Evolutionary analysis of a newly isolated Banna virus strain from Yunnan, China.

Banna virus (BAV) is a typical arbovirus whose infection is associated with fever and viral encephalitis. The whole genome of BAV is composed of 12 RNA segments. BAVs, which have been divided into three genotypes (A, B, and C) based on phylogenetic analysis of segment 12 or segment 9, are currently undergoing rapid evolution. Recent studies have shown that BAV variation can exceed intraspecific limits and generate novel viruses. In the current study, a new BAV strain, named 113c5, was isolated from Culex tritaeniorhynchus and found to be a member of genotype A2 based on phylogenetic analysis of segments 9 and 12. The complete genome sequence of the new BAV strain described in the current study might contribute to the surveillance of evolutionary characteristics of BAVs.

The Effect of Immunosuppressive Adjuvant Kynurenine on Type 1 Diabetes Vaccine.

Inducing antigen-specific tolerance is a promising treatment for preventing or reversing Type 1 diabetes (T1D). In contrast to a vaccine that induces immune responses against pathogens, a tolerogenic vaccine can suppress immunity against antigens causing diseases by administrating a mixture of self-antigens with an adjuvant that decreases the strength of antigen-specific response. Kynurenine (Kyn) is an endogenous substance that can inhibit the natural killer cell and T cell proliferation and promote the differentiation of naïve T cells into regulatory T cells (Tregs). In this study, we evaluated the efficacy of Kyn as a novel suppressive adjuvant. Kyn was co-immunized with GAD65 phage vaccine to induce Treg cells and tolerogenic responses for the prevention of T1D in NOD mouse model. Mice were subcutaneously immunized two times with 1011 Pfu (100µL,1012 Pfu/ml) GAD65 phage vaccine doses mixed with 200 µg of Kyn. Serum antibodies and cytokines were detected by ELISA and electrochemiluminescence, respectively. Flow cytometry assay was used to analyze DC and Treg. MTS was used for the analysis of spleen lymphocyte proliferation. RNA sequencing was used to investigate mRNA and miRNA expression profiles in spleen lymphocytes. Compared to GAD65 phage vaccine alone, co-immunization of Kyn and GAD65 phage vaccine resulted in the prevention of hyperglycemia in 60% of mice for at least one month. Further, Kyn enhances GAD65-specific Th2-mediated immune responses; regulates the Th1/Th2 imbalance and increases the secretion of Th2 cytokines and the number of CD4+CD25+Foxp3+T cells; suppresses DC maturation and GAD65-specific T lymphocyte proliferation. Moreover, we integrated Kyn related miRNA and mRNA expression profiles obtained from the spleen lymphocyte RNA-sequencing which was stimulated by Kyn in vitro. These data provide an important basis for understanding the mechanisms underlying Kyn as an immunosuppressive adjuvant which regulated the immune response. These findings suggest that Kyn can serve as an effective suppressive adjuvant candidate for Type 1 diabetes vaccines.

Hepatitis A virus-induced hsa-miR-146a-5p attenuates IFN-ß signaling by targeting adaptor protein TRAF6.

Hepatitis A virus (HAV), a unique hepatotropic human picornavirus, is the causative agent of acute hepatitis A in humans. Some studies have shown that HAV antagonizes the innate immune response by disrupting interferon-beta (IFN-ß) signaling by viral proteins. However, whether microRNAs (miRNAs), a class of non-coding RNAs, are involved in the antagonism of IFN-ß induction upon HAV infection is still unclear. In this study, we investigated the effects and mechanisms by which HAV-induced miRNAs antagonize IFN-ß signaling. A variety of analytical methods, including miRNA microarray, RT-qPCR, dual-luciferase reporter assay, and Western blotting, were performed using HAV-infected cells. The results indicated that HAV infection upregulates the expression of hsa-miR-146a-5p, which in turn partially suppresses the induction of IFN-ß synthesis, thereby promoting viral replication. Mechanistically, TRAF6 (TNF receptor-associated factor 6), a key adaptor protein in the RIG-I/MDA5-mediated IFN-I signaling pathway, is targeted and degraded by hsa-miR-146a-5p. As TRAF6 is necessary for IFN-ß induction, inhibition of this protein attenuates IFN-ß signaling. Taken together, the results from this study indicated that HAV disrupts RIG-I/MDA5-mediated IFN-I signaling partially through the cleavage of the essential adaptor molecule TRAF6 via hsa-miR-146a-5p.

