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。     

儿童重症腺病毒肺炎的临床研究

目的:分析儿童重症腺病毒肺炎(SAP)的临床特点。方法:选择2011年01月至2015年01月广州市妇女儿童医疗中心重症监护室收治的被诊断为SAP的患儿53例。患儿入监护室后24 h内采集静脉血,检测血常规、血气、生化等指标,并结合病史与常规生化等检测指标同步给出PCIS评分。根据患儿的转归,将所有患儿分为生存组和死亡组进行比较分析。结果:53例重症腺病毒肺炎患儿,男女比例为3.4:1,2岁以下发病者48例,夏秋季发病共40例。死亡组患儿共8例(15.1%),其LDH、AST、Pa CO2水平、肺叶受累数量、并发症种类较存活组患儿明显升高,危重症评分(PCIS)、血小板、白蛋白、Pa O2水平、Pa O2/FIO2比值均较生存组显著降低(P均0.05)。结论:儿童SAP炎症反应重,常伴肺内外各种损害,早期的LDH、AST、白蛋白水平、血小板数量、Pa O2、Pa CO2、Pa O2/Fi O2及PCIS评分是疾病预后的早期预测指标,有助于临床医生对重症腺病毒肺炎患儿病情危重程度尽早作出正确判断。     

腺病毒7d基因型与7b基因型全基因组比较分析

为了解重庆地区流行的7型腺病毒(Human adenovirus 7,HAdV-7)基因型型别,从全基因组水平探讨HAdV-7b和HAdV-7d可能的致病性差异,收集儿童急性下呼吸道感染患儿的呼吸道分泌物标本分离培养,提取DNA后进行HAdV-7基因型别鉴定、限制性酶切分析和全基因组测序,再利用MEGA6比较分析。本研究在2009年6月~2013年1月间共收集4334份急性下呼吸道感染患儿的鼻咽抽吸物标本,随机选取2411份进行病毒分离培养,共有164份(164/2411,6.8%)鼻咽抽吸物HAdV阳性,其中HAdV-7 87例。限制性酶切分析显示,随机挑选的3株HAdV-7分离株均为HAdV-7d。CQ1198株行全基因组测序并与其他HAdV-7全基因组构建进化树,结果发现16株中国地区的HAdV-7为HAdV-7d基因型,且上述16株HAdV-7与7d2的同源性为99.9%,与7b的同源性为98.2%。相较于ak40株(HAdV-7b),CQ1198共有49处核苷酸替换,其中20处可导致非同义替换;4处核苷酸插入,其中24bp和18bp 2个片段插入分别位于病毒相关RNAⅡ(Virus-associated RNAⅡ,VA RNAⅡ)和L3的pⅥ区域。HAdV-7可有多个基因型致病,且一直处于进化过程中,其主要流行基因型由HAdV-7b变为HAdV-7d。应加强对HAdV分子流行病学监测,明确其进化规律,为HAdV疫苗研发提供分子流行病学依据。     

长春和北京地区引起婴幼儿肺炎的3型与7型腺病毒的分子流行病学研究

对长春和北京地区连续12年(1976年冬至1988年春)引起小儿肺炎的3、7型腺病毒102株标本,进行了限制性内切酶核酸电泳图谱分析。56株7型腺病毒经BamHⅠ、BclⅠ、BglⅠ、XbaⅠ、SmaⅠ、HindⅢ分析后,表现为两个基因组型——Ad7 b和Ad7 d。46株3型腺病毒被Bg1 Ⅱ、BamHⅠ酶解后,表现为 3个基因组型——Ad 3Ⅰ、Ad 3Ⅱ、Ad 3Ⅲ。各基因组型的分布情况是:56株7型腺病毒中,43株为Ad 7 b(76.8％),流行于1976年冬至1986年春;13株是Ad 7 d(23.2％),出现于1982年,与Ad 7 b共同流行;1986年～1988年分析的5株病毒都是Ad 7d。43株3型腺病毒中,Ad3Ⅰ42株(91.0％),分布于12年中;Ad 3Ⅱ、Ad 3Ⅲ各2株,散在分布。此结果表明,国内这12年中引起小儿肺炎的3型腺病毒至少有3个基因组型,7型腺病毒至少有两个基因组型。Ad3Ⅰ和Ad7 b是流行优势基因组型。但自80年代初开始出现Ad7 d以来,有逐年增多的趋势,最近两年的标本又都是Ad7 d,很可能它将取代Ad7 b而成为流行的优势基因组型.     

