Genomic analysis of a recombinant coxsackievirus A19 identified in Xinxiang, China, in 2019.

Coxsackievirus A19 (CV-A19) is an enterovirus belonging to the species Enterovirus C, and the prototype strain 8663 was isolated from a patient with Guillain-Barré syndrome in Japan. In this study, we determined the complete genome sequence of a CV-A19 isolate identified in a stool sample from a child with hand, foot, and mouth disease in Xinxiang, Henan, China, in 2019 and named it CV-A19 strain 2019103106/XX/CHN/2019 - 2019103106 for short. The genome of this virus consists of 7409 nucleotides, including a 6624-nucleotide open reading frame encoding a potential polyprotein precursor of 2207 amino acids. Compared with strain 8663, strain 2019103106 showed 85.1% nucleotide sequence identity in the complete genome and 85.6% identity in the VP1 coding region, reflecting their genetic divergence. Phylogenetic analysis of strain 2019103106 and other representative EV-C strains with sequences available in the GenBank database showed that CV-A19 strains could be grouped into two clusters based on the complete or 214-nucleotide partial VP1 coding regions, and 2019103106 belonged to cluster 1, with the closest relationship to CV-A19 strain SWG82 from Shandong, China. Phylogenetic trees based on the P2 and P3 coding regions highlighted the divergence between strains 2019103106 and 8663, implying that strain 2019103106 had undergone recombination. Further recombination analysis suggested that strains V18A-like CV-A1 and BBD26-like CV-A19 probably recombined to yield strain 2019103106. The present study points out the genetic diversity of CV-A19. It expands our understanding of the evolution of the CV-A19 genome, but more genome sequences of epidemic strains are needed to explain the phylogeny and evolutionary history of CV-A19 comprehensively.

Enterovirus 71 Activates GADD34 via Precursor 3CD to Promote IRES-Mediated Viral Translation.

Enterovirus 71 (EV71) is the major pathogen of hand, foot, and mouth disease. In severe cases, it can cause life-threatening neurological complications, such as aseptic meningitis and polio-like paralysis. There are no specific antiviral treatments for EV71 infections. In a previous study, the host protein growth arrest and DNA damage-inducible protein 34 (GADD34) expression was upregulated during EV71 infection determined by ribosome profiling and RNA-sequencing. Here, we investigated the interactions of host protein GADD34 and EV71 during infections. Rhabdomyosarcoma (RD) cells were infected with EV71 resulting in a significant increase in expression of GADD34 mRNA and protein. Through screening of EV71 protein we determined that the non-structural precursor protein 3CD is responsible for upregulating GADD34. EV71 3CD increased the RNA and protein levels of GADD34, while the 3CD mutant Y441S could not. 3CD upregulated GADD34 translation via the upstream open reading frame (uORF) of GADD34 5'untranslated regions (UTR). EV71 replication was attenuated by the knockdown of GADD34. The function of GADD34 to dephosphorylate eIF2α was unrelated to the upregulation of EV71 replication, but the PEST 1, 2, and 3 regions of GADD34 were required. GADD34 promoted the EV71 internal ribosome entry site (IRES) activity through the PEST repeats and affected several other viruses. Finally, GADD34 amino acids 563 to 565 interacted with 3CD, assisting GADD34 to target the EV71 IRES. Our research reveals a new mechanism by which GADD34 promotes viral IRES and how the EV71 non-structural precursor protein 3CD regulates host protein expression to support viral replication. IMPORTANCE Identification of host factors involved in viral replication is an important approach in discovering viral pathogenic mechanisms and identifying potential therapeutic targets. Previously, we screened host proteins that were upregulated by EV71 infection. Here, we report the interaction between the upregulated host protein GADD34 and EV71. EV71 non-structural precursor protein 3CD activates the RNA and protein expression of GADD34. Our study reveals that 3CD regulates the uORF of the 5'-UTR to increase GADD34 translation, providing a new explanation for how viral proteins regulate host protein expression. GADD34 is important for EV71 replication, and the key functional domains of GADD34 that promote EV71 are PEST 1, 2, and 3 regions. We report that GADD34 promotes viral IRES for the first time and this process is independent of its eIF2α phosphatase activity.

