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Coxsackievirus infections [keywords]
- Tear-mediated delivery of nanoparticles through transcytosis of the lacrimal gland. [JOURNAL ARTICLE]
- J Control Release 2014 Dec 16.
Rapid clearance from the tears presents a formidable obstacle to the delivery of peptide drugs to the eye surface. This impedes therapies for ocular infections, wound healing, and dry-eye disease that affect the vision of millions worldwide. To overcome this challenge, this manuscript explores a novel strategy to reach the ocular surface via receptor-mediated transcytosis across the lacrimal gland (LG), which produces the bulk of human tears. The LG abundantly expresses the coxsackievirus and adenovirus receptor (CAR); furthermore, we recently reported a peptide-based nanoparticle (KSI) that targets CAR on liver cells. This manuscript reports the unexpected finding that KSI both targets and transcytoses into the LG acinar lumen, which drains to tear ducts. When followed using ex vivo live cell imaging KSI rapidly accumulates in lumen formed by LG acinar cells. LG transduction with a myosin Vb tail, which is dominantly negative towards transcytosis, inhibits lumenal accumulation. Transcytosis of KSI was confirmed in vivo by confocal and TEM imaging of LG tissue following administration of KSI nanoparticles. These findings suggest that it is possible to target nanomaterials to the tears by targeting certain receptors on the LG. This design strategy represents a new opportunity to overcome barriers to ocular delivery.
- BPIFB3 Regulates Autophagy and Coxsackievirus B Replication through a Noncanonical Pathway Independent of the Core Initiation Machinery. [Journal Article]
- MBio 2014; 5(6)
Enteroviruses require autophagy to facilitate the formation of autophagosome (AP)-like double-membrane vesicles that provide the scaffolding for RNA replication. Here, we identify bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3) as a gene whose silencing greatly enhances coxsackievirus B (CVB) replication and induces dramatic alterations in the morphology of CVB-induced replication organelles. We show that BPIFB3 is associated with the endoplasmic reticulum (ER), and its silencing by RNA interference enhances basal levels of autophagy and promotes increased autophagy during CVB replication. Conversely, overexpression of BPIFB3 inhibits CVB replication, dramatically alters the morphology of LC3B-positive vesicles, and suppresses autophagy in response to rapamaycin. In addition, we found that, whereas silencing of core autophagy components associated with the initiation of APs in control cells suppressed CVB replication, silencing of these same components had no effect on CVB-induced autophagy or viral replication in cells transfected with BPIFB3 small interfering RNA. Based on these results, taken together, this study reports on a previously uncharacterized regulator of enterovirus infection that controls replication through a noncanonical pathway independent from the core autophagy initiation machinery.Coxsackievirus B (CVB) infections are commonly associated with dilated cardiomyopathy, a condition that accounts for nearly half of all heart transplants annually. During infection, CVB co-opts a cellular pathway, termed autophagy, to provide the membranes necessary for its replication. Autophagy is an evolutionarily conserved process by which cells ingest damaged organelles as a means of maintaining cell homeostasis. Here, we report on a novel regulator of autophagy, bactericidal/permeability-increasing protein (BPI) fold-containing family B, member 3 (BPIFB3), whose expression functions to restrict CVB replication by suppressing key steps in the authophagic process. We show that loss of BPIFB3 expression greatly enhances CVB replication while having no effect on replication of poliovirus, a closely related virus. Our results thus identify a novel host cell therapeutic target whose function could be targeted to alter CVB replication.
- Evaluation of monovalent and bivalent vaccines against lethal Enterovirus 71 and Coxsackievirus A16 infection in newborn mice. [JOURNAL ARTICLE]
- Hum Vaccin Immunother 2014 Aug 21.:e29823.
Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) have caused severe epidemics of hand, foot and mouth disease (HFMD) in the Asia Pacific in recent years, particularly in infants and young children. This disease has become a serious public health problem, as no vaccines or antiviral drugs have been approved for EV71 and CA16 infections. In this study, we compared four monovalent vaccines, including formalin-inactivated EV71 virus (iEV71), EV71 virus-like particles (VLPs) (vEV71), formalin-inactivated CVA16 virus (iCVA16) and CVA16 VLPs (vCVA16), along with two bivalent vaccines, including equivalent doses of formalin-inactivated EV71+CVA16 virus (iEV71+iCVA16) and EV71+CVA16 VLPs (vEV71+vCVA16). The IgG titers and neutralization antibodies titers demonstrated that there are no immune interference exists between the two immunogens of EV71 and CVA16. IgG subclass isotyping revealed that IgG1 and IgG2b were induced primarily in all vaccine groups. Furthermore, cross-neutralization antibodies were elicited in mouse sera against other sub-genotypes of EV71 and CVA16. In vivo challenge experiments showed that the immune sera from vaccinated animals could confer passive protection to newborn mice against lethal challenge with 14 LD50 of EV71 and 50 LD50 of CVA16. Our results indicated that bivalent vaccination is promising for HFMD vaccine development. With the advantage of having a better safety profile than inactivated virus vaccines, VLPs should be used to combine both EV71 and CVA16 antigens as a candidate vaccine for prevention of HFMD virus transmission.