2010-2019年北京地区腹泻患儿中人腺病毒41型Fiber基因分子特征和进化分析

人腺病毒41型(Human adenovirus 41,HAdV-41)是引起儿童腹泻的重要病原之一.为了解北京地区腹泻儿童中HAdV-41 Fiber基因的分子变异和遗传进化特征,选取2010-2019年首都儿科研究所附属儿童医院门诊腹泻患儿HAdV-41阳性的粪便标本,扩增Fiber全基因,使用多种生物信息学软件进行系统发生、遗传进化、种群演变和选择压力等分析.结果显示HAdV-41本地毒株存在1644碱基(547氨基酸)和1689碱基(562氨基酸)两种长度的Fiber基因,截短的15个氨基酸位于Fiber蛋白纤维轴区.Mann-Whitney U秩和检验提示检测到感染短Fiber基因的HAdV-41的患儿年龄更小(P=0.036).同源性分析结果显示Fiber基因的核苷酸和氨基酸序列同源性均大于97％,主要分为四个进化分支(Ⅰ、Ⅱ、Ⅲ和Ⅳ),其中Ⅰ和Ⅲ是主要的流行分支,分支Ⅰ主要在国内流行,而分支Ⅲ全球均有流行.HAdV-41共祖起源可追溯到1856年,平均进化速率每年为6.81×105碱基替换/位点,10年间理论有效种群一直比较平稳呈缓慢下降趋势.压力选择分析表明,一个正向和三个负向选择位点均位于Fiber蛋白的纤维轴区.上述结果表明2010-2019年间不同进化分支的HAdV-41在北京地区流行,进化速率稳定.Fiber蛋白轴区存在两种不同类型的氨基酸缺失.     

腺病毒载体的细胞导向性基因转移

腺病毒（ａｄｅｎｏｖｉｒｕｓ,Ａｄ）载体广泛应用于基因治疗,其主要特征之一是能高效地进行各种组织如肺上皮、肝、肌肉、胰岛、胆囊等的体内（ｉｎｖｉｖｏ）基因转移。然而,因其混杂的向性而引起的全身体内基因转导后治疗基因不受组织限制的表达,限制了它在基因治疗中的应用,因此希望改变腺病毒的天然向性,而使治疗基因导向转移到选择的细胞类型中。目前腺病毒载体细胞导向性的基因转移可通过（１）修饰腺病毒载体的细胞表面识别成分,限制腺病毒导向性感染选择的细胞类型;（２）腺病毒载体中导入组织特异性启动子,控制治疗基因…     

长春地区引起婴幼儿肺炎的3、7型腺病毒分子流行病学研究

本文对长春地区1976年—1988年引起小儿肺炎的135株3.7型腺病毒核酸进行了限制性内切酶图谱分析,结果表明:75株7型腺病毒经BamHⅠ、BelⅠ、BglⅠ、XbaⅠ、SmaⅠ、HindⅢ分析后表现为二个基因组型—7b和7d。其中60株7b(88％),流行于1976—1988年;15株7d(12％)自1982年出现逐年增加,到1987—1988年的5株都是7d。60株3型腺病毒被BglⅡ、BamHⅠ分析后表现为三个基因组型,我们用3Ⅰ、3Ⅱ、3Ⅲ代表它们,其中S6株3Ⅰ(93.3％)流行于1976—1888年;3Ⅱ、3Ⅲ散在分布。通过临床资料整理,发现该地区3.7型腺病毒的不同基因组型在毒力致病性上存在着差别。     

