Characterization of cis-Acting RNA Elements of Zika Virus by Using a Self-Splicing Ribozyme-Dependent Infectious Clone.

Zika virus (ZIKV) has caused significant outbreaks and epidemics in the Americas recently, raising global concern due to its ability to cause microcephaly and other neurological complications. A stable and efficient infectious clone of ZIKV is urgently needed. However, the instability and toxicity of flavivirus cDNA clones in Escherichia coli hosts has hindered the development of ZIKV infectious clones. Here, using a novel self-splicing ribozyme-based strategy, we generated a stable infectious cDNA clone of a contemporary ZIKV strain imported from Venezuela to China in 2016. The constructed clone contained a modified version of the group II self-splicing intron P.li.LSUI2 near the junction between the E and NS1 genes, which were removed from the RNA transcripts by an easy-to-establish in vitro splicing reaction. Transfection of the spliced RNAs into BHK-21 cells led to the production of infectious progeny virus that resembled the parental virus. Finally, potential cis-acting RNA elements in ZIKV genomic RNA were identified based on this novel reverse genetics system, and the critical role of 5'-SLA promoter and 5'-3' cyclization sequences were characterized by a combination of different assays. Our results provide another stable and reliable reverse genetics system for ZIKV that will help study ZIKV infection and pathogenesis, and the novel self-splicing intron-based strategy could be further expanded for the construction of infectious clones from other emerging and reemerging flaviviruses.IMPORTANCE The ongoing Zika virus (ZIKV) outbreaks have drawn global concern due to the unexpected causal link to fetus microcephaly and other severe neurological complications. The infectious cDNA clones of ZIKV are critical for the research community to study the virus, understand the disease, and inform vaccine design and antiviral screening. A panel of existing technologies have been utilized to develop ZIKV infectious clones. Here, we successfully generated a stable infectious clone of a 2016 ZIKV strain using a novel self-splicing ribozyme-based technology that abolished the potential toxicity of ZIKV cDNA clones to the E. coli host. Moreover, two crucial cis-acting replication elements (5'-SLA and 5'-CS) of ZIKV were first identified using this novel reverse genetics system. This novel self-splicing ribozyme-based reverse genetics platform will be widely utilized in future ZIKV studies and provide insight for the development of infectious clones of other emerging viruses.

Features of Ebola Virus Disease at the Late Outbreak Stage in Sierra Leone: Clinical, Virological, Immunological, and Evolutionary Analyses.

We performed Ebola virus disease diagnosis and viral load estimation for Ebola cases in Sierra Leone during the late stage of the 2014-2015 outbreak (January-March 2015) and analyzed antibody and cytokine levels and the viral genome sequences. Ebola virus disease was confirmed in 86 of 1001 (9.7%) patients, with an overall case fatality rate of 46.8%. Fatal cases exhibited significantly higher levels of viral loads, cytokines, and chemokines at late stages of infection versus early stage compared with survivors. The viruses converged in a new clade within sublineage 3.2.4, which had a significantly lower case fatality rate.

Effect of Hypokalemia on Functional Outcome at 3 Months Post-Stroke Among First-Ever Acute Ischemic Stroke Patients.

BACKGROUND Hypokalemia has been confirmed to be a predictor of adverse cardiovascular and renal outcomes. There is a paucity of studies focusing on the potential connection between the serum K+ level and the outcome after acute ischemic stroke (AIS). This study investigated whether hypokalemia in the acute stroke stage contributes to worse functional outcome in AIS patients. MATERIAL AND METHODS This retrospective cohort study included consecutive patients with first-ever AIS admitted between June 2015 and March 2016. Patients were divided into 2 groups: hypokalemia (K+ <3.5 mmol/L) and normokalemia (3.5 mmol/L ≤K+ ≤5.5 mmol/L). Primary outcome measure was poor outcome at 3 months (modified Rankin scale >2). Univariate and multivariate logistic regression analyses were used to assess the association between hypokalemia and poor outcome. Receiver operating curve (ROC) analysis was performed to determine the optimal cutoff point of serum K+ level for predicting poor outcome. RESULTS The percent of patients with poor outcome at 3 months was higher in the hypokalemic group (62.9%) than in the normokalemic group (45.5%). Hypokalemic patients tended to have lower fasting glucose at admission, lower Glasgow coma scale score, and longer time from symptom onset to treatment compared with normokalemic patients. Hypokalemia was associated with poor outcome at 3 months after adjusting for potential confounders (odds ratio=2.42, 95% confidence interval=1.21-4.86, P=0.013). ROC analysis showed that the optimal threshold for serum K+ level was 3.7 mmol/L. CONCLUSIONS Hypokalemia at the initial admission is associated with poor prognosis at 3 months in first-ever AIS patients.