- Towards broadly protective polyvalent vaccines against hand, foot and mouth disease. [JOURNAL ARTICLE]
- Microbes Infect 2014 Nov 29.
Hand, foot, and mouth disease (HFMD) caused by multiple enterovirus infections is a serious health threat to children in the Asia-Pacific region. This article reviews progresses in the development of vaccines for HFMD and discusses the need for polyvalent HFMD vaccines for conferring broad-spectrum protection.
- Protease 2A induces stress granule formation during coxsackievirus B3 and enterovirus 71 infections. [JOURNAL ARTICLE]
- Virol J 2014 Nov 20; 11(1):192.
BackgroundStress granules (SGs) are granular aggregates in the cytoplasm that are formed under a variety of stress situations including viral infection. Previous studies indicate that poliovirus, a member of Picornaviridae, can induce SG formation. However, the exact mechanism by which the picornaviruses induce SG formation is unknown.MethodThe localization of SG markers in cells infected with coxsackievirus B3 (CVB3) or enterovirus 71 (EV71) and in cells expressing each viral protein was determined via immunofluorescence assays or plasmid transfection. Eight plasmids expressing mutants of the 2A protease (2Apro) of CVB3 were generated using a site-directed mutagenesis strategy. The cleavage efficiencies of eIF4G by CVB3 2Apro, and its mutants were determined via western blotting assays.ResultsIn this study, we found that CVB3 infection induced SG formation, as evidenced by the co-localization of some accepted SG markers in viral infection-induced granules. Furthermore, we identified that 2Apro of CVB3 was the key viral component that triggered SG formation. A 2Apro mutant with the G122E mutation, which exhibited very low cleavage efficiency toward eIF4G, significantly attenuated its capacity for SG induction, indicating that the protease activity was required for 2Apro to initiate SG formation. Finally, we observed that SGs also formed in EV71-infected cells. Expression of EV71 2Apro alone was also sufficient to cause SG formation.ConclusionBoth CVB3 and EV71 infections can induce SG formation, and 2Apro plays a crucial role in the induction of SG formation during these infections. This finding may help us to better understand how picornaviruses initiate the SG response.
- Platelets interact with Coxsackieviruses B and have a critical role in the pathogenesis of virus-induced myocarditis. [JOURNAL ARTICLE]
- J Thromb Haemost 2014 Nov 13.
To further understand the role of platelets in the pathogenesis of viral infections we explored platelet interaction with Coxsackievirus B (CVB) 1 and 3. CVB is a group of viruses that cause the majority of human enterovirus-related viral myocarditis; their receptor (CAR) is expressed on the platelet surface and there is a well characterized CVB3-induced myocarditis murine model.Human platelets were infected with CVB1 and 3 and viruses were detected in pellets and in supernatants. C57BL/6J mice with or without platelet depletion were inoculated with CVB3 and peripheral blood and heart samples collected at different times post-infection.CVB1 and 3 RNA and a capsid protein were detected in infected platelets. Despite the fact that titration assays in Vero cells showed increasing infectivity titers over time, supernatants and pellets from infected platelets showed similar levels, suggesting that platelets were not susceptible to a replicative infectivity cycle. CVB binding was CAR-independent and resulted in P-selectin and phosphatidylserine (PS) exposure. CVB3-infected mice showed a rapid thrombocytopenia that correlated with an increase in platelet PS exposure and platelet-leukocyte aggregates without modification of platelet P-selectin expression or von Willebrand factor levels. Mortality, viremia, heart viral titers and myocarditis were significantly higher in platelet-depleted than normal animals. Type I IFN levels were not changed but IgG levels were lower in infected and platelet-depleted mice.Our data reveal that platelets play a critical role in host survival and immune response against CVB3 infection. This article is protected by copyright. All rights reserved.
- Elicitation of T cell responses by structural and non-structural proteins of coxsackievirus B4. [JOURNAL ARTICLE]
- J Gen Virol 2014 Nov 7.
Coxsackievirus B4 (CV-B4) belongs to the genus Enterovirus within family Picornaviridae. To investigate target proteins recognised by T-cells in human enterovirus B infections, viral-encoded structural (VP0 [VP4 and VP2], VP1, VP3) and non-structural (2A, 2B, 2C, 3C and 3D) proteins were expressed and purified in E. coli. Peripheral blood of 19 healthy adult donors was used to create enterovirus-specific T-cell lines by repeated stimulation with CV-B4 cell lysate antigen. T-cell lines responded in individual patterns, and responses to all purified proteins were observed. The most often recognised enteroviral protein was VP0, which is the fusion between the most conserved structural proteins, VP4 and VP2. T-cell responses to VP0 were detected in 15 of 19 (79%) donor lines. Non-structural 2C protein was recognised in 11 of 19 (58%) lines, and 11 of 19 (58%) lines also had a response to 3D protein. Furthermore, responses to other non-structural proteins (2A, 2B and 3C) were also detected. T-cell responses did not correlate clearly to the individual HLA-DR-DQ phenotype or the history of past coxsackie B virus infections of the donors.
