Genetic analysis of the VP1 region of human enterovirus 71 strains isolated in Fuyang,China, during 2008

Enterovirus 71 (EV71) is a common cause of Hand, foot, and mouth disease (HFMD) and may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity of EV71, we determined and analyzed the complete VP1 sequences (891 nucleotides) from nine EV71 strains isolated in Fuyang, China. We found that nine EV71 strains isolated were over 98% homologous at the nucleotide level and 93%-100% homologous to members of the C4 subgenogroup. At the amino acid level, these Fuyang strains were 99% -100% homologous to one another, 97%-100% homologous to members of the C4 subgenogroup, and the histidine(H) at amino acid position 22 was conserved among the Fuyang strains. The results indicate that Fuyang isolates belong to genotype C4, and an H at position 22 appears to be a marker for the Fuyang strains.     

Positive selection analysis of VP1 Genes of worldwide human enterovirus 71 viruses

Human enterovirus 71 viruses have been long circulating throughout the world. In this study, we performed a positive selection analysis of the VP1 genes of capsid proteins from Enterovirus 71 viruses. Our results showed that although most sites were under negative or neutral evolution, four positions of the VP1 genes were under positive selection pressure. This might account for the spread and frequent outbreaks of the viruses and the enhanced neurovirulence. In particular, position 98 might be involved in neutralizing antibodies, modulating the virus-receptor interaction and enhancing the virulence of the viruses. Moreover, both positions 145 and 241 might correlate to determine the receptor specificity. However, these positions did not display much difference in amino acid polymorphism. In addition, no position in the VP1 genes of viruses isolated from China was under positive selection.     

Immunity to avirulent enterovirus 71 and coxsackie A16 virus protects against enterovirus 71 infection in mice

In this study, we sought to determine whether intratypic and intertypic cross-reactivity protected against enterovirus 71 (EV71) infection in a murine infection model. We demonstrate that active immunization of 1-day-old mice with avirulent EV71 strain or coxsackie A16 virus (CA16) by the oral route developed anti-EV71 antibodies with neutralizing activity (1:16 and 1:2, respectively). Splenocytes from both EV71- and CA16-immunized mice proliferated upon EV71 or CA16, but not coxsackie B3 virus (CB3), antigen stimulation. Immunized mice became more resistant to virulent EV71 strain challenge than nonimmunized mice. There was an increase in the percentage of activated splenic T cells and B cells in the immunized mice 2 days after EV71 challenge. The CA16 immune serum reacted with EV71 antigens in an enzyme-linked immunosorbent assay and neutralized EV71 but not CB3 or poliovirus at a titer of 1:4. Passive immunization with the CA16 immune serum reduced the clinical score, diminished the organ viral load, and increased the survival rate of mice upon EV71 challenge. CB3 neither shared in vitro cross-reactivity with EV71 nor provided in vivo protection after both active and passive immunization. These results illustrated that live vaccine is feasible for EV71 and that intertypic cross-reactivity of enteroviruses may provide a way to determine the prevalence of EV71.     

Protection of neonatal mice from lethal enterovirus 71 infection by maternal immunization with attenuated Salmonella enterica serovar Typhimurium expressing VP1 of enterovirus 71

This study describes the potential use of attenuated Salmonella enterica serovar Typhimurium strains to express and deliver VP1 of enterovirus 71 (EV71) as a vaccination strategy to prevent EV71 infection in mice. When orally administered to BALB/c mice, both attenuated carrier strains, CNP101 and SL7207, were able to efficiently invade livers and spleens, while only the virulence plasmid-carrying strain SL7207 persisted for more than 30 days in these organs. A recombinant in vivo-regulated promoter expression plasmid expressing VP1 antigen of EV71 was constructed. The expression of the VP1, directed by the pagC promoter, in attenuated Salmonella was confirmed by Western blot hybridization. Both humoral and cellular immune responses were elicited in mice by oral immunization with such Salmonella-based VP1 vaccines. We evaluated the protective efficacy of the vaccines in mice using a maternal immunization protocol. With a lethal challenge, ICR newborn mice born to dams immunized with Salmonella-based VP1 vaccine showed a 50-60% survival; in contrast, none of the mice in the control group survived the challenge. Our data indicated that Salmonella-based VP1 subunit vaccines are a promising vaccine strategy in the prevention of EV71 infection.     