2003～2012年北京儿童急性呼吸道感染中腺病毒监测及流行型别分析

2003~2012年连续10年对北京地区儿科急性呼吸道感染患儿腺病毒(Adenovirus,ADV)的监测,了解儿科急性呼吸道感染患儿中ADV感染状况和流行型别。收集39 214份儿科急性呼吸道感染患儿标本(包括来自住院及重症监护患儿的32 016例鼻咽分泌物标本及门诊的咽拭子标本7 198例)进行病毒分离和/或免疫荧光法进行ADV检测,对其中848份毒株和/或阳性标本提取DNA,通过两套巢式聚合酶链反应(nested-PCR)进行ADV分型。首先用3、7型2对分型引物的PCR可确定3、7型ADV,对少数扩增阴性的标本或毒株再使用2对可扩增AF各组ADV的通用引物对其DNA六邻体片段进行扩增后直接测序,并与GenBank中的序列比较以确定其型别。在39 214份呼吸道标本中,经病毒分离和/或免疫荧光法确定为ADV阳性的标本884份,总阳性率为2.25%(884/39 214),其中住院患儿ADV检测阳性率为2.08%(665/32 016),门诊患儿为3.04%(219/7 198)。以ADV年检出率统计,2003~2012年10年期间只有2010年的阳性率较高,为3.69%,其余年份均在3.0%以下。对其中848份毒株或阳性标本的分型结果显示:3型ADV占53.18%(451/848),7型ADV占36.79%(312/848),2型占3.78%(32/848),55型占2.24%(19/848),1型占2.0%(17/848),5型占0.94%(8/848),14型占0.47%(4/848),6型占0.35%(3/848),4型占0.24%(2/848)。除2012年优势型别为7型,2003年和2007年为3、7型外,占23例(88.5%)。12例诊断为扁桃体炎患儿11例为ADV3(91.7%)感染。0~3岁年龄组ADV阳性检出率高于4岁以上年龄组。男女性别比为1.9:1。近10年中ADV3和ADV7为主要的流行型别。儿童ADV重症肺炎多是由ADV7造成的。由11和14型重组的55型ADV从2006年开始在北京儿童中出现。     

小鼠CCR7基因重组腺病毒的构建与鉴定

目的:构建含有小鼠趋化因子受体-7(CCR7)基因的重组腺病毒,为下一步转染不成熟树突状细胞(imDC)诱导免疫耐受研究奠定基础。方法:提取小鼠胸腺总RNA,应用逆转录PCR(RT-PCR)方法,以自行设计的带有酶切位点的引物,扩增获得CCR7基因全部序列,经过T-A克隆,酶切亚克隆到穿梭质粒pAdTrack-CMV上,在BJ5183菌内和pAdeasy-1同源重组,筛选阳性克隆,酶切鉴定,线性化后脂质体法转染HEK293细胞进行包装、PCR鉴定及扩增,得到含有CCR7基因的重组腺病毒,根据报告基因GFP测定病毒滴度。结果:成功构建小鼠CCR7基因重组腺病毒,病毒滴度为1×10~9U/mL。结论:该重组腺病毒载体的成功构建,为进一步研究携带CCR7基因的imDC的趋化迁移性等研究提供了一定的工作基础。     

Distinctiveness of the cagA Genotype in Children and Adults with Peptic Symptoms in South China

