Prevention of human enterovirus 71 infection by kappa carrageenan

Enterovirus 71 (EV 71), the newest member of Enteroviridae, is notorious for its etiological role in epidemics of the hand-foot-and-mouth disease, particularly in association with fatal neurological complications in young children. Searching for new and more effective agents against EV 71 infections has never relented as corresponding vaccines or antiviral drugs remain unavailable. Sulfated polysaccharides from seaweed are known to possess a broad range of biological activities across anti-virus, anti-tumor, immunomodulation, anti-coagulation, etc. In this study, we report kappa carrageenan also has a strong and effective anti-EV 71 activity able to reduce plaque formation, prevent viral replication before or during viral adsorption, as well as inhibit EV 71-induced apoptosis. In virus binding assay, kappa carrageenan was shown able to bind EV 71 firmly, forming carrageenan-viruses complexes, whereby the virus-receptor interaction is likely disrupted. Added together, kappa carrageenan may be an ideal candidate worthwhile to develop into anti-EV 71 agents.     

抗肠道病毒71型天然药物化学成分研究进展

目的： 人类肠道病毒71（EV71）是引起手足口病（hand-foot and mouth disease，HFMD）的主要病原体之一，多发生于婴幼儿。本文总结了近年来抗EV71天然药物中的单体有效成分及其作用机制方面的研究，以期为抗EV71的新药发现和药物设计提供参考。方法： 在PubMed、Medline、The Cochrane、Library、Embase、Elsevier、John Wiley等外文数据库及中国知网（CNKI）、万方等中文数据库进行文献检索。结果： 综述的33个化合物通过各种作用机制发生抗EV71作用。结论： 天然药物中存在抗EV71有效单体成分，部分化合物结构、作用机制明确，可为新药发现和药物设计提供参考。     

杨梅素体外抗EV71病毒作用的研究

目的初步探究杨梅素抗肠道病毒71型(EV71)的活性及其机制。方法采用EV71感染非洲绿猴肾细胞(Vero细胞)模型,采用致细胞病变(CPE)法和空斑实验观察杨梅素的抗病毒效果。以不同浓度的杨梅素处理细胞,结合结晶紫染色的方法检测杨梅素的细胞毒性。以免疫印迹法检测杨梅素对VP1蛋白表达的影响。应用RT-PCR的方法评价杨梅素对VP1基因表达的影响。结果杨梅素在2.5~20μmol·L^-1的浓度下预处理病毒能够显著抑制EV71感染诱导的细胞死亡,且这种抑制作用具有剂量依赖性,IC50为5.6μmol·L^-1。与病毒组相比,杨梅素在2.5~20μmol·L^-1的浓度下还能够显著降低病毒滴度。免疫印迹和RT-PCR的结果显示,杨梅素还能够明显降低病毒衣壳蛋白VP1的基因和蛋白表达。结论杨梅素具有显著的体外抗EV71病毒活性。     

2010年中国人免疫球蛋白中EV71中和抗体效价的比较研究

目的:了解2010年我国生产的静注人免疫球蛋白(IVIG)中抗-EV71中和抗体效价的情况,为治疗EV71引起的手足口病(HFMD)提供特异性治疗药物。方法:采用经典微量细胞病变法,应用近2年分离自中国大陆的2株肠道病毒71型(EV71)病毒株,对我国不同血液制品厂家生产的97批IVIG进行抗-EV71中和效价检测,比较不同批次IVIG的EV71中和效价。结果:19家生产企业生产的97批IVIG的EV71中和效价几何平均滴度(GMTs)为1∶180(1∶170～1∶191)。其中有7家生产企业生产的12批制品的中和效价大于1∶256,以S公司生产的20100406批制品效价最高为1∶543,以A公司生产的含有1∶256以上效价的IVIG批数最多(5批),使该公司生产的IVIG的平均EV71中和抗体效价显著高于其他10家企业(P<0.05)。进一步分析显示,该2个公司分别是通过对献血员的EV71中和效价进行筛选,挑选出高效价血浆和选择人群抗体水平较高地区作为血浆来源地进行投料,而制备出了含有高效价EV71中和抗体的IVIG。结论:本研究显示,生产企业只要对原料血浆进行合理控制,制备出含有高效价EV71中和抗体的...     