Crystal structures of enterovirus 71 (EV71) recombinant virus particles provide insights into vaccine design.

Hand-foot-and-mouth disease (HFMD) remains a major health concern in the Asia-Pacific regions, and its major causative agents include human enterovirus 71 (EV71) and coxsackievirus A16. A desirable vaccine against HFMD would be multivalent and able to elicit protective responses against multiple HFMD causative agents. Previously, we have demonstrated that a thermostable recombinant EV71 vaccine candidate can be produced by the insertion of a foreign peptide into the BC loop of VP1 without affecting viral replication. Here we present crystal structures of two different naturally occurring empty particles, one from a clinical C4 strain EV71 and the other from its recombinant virus containing an insertion in the VP1 BC loop. Crystal structure analysis demonstrated that the inserted foreign peptide is well exposed on the particle surface without significant structural changes in the capsid. Importantly, such insertions do not seem to affect the virus uncoating process as illustrated by the conformational similarity between an uncoating intermediate of another recombinant virus and that of EV71. Especially, at least 18 residues from the N terminus of VP1 are transiently externalized. Altogether, our study provides insights into vaccine development against HFMD.

Rational design of thermostable vaccines by engineered peptide-induced virus self-biomineralization under physiological conditions.

The development of vaccines against infectious diseases represents one of the most important contributions to medical science. However, vaccine-preventable diseases still cause millions of deaths each year due to the thermal instability and poor efficacy of vaccines. Using the human enterovirus type 71 vaccine strain as a model, we suggest a combined, rational design approach to improve the thermostability and immunogenicity of live vaccines by self-biomineralization. The biomimetic nucleating peptides are rationally integrated onto the capsid of enterovirus type 71 by reverse genetics so that calcium phosphate mineralization can be biologically induced onto vaccine surfaces under physiological conditions, generating a mineral exterior. This engineered self-biomineralized virus was characterized in detail for its unique structural, virological, and chemical properties. Analogous to many exteriors, the mineral coating confers some new properties on enclosed vaccines. The self-biomineralized vaccine can be stored at 26 °C for more than 9 d and at 37 °C for approximately 1 wk. Both in vitro and in vivo experiments demonstrate that this engineered vaccine can be used efficiently after heat treatment or ambient temperature storage, which reduces the dependence on a cold chain. Such a combination of genetic technology and biomineralization provides an economic solution for current vaccination programs, especially in developing countries that lack expensive refrigeration infrastructures.

Human enterovirus 71 uncoating captured at atomic resolution.

