Geminiviruses cause substantial crop yield losses worldwide. The replication initiator protein (Rep) encoded by geminiviruses is indispensable for geminiviral replication. The Rep protein encoded by beet severe curly top virus (BSCTV, genus Curtovirus, family Geminiviridae) induces VARIANT IN METHYLATION 5 (VIM5) expression in Arabidopsis leaves upon BSCTV infection. VIM5 functions as a ubiquitination-related E3 ligase to promote the proteasomal degradation of methyltransferases, resulting in reduction of methylation levels in the BSCTV C2-3 promoter. However, the specific domains of Rep responsible for VIM5 induction remain poorly characterized. Although Rep proteins from several geminiviruses act as viral suppressors of RNA silencing (VSRs), whether BSCTV Rep also possesses VSR activity remains to be illustrated. In this study, we employed a transient expression system in the 16c-GFP transgenic and the wild-type Nicotiana benthamiana plants to analyze the VSR and the VIM5-inducing activities of different truncated Rep proteins haboring distinct domains. We found that the N-terminal domain (amino acids 1-180) of Rep suppressed GFP silencing in 16c-GFP transgenic N. benthamiana leaves. The minimal N-terminal fragment (amino acids 1-104) induced VIM5 expression upon co-infiltration, while C-terminal truncations lacked VIM5-inducing activity. Our results indicate that the N-terminal domain of Rep encoded by BSCTV mediates the suppression of RNA silencing and induces VIM5 expression. Thus, our findings contribute to a better understanding of interactions between geminiviral Rep and plant hosts.
Geminiviridae/genetics* ; Nicotiana/metabolism* ; Arabidopsis/metabolism* ; RNA Interference ; Viral Proteins/metabolism* ; Arabidopsis Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Protein Domains ; Plant Diseases/virology* ; Methyltransferases/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; DNA Helicases/genetics*

Influenza C virus is an important respiratory pathogen not only infecting people, but also pigs, dogs, and other animals. Polymerase is central to the replication of influenza C virus and is an important target for studying the mechanism of viral replication. However, there is no commercial monoclonal antibody (MAb) targeting influenza C virus polymerase, which hampers the development of relevant research to some extent. In order to prepare MAb targeting the polymerase basic protein 2 (PB2) of influenza C virus, influenza C virus RNA-dependent RNA polymerase (RdRp, consists of PB1, PB2 and P3) was co-immunoprecipitated with Flag-tagged human acidic nuclear phosphoprotein 32A (huANP32A-Flag) from 293T cells based on the interaction between huANP32A and influenza virus RdRp. The purified RdRp was used as antigen to immunize BALB/c mice. Six positive hybridoma cell lines (7B11-5, 8A4-5, 13D9-6, 8D4-1, 8D4-3, 9F9-4) that stably secrete and recognize PB2 MAb were screened by indirect ELISA and Western blotting. The subtypes of MAb 7B11-5, 8A4-5, 8D4-1 and 8D4-3 antibody were identified as IgG1, the subtypes of MAb 13D9-6 and 9F9-4 were IgG2a and IgG3, respectively. All the light chains of the MAbs were κ chain. A hybridoma cell line 8D4-1 with high titer was further selected to prepare ascites. The titer of mouse ascites antibody was determined to be 1:64 000. Western blotting results showed that the MAb 8D4-1 had a specific immune response with ICV PB2; laser confocal assay showed that the prepared MAb 8D4-1 accurately detected the subcellular localization of PB2 subunits. Moreover, ICV RdRp was highly enriched by ANP32A. The high specific of the prepared PB2 MAb 8D4-1 may facilitate the polymerase detection, structural analysis and mechanism study of influenza C virus.
Animals ; Antibodies, Monoclonal/metabolism* ; Ascites ; Humans ; Influenzavirus C/metabolism* ; Mice ; Nuclear Proteins/metabolism* ; RNA-Binding Proteins ; RNA-Dependent RNA Polymerase/genetics* ; Viral Proteins/metabolism* ; Virus Replication

