S/D处理结合低pH放孵灭活静注人免疫球蛋白

在低ｐＨ静脉注射用人免疫球蛋白（ＩＶＩＧ）的制备中,采用有机溶剂／表面活性剂处理和低ｐＨ放孵（２３°Ｃ～２５°Ｃ,２１天）进行病毒灭活,以提高ＩＶＩＧ的安全性,两法累积灭活效果为：＞８ｌｏｇＨＩＶ－Ⅰ;＞１１．３ＬｏｇＶＳＶ和＞１０．８ＬｏｇＳｉｎｄｂｉｓ。     

静注丙球低pH孵放灭活病毒效果验证

以水泡性口炎病毒（ＶＳＶ）为指示病毒,考察了低ｐＨ孵放不同时间对低温乙醇法生产的静注丙球（ＩＶＩＧ）中ＶＳＶ的灭活效果,并对不同厂家及不同批号ＩＶＩＧ中病毒灭活情况进行了比较。结果表明,液体ＩＶＩＧ在ＰＨ４．１±０．３,２０－２５℃,孵放２１天可灭活ＶＳＶ达６Ｌｏｇｓ以上（即低于实验检测限）,但不同厂家及不同批号的ＩＶＩＧ在病毒灭活的发生上有所不同     

静脉注射免疫球蛋白制备中的病毒灭活

在静脉注射免疫球蛋白（ＩＶＩＧ）的制备中,采用有机溶剂结合表面活性剂（Ｓ／Ｄ）处理或６０℃１０小时液态加热（巴氏灭活法）的方法对组分Ⅱ（ＩｇＧ）进行病毒灭活处理,灭活效果用指示病毒进行了评价,结果表明：Ｓ／Ｄ法可有效灭活脂包膜病毒ＶＳＶ和Ｓｉｎｄｂｉｓ,巴氏灭活法则对上述病毒以及Ｖａｃｃｉｎｉａ和Ｅｃｈｏ病毒均有较好的灭活作用。经病毒灭活处理的免疫球蛋白,在理化及生物学特性上基本未受到不利影响,用两种方法分别处理组分Ⅱ后制备的ＩＶＩＧ,其主要特性指标均符合该制品的有关质量规定。     

鸡传染性支气管炎病毒S1基因在昆虫细胞中表达水平初探

将含有鸡传染性支气管炎病毒 Ｓ１ 基因ｃ Ｄ Ｎ Ａ 的重组转移质粒ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３ Ｓ１ ． Ｈｏｌｔｅ 和ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３／４ Ｓ１ ． Ｈｏｌｔｅ 分别与粉纹夜蛾核型多角体病毒 Ｔｎ Ｎ Ｐ Ｖ Ｓ Ｖ Ｉ－ Ｇ Ｄ Ｎ Ａ（ Ｏ Ｃ Ｃ－ ,ｇａｌ＋ ） 共转染草地夜蛾（ Ｓｆ９） 细胞,经空斑纯化得到重组病毒 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 和 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 。将重组毒株分别感染 Ｔｎ５ Ｂ１ 细胞,并进行 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 与 Ｗｅｓｔｅｒｎｂｌｏｔ 检测。结果表明, Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 在感染的细胞中高效表达了 Ｓ１ 蛋白, Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 凝胶薄层色谱分析结果显示,感染病毒后７２ ｈ Ｓ１ 蛋白的表达量占细胞内总蛋白量的３５ ．８ ％ ,而 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 感染的细胞内检测不出 Ｓ１ 蛋白。经分析认为这一差异主要来自 Ｓ１ 基因翻译起始位点及其附近的周围环境。     

