板层人工角膜辐照法病毒灭活验证方法的建立及灭活效果验证

目的建立板层人工角膜钴-60照射病毒灭活验证方法,用该方法对脂包膜病毒灭活效果进行验证。方法以伪狂犬病毒(pseudorabies virus,PRV)为指示病毒,将干燥的板层人工角膜浸泡在高滴度病毒液中,在2~8℃浸泡5 h,经剪碎、研磨等步骤后4℃放置12 h,确立角膜吸附和释放病毒的条件。之后,将吸附病毒并呈干燥状态的角膜以0、5、10、15、20、25 k Gy辐照剂量进行钴-60辐照,以确立的条件释放病毒并进行滴定,考察灭活效果。结果板层角膜在2~8℃浸泡5 h可吸附足量病毒;病毒滴度可达到4 lg值以上,满足病毒灭活验证的要求。样品经25 k Gy剂量钴-60照射后灭活PRV为2.75~3.25 lg TCID50/100μL,灭活后的样品在敏感细胞上盲传3代均未出现细胞病变。结论成功建立了板层人工角膜病毒灭活验证的方法,并验证采用钴-60辐照法对PRV有较好的灭活效果。     

腮腺炎病毒三种实验室常规灭活方法效果评价

探究几种实验室常规消毒灭活方法对腮腺炎病毒的灭活效果,为实验室进行腮腺炎病毒消毒工作提供基础数据。本研究采用热灭活、紫外线灭活和化学试剂灭活三种方法对腮腺炎病毒进行灭活处理,灭活处理后腮腺炎病毒采用细胞培养技术进行病毒滴定,评估上述灭活方法对腮腺炎病毒的灭活效果。热灭活方法显示,腮腺炎病毒对热敏感,随温度升高和热灭活时间增长对腮腺炎病毒灭活有明显加强作用。65℃30min、60min和70℃10min、30min、60min可有效灭活腮腺炎病毒;紫外线灭活方法显示,紫外线照射15min,30min,60min均可有效灭活涂抹在平皿表面的腮腺炎病毒,但对毒株液倾倒导致的MuV液体不能达到完全灭活效果;化学试剂灭活方法显示,三氯异氰泡腾片、1%过氧化氢、3%过氧化氢和75%乙醇根据试剂说明书处理后病毒均能有效灭活腮腺炎病毒。三种灭活方法在一定条件下均能实现对腮腺炎病毒的灭活,可以作为实验室常规灭活腮腺炎病毒方法。建议使用65℃30min或70℃10min对腮腺炎病毒进行有效灭活。紫外线适用于对腮腺炎病毒进行表面消毒,但对液滴状病毒污染建议使用化学试剂进行有效灭活。     

干热法对人血浆血管性假血友病因子冻干品中病毒灭活效果的观察

目的探讨干热灭活法(dry heat treatment)对血管性假血友病因子(von willebrand factor,v WF)中猪细小病毒(porcine parvovirus,PPV)和脑心肌炎病毒(encephalomyocarditis virus,EMCV)的灭活效果。方法以PPV或EMCV作为灭活指示病毒与v WF混合,并分别在(80±1)℃4、8、12、24、36、48、72 h,(90±1)℃4、12、24、36、48 h以及(99±1)℃5、15、30 min进行干热灭活处理。将在干热灭活前以及干热灭活后不同温度时间段的v WF样品接种ST细胞(swine testis)或Vero细胞培养,观察细胞病变(cytopathic effect,CPE),用Reed-Muench法计算残余PPV和EMCV的病毒滴度。对无CPE的样品进行盲传3代培养,验证其灭活效果,并检测干热处理前后的v WF活性。结果v WF中EMCV滴度在(80±1)℃、(90±1)℃以及(99±1)℃灭活条件下,均下降超过4.0 lg TCID_(50)/0.1 m L,v WF中PPV的滴度下降也均超过4.0 lg TCID_(50)/0.1m L。干热法灭活后v WF的活性下降20%,质量稳定。结论干热法可以灭活v WF中的PPV以及EMCV,且对样品的活性影响在允许范围之内。     

S／D处理血浆过程中的脂包膜病毒灭活试验观察

通过有机溶剂/去污剂对血浆中指示病毒VSV灭活的观察评估有机溶剂/去污剂对脂包膜病毒死活的效果。血浆样品与VSV病毒按9:1混合,然后用1％TNBP/1％ Triton X-100在30℃处理4h,测定开始样品中的病毒总量和S/D处理后不同时间取样内的病毒总量。实验中样品内加入1％TNBP/1％ Triton X-10015min后VSV病毒已全部灭活,灭活效果≥6.0log。按所述S/D处理方法可以完全灭活血浆内所有的脂包膜病毒而没有主要血浆蛋白的损失。     