Efficacy of a coxsackievirus A6 vaccine candidate in an actively immunized mouse model.

Coxsackievirus A6 (CV-A6) has been emerging as a major pathogen of hand, foot and mouth disease (HFMD). Study on the pathogenesis of CV-A6 infection and development of vaccines is hindered by a lack of appropriate animal models. Here, we report an actively immunized-challenged mouse model to evaluate the efficacy of a Vero-cell-based, inactivated CV-A6 vaccine candidate. The neonatal Kunming mice were inoculated with a purified, formaldehyde-inactivated CV-A6 vaccine on days 3 and 9, followed by challenging on day 14 with a naturally selected virulent strain at a lethal dose. Within 14 days postchallenge, all mice in the immunized groups survived, while 100% of the Alum-only inoculated mice died. Neutralizing antibodies (NtAbs) were detected in the serum of immunized suckling mice, and the NtAb levels correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak in the immunized mice compared with those in Alum-only inoculated control mice. Elevated levels of interleukin-4, 6, interferon γ and tumour necrosis factor α were also observed in Alum-only control mice compared with immunized mice. Importantly, the virulent CV-A6 challenge strain was selected quickly and conveniently from a RD cell virus stock characterized with the natural multi-genotypes. The virulent determinants were mapped to V124M and I242 V at VP1. Together, our results indicated that this actively immunized mouse model is invaluable for future studies to develop multivalent vaccines containing the major component of CV-A6 against HFMD.

Antiviral peptides against Enterovirus A71 causing hand, foot and mouth disease.

The emergence of new and resistant viruses is a serious global burden. Conventional antiviral therapy with small molecules has led to the development of resistant mutants. In the case of hand, foot and mouth disease (HFMD), the absence of a US-FDA approved vaccine calls for urgent need to develop an antiviral that could serve as a safe, potent and robust therapy against the neurovirulent Enterovirus A71 (EV-A71). Natural peptides such as lactoferrin, melittin and synthetic peptides such as SP40, RGDS and LVLQTM have been studied against EV-A71 and have shown promising results as potent antivirals in pre-clinical studies. Peptides are considered safe, efficacious and pose fewer chances of resistance. Poor pharmacokinetic features of peptides can be overcome by the use of chemical modifications to improve in vivo delivery particularly by oral route. The use of nanotechnology can remarkably assist in the oral delivery of peptides and enhance stability in vivo. This can greatly increase patient compliance and make it more attractive as antiviral therapy.

Aquaporin­4 deletion ameliorates enterovirus 71 infection in mice.

Aquaporin-4 (AQP4) is a major water channel of the central nervous system. The present study was designed to determine whether AQP4 deletion could ameliorate enterovirus (EV) 71 infection­induced hand, foot and mouth disease (HFMD) by inhibiting inflammation and apoptosis in mice. EV 71 strains were injected into neonatal BALB/c mice to induce HFMD. Western blotting and ELISA were used to measure the protein expression and cytokine levels. The levels of AQP4 mRNA and protein in the brain were increased in EV 71­infected mice, while the survival rate and health score were improved in AQP4­knockout (KO) mice with EV 71 infection. The EV 71 infection­induced increases of tumor necrosis factor­α, interleukin (IL)­1ß, IL­6, monocyte chemotactic protein­1, interferon (IFN)­α and IFN­Î³ in plasma and brain were inhibited in AQP4­KO mice. AQP4 deletion reversed the decreased levels of Bcl2 and Bcl2/Bax, and the increased levels of Bax induced by EV 71 infection in the brain. These results demonstrated that AQP4 deletion ameliorated EV 71 induced­HFMD via inhibiting inflammation and apoptosis in mice.

The surveillance of the epidemiological and serotype characteristics of hand, foot, mouth disease in Neijiang city, China, 2010-2017: A retrospective study.