Phylogenetic and genetic analyses of the emerging Nipah virus from bats to humans.

Little is known about the genetic features of Nipah virus (NiV) associated with virulence and transmission. Herein, phylogenetic and genetic analyses for all available NiV strains revealed sequence variations between the two genetic lineages of NiV with pathogenic differences, as well as among different strains within Bangladesh lineage. A total of 143 conserved amino acid differences, distributed among viral nucleocapsid (N), phosphoprotein (P), matrix protein (M), fusion protein (F) and glycoprotein (G), were revealed. Structural modeling revealed one key substitution (S3554N) in the viral G protein that might mediate a 12-amino-acid structural change from a loop into a ß sheet. Multiple key amino acids substitutions in viral G protein were observed, which may alter viral fitness and transmissibility from bats to humans.

Comprehensive Analysis of the Expression Profiles of Long Non-Coding RNAs with Associated ceRNA Network Involved in the Colon Cancer Staging and Progression.

Long non-coding RNAs (lncRNAs) act as competing endogenous RNAs (ceRNAs) to compete with microRNAs (miRNAs) in cancer occurrence and development. However, the differential expression of RNAs and their ceRNA network during the development of colon cancer (CC) remains unclear. This study was aimed at comprehensive analysis of the lncRNAs and their ceRNA networks associated with CC. Whole transcriptome sequencing was performed on colorectal and adjacent normal tissues at different pathological stages. Forty-nine lncRNAs were differently expressed between the CC tissues and their adjacent normal tissues at all stages. Aberrant expression of lncRNA CDKN2B-AS1 and lncRNA MIR4435-2HG was confirmed by TCGA database. Moreover, 14 lncRNAs were differentially expressed between early and advance stages of the tumor tissues, and 117 miRNAs were specifically expressed in stage III & IV. Weighted gene co-expression network analysis of 17105 differently expressed mRNAs revealed that the mRNAs shown in module pink, midnight blue, black, and light cyan were related to TNM and pathological stage, and that these mRNAs were enriched in cancer related functions and pathways. As DElncRNA showed a trend of change similar to that of the DEmRNA and opposite to that of DEmiRNA, ceRNA network was constructed with 3 DEmiRNAs, 5 DElncRNAs, and 130 DEmRNAs. Real time PCR revealed that expression of MEG3 was decreased in the tumor tissues belonging to stage III and IV as compared to that in stage I. Moreover, hsa-miR-324-5p was upregulated, while FGFR3, PLCB4, and IKBKB were downregulated in the tumor tissues as compared to that in the adjacent normal tissues. Thus, this study revealed differentially expressed lncRNA between different stages of CC as well as suggested that lncRNA CDKN2B-AS1, MIR4435-2HG, and MEG3 may act as diagnostic biomarkers for the development of CC.

Full-length genome and molecular characterization of dengue virus serotype 2 isolated from an imported patient from Myanmar.

BACKGROUND: Dengue is the most common mosquito-borne infection worldwide and a serious threat to global public health. Sporadic dengue virus serotype 2 (DENV-2) imported cases from Myanmar have been documented almost every year in Yunnan Province of China since 2005. However, the complete genome sequences of DENV-2 isolates imported from Myanmar are not available. METHODS: The full-length genome of the DENV-2 strain (YNPE2), isolated from an imported case from Myanmar in 2013, was identified by the next-generation sequencing. The extreme ends of the viral genome were validated by 5'/3' RACE and Sanger sequencing. Furthermore, phylogenetic, recombination and selection pressure analyses were conducted for the molecular characterization of YNPE2 strain. RESULTS: Whole-genome sequencing revealed that the full-length sequence of YNPE2 strain was 10,724 bases, with an open reading frame encoding for 3391 amino acids. The YNPE2 strain had 99.0% nucleotide identity and 99.8% amino acid identity with two closely related strains, ThD2_0078_01 strain (DQ181797) and DENV-2/TH/BID-V2157/200 strain (FJ639832). The phylogenetic analysis suggested that the YNPE2 strain belonged to Asian I genotype and was likely derived from Thailand strain (DQ181797). Moreover, selection pressure analysis revealed two amino acid sites of the NS4B and NS5 proteins, with important evidence of positive selection. CONCLUSION: This study revealed the first complete genome sequence and molecular characterization of a DENV-2 strain (YNPE2) isolated from an imported case from Myanmar, thus providing a valuable reference genome source for future surveillance, epidemiology and vaccine development of DENV-2 virus in Yunnan, China.

