Targeting Ser78 phosphorylation of Hsp27 achieves potent antiviral effects against enterovirus A71 infection.

A positive-sense (+) single-stranded RNA (ssRNA) virus (e.g. enterovirus A71, EV-A71) depends on viral polypeptide translation for initiation of virus replication after entry. We reported that EV-A71 hijacks Hsp27 to induce hnRNP A1 cytosol redistribution to initiate viral protein translation, but the underlying mechanism is still elusive. Here, we show that phosphorylation-deficient Hsp27-3A (Hsp27S15/78/82A) and Hsp27S78A fail to translocate into the nucleus and induce hnRNP A1 cytosol redistribution, while Hsp27S15A and Hsp27S82A display similar effects to the wild type Hsp27. Furthermore, we demonstrate that the viral 2A protease (2Apro) activity is a key factor in regulating Hsp27/hnRNP A1 relocalization. Hsp27S78A dramatically decreases the IRES activity and viral replication, which are partially reduced by Hsp27S82A. However, Hsp27S15A displays the same activity as the wild-type Hsp27. Peptide S78 potently suppresses EV-A71 protein translation and reproduction through blockage of EV-A71-induced Hsp27 phosphorylation and Hsp27/hnRNP A1 relocalization. A point mutation (S78A) on S78 impairs its inhibitory functions on Hsp27/hnRNP A1 relocalization and viral replication. Taken together, we demonstrate the importance of Ser78 phosphorylation of Hsp27 regulated by virus infection in nuclear translocation, hnRNP A1 cytosol relocation, and viral replication, suggesting a new path (such as peptide S78) for target-based antiviral strategy.

EV71 5'UTR interacts with 3D protein affecting replication through the AKT-mTOR pathway.

BACKGROUND: EV71 is one of the important pathogens of Hand-foot-and-mouth disease (HFMD), which causes serious neurological symptoms. Several studies have speculated that there will be interaction between 5'UTR and 3D protein. However, whether 5'UTR interacts with the 3D protein in regulating virus replication has not been clarified. METHODS: Four 5'UTR mutation sites (nt88C/T, nt90-102-3C, nt157G/A and nt574T/A) and two 3D protein mutation sites (S37N and R142K) were mutated or co-mutated using virulent strains as templates. The replication of these mutant viruses and their effect on autophagy were determined. RESULTS: 5'UTR single-point mutant strains, except for EGFP-EV71(nt90-102-3C), triggered replication attenuation. The replication ability of them was weaker than that of the parent strain the virulent strain SDLY107 which is the fatal strain that can cause severe neurological complications. While the replication level of the co-mutant strains showed different characteristics. 5 co-mutant strains with interaction were screened: EGFP-EV71(S37N-nt88C/T), EGFP-EV71(S37N-nt574T/A), EGFP-EV71(R142K-nt574T/A), EGFP-EV71(R142K-nt88C/T), and EGFP-EV71(R142K-nt157G/A). The results showed that the high replicative strains significantly promoted the accumulation of autophagosomes in host cells and hindered the degradation of autolysosomes. The low replicative strains had a low ability to regulate the autophagy of host cells. In addition, the high replicative strains also significantly inhibited the phosphorylation of AKT and mTOR. CONCLUSIONS: EV71 5'UTR interacted with the 3D protein during virus replication. The co-mutation of S37N and nt88C/T, S37N and nt574T/ A, R142K and nt574T/A induced incomplete autophagy of host cells and promoted virus replication by inhibiting the autophagy pathway AKT-mTOR. The co-mutation of R142K and nt88C/T, and R142K and nt157G/A significantly reduced the inhibitory effect of EV71 on the AKT-mTOR pathway and reduced the replication ability of the virus.

Cordycepin Inhibits Enterovirus A71 Replication and Protects Host Cell from Virus-Induced Cytotoxicity through Adenosine Action Pathway.

