肠道病毒71型感染动物模型的应用与局限

肠道病毒71型(enterovirus 71,EV71)是一种被忽视的热带传染病——手足口病的主要病原体之一,过去15年在亚太地区引起了多次手足口病暴发。由于脊髓灰质炎病毒的有效控制,EV71已成为最重要的嗜神经肠道病毒,其严重的神经系统并发症威胁着儿童健康。合适的动物模型可帮助更好地了解EV71神经致病机制,并有利于开发有效的疫苗和治疗药物。本文就EV71已建立的3类主要动物模型(非人灵长类动物模型、小鼠适应性模型及转基因小鼠模型)的特征、应用与局限进行综述。     

Discovery of 3-benzyl-1,3-benzoxazine-2,4-dione analogues as allosteric mitogen-activated kinase kinase (MEK) inhibitors and anti-enterovirus 71 (EV71) agents

Enterovirus 71 (EV71) is a kind of RNA virus and one of the two causes of Hand, foot and mouth disease (HFMD). Inhibitors that target key components of Ras/Raf/MEK/ERK pathway in host cells could impair replication of EV71. A series of 3-benzyl-1,3-benzoxazine-2,4-diones were designed from a specific MEK inhibitor G8935, by replacing the double bond between C3 and C4 within the coumarin scaffold with amide bond. One compound (9f) showed submicromolar inhibitory activity among the 12 derivatives. Further optimization on 9f led to two active compounds (9k and 9m) with nanomolar bioactivities (55 nM and 60 nM). The results of enzymatic assays also demonstrated that this series of compounds were allosteric inhibitors of unphosphorylated MEK1. The binding mode of compound 9k was predicted by molecular dynamic simulation and the key interactions were same as published MEK1/2 allosteric inhibitors. In the cell-based assays, compounds 9k and 9m could effectively suppress the ERK1/2 pathway, expression of EV71 VP1, and EV71 induced cytopathic effect (CPE) in rhabdomyosarcoma (RD) cells.     

新型病毒衣壳结合剂 哌嗪新衍生物抑制肠道病毒71型的复制

目的研究哌嗪新衍生物体外对肠道病毒71型(EV71)的抑制作用。方法培养EV71感染的人横纹肌肉瘤RD细胞,建立体外病毒感染模型,将哌嗪新衍生物作用于RD细胞,通过Western blot、Real-time PCR和病毒滴度检测细胞内EV71病毒蛋白和mRNA的表达水平及培养基上清中子代病毒颗粒的数量;并且观察细胞病变效应(CPE),采用CCK-8法检测哌嗪新衍生物对细胞活性的影响。结果哌嗪新衍生物VP1-4浓度为5μg/mL能够显著抑制RD细胞中EV71 VP1蛋白的表达,EV71 mRNA水平降低了(93.8±3.1)%,IC_(50)约为0.016μg/mL,培养基上清病毒滴度降低了(51.1±0.8)%;而且VP1-4能够减缓EV71病毒感染引起的CPE。此外,化合物VP1-4 CC_(50)200μg/mL,说明细胞毒性低,安全性较高。结论本研究证实VP1-4能有效抑制EV71的复制,可以作为先导化合物开展进一步研究。     

Nasal Staphylococcus aureus and S. pseudintermedius carriage in healthy dogs and cats: a systematic review of their antibiotic resistance,virulence and genetic lineages of zoonotic relevance

The molecular ecology of Staphylococcus aureus, Staphylococcus pseudintermedius and their methicillin-resistant strains in healthy dogs and cats could serve as good models to understand the concept of bacterial zoonosis due to animal companionship. This study aims to provide insights into pooled prevalence, genetic lineages, virulence and antimicrobial resistance (AMR) among healthy dogs and cats. Original research and brief communication articles published from 2001 to 2021 that reported the nasal detection of S. aureus and S. pseudintermedius in healthy dogs and cats in the community, homes and outside veterinary clinics were examined and analysed. Forty-nine studies were eligible and included in this systematic review. The pooled prevalence of nasal carriage of S. aureus/methicillin-resistant S. aureus (MRSA) in healthy dogs and cats were 10.9% (95% CI: 10.1–11.9)/2.8% (95% CI: 2.4–3.2) and 3.2% (95% CI: 1.9–4.8)/0.5% (95% CI: 0.0–1.1), respectively. Conversely, the pooled prevalence of S. pseudintermedius/methicillin-resistant S. pseudintermedius (MRSP) in healthy dogs and cats were 18.3% (95% CI: 17.1–19.7)/3.1% (95% CI: 2.5–3.7) and 1.3% (95% CI: 0.6–2.4)/1.2% (95% CI: 0.6–2.3), respectively. Although highly diverse genetic lineages of S. aureus were detected in healthy dogs and cats, MSSA-CC1/CC5/CC22/CC45/CC121/CC398 and MRSA-CC5/CC93/CC22/CC30 were mostly reported in dogs; and MSSA-CC5/CC8/CC15/CC48 and MRSA-CC22/CC30/CC80 in cats. Of note, MSSA-CC398 isolates (spa-types t034 and t5883) were detected in dogs. Genetic lineages often associated with MSSP/MRSP were ST20/ST71, highlighting the frequent detection of the epidemic European MRSP-ST71 clone in dogs. S. aureus isolates carrying the luk-S/F-PV, tst, eta, etb and etd genes were seldomly detected in dogs, and luk-S/F-PV was the unique virulence factor reported in isolates of cats. S. pseudintermedius isolates harbouring the luk-S/F-I, seint and expA genes were frequently found, especially in dogs. High and diverse rates of AMR were noted, especially among MRSA/MRSP isolates. There is a need for additional studies on the molecular characterization of isolates from countries with under-studied nasal staphylococci isolates.     