稀土La~（3＋）和维生素C对黄瓜PSⅡ颗粒放氧活性机理影响的初探

用１０μｇ／ｇＬａ￣（３＋）、１．０％Ｖｃ、１０μｇ／ｇＬａ￣（３＋）＋１·０％Ｖｃ、蒸馏水四种处理,喷施正处在衰老过程中的黄瓜叶片,一周后提取ＰＳⅡ颗粒,通过ＳＤＳ－ＰＡＧＥ、薄层扫描和Ｃｌａｒｋ测氧等测试技术,发现处理与对照的ＰＳⅡ颗粒在与放氧活性密切相关的３３、２３、１７ｋＤ三肽的相对总含量上有变化,１０μｇ／ｇＬａ￣（３＋）处理比对照减少２８．９％,而１．０％Ｖｃ和１０μｇ／ｇＬａ￣（３＋）＋１．０％Ｖｃ处理的与对照接近。前者的放氧活性降低３７．３％,后者的放氧活性增加６２．４％,是对照的１．６倍,共同处理的结果与对照相近。可见,在光合放氧中,１．０％Ｖｃ与１０μｇ／ｇＬａ￣（３＋）拮抗。而且,３３、２３、１７ｋＤ三肽总量的相对减少与放氧活性的高低呈正相关。     

大鼠肥厚心肌缩短速度和肌凝蛋白重链同功酶谱的变化

用聚丙烯酰胺电泳分离并测定了大鼠左室肌凝蛋白ＡＴＰ酶活性依次降低的同功酶Ｖ_１,Ｖ_２和Ｖ_３的百分比（ＭＩ谱）,从乳头肌力－速度曲线读取心肌最大缩短速度（Ｖ_（ｍａｘ））,观察到：（１）正常大鼠出现增龄性Ｖ_１向Ｖ_３迁移和Ｖ_（ｍａｘ）下降,与８周龄组（Ｓ_０）相比,１６周和２４周龄组（Ｓ_８和Ｓ_（１６））的Ｖ_１／Ｖ_３比,分别下降３８．９％和６１．０％;Ｖ_（ｍａｘ）下降８．３％和１３．３％。（２）高血压肥厚心肌ＭＩ谱的迁移和Ｖ_（ｍａｘ）下降的程度大大超过增龄效应：高血压８周和１６周组（Ｈ_８和Ｈ_（１６））的Ｖ_１／Ｖ_３比值较术前对照Ｓ_０分别下降８４．４％和９３．５％,较同龄假手术对照Ｓ_８和Ｓ_（１６）组也分别低７４．５％和８３．３％,而Ｖ_（ｍａｘ）则比Ｓ_０组下降３３．３％和４８．３％。（３）６组４８只大鼠结果相关分析表明,Ｖ_（ｍａｘ）与Ｖ_１％高度正相关,与Ｖ_３％高度负相关。（Ｐ均小于０．０１）。上述结果提示：高血压肥厚心肌收缩速度明显下降,其主要生化机制似与同功酶谱由Ｖ_１优势向Ｖ_３迁移有关。     

用黄瓜花叶病毒卫星RNA生防制剂大面积防治麦茬辣椒病毒病

用黄瓜花叶病毒（ＣＭＶ）卫星ＲＮＡ生防制剂Ｓ＿（５２）免疫辣椒,能防治辣椒由ＣＭＶ引起的病毒病。用Ｓ＿（５２）免疫接种辣椒的大田对比试验,防效达５９．５－７１．０％,产量比对照增加２６．１－３２．２％,产值增加３３．５－４５．２％。小区对比试验,用Ｓ＿（５２）免疫接种５０天的辣椒,防效达７１．３－９２．９％,免疫接种８０天,防效达５５．９－７８．５％,产量比对照增加３６．０－６５．３％,产值增加４９．３－９３．５％,还有刺激生长,促进早熟和增强对真菌、细菌病害抵抗作用。     

黑龙江省及长春市烟草病毒病的种类鉴定

１９９１－１９９３年在黑龙江省主要烟区的１１个县及长春市采样,得１２９个毒株。经鉴别寄主测定,抗血清反应（板式酶联法或斑点酶联法）及电镜或免疫电镜观察,有ＴＭＶ（４３．４％）,ＰＶＹ（１７．８％）,ＣＭＶ（３．９％）,ＴＲＶ（０．８％）,ＴＳＷＶ等病毒,ＴＭＶ与ＰＶＹ混合侵染的占１０．１％,ＰＶＹ与其它病毒混合侵染的占１１．６％,另有５个标样为马铃薯Ｙ病毒组成员,１０个为未知。     

