一起腺病毒引起的急性呼吸道感染暴发疫情的调查研究

探讨2016年12月深圳市光明新区某小学一起急性呼吸道病毒感染暴发疫情的流行病学及病原学特点。采用相应的流行病学个案调查表对疫情进行现场调查,采集患者咽拭子标本,使用实时荧光定量PCR方法对标本进行流感样病例筛查,筛查后的阴性标本进行呼吸道病毒核酸检测,用腺病毒六邻体蛋白基因作为靶基因对腺病毒初筛阳性标本进行扩增并测序,测序结果在GenBank上进行序列比较后使用BLAST和MEGA 5.0软件进行分析。该起疫情首例病例发病时间为2016年12月2日,末例病例发病时间为2016年12月17日,共发病34例,期间出现3次发病高峰,患者均为学生,其中男女生各17例,男女比例1∶1。患者体温均39℃,大部分有头晕、头痛症状,少数伴有咽痛、咳嗽症状。发病的四个班级间的罹患率差异有统计学意义。共采集8份咽拭子标本,经实时荧光定量PCR检测均为腺病毒阳性,其中1份合并鼻病毒阳性。8份标本用分型引物检测并分析序列确定均为腺病毒E亚属4型。此次急性呼吸道感染暴发疫情为连锁式传播,由腺病毒4型引起,毒株序列与深圳、国内其他城市分离的腺病毒4型毒株序列具有高度同源性,提示该毒株可用于疫苗株筛选。早期发现可疑病例并采取隔离措施是预防腺病毒感染暴发的最佳手段。     

浙江省2019年儿童腺病毒肺炎流行病学特征分析

为了解浙江地区腺病毒肺炎的流行病学和病原学特征,本研究统计分析了2019年1月至2019年12月浙江大学医学院附属儿童医院腺病毒肺炎患儿的基本临床资料;并前瞻性的通过聚合酶链式反应(PCR)随机对部分的腺病毒阳性的肺炎患儿咽拭子进行基因扩增、测序和分型鉴定;进而构建系统发育树分析其核苷酸序列进化情况。本研究共检测咽拭子标本19650份,其中腺病毒阳性标本有4032份,腺病毒检出率为20.5%;春季和夏季腺病毒肺炎的患病率相对较高,患儿年龄主要在1~7岁。本研究所检测的99株腺病毒阳性标本经与GenBank数据库比对均为HAdV-3,通过Hexon基因全长系统进化分析得出,所检出的HAdV-3腺病毒均为Clade2进化分支。2019年浙江地区存在儿童HAdV-3肺炎的暴发流行,该病毒株未发生大的变异,均为Clade2进化分支。1~7岁的儿童为主要的易感人群。     

儿童呼吸道肺炎支原体感染的实验室诊断

目的同时用目前常用的几种诊断肺炎支原体（MP）感染的实验方法检测MP,相互比较,得出适于常规诊断MP感染的方法或组合。方法搜集我院于2006年10月至2007年5月间以呼吸道感染入院儿童病例75例,采集其咽拭子和双份血清标本,以多种方法检测有无MP感染：培养法、EIA法测抗原、PCR法测MP-DNA,ELISA法测MP特异性IgG、IgA型抗体以及捕获法ELISA测MP特异性IgM型抗体。结果上述75例儿童中,共计有12例感染MP。以此为基础,上述各方法的敏感度分别是：培养法（25％）、EIA法测抗原（8．3％）、PCR法测MP-DNA（75．0％）、ELISA法测MP特异性IgA型抗体（单份血清为0,双份血清为33．3％）、捕获法ELISA测MP特异性IgM型抗体（单份血清为66．7％,双份血清为100％）。特异度分别是：100％、96．8％、93．7％（93．7％）、98．4％（98．4％）。PCR法和捕获法ELISA测MP特异性IgM型抗体结合后敏感度和特异度分别达到100％和95．2％。结论PCR法测MP-DNA和捕获法ELISA测MP特异性IgM型抗体的组合可高效地诊断MP感染,因而可作为临床诊断MP感染的一个常规组合。     

2012年大连市流行性角结膜炎疫情的病原鉴定与基因特征分析

目的 鉴定一起大连市流行性角结膜炎疫情的病原体及型别.方法 采用荧光PCR方法针对30份眼拭标本进行检测,确定病原,并针对特定片段扩增产物测序进行分子定型.结果 21份标本荧光PCR检测结果为腺病毒阳性,核苷酸序列与人腺病毒15、29、56及其重组型高度同源,多重序列比对后构建的系统进化树上与人腺病毒56及其重组型位于同一分支.结论 本次疫情的致病病原为D亚属人腺病毒,疑为人腺病毒56型或其重组型.后续需要开展对五邻体(Penton)及纤突(Fi-ber)基因的全核苷酸序列测定及生物信息学分析最终确定型别.     

