Enterovirus Genome Research Articles

Identification of an enterovirus recombinant with a torovirus-like gene insertion during a diarrhea outbreak in fattening pigs.

Diarrhea outbreaks in pig farms have raised major concerns in Europe and USA, as they can lead to dramatic pig losses. During a suspected outbreak in Belgium of porcine epidemic diarrhea virus (PEDV), we performed viral metagenomics to assess other potential viral pathogens. Although PEDV was detected, its low abundance indicated that other viruses were involved in the outbreak. Interestingly, a porcine bocavirus and several enteroviruses were most abundant in the sample. We also observed the presence of a porcine enterovirus genome with a gene insertion, resembling a C28 peptidase gene found in toroviruses, which was confirmed using re-sequencing, bioinformatics, and proteomics approaches. Moreover, the predicted cleavage sites for the insertion suggest that this gene was being expressed as a single protein, rather than a fused protein. Recombination in enteroviruses has been reported as a major mechanism to generate genetic diversity, but gene insertions across viral families are rather uncommon. Although such inter-family recombinations are rare, our finding suggests that these events may significantly contribute to viral evolution.

Structure and function of RNA elements present in enteroviral genomes.

Enteroviruses are small RNA(+) viruses that encode one open reading frame flanked by two extensive noncoding regions carrying structural RNA regulatory elements that control replication and translation processes. For a long time the central, coding region was thought to remain single-stranded and its only function was supposed to be as the template for polyprotein synthesis. It turned out, however, that the protein coding region also encodes important RNA structures crucial for the viral life cycle and virus persistence in the host cells. This review considers the RNA structures in enteroviral genomes identified and characterized to date.

In Vivo Imaging with Bioluminescent Enterovirus 71 Allows for Real-Time Visualization of Tissue Tropism and Viral Spread.

Hand, foot, and mouth disease (HFMD) is a reemerging illness caused by a variety of enteroviruses. The main causative agents are enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and, most recently, coxsackievirus A6 (CVA6). Enterovirus infections can vary from asymptomatic infections to those with a mild fever and blisters on infected individuals' hands, feet, and throats to infections with severe neurological complications. Viral persistence for weeks postinfection (wpi) has also been documented by the demonstration of virus in children's stools. However, little is known about disease progression, viral spread, and tissue tropism of these viruses. These types of studies are limited because many recently developed mouse models mimic the severe neurological complications that occur in a small percentage of enterovirus infections. In the present study, we documented real-time EV71 infection in two different mouse strains by the use of in vivo imaging. Infection of BALB/c mice with a bioluminescent mouse-adapted EV71 construct (mEV71-NLuc) resulted in a lack of clinical signs of disease but in relatively high viral replication, as visualized by luminescence, for 2 wpi. In contrast, mEV71-NLuc infection of AG129 mice (alpha/beta and gamma interferon receptor deficient) showed rapid spread and long-term persistence of the virus in the brain. Interestingly, AG129 mice that survived infection maintained luminescence in the brain for up to 8 wpi. The results we present here will allow future studies on EV71 antiviral drug susceptibility, vaccine efficacy, transmissibility, and pathogenesis. IMPORTANCE We report here that a stable full-length enterovirus 71 (EV71) reporter construct was used to visualize real-time viral spread in AG129 and BALB/c mice. To our knowledge, this is the first report of in vivo imaging of infection with any member of the Picornaviridae family. The nanoluciferase (NLuc) gene, one of the smallest luciferase genes currently available, was shown to be stable in the EV71 genome for eight passages on rhabdomyosarcoma cells. Real-time visualization of EV71 infection in mice identified areas of tropism that would have been missed by traditional methods, including full characterization of EV71 replication in BALB/c mice. Additionally, the bioluminescent construct allowed for increased speed and sensitivity of cell culture assays and will allow future studies involving various degrees of enterovirus infection in mice, not just severe infections. Our data suggest that interferon plays an important role in controlling EV71 infection in the central nervous system of mice.

Detection and localization of viral infection in the pancreas of patients with type 1 diabetes using short fluorescently-labelled oligonucleotide probes.

