Development and characterization of a stable eGFP enterovirus 71 for antiviral screening

Shang, Baodi; Deng, Cheng-Lin; Ye, Han-Qing; Xu, Wenbo; Yuan, Zhiming; Shi, Pei Yong; Zhang, Bo

doi:10.1016/j.antiviral.2012.12.010

Cited by 56 publications

(60 citation statements)

References 29 publications

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“…WT and mutant viruses were produced by the electroporation of Vero cells with in vitro-transcribed RNA from an infectious cDNA clone, pACYC-EV71 (43). EV71 rabbit anti-VP1 polyantibody was provided by the group of Hua-Lin Wang (Wuhan Institute of Virology, Chinese Academy of Sciences) (43). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG was purchased from ProteinTech Group Inc.…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of Enterovirus 71 RNA-Dependent RNA Polymerase Complexed with Its Protein Primer VPg: Implication for a trans Mechanism of VPg Uridylylation

Chen

Wang

Shan

et al. 2013

J Virol

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g Picornavirus RNA replication is initiated by VPg uridylylation, during which the hydroxyl group of the third tyrosine residue of the virally encoded protein VPg is covalently linked to two UMP molecules by RNA-dependent RNA polymerase (RdRp; also known as 3D pol ). We previously identified site 311, located at the base of the palm domain of the enterovirus 71 (EV71) RdRp, to be the site for EV71 VPg binding and uridylylation. Here we report the crystal structure of EV71 3D pol complexed with VPg. VPg was anchored at the bottom of the palm domain of the 3D pol molecule and exhibited an extended V-shape conformation. The corresponding interface on 3D pol was mainly formed by residues within site 311 and other residues in the palm and finger domains. Mutations of the amino acids of 3D pol involved in the VPg interaction ( 3D L319A, 3D D320A, and 3D Y335A) significantly disrupted VPg binding to 3D pol , resulting in defective VPg uridylylation. In contrast, these mutations did not affect the RNA elongation activity of 3D pol . In the context of viral genomic RNA, mutations that abolished VPg uridylylation activity were lethal for EV71 replication. Further in vitro analysis showed that the uridylylation activity was restored by mixing VPg-binding-defective and catalysis-defective mutants, indicating a trans mechanism for EV71 VPg uridylylation. Our results, together with previous results of other studies, demonstrate that different picornaviruses use distinct binding sites for VPg uridylylation. Picornaviridae members make up one of the largest family of viruses and cause a wide range of infectious diseases in plants, animals, and humans. As the members of Picornaviridae, enterovirus 71 (EV71) and coxsackievirus (CV) are the major causative agents of hand, foot, and mouth disease (HFMD) in mainland China (1, 2). These agents caused over 1,000,000 infections and 900 deaths in 2010

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Section: Methodsmentioning

confidence: 99%

Crystal Structure of Enterovirus 71 RNA-Dependent RNA Polymerase Complexed with Its Protein Primer VPg: Implication for a trans Mechanism of VPg Uridylylation

Chen

Wang

Shan

et al. 2013

J Virol

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“…Vero (African green monkey kidney) cells were cultured in Dulbecco modified Eagle medium (DMEM; Invitrogen) with 10% fetal bovine serum (FBS), 100 U/ml of penicillin, and 100 g/ml of streptomycin. The wild-type (WT) recombinant viruses were prepared from the infectious cDNA clone of EV71 (22), stored as aliquots at Ϫ80°C, and used for antiviral activity assays and resistant virus selection. EV71 strain 5865/SIN/000009 (S41) (23) was used for an animal study.…”

Section: Methodsmentioning

confidence: 99%

Inhibition of Enterovirus 71 by Adenosine Analog NITD008

Deng

Yeo

et al. 2014

J Virol

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Enterovirus 71 (EV71) is a major viral pathogen in China and Southeast Asia. There is no clinically approved vaccine or antiviral therapy for EV71 infection. NITD008, an adenosine analog, is an inhibitor of flavivirus that blocks viral RNA synthesis. Here we report that NITD008 has potent antiviral activity against EV71. In cell culture, the compound inhibits EV71 at a 50% effective concentration of 0.67 M and a 50% cytotoxic concentration of 119.97 M. When administered at 5 mg/kg in an EV71 mouse model, the compound reduced viral loads in various organs and completely prevented clinical symptoms and death. To study the antiviral mechanism and drug resistance, we selected escape mutant viruses by culturing EV71 with increasing concentrations of NITD008. Resistance mutations were reproducibly mapped to the viral 3A and 3D polymerase regions. Resistance analysis with recombinant viruses demonstrated that either a 3A or a 3D mutation alone could lead to resistance to NITD008. A combination of both 3A and 3D mutations conferred higher resistance, suggesting a collaborative interplay between the 3A and 3D proteins during viral replication. The resistance results underline the importance of combination therapy required for EV71 treatment.