Genetic analysis of the P1 region of human enterovirus 71 strains and expression of the 55 F strainVP1 protein

Enterovirus 71 (EV71) is a member of the Entero-virus genus of the Picornaviridae family and is the major cause of Hand,foot,and mouth disease (HFMD) in children.Different strains from Gansu were clone...     

重叠延伸PCR法合成EV71 VP1-LTB融合基因及其原核表达

目的用重叠延伸PCR（overlap extension PCR）法获得人肠道病毒71型vp1与大肠埃希菌不耐热肠毒素B亚单位（ltb）的融合基因,构建EV71 VP1-LTB融合表达质粒并在原核系统表达。方法设计14对引物通过重叠延伸PCR技术合成vp1基因,以ETEC（44815）质粒DNA为模板,PCR扩增ltb基因,将vp1基因与ltb基因连接并测序,连接产物插入原核表达质粒pBEX,构建重组质粒pBEX-VP1-LTB,转化E.coliB1221（DE3）进行表达,SDS-PAGE及W estern blotting分析其表达。电转法将重组质粒转入双歧杆菌。结果合成的vp1基因全长891 bp,测序结果与预期相符,重组表达质粒pBEX-VP1-LTB经PCR及双酶切鉴定,表明构建正确;目的蛋白在E.coliBL21（DE3）中获得了表达,Western bloting分析结果表明该蛋白具有与EV71 VP1抗体的反应原性;电转法成功将重组质粒转入双歧杆菌。结论应用重叠延伸PCR技术成功构建了EV71 VP1-LTB融合表达质粒,并在E.coliBL21（DE3）中获得有生物学功能的VP1表达产物,重组质粒双歧杆菌转化及VP1-LTB融合蛋白的表达研究,为研制EV71分子内佐剂疫苗打下了基础。     

Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity

Enterovirus type 71 (EV71) causes hand, foot, and mouth disease (HFMD), which is mostly self-limited but may be complicated with a severe to fatal neurological syndrome in some children. Understanding the molecular basis of virus-host interactions might help clarify the largely unknown neuropathogenic mechanisms of EV71. In this study, we showed that human annexin II (Anx2) protein could bind to the EV71 virion via the capsid protein VP1. Either pretreatment of EV71 with soluble recombinant Anx2 or pretreatment of host cells with an anti-Anx2 antibody could result in reduced viral attachment to the cell surface and a reduction of the subsequent virus yield in vitro. HepG2 cells, which do not express Anx2, remained permissive to EV71 infection, though the virus yield was lower than that for a cognate lineage expressing Anx2. Stable transfection of plasmids expressing Anx2 protein into HepG2 cells (HepG2-Anx2 cells) could enhance EV71 infectivity, with an increased virus yield, especially at a low infective dose, and the enhanced infectivity could be reversed by pretreating HepG2-Anx2 cells with an anti-Anx2 antibody. The Anx2-interacting domain was mapped by yeast two-hybrid analysis to VP1 amino acids 40 to 100, a region different from the known receptor binding domain on the surface of the picornavirus virion. Our data suggest that binding of EV71 to Anx2 on the cell surface can enhance viral entry and infectivity, especially at a low infective dose.     

Generation and characterization of a protective mouse monoclonal antibody against human enterovirus 71