Enteroviruses, specifically of the Coxsackie B virus family, have been implicated in triggering islet autoimmunity and type 1 diabetes, but their presence in pancreata of patients with diabetes has not been fully confirmed.To detect the presence of very low copies of the virus genome in tissue samples from T1D patients, we designed a panel of fluorescently labeled oligonucleotide probes, each of 17-22 nucleotides in length with a unique sequence to specifically bind to the enteroviral genome of the picornaviridae family.With these probes enteroviral RNA was detected with high sensitivity and specificity in infected cells and tissues, including in FFPE pancreas sections from patients with T1D. Detection was not impeded by variations in sample processing and storage thereby overcoming the potential limitations of fragmented RNA. Co-staining of small RNA probes in parallel with classical immunstaining enabled virus detection in a cell-specific manner and more sensitively than by viral protein.

Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment.

Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 μmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 μmol/L) nor artemisinin (50 μmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication, thus is a candidate anti-HFMD drug.

Ambulatory Pediatric Surveillance of Hand, Foot and Mouth Disease as Signal of an Outbreak of Coxsackievirus A6 Infections, France, 2014-2015.

The clinical impact of enteroviruses associated with hand, foot and mouth disease (HFMD) is unknown outside Asia, and the prevalence of enterovirus A71 (EV-A71) in particular might be underestimated. To investigate the prevalence of enterovirus serotypes and the clinical presentations associated with HFMD in France, we conducted prospective ambulatory clinic-based surveillance of children during April 2014-March 2015. Throat or buccal swabs were collected from children with HFMD and tested for the enterovirus genome. Physical examinations were recorded on a standardized form. An enterovirus infection was detected in 523 (79.3%) of 659 children tested. Two epidemic waves occurred, dominated by coxsackievirus (CV) A6, which was detected in 53.9% of enterovirus-infected children. CV-A6 was more frequently related to atypical HFMD manifestations (eruptions extended to limbs and face). Early awareness and documentation of HFMD outbreaks can be achieved by syndromic surveillance of HFMD by ambulatory pediatricians and rapid enterovirus testing and genotyping.

Enterovirus but not Parvovirus B19 is associated with idiopathic dilated cardiomyopathy and endomyocardial CD3, CD68, or HLA‐DR expression

We assessed Enterovirus (EV) &Parvovirus B19 (PVB19) genomes and CD3, CD68&HLA-DR detection in dilated cardiomyopathies (DCM). EV&PVB19 genomes and CD3, CD68&HLA-DR were detected by PCR and immunohistochemistry assays in 115 endomyocardial biopsies obtained in 13 idiopathic DCM (iDCM) and 10 explained DCM (eDCM) patients. Results were compared with those of 47 atrial surgical samples (47 surgery controls) and 22 autoptic cardiac samples (11 healthy heart controls) (2008-2014, Reims, France). EV was detected in 23.1% of iDCM patients but not in eDCM and controls (P = 0.003) (viral load 803 copies/μg). PVB19 was detected in 76.9%, 80.0%, 63.6% and 78.2% of iDCM, eDCM, healthy heart and surgery controls (P = 0.99) with a mean viral load of 413, 346, 1,428, and 71 copies/μg. CD3, CD68 or HLA-DR were detected in 100 and 50% of EV and PVB19 "mono-infected" iDCM patients. EV was exclusively detected in iDCM cases in association with CD3, CD68, or HLA-DR indicating that EV could be an etiological cause in a subset of iDCM cases. By contrast the equal frequent detection of PVB19 in iDCM cases and controls without association with CD3, CD68, or HLA-DR suggested that PVB19 could be a bystander in many DCM cases. J. Med. Virol. 89:55-63, 2017. © 2016 Wiley Periodicals, Inc.

Domain I of the 5′ non-translated genomic region in coxsackievirus B3 RNA is not required for productive replication

Domain I is a cloverleaf-like secondary structure at the 5′ termini of all enterovirus genomes, comprising part of a cis-acting replication element essential for efficient enteroviral replication. 5′ genomic terminal deletions up to as much as 55% of domain I can occur without lethality following coxsackie B virus infections. We report here that the entire CVB structural domain I can be deleted without lethality.

A Novel Open and Infectious Form of Echovirus 1.