Enterovirus A71 (EV-A71) infection typically causes mild illnesses, such as hand-foot-and-mouth disease (HFMD), but occasionally leads to severe or fatal neurological complications in infants and young children. Currently, there is no specific antiviral treatment available for EV-A71 infection. Thus, the development of an effective anti-EV-A71 drug is required urgently. Cordycepin, a major bioactive compound found in Cordyceps fungus, has been reported to possess antiviral activity. However, its specific activity against EV-A71 is unknown. In this study, the potency and role of cordycepin treatment on EV-A71 infection were investigated. Results demonstrated that cordycepin treatment significantly reduced the viral load and viral ribonucleic acid (RNA) level in EV-A71-infected Vero cells. In addition, EV-A71-mediated cytotoxicity was significantly inhibited in the presence of cordycepin in a dose-dependent manner. The protective effect can also be extended to Caco-2 intestinal cells, as evidenced by the higher median tissue culture infectious dose (TCID50) values in the cordycepin-treated groups. Furthermore, cordycepin inhibited EV-A71 replication by acting on the adenosine pathway at the post-infection stage. Taken together, our findings reveal that cordycepin could be a potential antiviral candidate for the treatment of EV-A71 infection.

Enterovirus A71 does not meet the uncoating receptor SCARB2 at the cell surface.

Enterovirus A71 (EV-A71) infection involves a variety of receptors. Among them, two transmembrane protein receptors have been investigated in detail and shown to be critical for infection: P-selectin glycoprotein ligand-1 (PSGL-1) in lymphocytes (Jurkat cells), and scavenger receptor class B member 2 (SCARB2) in rhabdomyosarcoma (RD) cells. PSGL-1 and SCARB2 have been reported to be expressed on the surface of Jurkat and RD cells, respectively. In the work reported here, we investigated the roles of PSGL-1 and SCARB2 in the process of EV-A71 entry. We first examined the expression of SCARB2 in Jurkat cells, and detected it within the cytoplasm, but not on the cell surface. Further, using PSGL-1 and SCARB2 knockout cells, we found that although both PSGL-1 and SCARB2 are essential for virus infection of Jurkat cells, virus attachment to these cells requires only PSGL-1. These results led us to evaluate the cell surface expression and the roles of SCARB2 in other EV-A71-susceptible cell lines. Surprisingly, in contrast to the results of previous studies, we found that SCARB2 is absent from the surface of RD cells and other susceptible cell lines we examined, and that although SCARB2 is essential for infection of these cells, it is dispensable for virus attachment. These results indicate that a receptor other than SCARB2 is responsible for virus attachment to the cell and probably for internalization of virions, not only in Jurkat cells but also in RD cells and other EV-A71-susceptible cells. SCARB2 is highly concentrated in lysosomes and late endosomes, where it is likely to trigger acid-dependent uncoating of virions, the critical final step of the entry process. Our results suggest that the essential interactions between EV-A71 and SCARB2 occur, not at the cell surface, but within the cell.

VP1 codon deoptimization and high-fidelity substitutions in 3D polymerase as potential vaccine strategies for eliciting immune responses against enterovirus A71.

Enterovirus A71 (EV-A71) can induce severe neurological complications and even fatal encephalitis in children, and it has caused several large outbreaks in Taiwan since 1998. We previously generated VP1 codon-deoptimized (VP1-CD) reverse genetics (rg) EV-A71 viruses (rgEV-A71s) that harbor a high-fidelity (HF) 3D polymerase. These VP1-CD-HF rgEV-A71s showed lower replication kinetics in vitro and decreased virulence in an Institute of Cancer Research (ICR) mouse model of EV-A71 infection, while still retaining their antigenicity in comparison to the wild-type virus. In this study, we aimed to further investigate the humoral and cellular immune responses elicited by VP1-CD-HF rgEV-A71s to assess the potential efficacy of these EV-A71 vaccine candidates. Following intraperitoneal (i.p.) injection of VP1-CD-HF rgEV-A71s in mice, we observed a robust induction of EV-A71-specific neutralizing IgG antibodies in the antisera after 21 days. Splenocytes isolated from VP1-CD-HF rgEV-A71s-immunized mice exhibited enhanced proliferative activities and cytokine production (IL-2, IFN-γ, IL-4, IL-6, and TNF-α) upon re-stimulation with VP1-CD-HF rgEV-A71, as compared to control mice treated with adjuvant only. Importantly, administration of antisera from VP1-CD-HF rgEV-A71s-immunized mice protected against lethal EV-A71 challenge in neonatal mice. These findings highlight that our generated VP1-CD-HF rgEV-A71 viruses are capable of inducing both cellular and humoral immune responses, supporting their potential as next-generation EV-A71 vaccines for combating EV-A71 infection.IMPORTANCEEV-A71 can cause severe neurological diseases and cause death in young children. Here, we report the development of synthetic rgEV-A71s with the combination of codon deoptimization and high-fidelity (HF) substitutions that generate genetically stable reverse genetics (rg) viruses as potential attenuated vaccine candidates. Our work provides insight into the development of low-virulence candidate vaccines through a series of viral genetic editing for maintaining antigenicity and genome stability and suggests a strategy for the development of an innovative next-generation vaccine against EV-A71.