Complete Genome Sequence Analysis of an Imported Dengue Virus Serotype 1 Strain from Myanmar.

It has been determined that recent dengue virus epidemics in Yunnan, China, originated from Southeast Asian strains. Here, we report the first complete genome sequence and molecular characterization of the imported dengue virus serotype 1 strain YNPE1. Phylogenetic analysis revealed that strain YNPE1 belonged to genotype I.

Molecular characterization and phylogenetic analysis of a dengue virus serotype 3 isolated from a Chinese traveler returned from Laos.

BACKGROUND: Dengue virus (DENV) infection caused by international visitors has become a public health concern in China. Although sporadic imported cases of DENV have been documented in Yunnan, China since 2000, a complete genome sequence of dengue virus serotype 3 (DENV-3) imported from Laos is still not available. Here, we report the first complete genome sequence and genomic characterization of a DENV-3 strain (YNPE3) isolated from a patient returned from Laos. METHODS: Viral isolation from the patient's serum was performed using mosquitoes C6/36 cells. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for identification and serotyping of the virus. The complete sequence was determined by Sanger dideoxy sequencing. Homology analysis was implemented by NCBI-BLAST. Multiple sequence alignment was performed using MegAlign module of the Lasergene 7 software package DNASTAR. MFOLD software was used to predict the RNA secondary structure of 5' untranslated region (UTR) and 3' UTR. Phylogenetic analysis, which was based on envelope gene and complete coding sequence, was performed by Maximum-Likelihood method. RESULTS: RT-PCR analysis confirmed that the virus belonged to dengue virus serotype 3, which was named YNPE3 strain. The full-length genome of the YNPE3 strain was 10,627 nucleotides (nts) with an open reading frame (ORF) encoding 3390 amino acids. Strain YNPE3 shared 98.6-98.8% nucleotide identity with the closely related strains isolated in India (JQ922556, KU216209, KU216208). We observed the deletion of about 40 nts in the 5' UTR and 3' UTR of strain YNPE3, and 11 nts (ACGCAGGAAGT) insertion that was present in the 3' UTR of YNPE3. Compared with prototype strain H87, abundant amino acid substitutions in the YNPE3 strain were observed. Phylogenetic analysis revealed that the YNPE3 strain belonged to genotype III of DENV-3, and that it might be closely related with genotype III strains isolated in Laos and India. CONCLUSIONS: This is the first report of the complete genome sequence and molecular characterization of a DENV-3 isolate imported from Laos. The presented results can further promote disease surveillance, and epidemiological and evolutionary studies of the DENV-3 in Yunnan province of China.

Deep RNA Sequencing Reveals a Repertoire of Human Fibroblast Circular RNAs Associated with Cellular Responses to Herpes Simplex Virus 1 Infection.

BACKGROUND/AIMS: Circular RNAs (circRNAs), a new class of regulators of gene expression, are involved in diverse physiological and pathogenic processes. However, their role in cellular responses to virus infection is yet unclear. METHODS: A human lung fibroblast cell line was infected or mock infected by herpes simplex virus 1 (HSV-1). Deep RNA sequencing was used to profile the global changes in circRNAs, genes, and miRNAs following HSV-1 infection. Altered circRNAs, genes, and miRNAs were validated using quantitative reverse transcription polymerase chain reaction (RT-qPCR). An integration analysis of circRNAs, genes, and miRNAs was applied to investigate the putative function of the dysregulated circRNAs. RESULTS: A total of 536 circRNAs, 3,885 genes, and 207 miRNAs were significantly dysregulated after HSV-1 infection. An integration analysis of circRNAs, genes, and miRNAs revealed the alleged involvement of dysregulated circRNAs in cellular responses to HSV-1 infection via the circRNA-miRNA-gene regulatory axis. These genes regulated by circRNAs were enriched to NOD-like receptor/JAK-STAT signaling pathway, and pathways of apoptosis, cell cycle progression, and cell death, all of which may be implicated in the viral pathogenesis and cellular immunity. CONCLUSIONS: These data present a comprehensive view for circRNAs induced by HSV-1 and their interplay with miRNAs and genes during HSV-1 infection, thus offering new insights into the mechanisms of interactions between HSV-1 and the host.