One of the hallmarks of enterovirus genome delivery is the formation of an uncoating intermediate particle. Based on previous studies of mostly heated picornavirus particles, intermediate particles were shown to have externalized the innermost capsid protein (VP4) and exposed the N terminus of VP1 and to have reduced infectivity. Here, in addition to the native and intact particle type, we have identified another type of infectious echovirus 1 (E1) particle population during infection. Our results show that E1 is slightly altered during entry, which leads to the broadening of the major virion peak in the sucrose gradient. In contrast, CsCl gradient separation revealed that in addition to the light intact and empty particles, a dense particle peak appeared during infection in cells. When the broad peak from the sucrose gradient was subjected to a CsCl gradient, it revealed light and dense particles, further suggesting that the shoulder represents the dense particle. The dense particle was permeable to SYBR green II, it still contained most of its VP4, and it was able to bind to its receptor α2β1 integrin and showed high infectivity. A thermal assay further showed that the α2β1 integrin binding domain (I-domain) stabilized the virus particle. Finally, heating E1 particles to superphysiological temperatures produced more fragile particles with aberrant ultrastructural appearances, suggesting that they are distinct from the dense E1 particles. These results describe a more open and highly infectious E1 particle that is naturally produced during infection and may represent a novel form of an uncoating intermediate. In this paper, we have characterized a possible uncoating intermediate particle of E1 that is produced in cells during infection. Before releasing their genome into the host cytosol, enteroviruses go through structural changes in their capsid, forming an uncoating intermediate particle. It was shown previously that structural changes can be induced by receptor interactions and, in addition, by heating the native virion to superphysiological temperatures. Here, we demonstrate that an altered, still infectious E1 particle is found during infection. This particle has a more open structure, and it cannot be formed by heating. It still contains the VP4 protein and is able to bind to its receptor and cause infection. Moreover, we show that in contrast to some other enteroviruses, the receptor-virion interaction has a stabilizing effect on E1. This paper highlights the differences between enterovirus species and further increases our understanding of various uncoating forms of enteroviruses.

Therapeutic Use of Native and Recombinant Enteroviruses.

Research on human enteroviruses has resulted in the identification of more than 100 enterovirus types, which use more than 10 protein receptors and/or attachment factors required in cell binding and initiation of the replication cycle. Many of these “viral” receptors are overexpressed in cancer cells. Receptor binding and the ability to replicate in specific target cells define the tropism and pathogenesis of enterovirus types, because cellular infection often results in cytolytic response, i.e., disruption of the cells. Viral tropism and cytolytic properties thus make native enteroviruses prime candidates for oncolytic virotherapy. Copy DNA cloning and modification of enterovirus genomes have resulted in the generation of enterovirus vectors with properties that are useful in therapy or in vaccine trials where foreign antigenic epitopes are expressed from or on the surface of the vector virus. The small genome size and compact particle structure, however, set limits to enterovirus genome modifications. This review focuses on the therapeutic use of native and recombinant enteroviruses and the methods that have been applied to modify enterovirus genomes for therapy.

SiRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro.

Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2Apro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID50) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT–PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2Apro region of the EV71 genome exerted antiviral effects in vitro.

A Rapid Method for Engineering Recombinant Polioviruses or Other Enteroviruses.

The cloning of large enterovirus RNA sequences is labor-intensive because of the frequent instability in bacteria of plasmidic vectors containing the corresponding cDNAs. In order to circumvent this issue we have developed a PCR-based method that allows the generation of highly modified or chimeric full-length enterovirus genomes. This method relies on fusion PCR which enables the concatenation of several overlapping cDNA amplicons produced separately. A T7 promoter sequence added upstream the fusion PCR products allows its transcription into infectious genomic RNAs directly in transfected cells constitutively expressing the phage T7 RNA polymerase. This method permits the rapid recovery of modified viruses that can be subsequently amplified on adequate cell-lines.

Infectious Entry Pathway of Enterovirus B Species.

