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2011年26卷4期

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Review

Hand Foot and Mouth Disease Due to Enterovirus 71 in Malaysia

Kaw Bing Chua, Abdul Rasid Kasri

2011, 26(4): 221 doi: 10.1007/s12250-011-3195-8

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Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth. The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents. Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae. EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD), aseptic meningitis, encephalitis and poliomyelitis-like acute flaccid paralysis. A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997, and caused 41 deaths amongst young children. In late 2000, a recurrence of an outbreak of HFMD occurred in Malaysia with 8 fatalities in peninsular Malaysia. Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities. A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006. The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010. As with other HFMD outbreaks in Malaysia, both EV71 and CA16 were the main aetiological viruses isolated. In similarity with the HFMD outbreak in 2005, the isolation of CA16 preceded the appearance of EV71. Based on the VP1 gene nucleotide sequences, 4 sub-genogroups of EV71 (C1, C2, B3 and B4) co-circulated and caused the outbreak of hand, foot and mouth disease in peninsular Malaysia in 1997. Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak. EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003. In the 2005 outbreak, besides the EV71 strains of sub-genogroup C1, EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003. The four EV71 strains isolated from clinical specimens of patients with hand, foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5. Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia. Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years. In each of the past outbreaks, more than one sub-genogroup of the virus co-circulate.

Research Article

Epidemiology and Mutational Analysis of Global Strains of Crimean-Congo Haemorrhagic Fever Virus

Na Han, Simon Rayner

2011, 26(4): 229 doi: 10.1007/s12250-011-3211-z

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Crimean-Congo hemorrhagic fever (CCHF) is a severe illness with high fatality. Cases are reported in several countries in Africa, Europe, the Middle East, and Asia. Phylogenetic analyses based on the virus S (nucleocapsid), M (glycoprotein), and L (polymerase) genome segments sequences indicate distinct geographic lineages exist but their specific genetic characteristics require elucidation. In this work we collected all full length S segment sequences and generated a phylogenetic tree based on the alignment of these 62 samples. We then analyzed the alignment using entries from AAIndex, the Amino Acid Index database, to identify amino acid mutations that performed significant changes in charge, pka, hydropathy and side chain volume. Finally, we mapped these changes back to the tree and alignment to identify correlated mutations or sites that characterized a specific lineage. Based on this analysis we are able to propose a number of sites that appear to be important for virus function and which would be good candidates for experimental mutational analysis studies.

猪繁殖与呼吸综合征病毒逆转录环介导等温扩增检测方法的构建

张磊, 刘业兵, 陈蕾, 王建华, 宁宜宝

2011, 26(4): 252 doi: 10.1007/s12250-011-3185-x

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本研究构建了猪繁殖与呼吸综合症病毒(Porcine reproductive and respiratory syndrome virus ,PRRSV) 美洲型原型毒株与流行毒株的快速、特异的逆转录环介导等温扩增检测方法(Reverse transcription loop-mediated isothermal amplification ,RT-LAMP)。通过使用浊度仪对本研究构建的RT-LAMP检测体系的反应进行实时监控观察,结果显示:待检体系中存在2.5 TCID50 以上的PRRSV 的RNA核酸,利用本研究构建的RT-LAMP方法即可检出;并且该方法与PCV2、SIV、CSFV等病毒无交叉反应。使用本研究构建的RT-LAMP对42份田间样品进行检测,并与RT-PCR及PAM细胞体外分离鉴定 2种检测方法进行比较,结果显示:RT-LAMP检测出33份阳性样品,与PAM细胞分离鉴定检测结果一致;RT-PCR方法除3份阳性样品未检出,其余检测结果与RT-LAMP及PAM细胞分离鉴定一致。综上表明:本研究构建的RT-LAMP方法相对于RT-PCR方法具备更高的敏感性和特异性,对发展中国家的PRRSV 及猪高致病性蓝耳病(highly pathogenic PRRSV)检测有一定的实用价值。

抗重组VP28囊膜蛋白单克隆抗体的制备及在对虾白斑综合征病毒检测中的应用

侯崇林, 曹煜, 谢荣辉, 汪以真, 杜华华

2011, 26(4): 260 doi: 10.1007/s12250-011-3202-0

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以对虾白斑综合征病毒(White spot syndrome virus,WSSV)的全基因组为模板,扩增得到囊膜蛋白VP28基因,插入到表达载体pET-30a中并在大肠杆菌BL21菌株中进行表达得到重组VP28蛋白(rVP28),纯化的rVP28免疫Balb/c小鼠制备单克隆抗体。免疫电镜检测显示抗rVP28单克隆抗体可有效识别WSSV并特异性地结合在病毒粒子表面。斑点杂交试验表明抗rVP28单克隆抗体能快速并特异地检测患病螯虾鳃组织匀浆液中的WSSV,竞争定量PCR分析显示其检出的最低限度为104 拷贝。这暗示着抗rVP28单克隆抗体可用于WSSV的早期检测。