减蛋综合征病毒100K蛋白基因的克隆与序列分析

用常规方法提取减蛋综合征病毒（ＥＤＳＶ）中国分离株（ＡＡ２株）病毒ＤＮＡ,分别构建了限制性内切酶ＨｉｎｄⅢ、ＳｐｈⅠ、ＰｓｔⅠ水解片段的全基因文库,并对其中１００Ｋ蛋白基因的序列进行了分析。ＥＤＳＶ１００Ｋ蛋白基因位于减蛋综合征病毒基因组５５．７～６４．８物理图谱单位（ｍ．ｕ）,共２０９１个核苷酸（ｎｔ）,其编码产物由６９６个氨基酸（ａａ）组成,推测其分子量为７７．７ｋＤ。编码蛋白氨基酸同源性分析表明,ＥＤＳＶ１００Ｋ蛋白与人腺病毒（Ａｄ２、Ａｄ５、Ａｄ１２、Ａｄ４１）、Ⅰ群禽腺病毒（ＣＥＬＯ和ＦＡＶ１０）的同源性为３２．３～３４．４％之间,而与羊腺病毒（ＯＡＶ）的同源性达到５６．４％。     

用T-A载体克隆技术构建丙肝病毒C+E1区真核表达质粒pSVL-HCV/C+E1

用ＢａｍＨＩ和ＨｉｎｄＩＩ将丙肝病毒Ｃ＋Ｅ１ＤＮＡ片段从其克隆载体ｐＧＥＭ３ｚｆ－ＨＣＶ／Ｃ＋Ｅ１上切下,经Ｔａｑ酶补齐３’末端后插入到载体ｐＳＶＬ－Ｔ中,构建成丙肝病毒Ｃ＋Ｅ１真核表达载体ｐＳＶＬ－ＨＣＶ／Ｃ＋Ｅ１。本实验中重组效率达６４．７％（１１／１７）,正向插入为５０％（２／４）。     

Prevalence of Herpes and Respiratory Viruses in Induced Sputum among Hospitalized Children with Non Typical Bacterial Community-Acquired Pneumonia

Objective

Few comprehensive studies have searched for viruses in infants and young children with community-acquired pneumonia (CAP) in China. The aim of this study was to investigate the roles of human herpes viruses (HHVs) and other respiratory viruses in CAP not caused by typical bacterial infection and to determine their prevalence and clinical significance.

Methods

Induced sputum (IS) samples were collected from 354 hospitalised patients (infants, n = 205; children, n = 149) with respiratory illness (CAP or non-CAP) admitted to Wenling Hospital of China. We tested for HHVs and respiratory viruses using PCR-based assays. The epidemiological profiles were also analysed.

Results

High rate of virus detection (more than 98%) and co-infection (more than 80%) were found among IS samples from 354 hospitalised infants and children with respiratory illness in this study. Of 273 CAP samples tested, CMV (91.6%), HHV-6 (50.9%), RSV (37.4%), EBV (35.5%), HBoV (28.2%), HHV-7 (18.3%) and rhinovirus (17.2%) were the most commonly detected viruses. Of 81 non- CAP samples tested, CMV (63%), RSV (49.4%), HHV-6 (42%), EBV (24.7%), HHV-7 (13.6%) and HBoV (8.6%) were the dominant viruses detected. The prevalence of several viral agents (rhinovirus, bocavirus, adenovirus and CMV) among IS samples of CAP were significantly higher than that of non-CAP control group. We also found the prevalence of RSV coinfection with HHVs was also higher among CAP group than that of non-CAP control.

Conclusions

With sensitive molecular detection techniques and IS samples, high rates of viral identification were achieved in infants and young children with respiratory illness in a rural area of China. The clinical significance of rhinovirus, bocavirus, adenovirus and HHV (especially CMV) infections should receive greater attention in future treatment and prevention studies of CAP in infants and children.     

