Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles

Background

Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.

Principal Finding

In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10⁶ the tissue culture''s infectious dose (TCID₅₀) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10⁻⁵ was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24–28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35–38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176–190.

Conclusion

These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.     

Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates

Background

Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial.

Principal Finding

In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7–10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30–43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37°C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4°C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates.

Conclusion

These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.     

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.     

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.     

Characterization of an isotype-dependent monoclonal antibody against linear neutralizing epitope effective for prophylaxis of enterovirus 71 infection

Background

Enterovirus 71 (EV71) is the main causative agent of Hand, Foot and Mouth disease (HFMD) and is associated with severe neurologic complications and mortalities. At present, there is no vaccine or therapeutic available for treatment.

Methodology/Principal Finding

In this study, we generated two mAbs, denoted as mAb 51 and 53, both targeting the same linear epitope on VP1 capsid protein, spanning amino acids 215–219. In comparison, mAb 51 belonging to isotype IgM possesses neutralizing activity in vitro, whereas, mAb 53 belonging to isotype IgG1 does not have any neutralizing ability, even towards its homologous strain. When mAb 51 at 10 µg/g of body weight was administered to the 2-week-old AG129 mice one day prior to lethal challenge, 100% in vivo passive protection was observed. In contrast, the isotype control group mice, injected with an irrelevant IgM antibody before the challenge, developed limb paralysis as early as day 6 post-infection. Histological examination demonstrated that mAb 51 was able to protect against pathologic changes such as neuropil vacuolation and neuronal loss in the spinal cord, which were typical in unprotected EV-71 infected mice. BLAST analyses of that epitope revealed that it was highly conserved among all EV71 strains, but not coxsachievirus 16 (CA16).

Conclusion

We have defined a linear epitope within the VP1 protein and demonstrated its neutralizing ability to be isotype dependent. The neutralizing property and highly conserved sequence potentiated the application of mAb 51 and 53 for protection against EV71 infection and diagnosis respectively.     

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.     

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.     

Prevalence of 2009 pandemic influenza A (H1N1) virus antibodies, Tampa Bay Florida--November-December, 2009

Background

In 2009, a novel influenza virus (2009 pandemic influenza A (H1N1) virus (pH1N1)) caused significant disease in the United States. Most states, including Florida, experienced a large fall wave of disease from September through November, after which disease activity decreased substantially. We determined the prevalence of antibodies due to the pH1N1 virus in Florida after influenza activity had peaked and estimated the proportion of the population infected with pH1N1 virus during the pandemic.

Methods

During November-December 2009, we collected leftover serum from a blood bank, a pediatric children''s hospital and a pediatric outpatient clinic in Tampa Bay Florida. Serum was tested for pH1N1 virus antibodies using the hemagglutination-inhibition (HI) assay. HI titers ≥40 were considered seropositive. We adjusted seroprevalence results to account for previously established HI assay specificity and sensitivity and employed a simple statistical model to estimate the proportion of seropositivity due to pH1N1 virus infection and vaccination.

Results

During the study time period, the overall seroprevalence in Tampa Bay, Florida was 25%, increasing to 30% after adjusting for HI assay sensitivity and specificity. We estimated that 5.9% of the population had vaccine-induced seropositivity while 25% had seropositivity secondary to pH1N1 virus infection. The highest cumulative incidence of pH1N1 virus infection was among children aged 5–17 years (53%) and young adults aged 18–24 years (47%), while adults aged ≥50 years had the lowest cumulative incidence (11–13%) of pH1N1 virus infection.

Conclusions

After the peak of the fall wave of the pandemic, an estimated one quarter of the Tampa Bay population had been infected with the pH1N1 virus. Consistent with epidemiologic trends observed during the pandemic, the highest burdens of disease were among school-aged children and young adults.     

H1N1pdm influenza infection in hospitalized cancer patients: clinical evolution and viral analysis

Background

The novel influenza A pandemic virus (H1N1pdm) caused considerable morbidity and mortality worldwide in 2009. The aim of the present study was to evaluate the clinical course, duration of viral shedding, H1N1pdm evolution and emergence of antiviral resistance in hospitalized cancer patients with severe H1N1pdm infections during the winter of 2009 in Brazil.

