江苏省不明原因轻型呼吸道传染病暴发的病因学研究

自2004年4月18日起至6月末,江苏省东台市发生以发热、咽部充血、扁桃体肿大为临床表现的原因不明的疫情暴发,发病人群以中小学生、幼儿为主,地域以许河镇、新街镇以及临近乡镇为主。疫情涉及东台市9个镇,合计报告709例,分布在75个学校,240个班级。暴发的特点为传染性强,短期内出现大量的患者,发病有班级聚集性特点,未发现成人感染。临床表现以发烧（100％）、咽部充血（91．40％）、扁桃体肿大（60．22％）、咽痛（50．00％）为主;X线检查可发现肺纹理增强（90．3％）。用Hep-2细胞从患儿20份咽拭子标本中分离到12株病毒,经PCR扩增腺病毒L3部分基因及其PCR产物的序列测定和分析,证实这12株病毒为腺病毒3型;对28份咽拭子标本直接进行DNA提取和PCR扩增,有17份标本PCR扩增L3部分基因呈阳性,经序列测定和分析,也为腺病毒3型。这29个阳性标本L3基因的1446个核苷酸的序列与GenBank上所发表的腺病毒3型序列同源性高达99,5％。双份血IgG检测：对江苏省采集10份患儿急性期和恢复期双份血清,以从患儿咽拭子分离到的腺病毒3型作为抗原,进行病毒抗体测定,6份患儿的双份血清腺病毒总IgG滴度呈4倍以上增高。患儿急性期血IgA检测：对暴发疫情34例患儿急性期血清（发病时间为1～3天）进行腺病毒IgA抗体测定,结果有9份标本为阳性,1份标本为可疑阳性,阳性率为26．47％。IgA阳性率低可能与血清采集时间过早有关,机体还未全部产生IgA。另外,还对江苏省送检的从患儿血培养基培养的细菌毒株进行了鉴定,对其16S rRNA基因进行了基因扩增及序列分析,序列测定和分析提示为A组M3型化脓性链球菌。实验研究结果并结合此次疫情的流行病学和患儿的临床表现,证实腺病毒3型是引起此次儿童不明原因轻型呼吸道传染病暴发的病原体,部分病例伴有原发或继发化脓性链球菌感染。     

辽宁省2012年至2013年呼吸道腺病毒型别鉴定

目的基于国家重大传染病防治科技重大专项项目平台,对2012年1月至2013年12月间辽宁省采集的发热伴呼吸道症状患者标本进行检测,了解腺病毒的流行情况并对其型别进行分析。方法采集具有发热伴呼吸道症状患者的咽拭标本470份,经多重PCR检测,检出阳性标本30份,选取阳性标本进行病毒分离,共获得人类腺病毒(Human Adenovirus,HAdv)毒株20株。将所得腺病毒的核酸进行巢式PCR扩增并进行测序,获得Hexon基因序列。将基因序列在NCBI进行Blast比对。结果在分离的得到的20株腺病毒毒株中包含HAdv-2型6株,HAdv-3型11株,HAdv-5型2株,HAdv-14型1株。结论2012至2013年间,辽宁省引发发热伴呼吸道症状的腺病毒型别以2型和3型为主,同时也有少量其他型别检出。     

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。     

上海地区部分婴幼儿腹泻标本中诺如病毒基因分析

目的了解2006年上海哨点医院婴幼儿疑似病毒性腹泻散发病例中诺如病毒基因型别和基因特征。方法应用反转录-聚合酶链反应(RT-PCR)扩增标本中诺如病毒核酸,测序后使用DNAstar基因分析软件与Genbank中的参考株进行核酸序列分析。结果13份测序结果均属于诺如病毒GⅡ遗传组,以GⅡ.4基因型为主。其中2份核酸序列与2006年德国汉堡毒株序列同源性相近,分别为86.2%和88.2%;2份核酸序列与2006年荷兰乌得勒支毒株序列同源性相近,均为83.9%;其余9份核酸序列与2004年中国广西、2004年和2005年日本东京毒株序列同源性相近,为87.3%～97.5%。结论2006年上海哨点医院婴幼儿疑似病毒性腹泻标本中存在诺如病毒GⅡ.4基因型散发病例,不同病毒株之间差异较大。     

