EV71病毒核酸快速检测方法的比较

PCR方法检测肠道病毒71型(EV71)的分子诊断技术十分快速敏感。目前已有多篇文献报道了不同的检测EV71的PCR方法,但对不同方法检测结果的比较还少有报道。本研究将检测肠道病毒通用5’UTR基因的3种定量PCR(rRT-PCR)方法和1种普通PCR(cRT-PCR)方法和特异性检测EV71的2种rRT-PCR和1种cRT-PCR方法进行特异性和敏感性比较评估。所有试验方法都有较好的特异性,无交叉反应。核酸最低检测限值为从8.19×101～8.19×105个拷贝,rRT-PCR比cRT-PCR更敏感。所有的rRT-PCR方法检测50份临床标本时都较敏感。     

我国分离的肠道病毒71型(SHZH03病毒株)全基因组核苷酸序列分析

对肠道病毒71型(enterovirus 71,EV71)中国(深圳)分离株SHZH03进行了全基因组(未包括多聚腺苷尾)7406个碱基的核苷酸序列测定.结果表明,SHZH03株与其它肠道病毒71型毒株相比,在编码区没有核苷酸的缺失和插入,其5′UTR和3′UTR区的长度和序列有一定的差异.核苷酸同源性比较结果表明,在P1区SHZH03株与SHZH98株、中国台湾流行株(TW2086、TW2272)的同源性较高(分别为92.5%,90.1%和87.9%),与新加坡流行株SIN5666、SIN5865及标准株MS、BrCr的同源性则在81%左右,而与Coxsackievirus A16(Cox.A16)的同源性最低(63.6%).氨基酸同源性比较结果表明,在P1区SHZH03株与Cox. A16的同源性最低,但在P2和P3区SHZH03株与Cox.A16的同源性最高.P1区的遗传进化分析表明,SHZH03株和中国台湾1998年流行的EV71毒株的亲缘关系较近,属于同一型(genogroup),而与标准株BrCr和MS的亲缘关系较远.上述结果有助于肠道病毒71型的基础研究和中国对于EV71所致疾病的预防.     

Identification of Site-Specific Adaptations Conferring Increased Neural Cell Tropism during Human Enterovirus 71 Infection

Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP1₉₇ Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient.     

Glucose Homeostasis Is Important for Immune Cell Viability during Candida Challenge and Host Survival of Systemic Fungal Infection

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A non-mouse-adapted enterovirus 71 (EV71) strain exhibits neurotropism, causing neurological manifestations in a novel mouse model of EV71 infection

Enterovirus 71 (EV71) is a neurotropic pathogen that has been consistently associated with the severe neurological forms of hand, foot, and mouth disease. The lack of a relevant animal model has hampered our understanding of EV71 pathogenesis, in particular the route and mode of viral dissemination. It has also hindered the development of effective prophylactic and therapeutic approaches, making EV71 one of the most pressing public health concerns in Southeast Asia. Here we report a novel mouse model of EV71 infection. We demonstrate that 2-week-old and younger immunodeficient AG129 mice, which lack type I and II interferon receptors, are susceptible to infection with a non-mouse-adapted EV71 strain via both the intraperitoneal (i.p.) and oral routes of inoculation. The infected mice displayed progressive limb paralysis prior to death. The dissemination of the virus was dependent on the route of inoculation but eventually resulted in virus accumulation in the central nervous systems of both animal groups, indicating a clear neurotropism of the virus. Histopathological examination revealed massive damage in the limb muscles, brainstem, and anterior horn areas. However, the minute amount of infectious viral particles in the limbs from orally infected animals argues against a direct viral cytopathic effect in this tissue and suggests that limb paralysis is a consequence of EV71 neuroinvasion. Together, our observations support that young AG129 mice display polio-like neuropathogenesis upon infection with a non-mouse-adapted EV71 strain, making this mouse model relevant for EV71 pathogenesis studies and an attractive platform for EV71 vaccine and drug testing.     

