭活且保证流感病毒表面主要抗原的完整性.结果 采用改良后的β-丙内酯灭活法对流感病毒鸡胚高产母本株A/PR8/34株和X-157株灭活效果良好,鸡胚3代安检合格.灭活前后病毒血凝滴度不变.结论 改良后的β-丙内酯灭活法可用于流感病毒鸡胚高产母本株的灭活.     

狂犬病病毒体外生存活力研究

目的了解狂犬病病毒在体外组织和体液样本中的生存能力。方法通过病毒滴度测定、直接免疫荧光、RT-PCR和病毒分离实验室技术手段对体外组织和悬液中狂犬病病毒生存活力进行验证。结果随着温度升高, 脑组织和悬液中狂犬病病毒活力逐渐下降, 在56℃, 30 min后病毒即失去感染力, 在37℃时, 组织中病毒活力可保持7 d;在pH9.6时, 1 h后无法检测病毒活力;终浓度30%以上乙醇, 500 mg/L以上次氯酸钠和100 mg/L以上苯扎溴铵对悬液中狂犬病病毒作用3 min后即可完全灭活;80%丙酮对组织中狂犬病病毒灭活作用最强, 在浸泡4 h后样本即无法进行病毒分离。结论狂犬病病毒不耐高温, 在pH6.8～7.4环境中较为稳定, 常用消毒剂即可对病毒发挥作用;本实验旨在为狂犬病病毒的体外生存活力提供详细数据, 为下一步狂犬病防控工作开展提供理论支持。     

增强型绿色荧光蛋白标记的重组丙型肝炎病毒体外细胞培养系统的建立

目的 构建一个易检测且灵敏度高的丙型肝炎病毒(HCV)细胞感染模型,为HCV致病机制的研究和抗病毒药物的筛选提供一个有效的体外细胞培养体系.方法 利用重组PCR技术在HCV的非结构蛋白NS5A的C端引入优势突变点V2440L,然后在其RsrⅡ酶切位点插入增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因,基因测序及酶切鉴定重组基因序列构建成功后,体外转录获得RNA,然后转染入肝癌细胞系Huh7.5,用Western blot检测EGFP与NS5A融合蛋白,用细胞免疫荧光(IFA)检测病毒复制水平和EGFP表达情况.用RT-PCR检测转染细胞培养液不同时间点的HCV RNA水平.用IFN-α鉴定该系统用于抗丙型肝炎药物筛选的可行性.结果 在重组病毒JFH1-2440-EGFP RNA转染的细胞内可检测到EGFP的表达,EGFP表达水平与病毒复制水平一致.转染细胞的培养上清液能感染新的Huh7.5细胞,IFA结果显示转染后第9天培养上清液的病毒滴度为104 FFU/ml,提示JFH1-2440-EGFP RNA转染的细胞能释放有传染性的HCV病毒颗粒.RT-PCR检测结果显示转染细胞上清液中的HCV RNA在转染72 h后可达到3.06×105拷贝/ml,第9天可达到7.96×106拷贝/ml.EGFP的表达呈干扰素浓度依赖性.结论 构建的重组病毒HCVJFH1-2440-EGFP体外细胞培养系统具有经济、快速、敏感等优点,为HCV致病机制的研究和抗病毒药物的筛选提供了一个有效的工具.     

新生隐球菌墨汁染色检查实验的改进

目的为消除生物安全隐患,让学生能安全高效地完成新生隐球菌墨汁染色检查实验。方法分别用10％EP醛或56℃水浴5rain对腹腔液、脑脊液中的新生隐球菌进行灭活处理,使其不再具有活性和致病性,镜下观察其形态的改变。结果用10％甲醛处理过的新生隐球菌,其荚膜形态已不能观察,而经56℃水浴灭活的新生隐球菌,其荚膜形态没有发生变化。结论新生隐球菌腹腔液或脑脊液采用56℃水浴5min即可灭活而不影响其荚膜形态,为学生安全高效地完成新生隐球菌墨汁染色检查实验提供了条件。     

Comparative study of inactivation of herpes simplex virus types 1 and 2 by commonly used antiseptic agents.