- A preclinical study on the efficacy and safety of a new vaccine against Coxsackievirus B1 reveals no risk for accelerated diabetes development in mouse models. [JOURNAL ARTICLE]
- Diabetologia 2014 Nov 5.
Enterovirus infections have been implicated in the aetiology of autoimmune type 1 diabetes. A vaccine could be used to test the causal relationship between enterovirus infections and diabetes development. However, the development of a vaccine against a virus suspected to induce an autoimmune disease is challenging, since the vaccine itself might trigger autoimmunity. Another challenge is to select the enterovirus serotypes to target with a vaccine. Here we aimed to evaluate the function and autoimmune safety of a novel non-adjuvanted prototype vaccine to Coxsackievirus serotype B1 (CVB1), a member of the enterovirus genus.A formalin-inactivated CVB1 vaccine was developed and tested for its immunogenicity and safety in BALB/c and NOD mice. Prediabetic NOD mice were vaccinated, infected with CVB1 or mock-treated to compare the effect on diabetes development.Vaccinated mice produced high titres of CVB1-neutralising antibodies without signs of vaccine-related side effects. Vaccinated mice challenged with CVB1 had significantly reduced levels of replicating virus in their blood and the pancreas. Prediabetic NOD mice demonstrated an accelerated onset of diabetes upon CVB1 infection whereas no accelerated disease manifestation or increased production of insulin autoantibodies was observed in vaccinated mice.We conclude that the prototype vaccine is safe and confers protection from infection without accelerating diabetes development in mice. These results encourage the development of a multivalent enterovirus vaccine for human use, which could be used to determine whether enterovirus infections trigger beta cell autoimmunity and type 1 diabetes in humans.
- Review of Enterovirus 71 Vaccines. [JOURNAL ARTICLE]
- Clin Infect Dis 2014 Oct 28.
Enterovirus 71 (EV71) and coxsackieviruses are the major causative agents of hand, foot, and mouth disease (HFMD) outbreaks worldwide and have a significant socioeconomic impact, particularly in Asia. Formalin-inactivated (FI) EV71 vaccines evaluated in human clinical trials in China, Taiwan, and Singapore were found to be safe and to elicit strong neutralizing antibody responses against EV71 currently circulating in Asia. The results from 3 different phase 3 clinical trials performed in young children (6-60 months) indicate that the efficacy of FI-EV71 vaccines is >90% against EV71-related HFMDs and >80% against EV71-associated serious diseases, but the vaccines did not protect against coxsackievirus A16 infections. Here we discuss the critical factors affecting EV71 vaccine product registration, including clinical epidemiology, antigenic shift issues in cross-protection and vaccine strain selection, standardized animal models for potency testing, and cost-effective manufacturing processes for potential incorporation of FI-EV71 vaccine into Expanded Programme on Immunization vaccines.
- Emergence of enterovirus 71 C4a in Denmark, 2009 to 2013. [Journal Article]
- Euro Surveill 2014; 19(38)
Enterovirus (EV) 71 has emerged as a primary cause of severe neurologic enterovirus infection in the aftermath of the global polio eradication effort. Eleven subgenotypes of EV71 exist, the C4 subgenotype being associated with large outbreaks in Asia with high mortality rates. This subgenotype has rarely been reported in Europe. In the period between 1 January 2009 and 31 December 2013 a total of 1,447 EV positive samples from 1,143 individuals were sent to the Statens Serum Institute (SSI), and 938 samples from 913 patients were genotyped at the Danish National World Health Organization Reference laboratory for Poliovirus at SSI. Echovirus 6 (E06) (n=141 patients), echovirus 30 (E30) (n=114), coxsackievirus A6 (CA06) (n=96) and EV71 (n=63) were the most prevalent genotypes. We observed a shift in circulating EV71 subgenotypes during the study period, with subgenotype C4 dominating in 2012. A total of 34 EV71 patients were found to be infected with strains of the C4 subgenotype, and phylogenetic analysis revealed that they belonged to the C4a lineage. In our study, the proportions of cases with cerebral and/or sepsis-like symptoms were similar in those affected by C4a (19/34) and those with C1 and C2 (15/35). The majority (n=30) of the 34 EV71 C4 cases were children≤5 years of age, and males (n=22) were over-represented. Continued EV surveillance is required to monitor the spread of EV71 C4 in Denmark and the rest of Europe.