2308、M28、S1330、16M四株布鲁氏菌灭活参数研究

【目的】比较不同灭活方法对布鲁氏菌灭活的效果,确定灭活参数,为制备布鲁氏菌灭活抗原提供参考。【方法】将4株布鲁氏菌参考强毒株2308(牛种)、M28(羊种)、S1330(猪种)以及16M(羊种)分别经大豆酶消化蛋白胨(TSA)培养基培养繁殖后,用生理盐水制成(4-8)×1010 CFU/m L的菌悬液,分成等份于80 oC灭活不同时间,另将同样浓度的菌悬液分别用不同浓度甲醛于37 oC灭活不同时间,通过灭活检验,确定灭活效果。取经甲醛和热灭活的16M抗原,分别以1×1010 CFU/只剂量皮下注射1.5-2.0 kg家兔2只,免疫6周内,每周采血用虎红平板凝集试验(RBT)和试管凝集试验(SAT)测定抗体效价。【结果】80 oC、5 min可灭活2308、S1330和16M三种菌株,80 oC、10 min可灭活全部4种菌株。0.2%甲醛灭活7 d,4种试验菌株均不能被彻底灭活;0.4%甲醛在12 h内只能灭活16M,72 h可灭活M28;0.4%甲醛灭活2308和S1330两次试验结果差异较大。0.6%甲醛可在72 h内灭活4种试验菌。不同方法灭活的16M抗原免疫家兔后,其血清抗体虎红平板凝集和试管凝集效价消长趋势基本一致,甲醛灭活的抗原免疫原性略高于热灭活抗原。【结论】80 oC热灭活和0.6%甲醛灭活均可用于对布鲁氏菌的灭活,且不影响布鲁氏菌的免疫原性。     

低pH孵放、干热处理冻干静脉注射人免疫球蛋白的实验研究

主要介绍冻干静脉注射人免疫球蛋白 (IVIG)分别通过 60℃ 72h,80℃ 72h ,低pH孵放 2 1d处理后 ,IVIG的各项理化及生物活性的变化 ,以及IVIG经过低pH孵放的灭活病毒情况。实验结果表明 ,处理后的IVIG其IgG各组份相对含量、抗补体活性 (ACA)、前激肽释放酶激活剂 (PKA)、白喉抗体、抗 HBs等结果 ,除ACA ,PKA略有下降 ,其他指标无明显变化 ,各项指标均符合 2 0 0 0版《中国生物制品规程》 ;加热后没有新抗原产生。低pH孵放 2 1d通过加指示病毒的实验 ,病毒灭活效果达到 7.0 0logTCID50 / 0 .1mL以上 ,灭活病毒有效。     

安全、便捷技术再生一次性医用口罩的实验研究

在防控新型冠状病毒(2019 novel coronavirus, 2019-nCoV)疫情中,为了减少病毒的传播,一次性医用口罩是普通民众必不可缺少的防护品。然而,随着2019-nCoV的蔓延,口罩短缺现象严重。本研究旨在探讨一次性医用外科口罩(口罩)的再生方法,以达到既有个人防护的效果又能节省资源。用流行性感冒病毒(简称流感)模拟2019-nCoV污染口罩,采用常用的恒温烘箱干烤及电吹风机热风处理2种方法,对表面污染有流感病毒的医用口罩进行病毒灭活,洗脱口罩上已灭活处理的病毒,感染Mardin-Darby狗肾细胞(Mardin-Darby canine kidney cell,MDCK细胞),观察细胞病变并定量检测病毒基因组拷贝数以评价病毒灭活效果。同时采用抽滤系统和PM2.5监测仪对以2种相似热灭活方式处理过的口罩滤过截留PM2.5的效果进行评价。结果显示电吹风机30 min热风处理后病毒基因组拷贝数降低至原来的1/1 000 000～1/10 000 000,接近未感染组,但烘箱56 ℃ 30 min干热处理仅灭活部分病毒。2种热灭活方式对口罩的PM2.5滤过截留效果无显著影响。本研究提供了一个安全、便捷处理一次性医用外科口罩的方法,为个人防护用品口罩表面污染病毒的灭活及其再生利用提供了科学依据。然而,应注意的是在口罩匮乏的非常时期,普通人群可采用该简便技术再生口罩后再次使用,但该方法再生的口罩不适合密切接触患者的人群、医护人员及实验室工作人员使用。     