Hand, foot, and mouth disease (HFMD) is well recognized as one of the major threats to children's health globally. The increasing complexity of the etiology of HFMD still challenges disease control in China. There is little surveillance of the molecular epidemiological characteristics of the enteroviruses (EVs) that cause HFMD in Neijiang city or the Sichuan Basin area in Southwest China. In this study, demographic and epidemiological information for 14,928 probable HFMD cases was extracted and analyzed to describe the epidemic features of HFMD in Neijiang city from Jan 2010 to Dec 2017. The swab samples of select probable HFMD cases from 2012 to 2017 were tested by reverse transcription (RT) real-time PCR to identify the serotype distribution of EVs, and 110 randomly selected RT-real-time PCR positive samples were then amplified and analyzed for the VP1 or VP4 regions of EVs to further analyze the phylogenetic characteristics of the circulating strains in this area. The eight-year average annual incidence was 49.82 per 100,000 in Neijiang. The incidence rates varied between 19.51 and 70.73 per 100,000, demonstrating peaks of incidence in even-number years (2012, 2014 and 2016). The median age of the probable cases was 27 months and the interquartile range (25th to 75th percentile) of ages for the probable HFMD cases was between 14 and 42 months. The male-to-female ratio of the probable HFMD cases was 1.47:1, and scattered children were the major population classification (81.7%). Two epidemic peaks were observed: one major peak between April and July and the other lesser peak between October and December. Of 6513 probable cases tested with RT-real-time PCR, 4015 (61.6%) were positive for enterovirus with the serotype distribution as follows: EV71+, 30.1% (n = 1210); CV-A16+, 28.7% (n = 1154) and a sole pan-enterovirus+, 41.1% (n = 1651). A total of 91 cases (82.7%, 91/110) were successfully amplified and underwent phylogenetic analysis: all EV71+ cases were C4a serotype (n = 23/30); all CV-A16+ cases were B2b serotype (n = 24/30); of 42 sole pan-enterovirus+ samples, 20 were CV-A6, 14 were CV-A10 and the rest within this group were CV-A4 (n = 4), CV-A8 (n = 2), CV-A9 (n = 1) and CV-B3 (n = 1). Our findings provide important evidence that aids the improvement of strategies for vaccination against HFMD and comprehensive disease control in China.

High Permissiveness for Genetic Exchanges between Enteroviruses of Species A, including Enterovirus 71, Favors Evolution through Intertypic Recombination in Madagascar.

Human enteroviruses of species A (EV-A) are the leading cause of hand-foot-and-mouth disease (HFMD). EV-A71 is frequently implicated in HFMD outbreaks and can also cause severe neurological manifestations. We investigated the molecular epidemiological processes at work and the contribution of genetic recombination to the evolutionary history of EV-A in Madagascar, focusing on the recently described EV-A71 genogroup F in particular. Twenty-three EV-A isolates, collected mostly in 2011 from healthy children living in various districts of Madagascar, were characterized by whole-genome sequencing. Eight different types were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent recent intra- and intertypic genetic exchanges between the noncapsid sequences of Madagascan EV-A isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination, with one isolate displaying a mosaic genome resulting from recent genetic exchanges with Madagascan coxsackieviruses A7 and possibly A5 and A10 or common ancestors. The engineering and characterization of recombinants generated from progenitors belonging to different EV-A types or EV-A71 genogroups with distantly related nonstructural sequences indicated a high level of permissiveness for intertypic genetic exchange in EV-A. This permissiveness suggests that the primary viral functions associated with the nonstructural sequences have been highly conserved through the diversification and evolution of the EV-A species. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify surveillance of EV-A circulation and HFMD cases to prevent possible outbreaks due to emerging strains.IMPORTANCE Human enteroviruses of species A (EV-A), including EV-A71, are the leading cause of hand-foot-and-mouth disease (HFMD) and may also cause severe neurological manifestations. We investigated the circulation and molecular evolution of EV-A in Madagascar, focusing particularly on the recently described EV-A71 genogroup F. Eight different types, collected mostly in 2011, were identified, highlighting the local circulation and diversity of EV-A. Comparative genome analysis revealed evidence of frequent genetic exchanges between the different types of isolates. The three EV-A71 isolates had different evolutionary histories in terms of recombination. The engineering and characterization of recombinants involving progenitors belonging to different EV-A types indicated a high degree of permissiveness for genetic exchange in EV-A. No outbreak of disease due to EV-A has yet been reported in Madagascar, but the diversity, circulation, and evolution of these viruses justify the surveillance of EV-A circulation to prevent possible HFMD outbreaks due to emerging strains.