2004年冬至2005年春引起北京地区儿童肺炎的腺病毒基因特点分析

为了解北京地区腺病毒的流行毒株,从北京儿童医院收集25份儿童肺炎患者的痰洗液标本(2004年冬到2005年春),患者的年龄在2个月到2岁,临床表现以持续高热、咳喘和气促为主。对25份标本分别用Hep-2细胞进行了病毒分离培养、腺病毒属特异性引物PCR扩增以及PCR产物的序列测定分析。结果发现25份标本中有9份PCR扩增阳性,其中2份病毒培养物观察到典型的细胞病变;将9份PCR阳性产物纯化后进行序列测定,结果7份标本核苷酸序列完全一致,同源性100%,并与GenBank发表的序列相比较,发现与腺病毒7型最为接近,同源性皆为99.7%;2份与腺病毒3型最接近,同源性为99.6%。结果表明,此次病毒分离株为腺病毒B亚群。北京儿童医院2004年12月至2005年3月腺病毒引起的肺炎主要以7型和3型为主,7型占优势,与国内其它地区的情况相似。     

IgA肾病患者血液、尿液及咽拭子标本中穿通支原体的分离检出

目的探讨IgA肾病患者血液、尿液及咽拭子标本中穿通支原体（Mycoplasma penetrans,Mp）的分离检出率以及与病理型别相关性。方法采用分离培养法,共计从26例IgA肾病患者血液、尿液及咽拭子标本及38例正常对照相应标本中进行穿通支原体分离检测,对培养阳性标本用穿通支原体套式PCR进行证实。结果在11例（42．3％）患者血液与尿液或（和）咽拭子中同时分离到穿通支原体,单独尿液或咽拭子标本阳性分别为1例（3．8％）与7例（26．9％）。26例IgA肾病患者血液、尿液及咽拭子穿通支原体的分离检出率分别为42．3％、23．1％与57．7％;与38例正常对照组血液、尿液及咽拭子检出0例、2例（5．3％）与7例（18．4％）相比较,差异有非常显著性（P〈0．01）,在正常对照组中无2种以上标本同时检出穿通支原体。结论穿通支原体在ISA肾病患者的血液、尿液与咽拭子标本中均有较高的检出率且与病理型别有一定的相关性。     

1973—1975年北京地区婴幼儿病毒性肺炎的病原学研究

1973一1975年连续三个冬、春我们在北京地区对85例临床诊断为病毒性肺炎的婴幼儿病例进行了病原学研究。从59例的咽拭子分离出腺病毒,主要是7型,其次是3型。还从合并有胸膜炎的14例中10例的胸腔积液分离出型别相同的腺病毒。同型腺病毒也从5例死亡病儿的肺组织分离出来。从l例的咽拭子分离出I型副流感病毒。从60例得到急性期和恢复期双份血清进行了血清学诊断。在测定中和抗体的47例,7型或3型腺病毒中和抗体都显著升高。在测定补体结合抗体的60例中,40例的补体结合抗体也有明显的增长。从咽拭子、胸腔积液和肺组织分离出来的腺病毒代表株与同型腺病毒原型株的电子显微镜观察和比较表明,其形态一致。     

国内首次自患者标本中同时分离的登革2型和3型病毒的鉴定

采用间接免疫荧光方法 ,检测患者血清标本中的抗登革病毒IgM和IgG抗体 ;同时将病人急性期血清接种C6 36细胞进行病毒分离。从分离的病毒悬液中提取RNA ,进行RT PCR扩增和序列测定。结果显示 ,该患者血清中存在抗登革病毒的IgM和IgG抗体。从病人血清中分离的病毒 ,经RT PCR和序列测定证实为登革 2型和 3型病毒的特异序列。表明该患者为登革 2型和 3型病毒混合感染     

肠道病毒EV71 IgM抗体参考品的建立

目的建立肠道病毒EV71 IgM抗体检测用参考血清盘。方法通过收集手足口病感染早期患者的咽拭子和血清,对咽拭子病毒进行分离、鉴定、基因分型,通过血清中和试验及酶联免疫吸附试验(捕获法)的验证,确定EV71 IgM抗体阳性样品,组建EV71 IgM抗体参考血清盘,并经3家实验室对该参考血清盘进行协同标定和确认。结果 12名患儿的咽拭子中均分离到EV71病毒,在RD细胞上引起细胞病变。通过套式PCR扩增、序列测定后进化树分析确认均为流行的C4基因亚型。每名患儿的双份血清均可中和EV71病毒,抗体效价为1∶512~1∶2048。通过捕获法进行EV71 IgM抗体检测结果均为阳性。以此12份样品为原料制备EV71 IgM抗体阳性参考品,同时以12份EV71 IgM抗体阴性血清制备阴性参考品,建立了由12份阳性参考品、12份阴性参考品、1份最低检出限参考品和1份精密性参考品组成的肠道病毒EV71 IgM抗体参考血清盘。通过3家实验室协同标定,各实验室间差异均无统计学意义。结论建立了EV71 IgM抗体检测参考血清盘,为相关检测试剂的质量控制和评价提供了参考标准。     

