呼吸道合胞病毒的分子流行病学_英文_

呼吸道合胞病毒的分子流行病学_英文_ Molecular epidemiology of respiratory syncytial virus KONG Xiaohui 孔晓慧 , SHOU Haochang 寿好长 , L IU Chunyan 刘春艳 and J IANG Zaifang 江载芳 Keywo rd s : resp irat ory syncytial virus 〃s ubgr oup 〃ge n otyp e To determine the epide miolo gic p attern of subgro up s A a nd B a nd ge notyp e s of re spirato ry O bj e c ti v e ( ) syncytial viru s R SVduring two no nco ntinuo u s epide mic s during 1990 - 1991 a nd 1997 - 1998 in B eijing1 ( ) Me t h o d s Na sop harynge al secretio n N P S sa mple s of R SV po sitive o r R SV isolate s te ste d by indirect ( ) immunofluo re sc e nc e IIFa s say were cla s sifie d into subgro up s A a nd B1 Isolate s of R SV were divide d into at le a st six diff ere nt line a ge s , de signate d N P12N P6 , by re strictio n mapping of the N ge ne1 Np 1 , 3 a nd 6 were give n by subgro up B isolate s , while N P2 , 4 a nd 5 were give n by subgro up A isolate s1 Strain s of subgro up A were f urther subdivide d into six line a ge s SHL12SHL6 o n the ba sis of the SH ge ne se que nc e1 SH line a ge s were clo sely relate d to e ac h other a nd to N P12N P61 Strain s of SHL1 , 3 a nd 4 were clo sely relate d a nd belo nge d to N P2 , SHL2 a nd 6 to N P4 , a nd SHL5 to N P51 ( ) Of 145 R SV N P S sa mple s fro m the 1997 - 1998 epide mic , 83 5712 %were of subgro up B R SV Re s ult s ( ) po sitive , 62 4218 %of subgro up A R SV po sitive1 The rate of o cc urre nc e of subgro up A to B strain s wa s a bo ut 1?1131 Two of 10 isolate s during the epide mic were subgro up A strain s , where a s 8 were subgro up B strain s1 The rate of o cc urre nc e of subgro up A to B strain s wa s 1?41 Eight subgro up A strain s of 10 isolate s fro m the 1990 - 1991 epide mic were do mina nt ; the p ropo rtio n of subgro up A to B strain s wa s 4?11 With 10 R SV isolate s in 1997 - 1998 , all 2 subgro up A strain s gave N ge ne fra gme nt re strictio n p attern N P4 , a nd f ell into SH line a ge SHL2 , where a s 8 subgro up B strain s all belo nge d to N P31 All 8 subgro up A isolate s fro m the 1990 - 1991 epide mic gave p attern N P4 , a nd f ell into SHL2 , while 2 subgro up B strain s all belo nge d to N P31 The cla s sific atio n of subgro up s A a nd B de duc e d fro m N P p attern s co rre spo nde d to the definitio n of the se subgro up s by mo no clo nal a ntibo die s1 The se o b servatio n s co nfirm that subgro up s A a nd B o r multiple line a ge s of R SV co2 C o n cl u s i o n s circ ulate d in B eijing , but diff ere nt ge no me typ e s p re do minate d e ac h ye ar1 Mo reo ver , very similar viru se s were isolate d up to mo re tha n 5 ye ar s a go , indic ating that de spite app are nt diver sity of the subgro up A strain s , the sep arate line a ge s might be relatively sta ble1 ( ) Chin Med J 2001 ; 114 4: 3642368 2 ,3 be distinct at the nucleotide sequence level1The ( )is a major cause of lower Respiratory syncytial virus RSV ( ) nucleocap sid N protein was found to be the most tract infection in infants and vulnerable adults1 Annual conserved between the subgroup s , showing 96 % amino acid epidemics of the virus occur around the world , taking place ( ) identity between two subgroup s , while the attachment Gin the winter months in temperate climates1 Epidemics of protein was the most variable , showing 53 % amino acid RSV cause severe pressure on the provision of hospital beds , identity between two subgroup s , with the small hydrophobic and the prevention of spread of the virus within hospitals is a ( ) SHprotein gene being intermediate , showing 76 % amino major problem1 As yet , there is no effective vaccine1 acid identity1 Isolates of subgroup s A and B of RSV can be further subdivided into distinct groupings or lineages on the RSV is unusual in that it can infect babies in the presence of basis of three criteria : restriction mapping of part of the N maternal antibody and can then reinfect individuals gene , nucleotide of sequencing part of the SH gene throughout their lives , although second and subsequent 4and nucleotide sequencing of the G protein gene1 infections are usually less severe than the primary infection1 These lineages appear to be distributed worldwide , but the Reinfection could in part be due to variability of the virus1 RSV strains can be divided into two distinct antigenic subgroup s A and B , on the basis of their reactions with Beijing Children’s Hospital , Capital University of Medical Sciences , 1 ( )monoclonal antibodies1The subgroup s have been shown to Beijing 100045 , China Kong XH , Shou HC , Liu CY and Jiang ZF significance in terms of degree of virulence and immunity at RNAgents system protocol1 RNAgents , a commercially the individual and community level has not been ( ) available kit Promega , USAwas used1 determined1 PCR In this paper we confirmed , by restriction mapping of part of cDNA synthesis followed by PCR amplification was carried μμout using 20g total cell RNA in a final volume of 50l the N gene and sequencing of part of the SH gene , the ( ) containing 30 mmol/ L Tris2HCl p H 813 , 50 mmol/ L variability of RSV strains during two unconsecutive KCl , 4 mmol/ L MgCl, 015 mmol/ L each dNTP , 2 epidemics during 1990 - 1991 and 1997 - 1998 , therefore μ()10 mmol/ L dithiothreitol , 011g each primer , 2 units U facilitating our understanding of the molecular epidemiology ( ) superstar reverse transcriptase Gibeco and 2 U Taq of the virus1 polymerase , overlaid with liquid paraffin1 Using a METHODS ( ) programmable thermal controller dry2block, the reaction was incubated at 37 ?1 hour followed by 30 cycles of 93 ? Viruses for 115 min , 55 ?for 115 min and 72 ?for 115 min1 PCR in HEP22 cells in Twenty RSV strains were propagated ( products were analyzed by agarose gel electrophoresis Figs1 () Dulbecco’s modified eagle medium with glucose J apan1 ) 1 and 21 Strains of virus used in this study were isolated from samples () of nasopharyngeal secretions NPSin the Virus Laboratory , Beijing Children’s Hospital , Beijing , China1 For study of two unconsecutive epidemics in 1990 - 1991 and 1997 - 1998 , samples of NPS were collected from children with respiratory tract infection admitted to Beijing Children’s Hospital , Beijing , China1 The isolation procedure was carried out according to standard methods1 Briefly , NPS from patients were inoculated into HEP22 cell tubes1 Cultures were incubated at 35 ? and checked daily for ( ) typical cytopathic effect CPE , and harvested when cultures showed 3 + CPE1 Diagnosis of RSV infection was Fig1 11 Restriction mapping of PCR products of part of the N gene1 M : ( ) made by indirect immunofluorescence IIFdirectly on cells PCR product marker ; Lane 1 : positive control ; Lane 2 : 278 bp N gene isolated from NPS1 IIF was carried out using a commercial fragment ; Lane 3 : Hind ?; Lane 4 : Bgl ?; Lane 5 : Hae ?; and ( ) kit Chemical , USA1 All RSV positive NPS and HEP22 Lane 6 : Rsa ?1 cultures were prepared for slides and kept at - 70 ? until further studied1 Subtyping of RSV Typing of subgroup s A and B was determined by IIF1 Slides of RSV were made from infected HEP22 or positive NPS cells1 Cells were washed with phosphate2buffed saline ( ) PBSand