辛酸盐灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证一定浓度的辛酸盐对某一厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG在辛酸钠(0.7±0.2mmol/g蛋白)、pH(5.1±0.1)、29.5~30.5℃,孵放90min可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥4.00~4.12和≥5.25~5.75log TCID50/0.1ml。因此,辛酸盐是一种安全、有效、快速的灭活脂包膜病毒的灭活剂。     

低pH孵放法灭活静注人免疫球蛋白中脂包膜病毒效果验证的研究

选用不同核酸类型的脂包膜病毒,其中RNA病毒为水疱性口炎病毒(VSV),DNA病毒为伪狂犬病毒(PRV),将两种指示病毒分别用于验证低pH孵放法对不同厂家生产的人血静脉注射用丙种球蛋白(IVIG)的病毒灭活效果。结果表明,液体IVIG的pH值为3.8～4.4,在23～25℃环境中,孵放21天可灭活VSV和PRV,两种指示病毒的灭活效果分别为≥5.50～6.62和≥5.38～6.62logTCID50/0.1ml。因此,低pH孵放法是一种安全、有效且简便实用的灭活脂包膜病毒的方法。     

表面活性素体外抗伪狂犬病毒活性研究

研究了表面活性素（surfactin）体外抗伪狂犬病毒（Pseudorabies Virus,PRV）效果。观察表面活性素的细胞毒性、对PRV直接灭活作用、抗PRV吸附作用及对PRV生物合成抑制作用。结果表明表面活性素对猪肾（porcinekidney,PK-15）细胞的TD50和TD0分别为31．25、4．03μg／mL;具有直接灭活PRV效果,不具有抗PRV吸附作用,对PRV生物合成无显著影响.     

Inactivation kinetics of model and relevant blood-borne viruses by treatment with sodium hydroxide and heat.

To determine the efficacy of a clean-in-place system for the inactivation of viruses present in human plasma, the effect of 0.1 M sodium hydroxide at 60 degrees C on viral infectivity was investigated. Inactivation of the following model and relevant viruses were followed as a function of time: human hepatitis A virus (HAV), canine parvovirus (CPV; a model for human parvovirus B-19) pseudorabies virus (PRV, a model for hepatitis B virus), and bovine viral diarrhoea virus (BVDV, a model for hepatitis C virus and human immunodeficiency virus). Infectivity of CPV was determined by a novel in situ EIA method which will prove useful for studies to validate parvovirus inactivation or removal. Infectivity of BVDV, PRV and CPV were shown to be reproducibly inactivated below the limit of detection by 0.1 M NaOH at 60 degrees C within 30 s. HAV was inactivated to below the limit of detection within 2 min. Treatment with heat alone also resulted in some log reduction for all viruses tested except for CPV which remained unaffected after heating at 60 degrees C for 16 min. Treatment of HAV with hydroxide alone (up to 1.0 m) at 15 degrees C did not lead to rapid inactivation. Collectively, these data suggest that 0.1 M NaOH at 60 degrees C for two min should be sufficient to inactivate viruses present in process residues.     

热力对冻干IgG免疫原性和免疫反应性的影响

冻干ＩｇＧ经８０℃２小时和１００℃３０分钟干热处理后,其抗原决定簇未见明显改变,活性抗原量与对照相同,抗－ＨＢｓ和抗人ＩｇＧ等抗体活性未受明显影响。而８０℃７２小时和１００℃２小时干热剂量对ＩｇＧ的免疫学活性影响较大     

Selective inactivation of the infectivity of freeze-dried Sendai virus by heat

The infectivity of freeze-dried Sendai virus was destroyed after heating at 100 ° C for 20 min while the hemagglutinin (HA) titer and the hemolytic (HL) activity were not affected. The HA titer was unaltered after heating at up to 140 ° C for 30 min. The HL activity was increased after freeze-drying, further increased after heating of freeze-dried virus at 115 ° C for 20 min, but was destroyed after heating for 30 min at 140 ° C.The selective heat inactivation of freeze-dried Sendai virus could be of use in the production of myxovirus vaccines and inactivated virus for cell-fusion studies.     