腹泻犬中细小病毒携带情况以及VP2基因进化分析

为了解四川省部分地区腹泻犬中细小病毒的感染情况以及VP2基因的遗传变异情况。从四川省部分地区收集了50例疑似CPV感染的腹泻犬粪便,对标本采用PCR扩增VP2,并对VP2基因全序列进行测序分析。PCR检测阳性标本19份。序列分析显示,15份扩增出VP2基因全序列标本均为CPV-2a型,聚类分析显示全部序列聚类在同一分支,本次研究的四川省部分地区流行的CPV均为CPV-2a型。可推测目前四川省部分地区所常见的CPV流行株依然以CPV-2a型为主。本次试验所扩增的15份CPV阳性标本与国内外传统毒株有着极高的同源性,提示目前四川省部分地区CPV还未出现重大的变异情况。     

山东省急性出血性结膜炎病原学鉴定及其柯萨奇A24病毒基因特征分析

为明确2010年引起山东省青岛市、临沂市急性出血性结膜炎(Acute hemorrhagic conjunctivitis,AHC)流行的病原,采集26例患者的眼结膜拭子标本,分别采用实时荧光定量PCR(Real time-PCR)和细胞培养方法进行检测。Real time-PCR结果显示,17份标本柯萨奇病毒A组24型(Coxsackievirus A24,CVA24)阳性,阳性率为65.39%,肠道病毒70型(Enterovirus 70,EV70)和腺病毒均为阴性;使用Hep-2细胞共分离到10株病毒,通过VP1区基因扩增及核苷酸序列测定,10株病毒均鉴定为CVA24,同源性分析显示其核苷酸和氨基酸同源性分别为99.3%～100.0%和99.5%～100.0%,在系统进化树上聚集成一簇,本次分离的毒株与山东省急性弛缓性麻痹(AFP)病例中分离到的毒株序列差异较大,分别属于5个不同的进化分支。提示CVA24可能为引起两地AHC流行的病原,且属于同一传播链。     

西藏地区儿童急性呼吸道感染中呼吸道合胞病毒的初步研究

本研究为了解西藏地区儿童急性呼吸道感染中呼吸道合胞病毒(Respiratory syncytial virus,RSV)及基因型别。首先采用直接免疫荧光法检测2011年4～7月西藏自治区人民医院儿科病房因急性呼吸道感染住院患儿的鼻咽分泌物标本中7种常见的呼吸道病毒及人类偏肺病毒(Human metapneumovirus,hMPV)的抗原。然后对RSV抗原阳性的标本分别提取RNA,用逆转录-巢式聚合酶链反应法(Nest-PCR)确定RSV型别,同时用实时荧光PCR(Real-Time PCR)方法进行验证。再通过对G蛋白基因PCR扩增产物序列测定确定RSV的基因型。通过与GenBank中不同地区RSV分离株的G蛋白基因序列比对,了解西藏地区RSV G蛋白的结构特点及变异情况。结果表明,从167例标本中检测出呼吸道病毒抗原阳性的为65例,总阳性率为38.9%(65/167),其中RSV 45例,占阳性标本的69.2%(45/65),对其中42例RSV阳性标本进行了PCR分型,其中40例为A亚型,2例为B亚型。对7株A亚型RSV G蛋白基因PCR产物测序结果显示,全部为GA2基因型。西藏RSV与RSV原型株A2株核苷酸的同源性为90.7%～91.8%,氨基酸的同源性只有86.5%～87.2%。氨基酸的变异主要集中在胞外区一个高度保守序列的两端。7株西藏A亚型RSV G蛋白的核苷酸序列与GenBank中不同的RSV分离株相比同源性为90.7%～91.8%。西藏地区2011年春季小儿急性呼吸道感染的病毒病原主要为呼吸道合胞病毒,A亚型是2011年西藏地区的流行优势型别,其G蛋白胞外区基因具有较高的变异性。     

