上海市人群Echo30病毒性脑膜炎隐性感染调查

为了了解上海市不同年龄组人群的Echo30病毒隐性感染情况及IgG抗体阳性率分布。采集上海市412份不同年龄组人群血清,用间接酶联免疫吸附试验(ELISA)检测血清中的Echo30IgG抗体。发现受检普通人群血清中Echo30IgG抗体阳性率为25.8%。其中1岁以下婴幼儿中未见抗体阳性者,15岁以下儿童抗体阳性率较低(10%～16.7%),15岁以上人群抗体阳性率水平明显升高(45.0%～46.7%)。孕妇与普通人群抗体阳性率无显著性差异。研究结果提示上海市人群中存在隐性感染者,人群通过自然感染获得免疫保护,15岁以下儿童为Echo30感染及发病的高危人群,母体传递给婴幼儿的抗体水平较低,不能为婴幼儿提供先天性免疫保护。     

Elucidation of Echovirus 30's Origin and Transmission during the 2012 Aseptic Meningitis Outbreak in Guangdong,China, through Continuing Environmental Surveillance

An aseptic meningitis outbreak occurred in Luoding City of Guangdong, China, in 2012, and echovirus type 30 (ECHO30) was identified as the major causative pathogen. Environmental surveillance indicated that ECHO30 was detected in the sewage of a neighboring city, Guangzhou, from 2010 to 2012 and also in Luoding City sewage samples (6/43, 14%) collected after the outbreak. In order to track the potential origin of the outbreak viral strains, we sequenced the VP1 genes of 29 viral strains from clinical patients and environmental samples. Sequence alignments and phylogenetic analyses based on VP1 gene sequences revealed that virus strains isolated from the sewage of Guangzhou and Luoding cities matched well the clinical strains from the outbreak, with high nucleotide sequence similarity (98.5% to 100%) and similar cluster distribution. Five ECHO30 clinical strains were clustered with the Guangdong environmental strains but diverged from strains from other regions, suggesting that this subcluster of viruses most likely originated from the circulating virus in Guangdong rather than having been more recently imported from other regions. These findings underscore the importance of long-term, continuous environmental surveillance and genetic analysis to monitor circulating enteroviruses.     

Complete Genome Sequence Analysis of Human Echovirus Type 30 Isolated in China

We report here the complete genome sequence of a human echovirus type 30 strain ECV30/GX10/05 isolated in Guangxi, China, in 2010. Phylogenetic analysis showed that ECV30/GX10/05 was closely related to a Korean strain isolated in 2008. The sequence information will help in an understanding of the molecular epidemiology and evolution of echovirus.     

Phosphoinositide Turnover Associated with Synaptic Transmission

Although pharmacological stimulation of a wide variety of transmitter receptors triggers phosphoinositide (PI) turnover, little is known about the type of synaptic activity required to activate this system. To investigate this question, we have used primary cultures of embryonic cortical neurons, which develop functional glutamate and GABA synapses during maturation in vitro. Mature cultures display spontaneous synaptic activity that is totally suppressed by tetrodotoxin (TTX). PI turnover, assayed by the lithium-sensitive accumulation of [3H]CDP-diacylglycerol, was readily detected under basal conditions and was abolished by TTX. Increased excitatory synaptic activity induced by picrotoxin, an antagonist of GABAA receptor-mediated inhibition, further stimulated PI turnover. Similar results were obtained when PI turnover was assayed using [3H]inositol labeling. With either assay, the magnitude of synaptically induced PI turnover was comparable to maximal responses produced by muscarinic receptor stimulation. Although a component of the spontaneous synaptic currents is sensitive to N-methyl-D-aspartate (NMDA)-preferring glutamate receptor antagonists, blockade of NMDA receptors did not affect PI turnover associated with synaptic transmission. To assess the time course of synaptically mediated PI turnover, the amplitude and duration of spontaneous synaptic currents were reduced by lowering the extracellular Ca2+ concentration from 2.25 to 0.5 mM, a maneuver that suppresses basal PI turnover. Increases in PI turnover were detected as early as 5 min following restoration of the extracellular Ca2+ concentration to 2.25 mM. Taken together, these findings indicate that activation of the PI system is associated with physiological levels of glutamatergic synaptic transmission.     