Vitamin D receptor protects glioblastoma A172 cells against Coxsackievirus A16 infection induced cell death in the pathogenesis of hand, foot, and mouth disease.

Hand, foot, and mouth disease (HFMD) was one of the most common children illnesses. Coxsackievirus A16 was one of the major pathogens that cause HFMD. However, the role of vitamin D underlying this common illness has not been elucidated. Our study examined that vitamin D levels was significantly lower in 33 HFMD patients, compared to 36 healthy children. Unexpectedly, both mRNA and protein expression of VDR were significantly decreased in CA16 infected glioblastoma A172 cells. And overexpression of VDR or vitamin D treatment in CA16 infected glioblastoma A172 cells could reverse the CA16 infection induced cell death, apoptosis or mitochondrial membrane rupture. Therefore, our study, for the first time, demonstrated that vitamin D and VDR could associate with the pathogenesis of HFMD. Thus might provide useful information for HFMD prevention and treatments.

ARRDC4 regulates enterovirus 71-induced innate immune response by promoting K63 polyubiquitination of MDA5 through TRIM65.

Enterovirus 71 (EV71) is the main causative agent of hand, foot and mouth disease (HFMD), which induces significantly elevated levels of cytokines and chemokines, leading to local or system inflammation and severe complications, whereas the underlying regulatory mechanisms and the inflammatory pathogenesis remain elusive. ARRDC4 is one member of arrestins family, having important roles in glucose metabolism and G-protein-coupled receptors (GPCRs) related physiological and pathological processes, however, the function of ARRDC4 in innate immune system is largely unknown. Here we identified that ARRDC4 expression was increased after EV71 infection in THP-1-derived macrophages and verified in EV71-infected HFMD patients and the healthy candidates. The expression level of ARRDC4 was positively correlated with the serum concentration of IL-6, TNF-α and CCL3 in clinical specimens. ARRDC4 interacted with MDA5 via the arrestin-like N domain, and further recruited TRIM65 to enhance the K63 ubiquitination of MDA5, resulting in activation of the downstream innate signaling pathway and transcription of proinflammatory cytokines during EV71 infection. Our data highlight new function of ARRDC4 in innate immunity, contributing to the better understanding about regulation of MDA5 activation after EV71 infection, and also suggest ARRDC4 may serve as a potential target for intervention of EV71-induced inflammatory response.

[Molecular epidemiology of hand-foot-mouth disease associated pathogen Coxsackievirus A10 identified in Fujian province, 2011-2014].

OBJECTIVE: To study the molecular epidemiology of hand-foot-mounth disease (HFMD) associated Coxsackievirus A10 (Cox A10) identified in Fujian province. METHODS: A total of 1 525 specimens from non-EV71 non-Cox A16 HFMD patients were collected during 2011-2014. Isolated virus strains were identified and sub-typed. Full-length coding regions for the VP1 gene of the predominant serotype Cox A10 isolates were amplified and sequenced. RESULTS: Among the 407 non-EV71 non-Cox A16 HFMD cases confirmed by virus isolation and molecular subtyping, 103 (25.3%) were caused by Cox A10, accounting for 11.0%, 6.0%, 18.4% and 9.2% among the HFMD-associated entero-viruses identified in 2011, 2012, 2013 and 2014, respectively, in Fujian province. Compared to the general features observed in the HFMD epidemics, no differences on the Cox A10-specificity rates were observed among factors as geographical origins, gender or age groups, but all with high rates of severity. Data from the nucleotide sequence analyses on VP1 genes showed low homology levels of 76.0%-77.1% among Cox A10 strains from Fujian province, in contrast to the prototype Cox A10 strain, but with high levels of homology in the amino acid sequences (91.9%-93.6%). RESULTS from the Phylogenetic analysis also indicated that Cox A10 isolates from Fujian province were distinct from the prototype strain or other isolates from other countries but was homologous to domestic strains, but the Fujian isolates clustered into multiple branches. CONCLUSIONS: Cox A10 remained one of the predominant serotypes of HFMD in Fujian province. Cox A10 isolates identified in Fujian province were co-circulating and co-evolving with other domestic strains.

