Mutations in the non-structural protein region contribute to intra-genotypic evolution of enterovirus 71

Background

Clinical manifestations of enterovirus 71 (EV71) range from herpangina, hand-foot-and-mouth disease (HFMD), to severe neurological complications. Unlike the situation of switching genotypes seen in EV71 outbreaks during 1998–2008 in Taiwan, genotype B5 was responsible for two large outbreaks in 2008 and 2012, respectively. In China, by contrast, EV71 often persists as a single genotype in the population and causes frequent outbreaks. To investigate genetic changes in viral evolution, complete EV71 genome sequences were used to analyze the intra-genotypic evolution pattern in Taiwan, China, and the Netherlands.

Results

Genotype B5 was predominant in Taiwan’s 2008 outbreak and was re-emergent in 2012. EV71 strains from both outbreaks were phylogenetically segregated into two lineages containing fourteen non-synonymous substitutions predominantly in the non-structural protein coding region. In China, genotype C4 was first seen in 1998 and caused the latest large outbreak in 2008. Unlike shifting genotypes in Taiwan, genotype C4 persisted with progressive drift through time. A majority of non-synonymous mutations occurred in residues located in the non-structural coding region, showing annual increases. Interestingly, genotype B1/B2 in the Netherlands showed another stepwise evolution with dramatic EV71 activity increase in 1986. Phylogeny of the VP1 coding region in 1971–1986 exhibited similar lineage turnover with genotype C4 in China; however, phylogeny of the 3D-encoding region indicated separate lineage appearing after 1983, suggesting that the 3D-encoding region of genotype B2 was derived from an unidentified ancestor that contributed to intra-genotypic evolution in the Netherlands.

Conclusions

Unlike VP1 coding sequences long used for phylogenetic study of enteroviruses due to expected host immune escape, our study emphasizes a dominant role of non-synonymous mutations in non-structural protein regions that contribute to (re-)emergent genotypes in continuous stepwise evolution. Dozens of amino acid substitutions, especially in non-structural proteins, were identified via genetic changes driven through intra-genotypic evolution worldwide. These identified substitutions appeared to increase viral fitness in the population, affording valuable insights not only for viral evolution but also for prevention, control, and vaccine against EV71 infection.     

Population Dynamics and Genetic Diversity of C4 Strains of Human Enterovirus 71 in Mainland China, 1998–2010

Background

Since 1997, several countries within the Asian Pacific region have been affected by one or more massive outbreaks of Hand Foot and Mouth Disease (HFMD). Virus typing experiments revealed that these outbreaks were caused by strains of human enterovirus 71 (EV71) belonging to several different, recently emerged subgenogroups. In mainland China, a different situation was observed. The first outbreak, localized in Shangdong Province, was reported in 2007, and was followed by a wide-spread outbreak in mainland China in 2008. Since then, numbers of reported HFMD cases have been persistently high.

Methodology/Principal Findings

To gain insight in the epidemiological behavior of EV71 in China, we studied genetic diversity and EV71 population dynamics to address whether the increase in number of reported EV71 infections reflects a real increase in viral spread or is just the result of increased awareness and surveillance. We used systematically collected VP1 gene sequences of 257 EV71 strains collected in Guangdong province from 2008 to 2010 as part of HFMD surveillance activities, and supplemented them with 305 GenBank EV71 reference stains collected in China from 1998 to 2010. All isolates from Guangdong Province belonged to subgenogroup C4. Viral population dynamics indicated that the increased reporting of HFMD in China since 2007 reflects a real increase in viral spread and continued replacement of viral lineages through time. Amino acid sequence comparisons revealed substitution of amino acid in residues 22, 145 and 289 through time regularly with the VP1 gene of EV71 strains isolated in mainland China from 1998 to 2010.

Conclusions

EV71 strains isolated in mainland China mainly belonged to subgenogroup C4. There was exponential growth of the EV71 virus population in 2007 and 2008. There was amino acid substitution through time regularly with the VP1 gene which possibly increased viral spread and/or ability of the virus to circulate persistently among the Chinese population.     

Seroprevalence of Human Enterovirus 71 and Coxsackievirus A16 in Guangdong,China, in Pre- and Post-2010 HFMD Epidemic Period

Background

Human Enterovirus 71 and Coxsackie A16 have caused many outbreaks in the last decade in mainland China, resulting in thousands of fatal cases. Seroepidemiology which provides important information to document population immunity is rare in China.