Age-associated changes in immune cells have been linked to an increased risk for infection. However, a global and detailed characterization of the changes that human circulating immune cells undergo with age is lacking. Here, we combined scRNA-seq, mass cytometry and scATAC-seq to compare immune cell types in peripheral blood collected from young and old subjects and patients with COVID-19. We found that the immune cell landscape was reprogrammed with age and was characterized by T cell polarization from naive and memory cells to effector, cytotoxic, exhausted and regulatory cells, along with increased late natural killer cells, age-associated B cells, inflammatory monocytes and age-associated dendritic cells. In addition, the expression of genes, which were implicated in coronavirus susceptibility, was upregulated in a cell subtype-specific manner with age. Notably, COVID-19 promoted age-induced immune cell polarization and gene expression related to inflammation and cellular senescence. Therefore, these findings suggest that a dysregulated immune system and increased gene expression associated with SARS-CoV-2 susceptibility may at least partially account for COVID-19 vulnerability in the elderly.
Adult ; Aged ; Aged, 80 and over ; Aging ; genetics ; immunology ; Betacoronavirus ; CD4-Positive T-Lymphocytes ; metabolism ; Cell Lineage ; Chromatin Assembly and Disassembly ; Coronavirus Infections ; immunology ; Cytokine Release Syndrome ; etiology ; immunology ; Cytokines ; biosynthesis ; genetics ; Disease Susceptibility ; Flow Cytometry ; methods ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Gene Rearrangement ; Humans ; Immune System ; cytology ; growth & development ; immunology ; Immunocompetence ; genetics ; Inflammation ; genetics ; immunology ; Mass Spectrometry ; methods ; Middle Aged ; Pandemics ; Pneumonia, Viral ; immunology ; Sequence Analysis, RNA ; Single-Cell Analysis ; Transcriptome ; Young Adult

It was widely known that retinoic acid inducible gene I (RIG-I) functions as a cytosolic pattern recognition receptor that initiates innate antiviral immunity by detecting exogenous viral RNAs. However, recent studies showed that RIG-I participates in other various cellular activities by sensing endogenous RNAs under different circumstances. For example, RIG-I facilitates the therapy resistance and expansion of breast cancer cells and promotes T cell-independent B cell activation through interferon signaling activation by recognizing non-coding RNAs and endogenous retroviruses in certain situations. While in hepatocellular carcinoma and acute myeloid leukemia, RIG-I acts as a tumor suppressor through either augmenting STAT1 activation by competitively binding STAT1 against its negative regulator SHP1 or inhibiting AKT-mTOR signaling pathway by directly interacting with Src respectively. These new findings suggest that RIG-I plays more diverse roles in various cellular life activities, such as cell proliferation and differentiation, than previously known. Taken together, the function of RIG-I exceeds far beyond that of a pattern recognition receptor.
Animals ; DEAD Box Protein 58 ; genetics ; metabolism ; Mice ; RNA, Viral ; genetics ; metabolism ; STAT1 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; genetics ; physiology

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.
Animals ; Connectin/genetics* ; Cricetinae ; Eukaryotic Initiation Factor-4G/genetics* ; Gene Expression Regulation/genetics* ; Gene Silencing ; Growth Hormone/genetics* ; Hepatitis B Surface Antigens/genetics* ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/virology* ; Humans ; Hydro-Lyases/metabolism* ; Male ; Pregnancy-Specific beta 1-Glycoproteins/genetics* ; RNA, Viral/analysis* ; Spermatozoa/virology* ; Trans-Activators/genetics* ; Transcription, Genetic ; Transfection ; Viral Regulatory and Accessory Proteins