Methods

We performed a prospective single-center cohort study in a cancer center in Rio de Janeiro, Brazil. Hospitalized patients with cancer and a confirmed diagnosis of influenza A H1N1pdm were evaluated. The main outcome measures in this study were in-hospital mortality, duration of viral shedding, viral persistence and both functional and molecular analyses of H1N1pdm susceptibility to oseltamivir.

Results

A total of 44 hospitalized patients with suspected influenza-like illness were screened. A total of 24 had diagnosed H1N1pdm infections. The overall hospital mortality in our cohort was 21%. Thirteen (54%) patients required intensive care. The median age of the studied cohort was 14.5 years (3–69 years). Eighteen (75%) patients had received chemotherapy in the previous month, and 14 were neutropenic at the onset of influenza. A total of 10 patients were evaluated for their duration of viral shedding, and 5 (50%) displayed prolonged viral shedding (median 23, range = 11–63 days); however, this was not associated with the emergence of a resistant H1N1pdm virus. Viral evolution was observed in sequentially collected samples.

Conclusions

Prolonged influenza A H1N1pdm shedding was observed in cancer patients. However, oseltamivir resistance was not detected. Taken together, our data suggest that severely ill cancer patients may constitute a pandemic virus reservoir with major implications for viral propagation.     

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.     

IL28B SNP rs12979860 is a critical predictor for on-treatment and sustained virologic response in patients with hepatitis C virus genotype-1 infection

Background

Single nucleotide polymorphisms (SNPs) of interleukin-28B (IL28B) have received considerable interest for their association with sustained virological response (SVR) when treating patients of genotype-1 hepatitis C virus (GT1-HCV) chronic infection with pegylated interferon and ribavirin (PegIFN/RBV). This study was to investigate the predictive power of IL28B SNPs for on-treatment responses and SVR in treatment-naïve patients with GT1-HCV chronic infection.

Methodology/Principal Findings

We analyzed ten SNPs of IL28B in 191 treatment-naïve patients with GT1-HCV chronic infection who received PegIFN/RBV. In these patients, rapid virological response (RVR), early virological response (EVR) and SVR were achieved in 69.6%, 95.8% and 68.6% of the patients, respectively. Multivariate analysis (odds ratio; 95% confidence interval; P value) indicated age (0.96; 0.93–0.99; 0.012), low baseline viral load (4.65; 2.23–9.66; <0.001) and CC genotype of rs12979860 (7.74; 2.55–23.53; <0.001) but no other SNPs were independent predictors for SVR. In addition, none of the ten SNPs examined were associated with baseline viral load and stages of liver fibrosis. Regarding RVR, low baseline viral load (2.83; 1.40–5.73; 0.004) and CC genotype of rs12979860 (10.52; 3.45–32.04; <0.001) were two critical predictors. As for EVR, only CC genotype of rs12979860 (36.21; 6.68–196.38; <0.001) was the predictor. Similarly, for end of treatment response (ETR), CC genotype of rs12979860 (15.42; 4.62–51.18; <0.001) was the only predictor. For patients with RVR, only low baseline viral load (3.90; 1.57–9.68; 0.003) could predict the SVR. For patients without RVR, only rs12979860 (4.60; 1.13–18.65; 0.033) was the predictor for SVR.

Conclusions/Significance

rs12979860 is the critical predictor for RVR, EVR, ETR and SVR in treatment-naïve patients of GT1-HCV chronic infection. Furthermore, this SNP is the only predictor for SVR in patients without RVR. These results have provided evidence that rs12979860 is the ideal IL28B SNP for genetic testing in treating patients of GT1-HCV chronic infection.     

Epstein-Barr virus infection and sporadic breast cancer risk: a meta-analysis

Background

A large number of epidemiological studies have evaluated the association between Epstein-Barr virus infection and breast carcinoma risk but results have been inconsistent.

Methodology

Research using the polymerase chain reaction technique for detecting the Epstein-Barr virus was selected; 24 studies and 1535 cases were reviewed. Information on the study populations, sample types, publication calendar period and histological types of breast carcinoma were collected. An unconditional logistic regression model was used to analyze potential parameters related to the Epstein-Barr virus prevalence. A Kappa test was used to evaluate the consistency in detecting different Epstein-Barr virus DNA regions. Nine studies that included control groups and 1045 breast cancer cases were adopted in this meta-analysis.