then centrifuged at 1000 r/ min for 10 min at room temperature1 For each isolate of NPS , drop s of the ingected cell suspension were placed in the wells of a slide , then air dried , and acetone fixed for 10 min at room temperature1 Indirect immunofluorescence of fixed cells was () performed with anti2nucleoprotein NP, anti2fusion protein ( ) ( ) F, and anti2attachment protein Gsubgroup s A and B2 specific antibodies as the first antibodies and an anti2rabbit Fig1 21 PCR products of part of the SH gene1 M : PBR322 Hinf1 fluorescein isothiocyanate conjugate as the second antibody1 marker ; Lane 1 : negative control ; Lanes 2 and 3 : 286 bp SH gene fragment1 RSV2specific monoclonal antibodies were provided by the Department of Virology , State Bakteriologiska Laboratorium , Stockholm , Sweden1 PCR primers N gene RNA extraction Primer 1 : 5’GGAACAAGTTGTGAGGTTTATGAATATGC3’1 Total RNA was extracted from infected HEP22 cells by the guanidine isothiocyanate method according to the modified Primer 2 : 5’CTTCTGCTGTCAGTCTA GTACACTGTAGT3’1 Primers 1 and 2 were designed to amplify between strains of RSV examined1 Primers 3 and 4 amplified a fragment of 281 bp from subgroup A strains of human RSV nucleotides 858 and 1135 of the human RSV N gene1 only1 These sizes of PCR products obtained correlated with those predicted from the design of the primers1 S H gene Primer 3 : 5 ’GGATCCC GGGGCAAATAATCATTGGA 2 GG3’1 Ta ble 11 Subgrouping of RSV positive NPS samples or isolates by IIF RSV positive NPS/ isolates Subgroup A Subgroup B Period ( )( )( ) n n % n % Primer 4 : 5’AAGCTTGCTATGTGTTGACTCGAGC3 ’1 ()()()1990111 - 199114 10 10 8 802 2010 isolates (())()62 4218 83 5712 1997111 - 199815 145 NPS Primers 3 and 4 were designed to amplify between (())()2 8010 8 2010 10 isolates nucleotides 1 and 281 of the SH gene of subgroup A , strain 5 A21 Restriction ma pping of N gene fragments Purif ying of PCR products As mentioned earlier , primers 1 and 2 gave a 278 bp A commercial kit was provided to purify double2stranded fragment from all RSV strains tested1 Table 2 shows the ( ) PCR amplified DNA Promega1 To purify PCR products of various patterns of restriction sites detected in the RSV the N and SH genes , we used the agarose method according epidemic isolates1 Six different patterns have been found , to the manufacturer’s protocol1 numbered NP1 to NP61 All 8 RSV subgroup A isolates obtained during the 1990 - 1991 epidemic gave N gene Sequencing of PCR products () fragment restriction pattern NP4 8010 %, whereas 2 RSV Purified PCR products of the SH gene of subgroup A strains ( ) subgroup B strains give pattern NP3 2010 %1 Also , 2 were submitted to National Center for DNA Sequencing and RSV subgroup A isolates in the 1997 - 1998 epidemic Analysing , Military Academy of Medical Science for () belonged to restriction pattern NP4 2010 %, while 8 RSV sequencing1 ( ) subgroup B strains pattern NP3 8010 %1 Other N gene restriction patterns , such as NP1 , NP2 , NP5 and NP6 , Restriction ma pping of PCR products Purified PCR products of the N gene were then restricted were not detected during these two epidemics1 The with Hind ?, Pst ?, Bgl ?, Hae ?, Rsa ? using classification of subgroup s A and B deduced from NP ( ) buffers supplied by the manufactures Fig1 1 1 These patterns was corresponded to the definition of these subgroup s by monoclonal antibodies1 restriction enzymes were chosen with reference to the sequences genes of subgroup s A and B of the protein 5 RSV1 Ta ble 21 N gene fragment restriction patterns Restriction enzyme NP A or B RESUL TS 33 group subgroup Hind ? Pst ? Bgl ? Hae ? Rsa ? Hae ? 