Methodology/Principal Findings

A cross sectional study of Enterovirus 71 (EV71) and Coxsackie A16 (CA16) seroprevalence was carried out in Guangdong, China, pre- and post- the 2010 hand, foot and mouth disease (HFMD) epidemic period. The levels of EV71 and CA16 specific antibodies were evaluated by a microneutralization test and the geometric mean titer (GMT) was calculated and compared. Our results indicated frequent infection by EV71 and CA16 in Guangdong before the 2010 epidemic. Only EV71 neutralizing antibody but not CA16 seroprevalence was significantly increased after the 2010 HFMD epidemic. Children less than 3 years old especially those aged 2 years showed the lowest positive rates for EV71 and CA16 NA before epidemic and the most significantly increased EV71 seroprevalence after epidemic. CA16 GMT values declined after the 2010 epidemic.

Conclusions

These results indicate EV71 was the major pathogen of HFMD in Guangdong during the 2010 epidemic. The infection occurs largely in children less than 3 years, who should have first priority to receive an EV71 vaccine.     

Incidence rates of enterovirus 71 infections in young children during a nationwide epidemic in Taiwan, 2008-09

Objective

Enterovirus 71 (EV71) is causing life-threatening outbreaks in tropical Asia. In Taiwan and other tropical Asian countries, although nationwide EV71 epidemics occur cyclically, age-specific incidence rates of EV71 infections that are critical to estimate disease burden and design vaccine trials are not clear. A nationwide EV71 epidemic occurred in 2008–09 in Taiwan, which provided a unique opportunity to estimate age-specific incidence rates of EV71 infections.

Study Design

We prospectively recruited 749 healthy neonates and conducted follow-ups from June 2006 to December 2009. Sera were obtained from participants at 0, 6, 12, 24, and 36 months of age for measuring EV71 neutralizing antibody titers. If the participants developed suspected enterovirus illnesses, throat swabs were collected for virus isolation.

Results

We detected 28 EV71 infections including 20 symptomatic and 8 asymptomatic infections. Age-specific incidence rates of EV71 infection increased from 1.71 per 100 person-years at 0–6 months of age to 4.09, 5.74, and 4.97 per 100 person-years at 7–12, 13–24, and 25–36 months of age, respectively. Cumulative incidence rate was 15.15 per 100 persons by 36 months of age, respectively.

Conclusions

Risk of EV71 infections in Taiwan increased after 6 months of age during EV71 epidemics. The cumulative incidence rate was 15% by 36 months of age, and 29% of EV71 infections were asymptomatic in young children.     

Phylogenetic evidence for inter-typic recombination in the emergence of human enterovirus 71 subgenotypes

Background  

Human enterovirus 71 (EV-71) is a common causative agent of hand, foot and mouth disease (HFMD). In recent years, the virus has caused several outbreaks with high numbers of deaths and severe neurological complications. Several new EV-71 subgenotypes were identified from these outbreaks. The mechanisms that contributed to the emergence of these subgenotypes are unknown.     

Display of VP1 on the surface of baculovirus and its immunogenicity against heterologous human enterovirus 71 strains in mice

Background

Human Enterovirus 71 (EV71) is a common cause of hand, foot and mouth disease (HFMD) in young children. It is often associated with severe neurological diseases and has caused high mortalities in recent outbreaks across the Asia Pacific region. Currently, there is no effective vaccine and antiviral agents available against EV71 infections. VP1 is one of the major immunogenic capsid protein of EV71 and plays a crucial role in viral infection. Antibodies against VP1 are important for virus neutralization.

Methodology/Principal Finding

In the present study, infectious EV71 viruses were generated from their synthetic complementary DNA using the human RNA polymerase I reverse genetics system. Secondly, the major immunogenic capsid protein (VP1) of EV71-Fuyang (subgenogroup C4) was displayed on the surface of recombinant baculovirus Bac-Pie1-gp64-VP1 as gp64 fusion protein under a novel White Spot Syndrome Virus (WSSV) immediate early ie1 promoter. Baculovirus expressed VP1 was able to maintain its structural and antigenic conformity as indicated by immunofluorescence assay and western blot analysis. Interestingly, our results with confocal microscopy revealed that VP1 was able to localize on the plasma membrane of insect cells infected with recombinant baculovirus. In addition, we demonstrated with transmission electron microscopy that baculovirus successfully acquired VP1 from the insect cell membrane via the budding process. After two immunizations in mice, Bac-Pie1-gp64-VP1 elicited neutralization antibody titer of 1∶64 against EV71 (subgenogroup C4) in an in vitro neutralization assay. Furthermore, the antisera showed high cross-neutralization activities against all 11 subgenogroup EV71 strains.

