Complete sequence analyses of enterovirus 71 strains from fatal and non-fatal cases of the hand,foot and mouth disease outbreak in Singapore (2000)

Enterovirus 71 (EV71) is a major aetiological agent of hand, foot and mouth disease (HFMD). In recent years, several outbreaks in East Asia were associated with neurological complications and numerous deaths. An outbreak in Singapore in October 2000 afflicted thousands of children, resulting in four fatal cases from three of whom EV71 was isolated. The genomes of two representative EV71 strains isolated from a fatal case and a surviving patient were completely sequenced, and their nucleotide and amino acid sequences compared with known EV71 strains. The two outbreak strains were classified under genogroup B, together with those previously isolated in Singapore, Malaysia and Japan. Comparative sequence analysis of the two Singapore strains revealed 99% nucleotide similarity, while their deduced amino acid sequences were almost identical except for residue 1506 in the 3A non-structural region. Given that the outbreak involved closely related genetic variants of EV71, the broad spectrum of disease severity may be attributed to critical factors such as varying viral inoculation doses or differing host immune responses following infection, but is less likely to be due to the emergence of EV71 strains with heightened virulence.     

The LIMP-2/SCARB2 Binding Motif on Acid β-Glucosidase: BASIC AND APPLIED IMPLICATIONS FOR GAUCHER DISEASE AND ASSOCIATED NEURODEGENERATIVE DISEASES*

The acid β-glucosidase (glucocerbrosidase (GCase)) binding sequence to LIMP-2 (lysosomal integral membrane protein 2), the receptor for intracellular GCase trafficking to the lysosome, has been identified. Heterologous expression of deletion constructs, the available GCase crystal structures, and binding and co-localization of identified peptides or mutant GCases were used to identify and characterize a highly conserved 11-amino acid sequence, DSPIIVDITKD, within human GCase. The binding to LIMP-2 is not dependent upon a single amino acid, but the interactions of GCase with LIMP-2 are heavily influenced by Asp³⁹⁹ and the di-isoleucines, Ile⁴⁰² and Ile⁴⁰³. A single alanine substitution at any of these decreases GCase binding to LIMP-2 and alters its pH-dependent binding as well as diminishing the trafficking of GCase to the lysosome and significantly increasing GCase secretion. Enterovirus 71 also binds to LIMP-2 (also known as SCARB2) on the external surface of the plasma membrane. However, the LIMP-2/SCARB2 binding sequences for enterovirus 71 and GCase are not similar, indicating that LIMP-2/SCARB2 may have multiple or overlapping binding sites with differing specificities. These findings have therapeutic implications for the production of GCase and the distribution of this enzyme that is delivered to various organs.     

Functional interpretation and structural insights of Arabidopsis lyrata cytochrome P450 CYP71A13 involved in auxin synthesis

Cytochrome P450 CYP71A13 of Arabidopsis lyrata is a heme protein involved in biosynthesis of indole-3-acetonitrile which leads to the formation of indolyl-3-acetic acid. It catalyzes a unique reaction: formation of a carbon-nitrogen triple bond and dehydration of indolyl-3-acetaldoxime. Homology model of this 57 kDa polypeptide revealed that the heme existed between H-helix and J- helix in the hydrophobic pocket, although both helixes are involved in catalytic activity, where Gly305 and Thr308, 311 of H- helix were involved in its stabilization. The substrate indole-3-acetaldoxime was tightly fitted into the substrate pocket with the aromatic ring being surrounded by amino acid residues creating a hydrophobic environment. The smaller size of the substrate binding pocket in cytochrome P450 CYP71A13 was due to the bulkiness of the two amino acid residues Phe182 and Trp315 pointing into the substrate binding cavity. The apparent role of the heme in cytochrome P450 CYP71A13 was to tether the substrate in the catalysis by indole-3-acetaldoxime dehydratase. Since the crystal structure of cytochrome P450 CYP71A13 has not yet been solved, the modeled structure revealed mechanism of substrate recognition and catalysis.     