Background:  Helicobacter pylori infection is different between children and adults, not only in infection rate but also in virulence genotypes. However, the 3' region of CagA, important in stomach carcinogenesis, still remains unclear in children. The present study aims to compare the frequency of cagA and the distribution of its subtypes between children and adults in South China.
Materials and Methods:  One hundred and twenty-eight children and 99 adults with peptic symptoms were enrolled in our research. Histology, rapid urease test, and real-time polymerase chain reaction (PCR) assay were used to diagnose H. pylori infection. vacA s1 was detected by real-time PCR, and EPIYA motifs in the 3' region of CagA by conventional PCR and DNA sequencing.
Results:  H. pylori infection was diagnosed in 53 children and 62 adults. vacA s1 was identified in 90.6% and 91.9% of infected children and adults, respectively. Furthermore, cagA was identified in 73.6% and 82.3% of infected children and adults, respectively. No patient with multiple cagA subtypes was observed. A higher prevalence of more virulent cagA genotype was found in children compared to adults ( p <  .05). Thirty-eight of 39 (97.4%) cagA -positive children were found to have EPIYA-ABD and only one (2.6%) with EPIYA-ABC. In adults, four types of EPIYA motifs – ABC (29.4%), ABD (64.7%), ABAB (2%), and AAD (3.9%) – were identified, and the ABD type was found more commonly in severe diseases, such as atrophic gastritis (53.3%) and gastric cancer (71.4%).
Conclusion:  cagA genotypes in children and in adults are different, and EPIYA-ABD may have potential clinical implication in the development of gastric cancer in South China.     

吉林地区HFRS病原体基因型分析

为调查吉林地区褐家鼠中汉坦病毒病原体基因型及其分布情况,采用免疫荧光法检测吉林市区及吉林市所属各县市收集的鼠肺标本,对汉坦病毒抗原阳性标本以巢式RT-PCR法扩增S基因片段上的核苷酸序列并测序,将扩增片段的核苷酸序列与已知病毒序列进行同源性分析及系统进化树分析.序列分析发现吉林地区褐家鼠携带的汉坦病毒均为汉城病毒,S3...     

Molecular Characterization of Acanthamoeba spp. Occurring in Water Bodies and Patients in Poland and Redefinition of Polish T16 Genotype

Acanthamoeba genus is divided into 20 genotypes (T1–T20) on the basis of the gene encoding 18S rRNA sequence. Using of at least 2 kbp gene fragments is strongly recommended to identify new genotypes and 5% difference is commonly used as a criterion of new genotypes, however, this value is questionable. In this paper, Polish Acanthamoeba strains described earlier on the basis of ~850 bp Ami fragment of 18S rRNA gene as T4, T11 and a new T16 genotype, have been analyzed using near‐complete sequence of the gene. This analysis was needed because the Ami fragment does not reveal full variability within 18S rRNA gene. Phylogenetic analysis based on Ami fragment is biased by artifacts in the construction of the tree, so the fragment should not be used for identification of new putative Acanthamoeba genotypes. The analysis confirmed that the Polish sequences represent T4 and T11 genotypes and that the strains described earlier as T16 genotype are in fact a new subgroup of the T20 genotype and that this genotype should be divided into two subgroups: T20a (two strains described by [J. Eukaryot. Microbiol. 62 (2015) 69]) and T20b (11 Polish strains described in this study). The T20b subgroup was isolated from both clinical samples and water bodies used by people as bathing places and there is a risk of infection for humans during contact with water.     

我国汉坦病毒基因型和基因亚型的分布研究

为了搞清全国汉坦病毒的基因型和亚型的分布情况,广泛收集了全国各地汉坦病毒毒株、阳性病人血清和阳性鼠肺,并应用RT-PCR的方法,应用汉坦病毒型特异性引物,对这些不同来源的阳性标本中汉坦病毒型特异性M和S片段进行扩增和测序,并与其它已知病毒序列进行比较,以明确其型别和亚型及其在全国的分布情况.结果表明:我国HFRS各疫区仍然为HTNV和SEOV两型病毒,但亚型分布差异较大,其中HTNV可分为9个亚型,SEOV则有4～6个亚型.Q32株的部分M和S片段分属于H9和H2亚型,是一个基因重排病毒,而Nc167株在系统发生上与其它HTNV明显不同,比较核苷酸序列发现,其M片段与其它HTNV的同源性在71.3%～76.7%之间,S片段与其它HTNV的同源性只有52.3%～57.8%,可能是一个新型病毒.     