A comparative study of the different reactions of herpes simplex virus types 1 and 2 to Lysol, Listerine, bleach, rubbing alcohol, Alcide disinfectant (Alcide Corp., Westport, Conn.), and various pHs, temperatures, and UV light exposures was performed. Both types of stock virus (titers of approximately 10(6) and 10(5.5) for types 1 and 2, respectively) were inactivated by 0.5% Lysol in 5 min; by Listerine (1:1 mixtures) in 5 min; by 2,000 ppm (2,000 microliters/liter) of bleach in 10 min; by rubbing alcohol (1:1 mixtures) at zero time; by Alcide disinfectant (0.2 ml of virus plus 2.0 ml of Alcide) at zero time; by pHs 3, 5, and 11 in 10 min; and by a temperature of 56 degrees C in 30 min. A germicidal lamp (model G30TB; General Electric Co., Schenectady, N.Y.) (30 W) at a distance of 48 cm failed to completely inactivate the two types in 15 min. Type 1 showed slightly more resistance to Listerine and bleach and significantly more resistance to heat; moreover, pH 9 did not affect the infectivity of either type after 10 min.     

HPLC法测定注射用泮托拉唑钠中EDTA-2Na的含量

目的 建立高效液相色谱（HPLC）法测定注射用泮托拉唑钠中EDTA-2Na的含量。方法 选择固定相为十八烷基硅烷键合硅胶（直径为5 μm）,尺寸为250 mm×4.6 mm色谱柱,以乙腈-磷酸盐缓冲液（15∶85,v/v,用磷酸调节pH至2.5±0.1）为流动相,流速为1.0 ml/min,检测波长为255 nm,柱温为35 ℃。结果 EDTA-2Na浓度在2.0~32.0 μg/ml范围内线性方程为A=2.317×104C-1.235×103（r=0.9998）,EDTA-2Na的平均回收率为100.40%,RSD 为0.92%（n=9）。结论 本方法简便、可靠、准确度高、重复性好,可用于测定注射用泮托拉唑钠中EDTA-2Na的含量。     

Detection of anti-hepatitis C virus effects of interferon and ribavirin by a sensitive replicon system

Although combination therapy with interferon and ribavirin has improved the treatment for chronic hepatitis C virus (HCV) infection, the detailed anti-HCV effect of ribavirin in clinical concentrations remains uncertain. To detect the anti-HCV effect of ribavirin in lower concentrations, a sensitive and accurate assay system was developed using the reporter replicon system with an HCV genotype 2a subgenomic replicon (clone JFH-1) that exhibits robust replication in various cell lines. This reporter replicon was generated by introducing the luciferase reporter gene (instead of the neomycin resistance gene) into the subgenomic JFH-1 replicon. To assess the replication of this reporter replicon, luciferase activity was measured serially up to day 3 after transient transfection of Huh7 cells. The luciferase activity increased exponentially over the time course of the experiment. After adjustment for transfection efficiency and transfected cell viability, the impacts of interferon and ribavirin were determined. The administration of interferon and ribavirin resulted in dose-dependent suppression of replicon RNA replications. The 50% inhibitory concentration of interferon and ribavirin was 1.80 IU/ml and 3.70 microg/ml, respectively. In clinical concentrations, replications were reduced to 0.09% and 53.74% by interferon (100 IU/ml) and ribavirin (3 microg/ml), respectively. Combination use of ribavirin and interferon enhanced the anti-HCV effect of interferon by 1.46- to 1.62-fold. In conclusion, we developed an accurate and sensitive replicon system, and the antivirus effect of interferon and ribavirin was easily detected within their clinical concentrations by this replicon system. This system will provide a powerful tool for screening new antiviral compounds against HCV.     