快速获取55型腺病毒基因组序列的方法

【目的】建立快速获取55型腺病毒的全长基因组序列的方法。【方法】根据55型腺病毒的基因组特点,设计覆盖55型腺病毒基因组序列的12对引物,分别以55型腺病毒DNA为模板,扩增得到12个PCR产物,通过对12个PCR产物测序及序列拼接,获得55型腺病毒的全长基因组序列。【结果】从本院急性上呼吸道感染者咽拭子标本中分离得到一株55型腺病毒毒株SF04/SC/2016,以其DNA为模板扩增成功获得12个PCR产物,对其进行测序,并对12段序列进行拼接得到55型腺病毒的全长基因组序列,与已报到的各型腺病毒序列进行比对,采用邻位相连法构建系统发育进化树,所得序列与55型腺病毒处于同一分支,进一步确认该病原体为55型腺病毒。【结论】研究公布的序列和方法,能够实现更方便对腺病毒的快速测序,为揭示55型腺病毒的进化特点及制订疾病防控策略提供了有效手段。     

Evidence for the involvement of unsaturated fatty acids in initiating chromosome replication in Escherichia coli

The analogue 3-decynoyl-N-acetylcysteamine inhibits the synthesis of unsaturated fatty acids in Escherichia coli, resulting in the accumulation of saturated fatty acids in the membrane (Kass, 1968).In the presence of this analogue, DNA, RNA and protein synthesis continue at a linear rate for approximately two doubling times, and then cease. On the other hand, the analogue will inhibit the formation of new replication forks (premature initiation), which normally arise as a result of thymine starvation.Unlike other temperature-sensitive DNA mutants, mutants that are defective in initiating DNA replication (dnaA or dnaC) are unable to replicate DNA at a permissive temperature if they terminate replication at 42 °C in the presence of 3-decynoyl-N-acetylcysteamine.When replication is terminated at 42 °C, cultures of dnaA or dnaC mutants normally will reinitiate replication upon lowering the temperature to 30 °C. For each mutant this reinitiation is characterized by a particular temperature sensitivity. Such mutants become more temperature sensitive if the temperature is lowered in the presence of 3-decynoyl-N-acetylcysteamine. All the effects of this analogue can be reversed by the addition of unsaturated fatty acids.These results are interpreted using a model in which replication is initiated at a particular lipid site on the membrane. In the absence of unsaturated fatty acids functional lipid sites are not made. Functional sites, however, can be used again provided they are not inactivated by interaction with an inactive dnaA or dnaC product.     

3-Oxoecdysteroid reductases in Manduca sexta midgut

The 80,000g supernatant o larval midgut homogenates of the tobacco hornworm, Manduca sexta, was fractionated by affinity chromatography on Blue Sepharose CL-6B and by anion exchange chromatography on Q Sepharose. Both methods resolved one major 3-oxoecdysteroid 3α-reductase and three major 3-oxoecdysteroid 3β-reductases. The 3β-reducates reacted only with BADPH as cosubstrate. The 3α-reductase was active with both NADPH and NADH, and the NADPH/NADH activity ratio increased with the NaCl concentration (0–0.5 M) in the incubation mixtures. The 3-α-reductase and one of the 3-β-reductases showed very similar chromatographic properties, and their isoelectric points were 5.2 and 5.8, respectively. © 1992 Wiley-Liss, Inc.     

The mutational spectrum of procarbazine in Drosophila melanogaster.

The antineoplastic agent Procarbazine was tested for the induction of genetic damage in Drosophila melanogaster. The compound was administered to adult males by oral application. The following types of genetic damage were measured: (1) sex-linked recessive lethals; (2) dominant lethals; (3) total and partial sex-chromosome loss; and (4) translocations. Procarbazine is highly mutagenic in causing recessive lethal mutations in all stages of spermatogenesis. In sperm a clear-cut concentration-effect relationship is not apparent, but in spermatids such a relationship is obtained for mutation induction at low levels of procarbazine exposure, while at high concentrations the induction of recessive lethals is not a function of concentration. A low induction of total sex-chromosome loss (X,Y) and dominant lethals was observed in metabolically active germ cells (spermatids), but procarbazine failed to produce well-defined breakage events, such as partial sex-chromosome loss (YL,YS) and II-III translocations. The results obtained in Drosophila melanogaster are discussed and compared with the mutational pattern reported in the mouse after procarbazine treatment.     