Inhibition of enterovirus 71 replication by chrysosplenetin and penduletin

In recent years, enterovirus 71 (EV71) infections have caused an increasing epidemic in young children, accompanying with more severe nervous system disease and more deaths. Unfortunately, there is no specific medication for it so far. Here we investigated the anti-EV71 activity of chrysosplenetin and penduletin, two o-methylated flavonols isolated from the leaves of Laggera pterodonta. These two compounds were found to have strong activity in vitro against EV71 with low cytotoxicity. In the cytopathic effect (CPE) inhibition assays, both plaque reduction assay and virus yield inhibition assay, the compounds showed a similar 50% inhibitory concentration (IC(50)) value of about 0.20 μM. The selectivity indices (SI) of chrysosplenetin and penduletin were 107.5 and 655.6 in African green monkey kidney (Vero) cells, and 69.5 and 200.5 in human rhabdomyosarcoma (RD) cells, accordingly. The preliminary mechanism analysis indicates that they function not through blocking virus entry or inactivating virus directly but inhibiting viral RNA replication. In the time-of-addition assay, both compounds inhibited progeny virus production and RNA replication by nearly 100% when introduced within 4h post infection. In addition to EV71, both compounds inhibited several other human enteroviruses with similar efficacy. These findings provide a significant lead for the discovery of anti-EV71 drug.     

Immunogenicity of enterovirus 70 capsid protein VP1 and its non-overlapping N- and C-terminal fragments

Currently no practical treatment method or effective virus vaccine is available for acute hemorrhagic conjunctivitis (AHC) caused by enterovirus 70 (EV70). Antibodies to UV-inactivated EV70 (J670/71 epidemic isolate) and to the inclusion bodies of recombinant proteins of full-length EV70 VP1 (GST-VP1m), its non-overlapping terminal fragments N138 (1-138 aa) and C170 (141-310 aa) (or GST-N138m and GST-C170) were developed in rabbits. The anti-EV70 neutralizing activities of the rabbit sera were determined by standard neutralization assays. The antibodies to UV-inactivated EV70, were immuno-reactive with EV70 capsid proteins VP1 and VP3 of four EV70 epidemic isolates (KW/97, T260/74, J670/71 and AE/72) in Western-blot analysis, and immunoprecipitated the capsid proteins VP1 and VP3 from the cell lysates of virus-infected human Chang's conjunctival (HCC) cells. The antibodies to GST-VP1m, GST-N138m and GST-C170, immunoprecipitated only the VP1 proteins of the four EV70 isolates. Anti-EV70 J670/71 antibodies and the antibodies to the three recombinant VP1 proteins were all capable of immunoprecipitating EV70 whole-virus of the four EV70 epidemic isolates grown in HCC cells. The anti-EV70 virion antibodies neutralized EV70 isolates with titers of 6000-10,000 units/ml while the antibodies to GST-VP1m, GST-N138m or GST-C170 neutralized EV70 isolates with titers of 20-320units/ml. The results suggest that (a) immunization with bacterially produced recombinant EV70 VP1 and its non-overlapping N- and C-terminal fragments, was capable of eliciting EV70-neutralizing antibodies; (b) the neutralization titers of antibodies to the recombinant VP1 proteins were lower than that of antibodies to the UV-inactivated EV70 virions; and (c) the non-overlapping N138 and C170 fragments of EV70 VP1 both harbor independent anti-EV70 neutralization antigenic sites.     

Developments towards antiviral therapies against enterovirus 71

Enterovirus 71 (EV71) has emerged as a clinically important neurotropic virus that can cause acute flaccid paralysis and encephalitis, leading to cardiopulmonary failure and death. Recurring outbreaks of EV71 have been reported in several countries. The current lack of approved anti-EV71 therapy has prompted intense research into antiviral development. Several strategies--ranging from target-based chemical design to compound library screenings--have been employed, while others revisited compound series generated from antiviral developments against poliovirus and human rhinoviruses. These efforts have given rise to a diversity of antiviral candidates that include small molecules and non-conventional nucleic-acid-based strategies. This review aims to highlight candidates with potential for further clinical development based on their putative modes of action.     