利用GP5蛋白制备抗PRRSV的单克隆抗体

田宏, 吴锦艳, 陈妍, 何建辉, 尚佑军, 刘湘涛

2011, 26(4): 267 doi: 10.1007/s12250-011-3198-5

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用纯化的蓝耳病病毒GP5表位重组蛋白为抗原免疫6~8周龄的雌性BALB / c小鼠, 经过3次免疫后, 取其脾细胞与Sp2/0骨髓瘤细胞融合。采用有限稀释法和间接ELISA 克隆和筛选阳性杂交瘤细胞株, 用SDS- PAGE电泳、间接ELISA以及中和试验对所获得的mAb的特异性进行鉴定。结果成功获得2株能稳定传代并分泌抗PRRVS 抗体的杂交瘤细胞株, 分别命名为:8C9和4B4 其分泌的mAb为IgG1和IgG2a亚类, 它们均能特异性的识别GP5重组蛋白和PRRVS全病毒, 其腹水效价在1:104~1:105。中和试验表明该mAb能很好地识别灭活的PRRVS病毒, 中和效价达1:512以上。交叉试验表明该mAb具有高度特异性, 与CSFV及SVDV无交叉反应, 证明所获得的mAb均完全针对GP5抗原决定簇。蓝耳病病毒GP5 mAb的成功制备, 为进一步研究和开发新型蓝耳病的检测方法和抗原表位奠定了基础。

利用A Type FMDV蛋白制备抗Asia I口蹄疫病毒的单克隆抗体

林彤, 李菁, 邵军军, 独军政, 丛国正, 高闪电, 常惠芸

2011, 26(4): 273 doi: 10.1007/s12250-011-3199-4

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用纯化的A型口蹄疫灭活全病毒为抗原免疫6~8周龄的雌性BALB / c小鼠, 经过3次免疫后, 取其脾细胞与Sp2 /0骨髓瘤细胞融合。采用有限稀释法和间接EL ISA 克隆和筛选阳性杂交瘤细胞株, 用SDS-PAGE电泳、间接EL ISA以及中和试验对所获得的mAb的特异性进行鉴定。结果成功获得2株能稳定传代并分泌抗A型口蹄疫病毒的杂交瘤细胞株, 分别命名为: 7B11、4H4, 其分泌的mAb为IgG1 (7B11)和IgG2a (8H4)亚类, 它们均能特异性的识别A 型FMDV重组蛋白和A型灭活口蹄疫全病毒, 其腹水效价在1∶104~1∶105。中和试验表明该mAb能很好地识别灭活的FMDV, 中和效价达1∶512以上。交叉试验表明该mAb具有高度特异性, 型间无交叉反应, 证明所获得的mAb均完全针对A FMDV抗原决定簇。A口蹄疫病毒mAb的成功制备, 为进一步研究和开发新型口蹄疫的检测方法和抗原表位奠定了基础。

海南BBTV satellite DNA的生物信息学分析

余乃通, 冯团诚, 张雨良, 王健华, 刘志昕

2011, 26(4): 279 doi: 10.1007/s12250-011-3196-7

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以本实验前期提交至Genbank的BBTV Haikou 2 satellite DNA序列(HQ616080)为研究对象,利用生物信息学的方法预测DNA序列结构特征和编码蛋白的理化性质和功能分析。结果表明,Primer Premier 5.0对satellite DNA UTR进行分析,发现其存在12个基序;对编码蛋白的理化性质、结构组成、信号肽、磷酸化、二级结构、三级结构与功能结构域等作了较为详细的分析与预测,并与BBTV DNA1组份Rep蛋白做比较。本文对海南BBTV satellite DNA组分的功能预测,为研究BBTV的复制过程,侵染的机理打下了重要的基础。

Hemorrhagic Fever with Renal Syndrome Complicated by Orchitis

Edmond Puca, Arben Pilaca, Pellumb Pipero, Silva Bino, Majlinda Kote,

2011, 26(4): 285 doi: 10.1007/s12250-011-3182-0

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Hemorrhagic fever with renal syndrome (HFRS) is a disease caused by viruses of the family Bunyaviridae, genus Hantavirus. HFRS from Dobrava virus (DOBV) is a seldom reported disease in Albania. Clinically HFRS is manifested as mild, moderate, or severe. Therefore, the number of cases of Hantavirus’ infection may be underestimated, and should be included in the differential diagnosis of many acute infections, hematologic diseases, acute abdominal diseases and renal diseases complicated by acute renal failure. We report here an atypical presentation of HFRS from Dobrava virus complicated by orchitis with a positive outcome.

Cover story

Putative Phosphorylation Sites On WCA Domain of HA2 Is Essential For Helicoverpa armigera Single Nucleopolyhedrovirus Replication*

Yi-pin Lv, Qian Wang, Chun-chen Wu, Rong-juan Pei, Yuan Zhou,

2011, 26(4): 245 doi: 10.1007/s12250-011-3189-6

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Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality. The Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain, in which phosphorylation status are supposed to be critical in respect to actin polymerization. In the present study, two putative phosphorylation sites (232Thr and 250Ser) and a highly conserved Serine (245Ser) on the WCA domain of HA2 were mutated, and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome. Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at 245Ser can produce infectious virions, both 232Thr and 250Ser mutations were lethal to the virus. However, actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus, which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.