我国新分离ECHO30病毒VP1序列分析

测定了引起2003年苏北地区无菌性脑膜炎暴发流行的病因病毒FDJS03分离株的VP1基因序列,并与国外同型流行毒株做比较,以了解本流行株的分子生物学特点及遗传变异规律。随机选取FDJS03分离毒株中的4株,用肠道病毒、VP1序列的特异性引物008／011进行RT-PCR,扩增产物经凝胶纯化后测序。将序列输入GenBank,用BLAgr程序进行核苷酸和氨基酸序列比对;选取32株不同地区不同年代的ECHO30分离毒株,在PHYLIP3．573C和TREE-PUZZLE5．0中构建进化树,比较它们完整VP1序列(876nt)的进化关系。核苷酸和氨基酸同源性比较结果证明：4株分离病毒均为ECHO30。进化树分析显示：本次FDJS03分离株与欧美20世纪70、80年代流行株亲缘关系最近,但自成一簇,与国外毒株仍然有地区差别。ECHO30的VP1基因进化有时间效应,但存在地区差异。本次流行的病原可能是单一基因型的ECHO30病毒。     

基于RNA-seq技术研究埃可病毒30型感染RD细胞前后的差异表达基因

埃可病毒30型(Echovirus 30,E30)是一种全球传播的B组肠道病毒,常与无菌性脑膜炎等疾病暴发有关,分析E30在感染人横纹肌肉瘤(Human rhabdomyosarcoma,RD)细胞前后的差异表达基因有助于了解该病毒的复制周期以及宿主感染机制。本研究通过转录组测序技术探究E30感染RD细胞前后的基因表达谱变化,共检测到的1281个差异表达基因,其中包括730个下调基因和551个上调基因。基因本体论(Gene Ontology,GO)和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)富集分析表明,显著差异表达基因主要参与细胞受体信号通路的调节、炎症反应、免疫细胞活化、调控细胞生命周期等。利用荧光定量PCR(Realtime quantitative PCR,qPCR)对其中9个与炎症和免疫反应相关的差异表达基因进行验证,发现DEAD-box解旋酶3(DEAD-box RNA helicase 3,DDX3)表达上调,这与转录组学分析一致。利用RK-33(DDX3的小分子抑制剂)靶向抑制DDX3的表达,发现RK-33能够抑制E30的复制,并且qPCR结果显示在抑制DDX3的表达后,GTP酶激活蛋白结合蛋白1(GTPase-activating protein-binding protein1,G3BP1)和干扰素调节因子3(Interferon Regulatory Factor 3,IRF3)的表达也出现不同程度地降低。本研究的结果提示DDX3表达可能影响E30复制,这一发现为进一步探索E30在感染宿主过程中的分子机制奠定基础。     

一起传染病暴发中肠道病毒血清型鉴定和ECHO30基因特征分析

2003年5～9月,山东省泰安市发生了由肠道病毒(Enterovirus,EV)感染所致的传染病暴发,临床症状以手足口病(HFMD)为主,同时有心肌炎和无菌性脑膜炎等中枢神经系统症状患者也占较大比例。131份病人(粪便、咽拭子、脑脊液)标本中共分离到EV62株,其中ECHO1939株,EV716株,ECHO304株,其它肠道病毒13株。4株ECHO30病毒中的2株分离自2个患者的粪便标本,但用WHO肠道组合血清中和试验未能定出型别。另外2株分离自同一患者的粪便和脑脊液标本。病原学分析表明,ECHO30是引起该患者无菌性脑膜炎的病原。抗E—CHO30标准株的血清中和这4株病毒的滴度低于标准株5～20倍。VP1区全基因序列测定和同源性比较分析表明,4株ECHO30分离株病毒核苷酸同源性在98．0％～98．5％,氨基酸同源性在98．9％～99．3％,提示这4株病毒来源于同一传播链,2003年5～9月ECHO30在该地区可能有局部流行。系统进化树分析表明,ECHO30病毒可以划分为6个基因型,其中基因型1～5为GenBank中已发表的ECHO30分离株,山东分离株与其它5个基因型成员核苷酸差异分别在9．4％～24．4％,在进化树上形成了较独立的分支,是一个新基因型,将其划分为第6基因型。     

Population Turnover in Remote Oceania Shortly after Initial Settlement

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Beta Diversity of Plant-Pollinator Networks and the Spatial Turnover of Pairwise Interactions

Interactions between species form complex networks that vary across space and time. Even without spatial or temporal constraints mutualistic pairwise interactions may vary, or rewire, across space but this variability is not well understood. Here, we quantify the beta diversity of species and interactions and test factors influencing the probability of turnover of pairwise interactions across space. We ask: 1) whether beta diversity of plants, pollinators, and interactions follow a similar trend across space, and 2) which interaction properties and site characteristics are related to the probability of turnover of pairwise interactions. Geographical distance was positively correlated with plant and interaction beta diversity. We find that locally frequent interactions are more consistent across space and that local flower abundance is important for the realization of pairwise interactions. While the identity of pairwise interactions is highly variable across space, some species-pairs form interactions that are locally frequent and spatially consistent. Such interactions represent cornerstones of interacting communities and deserve special attention from ecologists and conservation planners alike.     