Conclusion

Our results illustrated that Bac-Pie1-gp64-VP1 retained native epitopes of VP1 and acted as an effective EV71 vaccine candidate which would enable rapid production without any biosafety concerns.     

Neutralizing Antibodies Induced by Recombinant Virus-Like Particles of Enterovirus 71 Genotype C4 Inhibit Infection at Pre- and Post-attachment Steps

Background

Enterovirus 71 (EV71) is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia–Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear.

Methods/Findings

In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs). Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC) located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment.

Conclusions

Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.     

Streamlined design of a self-inactivating feline immunodeficiency virus vector for transducing ex vivo dendritic cells and T lymphocytes

Background

Enterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and construct a DNA vaccine against Enterovirus 71 using the viral capsid protein (VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo.

Methods

The VP1 gene of EV71 from two local outbreak isolates were amplified using PCR and then inserted into a eukaryotic expression vector, pVAX1. The 3.9 kb recombinant constructs were transformed into competent E. coli cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in Vero cells. Subsequently, in the in vivo studies, female Balb/c mice were immunized with the DNA vaccine constructs. Enzyme Linked Immunosorbent Assay (ELISA) and virus neutralizing assay were performed to detect the presence of anti-VP1 IgG in mice and its neutralizing effect against the EV71.

Results

The pVAX1 vector was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame. The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting. The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster. The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71.

Conclusion

The promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccine.     

Comparative Study of the Cytokine/Chemokine Response in Children with Differing Disease Severity in Enterovirus 71-Induced Hand,Foot, and Mouth Disease

Background

Enterovirus 71 (EV71) infection can lead to a rapidly progressing, life-threatening, and severe neurological disease in young children, including the development of human hand, foot, and mouth disease (HFMD). This study aims to further characterize the specific immunological features in EV71–mediated HFMD patients presenting with differing degrees of disease severity.

Methodology

Comprehensive cytokine and chemokine expression were broadly evaluated by cytokine antibody array in EV71–infected patients hospitalized for HFMD compared to Coxsackievirus A16-infected patients and age-matched healthy controls. More detailed analysis using Luminex-based cytokine bead array was performed in EV71–infected patients stratified into diverse clinic outcomes. Additionally, immune cell frequencies in peripheral blood and EV71–specific antibodies in plasma were also examined.

Principal Findings

Expression of several cytokines and chemokines were significantly increased in plasma from EV71–infected patients compared to healthy controls, which further indicated that: (1) GM-CSF, MIP-1β, IL-2, IL-33, and IL-23 secretion was elevated in patients who rapidly developed disease and presented with uncomplicated neurological damage; (2) G-CSF and MCP-1 were distinguishably secreted in EV71 infected very severe patients presenting with acute respiratory failure; (3) IP-10, MCP-1, IL-6, IL-8, and G-CSF levels were much higher in cerebrospinal fluid than in plasma from patients with neurological damage; (4) FACS analysis revealed that the frequency of CD19⁺HLADR⁺ mature B cells dynamically changed over time during the course of hospitalization and was accompanied by dramatically increased EV71–specific antibodies. Our data provide a panoramic view of specific immune mediator and cellular immune responses of HFMD and may provide useful immunological profiles for monitoring the progress of EV71–induced fatal neurological symptoms with acute respiratory failure.     

Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model

Background

We previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice.

Results

We demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH₄Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice.

Conclusions

In this study, we reveal that EV71 infection of suckling mice induces an amphisome formation accompanied with the autophagic flux in the brain tissues. Autophagy induced by EV71 promotes viral replication and EV71-related pathogenesis.     

Epidemiology and seroepidemiology of human enterovirus 71 among Thai populations

Background

Human enterovirus 71 (EV71) is an important pathogen caused large outbreaks in Asian-Pacific region with severe neurological complications and may lead to death in young children. Understanding of the etiological spectrum and epidemic changes of enterovirus and population’s immunity against EV71 are crucial for the implementation of future therapeutic and prophylactic intervention.