Assessment of viral inactivation during pH 3.3 pepsin digestion and caprylic acid treatment of antivenoms

Antivenoms are manufactured by the fractionation of animal plasma which may possibly be contaminated by infectious agents pathogenic to humans. This study was carried out to determine whether pre-existing antivenom production steps, as carried out by EgyVac in Egypt, may reduce viral risks. Two typical manufacturing steps were studied by performing down-scaled viral inactivation experiments: (a) a pH 3.3 pepsin digestion of diluted plasma at 30 degrees C for 1h, and (b) a caprylic acid treatment of a purified F(ab')2 fragment fraction at 18 degrees C for 1h. Three lipid-enveloped (LE) viruses [bovine viral diarrhoea virus (BVDV), pseudorabies virus (PRV), and vesicular stomatitis virus (VSV)] and one non-lipid-enveloped (NLE) virus [encephalomyocarditis virus (EMC)] were used as models. Kinetics of inactivation was determined by taking samples at 3 time-points during the treatments. The pH 3.3 pepsin digestion resulted in complete clearance of PRV (>7.0 log(10)) and in almost complete reduction of VSV (>4.5 but < or =6.4 log(10)), and in a limited inactivation of BVDV (1.7 log(10)). EMC inactivation was > or =2.5 but < or =5.7 log(10). The caprylic acid treatment resulted in complete inactivation of the 3 LE viruses tested: BVDV (>6.6 log(10)), PRV (>6.6 log(10)), and VSV (>7.0 log(10)). For EMC no significant reduction was obtained (0.7 log(10)). Cumulative reduction was >13.6, >11.5, >8.3 and > or =2.5 for PRV, VSV, BVDV and EMC, respectively. Therefore the current manufacturing processes of at least some animal antisera already include production steps that can ensure robust viral inactivation of LE viruses and moderate inactivation of a NLE virus.     

No influence of collagenous proteins of Achilles tendon, skin and cartilage on the virus-inactivating efficacy of peracetic acid–ethanol

The risk of transmitting human pathogenic viruses via allogeneic musculoskeletal tissue transplants is a problem requiring effective inactivation procedures. Virus safety of bone transplants was achieved using peracetic acid (PAA)-ethanol sterilisation. Proteins are known to have an adverse effect on the virus-inactivating capacity of PAA. Therefore we investigated virus inactivation by PAA in collagenous tissues. Achilles tendon, skin and cartilage were cut into small pieces, lyophilised and contaminated with pseudorabies virus (PRV) or porcine parvovirus (PPV). The inactivating capacity of PAA-ethanol was investigated by determining virus titres in the supernatant or the tissue pellet at different time-points. In all virus-contaminated tissue samples treatment for 10 min with PAA-ethanol resulted in titre reductions by a factor of >10(3). PRV was rapidly inactivated below the detection limit (< or =2.8 x 10(1) TCID(50)/ml). After 240 min a reduction by a factor of >10(4) was obtained for PPV in all samples, but a residual infectivity remained. Collagenous proteins of Achilles tendon, skin and cartilage had no adverse effect on the virus-inactivating capacity of PAA. PAA-ethanol used in the production process at the Charité tissue bank can therefore be recommended for treatment of non-osseous musculoskeletal tissues.     

水溶性红曲中MonacolinK的稳定性

对红曲中Monacolin K（MK）在水溶液中的稳定性进行研究,考察不同加热温度、时间、pH、盐、维生素C（VC）、氨基酸、蔗糖质量浓度、光照和存放时间对其稳定性的影响。结果表明：100℃保温30min,水溶液中Monacolin K的浓度比20℃保温相同时间仅下降了4．6％。热处理、长时间光照对水溶液中Monacolin K有轻微的破坏作用：pH〈5．0,盐、VC、氨基酸的添加可降低水溶液Monacolin K溶解度。pH〈4．0可使酸型Monacolin K转化为内酯型。在Monacolin K水溶液中添加30g/L蔗糖可使水溶液中Monacolin K饱和质量浓度增至632．9mg／L。     

Toxicity of crude extracellular products of Aeromonas hydrophila in tilapia, Tilapianilotica

Extracellular products (ECP) secreted from Aeromonas hydrophila with haemolytic andproteolytic activity were studied with respect to temperature and time of incubation as well as thelethal toxicity on tilapia, Tilapia nilotica . The highest production of the haemolysin productwas achieved when Aer. hydrophila was grown at 35°C for 30 h. Tilapia erythrocytewas found to be more susceptible than sheep erythrocyte for determining the haemolytic activity.The haemolytic activity against tilapia erythrocyte was completely inactivated after heating theECP at 60°C for 10 min or 55°C for 15 min. The proteolytic activity was maximized whenthe bacterium was grown at 30°C for 36 h. Complete inactivation of the protease enzyme wasperformed after heating the ECP at 80°C for 10 min or 70°C for 15 min. Aeromonashydrophila was found to produce haemolytic and proteolytic exotoxin lethal to tilapia (LD₅₀ 2·1 × 10⁴ cell/fish), as well as heat stable unknown virulent factors thatwere responsible for 20% mortality. The lethality of ECP was decreased by heating andcompletely inactivated by boiling at 100°C for 10 min.