An enterovirus A71 virus-like particle with replaced loops confers partial cross-protection in mice.

Enterovirus A71 (EV-A71), coxsackievirus A16 (CV-A16), and CV-A10 belong to the main prevailing types causing hand-foot-and-mouth disease. Since EV-A71 monovalent vaccine does not confer cross-protection, developing a multivalent vaccine is essential. In this study, a trivalent chimeric virus-like particle of EV-A71 (EV-A71-VLPCHI3) was constructed based on EV-A71-VLP backbone by replacing the corresponding surface loops with CV-A16 VP1 G-H, CV-A10 VP1 B-C and E-F loops, which are critical for immunogenic neutralization. The baculovirus-insect cell expression system was employed for EV-A71-VLPCHI3 production. EV-A71-VLPCHI3 was purified by sucrose density gradient and observed by transmission electron microscopy. The immunogenicity and protective efficacy of EV-A71-VLPCHI3 were evaluated in mice. Our results revealed that EV-A71-VLPCHI3 had a similar morphology to inactivated EV-A71 particles and could induce specific IgG antibodies against EV-A71, CV-A16 and CV-A10 in mice. More importantly, EV-A71-VLPCHI3 enhanced cross-reactive protection against CV-A16 and CV-A10, by 20 % and 40 %, compared to inactivated EV-A71 counterparts, respectively. In conclusion, the successful construction of EV-A71-VLPCHI3 suggested that loop-dependent heterologous protection could be transferred by loops replacement on the surface of viral capsid. This strategy may also supplement the development of multivalent vaccines against other infectious viral diseases.

Coxsackievirus A6 2C protein antagonizes IFN-ß production through MDA5 and RIG-I depletion.

IMPORTANCE: Coxsackievirus A6 (CV-A6) is a major emerging pathogen associated with atypical hand, foot, and mouth disease and can cause serious complications such as encephalitis, acute flaccid paralysis, and neurorespiratory syndrome. Therefore, revealing the associated pathogenic mechanisms could benefit the control of CV-A6 infections. In this study, we demonstrate that the nonstructural 2CCV-A6 suppresses IFN-ß production, which supports CV-A6 infection. This is achieved by depleting RNA sensors such as melanoma differentiation-associated gene 5 and retinoic acid-inducible gene I (RIG-I) through the lysosomal pathway. Such a function is shared by 2CEV-A71 and 2CCV-B3 but not 2CCV-A16, suggesting the latter might have an alternative way to promote viral replication. This study broadens our understanding of enterovirus 2C protein regulation of the RIG-I-like receptor signaling pathway and reveals a novel mechanism by which CV-A6 and other enteroviruses evade the host innate immune response. These findings on 2C may provide new therapeutic targets for the development of effective inhibitors against CV-A6 and other enterovirus infections.

Identification of Critical Amino Acids of Coxsackievirus A10 Associated with Cell Tropism and Viral RNA Release during Uncoating.

