A 3-month-old neonatal rhesus macaque HFMD model caused by coxsackievirus B1 infection and viral tissue tropism.

Coxsackievirus B1 (CVB1), an enterovirus with multiple clinical presentations, has been associated with potential long-term consequences, including hand, foot, and mouth disease (HFMD), in some patients. However, the related animal models, transmission dynamics, and long-term tissue tropism of CVB1 have not been systematically characterized. In this study, we established a model of CVB1 respiratory infection in rhesus macaques and evaluated the clinical symptoms, viral load, and immune levels during the acute phase (0-14 days) and long-term recovery phase (15-30 days). We also investigated the distribution, viral clearance, and pathology during the long-term recovery period using 35 postmortem rhesus macaque tissue samples collected at 30 days postinfection (d.p.i.). The results showed that the infected rhesus macaques were susceptible to CVB1 and exhibited HFMD symptoms, viral clearance, altered cytokine levels, and the presence of neutralizing antibodies. Autopsy revealed positive viral loads in the heart, spleen, pancreas, soft palate, and olfactory bulb tissues. HE staining demonstrated pathological damage to the liver, spleen, lung, soft palate, and tracheal epithelium. At 30 d.p.i., viral antigens were detected in visceral, immune, respiratory, and muscle tissues but not in intestinal or neural tissues. Brain tissue examination revealed viral meningitis-like changes, and CVB1 antigen expression was detected in occipital, pontine, cerebellar, and spinal cord tissues at 30 d.p.i. This study provides the first insights into CVB1 pathogenesis in a nonhuman primate model of HFMD and confirms that CVB1 exhibits tissue tropism following long-term infection.

A Coxsackievirus B1-mediated nonlytic Extracellular Vesicle-to-cell mechanism of virus transmission and its possible control through modulation of EV release.

Like most non-enveloped viruses, CVB1 mainly uses cell lysis to spread. Details of a nonlytic virus transmission remain unclear. Extracellular Vesicles (EVs) transfer biomolecules between cells. We show that CVB1 entry into HeLa cells results in apoptosis and release of CVB1-induced 'medium-sized' EVs (CVB1i-mEVs). These mEVs (100-300 nm) harbour CVB1 as shown by immunoblotting with anti-CVB1-antibody; viral capsids were detected by transmission electron microscopy and RT-PCR revealed CVB1 RNA. The percentage of mEVs released from CVB1-infected HeLa cells harbouring virus was estimated from TEM at 34 %. Inhibition of CVB1i-mEV production, with calpeptin or siRNA knockdown of CAPNS1 in HeLa cells limited spread of CVB1 suggesting these vesicles disseminate CVB1 virions to new host cells by a nonlytic EV-to-cell mechanism. This was confirmed by detecting CVB1 virions inside HeLa cells after co-culture with CVB1i-mEVs; EV release may also prevent apoptosis of infected cells whilst spreading apoptosis to secondary sites of infection.

Salt Enhances the Thermostability of Enteroviruses by Stabilizing Capsid Protein Interfaces.

Enteroviruses are common agents of infectious disease that are spread by the fecal-oral route. They are readily inactivated by mild heat, which causes the viral capsid to disintegrate or undergo conformational change. While beneficial for the thermal treatment of food or water, this heat sensitivity poses challenges for the stability of enterovirus vaccines. The thermostability of an enterovirus can be modulated by the composition of the suspending matrix, though the effects of the matrix on virus stability are not understood. Here, we determined the thermostability of four enterovirus strains in solutions with various concentrations of NaCl and different pH values. The experimental findings were combined with molecular modeling of the protein interaction forces at the pentamer and the protomer interfaces of the viral capsids. While pH only had a modest effect on thermostability, increasing NaCl concentrations raised the breakpoint temperatures of all viruses tested by up to 20°C. This breakpoint shift could be explained by an enhancement of the van der Waals attraction forces at the two protein interfaces. In comparison, the (net repulsive) electrostatic interactions were less affected by NaCl. Depending on the interface considered, the breakpoint temperature shifted by 7.5 or 5.6°C per 100-kcal/(mol·Å) increase in protein interaction force.IMPORTANCE The genus Enterovirus encompasses important contaminants of water and food (e.g., coxsackieviruses), as well as viruses of acute public health concern (e.g., poliovirus). Depending on the properties of the surrounding matrix, enteroviruses exhibit different sensitivities to heat, which in turn influences their persistence in the environment, during food treatment, and during vaccine storage. Here, we determined the effect of NaCl and pH on the heat stability of different enteroviruses and related the observed effects to changes in protein interaction forces in the viral capsid. We demonstrate that NaCl renders enteroviruses thermotolerant and that this effect stems from an increase in van der Waals forces at different protein subunits in the viral capsid. This work sheds light on the mechanism by which salt enhances virus stability.