UNLABELLED: Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children, and structural characterization of EV71 during its life cycle can aid in the development of therapeutics against HFMD. Here, we present the atomic structures of the full virion and an uncoating intermediate of a clinical EV71 C4 strain to illustrate the structural changes in the full virion that lead to the formation of the uncoating intermediate prepared for RNA release. Although the VP1 N-terminal regions observed to penetrate through the junction channel at the quasi-3-fold axis in the uncoating intermediate of coxsackievirus A16 were not observed in the EV71 uncoating intermediate, drastic conformational changes occur in this region, as has been observed in all capsid proteins. Additionally, the RNA genome interacts with the N-terminal extensions of VP1 and residues 32 to 36 of VP3, both of which are situated at the bottom of the junction. These observations highlight the importance of the junction for genome release. Furthermore, EV71 uncoating is associated with apparent rearrangements and expansion around the 2- and 5-fold axes without obvious changes around the 3-fold axes. Therefore, these structures enabled the identification of hot spots for capsid rearrangements, which led to the hypothesis that the protomer interface near the junction and the 2-fold axis permits the opening of large channels for the exit of polypeptides and viral RNA, which is an uncoating mechanism that is likely conserved in enteroviruses. IMPORTANCE: Human enterovirus 71 (EV71) is the major causative agent of severe hand-foot-and-mouth diseases (HFMD) in young children. EV71 contains an RNA genome protected by an icosahedral capsid shell. Uncoating is essential in EV71 life cycle, which is characterized by conformational changes in the capsid to facilitate RNA release into host cell. Here we present the atomic structures of the full virion and an uncoating intermediate of a clinical C4 strain of EV71. Structural analysis revealed drastic conformational changes associated with uncoating in all the capsid proteins near the junction at the quasi-3-fold axis and protein-RNA interactions at the bottom of the junction in the uncoating intermediate. Significant capsid rearrangements also occur at the icosahedral 2- and 5-fold axes but not at the 3-fold axis. Taking the results together, we hypothesize that the junction and nearby areas are hot spots for capsid breaches for the exit of polypeptides and viral RNA during uncoating.

Generation and characterization of a protective mouse monoclonal antibody against human enterovirus 71.

Human enterovirus 71 (EV71) infection has emerged as a major threat to children; however, no effective antiviral treatment or vaccine is currently available. Antibody-based treatment shows promises to control this growing public health problem of EV71 infection, and a few potent monoclonal antibodies (mAbs) targeting viral capsid protein have been well described. Here, we generated an EV71-specific mouse mAb 2G8 that conferred full protection against lethal EV71 challenge in a suckling mouse model. 2G8 belonged to IgM isotype and neutralized EV71 at the attachment stage. Biochemical assays mapped the binding epitope of 2G8 to the SP70 peptide, which spanning amino acid residues 208-222 on the VP1 protein. Alanine scanning mutagenesis defined the essential roles of multiple residues, including Y208, T210, G212, K215, K218, L220, E221, and Y222, for 2G8 binding. Then, a panel of single mutation was individually introduced into the EV71 infectious clone by reverse genetics, and three mutant viruses, K215A, K218A, and L220A, were successfully recovered and characterized. Biochemical and neutralization assays revealed that K218A mutant partially escaped 2G8 neutralization, while L220A completely abolished 2G8 binding and neutralization. In particular, neutralization assays with human sera demonstrated that K218A and L220A substitutions are also critical for antibody neutralization in natural infection population. These findings not only generate a protective mAb candidate with therapeutic potential but also provide insights into antibody-mediated EV71 neutralization mechanism.

Recombinant tandem multi-linear neutralizing epitopes of human enterovirus 71 elicited protective immunity in mice.

BACKGROUND: Human Enterovirus 71 (EV71) has emerged as the leading cause of viral encephalitis in children, especially in the Asia-Pacific regions. EV71 vaccine development is of high priority at present, and neutralization antibodies have been documented to play critical roles during in vitro and in vivo protection against EV71 infection. RESULTS: In this study, a novel strategy to produce EV71 vaccine candidate based on recombinant multiple tandem linear neutralizing epitopes (mTLNE) was proposed. The three well identified EV71 linear neutralizing epitopes in capsid proteins, VP1-SP55, VP1-SP70 and VP2-SP28, were sequentially linked by a Gly-Ser linker ((G4S)3), and expressed in E.coli in fusion with the Trx and His tag at either terminal. The recombinant protein mTLNE was soluble and could be purified by standard affinity chromatography. Following three dosage of immunization in adult mice, EV71-specific IgG and neutralization antibodies were readily induced by recombinant mTLNE. IgG subtyping demonstrated that lgG1 antibodies dominated the mTLNE-induced humoral immune response. Especially, cytokine profiling in spleen cells from the mTLNE-immunized mice revealed high production of IL-4 and IL-6. Finally, in vivo challenge experiments showed that passive transfer with anti-mTLNE sera conferred full protection against lethal EV71 challenge in neonatal mice. CONCLUSION: Our results demonstrated that this rational designed recombinant mTLNE might have the potential to be further developed as an EV71 vaccine in the future.