1 2 B NP1 - - - - + - Temporal patterns of RSV subgroups NP2 - - - + + - A The total numbers of RSV2positive specimens recorded from B NP3 - - + - + - A the two epidemics , the relative proportions of subgroup s A NP4 - - + + + - NP5 + - - + + - A and B , and the numbers of isolates analyzed are given in A NP6 - - + + + + Table 11 During the 1997 - 1998 period , the subgroup s of 3 Two separate Hae ? sites which can be distinguished by the size of fragment () 145 RSV NPS samples were determined : 83 5712 %were given1 ( ) of subgroup B strain positive , 62 4218 %of subgroup A strain positive1 The rate of occurrence of subgroup A to B Sequence of S H genes of RSV stra ins of subgroup A strains was about 1 ?1131 In the meantime , 10 isolates The 5 ’part of the SH gene2specific PCR products was ()obtained during the epidemic were subgrouped : 2 2010 % sequenced for 10 RSV subgroup A isolates during the 1990 ( )10 % were subgroup A strains , whereas 8 80were - 1991 and 1997 - 1998 epidemics1 The nucleotide subgroup B strains1 During 1990 - 1991 , subgroup s of 10 ( ) sequences for 5’end nucleotides 22 - 156of SH genes of isolates from the 1990 - 1991 epidemic were defined : 8 RSV subgroup A isolates fall into four distinct lineages () 8010 %subgroup A strains were dominant , the proportion ( )Fig1 3 and were determined by direct sequencing of PCR of subgroup A to B strains was 4?11 products1 All 10 isolates fell into SH lineage SHL21 In addition , all the isolates that fell into SH lineage SHL2 were PCR of RSV of the N gene restriction pattern NP41 Primers 1 and 2 gave a PCR product of 278 bp from all New York , Subgroup A isolates predominated for 9 years , subgroup B strains predominated for 2 years , and subgroup A and B strains were equally distributed for 4 years1 Among the five epidemics of RSV from 1987 to 1992 in Canada , subgroup A strains occurred at least three times as often in all years except 1988 - 19891 One characteristic of the epidemiology of RSV outbreaks that might be explained by strain differences is the year2to2year variation in severity1 It is possible that one strain is consistently more pathogenic , or a shift in strains would make a strain transiently more pathogenic1 But the severity of RSV outbreaks was not linked to either of the two subgroup s1 Part of this variation may be explained by the circulation of different subgroup s1 Some findings suggest differences in pathogenicity among subgroup s1 Also , in Beijing , the epidemic starts early in alternate Fig1 31 Nucleotide sequences of part of the SH gene from lineages years , and the peak months are December and J anuary1 The SHL1 to SHL41 epidemic starts late in other years , and the peak months are ( ) March and April unpublished data1 The occurrence of D ISCUSSIO N subgroup A and B strains of RSV in Beijing follows this pattern1 RSV strains can readily be divided into two antigenic subgroup s A and B , and then further classified into RSV genotypes have a worldwide distribution and viruses lineages1 The relatedness of SH lineages to each other and to isolated in distant places and in slightly different years may the N gene restriction pattern is shown in Fig1 41 This be more related than viruses isolated in the same place on showns that SH lineages are closely related to each other and two consecutive days1 Moreover , most epidemics are to NP126 : strains of SHL1 , 3 and 4 , are closely related and produced by viruses classified into more than one genotype1 6belong to NP2 , SHL2 and 6 to NP4 , and SHL5 to NP51 Examination of a