Molecular characterization of an imported dengue virus serotype 4 isolate from Thailand.

The epidemic of dengue virus infections has spread markedly in Yunnan province of China in recent years due to an increase in the number of imported dengue cases. To the best of our knowledge, the present study is the first to report a whole genome sequence and molecular characterization of an imported DENV-4 isolate from Thailand. The current strain, 2013JH285, has an RNA genome of 10,772 nucleotides that shares 99.0% nucleotide and 99.7% amino acid sequence identity with the 2013 Thailand strain CTI2-13. Phylogenetic analysis of the whole genome sequence revealed that the 2013JH285 strain belongs to genotype I of DENV-4. Recombination analysis suggested that the 2013JH285 strain originated from inter-genotypic recombination of DENV-4 strains. The new complete DENV-4 genome sequence reported here might contribute to further understanding of the molecular epidemiology and disease surveillance of DENV-4 in China.

Unique expression signatures of circular RNAs in response to DNA tumor virus SV40 infection.

Circular RNAs (circRNAs), identified as a class of widely expressed endogenous regulatory RNAs, are involved in diverse physiological and pathological processes. However, their role in viral pathogenesis and cellular antiviral response remains unexplored. In this study, a potent DNA tumor virus, simian virus 40 (SV40), was used as a model to investigate the viral influences on cellular circRNA transcriptome. Using RNA-seq, 15,241 putative circRNAs were identified de novo from 5,057 parental genes in monkey kidney-derived Vero cells. The expression of selected circRNAs was confirmed by reverse transcription-polymerase chain reaction and Sanger sequencing. Further analysis showed that most circRNAs comprised multiple exons, and most parental genes produced multiple circRNA isoforms. A total of 134 significantly dysregulated circRNAs, including 103 upregulated and 31 downregulated circRNAs, were found after SV40 infection. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that various cancer-related pathways, including p53 and Wnt pathway, could be affected by SV40 infection via the alteration of the circRNA hosting genes. Moreover, diverse cellular immune pathways, including Toll-like receptor pathway and Janus kinase-signal transducer and activator of transcription pathway, could also be affected by SV40 infection. An integrated circRNA-miRNA-gene analysis suggested the putative function of circRNAs as cellular and viral miRNA decoys to indirectly regulate the gene expression during SV40 infection. This study presented the first comprehensive expression and functional profile of circRNAs in response to SV40 infection, thus providing new insights into the mechanisms of viral pathogenesis and cellular immune response.

Comprehensive profiling and characterization of cellular miRNAs in response to hepatitis A virus infection in human fibroblasts.

Hepatitis A virus (HAV), the causative agent of acute hepatitis, grows slowly without causing any cytopathic effect (CPE) and lead to a persistent infection in the fibroblasts in vitro. miRNAs play a key role in the viral pathogenesis and virus-host interactions. In this study, the comprehensive miRNA expression profiles of HAV-infected and uninfected fibroblasts were investigated by sRNA-seq and validated by RT-qPCR. The results showed that a total of 94 miRNAs were differentially expressed during HAV infection, including 11 up-regulated miRNAs and 83 down-regulated miRNAs. RT-qPCR analysis showed the expression levels of specific miRNAs were consistent with sRNA-seq data. Further, target prediction analysis showed 729 putative target genes that included many immune-related transcripts were revealed. The GO enrichment analysis and the KEGG pathway analysis of the target genes showed that various biological pathways, including JAK-STAT cascade, type I interferon signaling pathway could be affected by HAV infection by the alteration of host miRNAs. The core regulatory relationship between miRNAs and their targets were revealed by miRNA-gene-network. Collectively, this study provides an overall analysis of miRNA profile in cell culture infected with HAV. The present results imply the alteration of miRNAs expression induced by HAV infection which may be related to the establishment of persistent HAV infection and might provide new clues for understanding the persistent HAV infections in vitro and the unique biological characteristics associated with HAV during infection.

Small-Activating RNA Can Change Nucleosome Positioning in Human Fibroblasts.