[Expression of Type-Ι Interferon Production Pathway-Related Genes Induced by Infection Due to Enterovirus 71 or Coxsackievirus A16 in Normal Human Airway Epithelial Cells].

Hand, foot, and mouth disease(HFMD)is caused by mainly enterovirus 71(EV-A71)and coxsackievirus A16(CV-A16),and is a serious healthcare problem worldwide.EV-A71 infection is thought to progress readily to serious complications whereas CV-A16 infection, in general, results in mild symptoms and presents repeatedly. However, the underlying mechanisms leading to these differences are not known. We compared changes in expression of type-I interferon(IFN-I)-related genes in normal human bronchial epithelial(16HBE) cells. Gene-expression levels of TLR3,MAVS,MDA5,MyD88,IRF7,IFNαand IFNßwere elevated significantly after EVA71 infection.MDA5expression was increased markedly, and that of TLR3 and IRF3was decreased obviously after CV-A16 infection, but that of MAVS,MyD88,IFNαand IFNßdid not show significant differences. Viral copy number and viral titers suggested that CV-A16 replicates more efficiently than EV-A71 in 16HBE.These results suggest that IFN-I production pathway-related genes in response to infection by EV-A71 and CV-A16 have notable discrepancies. Such information could shine a light on the different manifestations caused by EV-A71 and CV-A16,and the mechanism of repeat infection by CV-A16.

Association of interleukin-6-572C/G gene polymorphism and serum or cerebrospinal fluid interleukin-6 level with enterovirus 71 encephalitis in Chinese Han patients with hand, foot, and mouth disease.

Interleukin-6 (IL-6), as one of pro-inflammatory cytokines, plays a key role in Enterovirus 71 (EV71) encephalitis. We investigated the association of IL-6-572C/G polymorphism and serum or cerebrospinal fluid (CSF) IL-6 level with EV71 encephalitis in patients with hand, foot, and mouth disease (HFMD). This study was carried out in 59 Chinese Han patients with EV71 encephalitis, 128 EV71-related HFMD without complications, and 232 controls. The IL-6-572C/G polymorphism was detected by polymerase chain reaction-restricted fragment length polymorphism gene analysis. Serum or CSF IL-6 levels were determined using a commercial enzyme-linked immunosorbent assay. The patients with EV71 encephalitis had a higher frequency of IL-6-572GG/GC genotype compared to the patients with EV71-related HFMD without encephalitis complications (40.7 vs. 15.6 %, odds ratio (OR)=3.70, 95 % confidence interval (CI)=1.83-7.50, p=0.001). Similarly, the frequency of IL-6-572 G allele among the patients with EV71 encephalitis was also higher than that of patients with EV71-related HFMD without encephalitis complications (23.7 vs. 8.6 %, OR=3.31, 95 % CI=1.80-6.08, p<0.001). Serum IL-6 levels in G carries (CG + GG) (195.1 ± 11.8 pg/ml) elevated significantly compared to CC homozygotes (167.7 ± 6.7 pg/ml) in EV71-infected patients (p<0.001), but no significant differences were observed in CSF IL-6 levels among different genotypes in patients with EV71 encephalitis. Furthermore, G carriers (GG + GC) (10.6 ±.29 mg/l) had significantly higher blood CRP levels compared to CC homozygotes (9.31 ± 1.93 mg/l) in patients with EV71 encephalitis (p=0.005). These findings suggested that IL-6-572 G allele was significantly associated with the susceptibility to EV71 encephalitis in Chinese Han patients, and IL-6-572 G allele might elevate the risk to EV71 encephalitis.

The significance of Notch ligand expression in the peripheral blood of children with hand, foot and mouth disease (HFMD).