large number of RSV strains from the two epidemics in Beijing revealed that , far from the epidemics being homogeneous , viruses of several different genotypes were present simultaneously1 The epidemics were made up of at least 3 different lineages of the virus , two subgroup A and one subgroup B1 Although the analysis of the two epidemics in the same area showed similar mixtures of RSV genotypes , not all genotypes were present in each epidemic and the predominant genotype varied from year to year1 The observation that some of the isolates examined from this ( ) epidemic lineage SHL2were very similar to the strains isolated some 6 years previously , indicated that despite the ( Fig1 41 Dendrogram to show relatedness of subgroup A lineages Cane ) and Pringe , 19911 apparent diversity of the RSV subgroup A strains , the separate lineages may be relatively stable1 Several recent studies from the USA , Canada , UK and 9 J apan have shown that during most epidemics , both Our findings were concordant with the results of Johansen6 subgroup A and B strains tend to cocirculate simultaneously and Canewho studied RSV prevalence during most but the relative proportions may vary in different years : epidemics1 Cane analyzed the variability of RSV isolates during an epidemic , subgroup A or subgroup B strains may during the period 1988 - 1991 in Birmingham area , dominate , or both may be equally distributed1 Among the Finland , Germany , Malaysia , Uruguay and two other cities two epidemics of RSV that we studied , subgroup A and B (in UK1 Viruse of six different genotypes four subgroup A ) strains cocirculated1 These findings agree with the data of and two subgroup Bwere recovered during the 1988 - 1991 78 epidemic in Birmingham1 In addition , different genotypes and Thomaswho reported the occurrence of Mufson predominated each year but not all lineages were detected both subgroup A and B strains during RSV epidemics1 everywhere1 A similar analysis of isolates from other places ( ) During 15 consecutive years 1975 - 1990in Rochester , REFERENCES showed that similar viruses were circulating throughout the world1 SHL2 lineages were also present among isolates from Anderson LJ , Hierholzer J C , Tsou C , et al1 Antigenic 11 Newcastle , Hannover , and Kuala Lumpur1 Some of the characterization of respiratory syncytial virus strains with genotypes found seemed related to strains isolated 7 years monoclonal antibodies1 J Infect Dis 1985 ;151 :62626331 ago in other parts of the world1 This indicates that the 21 Johnson PR , Spriggs MK , Olmsted RA , et al1 The G temporal fluctuation in predominance of genotypes glycoprotein of human respiratory syncytial virus of subgroup A presumably caused by selective pressure exerted by host and B : extensive sequence divergence between antigenically immunity is due to the favoring of strains from a pool of related proteins1 Proc Natl Acad USA 1987 ;84 :5625256291 ( ) ( ) 31 Johnson PR , Collins PL1 The 1B NS2 , 1C NS1 and N globally circulating , genetically relatively stable genotypes , ( ) proteins of human respiratory syncytial virus RSVof subgroups rather than a molecular evolution in strains induced or A and B : sequence conservation and divergence within RSV directed by immunoselective pressure1 genomic RNA1 J Gen Virol 1989 ;70 :1539215471 41 Cane PA , Pringle CR1 Molecular epidemiology of respiratory In our study of the two epidemics , RSV positive specimens syncytial virus : rapid identification of subgroup A lineages1 J were collected from hospitalized children and so represent Virol Methods 1992 ;40 :29723061 only