Population dynamics of mitochondria II. Turnover and ageing of rat liver mitochondria

Membrane-bound multi-protein complexes in mitochondria are provisionally classified into four categories based on possible mechanism of their assembly and degradation. These mechanisms may be investigated by the use of pulse-labeled radioactive markers which are not re-utilizable. Age dependent assembly is defined as that mechanism by which one or more of the pulse-labeled subunits are assembled into a complex, only while this complex is assembled. If the labeled sub-units can be taken up by the complex randomly during its life-span, then the mechanism is called age-independent assembly. Age-dependent degradation was defined as that mechanism by which the labeled subunits are decomposed, only when the complex is being degraded as an entity. If the labeled subunits are decomposed randomly, the mechanism is called age-independent degradation. Four categories are made by combining each of the assembly and degradation mechanisms. A differential equation was obtained to describe the fate of labeled sub-units that follow the age-dependent assembly and age-dependent degradation. Also derived was an equation for the age-independent assembly and age-dependent degradation. The other two categories which involve the age-independent degradation after age-dependent or age-independent assembly are described by single exponential kinetics. Practical application of the equations is illustrated with the use of experimental data on mitochondrial turnover found in the literature which suggests that the pulse-labeled proteins in rat liver mitochondria may follow the age-dependent assembly and degradation. The present attempts to introduce the concept of ageing into multi-protein complexes in mitochondria are the extensions of the steady state theory of mutation by Eyring & Stover (1970).     

Numbers, Turnover, and Social Structure of the Isle Royale Wolf Population

Observations of wolves on Isle Royale are reported for 1961–66,with interpretations including the earlier 3-year period describedby Mech (1966). On this 210-square-mile island the fully protectedwolf population varied from approximately 22 to 28 in midwinter.The major and minor foods were moose and beaver, respectively. The main pack varied in number between 11 and 22 with aboutthree breeding pairs believed present. The population remainedrelatively stable; mating occurred every winter; and adult mortalityappeared to be low. High mortality among pups seemed to be thepoint of population control. Socio-economic factors may havecontrolled the size of the large pack. Availability of foodduring the period of parturition and rearing probably was criticalto survival of young. Recruitment of young appeared to take place in years of highproduction of moose calves. Numbers in the large pack probablywere curtailed through the progressive exclusion of aged andsocially subordinate individuals. Under harassment these animalsseparated and became pack-following scavengers, then probablytrue loners ranging outside the area used by the pack. Smalleraggregations of two or three non-breeders were seen each winter,as were the loners, some of which appeared thin and weak. The only known breeding outside the big pack was in a groupof five present in the winter of 1965. This group was probablya family unit which separated from the main pack. A year laterthe male had disappeared, and remains of a pup, probably theirs,were found. In the winter of 1966 the alpha male of the largepack became lame and apparently was killed. This backgroundappears favorable for further changes in social organization.     

Evolutionary Dynamics and Temporal/Geographical Correlates of Recombination in the Human Enterovirus Echovirus Types 9, 11, and 30