Coxsackievirus B1 reveals strain specific differences in plasmacytoid dendritic cell mediated immunogenicity.

Enterovirus infections are usually mild but can also cause severe illnesses and play a role in chronic diseases, such as cardiomyopathies and type 1 diabetes. Host response to the invading virus can markedly modulate the course of the infection, and this response varies between individuals due to the polymorphism of immune response genes. However, it is currently not known if virus strains also differ in their ability to stimulate the host immune system. Coxsackievirus B1 (CBV1) causes severe epidemics in young infants and it has recently been connected with type 1 diabetes in seroepidemiological studies. This study evaluated the ability of different field isolates of CBV1 to induce innate immune responses in PBMCs. CBV1 strains differed markedly in their capacity to induce innate immune responses. Out of the 18 tested CBV1 strains two induced exceptionally strong alpha interferon (IFN-α) response in PBMC cultures. The responding cell type was found to be the plasmacytoid dendritic cell. Such a strong innate immune response was accompanied by an up-regulation of several other immune response genes and secretion of cytokines, which modulate inflammation, and adaptive immune responses. These results suggest that enterovirus-induced immune activation depends on the virus strain. It is possible that the immunotype of the virus modulates the course of the infection and plays a role in the pathogenesis of chronic immune-mediated enterovirus diseases.

Screening of a new candidate coxsackievirus B1 vaccine strain based on its biological characteristics.

Coxsackievirus B1 (CVB1) is one of the significant pathogens causing viral myocarditis, hand, foot, and mouth disease (HFMD), and aseptic meningitis, and it has been associated with type 1 diabetes (T1DM). No effective antiviral drugs against CVB1 infection or preventive vaccines are available. Due to the success of two inactivated vaccines against enterovirus 71 and poliovirus, an inactivated Vero cell-based CVB1 vaccine could be developed. In this study, we isolated a high-growth CVB1 virus strain KM7 in Vero cells and developed a Vero-adapted vaccine candidate strain KM7-X29 via three rounds of plaque purification and serial passages. The KM7-X29 strain was grouped into the GII sub-genotype, which belonged to the Chinese epidemic strain and grew to a titer of more than 107 CCID50/ml in Vero cells. The inactivated CVB1 vaccine produced by the KM7-X29 strain induced an effective neutralizing antibody response in BALB/c mice, and maternal antibodies were able to provide a 100% protective effect against lethal challenges with a CVB1 strain in suckling BALB/c mice. Thus, the KM7-X29 strain might be used as a new candidate coxsackievirus B1 vaccine strain. The neonatal murine model of CVB1 infection will contribute to the development of the CVB1 vaccine.

Development of a rapid neutralization assay for the detection of neutralizing antibodies against coxsackievirus B1.

Coxsackievirus B1 (CVB1) is a major pathogen that causes viral myocarditis and aseptic meningitis and is implicated as a cause of type 1 diabetes mellitus. The rapid detection of neutralizing antibodies can help in the prevention and diagnosis of viral infection. The traditional cytopathic effect (CPE)-based neutralization assay (Nt-CPE) is time-consuming and labor-intensive. In this study, an efficient neutralization test based on an enzyme-linked immunospot assay and a monoclonal antibody 2E6 against CVB1 (Nt-Elispot) was developed. In this optimal Nt-Elispot, a multiplicity of infection (MOI) of 1 per well was set as the infection dose, and an incubation time of 18 hours was selected as the checkpoint. Compared with Nt-CPE, Nt-Elispot significantly shortened the detection period and displayed a good correlation with it. This established CVB1 Nt-Elispot could be applied to efficiently screen neutralizing antibodies and evaluate the level of NAb against CVB1 in large cohorts.