Coxsackievirus A10 (CV-A10) is a prevailing causative agent of hand-foot-mouth disease, necessitating the isolation and adaptation of appropriate strains in cells allowed for human vaccine development. In this study, amino acid sequences of CV-A10 strains with different cell tropism on RD and Vero cells were compared. Various amino acids on the structural and non-structural proteins related to cell tropism were identified. The reverse genetic systems of several CV-A10 strains with RD+/Vero- and RD+/Vero+ cell tropism were developed, and a set of CV-A10 recombinants were produced. The binding, entry, uncoating, and proliferation steps in the life cycle of these viruses were evaluated. P1 replacement of CV-A10 strains with different cell tropism revealed the pivotal role of the structural proteins in cell tropism. Further, seven amino acid substitutions in VP2 and VP1 were introduced to further investigate their roles played in cell tropism. These mutations cooperated in the growth of CV-A10 in Vero cells. Particularly, the valine to isoleucine mutation at the position VP1-236 (V1236I) was found to significantly restrict viral uncoating in Vero cells. Co-immunoprecipitation assays showed that the release of viral RNA from the KREMEN1 receptor-binding virions was restricted in r0195-V1236I compared with the parental strain r0195 (a RD+/Vero+ strain). Overall, this study highlights the dominant effect of structural proteins in CV-A10 adaption in Vero cells and the importance of V1236 in viral uncoating, providing a foundation for the mechanism study of CV-A10 cell tropism, and facilitating the development of vaccine candidates.

Targeting HNRNPA2B1 inhibits enterovirus 71 replication in SK-N-SH cells.

OBJECTIVE: To investigate the effect of heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) on the replication of enterovirus 71 (EV-71) in SK-N-SH cells. METHODS: The mRNA and protein expression of HNRNPA2B1 in SK-N-SH cells were detected by real-time quantitative PCR (qRT-PCR) and western blotting (WB), respectively. WB was used to detect HNRNPA2B1 protein expression in the nucleus and cytosol. The localization of HNRNPA2B1 protein in the nucleus and cytosol was detected by immunofluorescence (IF). The expression of HNRNPA2B1 was inhibited by small interfering RNA (si-HNRNPA2B1). Viral RNA, viral structural protein VP1, and viral titer were detected by qRT-PCR, WB, and viral dilution counting, respectively. RESULTS: EV-71 infection significantly upregulates the expression of HNRNPA2B1 in SK-N-SH cells. EV-71 infection promotes HNRNPA2B1 nucleus-cytoplasm redistribution. Down-regulation of HNRNPA2B1 expression significantly inhibited EV-71 replication. CONCLUSION: HNRNPA2B1 protein redistributed from nucleus to cytoplasm and is highly expressed in the cytoplasm during EV-71 infection. Inhibition of HNRNPA2B1 levels effectively inhibits EV-71 replication in SK-N-SH cells.

Molecular epidemiology and clinical characteristics of enteroviruses associated HFMD in Chengdu, China, 2013-2022.

OBJECTIVES: This study aims to investigate molecular epidemiology and clinical characteristics of enterovirus associated hand-foot-mouth disease (HFMD) in Chengdu, China, 2013-2022. Monitoring the molecular epidemiology and clinical features of HFMD for up to 10 years may provide some ideas for future protection and control measures. METHODS: We conducted a retrospective analysis of the medical records of all patients with laboratory-confirmed HFMD-related enterovirus infection at the West China Second University Hospital from January 2013 to December 2022. We described the characteristics in serotype, age, sex distribution and hospitalization of enterovirus infection cases using data analysis and graphic description. RESULTS: A total of 29,861 laboratory-confirmed cases of HFMD-related enterovirus infection were reported from 2013 to 2022. There was a significant reduction in the number and proportion of EV-A71 cases after 2016, from 1713 cases (13.60%) in 2013-2015 to 150 cases (1.83%) in 2017-2019. During the COVID-19 pandemic, EV-A71 cases even disappeared. The proportion of CV-A16 cases decreased from 13.96% in 2013-2015 to 10.84% in 2017-2019 and then to 4.54% in 2020-2022. Other (non-EV-A71 and non-CV-A16) serotypes accounted for 95.45% during 2020-2022, with CV-A6 accounting for 50.39% and CV-A10 accounting for 10.81%. Thus, CV-A6 and CV-A10 became the main prevalent serotypes. Furthermore, There was no significant difference in the enterovirus prevalence rate between males and females. The hospitalization rate of EV-A71 patients was higher that of other serotypes. In general, the proportion of HFMD hospitalizations caused by other pathogens except for EV-A71, CV-A16, CV-A10 and CV-A16 was second only to that caused by EV-A71. The proportion of children over 4 years old infected with enterovirus increased. CONCLUSION: The incidence of HFMD associated with enterovirus infection has decreased significantly and CV-A6 has been the main pathogen of HFMD in Chengdu area in recent years. The potential for additional hospitalizations for other untested enterovirus serotypes suggested that attention should also be paid to the harms of infections with unknown enterovirus serotypes. Children with HFMD were older. The development of new diagnostic reagents and vaccines may play an important role in the prevention and control of enterovirus infection.