Design, synthesis, and antipicornavirus activity of 1-[5-(4-arylphenoxy)alkyl]-3-pyridin-4-ylimidazolidin-2-one derivatives

A series of pyridylimidazolidinone derivatives was synthesized and tested in vitro against enterovirus 71 (EV71). On the basis of compound 33 (DBPR103), introduction of a methyl group at the 2- or 3-position of the linker between the imidazolidinone and the biphenyl resulted in markedly improved antiviral activity toward EV71 with IC(50) values of 5.0 nM (24b) and 9.3 nM (14a), respectively. Increasing the branched chain to propyl resulted in a progressive decrease in activity, while inserting different heteroatoms entirely rendered the compound only weakly active. The introduction of a bulky group (cyclohexyl, phenyl, or benzyl) led to loss of activity against EV71. The 4-chlorophenyl moiety in 14a was replaced with bioisosteric groups such as oxadiazole (28a-d) or tetrazole (32a,b), dramatically improving anti-EV71 activity and selectivity indices. Compounds 14a, 24b, 28b, 28d, and 32a exhibited a strong activity against lethal EV71, and no apparent cellular toxicity was observed. Three of the more potent imidazolidinone compounds, 14a, 28b, and 32b, were subjected to a large group of picornaviruses to determine their spectrum of antiviral activity.     

Curcumin inhibits the replication of enterovirus 71 in vitro

Human enterovirus 71 (EV71) is the main causative pathogen of hand, foot, and mouth disease (HFMD) in children. The epidemic of HFMD has been a public health problem in Asia-Pacific region for decades, and no vaccine and effective antiviral medicine are available. Curcumin has been used as a traditional medicine for centuries to treat a diversity of disorders including viral infections. In this study, we demonstrated that curcumin showed potent antiviral effect again EV71. In Vero cells infected with EV71, the addition of curcumin significantly suppressed the synthesis of viral RNA, the expression of viral protein, and the overall production of viral progeny. Similar with the previous reports, curcumin reduced the production of ROS induced by viral infection. However, the antioxidant property of curcumin did not contribute to its antiviral activity, since N-acetyl-l-cysteine, the potent antioxidant failed to suppress viral replication. This study also showed that extracellular signal-regulated kinase (ERK) was activated by either viral infection or curcumin treatment, but the activated ERK did not interfere with the antiviral effect of curcumin, indicating ERK is not involved in the antiviral mechanism of curcumin. Unlike the previous reports that curcumin inhibited protein degradation through ubiquitin–proteasome system (UPS), we found that curcumin had no impact on UPS in control cells. However, curcumin did reduce the activity of proteasomes which was increased by viral infection. In addition, the accumulation of the short-lived proteins, p53 and p21, was increased by the treatment of curcumin in EV71-infected cells. We further probed the antiviral mechanism of curcumin by examining the expression of GBF1 and PI4KB, both of which are required for the formation of viral replication complex. We found that curcumin significantly reduced the level of both proteins. Moreover, the decreased expression of either GBF1 or PI4KB by the application of siRNAs was sufficient to suppress viral replication. We also demonstrated that curcumin showed anti-apoptotic activity at the early stage of viral infection. The results of this study provide solid evidence that curcumin has potent anti-EV71 activity. Whether or not the down-regulated GBF1 and PI4KB by curcumin contribute to its antiviral effect needs further studies.KEY WORDS: Curcumin, Enterovirus 71, Viral replication, GBF1, PI4KB, Ubiquitin–proteasome system, ApoptosisAbbreviations: CVB, coxsackieviurs B; DCFH-DA, dichloro-dihydro-fluorescein diacetate; ERK, extracellular signal-regulated kinase; EV71, enterovirus 71; GBF1, Golgi brefeldin A resistant guanine nucleotide exchange factor 1; GEF, guanine nucleotide exchange factor; HBV, hepatitis B virus; HCV, hepatitis C virus; HFMD, hand, foot, and mouth disease; HIV, human immunodeficiency virus; HPV, human papillomavirus; NAC, N-acetyl-l-cysteine; PARP-1, poly(ADP-ribose) polymerase; PGC-1α, peroxisome proliferator-activated receptor-gamma co-activator 1 alpha; p.i., post-infection; PI4KB, phosphatidylinositol 4-kinase class III catalytic subunit β; PI4P, phosphatidylinositol 4-phosphate; ROS, reactive oxygen species; siRNA, small interfering RNA; SLLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; UPS, ubiquitin–proteasome system     