Human enterovirus 71 (EV71) infection has emerged as a major threat to children; however, no effective antiviral treatment or vaccine is currently available. Antibody-based treatment shows promises to control this growing public health problem of EV71 infection, and a few potent monoclonal antibodies (mAbs) targeting viral capsid protein have been well described. Here, we generated an EV71-specific mouse mAb 2G8 that conferred full protection against lethal EV71 challenge in a suckling mouse model. 2G8 belonged to IgM isotype and neutralized EV71 at the attachment stage. Biochemical assays mapped the binding epitope of 2G8 to the SP70 peptide, which spanning amino acid residues 208–222 on the VP1 protein. Alanine scanning mutagenesis defined the essential roles of multiple residues, including Y208, T210, G212, K215, K218, L220, E221, and Y222, for 2G8 binding. Then, a panel of single mutation was individually introduced into the EV71 infectious clone by reverse genetics, and three mutant viruses, K215A, K218A, and L220A, were successfully recovered and characterized. Biochemical and neutralization assays revealed that K218A mutant partially escaped 2G8 neutralization, while L220A completely abolished 2G8 binding and neutralization. In particular, neutralization assays with human sera demonstrated that K218A and L220A substitutions are also critical for antibody neutralization in natural infection population. These findings not only generate a protective mAb candidate with therapeutic potential but also provide insights into antibody-mediated EV71 neutralization mechanism.     

肠道病毒71型的功能基因组学研究进展

肠道病毒71型（enterovirus type 71,EV71）感染通常引起婴幼儿手足口病（hand,foot and mouth disease,HFMD）,但少数可引起无菌性脑膜炎（asepic meningitis）、脑炎（encephalitis）和类脊髓灰质炎的麻痹性疾病（poliomyelitis-like paralysis）等严重的神经系统疾病。功能基因组学研究对于探讨EV71的感染及复制过程、药物及疫苗的研制具有重大意义。该文就EV71的基因组结构及其功能的研究进展作简要的概述。     

Recombinant VP1 protein expressed in Pichia pastoris induces protective immune responses against EV71 in mice

Human enterovirus 71 (EV71) is one of the major causative agents of hand, foot and mouth disease and is also associated with serious neurological diseases in children. Currently, there are no effective antiviral drugs or vaccines against EV71 infection. VP1, one of the major immunogenic capsid proteins of EV71, is widely considered to be the candidate antigen for an EV71 vaccine. In this study, VP1 of EV71 was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast Pichia pastoris, and purified by Ni–NTA affinity chromatography. Immunogenicity and vaccine efficacy of the recombinant VP1 were assessed in mouse models. The results showed that the recombinant VP1 could efficiently induce anti-VP1 antibodies in BALB/c mice, which were able to neutralize EV71 viruses in an in vitro neutralization assay. Passive protection of neonatal mice further confirmed the prophylactic efficacy of the antisera from VP1 vaccinated mice. Furthermore, VP1 vaccination induced strong lymphoproliferative and Th1 cytokine responses. Taken together, our study demonstrated that the yeast-expressed VP1 protein retained good immunogenicity and was a potent EV71 vaccine candidate.     

Inhibition of enterovirus 71 (EV-71) infections by a novel antiviral peptide derived from EV-71 capsid protein VP1

Enterovirus 71 (EV-71) is the main causative agent of hand, foot and mouth disease (HFMD). In recent years, EV-71 infections were reported to cause high fatalities and severe neurological complications in Asia. Currently, no effective antiviral or vaccine is available to treat or prevent EV-71 infection. In this study, we have discovered a synthetic peptide which could be developed as a potential antiviral for inhibition of EV-71. Ninety five synthetic peptides (15-mers) overlapping the entire EV-71 capsid protein, VP1, were chemically synthesized and tested for antiviral properties against EV-71 in human Rhabdomyosarcoma (RD) cells. One peptide, SP40, was found to significantly reduce cytopathic effects of all representative EV-71 strains from genotypes A, B and C tested, with IC(50) values ranging from 6-9.3 μM in RD cells. The in vitro inhibitory effect of SP40 exhibited a dose dependent concentration corresponding to a decrease in infectious viral particles, total viral RNA and the levels of VP1 protein. The antiviral activity of SP40 peptide was not restricted to a specific cell line as inhibition of EV-71 was observed in RD, HeLa, HT-29 and Vero cells. Besides inhibition of EV-71, it also had antiviral activities against CV-A16 and poliovirus type 1 in cell culture. Mechanism of action studies suggested that the SP40 peptide was not virucidal but was able to block viral attachment to the RD cells. Substitutions of arginine and lysine residues with alanine in the SP40 peptide at positions R3A, R4A, K5A and R13A were found to significantly decrease antiviral activities, implying the importance of positively charged amino acids for the antiviral activities. The data demonstrated the potential and feasibility of SP40 as a broad spectrum antiviral agent against EV-71.     