Complete genome sequence analysis of human echovirus type 30 isolated in China.

We report here the complete genome sequence of a human echovirus type 30 strain ECV30/GX10/05 isolated in Guangxi, China, in 2010. Phylogenetic analysis showed that ECV30/GX10/05 was closely related to a Korean strain isolated in 2008. The sequence information will help in an understanding of the molecular epidemiology and evolution of echovirus.

Virus-like particles produced in Saccharomyces cerevisiae elicit protective immunity against Coxsackievirus A16 in mice.

Hand, foot, and mouth disease (HFMD) has caused significant morbidity and mortality in the Asia-Pacific regions, particularly in infants and young children. Coxsackievirus A16 (CA16) represents one of the major causative agents for HFMD, and the development of a safe and effective vaccine preventing CA16 infections has become a public health priority. In this study, we have developed a yeast system for the production of virus-like particles (VLPs) for CA16 by co-expressing P1 and 3CD of CA16 in Saccharomyces cerevisiae. These VLPs exhibit similarity in both protein composition and morphology as empty particles from CA16-infected cells. Immunization with CA16 VLPs in mice potently induced CA16-specific IgG and neutralization antibodies in a dose-dependent manner. IgG subclass isotyping revealed that IgG1 and lgG2b were dominantly induced by VLPs. Meanwhile, cytokine profiling demonstrated that immunization with VLPs significantly induced the secretion of IFN-γ, indicating potent cellular immune response. Furthermore, in vivo challenge experiments showed that passive immunization with anti-VLPs sera conferred full protection against lethal CA16 challenge in neonate mice. Taken together, our data demonstrated that VLPs produced in yeast might have the potential to be further developed as a vaccine candidate against HFMD.

Unequal Efficacy of Different Infarct Location in Predicting Futile Recanalization of Patients With Acute Ischemic Stroke.

Objectives: Endovascular thrombectomy (EVT) is a standard treatment for acute ischemic stroke (AIS) caused by large vessel occlusion, while futile recanalization is the main factor influencing the prognosis. The present study aimed to investigate the efficacy of different infarct sites in predicting futile recanalization of patients with AIS. Methods: Data were obtained from two multicenter, prospective, randomized, and controlled trials, which were concurrently conducted in China. Cases achieving a successful recanalization and with complete data of preoperative Alberta Stroke Program Early CT score (ASPECTS) and 90-day follow-up were included. The ASPECTS subregions were used to mark different infarct locations in the two cerebral hemispheres. First, the distribution of each ASPECTS subregion in the left and right hemispheres and the whole brain was analyzed, respectively. Then, the regions associated with futile recanalization were initially assessed by a univariate model. Afterward, a multivariate logistic regression model was used to identify the efficacy of different infarct sites in predicting futile recanalization. Results: A total of 336 patients were included in this study with a median age of 65 years (IQR: 51-74), of whom 210 (62.50%) patients were male, and 189 (56.25%) met the definition of futile recanalization. The correlation between each ASPECTS subregion and poor outcome was different when it was restricted to a specific cerebral hemisphere. Moreover, in the left hemisphere, the internal capsule region (OR: 1.42, 95%CI: 1.13-1.95, P = 0.03) and the M3 region (OR: 2.26, 95%CI: 1.36-3.52, P = 0.001), and in the right hemisphere, M6 region (OR: 2.24, 95%CI: 1.32-3.36, P = 0.001) showed significantly higher efficacy in predicting futile recanalization. Conclusion: The efficacy of different infarct locations in predicting futile recanalization is different. Different preoperative patterns of the high-efficiency regions in the infarction core or penumbra can guide the thrombectomy decision-making.

Development and evaluation of a reverse transcription-loop-mediated isothermal amplification assay for rapid detection of enterovirus 71.