RNA activation (RNAa) is a mechanism of positive gene expression regulation mediated by small-activating RNAs (saRNAs), which target gene promoters and have been used as tools to manipulate gene expression. Studies have shown that RNAa is associated with epigenetic modifications at promoter regions; however, it is unclear whether these modifications are the cause or a consequence of RNAa. In this study, we examined changes in nucleosome repositioning and the involvement of RNA polymerase II (RNAPII) in this process. We screened saRNAs for OCT4 (POU5F1), SOX2, and NANOG, and identified several novel saRNAs. We found that nucleosome positioning was altered after saRNA treatment and that the formation of nucleosome-depleted regions (NDRs) contributed to RNAa at sites of RNAPII binding, such as the TATA box, CpG islands (CGIs), proximal enhancers, and proximal promoters. Moreover, RNAPII appeared to be bound specifically to NDRs. These results suggested that changes in nucleosome positions resulted from RNAa. We thus propose a hypothesis that targeting promoter regions using exogenous saRNAs can induce the formation of NDRs, exposing regulatory binding sites to recruit RNAPII, a key component of preinitiation complex, and leading to increased initiation of transcription.

Hepatitis A virus-encoded miRNAs attenuate the accumulation of viral genomic RNAs in infected cells.

The establishment of persistent infection with hepatitis A virus (HAV) is the common result of most HAV/cell culture systems. Previous observations show that the synthesis of viral RNAs is reduced during infection. However, the underlying mechanism is poorly understood. We characterized three HAV-encoded miRNAs in our previous study. In this study, we aim to investigate the impact of these miRNAs on the accumulation of viral RNAs. The results indicated that the synthesis of viral genomic RNAs was dramatically reduced (more than 75 % reduction, P < 0.05) when transfected with one or two viral miRNA mimics. Conversely, they were significantly increased (more than 3.3-fold addition, P < 0.05) when transfected with one or two viral miRNA inhibitors. The luciferase reporter assay of miRNA targets showed that viral miRNAs were fully complementary to specific sites of the viral plus or minus strand RNA and strongly inhibited their expressions. Further data showed that the relative abundance of viral genomic RNA fragments that contain miRNA targets was also dramatically reduced (more than 80 % reduction, P < 0.05) when viral miRNAs were overexpressed with miRNA mimics. In contrast, they were significantly increased (approximately 2-fold addition, P < 0.05) when viral miRNAs were inhibited with miRNA inhibitors. In conclusion, these data suggest a possible mechanism for the reduction of viral RNA synthesis during HAV infection. Thus, we propose that it is likely that RNA virus-derived miRNA could serve as a self-mediated feedback regulator during infection.

Influence of miR-30b regulating humoral immune response by genetic difference.

Investigation of genetic difference will be beneficial to researchers to understand the origins and nature of diseases. Previous studies have revealed that L-kynurenine (L-Kyn) level was changed significantly in patient with cancer and that miR-30b play different role in tumor cells and immune cells. Moreover, it has been also conformed that miR-30b involved in the process of L-Kyn-mediated suppression of humoral immune responses induced by lipopolysaccharide (LPS) in human normal B cells separated from volunteers' peripheral blood. Nevertheless, the miR-30b role regulating humoral immune response in B lymphoma cells has been still unclear due to the genetic difference between normal cells and tumor cells. The current study demonstrated that the selected concentration of L-Kyn (100, 1000 µM) significantly reduced the immunoglobulin M secretion induced by LPS when compared with the control group in B lymphoma, CH12.LX, and BCL-1 cells, which had, at least, incomplete dependence on Aryl hydrocarbon receptor, the receptor of L-Kyn. In addition, although L-Kyn (100 µM) significantly attenuated the expression of miR-30b in BCL-1 cells rather than in CH12.LX cells, no significant differences in the strength of L-Kyn-mediated suppression of humoral immune responses induced by LPS were detected by enzyme-linked immunosorbent assay between the LPS (10 µg/ml) + L-Kyn (100 µM) group and the LPS (10 µg/ml) + L-Kyn (100 µM) + miR-30b mimics/miR-30b inhibitor group in CH12.LX and BCL-1 cells, respectively. Further data also showed that mouse Bach2 mRNA was a novel target of miR-30b. These results suggest that genetic difference among cells has a great influence on the miR-30b role in the process of L-Kyn-mediated suppression of humoral immune responses induced by LPS.