Enterovirus B species (EV-B) are responsible for a vast number of mild and serious acute infections. They are also suspected of remaining in the body, where they cause persistent infections contributing to chronic diseases such as type I diabetes. Recent studies of the infectious entry pathway of these viruses revealed remarkable similarities, including non-clathrin entry of large endosomes originating from the plasma membrane invaginations. Many cellular factors regulating the efficient entry have recently been associated with macropinocytic uptake, such as Rac1, serine/threonine p21-activated kinase (Pak1), actin, Na/H exchanger, phospholipace C (PLC) and protein kinase Cα (PKCα). Another characteristic feature is the entry of these viruses to neutral endosomes, independence of endosomal acidification and low association with acidic lysosomes. The biogenesis of neutral multivesicular bodies is crucial for their infection, at least for echovirus 1 (E1) and coxsackievirus A9 (CVA9). These pathways are triggered by the virus binding to their receptors on the plasma membrane, and they are not efficiently recycled like other cellular pathways used by circulating receptors. Therefore, the best “markers” of these pathways may be the viruses and often their receptors. A deeper understanding of this pathway and associated endosomes is crucial in elucidating the mechanisms of enterovirus uncoating and genome release from the endosomes to start efficient replication.

Enterovirus infections in hospitals of Ile de France region over 2013

BackgroundThe monitoring and genotyping of Enterovirus (EV) infections can help to associate particular or severe clinical manifestations with specific EV types and to identify the aetiology of infectious outbreaks. ObjectivesTo describe the epidemiological features of EV infections diagnosed during the year 2013 in the Greater Paris area (Ile de France). Study designDuring 2013, 2497 samples taken from 470 patients in 33 hospitals of Ile-de France were tested for EV genome by RT-PCR. EV genotyping was performed by the National Reference Centre (NRC) laboratories. EV infections were retrospectively reviewed by retrieving clinical and genotyping data from the NRC database. ResultsOf the 2497 samples, 490 (19.6%) was positive for EV genome detection. These EV infections represented 88.7% and 24.1%, respectively, of all reported regional and national infections. Twenty-seven different genotypes were identified. Echovirus 30 (E-30) accounted for 54.1% of all characterized strains and caused a large outbreak. Four severe neonatal infections were reported, of which two were caused by EV-A71. Respiratory infections involving EV-D68 were observed in two adults. One fatal case of Coxsackievirus A2-associated myocarditis was reported. ConclusionMonitoring EV infections in combination with EV genotyping via the French EV network characterized the epidemiology of EV infections in the Ile de France region in 2013 and documented severe EV infections associated with EV-A71 or CV-A2.

Enterovirus meningitis in Tunisia (Monastir, Mahdia, 2011–2013): identification of virus variants cocirculating in France

Acute enterovirus (EV) meningitis is a frequent cause of hospitalisation, and over 100 EV serotypes may be involved. A total of 215 patients of all ages with meningitis signs were investigated in 2 Tunisian hospitals. Their cerebrospinal fluid (CSF) was analysed retrospectively for EVs with a TaqMan real-time RT-qPCR. The virus strains were typed, and their evolutionary relationships were determined by Bayesian phylogenetic methods. An EV genome was detected in 21/215 patients (9.8%). The CSF viral loads ranged from 3.27 to 5.63 log10 genome copies/mL. The strains were identified in 13/21 patients and assigned to EV-B types. Viruses identified in Tunisian patients were genetically related to variants detected in France. The viral loads were similar in Tunisian and French patients for most EV types. The phylogenetic data and viral loads determined in Tunisian and French patients suggest that close EV variants were involved in aseptic meningitis in the 2 countries over a same period.

Comparison of Two Concentration Methods for the Molecular Detection of Enteroviruses in Raw and Treated Sewage.

Human enteric viruses are a major causative agent of emerging waterborne diseases and constitute a serious public health concern. Environmental contamination occurs through discharge of waste materials from infected persons. Methods for viral detection should be developed to detect low infective dose of enteric viruses in environment. In this study, we aimed at comparing two concentration methods for the detection of naturally occurring enteroviruses in raw and treated sewage. In the first method, polyethylene glycol is used to concentrate viral particles from the collected samples. The second method is based on ultracentrifugation of viral particles at high speed (110,000×g). Genomes of enteroviruses were quantified by the quantitative real-time PCR method in raw and treated sewage samples. PEG-based method yielded higher genomic copies of enteric viruses (with an average of 5.9 log10 genomic copies/100 mL) when applied to raw sewage samples. While the ultracentrifugation assay in the second method decreases genomic copies number (with an average of 5.4 log10 genomic copies/100 mL). The recovery differences between the two methods were not significant when applied to clean samples (treated sewage). This could be explained by the presence of inhibitors, which interfere with qRT-PCR, in less quantity comparatively to raw sewage. PEG-based method would be more accurate for samples with high-organic matter load. This report emphasizes the importance of matrices nature on the recovery of enteroviruses from sewage samples. This should be taken into consideration for establishing standardized virological assays to ensure the virological quality control of discharged water in environment.