白介素18启动子区基因多态性与儿童EV71感染相关性研究

目的:研究白介素18基因启动子多态性与儿童EV71感染遗传易感性的关系.方法:收集EV71感染患儿177例,单纯HFMD组127例,HFMD并脑炎组50例,提取外周血DNA,用序列特异性引物-聚合酶链反应(SSP-PCR)技术及基因测序法检测IL-18启动子区-137G/C、-607A/C位点的基因多态性.结果:EV71感染患儿与健康儿童IL-18-607基因型以CA为主,AA、CC次之,EV71感染患儿AA基因型、A等位基因分布频率显著高于健康儿童.EV71感染HFMD并脑炎组患儿AA基因型分布显著高于单纯HFMD组患儿,差异有统计学意义.EV71感染患儿与健康儿童IL-18-137基因型以CC为主,CG次之,GG占少数.该位点基因型及等位基因在EV71感染组与健康儿童、单纯HFMD与HFMD并脑炎组的分布无显著差异.结论:IL-18基因多态性与EV71感染相关,IL-18-607AA基因型、A等位基因携带儿童更易感染EV71病毒,且AA基因型患儿易并发脑炎,-607AA基因型可能为EV71感染的易感基因型.-137C/G位点基因多态性与EV71感染无相关性.     

肠道病毒71型安徽、河南株的分离与VP1序列进化分析

旨在研究手足口病患者中肠道病毒71型分离株的病毒基因型特征。采集手足口病患者的粪便标本,进行病毒分离和逆转录-聚合酶链式反应(RT-PCR)特异性扩增进行鉴定,同时选取其中9株EV71分离株,对其抗原决定簇部位VP1区进行核酸序列测定,并参考EV71 A、B、C各基因型的参考株和以往中国EV71的分离株进行同源分析和构建系统发生树。结果显示,所分析的9株病毒株均为C4亚型,3株安徽株H7、H8和H9的VP1序列相似度很高(≥98.8%,其中H7、H9的相似度为100.0%),4株河南株H3、H4、H5和H6相似度较高(≥98.4%,H3、H4和H5≥99.6%,其中H3、H4的相似度为100.0%),它们同河南株H1、H5的相似度也较高(≥97.2%),河南株H2虽然与其他河南株具有较高的序列相似度,但进化分析表明,其与安徽株同源性较高。结果表明,安徽株H7、H8和H9株变异速率明显加快,这可能导致了手足口病在安徽省的率先爆发与大流行,河南株H2最初可能由安徽传入河南。     

Hand foot and mouth disease due to enterovirus 71 in Malaysia

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 41deaths amongst young children.In late 2000,a recurrence of an outbreak of HFMD occurred in Malaysia with S 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 VP 1 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.     

Sterol chemical configuration influences the thermotropic phase behaviour of dipalmitoylphosphatidylcholine bilayers containing 5α-cholestan-3β- and 3α-ol

It is commonly believed that all membrane sterols are rigid all-trans ring systems with a fully extended alkyl side-chain and that they similarly influence phospholipid bilayer physical properties. Here, we report the sterol concentration-dependent, thermotropic phase behaviour of binary dipalmitoylphosphatidylcholine (DPPC)/sterol mixtures containing two similar 5α-H sterols with different functional group orientations (3α-OH or 3β-OH), which adopt an ideal all-trans planar ring conformation but lack the deformed ring B conformation of cholesterol (Chol) and epicholesterol (Echol), using differential scanning calorimetry (DSC). Our deconvolution of the DSC main phase transition endotherms show differences in the proportions of sterol-poor (sharp) and sterol-rich (broad) domains in the DPPC bilayer with increasing sterol concentration, which delineate gel/liquid-crystalline (P_β′/L_α) and disordered gel (L_β)/liquid-ordered (l_o) phase regions. There are similarities in the DPPC main phase transition temperature, cooperativity and enthalpy for each 3β-ol and 3α-ol pair with increasing sterol concentration and differences in the parameters obtained for both the sterol-poor and sterol-rich regions. The sterol-poor domain persists over a greater concentration range in both 3α-ol/DPPC mixtures, suggesting that either those domains are more stable in the 3α-ols or that those sterols are less miscible in the sterol-rich domain. Corresponding parameters for the sterol-rich domain show that at sterol concentrations up to 20 mol%, the 5α-H,3β-ol is more effective at reducing the phase transition enthalpy of the broad component () than Chol, but is less effective at higher concentrations. Although mixtures containing Echol and 5α-cholestan-3α-ol have similar positive slopes below 7 mol% sterol, suggesting that they abolish the L_β/l_o phase transition equally effectively at low concentrations, Echol is more effective than the saturated 3α-ol at higher sterol concentrations. A comparison of obtained for the saturated and unsaturated pairs suggests that the latter sterols stabilize the l_o phase and broaden and abolish the DPPC main phase transition more effectively than the saturated sterols at physiologically relevant concentrations, supporting the idea that the double bond of Chol and Echol promotes greater sterol miscibility and the formation of l_o phase lipid bilayers relative to corresponding saturated sterols in biological membranes.