The neuropathological mechanism of EV-A71 infection attributes to inflammatory pryoptosis and viral replication via activating the hsa_circ_0045431/ hsa_miR_584/NLRP3 regulatory axis.

Neuropathological damage has been considered to be the main cause of death from EV-A71 infection, but the underlying mechanism has not been elucidated. Pyroptosis, a new form of inflammatory programmed cell death, has been verified to be involved in the pathogenesis of various viruses. circRNAs are a novel type of endogenous noncoding RNA gaining research interest in recent years, especially their special roles in the process of virus infection. Thus, in this study, we combined EV-A71, pyroptosis and circRNA to find a breakthrough in the pathogenesis of EV-A71 infection. Firstly, whether EV-A71 infection leaded to pyroptosis formation was examined by a series detection of cell death, cell viability, LDH release, caspase 1 activity, the expression levels of pyroptosis-related molecules and the concentrations of IL-1ß and IL-18. Secondly, high-throughput sequencing of circRNAs was carried out to excavate the circRNA-miRNA-mRNA regulatory axis which might be associated with pyroptosis formation. Finally, the gain- and loss-of-functional experiments were further conducted to identify their functions. Our results showed that EV-A71 infection caused pyroptosis formation in SH-SY5Y cells. The circRNA sequencing analyzed the differentially expressed circRNAs and their possible functions. It was found that the hsa_circ_0045431/hsa_miR_584/NLRP3 regulatory axis might be involved in pyroptosis formation during EV-A71 infection. Then, hsa_circ_0045431 sponged hsa_miR_584 and hsa_miR_584 directly targeted NLRP3 were validated by IF, dual-luciferase, qRT-PCR and WB assays. Functional experiments were performed to further uncover that the up-regulation of hsa_circ_0045431 and NLRP3 promoted the inflammatory pyroptosis and viral replication, while the up-regulation of hsa_miR_584 suppressed the inflammatory pyroptosis and viral replication, and vice versa. Collectively, our study demystified that EV-A71 infection induced pyroptosis formation by activating hsa_circ_0045431/hsa_miR_584/NLRP3 regulatory axis, which could further effect viral replication. These findings provided novel insights into the pathogenesis of EV-A71 infection, and meanwhile revealed that the hsa_circ_0045431/ hsa_miR_584/NLRP3 regulatory axis can serve as a potential biological therapeutic target for EV-A71 infection.

SHFL inhibits enterovirus A71 infection by triggering degradation of viral 3Dpol protein via the ubiquitin-proteasome pathway.

Enterovirus A71 (EV-A71) is a highly contagious virus that poses a major threat to global health, representing the primary etiological agent for hand-foot and mouth disease (HFMD) and neurological complications. It has been established that interferon signaling is critical to establishing a robust antiviral state in host cells, mainly mediated through the antiviral effects of numerous interferon-stimulated genes (ISGs). The host restriction factor SHFL is a novel ISG with broad antiviral activity against various viruses through diverse underlying molecular mechanisms. Although SHFL is widely acknowledged for its broad-spectrum antiviral activity, it remains elusive whether SHFL inhibits EV-A71. In this work, we validated that EV-A71 triggers the upregulation of SHFL both in cell lines and in a mouse model. Knockdown and overexpression of SHFL in EVA71-infected cells suggested that this factor could markedly suppress EV-A71 replication. Our findings further revealed an intriguing mechanism of SHFL that it could interact with the nonstructural proteins 3Dpol of EV-A71 and promoted the degradation of 3Dpol through the ubiquitin-proteasome pathway. Furthermore, the zinc-finger domain and the 36 amino acids (164-199) of SHFL were crucial to the interaction between SHFL and EV-A71 3Dpol . Overall, these findings broadened our understanding of the pivotal roles of SHFL in the interaction between the host and EV-A71.