In vitro antiviral activities of fruit extract from Lycium barbarum and methylxanthines extracted from Pu-erh and Bancha tea leaves.

INTRODUCTION: Based on traditional medicine, many countries use various plant products (fruits, leaves and other plant parts) as food supplements or in the form of tea. The use of these plant sources has been established through the years of use and the proven benefits of their ingredients to improve human health.

Development of A Neonatal Mouse Model for Coxsackievirus B1 Antiviral Evaluation.

Coxsackievirus B1 (CVB1) is a leading causative agent of severe infectious diseases in humans and has been reported to be associated with outbreaks of aseptic meningitis, myocarditis, and the development of chronic diseases such as type 1 diabetes mellitus (T1DM). There is no approved vaccine or effective antiviral therapy to treat CBV1 infection. And animal models to assess the effects of antiviral agents and vaccine remain limited. In this study, we established a neonatal mouse model of CVB1 using a clinically isolated strain to characterize the pathological manifestations of virus infection and to promote the development of vaccines and antiviral drugs against CVB1. One-day-old BALB/c mice were susceptible to CVB1 infection by intraperitoneal injection. Mice challenged with CVB1 at a low dose [10 median tissue culture infective dose (TCID50)] exhibited a series of clinical symptoms, such as inactivity, emaciation, limb weakness, hair thinning, hunching and even death. Pathological examination and tissue viral load analysis showed that positive signals of CVB1 were detected in the heart, spinal cord, limb muscle and kidney without pathological damage. Particularly, CVB1 had a strong tropism towards the pancreas, causing severe cellular necrosis with inflammatory infiltration, and was spread by viraemia. Notably, the monoclonal antibody (mAb) 6H5 and antisera elicited from CVB1-vaccinated mice effectively protected the mice from CVB1 infection in the mouse model. In summary, the established neonatal mouse model is an effective tool for evaluating the efficacy of CVB1 antiviral reagents and vaccines.

Mt10-CVB3 Vaccine Virus Protects against CVB4 Infection by Inducing Cross-Reactive, Antigen-Specific Immune Responses.

Group B coxsackieviruses (CVB) containing six serotypes, B1-B6, affect various organs, and multiple serotypes can induce similar diseases such as myocarditis and pancreatitis. Yet, no vaccines are currently available to prevent these infections. Translationally, the derivation of vaccines that offer protection against multiple serotypes is highly desired. In that direction, we recently reported the generation of an attenuated strain of CVB3, termed Mt10, which completely protects against both myocarditis and pancreatitis induced by the homologous wild-type CVB3 strain. Here, we report that the Mt10 vaccine can induce cross-protection against multiple CVB serotypes as demonstrated with CVB4. We note that the Mt10 vaccine could induce cross-reactive neutralizing antibodies (nABs) against both CVB1 and CVB4. In challenge studies with CVB4, the efficacy of the Mt10 vaccine was found to be 92%, as determined by histological evaluation of the heart and pancreas. Antibody responses induced in Mt10/CVB4 challenged animals indicated the persistence of cross-reactive nABs against CVB1, CVB3, and CVB4. Evaluation of antigen-specific immune responses revealed viral protein 1 (VP1)-reactive antibodies, predominantly IgG2a, IgG2b, IgG3, and IgG1. Similarly, by using major histocompatibility complex class II tetramers, we noted induction of VP1-specific CD4 T cells capable of producing multiple T cell cytokines, with interferon-γ being predominant. Finally, none of the vaccine recipients challenged with CVB4 revealed the presence of viral nucleic acid in the heart or pancreas. Taken together, our data suggest that the Mt10 vaccine can prevent infections caused by multiple CVB serotypes, paving the way for the development of monovalent CVB vaccines to prevent heart and pancreatic diseases of enteroviral origin.