Epitope-Based Vaccine Target Screening against Highly Pathogenic MERS-CoV: An In Silico Approach Applied to Emerging Infectious Diseases.

Middle East respiratory syndrome coronavirus (MERS-CoV) with pandemic potential is a major worldwide threat to public health. However, vaccine development for this pathogen lags behind as immunity associated with protection is currently largely unknown. In this study, an immunoinformatics-driven genome-wide screening strategy of vaccine targets was performed to thoroughly screen the vital and effective dominant immunogens against MERS-CoV. Conservancy and population coverage analysis of the epitopes were done by the Immune Epitope Database. The results showed that the nucleocapsid (N) protein of MERS-CoV might be a better protective immunogen with high conservancy and potential eliciting both neutralizing antibodies and T-cell responses compared with spike (S) protein. Further, the B-cell, helper T-cell and cytotoxic T lymphocyte (CTL) epitopes were screened and mapped to the N protein. A total of 15 linear and 10 conformal B-cell epitopes that may induce protective neutralizing antibodies were obtained. Additionally, a total of 71 peptides with 9-mer core sequence were identified as helper T-cell epitopes, and 34 peptides were identified as CTL epitopes. Based on the maximum HLA binding alleles, top 10 helper T-cell epitopes and CTL epitopes that may elicit protective cellular immune responses against MERS-CoV were selected as MERS vaccine candidates. Population coverage analysis showed that the putative helper T-cell epitopes and CTL epitopes could cover the vast majority of the population in 15 geographic regions considered where vaccine would be employed. The B- and T-cell stimulation potentials of the screened epitopes is to be further validated for their efficient use as vaccines against MERS-CoV. Collectively, this study provides novel vaccine target candidates and may prompt further development of vaccines against MERS-CoV and other emerging infectious diseases.

Inferring Protective CD8+ T-Cell Epitopes for NS5 Protein of Four Serotypes of Dengue Virus Chinese Isolates Based on HLA-A, -B and -C Allelic Distribution: Implications for Epitope-Based Universal Vaccine Design.

Dengue is one of the most globally serious vector-borne infectious diseases in tropical and subtropical areas for which there are currently no effective vaccines. The most highly conserved flavivirus protein, NS5, is an indispensable target of CD8+ T-cells, making it an ideal vaccine design target. Using the Immune Epitope Database (IEDB), CD8+ T-cell epitopes of the dengue virus (DENV) NS5 protein were predicted by genotypic frequency of the HLA-A,-B, and-C alleles in Chinese population. Antigenicity scores of all predicted epitopes were analyzed using VaxiJen v2.0. The IEDB analysis revealed that 116 antigenic epitopes for HLA-A (21),-B (53), and-C (42) had high affinity for HLA molecules. Of them, 14 had 90.97-99.35% conversancy among the four serotypes. Moreover, five candidate epitopes, including 200NS5210 (94.84%, A*11:01), 515NS5525 (98.71%, A*24:02), 225NS5232 (99.35%, A*33:03), 516NS5523 (98.71%, A*33:03), and 284NS5291 (98.06%, A*33:03), were presented by HLA-A. Four candidate epitopes, including 234NS5241 (96.77%, B*13:01), 92NS599 (98.06%, B*15:01, B*15:02, and B*46:01), 262NS5269 (92.90%, B*38:02), and 538NS5547 (90.97%, B*51:01), were presented by HLA-B. Another 9 candidate epitopes, including 514NS5522 (98.71%, C*01:02), 514NS5524 (98.71%, C*01:02 and C*14:02), 92NS599 (98.06%, C*03:02 and C*15:02), 362NS5369 (44.84%, C*03:04 and C*08:01), 225NS5232 (99.35%, C*04:01), 234NS5241(96.77%, C*04:01), 361NS5369 (94.84%, C*04:01), 515NS5522 (98.71%, C*14:02), 515NS5524 (98.71%, C*14:02), were presented by HLA-C. Further data showed that the four-epitope combination of 92NS599 (B*15:01, B*15:02, B*46:01, C*03:02 and C*15:02), 200NS5210 (A*11:01), 362NS5369 (C*03:04, C*08:01), and 514NS5524 (C*01:02, C*14:02) could vaccinate >90% of individuals in China. Further in vivo study of our inferred novel epitopes will be needed for a T-cell epitope-based universal vaccine development that may prevent all four China-endemic DENV serotypes.