Lactoferrin inhibits enterovirus 71 infection by binding to VP1 protein and host cells

The antiviral activities of bovine lactoferrin (LF) against enterovirus 71 (EV71) were studied both in vitro and in vivo. LF protected both human rhabdomyosarcoma and neuroblastoma SK-N-SH cell lines from EV71 infection when it was added at the same time, before, or within 30min after EV71 infection. Using enzyme-linked immunosorbent assay-based binding assay and indirect fluorescent stain, we found that LF could bind to the target cells. Furthermore, it was found that LF could bind to the VP1 protein of EV71, which was blocked in the presence of anti-VP1 antibody. In addition, LF could induce IFN-alpha expression of SK-N-SH cells and inhibit EV71-induced IL-6 production. Finally, LF protected mice against lethal EV71 challenge. Taken together, these results suggest that LF can inhibit EV71 infection by interacting with both EV71 and host cells.     

肠道病毒71型灭活疫苗（Vero细胞）接种5年免疫持久性观察

目的 观察肠道病毒71型（enterovirus 71,EV71）疫苗III期临床试验接种5年的免疫持久性。方法 在III期临床试验免疫原性亚组中,对完成全程两针次免疫的免前抗EV71中和抗体阴性人群进行免疫后5年采血并检测抗EV71中和抗体,对0 d、56 d、8个月、14个月、26个月、64个月同时具有抗EV71中和抗体检测数据人群评价免疫持久性。结果 共614名受试者完成5年免疫持久性血样采集并获得中和抗体检测结果,其中490名受试者同时具有0天、56 d、8个月、14个月、26个月、64个月抗EV71中和抗体检测数据,疫苗组和安慰剂对照组分别为235名和255名。EV71疫苗免疫后5年,疫苗组抗EV71中和抗体阳性率（100.00%）和抗体几何平均滴度（geometric mean titer,GMT）（369.57）均高于对照组（69.02%和55.58）。分别以抗体滴度8、16、32为阳性判定标准,疫苗组阳性率（100%、99.57%、97.87%）均高于对照组（69.02%、61.96%、59.61%）。对不同年龄亚组进行分析,6~11、12~35月龄受试者免疫后疫苗组5年抗EV71中和抗体阳性（滴度≥8）率（均为100.00%）和抗体GMT（367.14、370.64）均高于对照组（66.67%、71.27%和53.43、63.66）。在EV71疫苗免疫后的各个时间点,疫苗组抗EV71中和抗体阳性率和GMT均高于对照组。结论 该EV71疫苗两针基础免疫后能够诱导良好的免疫原性,在免疫后5年依然保持较好的免疫持久性。     

Synthesis and antiviral activity of a series of novel N-phenylbenzamide and N-phenylacetophenone compounds as anti-HCV and anti-EV71 agents

A series of novel N-phenylbenzamide and N-phenylacetophenone compounds were synthesized and evaluated for their antiviral activity against HCV and EV71 (strain SZ-98). The biological results showed that three compounds (23, 25 and 41) exhibited considerable anti-HCV activity (IC₅₀=0.57–7.12 μmol/L) and several compounds (23, 28, 29, 30, 31 and 42) displayed potent activity against EV71 with the IC₅₀ values lower than 5.00 μmol/L. The potency of compound 23 (IC₅₀=0.57 μmol/L) was superior to that of reported compounds IMB-1f (IC₅₀=1.90 μmol/L) and IMB-1g (IC₅₀=1.00 μmol/L) as anti-HCV agents, and compound 29 possessed the highest anti-EV71 activity, comparable to the comparator drug pirodavir. The efficacy in vivo and antiviral mechanism of these compounds warrant further investigations.KEY WORDS: Synthesis, N-phenylbenzamide, N-phenylacetophenone, Anti-HCV, Anti-EV71, Agents     

Antiviral Activity of Hederasaponin B from Hedera helix against Enterovirus 71 Subgenotypes C3 and C4a

Enterovirus 71 (EV71) is the predominant cause of hand, foot and mouth disease (HFMD). The antiviral activity of hederasaponin B from Hedera helix against EV71 subgenotypes C3 and C4a was evaluated in vero cells. In the current study, the antiviral activity of hederasaponin B against EV71 C3 and C4a was determined by cytopathic effect (CPE) reduction method and western blot assay. Our results demonstrated that hederasaponin B and 30% ethanol extract of Hedera helix containing hederasaponin B showed significant antiviral activity against EV71 subgenotypes C3 and C4a by reducing the formation of a visible CPE. Hederasaponin B also inhibited the viral VP2 protein expression, suggesting the inhibition of viral capsid protein synthesis.These results suggest that hederasaponin B and Hedera helix extract containing hederasaponin B can be novel drug candidates with broad-spectrum antiviral activity against various subgenotypes of EV71.     