Recent Transmission and Prevalent Characterization of the Beijing Family Mycobacterium Tuberculosis in Jiangxi,China

The Beijing genotype is the most common type of tuberculosis in Jiangxi Province, China. The association of population characteristics and their prevalence in the development of recent transmission is still unclear. 1,433 isolates were subjected to drug-resistant tests and MIRU-VNTR analysis. We compared differences in demographic characteristics and drug resistance patterns between the Beijing and non-Beijing family strains. We also explored the association of the clustering rate with the Beijing genotype of Mycobacterium tuberculosis. The Beijing genotype was dominant (78.16%). The results of MIRU-VNTR showed that 775 of 1,433 strains have unique patterns, and the remaining gather into 103 clusters. A recent transmission rate was 31.54% (452/1,433). The Beijing genotype strains were more likely to spread among the recurrent population (p = 0.004), people less than 50 years of age (p = 0.02 or 0.003), and the personnel in the northern regions (p = 0.03). Drug resistance patterns did not show significant differences between Beijing and non-Beijing genotype isolates. Furthermore, we found that HIV-positive cases had a lower clustering rate (p = 0.001). Our results indicated that the recurrent population and people under 50 years of age were more likely to be infected with the Beijing genotype of M. tuberculosis. The strains from the Beijing family were easier to cluster compared to strains isolated from the non-Beijing family. Social activity and AIDS substantially impacted the clustering rate of the Beijing genotype of M. tuberculosis. Multidrug resistant M. tuberculosis affected Beijing genotype transmission.     

表达流行性感冒病毒HA基因非复制型重组腺病毒的构建及免疫效果的研究

通过RT-PCR扩增流行性感冒(流感)病毒HA基因,克隆至腺病毒穿梭载体pAd Track-MV,该重组质粒与腺病毒DNA共转化E．coli BJ5183,通过细菌内同源重组获得重组腺病毒DNA,将其转染293细胞获得重组腺病毒。PCR证实HA基因已整合至腺病毒基因组中,Western blot结果检测到重组病毒感染293细胞中HA的表达。重组病毒经滴鼻和灌胃两种途径免疫小鼠,结果2次免疫后滴鼻组和灌胃组均产生明显的免疫应答,血清IgG抗体滴度分别为1：10000和1：1000。除血清IgG外,还在肺灌洗液中检测到分泌型IgA。滴鼻组的免疫效果强于灌胃组。经小剂量攻毒实验显示,重组腺病毒保护率为100％。该文成功构建了表达流感病毒HA基因的非复制型重组腺病毒,重组病毒免疫小鼠可产生较好的免疫效果。     

Genotype by environment interactions in winter survival in red deer

1. The extent to which environmental heterogeneity interacts with genetic hetero geneity to affect individual fitness within populations has the potential to affect the dynamics of natural populations and the amount of genetic variation maintained in natural populations, yet is a relatively poorly investigated topic in either ecology or evolutionary biology.
2. Many individual-based studies are precluded from such investigations by the practical problems of measuring heritability of traits affecting fitness and the difficulties of experimental manipulation of the study population. One way of demonstrating how commonly genotype by environmental interactions affect fitness, though not their overall importance in determining fitness, is to investigate fitness in a population subdivided by genotype at one or more marker loci.
3. We analyse data on calf winter survival from a population of red deer from the Isle of Rum, Scotland. Data on individual survival, environmental fluctuations and genotype at 13 loci were collected from 1982 to 1994.
4. We found associations between survival over the first winter of life and calf genotype at two out of three allozyme loci and five out of 10 microsatellite loci. All of the results remained significant under randomization tests. Other genotypes that initially appeared to have an association with survival were rejected when bootstrapped, usually due to insufficient data or anomalies in the data.
5. Our results suggest that associations between fitness and genotype are common. Five out of the seven associations found involved interactions with environmental variables. Four of these showed density-dependent selection with different genotypes showing high survival at high population size compared to low population size and one interacted with autumn rainfall. In a sixth case, genotype interacted with sex.