BACKGROUND: Hand, foot and mouth disease (HFMD), a virus-induced infectious disease that usually affects infants and children, has an increased incidence in China in recent years. This study attempted to investigate the role of the Notch signaling pathway in the pathogenesis of HFMD. METHODS: Eighty-two children diagnosed with HFMD were enrolled into this study. The HFMD group was further divided into the uncomplicated HFMD and HFMD with encephalitis groups. The control group included 40 children who underwent elective surgery for treatment of inguinal hernias. RESULTS: Children with HFMD displayed significantly reduced CD3+, CD3+CD4+ and CD3+CD8+ cell subsets, but substantially enhanced CD3-CD19+ cell subset (p<0.05 versus control subjects). The expression levels of Notch ligands Dll1 and Dll4 in the peripheral blood of the HFMD group were significantly higher than those in the control group (p<0.05). There were statistically significant differences in CD3+, CD3+CD4+ and CD3-CD19+ cell subsets, but not in Notch ligand expression, between the uncomplicated HFMD and HFMD with encephalitis groups. Dll4 expression in HFMD subjects correlated negatively with the CD3+ and CD3+CD8+ cell subsets (p<0.05), but positively with the CD3-CD19+ cell subset (p<0.05). Furthermore, Dll4 expression in HFMD with encephalitis subjects correlated positively with total white blood cell (WBC) counts and total protein contents in cerebrospinal fluid (CSF) (p<0.05). CONCLUSIONS: The Notch ligand Dll4 exhibits a strong correlation with the CD3+, CD3+CD8+ and CD3-CD19+ cell subsets in children with HFMD, indicating that the Notch signaling may be involved in the development of HFMD by affecting the number and status of peripheral lymphocytes.

Differential interferon pathway gene expression patterns in Rhabdomyosarcoma cells during Enterovirus 71 or Coxsackievirus A16 infection.

Exposure of cells to type I interferon (IFN) induces an antiviral state that prevents viral infection, but viruses can utilize multiple tactics to antagonize the host immune system. Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are two major pathogens that cause hand, foot, and mouth disease (HFMD), which is prevalent among children. We found that both EV71 and CA16 have different reactions to type I IFN pretreatment and induction patterns of type I IFN on Rhabdomyosarcoma (RD) cells. Further, a human-α and ß IFN PCR array was employed to analyze the expressions of 84 genes related to the type I IFN pathway. We found significant up-regulation of multiple genes in the presence of type I IFN and differential regulation patterns during EV71 or CA16 infection in RD cells. For instance, EV71 infection repressed the JAK-STAT signaling pathway and interferon-stimulated gene (ISG) expression, whereas CA16 infection normally triggers the JAK-STAT pathway, leading to the expression of ISGs. Taken together, this study provides a comprehensive view of the differential impacts of EV71 and CA16 infection on 84 genes in the IFN pathway, shedding light on the different resistances of these viruses to type I IFN treatment and cytotoxic effects in RD cells.

A polymorphism in melanoma differentiation-associated gene 5 may be a risk factor for enterovirus 71 infection.

Enterovirus 71 (EV71) infection has a wide variety of clinical manifestations, from no symptoms to fatal disease. Host immune response may be a determinant of disease severity. We investigated the association of polymorphisms in three pattern recognition receptor (PRR) genes-toll-like receptor 3 (TLR3) (rs3775291), retinoic acid-inducible gene I (RIG-I) (rs10813831) and melanoma differentiation-associated gene 5 (MDA5) (rs1990760)-with the severity of EV71 infection. Polymorphisms of candidate genes in 87 EV71-infected patients and 57 asymptomatic controls were detected. Binary logistic regression analysis revealed statistically significant differences in polymorphism of MDA5 (rs1990760) between patients with severe EV71 infection and asymptomatic controls in an additive model (OR 0.424, 95% CI 0.213-0.845, p 0.015) and a dominant model (OR 0.256, 95% CI 0.103-0.635, p 0.003). Polymorphism of MDA5 (rs1990760) (OR 0.399, 95% CI 0.199-0.798, p 0.009) was found to be associated with the severity of EV71 infection with the analysis of ordinal logistic regression. These results indicated the association between MDA5 (rs1990760) polymorphism and an increased risk of a severe EV71 infection in Chinese children, which offers potential for investigating the innate immune mechanism of EV71 infection and identifying at-risk infants, for whom a preventive strategy may reduce the severity of EV71 infection.

lncRNA expression signatures in response to enterovirus 71 infection.