High Incidence of Multiple Viral Infections Identified in Upper Respiratory Tract Infected Children under Three Years of Age in Shanghai, China

Background

Upper respiratory tract infection (URTI) is a major reason for hospitalization in childhood. More than 80% of URTIs are viral. Etiological diagnosis of URTIs is important to make correct clinical decisions on treatment methods. However, data for viral spectrum of URTIs are very limited in Shanghai children.

Methods

Nasopharyngeal swabs were collected from a group of 164 children aged below 3 years who were hospitalized due to acute respiratory infection from May 2009 to July 2010 in Shanghai. A VRDAL multiplex PCR for 10 common respiratory viruses was performed on collected specimens compared with the Seeplex® RV15 ACE Detection kit for 15 respiratory viruses.

Results

Viruses were detected in 84 (51.2%) patients by VRDAL multiplex PCR, and 8 (4.9%) of cases were mixed infections. Using the Seeplex® RV15 ACE Detection kit, viruses were detected in 129 (78.7%) patients, 49 (29.9%) were co-infected cases. Identified viruses included 37 of human rhinovirus (22.6% of cases), 32 of influenza A virus (19.5%), 30 of parainfluenzavirus-2 (18.3%), 23 of parainfluenzavirus-3 (14.0%), 15 of human enterovirus (9.1%), 14 each of parainfluenzavirus-1, respiratory syncytial virus B and adenovirus (8.5%), 8 of coronavirus 229E/NL63 (4.9%), 6 of human bocavirus (3.7%), 5 each of influenza B virus and respiratory syncytial virus A (3.0%), 3 of parainfluenzavirus-4 (1.8%), 2 of coronavirus OC43/HKU1 (1.2%), and 1 human metapneumovirus (0.6%).

Conclusions

A high frequency of respiratory infections (78.7%) and co-infections (29.9%) was detected in children with acute respiratory infection symptoms in Shanghai. The Seeplex® RV15 ACE detection method was found to be a more reliable high throughput tool than VRDAL method to simultaneously detect multiple respiratory viruses.     

Is halogen content the most important factor in the removal of halogenated trace organics by MBR treatment?

This study investigated the relationship between physicochemical properties (namely halogen content and hydrophobicity) of halogenated trace organics and their removal efficiencies by a laboratory scale membrane bioreactor (MBR) under stable operating conditions. The reported results demonstrated a combined effect of halogen content and hydrophobicity on the removal. Compounds with high halogen content (>0.3) were well removed (>85%) when they possessed high hydrophobicity (Log D>3.2), while those with lower Log D values were also well removed if they had low halogen content (<0.1). General indices such as the BIOWIN index (which is based on only biodegradation) or a more specific index such as the halogen content (which captures a chemical aspect) appeared insufficient to predict the removal efficiency of halogenated compounds in MBR. Experimental data confirmed that the ratio of halogen content and Log D, which incorporates two important physico-chemical properties, is comparatively more suitable.     

A Novel Simple Assay System to Quantify the Percent HCV-RNA Levels of NS5A Y93H Mutant Strains and Y93 Wild-Type Strains Relative to the Total HCV-RNA Levels to Determine the Indication for Antiviral Therapy with NS5A Inhibitors

Aim

Oral treatment with asunaprevir and daclatasvir has been reported to yield a SVR ratio of 80% in patients with genotype 1b HCV infection, however, treatment failure has been reported, especially in patients with HCV strains showing the NS5A-Y93H mutation at baseline. An assay system to detect such strains was established to facilitate selection of appropriate candidates for this antiviral therapy.

Methods

Primer sets and 2 types of cycling probe mixtures were designed, and real-time PCR was performed with HCV-RNA purified from 332 patients with genotype 1b HCV infection, and the results were compared with those obtained by direct sequencing.