Results

A total of 1,182 patients who presented with the symptoms of hand foot and mouth disease (67.3%) or herpangina (HA) (16.7%) and admitted to the hospitals during 2008-2013 were tested for enterovirus using pan-enterovirus PCR targeting 5′-untranslated region and specific PCR for viral capsid protein 1 gene. Overall, 59.7% were pan-enterovirus positive comprising 9.1% EV71 and 31.2% coxsackievirus species A (CV-A) including 70.5% CV-A6, 27.6% CV-A16, 1.1% CV-A10, and 0.8% CV-A5. HFMD and HA occurred endemically during 2008-2011. The number of cases increased dramatically in June 2012 with the percentage of the recently emerged CV-A6 significantly rose to 28.4%. Co-circulation between different EV71 genotypes was observed during the outbreak. Total of 161 sera obtained from healthy individuals were tested for neutralizing antibodies (NAb) against EV71 subgenotype B5 (EV71-B5) using microneutralization assay. The seropositive rate of EV71-B5 was 65.8%. The age-adjusted seroprevalence for individuals was found to be lowest in children aged >6 months to 2 years (42.5%). The seropositive rate remained relatively low in preschool children aged > 2 years to 6 years (48.3%) and thereafter increased sharply to more than 80% in individuals aged > 6 years.

Conclusions

This study describes longitudinal data reflecting changing patterns of enterovirus prevalence over 6 years and demonstrates high seroprevalences of EV71-B5 NAb among Thai individuals. The rate of EV71 seropositive increased with age but without gender-specific significant difference. We identified that relative lower EV71 seropositive rate in early 2012 may demonstrate widely presented of EV71-B5 in the population before account for a large outbreak scale epidemic occurred in 2012 with due to a relatively high susceptibility of the younger population.     

Formalin-Inactivated EV71 Vaccine Candidate Induced Cross-Neutralizing Antibody against Subgenotypes B1, B4, B5 and C4A in Adult Volunteers

Background

Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia. No effective EV71 vaccine is available. A randomized and open-label phase I clinical study registered with ClinicalTrials.gov #NCT01268787, aims to evaluate the safety, reactogenicity and immunogenicity of a formalin-inactivated EV71 vaccine candidate (EV71vac) at 5- and 10-µg doses. In this study we report the cross-neutralizing antibody responses from each volunteer against different subgenotypes of EV71 and CVA16.

Methods

Sixty eligible healthy adults were recruited and vaccinated. Blood samples were obtained on day 0, 21 and 42 and tested against B1, B4, B5, C2, C4A, C4B and CVA16 for cross-neutralizing antibody responses.

Results

The immunogenicity of both 5- and 10- µg doses were found to be very similar. Approximately 45% of the participants had <8 pre-vaccination neutralization titers (Nt) against the B4 vaccine strain. After the first EV71vac immunization, 95% of vaccinees have >4-fold increase in Nt, but there was no further increase in Nt after the second dose. EV71vac induced very strong cross-neutralizing antibody responses in >85% of volunteers without pre-existing Nt against subgenotype B1, B5 and C4A. EV71vac elicited weak cross-neutralizing antibody responses (∼20% of participants) against a C4B and Coxsackie virus A16. Over 90% of vaccinated volunteers did not develop cross-neutralizing antibody responses (Nt<8) against a C2 strain. EV71vac can boost and significantly enhance the neutralizing antibody responses in volunteers who already had pre-vaccination antibodies against EV71 and/or CVA16.

Conclusion

EV71vac is efficient in eliciting cross-neutralizing antibody responses against EV71 subgenotypes B1, B4, B5, and C4A, and provides the rationale for its evaluation in phase II clinical trials.

Trial Registration

ClinicalTrials.gov __NCT01268787     

Norepinephrine and Epinephrine Enhanced the Infectivity of Enterovirus 71

Background

Enterovirus 71 (EV71) infections may be associated with neurological complications, including brainstem encephalitis (BE). Severe EV71 BE may be complicated with autonomic nervous system (ANS) dysregulation and/or pulmonary edema (PE). ANS dysregulation is related to the overactivation of the sympathetic nervous system, which results from catecholamine release.