Genetic Adaptation of Coxsackievirus B1 during Persistent Infection in Pancreatic Cells.

Coxsackie B (CVB) viruses have been associated with type 1 diabetes. We have recently observed that CVB1 was linked to the initiation of the autoimmune process leading to type 1 diabetes in Finnish children. Viral persistency in the pancreas is currently considered as one possible mechanism. In the current study persistent infection was established in pancreatic ductal and beta cell lines (PANC-1 and 1.1B4) using four different CVB1 strains, including the prototype strain and three clinical isolates. We sequenced 5' untranslated region (UTR) and regions coding for structural and non-structural proteins and the second single open reading frame (ORF) protein of all persisting CVB1 strains using next generation sequencing to identify mutations that are common for all of these strains. One mutation, K257R in VP1, was found from all persisting CVB1 strains. The mutations were mainly accumulated in viral structural proteins, especially at BC, DE, EF loops and C-terminus of viral capsid protein 1 (VP1), the puff region of VP2, the knob region of VP3 and infection-enhancing epitope of VP4. This showed that the capsid region of the viruses sustains various changes during persistency some of which could be hallmark(s) of persistency.

Fulminant type 1 diabetes mellitus associated with Coxsackievirus type B1 infection during pregnancy: a case report.

BACKGROUND: Fulminant type 1 diabetes is characterized by an intrinsic insulin deficiency resulting from the severe destruction of pancreatic ß cells and it rapidly leads to ketoacidosis. However, the association between fulminant type 1 diabetes in pregnancy and specific viral infections has not been reported. CASE PRESENTATION: The patient in this study was a 31-year-old Japanese woman, and at 30 weeks of pregnancy she was admitted with marked fatigue. Fetal bradycardia was noted, and the child was delivered by emergency cesarean section but was stillborn. The maternal blood sugar level was high (427 mg/dL), but the glycated hemoglobin value was 6.2%; therefore, fulminant type 1 diabetes was suspected. Serum antibody testing confirmed a Coxsackievirus B1 infection. The patient in this case had fulminant type 1 diabetes in pregnancy associated with Coxsackievirus B1. CONCLUSION: This case highlights that fulminant type 1 diabetes in pregnancy may be associated with Coxsackievirus B1 infection.

Optimized production and purification of Coxsackievirus B1 vaccine and its preclinical evaluation in a mouse model.

Coxsackie B viruses are among the most common enteroviruses, causing a wide range of diseases. Recent studies have also suggested that they may contribute to the development of type 1 diabetes. Vaccination would provide an effective way to prevent CVB infections, and the objective of this study was to develop an efficient vaccine production protocol for the generation of novel CVB vaccines. Various steps in the production of a formalin-inactivated Coxsackievirus B1 (CVB1) vaccine were optimized including the Multiplicity Of Infection (MOI) used for virus amplification, virus cultivation time, type of cell growth medium, virus purification method and formulation of the purified virus. Safety and immunogenicity of the formalin inactivated CVB1 vaccine was characterized in a mouse model. Two of the developed methods were found to be optimal for virus purification: the first employed PEG-precipitation followed by gelatin-chromatography and sucrose cushion pelleting (three-step protocol), yielding 19-fold increase in virus concentration (0.06µg/cm2) as compared to gold standard method. The second method utilized tandem sucrose pelleting without a PEG precipitation step, yielding 83-fold increase in virus concentration (0.24µg/cm2), but it was more labor-intensive and cannot be efficiently scaled up. Both protocols provide radically higher virus yields compared with traditional virus purification protocols involving PEG-precipitation and sucrose gradient ultracentrifugation. Formalin inactivation of CVB1 produced a vaccine that induced a strong, virus-neutralizing antibody response in vaccinated mice, which protected against challenge with CVB1 virus. Altogether, these results provide valuable information for the development of new enterovirus vaccines.