MicroRNA expression profile of human umbilical vein endothelial cells in response to coxsackievirus A10 infection reveals a potential role of miR-143-3p in maintaining the integrity of the blood-brain barrier.

Coxsackievirus A10 (CV-A10) has been one of the main etiologies of hand, foot, and mouth disease (HFMD) epidemics in recent years and can cause mild to severe illness and even death. Most of these severe and fatal cases were closely associated with neurological impairments, but the potential mechanism of neuropathological injury triggered by CV-A10 infection has not been elucidated. MicroRNAs (miRNAs), implicated in the regulation of gene expression in a post-transcriptional manner, play a vital role in the pathogenesis of various central nervous system (CNS) diseases; therefore, they serve as diagnostic biomarkers and are emerging as novel therapeutic targets for CNS injuries. To gain insights into the CV-A10-induced regulation of host miRNA-processing machinery, we employed high-throughput sequencing to identify differentially expressed miRNAs in CV-A10-infected human umbilical vein endothelial cells (HUVECs) and further analyzed the potential functions of these miRNAs during CV-A10 infection. The results showed that CV-A10 infection could induce 189 and 302 significantly differentially expressed miRNAs in HUVECs at 24 and 72 hpi, respectively, compared with the uninfected control. Moreover, the expression of four selected miRNAs and their relevant mRNAs was determined to verify the sequencing data by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) methods. After that, gene target prediction and functional annotation revealed that the targets of these dysregulated miRNAs were mostly enriched in cell proliferation, signal transduction, cAMP signalling pathway, cellular response to interleukin-6, ventral spinal cord interneuron differentiation, negative regulation of glial cell differentiation, neuron migration, positive regulation of neuron projection development, etc., which were primarily involved in the processes of basic physiology, host immunity, and neurological impairments and further reflected vital regulatory roles of miRNA in viral pathogenicity. Finally, the construction of a miRNA-regulated network also suggested that the complex regulatory mechanisms mediated by miRNAs might be involved in viral pathogenesis and virus-host interactions during CV-A10 infection. Furthermore, among these dysregulated miRNAs, miR-143-3p was demonstrated to be involved in the maintenance of blood-brain barrier (BBB) integrity.

miR-342-5p targets CTNNBIP1 to promote enterovirus 71 replication.

OBJECTIVE: The aim of this research was to explore the role of miR-342-5p in EV71 replication. METHODS: Peritoneal injection of EV71 (107 TCID50/mL) at 50, 100, and 150 µL was conducted to infect 12-day-old suckling mice (n = 10 per group), and clinical scores and survival rates were recorded during a 6-day trial duration and followed by transcriptome sequencing of collected spinal cord tissues. The differential miRNAs and target genes of the infected and uninfected EV71 mice were analyzed. The miR-342 and CTNNBIP1 binding sites were detected using a dual luciferase reporter assay. Cell viability was detected by CCK-8. RT-qPCR, Western blot, immunofluorescence, and immunohistochemistry assays were conducted to detect VP1 protein levels. RESULTS: Transcriptome sequencing analyses know that the Wnt pathway played a role in EV71 infection, and the CTNNBIP1 gene in this pathway was the target gene of miR-342-5p. Whether in HMC3 cells or in the spinal cord tissue from the suckling mice, high levels of miR-342-5p markedly promoted EV71 VP1 mRNA and protein expression, elevated TNF-α, IL-6, and IL-10 levels, and inhibited IFN-ß levels. In addition, highly expressed miR-342-5p destroyed neuronal structure in spinal cord tissues and reduced the number of glial cells. Highly expressed CTNNBIP1 blocked the promotion of miR-342-5p in EV71 replication, and inhibited TNF-α, IL-6, and IL-10 levels, whereas elevated IFN-ß levels. This mechanism is that miR-342-5p can target the CTNNBIP1 3' UTR region, inhibit its expression and reduce its binding to CTNNB1, thus enhancing the interaction between CTNNB1 and TCF4 and activating the Wnt pathway-mediated type I interferon response. CONCLUSION: In nerve cells and tissues, the overexpression of miR-342-5p promoted the replication of EV71 and attenuated the innate immune response to antiviral disease via Wnt/CTNNB1 signaling pathway.