Objective

The aims of this study were to explore the effects of catecholamines on severe EV71 infection and to investigate the changes in the percentages of EV71-infected cells, virus titer, and cytokine production on the involvement of catecholamines.

Study Design

Plasma levels of norepinephrine (NE) and epinephrine (EP) in EV71-infected patients were measured using an enzyme-linked immunoassay. The expression of adrenergic receptors (ADRs) on RD, A549, SK-N-SH, THP-1, Jurkat and human peripheral blood mononuclear cells (hPBMCs) were detected using flow cytometry. The percentages of EV71-infected cells, virus titer, and cytokine production were investigated after treatment with NE and EP.

Results

The plasma levels of NE and EP were significantly higher in EV71-infected patients with ANS dysregulation and PE than in controls. Both α_1A- and β₂-ADRs were expressed on A549, RD, SK-N-SH, HL-60, THP-1, Jurkat cells and hPBMCs. NE treatment elevated the percentages of EV71-infected cells to 62.9% and 22.7% in THP-1 and Jurkat cells, respectively. Via treatment with EP, the percentages of EV71-infected cells were increased to 64.6% and 26.9% in THP-1 and Jurkat cells. The percentage of EV71-infected cells increased upon NE or EP treatment while the α- and β-blockers reduced the percentages of EV71-infected cells with NE or EP treatment. At least two-fold increase in virus titer was observed in EV71-infected A549, SK-N-SH and hPBMCs after treatment with NE or EP. IL-6 production was enhanced in EV71-infected hPBMCs at a concentration of 10² pg/mL NE.

Conclusion

The plasma levels of NE and EP elevated in EV71-infected patients with ANS dysregulation and PE. Both NE and EP enhanced the percentages of infected cells and virus titers in EV71 infection in vitro. NE and EP may play a role in the pathogenesis of EV71 BE complicated with ANS dysregulation and PE.     

Exhaled breath condensate sampling is not a new method for detection of respiratory viruses

Background

Human enterovirus 71 (EV71) has emerged as a significant cause of acute encephalitis and deaths in young children. The clinical manifestations caused by EV71 varied from mild hand, foot and mouth disease to severe neurological complications and deaths, but its pathogenesis remains elusive. Antibody dependent enhancement (ADE) infection has been reported in various viruses and has been shown to contribute to disease severity.

Results

In this study, the presence of sub-neutralizing antibody was demonstrated to enhance EV71 infection in THP-1 cells and increase the mortality of EV71 infection in a suckling mouse model. Further, a secondary infection model was established to characterize the correlation between ADE and disease severity, and primary asymptomatic EV71 infection was shown to increase the mortality of the secondary EV71 infection in suckling mice.

Conclusions

Together, these in vitro and in vivo experiments strongly supported the hypothesis of ADE infection of EV71. The present findings indicate ADE might contribute to the pathogenesis of severe EV71 infection, and raise practical issues of vaccine development and antibody-based therapy.     

Seroepidemiology and Molecular Epidemiology of Enterovirus 71 in Russia

Enterovirus 71 (EV71) is an emerging human pathogen causing massive epidemics of hand, foot and mouth disease with severe neurological complications in Asia. EV71 also circulates in Europe, however it does not cause large outbreaks. The reason for distinct epidemiological patterns of EV71 infection in Europe and Asia and the risk of EV71 epidemic in Europe and Russia remain unknown. Seroepidemiology of EV71 and molecular epidemiology of occasional EV71 isolates were studied to explore circulation of EV71 in Russia. In six regions of Russian Federation, seroprevalence of EV71 in sera collected in 2008 ranged from 5% to 20% in children aged 1–2 years and from 19% to 83% in children aged 3–5 years. The seroprevalence among elder children was significantly higher (41–83% vs. 19–27%) in Asian regions of Russia. EV71 strains identified in Russia in 2001–2011 belonged to subtypes C1 and C2, while genotype C4 that was causing epidemics in Asia since 1998 emerged in 2009 and became dominant in 2013.     

Exogenous interleukin-6, interleukin-13, and interferon-gamma provoke pulmonary abnormality with mild edema in enterovirus 71-infected mice

Background

Neonatal mice developed neurological disease and pulmonary dysfunction after an infection with a mouse-adapted human Enterovirus 71 (EV71) strain MP4. However, the hallmark of severe human EV71 infection, pulmonary edema (PE), was not evident.