Construction and characterization of infectious cDNA clones of enterovirus 71(EV71)

<正>Dear Editor,Enterovirus 71(EV71),a member of the genus Enterovirus of the family Picornaviridae,is a single-stranded,positive-sense RNA virus that usually causes mild handfoot-mouth disease(HFMD)in children,with symptoms such as fever,diarrhea,and herpangina(Liu et al.,2013).However,certain strains of EV71 infection can cause severe neurological complications,such as SDLY107(Sun et al.,2014).EV71 is classified into three distinct genotypes(A–C);the B and C genotypes are further divided into B1–B5 and C1–C5 genotypes,respectively,based on     

Reverse Genetic Analysis of Adaptive Mutations within the Capsid Proteins of Enterovirus 71 (EV-A71) Strains Necessary for Infection of CHO-K1 Cells

正Dear Editor,Previous studies had described the adaptation of enterovirus 71 (EV-A71) strains that enabled entry and viral replication in Chinese Hamster Ovary (CHO) cell line(Zaini and Mc Minn 2012; Zaini et al. 2012). These adapted     

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.     

Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen multiplication and promote survival, facilitating pathogen transmission.     

UNC93B1 and Nucleic Acid-sensing Toll-like Receptors Mediate Host Resistance to Infection with Leishmania major

The mammalian homolog B1 of Unc-93 Caenorhabditis elegans known as UNC93B1 is a chaperone protein that mediates translocation of the nucleic acid-sensing Toll-like receptors (TLRs) from the endoplasmic reticulum to the endolysosomes. The triple deficient (UNC93B1 mutant) mice have a functional single point mutation in the UNC93B1 that results in non-functional TLR3, TLR7, and TLR9. Herein, we demonstrate that UNC93B1 mutant mice, in the C57BL/6 (resistant) genetic background, are highly susceptible to Leishmania major infection. Enhanced swelling of the footpad was associated with high levels of interleukin 10, decreased levels of interferon γ, and increased parasitism. None of the single TLR3, TLR7, and TLR9 knock-out (KO) mice resemble the UNC93B1 mutant phenotype upon infection with L. major. Whereas the double TLR7/TLR9 KO showed a partial phenotype, the triple TLR3/TLR7/TLR9 KO mice were as susceptible as the UNC93B1 mutant mice, when infected with Leishmania parasites. Finally, we demonstrate that treatment with either anti-interleukin 10 receptor monoclonal antibody or recombinant interleukin 12 restored a robust anti-parasite T_H1 response and reverted the susceptible phenotype of UNC93B1 mutant mice. Altogether, our results indicate the redundant and essential role of nucleic acid-sensing TLR3, TLR7 and TLR9 in inducing interleukin 12, development of a T_H1 response, and resistance to L. major infection in resistant C57BL/6 mice.     

Multiple Host Kinases Contribute to Akt Activation during Salmonella Infection

SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P₂/PI(3–5) P₃ by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P₂/PI(3–5) P₃ appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.     

Multivariate Statistical Analyses Demonstrate Unique Host Immune Responses to Single and Dual Lentiviral Infection

Background

Feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) are recently identified lentiviruses that cause progressive immune decline and ultimately death in infected cats and humans. It is of great interest to understand how to prevent immune system collapse caused by these lentiviruses. We recently described that disease caused by a virulent FIV strain in cats can be attenuated if animals are first infected with a feline immunodeficiency virus derived from a wild cougar. The detailed temporal tracking of cat immunological parameters in response to two viral infections resulted in high-dimensional datasets containing variables that exhibit strong co-variation. Initial analyses of these complex data using univariate statistical techniques did not account for interactions among immunological response variables and therefore potentially obscured significant effects between infection state and immunological parameters.

Methodology and Principal Findings

Here, we apply a suite of multivariate statistical tools, including Principal Component Analysis, MANOVA and Linear Discriminant Analysis, to temporal immunological data resulting from FIV superinfection in domestic cats. We investigated the co-variation among immunological responses, the differences in immune parameters among four groups of five cats each (uninfected, single and dual infected animals), and the “immune profiles” that discriminate among them over the first four weeks following superinfection. Dual infected cats mount an immune response by 24 days post superinfection that is characterized by elevated levels of CD8 and CD25 cells and increased expression of IL4 and IFNγ, and FAS. This profile discriminates dual infected cats from cats infected with FIV alone, which show high IL-10 and lower numbers of CD8 and CD25 cells.

Conclusions

Multivariate statistical analyses demonstrate both the dynamic nature of the immune response to FIV single and dual infection and the development of a unique immunological profile in dual infected cats, which are protected from immune decline.