Results

Both the wild-type and mutant strains were quantified, with a threshold of 4.0 Log copies/mL, in 295 of the 332 patients (88.9%), and the percentage of the mutant strains relative to the total HCV-RNA level in the serum was calculated. The percentage was 0% in 237 patients (80.3%) and 100% in 23 patients (7.8%), identical to the results of direct sequencing. Both wild-type and mutant strains were detected in the remaining 35 patients (11.9%), at levels between 1% and 99%, despite the mutant strains having been undetectable by direct sequencing in 11 patients with percentages of these strains of less than 25%.

Conclusion

A novel assay system to quantify the percent RNA of Y93H mutant strains relative to the total HCV-RNA level was established. This system may be useful to determine the indication for treatment with NA5A inhibitors in patients with HCV.     

Inactivation of lipid enveloped viruses by octanoic Acid treatment of immunoglobulin solution.

Inactivation of lipid enveloped viruses by treatment with octanoic acid has been investigated for three intravenous immunoglobulin preparations, using Human Immunodeficiency Virus, Bovine Viral Diarrhoea Virus, Sindbis Virus and Pseudorabies Virus as test viruses. At a concentration of 7.45 g octanoic acid per kg solution complete inactivation of lipid enveloped viruses to below detectable level (>5.36, >4.68, >6.25 and >5.55 log(10), respectively) was achieved within the first minutes of treatment. Octanoic acid treatment as described here, has been demonstrated as an effective and rapid virus inactivation procedure, which shows high robustness at the tested ranges of temperature, pH and protein content of the test material. However, pH must be considered as a critical parameter of treatment, as octanoic acid fails to inactivate lipid coated viruses at basic pH. At suitable conditions, e.g. pH<6.0 and a concentration of >3.7 g/kg, octanoic acid treatment gives reliable and highly effective inactivation of lipid enveloped viruses.     

Neutralizing antibodies against endogenous interferon in myasthenia gravis patients

Anti-cytokine antibodies (Abs) play an important role in the regulation of the immune response, both under normal conditions and in several autoimmune and neoplastic disorders. In the present study, we have investigated the occurrence and the clinical significance of natural neutralizing Abs (NAbs) against interferons (IFNs) alpha, beta, and gamma, as detected by bioassay, in 52 patients with myasthenia gravis (MG), and 43 sex- and age-matched healthy individuals. Patients showing titres > or = 1.3 Log t(1/10), confirmed in 2 consecutive samples collected two months apart, were considered positive. NAbs against any of the IFNs were not detected in healthy subjects. Of the 52 MG patients, 11 (21.1%) had NAbs against IFNalpha and three (5.8%) had NAbs against IFNbeta. None of these patients was found to be positive for NAbs against IFNgamma. Of the patients positive for NAbs against IFNalpha, eight (15.4%) had NAbs at titres > or =2 Log t(1/10). A positive association was observed between high titres of NAbs and the presence of thymoma. These data suggest the presence of a generalized activation of the humoral response in MG.     

Evaluation of viral clearance in the production of HPV-16 L1 virus-like particles purified from insect cell cultures

Biopharmaceutical products produced from cell cultures have a potential for viral contamination from cell sources or from adventitious introduction during production. The objective of this study was to assess viral clearance in the production of insect cell-derived recombinant human papillomavirus (HPV)-16 type L1 virus-like particles (VLPs). We selected Japanese encephalitis virus (JEV), bovine viral diarrhea virus (BVDV), and minute virus of mice (MVM) as relevant viruses to achieve the aim of this study. A downstream process for the production of purified HPV-16 L1 VLPs consisted of detergent lysis of harvested cells, sonication, sucrose cushion centrifugation, and cesium chloride (CsCl) equilibrium density centrifugation. The capacity of each purification/treatment step to clear viruses was expressed as reduction factor by measuring the difference in log virus infectivity of sample pools before and after each process. As a result, detergent treatment (0.5% v/v, Nonidet P-40/phosphate-buffered saline) was effective for inactivating enveloped viruses such as JEV and BVDV, but no significant reduction (< 1.0 log(10)) was observed in the non-enveloped MVM. The CsCl equilibrium density centrifugation was fairly effective for separating all three relevant adventitious viruses with different CsCl buoyant density from that of HPV-16 L1 VLPs (JEV, BVDV, and MVM = 4.30, 3.10, > or = 4.40 log(10) reductions). Given the study conditions we used, overall cumulative reduction factors for clearance of JEV, BVDV, and MVM were > or = 10.50, > or = 9.20, and > or = 6.40 log(10) in 150 ml of starting cell cultures, respectively.     