Methods

To test whether EV71-induced PE required a proinflammatory cytokine response, exogenous pro-inflammatory cytokines were administered to EV71-infected mice during the late stage of infection.

Results

After intracranial infection of EV71/MP4, 7-day-old mice developed hind-limb paralysis, pulmonary dysfunction, and emphysema. A transient increase was observed in serum IL-6, IL-10, IL-13, and IFN-γ, but not noradrenaline. At day 3 post infection, treatment with IL-6, IL-13, and IFN-γ provoked mild PE and severe emphysema that were accompanied by pulmonary dysfunction in EV71-infected, but not herpes simplex virus-1 (HSV-1)-infected control mice. Adult mice did not develop PE after an intracerebral microinjection of EV71 into the nucleus tractus solitarii (NTS). While viral antigen accumulated in the ventral medulla and the NTS of intracerebrally injected mice, neuronal loss was observed in the ventral medulla only.

Conclusions

Exogenous IL-6, IL-13, and IFN-γ treatment could induce mild PE and exacerbate pulmonary abnormality of EV71-infected mice. However, other factors such as over-activation of the sympathetic nervous system may also be required for the development of classic PE symptoms.     

The Association between Enterovirus 71 Infections and Meteorological Parameters in Taiwan

Background

Enterovirus 71 (EV71) infections are a significant cause of neurological disorder and death in children worldwide. Seasonal variations in EV71 infections have been recognized, but the mechanisms responsible for this phenomenon remain unknown. The purpose of this study was to examine the relationship between meteorological parameters and EV71 infection.

Methods and Findings

We analyzed the number of EV71 infections and daily climate data collected in Taiwan between 1998 and 2008 and used Poisson regression analysis and case-crossover methodology to evaluate the association between weather variability and the incidence of EV71 infection. A total of 1,914 EV71-infected patients were reported between 1998 and 2008. The incidence of EV71 infections reflected significant summertime seasonality (for oscillation, p<0.001). The incidence of EV71 infections began to rise at temperatures above 13°C (r² = 0.76, p<0.001); at temperatures higher than approximately 26°C (r² = 0.94, p<0.05), the incidence began to decline, producing an inverted V-shaped relationship. The increase in the incidence with increasing relative humidity was positive and linear (r² = 0.68, p<0.05). EV71 infection was most highly correlated with temperature and relative humidity in the period that likely preceded the infection.

Conclusion

Our study provides quantitative evidence that the rate of EV71 infection increased significantly with increasing mean temperature and relative humidity in Taiwan.     

The Interplays between Autophagy and Apoptosis Induced by Enterovirus 71

Background

Enterovirus 71 (EV71) is the causative agent of human diseases with distinct severity, from mild hand, foot and mouth disease to severe neurological syndromes, such as encephalitis and meningitis. The lack of understanding of viral pathogenesis as well as lack of efficient vaccine and drugs against this virus impedes the control of EV71 infection. EV71 virus induces autophagy and apoptosis; however, the relationship between EV71-induced autophagy and apoptosis as well as the influence of autophagy and apoptosis on virus virulence remains unclear.

Methodology/Principal Findings

In this study, it was observed that the Anhui strain of EV71 induced autophagy and apoptosis in human rhabdomyosarcoma (RD-A) cells. Additionally, by either applying chemical inhibitors or knocking down single essential autophagic or apoptotic genes, inhibition of EV71 induced autophagy inhibited the apoptosis both at the autophagosome formation stage and autophagy execution stage. However, inhibition of autophagy at the stage of autophagosome and lysosome fusion promoted apoptosis. In reverse, the inhibition of EV71-induced apoptosis contributed to the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to LC3-II and degradation of sequestosome 1 (SQSTM1/P62). Furthermore, the inhibition of autophagy in the autophagsome formation stage or apoptosis decreased the release of EV71 viral particles.

Conclusions/Significance

In conclusion, the results of this study not only revealed novel aspect of the interplay between autophagy and apoptosis in EV71 infection, but also provided a new insight to control EV71 infection.     

Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system

Background

Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection.

Principal Finding

In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >10⁶ TCID₅₀/mL by 6 days post infection when a MOI of 10⁻⁵ was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225).

Conclusion

These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.