Quantification of human infection risk caused by rotavirus in surface waters from Córdoba, Argentina

Fecal contamination of water is a worrying problem because it is associated with the transmission of enteric pathogenic microorganisms that can cause many infectious diseases. In this study, an environmental survey was conducted to assess the level of viral contamination by viable enterovirus and rotavirus genome in two recreational rivers (Suquía and Xanaes) of Córdoba, Argentina. Quantitative microbial risk assessment (QMRA) was calculated to estimate the risk of rotavirus infection. Water sampling was carried out during a one-year period, the presence of total and fecal coliforms was determined and water samples were then concentrated for viral determination. Cell culture and indirect immunofluorescence were applied for enterovirus detection and RT-qPCR for rotavirus quantification. Coliform bacteria levels found in Suquía River often far exceeded the guideline limits for recreational waters. The Xanaes exhibited a lower level of bacterial contamination, frequently within the guideline limits. Enterovirus and rotavirus were frequently detected in the monitoring rivers (percentage of positive samples in Suquía: 78.6% enterovirus, 100% rotavirus; in Xanaes: 87.5% enterovirus, 18.7% rotavirus). Rotavirus was detected at a media concentration of 5.7×105 genome copies/L (gc/L) in the Suquía and 8.5×100gc/L in the Xanaes. QMRA revealed high risk of rotavirus infection in the Suquía, at sampling points with acceptable and non-acceptable bacteria numbers. The Xanaes showed significantly lower health risk of rotavirus infection but it proved to be a public health hazard. The viral occurrence was not readily explained by the levels of bacteria indicators, thus viral monitoring should be included to determine microbiological water quality. These findings provide the first data of QMRA for recreational waters in Argentina and reveal the need for public awareness of the health implications of the use of the river waters.

Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation.

Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5' untranslated region (5'UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially enhance the translation of virus protein. To our knowledge, this is the first report that describes Sam68 actively participating in the life cycle of EV71 at a molecular level. These studies will not only improve our understanding of the replication of EV71 but also have the potential for aiding in developing a therapeutic strategy against EV71 infection.

Virological and epidemiological analysis of coxsackievirus A24 variant epidemic of acute hemorrhagic conjunctivitis in Okinawa, Japan, in 2011.

BackgroundAcute hemorrhagic conjunctivitis (AHC) is a highly contagious enterovirus infection of the conjunctiva and cornea. Coxsackievirus A24 variant (CA24v) is one of its etiological agents. We report a clinical, epidemiological, and virological analysis of a large epidemic of AHC that occurred from May to September, 2011, in Okinawa, Japan.MethodsClinical and epidemic aspects were evaluated for 435 AHC patients (348 bilateral and 87 unilateral, 783 eyes). Virological studies were carried out on nine isolates from ten patients. Virus isolation and direct detection of the enterovirus genome by the reverse transcription polymerase chain reaction method and complete nucleotide sequencing of the VP1 gene and phylogeny-based classification using the VP4 sequences were carried out.ResultsThe 11–15-year age group comprised the highest (62.0%) proportion of cases among all age groups. Conjunctival hyperemia was present in all patients, and subconjunctival hemorrhage, superficial punctate keratitis, and preauricular lymphadenopathy were present in 25.4%, 10.3%, and 7.8% of eyes, respectively. CA24v was isolated from the epidemic strain, and phylogenetic analysis based on a fragment of the VP1 gene showed 96%–97% identity between the current strain and the recent China/GD01/2010 strain.ConclusionThese findings demonstrate that the clinical and epidemiological features of AHC observed in this study were similar to those of the past epidemic in the same region. It should be noted that sequential outbreaks of AHC due to CA24v might occur in the same location after a considerable period of time, and public health precautions are necessary to control this explosive epidemic.

Phosphatidylserine Vesicles Enable Efficient En Bloc Transmission of Enteroviruses

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.