5种不同细胞对柯萨奇病毒A组16型和肠道病毒71型的敏感性研究

 目的   评价肠道病毒研究中常用的5种细胞对柯萨奇病毒A组16型（coxsackievirus A16,CA16）和肠道病毒71型（enterovirus 71,EV71）的敏感性,为分离和培养CA16和EV71提供实验室依据。方法  用分离的各15株CA16和EV71分别感染RD、Vero、KMB17、Hep2和L20B细胞,每天观察细胞病变情况,按Karber法计算病毒感染性滴度。采用不同的统计学方法（包括方差分析和卡方检验）比较5种细胞对CA16和EV71的敏感性差异。 结果   5种细胞的CA16和EV71病毒感染性滴度间的差异具有统计学意义（CA16：F＝18.481,P＝0.000;EV71：χ²＝63.106,P＝0.000）。在5种细胞中,RD细胞对2种病毒的敏感性最高,CA16和EV71的病毒感染性滴度分别可达7.8和8.2 lgCCID₅₀/ml,且均于接种细胞后48 h达增殖高峰,第4天进入增殖平台期;2种病毒在Hep2和L20B细胞中的病毒感染性滴度均较低,CA16分别为5.2和4.2 lgCCID₅₀/ml,EV71分别为4.9和4.0 lgCCID₅₀/ml,且2种病毒在接种后第6~7天才达增殖高峰,但仅L20B细胞中的2种病毒于接种后第7天进入增殖平台期;CA16在Vero和KMB17细胞中的病毒感染性滴度分别为6.9和7.3 lgCCID₅₀/ml,EV71分别为7.1和6.8 lgCCID50/ml,2种病毒均在接种Vero和KMB17细胞后第3天达到增殖高峰,第5天进入增殖平台期。 结论   RD细胞对CA16和EV71的敏感性最好,其次是Vero和KMB17细胞,而Hep2和L20B细胞并非是分离和培养这2种病毒的理想细胞。     

RNA干扰技术抗肠道病毒71型复制

目的肠道病毒71型(Enterovirus 71,EV 71)是手足口病(hand,foot and mouth disease,HFMD)主要的病原体之一。研究RNA干扰技术抑制肠道病毒71型复制。方法以RNA干扰技术(RNAi)为干预手段,抑制病毒EV 71在人横纹肌肉瘤(rhabdomyosarcoma,RD)细胞中的复制。通过Western blot、Real-time PCR和病毒滴度3种方法验证,其抑制效果体现在感染细胞内部病毒RNA,病毒蛋白质的表达水平和培养基上清中子代病毒颗粒的数量上。结果靶向病毒基因组5'UTR和VP2的siRNAs具有明显的病毒抑制效应。结论此研究证实RNAi方法具有特异性抑制病毒复制的潜能和可行性。     

鱼腥草不同溶剂提取物的抗病毒活性研究

目的 研究鱼腥草不同溶剂提取物体外抗手足口病毒（enterovirus type 71,EV71）、单纯疱疹病毒1型（herpes simplex virus type 1,HSV-1）、呼吸道合胞病毒（respiratory syncytial virus,RSV）、柯萨奇病毒B3型（coxsackie virus type B3,CV-B3）、柯萨奇病毒B5型（coxsackie virus type B5,CV-B5）的活性。方法 采用细胞体外培养技术,建立不同病毒感染模型,用鱼腥草不同溶剂提取物进行治疗,通过细胞融合病变观察法以及MTT比色法检测得到治疗指数（therapeutic index,TI）,以此判定其体外抗病毒活性。结果 体外抗病毒试验结果证明,鱼腥草不同溶剂提取物对HSV-1、EV71抗病毒作用较强,对RSV、CV-B3、CV-B5的效果甚微或无明显作用。鱼腥草醇提物中黄酮类成分含量较高,抗病毒活性相对较高;30%,50%,75%乙醇提取物以及水提醇沉处理沉淀物对HSV-1的TI值分别是81.68,67.23,41.91,32.61,均高于阳性对照阿昔洛韦TI值23.43,显示鱼腥草水醇提取物抗HSV-1的活性较强;75%,50%,30%乙醇提取物对EV71的TI值分别是19.58,20.13,18.84,效果较为显著。结论 研究证实鱼腥草提取物对HSV-1、EV71的抗病毒活性较高,可一定程度为临床应用鱼腥草治疗这2种病毒感染引发的疾病提供参考依据。     