肠道病毒71型致神经元病变的机制研究进展

中枢神经系统感染是由病原体侵犯中枢神经系统引起的一类具有较高的发病率和死亡率的疾病。病毒是引起中枢神经系统感染的重要病原体之一,其中肠道病毒71型在继发神经系统症状的重症手足口病患儿中较为常见。EV71致神经元病变是其感染中枢神经系统的基础,阐明肠道病毒71型致神经元病变的机制,不仅可以促进基础病毒学研究,也能为抗病毒药物的开发提供思路,对临床肠道病毒71型致中枢神经系统感染的治疗提供支持。本文主要从肠道病毒71型侵入神经元的受体途径、损伤神经元的线粒体途径、诱导凋亡与自噬、感染胶质细胞后对神经元的旁观者效应、免疫病理机制以及病毒自身因素等多个方面,对肠道病毒71型致神经元病变机制展开综述。     

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).     

A new active vitamin D analog,ED-71, causes increase in bone mass with preferential effects on bone in osteoporotic patients

As a candidate for active vitamin D analogs that have selective effects on bone, 1alpha,25-dihydroxy-2beta-(3-hydroxypropoxy)vitamin D3 (ED-71) has been synthesized and is currently under clinical trials. In ovariectomized rat model for osteoporosis, ED-71 caused an increase bone mass at the lumbar vertebra to a greater extent than 1alpha-hydroxyvitamin D3 (alfacalcidol), while enhancing calcium absorption and decreasing serum parathyroid hormone levels to the same degree as alfacalcidol. ED-71 lowered the biochemical and histological parameters of bone resorption more potently than alfacalcidol, while maintaining bone formation markers.An early phase II clinical trial was conducted with 109 primary osteoporotic patients. The results indicate that oral daily administration of ED-71 (0.25, 0.5, 0.75, and 1.0 microgram) for 6 months increased lumbar bone mineral density in a dose-dependent manner without causing hypercalcemia and hypercalciuria. ED-71 also exhibited a dose-dependent suppression of urinary deoxypyridinoline with no significant reduction in serum osteocalcin. These results demonstrate that ED-71 has preferential effects on bone with diminished effects on intestinal calcium absorption. ED-71 offers potentially a new modality of therapy for osteoporosis with selective effects on bone.     

Synthesis of (20S)-[7,7,21,21-2H4]-3beta-(tert-butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol,a useful intermediate for the preparation of deuterated isotopomers of sterols

(20S)-[7,7,21,21-2H(4)]-3beta-(tert-Butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol, an intermediate for the preparation of deuterated isotopomers of sterols to be used as standards for biomedical studies, was prepared by reduction with dichloroaluminum deuteride of ethyl (20S)-3beta-(tert-butyldimethylsilanyloxy)-7-oxo-pregn-5-en-20-carboxylate. Using controlled experimental conditions, it has also been shown that the dichloroaluminum hydride reduction of a 7-keto steroid affords the corresponding 7beta-hydroxy derivative in a highly stereoselective manner.     

Antiviral effects of two Ganoderma lucidum triterpenoids against enterovirus 71 infection

Enterovirus 71 (EV71) is a major causative agent for hand, foot and mouth disease (HFMD), and fatal neurological and systemic complications in children. However, there is currently no clinical approved antiviral drug available for the prevention and treatment of the viral infection. Here, we evaluated the antiviral activities of two Ganoderma lucidum triterpenoids (GLTs), Lanosta-7,9(11),24-trien-3-one,15;26-dihydroxy (GLTA) and Ganoderic acid Y (GLTB), against EV71 infection. The results showed that the two natural compounds display significant anti-EV71 activities without cytotoxicity in human rhabdomyosarcoma (RD) cells as evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay. The mechanisms by which the two compounds affect EV71 infection were further elucidated by three action modes using Ribavirin, a common antiviral drug, as a positive control. The results suggested that GLTA and GLTB prevent EV71 infection through interacting with the viral particle to block the adsorption of virus to the cells. In addition, the interactions between EV71 virion and the compounds were predicated by computer molecular docking, which illustrated that GLTA and GLTB may bind to the viral capsid protein at a hydrophobic pocket (F site), and thus may block uncoating of EV71. Moreover, we demonstrated that GLTA and GLTB significantly inhibit the replication of the viral RNA (vRNA) of EV71 replication through blocking EV71 uncoating. Thus, GLTA and GLTB may represent two potential therapeutic agents to control and treat EV71 infection.     

肠道病毒71型分子生物学研究进展

肠道病毒7l 型( EV71) 是导致手足口病的主要病原体之一, 常引起多种与神经系统相关的严重疾病。自1969 年首次分离以来, 其感染已在世界范围内引起数次暴发与流行, 尤其是近几年在亚太地区呈上升趋势, 且出现越来越严重的神经系统症状。目前, 对于EV71 病毒结构及其所致严重神经系统症状的机制, 以及疫苗的开发等已进行了大量和深入的研究。