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“…After 72 h of incubation at 37˚C, a second layer of 1.2% agar containing the neutral red pH indicator was added. Following incubation at 37˚C for 12-24 h, images of the plaques were captured and the number of formed plaques was determined as described previously (16,18). The viral titer was calculated as pfu/ml.…”

Section: Methodsmentioning

confidence: 99%

“…Virus titer and morphology were determined via a bilayer plaque assay (16). A series of 1:10 dilutions were prepared by diluting 15 ml virus stock with 135 ml DMEM supplemented with 10% FBS, and 100 ml of each dilution was seeded into each well of a 6-well plate containing confluent Vero cells (1x10 6 cells/well; plated 1 day in advance) as described previously (18). Plates were incubated at 37˚C for 1 h before the first layer of 1.2% agar was added.…”

Section: Methodsmentioning

confidence: 99%

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Enterovirus 71 infection impairs the reproductive capacity of female mice

Qin

Yan

Chen

et al. 2017

Experimental and Therapeutic Medicine

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Abstract. Enterovirus 71 (EV71) is a major cause of hand, foot and mouth disease (HFMD); however, no clinically approved vaccine or antiviral treatment is currently available for EV71 infection. In the present study, a murine model of EV71 infection was constructed. The clinical isolates of EV71 were amplified in Vero cells and used to challenge adult mice via hydrodynamic injection (HI) and intraperitoneal injection (IP). Following two challenges, >50% of the mice succumbed to EV71 infection. Surviving female mice were identified to have impaired fertility and their litter sizes were significantly decreased compared with the control group. The antibody against EV71-VP1 persisted in the sera of female mice at a high titer for >2 years after challenge. The maternal antibody in the offspring sera also persisted for ~1 year and disappeared after ~2 years. Results from the present study suggest that a high titer of active EV71 was able to impair the reproductivity of adult female mice, and that high levels of maternal antibody persisted in the offspring and protected postnatal mice from EV71-induced mortality. The promising antigenicity, immunogenicity and reactogenicity of EV71 suggests that it a potential vaccine target that may be beneficial to the control of HFMD, through immunizing infants and women of reproductive age. IntroductionEnterovirus (EV) 71 is a member of the picornaviridae family of viruses that contain single-stranded positive-sense RNA (1), which primarily infects children <5 years old (2). Infants with EV71 infection develop sores on their hands, feet, buttocks and mouth; therefore, the infection is referred to as hand, foot and mouth disease (HFMD) (3). Several types of enterovirus have been indicated to cause HFMD in children; however, EV71 is commonly associated with severe complications of the nervous system in infants with HFMD and is therefore considered to be a major virulent pathogen and cause of mortalities in HFMD (4). Previous studies have indicated that a total of 488,955 HFMD cases and 126 HFMD-associated mortalities were reported in 2008 in China, of which EV71 infection was the primary cause (5). EV71 is a non-enveloped virus with a single-stranded positive-sense RNA genome that is ~7.5 kb in size and belongs to the enterovirus genus of the picornaviridae family (6). EV71 is classified into five genotypes as follows: A, B, C, D and F, and further subdivided into B0-B5 and C1-C5 subgenotypes (7). HFMD is an important public health issue and further understanding of the mechanisms underlying the development of HFMD are required to develop effective treatments. In mainland China, Taiwan and Singapore, EV71 whole-virus vaccines have been produced by various manufacturers. Two available inactivated EV71 vaccines have been approved by the China Food and Drug Administration, one produced by Sinovac Biotech Ltd. and the other by the Chinese Academy of Medical Science (all Beijing, China) (8-10). Currently, no effective treatment is available to treat severe EV71 infections, aside from symp...

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Development and characterization of a stable eGFP enterovirus 71 for antiviral screening

Cited by 56 publications

References 29 publications

Crystal Structure of Enterovirus 71 RNA-Dependent RNA Polymerase Complexed with Its Protein Primer VPg: Implication for a trans Mechanism of VPg Uridylylation

Crystal Structure of Enterovirus 71 RNA-Dependent RNA Polymerase Complexed with Its Protein Primer VPg: Implication for a trans Mechanism of VPg Uridylylation

Inhibition of Enterovirus 71 by Adenosine Analog NITD008

Enterovirus 71 infection impairs the reproductive capacity of female mice

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