a small fraction of the infections occurring in the Cane PA , Pringle CR1 Respiratory syncytial virus heterogenity 51 community1 It is therefore possible that other strains of RSV during an epidemic : analysis by limited nucleotide sequencing () ( ) SH gene and restriction mapping N gene 1 J Gen Virol were circulating simultaneously but were not recovered 1991 ;72 :34923571 because of their low virulence or decreased incidence1 Cane PA , Matthews DA , Pringle CR1 Analysis of relatedness of 61 subgroup A respiratory syncytial viruses isolated worldwide1 Virus In summary , this paper reports the classification of RSV by Res 1992 ;25 :152221 IIF , restriction mapping of part of the N gene and Mufson MA , Belshe RB , Orvell C , et al1 Respiratory syncytial 71 sequencing of part of the SH gene , thereby permitting virus epidemics : variable dominance of subgroups A and B examination of the molecular epidemiology of the virus1 strains among children , 1981 - 19861 J Infect Dis 1988 ; 157 : These observations confirmed that subgroup s A and B , or 14321481 Thomas E , Margach MJ , Orvell C , et al1 Respiratory syncytial 81 multiple lineages of RSV co2circulated , with the relative virus subgroups B dominance during one winter season between proportions of each changing each year , at least in 1987 and 1992 in Vancouver , Canada1 J Clin Microbiol 1994 ; hospitalized babies1 Very similar strains were obtained after 32 :23822421 an interval of 7 years , indicating that despite apparent Johansen J , Christensen LS , Hornsleth A , et al1 Restriction 91 diversity of the subgroup A strains , the separate lineages pattern variability of respiratory syncytial virus during three may be rather stable1 consecutive epidemics in Denmark1 APMIS 1997 ;105 :30323081 ( ) Received December 3 , 1999 本文编辑 : 钱寿初 顾 佳 呼吸道合胞病毒的分子流行病学 Molecular epidemiology of respiratory syncytial virus ( ) Chin Med J 2001 ; 114 4: 3642368 首都医科大学附属北京儿童医院 北京 100045孔晓慧 寿好长 刘春艳 江载芳 ( ) 目的 了解北京地区 1990 - 1991 和 1997 - 1998 两个非连续的流行年中呼吸道合胞病毒 RSVA 、B 亚型和基因 型的流行情况 。 ( ) () 方法 用间接免疫荧光法 IIF检测呼吸道合胞病毒阳性鼻咽分泌物 NPS标本或 RSV 分离株 ,划分 A 、B 亚型 。 根据 N 基因片段的限制性酶切图型将 RSV 分离株分成至少 6 个基因型 NP1 - 6 ,其中 NP1 、3 和 6 属于 B 亚型 , NP2 、4 和 5 属于 A 亚型 。根据 SH 基因片段的核苷酸序列将 A 亚型分离株进一步划分为 SH 基因型 SHL1 - 6 。 SHL 之间关系密切 ,并与 NP 有关 。SHL1 、3 和 4 紧密相关 ,属于 NP2 ; SHL2 和 6 属于 NP4 ; SHL5 属于 NP5 。 ( ) ( ) 结果 1997 冬季至 1998 年春季 145 份 RSV NPS 标本中 ,83 份 57 . 2 %为 B 亚型 ,62 份 42 . 8 %为 A 亚型 ,A :B 为 1 :1 . 3 。1997 - 1998 流行年所获的 10 株 RSV 分离株中 ,2 株为 A 亚型 ,8 株为 B 亚型 ,A :B 为 1 :4 。1990 - 1991流行年所获的 10 株毒株中 ,8 株为 A 亚型 ,2 株为 B 亚型 ,A :B 为 4 :1 。1997 - 1998 流行年分离到的 10 株 RSV 分 离株中 ,2 株 A 亚型均属 NP4 ,SHL2 ;8 株 B 亚型均为 NP3 。1990 - 1991 流行年分离得到的 10 株毒株中 8 株 A 亚 型均属 NP4 ,SHL2 ;而 2 株 B 亚型均为 NP3 。根据 NP 推导出的 A 、B 亚型划分与单克隆抗体定义的亚型一致 。 结论 北京地区 RSV A 、B 亚型或多个基因型可同时流行 ,但每个流行年的优势基因型可不同 。相距 5 年仍可分 离到相同的毒株 ,明尽管 A 亚型毒株之间变异明显 ,但是一些基因型仍然相当稳定 。 关键词 呼吸道合胞病毒亚型基因型 致心律失常性右室心肌病的临床研究和家系调查 Clinical and fa milial study of arrhythmogenic right ventricular cardiomyopathy ( ) Chin Med J 2001 ; 114 4: 3692373 南京医科大学第一附属医院心脏科 南京 210029单其俊 曹克将 黄元铸 廖铭扬 陈明龙李闻奇 邹建刚 朱必顺 马文珠 目的 研究致心律失常性右室心肌病的特点 。 () 方法 本文对 7 例致心律失常性右室心肌病 ARVC及其 3 个家系中的 34 个成员进行调查 。所有病人和家族成 员进行了病史调查 、临床检查 、心电图 、超声心动图和晚电位检查 。5 例 ARVC 行电生理检查 。 结果 所有调查的病人和家族成员超声心动图示左室结构正常 。14 例有异常临床表现者诊断为 ARVC 。其中 5例右室扩大伴弥漫性搏动减弱 ;8 例右室心尖区变薄 ,搏动减弱伴收缩期膨出 ;1 例右室下壁变薄 ,搏动减弱伴收 () () 缩期膨出 。25 例心电图异常 7 例病人 ,18 例成员。13 例晚电位阳性 6 例病人。5 例病人电生理检查诱发出 2 - 3 种左束支阻滞室性心动过速 。2 例病人在电生理检查时诱发室颤 。5 例病人右室一个或多个部位起搏阈值 明显增高或起搏无效 。一家系中 2 个成员猝死 。5 例病人记录到自发的左束支阻滞室性心动过速 。一例 ARVC () 同时伴有侏儒症 。一例病人有联律间期极短的多形性室速 。12 例 6 例病人有左束支阻滞室性早搏 。 结论 本研究结果提示 ARVC 是一遗传性疾病 ,家系调查有助于诊断和发现新病例 ,右室结构和功能的异常 、心