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。     

病毒分离和PCR-RFLP法对急性结膜炎标本中腺病毒感染及其型别的分析

目的分析2003年下半年收集的哈尔滨医科大学第一临床医学院急性结膜炎标本中的腺病毒(Adenovirus,AdV)感染情况及其型别。方法采集59例临床确诊为急性结膜炎患者的结膜拭子标本,在观察和分析接种细胞的病变(CPE)情况的基础上,采用聚合酶链式反应-限制性片段长度多态性分析(PCR—RFLP)法,特异性检测CPE阳性细胞中腺病毒核酸的存在情况,并对PCR阳性扩增产物进行病毒型别的分析。结果70．7％(41／59)的急性结膜炎标本接种于培养细胞后可以引起明显的CPE;其中,53．6％(22／41)的CPE阳性标本可以扩增出腺病毒核酸,RFLP分析证实68．18％(15／22)的腺病毒感染为Ad3型,31．82％(7／22)的腺病毒感染为Ad7。结论2003年下半年哈尔滨市急性结膜炎主要以腺病毒3、7型感染为主。提示结合病毒分离培养和PCR—RFLP方法,可以广泛应用于急性结膜炎相关的病毒感染及其型别的进一步分析与研究。     

Processing of vimentin occurs during the early stages of adenovirus infection.

Evidence is presented that a fraction of vimentin, a component of cytoskeleton recently found to be associated with intracytoplasmic, migrating adenovirus type 2 (Ad2), is processed into smaller polypeptides at early times after infection. The extent of vimentin cleavage appears to depend upon both the multiplicity of infection and the adenovirus serotype. Ad2, Ad5, Ad4, and Ad9 induced similar vimentin cleavage in infected cells, whereas Ad3, Ad7, and Ad12, for which most infecting particles are found sequestered within phagosomes, induced very little, if any, vimentin breakdown. This suggests that vimentin processing is in some way related to the number of virus particles migrating through the cytoplasm. Experiments performed in vitro and in vivo with adenovirus temperature-sensitive mutants H2 ts1 and H2 ts112 and UV-inactivated wild-type Ad2 indicated that vimentin processing is due to a nonvirion, cytoskeleton-associated, proteolytic enzyme activated by adenovirus and sharing characteristics with the protease described by Nelson and Traub (W.J. Nelson and P. Traub, J. Cell Sci. 57:25-49, 1982). The activity of this protease appears to be required for productive infection by adenovirus serotypes 2 and 5 (subgroup C), 4 (subgroup E), and 9 (subgroup D) but not by the oncogenic serotypes 3 and 7 (subgroup B) and 12 (subgroup A).     

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.     

Human adenovirus-host cell interactions: comparative study with members of subgroups B and C.

Host cell interactions of human adenovirus serotypes belonging to subgroups B (adenovirus type 3 [Ad3] and Ad7) and C (Ad2 and Ad5) were comparatively analyzed at three levels: (i) binding of virus particles with host cell receptors; (ii) cointernalization of macromolecules with adenovirions; and (iii) adenovirus-induced cytoskeletal alterations. The association constants with human cell receptors were found to be similar for Ad2 and Ad3 (8 x 10(9) to 9 x 10(9) M-1), and the number of receptor sites per cell ranged from 5,000 (Ad2) to 7,000 (Ad3). Affinity blottings, competition experiments, and immunofluorescence stainings suggested that the receptor sites for adenovirus were distinct for members of subgroups B and C. Adenovirions increased the permeability of cells to macromolecules. We showed that this global effect could be divided into two distinct events: (i) cointernalization of macromolecules and virions into endocytotic vesicles, a phenomenon that occurred in a serotype-independent way, and (ii) release of macromolecules into the cytoplasm upon adenovirus-induced lysis of endosomal membranes. The latter process was found to be type specific and to require unaltered and infectious virus particles of serotype 2 or 5. Perinuclear condensation of the vimentin filament network was observed at early stages of infection with Ad2 or Ad5 but not with Ad3, Ad7, and noninfectious particles of Ad2 or Ad5, obtained by heat inactivation of wild-type virions or with the H2 ts1 mutant. This phenomenon appeared to be a cytological marker for cytoplasmic transit of infectious virions within adenovirus-infected cells. It could be experimentally dissociated from vimentin proteolysis, which was found to be serotype dependent, occurring only with members of subgroup C, regardless of the infectivity of the input virus.     