EV71抗病毒药物及疫苗研究进展

手足口病(hand-foot and mouth disease,HFMD)作为全球关注的公共卫生疾病,亚洲地区频发,危害不容小觑.其传播途径广、传染性强、易引起并发症,致病株多样且易变异等特点,均为手足口病的治疗与防控带来挑战.肠道病毒71型(human enterovirus 71,EV71)作为手足口病主要的病原...     

Structural and functional analysis of a potent human neutralizing antibody against enterovirus A71

Science China Life Sciences - Enterovirus A71 (EV-A71) causes major outbreaks of hand, foot, and mouth disease (HFMD) in many countries, most frequently affecting children, and a small proportion...     

人肠道病毒71型与宿主相互作用研究进展

自1969年首次从美国加利福尼亚患者分离出人肠道病毒71型(Human enterovirus 71,HEV71)后,该病毒已在世界范围内造成多次暴发流行,近年来又在中国大陆、中国台湾及其他亚太地区流行。该肠道病毒主要引起婴幼儿手足口病,但在某些患儿可表现为严重的神经系统感染和呼吸、循环功能     

Antiviral activity of GuiQi polysaccharides against enterovirus 71 in vitro

In this study, we have investigated the antiviral activity of GuiQi polysaccharides（GQP） upon enterovirus 71 （EV71） in vitro. An assay using methyl thiazolyl tetrazolium（MTT）, and analyses of cytopathic effects（CPE） were used to examine the antiviral activity of GQP upon Vero cells infected with EV71. The results revealed that GQP at concentrations below 31.2 μg/mL exhibited significant antiviral effects upon EV71 when applied under three different experimental protocols. GQP was most strongly active in preventing the adsorption of EV71 to target cells and in this respect it was significantly more effective than ribavirin. In addition, it was clear that GQP could inhibit viral replication when added to cells 2 h after infection, but if added at the point of infection its effect was weak. GQP is considered to be less toxic than ribavirin, and may warrant further evaluation as a possible agent in the treatment of hand, foot and mouth disease（HFMD）.     

Antiviral effect of geraniin on human enterovirus 71 in vitro and in vivo

Human enterovirus 71 infection causes hand, foot and mouth disease in children under 6 years of age and has caused mortalities in large-scale outbreaks in the Asia-Pacific region. No effective vaccine or antiviral drugs currently exist against enterovirus 71 in the clinic. In this study, we investigated the antiviral effect of geraniin on enterovirus 71 both in vitro and in vivo. The results showed that geraniin effectively inhibited virus replication in rhabdomyosarcoma cells with an IC(50) of 10 μg/ml. Moreover, geraniin treatment of mice that were challenged with a lethal dose of enterovirus 71 resulted in a reduction of mortality, relieved clinical symptoms, and inhibited virus replication in muscle tissues. The results suggest that geraniin may be used as a potential drug for anti-enterovirus 71.     

Suppression of interleukin-6 increases enterovirus A71 lethality in mice

Background

Enterovirus A71 (EV-A71) infection can induce fatal encephalitis in young children. Clinical reports show that interleukin-6 (IL-6) levels in the serum and cerebrospinal fluid of infected patients with brainstem encephalitis are significantly elevated. We used a murine model to address the significance of endogenous IL-6 in EV-A71 infection.

Results

EV-A71 infection transiently increased serum and brain IL-6 protein levels in mice. Most importantly, absence of IL-6 due to gene knockout or depletion of IL-6 using neutralizing monoclonal antibody enhanced the mortality and tissue viral load of infected mice. Absence of IL-6 increased the damage in the central nervous system and decreased the lymphocyte and virus-specific antibody responses of infected mice.

Conclusions

Endogenous IL-6 functions to clear virus and protect the host from EV-A71 infection. Our study raises caution over the use of anti-IL-6 antibody or pentoxifylline to reduce IL-6 for patient treatment.