Identification and validation of a novel microRNA-like molecule derived from a cytoplasmic RNA virus antigenome by bioinformatics and experimental approaches.

BACKGROUND: It is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs (miRNAs) due to nucleus inaccessibility. Recent studies have described cytoplasmic RNA virus genome-derived miRNAs in West Nile virus (WNV) and Dengue virus (DENV). However, naturally occurring miRNAs derived from the antigenome of a cytoplasmic RNA virus have not been described. METHODS: Hepatitis A virus (HAV) was served as a model virus to investigate whether the antigenome of a cytoplasmic RNA virus would be processed into miRNAs or miRNA-like small RNAs upon infection. HAV antigenome was queried for putative miRNA precursors (pre-miRNA) with the VMir analyzer program. Mature miRNA prediction was performed using MatureBayes and Bayes-SVM-MiRNA web server v1.0. Finally, multiple experimental approaches, including cloning and sequencing-, RNAi-, plasmid-based miRNA expression- and luciferase reporter assays, were performed to identify and validate naturally occurring viral antigenome-derived miRNAs. RESULTS: Using human HAV genotype IA (isolate H2) (HAVH2), a virally encoded miRNA-like small RNA was detected on the antigenome and named hav-miR-N1-3p. Transcription of viral pre-miRNA in KMB17 and HEK293T cells led to mature hav-miR-N1-3p production. In addition, silencing of the miRNA-processing enzyme Dicer or Drosha caused a dramatic reduction in miRNA levels. Furthermore, artificial target of hav-miR-N1-3p was silenced by synthesized viral miRNA mimics and the HAVH2 naturally-derived hav-miR-N1-3p. CONCLUSION: These results suggested that the antigenome of a cytoplasmic RNA virus could be processed into functional miRNAs. Our findings provide new evidence supporting the hypothesis that cytoplasmic RNA viruses naturally encode miRNAs through cellular miRNA processing machinery.

Novel microRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection.

MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host-virus interactions. DNA viruses encode miRNAs that regulate the viral life cycle. However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery. Here, we provide a comprehensive genome-wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV; Hu/China/H2/1982), which is a typical cytoplasmic RNA virus. Using deep-sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav-miR-1-5p and hav-miR-2-5p. Both of the novel virally encoded miRNAs were clearly detected in infected cells. Analysis of Dicer enzyme silencing demonstrated that HAV-derived miRNA biogenesis is Dicer dependent. Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre-miRNAs) in host cells. Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post-transcriptional gene silencing (PTGS). Genomic location analysis revealed novel miRNAs located in the coding region of the viral genome. Overall, our results show that HAV naturally generates functional miRNA-like small regulatory RNAs during infection. This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus. These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do. These findings also contribute to improved understanding of host-RNA virus interactions mediated by RNA virus-derived miRNAs.

Attenuation of antigenic immunogenicity by kynurenine, a novel suppressive adjuvant.

A novel therapeutic strategy is required for autoimmune diseases characterized by the production of autoantibody, because current clinical strategies have limitations. Vaccination against autoimmune diseases is a feasible strategy because vaccines induce immune response memory and the antigen specificity. However, no suitable adjuvant is available to direct the immune response toward tolerance or suppression. In the current study, we evaluated whether kynurenine (Kyn) could serve as a novel suppressive adjuvant to decrease the humoral immune responses against hepatitis A virus (HAV) in the ICR mouse model in vivo and lipopolysaccharide (LPS) in B cells in vitro. The underlying mechanisms of Kyn-mediated suppression of LPS-induced IgM responses were explored. The results showed that Kyn significantly decreased HAV immunogenicity when co-administered with HAV, and that Kyn (100 µM/1000 µM) impaired IgM generation compared with that induced by LPS alone. We also demonstrated that microRNA30b (miR30b) played a critical role in the process of Kyn-mediated suppression of IgM responses induced by LPS, and that Bach2, a transcriptional repressor of B cell terminal differentiation, was a novel target of miR30b. These findings suggest that Kyn can serve as a novel and effective suppressive adjuvant for vaccines.