Propagation of Aleutian disease parvovirus in cell line CCC clone 81

Aleutian disease parvovirus (ADV), mutant Gorham of the Utah-1 strain, was grown and comparatively assayed in feline cell lines CRFK and CCC clone 81 at 31.8 degrees C. The maximum virus titres as determined by a fluorescent focus assay were found to be about 10(5) FFU/ml in CRFK at day 6 p. i. and 10(6) FFU/ml at day 4 p. i. in CCC clone 81 cells. Shifting of the incubation temperature from 31.8 to 37 degrees C led to a reduced virus production after three passages. The synchronization of the CCC clone 81 cells by 1 X 10(-3) M hydroxyurea followed by infection with low (less than or equal to 0.8) multiplicities of infection (MOI) did not significantly influence the virus titres. Several mammalian cell lines such as MiCl 1 (S+L-), Mv1-Lu, 64F3 clone 7 and FEF or fish cell lines such as BB and CHSE 114 developed abortive infections after inoculation with the temperature-sensitive mutant Gorham of the ADV strain Utah-1 (ADV-G). Three new isolates designated ADV-Sl1--ADV-Sl3 were isolated from spleen and blood lymphocytes and bone marrow cells of ADV-infected mink and were adapted to grow in CCC clone 81 cells at 31.8 degrees C with virus titres between 10(4) and 10(4.7) FFU/ml. ADV particle populations varying in their bouyant density between 1.32, 1.36 and 1.43 g/ml were isolated from infected cells and culture supernatants. By protein blotting and immunodetection two major protein components with apparent M. W. of 85 and 75 KD and three minor polypeptides of 33, 28.9 and 27.5 KD were detected.     

Prediction of pneumococcal conjugate vaccine effectiveness against invasive pneumococcal disease using opsonophagocytic activity and antibody concentrations determined by enzyme-linked immunosorbent assay with 22F adsorption

We compared the abilities of two serological readouts, antipolysaccharide IgG antibody concentrations and opsonophagocytic activity (OPA) titers, to predict the clinical effectiveness of the 7-valent pneumococcal conjugate vaccine (7vCRM) against invasive pneumococcal disease (IPD). We also assessed the accuracy of the previously established thresholds for GlaxoSmithKline's enzyme-linked immunosorbent assay with 22F adsorption (22F-ELISA) (≥0.2 μg/ml) and OPA assay (titer, ≥8) in predicting effectiveness. We showed that following a 3-dose 7vCRM primary vaccination, the serological response rates as determined using thresholds of ≥0.2 μg/ml IgG and an OPA titer of ≥8 corresponded well with overall effectiveness against IPD. In addition, the OPA assay seemed to better predict serotype-specific effectiveness than enzyme-linked immunoassay. Finally, when applied to post-dose-2 immune responses, both thresholds also corresponded well with the overall IPD effectiveness following a 2-dose 7vCRM primary vaccination. These results support the importance of the OPA assay in evaluating immune responses to pneumococcal conjugate vaccines.     

Impact of hepatitis C virus (HCV) genotypes on quantification of HCV RNA in serum by COBAS AmpliPrep/COBAS TaqMan HCV test, Abbott HCV realtime assay [corrected] and VERSANT HCV RNA assay

The VERSANT HCV RNA 3.0 (bDNA), COBAS AmpliPrep/COBAS TaqMan HCV, and Abbott ART HCV RealTime assays were compared for hepatitis C virus RNA quantification in 158 clinical specimens (genotypes 1 to 5). RNA values differed significantly between methods (P < 0.0001), and mean titer differences ranged from 0.01 to 0.50 log(10) IU/ml depending on the genotypes.     

Titration of avirulent Newcastle disease virus by the plaque assay method

Parameters of a plaque assay for avirulent strains of Newcastle disease virus (NDV) were optimized for reproducibility and optimum titer in LLC-MK₂ cells. Plaques were visible after 2 days and maximum virus titers were reached in 3 days. Virus titers were not affected by continued incubation through 6 days, although plaque size increased. An adsorption volume of 0.1 or 0.2 ml per 60 mm Petri dish was optimal, as was an adsorption time of 45 min. Trypsin (2.5 μg/ml) and magnesium sulfate (0.03 M) were essential requirements of the overlay medium and the presence of DEAE-dextran (0.02%) resulted in a 30% increase in titer. The use of cell monolayers, 1, 2, or 3 days old facilitated the performance of multiple assays per week and did not affect the virus titer.