Human enterovirus 71 (EV71) is the major etiological agent of hand, foot, and mouth disease (HFMD), which is a common infectious disease in young children and infants. EV71 can cause various clinical manifestations and has been associated with severe neurological complications; it has resulted in fatalities during recent outbreaks in Asian-Pacific regions since 1997. The early and rapid detection is critical for prevention and control of EV71 infection, since no vaccine or antiviral drugs are currently available. In this study, a simple and sensitive reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid detection of EV71. The detection limit of the RT-LAMP assay was approximately 0.01 PFU per reaction mixture, and no cross-reactive amplification with other enteroviruses was observed. The assay was evaluated further with 40 clinical specimens and exhibited 92.9% sensitivity and 100% specificity. This RT-LAMP assay may become a useful alternative in clinical diagnosis of EV71, especially in resource-limited hospitals or rural clinics of China and other countries in the Asian-Pacific region.

Antibody dependent enhancement infection of enterovirus 71 in vitro and in vivo.

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.

Producing infectious enterovirus type 71 in a rapid strategy.

BACKGROUND: Enterovirus 71 (EV71) is an etiologic agent of hand-foot-and-mouth disease (HFMD), and recent HFMD epidemics worldwide have been associated with a severe form of brainstem encephalitis associated with pulmonary edema and high case-fatality rates. EV71 contains a positive-sense single-stranded genome RNA of approximately 7400 bp in length which encodes a polyprotein with a single open reading frame (ORF), which is flanked by untranslated regions at both the 5' and 3' ends. RESULTS: A long distance RT-PCR assay was developed to amplify the full length genome cDNA of EV71 by using specific primes carrying a SP6 promoter. Then the in vitro synthesized RNA transcripts from the RT-PCR amplicons were then transfected into RD cells to produce the rescued virus. The rescued virus was further characterized by RT-PCR and indirect fluorescent-antibody (IFA) assay in comparison with the wild type virus. The rescued viruses were infectious on RD cells and neurovirulent when intracerebrally injected into suckling mice. CONCLUSIONS: Thus, we established a rapid method to produce the infectious full length cDNA of EV71 directly from RNA preparations and specific mutations can be easily engineered into the rescued enterovirus genome by this method.

Resveratrol promotes osteoblastic differentiation in a rat model of postmenopausal osteoporosis by regulating autophagy.

OBJECTIVE: Resveratrol is a natural polyphenolic compound that ameliorates postmenopausal osteoporosis by activating the estrogen receptor. Research has shown that resveratrol exhibits some type of estrogen receptor agonist activity, reducing the risk of breast cancer. However, its mechanism of action remains largely unknown. This study aims to investigate the effect of resveratrol on osteoblastic and osteoclastic differentiation and its potential role in the regulation of autophagy. METHODS: Sprague Dawley (SD) rats underwent ovariectomies (OVX) and were administered resveratrol (at 10, 20 or 40 mg/kg/d) for 8 weeks. The calcium content and the bone mineral density (BMD) were measured in the lumbar vertebrae (L3) and the right distal femur-tibia bone region. The osteoblasts and osteoclasts were isolated from rat lumbar vertebrae by enzyme digestion and bone marrow induction, respectively. The cells were then cultured with resveratrol in combination with bafilomycin or leupeptin to inhibit or activate autophagy, respectively. Western blotting was used to assess the differentiation markers and autophagy-related genes in the osteoblasts and osteoclasts. RESULTS: Compared to the sham group, the bone calcium content and BMD were significantly decreased in the OVX group (p < 0.05), while resveratrol attenuated these in a dose-dependent manner. In the osteoblasts, vascular endothelial growth factor (VEGF), and alpha-1 type I collagen (COL1A1) were markedly decreased, and in osteoclasts, the receptor activator of nuclear factor-κB ligand (RANKL) was increased in the OVX group, while resveratrol reversed this pattern in a dose-dependent manner. The inhibition of autophagy in osteoblasts and its activation in osteoclasts was observed in the OVX group. However, with resveratrol, this was reversed in a dose-dependent manner. CONCLUSION: Overall, resveratrol promotes osteoblastic differentiation and suppresses osteoclastic differentiation in a rat model with postmenopausal osteoporosis by regulating autophagy.