The relationship between virus evolution and recombination in species B human enteroviruses was investigated through large-scale genetic analysis of echovirus type 9 (E9) and E11 isolates (n = 85 and 116) from 16 European, African, and Asian countries between 1995 and 2008. Cluster 1 E9 isolates and genotype D5 and A E11 isolates showed evidence of frequent recombination between the VP1 and 3Dpol regions, the latter falling into 23 (E9) and 43 (E11) clades interspersed phylogenetically with 46 3Dpol clades of E30 and with those of other species B serotypes. Remarkably, only 2 of the 112 3Dpol clades were shared by more than one serotype (E11 and E30), demonstrating an extremely large and genetically heterogeneous recombination pool of species B nonstructural-region variants. The likelihood of recombination increased with geographical separation and time, and both were correlated with VP1 divergence, whose substitution rates allowed recombination half-lives of 1.3, 9.8, and 3.1 years, respectively, for E9, E11, and E30 to be calculated. These marked differences in recombination dynamics matched epidemiological patterns of periodic epidemic cycles of 2 to 3 (E9) and 5 to 6 (E30) years and the longer-term endemic pattern of E11 infections. Phylotemporal analysis using a Bayesian Markov chain Monte Carlo method, which placed recombination events within the evolutionary reconstruction of VP1, showed a close relationship with VP1 lineage expansion, with defined recombination events that correlated with their epidemiological periodicity. Whether recombination events contribute directly to changes in transmissibility that drive epidemic behavior or occur stochastically during periodic population bottlenecks is an unresolved issue vital to future understanding of enterovirus molecular epidemiology and pathogenesis.Human enteroviruses (HEV) are single-stranded, positive-sense RNA viruses in the virus family Picornaviridae. Infections with these viruses are enterically transmitted and normally cause subclinical or mild symptoms, but they are also capable of causing a wide array of often severe disease presentations, including aseptic meningitis, encephalitis, and acute flaccid paralysis. Echovirus type 9 (E9) and E11 are serotypes of species B enteroviruses and are among the most common etiological agents of aseptic meningitis (14). The first epidemiological study of E9 investigated isolates obtained from sick children in 1995 (24). E9 is chiefly associated with mild infections affecting children over the age of 5 years and teenagers, although it can cause more severe syndromes in neonates and immunosuppressed patients (14). E11 infections, which predominantly affect infants less than 1 year old, have been associated with large outbreaks of uveitis in infants (15, 21), sepsis, and other neonatal systemic illnesses with high mortality rates (14, 23).Circulating strains and genotypes of E9 (2, 8, 11, 14) and E11 (4, 7, 21) have been characterized in many different countries through analysis of the structural genome regions, principally VP1. In common with other mammalian RNA viruses, both E9 and E11 show rapid accumulation of nucleotide substitutions over time, but their epidemiologies are distinct. E9 is associated with widespread, large-scale seasonal outbreaks and displays a regular epidemic pattern of outbreaks occurring approximately every 3 years (8, 14). Outbreaks of E11 are less common, occur irregularly, and often last for several years (14, 25).In contrast to time-related diversification observed in the capsid-encoding regions of the HEV genome, nonstructural region genes are subject to frequent recombination (7, 18, 19, 26, 30), leading to the concept of separate, modular evolution of the structural and nonstructural regions of the HEV genome (20). Recombination in the nonstructural gene region of HEV is limited to members of the same species (27). In a recent large-scale investigation of echovirus type 30 (E30) molecular epidemiology, phylogenetic analysis of the 3Dpol region revealed the existence of a number of discrete, bootstrap-supported clades, allowing circulating E30 variants to be classified into a series of distinct recombinant forms (RFs) (22). Individual RFs became very rapidly disseminated over large geographical distances through repeated cycles of emergence, dominance, and disappearance in a 3- to 5-year cycle.In the current study, we carried out parallel investigations of the molecular epidemiology and evolution of clinically presenting isolates of E9 and E11 collected in 16 countries in Europe, central Asia, Southeast Asia, and West Africa over a 14-year period. By comprehensive sequencing of these isolates in the structural (VP1) and nonstructural (3Dpol) genome regions, it was possible to explore the dynamics of sequence diversification and recombination. The turnover of individual recombinant groups and the phylogeographical correlates of recombination were determined and compared to those of E30, providing new insights into the nature of the global spread of these important viral pathogens and the role of recombination in enteroviral evolution.     

Internalization of Echovirus 1 in Caveolae

Echovirus 1 (EV1) is a human pathogen which belongs to the Picornaviridae family of RNA viruses. We have analyzed the early events of infection after EV1 binding to its receptor alpha 2 beta 1 integrin and elucidated the route by which EV1 gains access to the host cell. EV1 binding onto the cell surface and subsequent entry resulted in conformational changes of the viral capsid as demonstrated by sucrose gradient sedimentation analysis. After 15 min to 2 h postinfection (p.i.) EV1 capsid proteins were seen in vesicular structures that were negative for markers of the clathrin-dependent endocytic pathway. In contrast, immunofluorescence confocal microscopy showed that EV1, alpha 2 beta 1 integrin, and caveolin-1 were internalized together in vesicular structures to the perinuclear area. Electron microscopy showed the presence of EV1 particles inside caveolae. Furthermore, infective EV1 could be isolated with anti-caveolin-1 beads 15 min p.i., confirming a close association with caveolin-1. Finally, the expression of dominant negative caveolin in cells markedly inhibited EV1 infection, indicating the importance of caveolae for the viral replication cycle of EV1.     

Sheep Movement Networks and the Transmission of Infectious Diseases

Background and Methodology

Various approaches have been used to investigate how properties of farm contact networks impact on the transmission of infectious diseases. The potential for transmission of an infection through a contact network can be evaluated in terms of the basic reproduction number, R ₀. The magnitude of R ₀ is related to the mean contact rate of a host, in this case a farm, and is further influenced by heterogeneities in contact rates of individual hosts. The latter can be evaluated as the second order moments of the contact matrix (variances in contact rates, and co-variance between contacts to and from individual hosts). Here we calculate these quantities for the farms in a country-wide livestock network: >15,000 Scottish sheep farms in each of 4 years from July 2003 to June 2007. The analysis is relevant to endemic and chronic infections with prolonged periods of infectivity of affected animals, and uses different weightings of contacts to address disease scenarios of low, intermediate and high animal-level prevalence.