肠道病毒71型结构蛋白和结构蛋白和非结构蛋白的分子生物学特性

  肠道病毒71型（enterovirus 71,EV71）和柯萨奇病毒A组16型感染导致的手足口病已在世界范围内引起数次大规模暴发和流行,成为婴幼儿健康的重大威胁。对EV71结构的分子生物学特征的研究有助于为EV71疫苗的研发提供重要依据,此文就EV71的几种重要结构蛋白和非结构蛋白在病毒感染过程中作用的研究现状做一综述。     

Ozone exposure in the culture medium inhibits enterovirus 71 virus replication and modulates cytokine production in rhabdomyosarcoma cells

In the present study, the effects of ozone exposure on enterovirus 71 (EV71) replication and related cytokine production were investigated. Rhabdomyosarcoma cells (RD) were exposed to 0.5, 1, 1.5 and 2 ppm ozone or filtered air under different exposure regimens before or after infection for 1 or 2 h. The results revealed that at a proper concentration of ozone, e.g., 1.5 or 2 ppm, ozone exposure restricted virus production, prolonged survival time of cells and modulated cytokine production related to EV71 infection. Upon exposure of non-infected cells to ozone at 1.5 or 2 ppm for 1 h, the production of IL-1β, IL-6 and TNF-α was primed and boosted by the subsequent EV71 infection, generating an inhibitory effect on EV71 replication during the post-infection period of 48 h. While infected cells were exposed to ozone for 2 h at 1.5 or 2 ppm, ozone did not affect cytokine production by RD cells in response to EV71 infection. The data showed that ozone effect on induction of cytokine was only found in uninfected cells. The ozone-induced cytokines produced prior to the onset of EV71 infection generated antiviral effects, which proved beneficial in suppressing the subsequent EV71 infection.     

微量细胞培养法在手足口病毒研究中的应用

目的建立应用于手足口病毒分离和对手足口EV71病毒抑制药物筛选的微量细胞培养方法。方法比较微量细胞培养结合real-timePCR和直接应用real-timePCR两种方法检测手足口病毒的差别,并应用微量细胞培养采用细胞病变效应法和MTT分析法,观察利巴韦林对EV71病毒的抑制作用。结果经微量细胞培养后EV71、CA16阳性检出率为90%和80%,但PE阴性样本20%检测为EV71阳性;利巴韦林浓度为0.4、0.2、0.1mg/mL时对EV71病毒有抑制作用,抑制率分别为30.28%、28.09%和29.16%。结论微量细胞培养在手足口病毒分离检测和对其抑制作用药物筛选研究中具有可行性,且其具有操作简便、省时省力等优点。     

Optimized development of a candidate strain of inactivated EV71 vaccine and analysis of its immunogenicity in rhesus monkeys

Enterovirus type 71 (EV71) is one of the main etiologic agents responsible for periodic epidemics of hand-foot-and-mouth disease (HFMD). The prevention and control of EV71 epidemics with effective anti-viral agents and vaccines is very important for public health. Because the pathogenesis of EV71 in the human body is not completely clear and genetic variations in the virus during its replication are difficult to control, we have focused on the development of an inactivated whole-virus vaccine. In this study, we screened 16 strains isolated from different areas of China and selected one strain for the development of an inactivated EV71 vaccine. The results of our study suggest that the FY-23K-B strain, which is a candidate strain for an EV71 inactivated vaccine, satisfied the requirements of vaccine production in terms of genetic stability, biological activity, and good immunogenicity. The experimentally inactivated vaccine produced using this strain was capable of inducing an immune response and offered protection to rhesus monkeys against future virus attacks.