Association between platelet distribution width and poor outcome of acute ischemic stroke after intravenous thrombolysis.

PURPOSE: The platelet distribution width (PDW) reflects the status of platelet activity and may be useful for early predictions of the clinical outcome of stroke patients. The purpose of the study was to determine the associations between PDW and clinical outcomes after intravenous thrombolysis in stroke patients. PATIENTS AND METHODS: Acute ischemic stroke patients who received intravenous treatment with recombinant tissue-type plasminogen activator were selected for inclusion in the retrospective cohort of this study. The relations between PDW at admission and clinical outcomes were analyzed, including a poor outcome as assessed using the modified Rankin Scale at 3 months, early neurological improvement, and any hemorrhage. The effect of PDW at admission on a poor outcome at 3 months was analyzed using a multivariable logistic regression model with adjustment for potential confounders. The optimal PDW cutoff for predicting poor outcome at 3 months was determined by analyzing the receiver operating characteristics curve. RESULTS: PDW was significantly higher for a good outcome than a poor outcome (p=0.005), with median (interquartile range) values of 16.2 (13.2-17.2) and 13.6 (12.5-15.9), respectively. PDW was also higher in patients with early neurological improvement than in patients without improvement (p=0.020) and did not differ between hemorrhage and nonhemorrhage patients. The association between PDW <16.05% and poor outcome remained in a multivariable logistic regression analysis, with an OR of 6.68 and a 95% CI of 1.69-26.49 (p=0.007). CONCLUSION: Results suggest a novel hypothesis that a lower PDW may be related with a poor outcome at 3 months after intravenous thrombolysis in acute ischemic stroke patients.

Immunization with truncated envelope protein of Zika virus induces protective immune response in mice.

The global spread of Zika virus (ZIKV) as well as its unexpected link to infant microcephaly have resulted in serious public health concerns. No antiviral drugs against ZIKV is currently available, and vaccine development is of high priority to prepare for potential ZIKV pandemic. In the present study, a truncated E protein with the N-terminal 90% region reserved (E90) from a contemporary ZIKV strain was cloned and expressed in Escherichia coli, purified by a Ni-NTA column, and characterized by Western blotting assays. Immunization with recombinant E90 induced robust ZIKV-specific humoral response in adult BALB/c mice. Passive transfer of the antisera from E90-immunized mice conferred full protection against lethal ZIKV challenge in a neonatal mice model. Our results indicate that recombinant ZIKV E90 described here represents as a promising ZIKV subunit vaccine that deserves further clinical development.

De Ritis ratio (AST/ALT) as an independent predictor of poor outcome in patients with acute ischemic stroke.

PURPOSE: The aspartate transaminase/alanine transaminase ratio (De Ritis ratio, AAR) was reported to be associated with patients' prognosis in certain diseases recently. The objective of the current study was to determine the association between the AAR at admission and poor outcome at 3 months in acute ischemic stroke (AIS) patients. PATIENTS AND METHODS: This retrospective cohort study included patients who experienced their first-ever AIS between June 2015 and March 2016. The primary outcome measure was a poor outcome at 3 months (modified Rankin Scale score >2). Multivariate logistic regression models were used to assess the relationship between AAR quartiles and clinical outcomes among the AIS patients. Receiver operating characteristic curve analysis was applied to identify the optimal cutoff for AAR in predicting the prognosis of AIS. RESULTS: In terms of the relationship between poor outcome and AAR, the adjusted odds ratio comparing the highest and lowest AAR quartiles was 2.15 (95% confidence interval =1.14-4.05). An AAR of 1.53 was identified as the optimal cutoff. In a prespecified subgroup analysis according to the time from symptom onset to treatment (>24 vs ≤24 hours), there was no significant difference in the effect of AAR >1.53 between the two groups. CONCLUSION: An increased AAR at admission is significantly associated with a poor outcome at 3 months in AIS patients.