Molecular detection of viruses in Kenyan bats and discovery of novel astroviruses,caliciviruses and rotaviruses

This is the first country-wide surveillance of bat-borne viruses in Kenya spanning from 2012–2015covering sites perceived to have medium to high level bat-human interaction. The objective of this surveillance study was to apply a non-invasive approach using fresh feces to detect viruses circulating within the diverse species of Kenyan bats. We screened for both DNA and RNA viruses;specifically, astroviruses(AstVs), adenoviruses(ADVs), caliciviruses(CalVs), coronaviruses(CoVs), flaviviruses, filoviruses, paramyxoviruses(PMVs), polyomaviruses(PYVs) and rotaviruses.We used family-specific primers, amplicon sequencing and further characterization by phylogenetic analysis. Except for filoviruses, eight virus families were detected with varying distributions and positive rates across the five regions(former provinces) studied. AstVs(12.83%), CoVs(3.97%), PMV(2.4%), ADV(2.26%), PYV(1.65%), CalVs(0.29%), rotavirus(0.19%) and flavivirus(0.19%). Novel CalVs were detected in Rousettus aegyptiacus and Mops condylurus while novel Rotavirus-A-related viruses were detected in Taphozous bats and R. aegyptiacus. The two Rotavirus A(RVA) strains detected were highly related to human strains with VP6 genotypes I2 and I16. Genotype I16 has previously been assigned to human RVA-strain B10 from Kenya only, which raises public health concern, particularly considering increased human-bat interaction.Additionally, 229E-like bat CoVs were detected in samples originating from Hipposideros bats roosting in sites with high human activity. Our findings confirm the presence of diverse viruses in Kenyan bats while providing extended knowledge on bat virus distribution. The detection of viruses highly related to human strains and hence of public health concern, underscores the importance of continuous surveillance.     

Dry-heat treatment process for enhancing viral safety of an antihemophilic factor VIII concentrate prepared from human plasma

Viral safety is a prerequisite for manufacturing clinical antihemophilic factor VIII concentrates from human plasma. With particular regard to the hepatitis A virus (HAV), a terminal dry-heat treatment (100 degrees for 30 min) process, following lyophilization, was developed to improve the virus safety of a solvent/detergent-treated antihemophilic factor VIII concentrate. The loss of factor VIII activity during dry-heat treatment was of about 5%. No substantial changes were observed in the physical and biochemical characteristics of the dry-heat-treated factor VIII compared with those of the factor VIII before dry-heat treatment. The dry-heat-treated factor VIII was stable for up to 24 months at 4oC. The dry-heat treatment after lyophilization was an effective process for inactivating viruses. The HAV, murine encephalomyocarditis virus (EMCV), and human immunodeficiency virus (HIV) were completely inactivated to below detectable levels within 10 min of the dry-heat treatment. Bovine herpes virus (BHV) and bovine viral diarrhea virus (BVDV) were potentially sensitive to the treatment. However porcine parvovirus (PPV) was slightly resistant to the treatment. The log reduction factors achieved during lyophilization and dry-heat treatment were > or =5.55 for HAV, > or =5.87 for EMCV, > or =5.15 for HIV, 6.13 for BHV, 4.46 for BVDV, and 1.90 for PPV. These results indicate that dry-heat treatment improves the virus safety of factor VIII concentrates, without destroying the activity. Moreover, the treatment represents an effective measure for the inactivation of non-lipid-enveloped viruses, in particular HAV, which is resistant to solvent/detergent treatment.