Outbreaks of hand, foot, and mouth disease caused by enterovirus 71 (EV71) have become considerable threats to the health of infants and young children. To identify the cellular long noncoding RNAs (lncRNAs) involved in the host response to EV71 infection, we performed comprehensive lncRNA and mRNA profiling in EV71-infected rhabdomyosarcoma cells through microarray. We observed the differential expression of more than 4800 lncRNAs during infection. Further analysis showed 160 regulated enhancer-like lncRNA and nearby mRNA pairs, as well as 313 regulated Rinn's lncRNA [M. Guttman I. Amit, M. Garber, C. French, M.F. Lin, D. Feldser, M. Huarte, O. Zuk, B.W. Carey, J.P. Cassady, M.N. Cabili, R. Jaenisch, T.S. Mikkelsen, T. Jacks, N. Hacohen, B.E. Bernstein, M. Kellis, A. Regev, J.L. Rinn, E.S. Lander. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458 (2009) 223-227, A.M. Khalil, M. Guttman, M. Huarte, M. Garber, A. Raj, D. Rivea Morales, K. Thomas, A. Presser, B.E. Bernstein, A. van Oudenaarden, A. Regev, E.S. Lander, J.L. Rinn. Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. Proc. Natl. Acad. Sci. USA 106 (2009) 11667-11672] and nearby mRNA pairs. The results provided information for further research on the prevention and treatment of EV71 infection, as well as on distinguishing severe and mild EV71 cases.

Comparative proteome analyses of host protein expression in response to Enterovirus 71 and Coxsackievirus A16 infections.

Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the main etiological agents of Hand, Foot and Mouth Disease (HFMD), a common disease among children and had caused several outbreaks in the Asia-Pacific region. Although being genetically close to each other, EV71 infection can cause serious and fatal neurological complications like encephalitis, myocarditis, acute flaccid paralysis (AFP) and aseptic meningitis, but not in CA16 infections. In this study, the cellular response of host cells infected with EV71 and CA16 was characterized and compared by 2-dimensional proteome analyses. A total of 16 proteins were identified to be differentially expressed in EV71 and CA16-infected host cells. Desmin and HSP27, both indirectly regulate the contraction of muscle cells, were significantly downregulated as a result of EV71 infection, suggesting a link to acute flaccid paralysis. The ability of EV71 to evade host immune system may be due to the downregulation of MHC-I synthesis proteins like protein disulfide isomerase A3 and calreticulin. Proteins such as nucleophosmin, nuclear ribonucleoprotein C, and eukaryotic translation initiation factor 2 were all downregulated significantly, suggesting the rapid shutting down of host translation machinery by EV71. These findings provide insight into the nature of high virulent EV71 infection as compared to CA16.

[The expression of CD95 and activated antigens in peripheral blood lymphocyte subsets in patients with hand foot and mouth disease (HFMD)].

OBJECTIVE: To investigate the expression of CD95 and special marker for activation of peripheral blood lymphocytes in patients with hand foot and mouth disease (HFMD) and its significance. METHODS: Immunofluorescent two-color flow cytometry was used to study the expression of CD95 and HLA-DR on lymphocytes in 58 patients with HFMD and 34 normal controls. RESULTS: Expression of CD3+ T cells was significantly lower in patients (63.82 +/- 7.74)% than that in controls (P < 0.001), meanwhile the expression of CD4+ T cells was (34.29 +/- 7.33)%, significantly lower than that of the controls (P < 0.005). The percentage of lymphocytes expressing HLA-DR in patients was (23.77 +/- 5.78)%, significantly higher than that of the controls (P < 0.005). Significant difference was observed in the expression of HLA- DR on CD8+ T cells in patients (1.34 +/- 1.12)% as compared with controls (P < 0.005). No significant difference in the expression of CD95 on lymphocytes was observed between patients and the controls (P > 0.05). CONCLUSION: The findings support that cellular immunodeficiency exists in patients and that lymphocytes were abnormally activated in the patients. The activation of peripheral blood T lymphocytes in patients mainly involves CD8 subset and it may play an important role in the immune response to antiviral infection.

Expression of an active foot-and-mouth disease virus RNA polymerase in Escherichia coli

The expression of a native form of the foot-and-mouth disease virus RNA polymerase was obtained. Two oligonucleotides of 66 base pairs were used to renuild the 5' end of the gene and to introduce the first methionine codon. The expression of the active polymerase in E. coli was achieved by inserting the gene before the tac promoter of the pKK223-3 plasmid