Conclusions

We found that 29.32% of the patients with breast carcinoma were infected with the Epstein-Barr virus. The prevalence of Epstein-Barr was highest in Asia (35.25%) and lowest in the USA (18.27%). Statistical analysis revealed a trend that showed lobular breast carcinoma might have the strongest association with Epstein-Barr virus infection. This meta-analysis showed a significant increase in breast malignancy risk in patients testing positive for the Epstein-Barr virus (OR = 6.29, 95% CI = 2.13–18.59). This result suggests that an Epstein-Barr virus infection is statistically associated with increased breast carcinoma risk.     

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.     

Seroprevalence of pandemic H1N1 antibody among health care workers in Hong Kong following receipt of monovalent 2009 H1N1 influenza vaccine

Background

Healthcare workers in many countries are recommended to receive influenza vaccine to protect themselves as well as patients. A monovalent H1N1 vaccine became available in Hong Kong in December 2009 and around 10% of local healthcare workers had received the vaccine by February 2010.

Methods

We conducted a cross-sectional study of the prevalence of antibody to pandemic (H1N1) 2009 among HCWs in Hong Kong in February–March 2010 following the first pandemic wave and the pH1N1 vaccination campaign. In this study we focus on the subset of healthcare workers who reported receipt of non-adjuvanted monovalent 2009 H1N1 vaccine (Panenza, Sanofi Pasteur). Sera collected from HCWs were tested for antibody against the pH1N1 virus by hemagglutination inhibition (HI) and viral neutralization (VN) assays.

Results

We enrolled 703 HCWs. Among 104 HCWs who reported receipt of pH1N1 vaccine, 54% (95% confidence interval (CI): 44%–63%) had antibody titer ≥1∶40 by HI and 42% (95% CI: 33%–52%) had antibody titer ≥1∶40 by VN. The proportion of HCWs with antibody titer ≥1∶40 by HI and VN significantly decreased with age, and the proportion with antibody titer ≥1∶40 by VN was marginally significantly lower among HCWs who reported prior receipt of 2007–08 seasonal influenza vaccine (odds ratio: 0.43; 95% CI: 0.19–1.00). After adjustment for age, the effect of prior seasonal vaccine receipt was not statistically significant.

Conclusions

Our findings suggest that monovalent H1N1 vaccine may have had suboptimal immunogenicity in HCWs in Hong Kong. Larger studies are required to confirm whether influenza vaccine maintains high efficacy and effectiveness in HCWs.     

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.     

Uptake and metabolism of the novel peptide angiotensin-(1-12) by neonatal cardiac myocytes

Background

Angiotensin-(1–12) [Ang-(1–12)] functions as an endogenous substrate for the productions of Ang II and Ang-(1–7) by a non-renin dependent mechanism. This study evaluated whether Ang-(1–12) is incorporated by neonatal cardiac myocytes and the enzymatic pathways of ¹²⁵I-Ang-(1–12) metabolism in the cardiac myocyte medium from WKY and SHR rats.

Methodology/Principal Findings

The degradation of ¹²⁵I-Ang-(1–12) (1 nmol/L) in the cultured medium of these cardiac myocytes was evaluated in the presence and absence of inhibitors for angiotensin converting enzymes 1 and 2, neprilysin and chymase. In both strains uptake of ¹²⁵I-Ang-(1–12) by myocytes occurred in a time-dependent fashion. Uptake of intact Ang-(1–12) was significantly greater in cardiac myocytes of SHR as compared to WKY. In the absence of renin angiotensin system (RAS) enzymes inhibitors the hydrolysis of labeled Ang-(1–12) and the subsequent generation of smaller Ang peptides from Ang-(1–12) was significantly greater in SHR compared to WKY controls. ¹²⁵I-Ang-(1–12) degradation into smaller Ang peptides fragments was significantly inhibited (90% in WKY and 71% in SHR) in the presence of all RAS enzymes inhibitors. Further analysis of peptide fractions generated through the incubation of Ang-(1–12) in the myocyte medium demonstrated a predominant hydrolytic effect of angiotensin converting enzyme and neprilysin in WKY and an additional role for chymase in SHR.

Conclusions/Significance

These studies demonstrate that neonatal myocytes sequester angiotensin-(1–12) and revealed the enzymes involved in the conversion of the dodecapeptide substrate to biologically active angiotensin peptides.