Single-tube multiplex PCR for rapid and sensitive diagnosis of subgenus B and other subgenera adenoviruses in clinical samples

We developed a new diagnostic method of subgenus (Sub) B adenovirus (Ad) in clinical samples using non-nested polymerase chain reaction (PCR). Sequences of the conserved hexon-coding region of representative strains of eight serotypes (3, 7, 11, 14, 16, 21, 34 and 35) of Sub B Ad were heterogeneous. In order to distinguish Ad serotype 3 (Ad 3) and Ad 7 from the other serotypes of Sub B Ad, and to differentiate Ad 3 and 7 from each other, 3 different downstream primers were designed based on the sequence heterogeneity. By a single-tube PCR method using a combination of 6 primers including the 3 new primers, Ads demonstrated to amplify 188, 206, 284, and 301 bp DNA fragments for Ad 3, Ad 7, other Sub B Ads, and non-Sub B Ads, respectively. A total of 114 clinical samples were selected to evaluate the direct applicability of our PCR. The results were compared with previous culture results. Sixty-seven out of 71 (94%) Sub B Ad culture-positive samples, and 15 out of 19 (79%) Sub C or E-positive samples amplified products of the expected size. Two of 20 (10%) culture-negative samples from pharyngoconjunctival fever patients were identified as Ad 3 by the PCR. Four samples, from which non-Ad viruses were isolated, were negative by the PCR. The present study might provide a rapid and sensitive diagnosis method for infections caused by Sub B Ads.     

Development and Application of a Real-time PCR Method for Pharmacokinetic and Biodistribution Studies of Recombinant Adenovirus

A replication-deficient recombinant adenovirus (Ad5-LFA-3/IgG₁) that encodes secreted LFA-3/IgG₁ was constructed for gene therapy treatment of psoriasis. The purpose of this study was to develop a real-time PCR method for pharmacokinetic and biodistribution studies of Ad5-LFA-3/IgG₁ within the circulation and organs. This method showed good specificity, sensitivity and reproducibility over a wide dynamic range of concentrations. Quantitative measurement of recombinant adenoviral DNA suggested that the level of Ad5-LFA-3/IgG₁ DNA in circulating blood peaked within 10 min following intravenous injection in rhesus macaques. Following this peak, the adenoviral DNA level dropped significantly to a very low level. Real-time PCR revealed that Ad5-LFA-3/IgG₁ DNA was enriched in the spleen, lung and liver after injection of the adenovirus into rats through the tail vein. The adenoviral DNA was barely detected in other tissues. These data provide important information for clinical trials of Ad5-LFA-3/IgG₁ and confirm the utility of the real-time PCR assay for monitoring gene therapy trials.     

Clinical significance of 4 patients with chronic hepatitis B achieving HBsAg clearance by treated with pegylated interferon alpha-2a for less than 1 year: a short report

This study describes the prevalence of culture-positive adenovirus serotypes in culture-positive respiratory specimens sent to the Central Public Health Laboratory, Toronto, Ontario, Canada for the period September 2007–June 2008. Total nucleic acid was extracted from virus cultures using an automated extraction method followed by polymerase chain reaction and Sanger sequencing of the adenovirus hexon gene hypervariable region 7. 73% of specimens (n = 70) were from patients ≤ 4 years of age. Of the 96 adenovirus isolates, the most common identified serotypes were serotype 3 (n = 44, 46%), serotype 2 (n = 25, 26%), serotype 1 (n = 17, 18%), and serotype 21 (n = 5, 5%). Adenovirus serotype 14 was not found in this study group. The leading serotype, Ad3, was identified throughout the duration of the study period. Molecular methods allow for the determination of circulating adenovirus serotypes and be used to document the spread of highly virulent adenoviral serotypes into a region.     

Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes

The common species C adenoviruses (serotypes Ad1, Ad2, Ad5, and Ad6) infect more than 80% of the human population early in life. Following primary infection, the virus can establish an asymptomatic persistent infection in which infectious virions are shed in feces for several years. The probable source of persistent virus is mucosa-associated lymphoid tissue, although the molecular details of persistence or latency of adenovirus are currently unknown. In this study, a sensitive real-time PCR assay was developed to quantitate species C adenovirus DNA in human tissues removed for routine tonsillectomy or adenoidectomy. Using this assay, species C DNA was detected in Ficoll-purified lymphocytes from 33 of 42 tissue specimens tested (79%). The levels varied from fewer than 10 to greater than 2 x 10(6) copies of the adenovirus genome/10(7) cells, depending on the donor. DNA from serotypes Ad1, Ad2, and Ad5 was detected, while the rarer serotype Ad6 was not. When analyzed as a function of donor age, the highest levels of adenovirus genomes were found among the youngest donors. Antibody-coated magnetic beads were used to purify lymphocytes into subpopulations and determine whether viral DNA could be enriched within any purified subpopulations. Separation of T cells (CD4/8- expressing and/or CD3-expressing cells) enriched viral DNA in each of nine donors tested. In contrast, B-cell purification (CD19-expressing cells) invariably depleted or eliminated viral DNA. Despite the frequent finding of significant quantities of adenovirus DNA in tonsil and adenoid tissues, infectious virus was rarely present, as measured by coculture with permissive cells. These findings suggest that human mucosal T lymphocytes may harbor species C adenoviruses in a quiescent, perhaps latent form.     