AAV-CRISPR-Cas13 eliminates human enterovirus and prevents death of infected mice.

BACKGROUND: RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice. METHODS: We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in vitro viral plaque assay and in vivo EV-A71 lethally-infected mouse model. FINDINGS: We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model. INTERPRETATION: Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections. FUNDING: Agency for Science, Technology and Research (A∗STAR) Assured Research Budget, A∗STAR Central Research Fund UIBR SC18/21-1089UI, A∗STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4.

Natural Oncolysis of Enterovirus 71 in Antitumor Therapy of Colorectal Cancer.

Colorectal cancer (CRC) is an intestinal malignant tumor with high morbidity and mortality worldwide. Inoperability or resistanance to radiation and chemotherapy occur in the conventional treatments against CRC. Oncolytic viruses (OVs) are one kind of virus that selectively infects and lyses cancer cells, which is considered to be a new anticancer therapy with biological and immune-based approaches. Enterovirus 71 (EV71), belonging to the enterovirus genus in the family Picornaviridae, is a single positive-stranded RNA virus. EV71 is transmitted in a fetal-oral route and infects gastrointestinal tract in infants. Here, EV71 is exploited to be a novel oncolytic virus in colorectal cancer. It is revealed that EV71 infection can selectively cause colorectal cancer cells cytotoxicity but not primary intestinal epithelial cells. Consistently, EV71 injection significantly inhibits tumor growth in nude mice xenografted colorectal cancer cells. In detail, EV71 infects colorectal cancer cells to repress the expression of Ki67 and B-cell leukemia 2 (Bcl-2) leading to the inhibition of cell proliferation, while activating the cleavage of poly-adenosine diphosphatase-ribose polymerase and Caspase-3 protein resulting in the promotion of cell apoptosis. The findings demonstrate the oncolytic feature of EV71 in CRC treatment and may provide a potential clue for clinical anticancer therapy.

Engineering Oncolytic Coxsackievirus A21 with Small Transgenes and Enabling Cell-Mediated Virus Delivery by Integrating Viral cDNA into the Genome.

Coxsackievirus A21 (CVA21) is a naturally occurring RNA virus that, in preclinical studies and clinical trials, has demonstrated promising potential in treating a range of malignancies. Other oncolytic viruses, such as adenovirus, vesicular stomatitis virus, herpesvirus, and vaccinia virus, all can be engineered to carry one or more transgenes for various purposes, including immune modulation, virus attenuation, and induction of apoptosis of tumor cells. However, it remained unknown whether CVA21 can express therapeutic or immunomodulatory payloads due to its small size and high mutation rate. Using reverse genetics techniques, we demonstrated that a transgene encoding a truncated green fluorescent protein (GFP) of up to 141 amino acids (aa) can be successfully carried in the 5' end of the coding region. Furthermore, a chimeric virus carrying an eel fluorescent protein, UnaG (139 aa), was also made and shown to be stable, and it maintained efficient tumor cell-killing activity. Similar to other oncolytic viruses, the likelihood of delivering CVA21 by the intravenous route is low due to issues like blood absorption, neutralizing antibodies, and liver clearance. To address this problem, we designed the CVA21 cDNA under the control of a weak RNA polymerase II promoter, and subsequently, a stable cell pool in 293T cells was made by integrating the resulting CVA21 cDNA into the cell genome. We showed that the cells are viable and able to persistently generate rCVA21 de novo. The carrier cell approach described here may pave the way to designing new cell therapy strategies by arming with oncolytic viruses. IMPORTANCE As a naturally occurring virus, coxsackievirus A21 is a promising oncolytic virotherapy modality. In this study, we first used reverse genetics to determine whether A21 can stably carry transgenes and found that it could express up to 141 amino acids of foreign GFP. The chimeric virus carrying another fluorescent eel protein UnaG (139 amino acids) gene also appeared to be stable over at least 7 passages. Our results provided guidance on how to select and engineer therapeutic payloads for future A21 anticancer research. Second, the challenges of delivering oncolytic viruses by the intravenous route hamper the broader use of oncolytic viruses in the clinic. Here, we used A21 to show that cells could be engineered to stably carry and persistently release the virus by harboring the viral cDNA in the genome. The approach we presented here may pave a new way for oncolytic virus administration using cells as carriers.

Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system.

Coxsackievirus A10 (CVA10) is one of enteroviral pathogens that cause the hand, foot, and mouth disease (HFMD). Since CVA10 was reported to be not easily propagated in the Vero cell culture, a feasible manufacture process for producing formalin-inactivated CVA10 vaccine is urgently needed. Several cell lines that commonly used for viral vaccine production was tested for CVA10 (M2014 strain) culture in this study, and our result showed that CVA10 could be easily propagated in the HEK293A cells. A serum-free HEK293A cell culture system was developed for CVA10 production and the yields have reached over 108 TCID50/mL. The biochemical and immunogenic properties of CVA10 particles obtained from this serum-free HEK293A culture were identical to our previous study. Two major particles of CVA10 were separated by ultracentrifugation, and only the infectious mature particles were capable of inducing CVA10 neutralizing antibody responses in the mouse immunogenicity studies. Additionally, we found that coxsackievirus A6 and enterovirus A71 could also be easily propagated using this serum-free HEK293A cell culture system. Our results provide a solution to overcome the obstacle in the propagation of CVA10 and facilitate the development of multivalent vaccines for prevention of HFMD.

The internal ribosome entry site determines the neurotropic potential of enterovirus A71.

The mechanisms underlying tissue-specific replication of enteroviruses are currently unclear. Although enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are both common pathogens that cause hand-foot-mouth disease, they display quite different neurotropic properties. Herein, we characterized the role of the internal ribosome entry site (IRES) in determining neurovirulence using an oral inoculation model of EV-A71. The receptor transgenic (hSCARB2-Tg) mice developed neurological symptoms after being infected with a mouse-adapted EV-A71 strain (MP4) via different administrative routes. Intragastric administration of the MP4 strain caused pathological changes such as neuronal loss and neuropil vacuolation, whereas replacing EV-A71 IRES with CV-A16 abolished the neuropathological phenotypes. The attenuated neurotropic potential of IRES-swapped EV-A71 was observed even in mice that received intraperitoneal and intracerebral inoculations. Fewer chimeric MP4 viral RNAs and proteins were detected in the mouse tissues, regardless of the inoculation route. Tissue-specific replication can be reflected in cell-based characterization. While chimeric MP4 virus replicated poorly in human intestinal C2BBe1 and neuroblastoma SH-SY5Y cells, its replication in susceptible rhabdomyosarcoma cells was not affected. Overall, our results demonstrated that the IRES determined the neurotropic potential of EV-A71 and CV-A16, emphasizing the importance of the IRES in tissue tropism, along with the host receptors.

Humoral and cellular immunogenicity and efficacy of a coxsackievirus A10 vaccine in mice.

Coxsackievirus A10 (CV-A10) has become one of the major pathogens of hand, foot and mouth disease (HFMD), and studies on the vaccine and animal model of CV-A10 are still far from complete. Our study used a mouse-adapted CV-A10 strain, which was lethal for 14-day-old mice, to develop an infected mouse model. Then this model was employed to establish an actively immunized-challenged mouse model to evaluate the efficacy of a formaldehyde-inactivated CV-A10 vaccine, which was prepared from a Vero cell-adapted strain. CV-A10 vaccine at a dose of 0.5 or 2.0 µg was inoculated intraperitoneally in neonatal Kunming mice on the third and ninth day. Then the mice were challenged on day 14. The survival rate of mice immunized with 0.5 or 2.0 µg vaccine were 90% and 100%, respectively, while all Alum-inoculated mice died. Compared to those in the two vaccinated groups, the Alum-inoculated mice showed severe pathological damage, strong viral protein expression and high viral loads. The antisera from vaccinated mice showed high level of neutralizing antibodies against CV-A10. Meanwhile, three potential T cell epitopes located at the carboxyl-terminal regions of the VP1 and VP3 were identified and exhibited CV-A10 serotype-specific. The humoral and cellular immunogenicity analysis showed that immunization with two doses of the vaccine elicited CV-A10 specific neutralizing antibody and T cell response in BALB/c mice. Collectively, these findings indicated that this actively immunized-challenged mouse model will be invaluable in future studies on CV-A10 pathogenesis and evaluation of vaccine candidates.