     

2007―2008年肠道病毒71型北京地区分离株的VP4基因序列分析

为了解2007 ― 2008 年北京地区流行的肠道病毒71 型( EV71) 是否存在基因序列变异及其与病毒毒力的关系, 我们选择2007 年分离的3 株EV71( 其中1 株分离自重症手足口病患儿的咽拭子标本, 其余2 株分离自普通手足口病患儿咽拭子标本) 和2008 年分离的5 株EV71( 其中3 株分离自重症手足口病患儿的咽拭子或鼻拭子标本, 2 株分离自普通手足口病患儿的疱疹液标本) , 提取基因组RNA, 经反转录-聚合酶链反应( RT-PCR) 扩增得到VP4 基因片段, 并进行核苷酸序列测定, 使用生物信息软件与GenBank 中的EV71 VP4 基因进行序列及病毒型别分析。结果表明, 所测得的8 株EV71 VP4 基因全长均为207 bp, 编码69 个氨基酸, 理论相对分子质量( Mr) 为7 ×10³。8 株EV71 病毒VP4 基因的核苷酸同源性在94% ～100% , 与GenBank 中其他EV71 病毒株VP4 的核苷酸同源性为82% ～100% , 与阜阳、深圳和台湾等地区流行的EV71 VP4 的核苷酸同源性比其他地区高。除了与印度报道的VP4 编码的氨基酸在第7 和54 位不同外( 印度株: 7 位蛋氨酸, 54位苏氨酸; 其余株7 位苏氨酸,54 位丙氨酸) , 这8 株EV71 VP4 编码的氨基酸序列之间以及与其他EV71 VP4编码的氨基酸同源性均为100%。8 株EV71 病毒VP4 与文献报道的3 株重症感染病毒株VP4 ( BrCr、MS 和NCKU9822) 核苷酸有较大差别, 而8 株病毒株中从重症感染( BJ97、BJ110B、BJ110Y 和BJ4243) 与轻症感染( BJ25、BJ47、BJ65 和BJ67) 分离到的毒株之间VP4 基因序列未见明显改变, 只有几个核苷酸存在差别。VP4 核苷酸序列的进化树分析表明, 这8 株EV71 均属于C4 亚型, 显示2007 ― 2008 年北京地区流行的EV71的VP4 基因相当保守, 分离自伴有神经系统感染的重症手足口病和普通手足口病患儿的EV71 的VP4 基因之间在核苷酸水平未出现同样的变异。结果提示, 近2 年来北京地区所流行的EV71 属C4 亚型。     

Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998

Enterovirus 71 (EV71) (genus Enterovirus, family Picornaviridae), a common cause of hand, foot, and mouth disease (HFMD), may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity and rate of evolution of EV71, we have determined and analyzed complete VP1 sequences (891 nucleotides) for 113 EV71 strains isolated in the United States and five other countries from 1970 to 1998. Nucleotide sequence comparisons demonstrated three distinct EV71 genotypes, designated A, B, and C. The genetic variation within genotypes (12% or fewer nucleotide differences) was less than the variation between genotypes (16.5 to 19.7%). Strains of all three genotypes were at least 94% identical to one another in deduced amino acid sequence. The EV71 prototype strain, BrCr-CA-70, isolated in California in 1970, is the sole member of genotype A. Strains isolated in the United States and Australia during the period from 1972 to 1988, a 1994 Colombian isolate, and isolates from a large HFMD outbreak in Malaysia in 1997 are all members of genotype B. Although strains of genotype B continue to circulate in other parts of the world, none have been isolated in the United States since 1988. Genotype C contains strains isolated in 1985 or later in the United States, Canada, Australia, and the Republic of China. The annual rate of evolution within both the B and C genotypes was estimated to be approximately 1.35 x 10(-2) substitutions per nucleotide and is similar to the rate observed for poliovirus. The results indicate that EV71 is a genetically diverse, rapidly evolving virus. Its worldwide circulation and potential to cause severe disease underscore the need for additional surveillance and improved methods to identify EV71 in human disease.