Strain differences in the induction of soluble and microsomal enzymes by phenobarbital and 3-methylcholanthrene

The ability of phenobarbital and 3-methylcholanthrene (3MC) to induce liver microsomal and soluble enzymes was compared in Sprague-Dawley and Long-Evans rats. 3MC increased the V for the aniline hydroxylase and stimulated the formation of the hemoprotein P₄₄₈ to a similar extent in the 2 strains of rats. On the other hand phenobarbital increased the V for the microsomal enzyme aniline hydroxylase and aminopyrine demethylase and enhanced the activity of the soluble enzyme aldehyde dehydrogenase only in Sprague-Dawley rats. It induced a more marked increase of cytochrome P₄₅₀ in the Sprague-Dawley than in the Long-Evans strain.     

Gender-related differences in the formation of skatole metabolites by specific CYP450 in porcine hepatic S9 fractions

Higher accumulation of skatole in the fat of male pigs compared with female pigs might be due to gender-related differences in the rate of skatole degradation. In the present study, skatole metabolites and cytochrome P450 (CYP450) isoforms involved in skatole metabolism were for the first time investigated in hepatic S9 fractions from six male and four female pigs (crossbred Landrace×Yorkshire dams and Duroc boar). Surprisingly, the rates of production of major skatole metabolites were similar in male and female pigs. The most abundant metabolite of skatole was 3-hydroxy-3-methyloxindole (HMOI) followed by 3-methyloxindole and indole-3-carbinol in both male and female S9 fractions. Concentrations of formed HMOI and 3-methyloxindole did not differ between the genders (P=0.124 for HMOI, and P=0.575 for 3-methyloxindole). Indole-3-carbinol formation was higher in S9 fractions from the females compared with male pigs (P=0.0001). Enzyme kinetic parameters were similar for both genders (P>0.05). In both male and female pigs, ellipticine, diallyl sulphide (DAS) and quercetin inhibited HMOI formation, confirming the involvement of CYP1A1 and CYP2E1. The formation of 3-methyloxindole was reduced in the presence of the CYP2E1 inhibitor DAS, and formation of indole-3-carbinol was reduced in the presence of CYP1A1 and CYP2A19 inhibitors. We found only minor differences in skatole metabolism between male and female pigs, particularly the involvement of CYP2C and CYP3A in indole-3-carbinol formation in female but not in male pigs. This is a very essential finding, suggesting the involvement of larger number of CYP450 isoforms in female pigs. On the other hand, indole-3-carbinol is a minor skatole metabolite, and the physiological significance of CYP2C and CYP3A involvement in its formation in female pigs, but not in male pigs, needs to be elucidated. Our results, however, should be interpreted with caution because of the low number of animals and possibility of breed and age effects on skatole metabolism.     

鼠源成纤维细胞生长因子-21对脂肪细胞糖代谢的作用

成纤维细胞生长因子-21(FGF-21)是FGF家族的成员之一．近年发现FGF-21是一种新的代谢调节因子．从小鼠肝脏克隆FGF-21 cDNA,经测序正确后亚克隆至具有羟胺切割位点的小泛素相关修饰物表达载体上,转化宿主菌Rosetta,得到的转化子经IPTG诱导后获得稳定、高效、可溶的表达产物．表达产物经羟胺切割、透析、复性、柱层析纯化后,在每升宿主菌中可获得4 mg纯度为95%的成熟鼠源FGF-21蛋白,利用葡萄糖氧化酶-过氧化物酶(POD-GOD)法在小鼠3T3-L1脂肪细胞中进行生物学活性检测．结果表明,鼠源FGF-21具有促进脂肪细胞吸收葡萄糖的作用,短期作用(1 h)与胰岛素相似,长期作用(8和12 h)明显优于胰岛素．这一结果为以鼠源FGF-21为模型进一步研究FGF-21的生物学活性及其在糖代谢方面的作用机理奠定了基础．     

Ecdysone oxidase and 3-oxoecdysteroid reductases in Manduca sexta: Developmental changes and tissue distribution

The activities of ecdysone oxidase (EO), 3-oxoecdysteroid 3α-reductase (3α-R), and 3-oxoecdysteroid 3β-reductase (3β-R) were determined for epidermis, hemolymph, and fat body of wandering fifth instar Manduca sexta larvae and for midguts of various developmental stages between 3 days after the last larval and 14 days after the pupal ecdysis. The larval midgut was the only organ showing substantial specific activities of EO and 3α-R, and both increased up to the seventh day after ecdysis. Hemolymph and fat body had only moderate to high 3β-R and low EO activites, and the epidermis did not contain significant activity of any of the enzymes. On the ninth day after the last larval ecdysis the larval midgut epithelium was replaced by a new pupal midgut epithelium. After this event only 3β-R was restored to high activities, whereas EO and 3α-R showed only low to marginal activities. It is concluded that only the larval midgut has a role in the inactivation of ecdysteroids by 3-epimerization. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.