Principal Findings and Conclusions

Analysis of networks of Scottish farms via sheep movements from July 2003 to June 2007 suggests that heterogeneities in movement patterns (variances and covariances of rates of movement on and off the farms) make a substantial contribution to the potential for the transmission of infectious diseases, quantified as R ₀, within the farm population. A small percentage of farms (<20%) contribute the bulk of the transmission potential (>80%) and these farms could be efficiently targeted by interventions aimed at reducing spread of diseases via animal movement.     

Population Cycles,Disease, and Networks of Ecological Knowledge

Wildlife populations in the northern reaches of the globe have long been observed to fluctuate or cycle periodically, with dramatic increases followed by catastrophic crashes. Focusing on the early work of Charles S. Elton, this article analyzes how investigations into population cycles shaped the development of Anglo-American animal ecology during the 1920s–1930s. Population cycling revealed patterns that challenged ideas about the “balance” of nature; stimulated efforts to quantify population data; and brought animal ecology into conversation with intellectual debates about natural selection. Elton used the problem of understanding wildlife population cycles to explore a central tension in ecological thought: the relative influences of local conditions (food supply, predation) and universal forces (such as climate change and natural selection) in regulating wild animal populations. He also sought patronage and built research practices and the influential Bureau of Animal Population around questions of population regulation during the 1930s. Focusing on disease as a local population regulator that could interact with global climatic influences, Elton facilitated an interdisciplinary and population-based approach in early animal ecology. Elton created a network of epidemiologists, conservationists, pathologists and mathematicians, who contributed to population cycle research. I argue that, although these people often remained peripheral to ecology, their ideas shaped the young discipline. Particularly important were the concepts of abundance, density, and disease; and the interactions between these factors and natural selection. However, Elton’s reliance on density dependence unwittingly helped set up conditions conducive to the development of controversies in animal ecology in later years. While ecologists did not come to consensus on the ultimate causes of population cycles, this phenomenon was an important early catalyst for the development of theory and practice in animal ecology.     

Raccoon Social Networks and the Potential for Disease Transmission

Raccoons are an important vector of rabies and other pathogens. The degree to which these pathogens can spread through a raccoon population should be closely linked to association rates between individual raccoons. Most studies of raccoon sociality have found patterns consistent with low levels of social connectivity within populations, thus the likelihood of direct pathogen transmission between raccoons is theoretically low. We used proximity detecting collars and social network metrics to calculate the degree of social connectivity in an urban raccoon population for purposes of estimating potential pathogen spread. In contrast to previous assumptions, raccoon social association networks were highly connected, and all individuals were connected to one large social network during 15 out of 18 months of study. However, these metrics may overestimate the potential for a pathogen to spread through a population, as many of the social connections were based on relatively short contact periods. To more closely reflect varying probabilities of pathogen spread, we censored the raccoon social networks based on the total amount of time spent in close proximity between two individuals per month. As this time criteria for censoring the social networks increased from one to thirty minutes, corresponding measures of network connectivity declined. These findings demonstrate that raccoon populations are much more tightly connected than would have been predicted based on previous studies, but also point out that additional research is needed to calculate more precise transmission probabilities by infected individuals, and determine how disease infection changes normal social behaviors.     

Population genetics of 30 INDELs in populations of Poland and Taiwan

The Investigator DIPplex^® kit (Qiagen) contain components for the simultaneous amplification and analysis of 30 biallelic autosomal INDELs and amelogenin. The objective of this study was to estimate the diversity of the 30 markers in Polish (N _P = 122) and Taiwanese (N _T = 126) population samples and to evaluate their usefulness in forensic genetics. All amplicon lengths were shorter than 160 base pairs. The DIPplex genotype distributions showed no significant deviation from Hardy–Weinberg rule expectations (Bonferroni corrected) except for DLH39 in the Taiwanese population. Among the Poles and the Taiwanese the mean observed heterozygosity values are 0.4385 and 0.4079, and the combined matching probability values are 7.98 × 10^?14 and 1.22 × 10^?11, respectively. The investigated marker set has been confirmed as a potential extension to standard short tandem repeat-based kits or a separate informative system for individual identification and kinship analysis. Eight INDELs have been selected as possible ancestry informative single-nucleotide polymorphisms for further analyses.