Generation and Immunity Testing of a Recombinant Adenovirus Expressing NcSRS2-NcGRA7 Fusion Protein of Bovine Neospora caninum

Neospora caninum is the etiologic agent of bovine neosporosis, which affects the reproductive performance of cattle worldwide. The transmembrane protein, NcSRS2, and dense-granule protein, NcGRA7, were identified as protective antigens based on their ability to induce significant protective immune responses in murine neosporosis models. In the current study, NcSRS2 and NcGRA7 genes were spliced by overlap-extension PCR in a recombinant adenovirus termed Ad5-NcSRS2-NcGRA 7, expressing the NcSRS2-NcGRA7 gene, and the efficacy was evaluated in mice. The results showed that the titer of the recombinant adenovirus was 10⁹TCID₅₀/ml. Three weeks post-boost immunization (w.p.b.i.), the IgG antibody titer in sera was as high as 1:4,096. IFN-γ and IL-4 levels were significantly different from the control group (P<0.01). This research established a solid foundation for the development of a recombinant adenovirus vaccine against bovine N. caninum.     

Group C adenovirus DNA sequences in human lymphoid cells.

Human peripheral blood lymphocytes from healthy adults, cord blood lymphocytes, and lymphoblastoid cell lines were screened by hybridization for the presence of group C adenovirus DNA sequences. In 13 of 17 peripheral blood lymphocyte samples from adults, 1 of 10 cord blood samples, and seven of seven lymphoblastoid cell lines tested, results were positive for Group C adenovirus DNA (adenovirus 1 [Ad1], Ad2, Ad5, or Ad6). About 1 to 2% of the lymphocytes carried 50 to 100 viral genome copies per positive cell, as estimated by in situ hybridization. Infectious virus representing all members of group C were recovered, but cultivation in the presence of adenovirus antibody did not cure the cells of free viral genomes. Viral DNA was found in B, T, and N cells but only in 1 of 10 cord blood samples. The results suggest that group C adenovirus infections in childhood result in the persistence of the viral genome in circulating lymphocytes.     

Adenovirus Serotype 30 Fiber Does Not Mediate Transduction via the Coxsackie-Adenovirus Receptor

Prior work by members of our laboratory and others demonstrated that adenovirus serotype 30 (Ad30), a group D adenovirus, exhibited novel transduction characteristics compared to those of serotype 5 (Ad5, belonging to group C). While some serotype D adenoviruses bind to the coxsackie-adenovirus receptor (CAR), the ability of Ad30 fiber to bind CAR is unknown. We amplified and purified Ad30 and cloned the Ad30 fiber by overlap PCR. Alignment of Ad30 fiber with Ad3, Ad35, Ad5, Ad9, and Ad17 revealed that Ad30, like Ad9 and Ad17, has a shortened fiber sequence relative to that of Ad5. The knob region of fiber was 45% identical to that of the Ad5 knob regions. We made a chimeric recombinant virus (Ad5GFPf30) in which the Ad5 fiber (amino acids [aa]47 to 582) was replaced with Ad30 fiber sequences (aa 46 to 372), and CAR-mediated viral entry was determined on CAR-expressing Chinese hamster ovary (CHO) cells. While CAR expression significantly increased Ad5GFP-mediated transduction in CHO cells (from 1 to 36%), it did not enhance Ad5GFPf30 gene transfer. Binding of radiolabeled Ad5GFPf30 or Ad30 wild-type virus was also not improved by the expression of CAR. These results suggest that Ad30 fiber is distinct from Ad5, Ad9, and Ad17 fibers in its inability to direct transduction via CAR.