From 2013 to 2015, the National Institute of Health, Pakistan, received 1,270 blood samples of suspected dengue cases reported from inpatient and outpatient departments of various hospitals in Khyber Pakhtunkhwa (KPK) province. In this study, we determined the circulating dengue virus (DENV) serotypes using real-time reverse transcriptase (RT)-PCR to understand the serotype-based epidemiology of DENV. All four serotypes (DENV-1 [6%], DENV-2 [33%], DENV-3 [47%], and DENV-4 [0.1%]) were found circulating during the study period. Our findings suggest the need for an active surveillance system coupled with the laboratory diagnosis, especially in the chronic endemic areas of the country. Public awareness programs are needed for effective control and prevention of outbreaks in the future.
Adolescent ; Adult ; Dengue/diagnosis/*epidemiology/virology ; Dengue Virus/genetics/*isolation & purification ; Disease Outbreaks ; Female ; Humans ; Male ; Middle Aged ; Pakistan/epidemiology ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Serogroup ; Young Adult

No abstract available.
Coronavirus Infections/*diagnosis/virology ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/isolation & purification ; Nasopharynx/virology ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Viral Envelope Proteins/genetics

The Epstein-Barr virus (EBV) is a gamma herpes virus associated with several types of malignancies. The EBV encodes viral microRNAs (miRNAs) that can target genes within cells. The EBV participates in signal transduction as well as the proliferation and differentiation of cells. How the target genes and functions of EBV-encoded miRNAs contribute to the pathogenesis of EBV is an important research topic. Some target genes have been validated since EBV-encoded miRNAs were discovered and, in this article, we summarize them and their functions.
Animals ; Epstein-Barr Virus Infections ; genetics ; metabolism ; virology ; Herpesvirus 4, Human ; genetics ; physiology ; Humans ; MicroRNAs ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism

Small interfering ribonucleic acid (siRNA)-induced RNA degradation can inhibit viral infection, and has been investigated extensively for its efficacy as antiviral therapy. The potential therapeutic role of lentiviral-mediated short hairpin ribonucleic acid (shRNA) to Newcastle disease virus (NDV) replication in vivo has been explored less often. We constructed two recombinant lentiviral vectors containing shRNA against the phosphoprotein (P) of the NDV, RNAi-341 and RNAi-671. Recombinant shRNA lentivirus vectors were co-transfected into 293T cells, along with helper plasmids, to package the recombinant shRNA lentivirus. Lentivirus-based shRNAs were titrated and transduced into NDV-susceptible chicken embryo fibroblasts (CEFs) and chick embryos. Antiviral activity against the NDV strain was evaluated by virus titration and real-time reverse transcription-polymerase chain reaction. RNAi-341 and RNAi-671 strongly suppressed transient expression of a FLAG-tagged P fusion protein in 293T cells. RNAi-341 and RNAi-671 NDV reduced virus titers by 66.6-fold and 30.6-fold, respectively, in CEFs 16 h after infection. RNAi-341 and RNAi-671 reduced virus titers in specific pathogen-free chick embryos by 99% and 98%, respectively, 48 h after infection. Both shRNAs inhibited accumulation of not only P-gene mRNA, but also nucleocapsid, M-, F-, HN-, and L-gene mRNA. RNAi-341 silenced P-gene mRNA more potently than RNAi-671. These results suggest that shRNAs silencing the P gene had substantial antiviral properties and inhibited NDV replication in CEFs and chick embryos.
Animals ; Chick Embryo ; Chickens ; Down-Regulation ; Fibroblasts ; virology ; Gene Targeting ; Lentivirus ; genetics ; metabolism ; Newcastle Disease ; virology ; Newcastle disease virus ; genetics ; physiology ; Phosphoproteins ; genetics ; metabolism ; Poultry Diseases ; virology ; RNA Interference ; RNA, Small Interfering ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

OBJECTIVETo investigate distinctive features in drug-resistant mutations (DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients.

METHODSForty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA.

RESULTSCompared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher's exact test, P<0.05). Considering 'majority resistant variants', 15 samples (19.48%) showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA.

CONCLUSIONCompared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.

Adult ; Antiviral Agents ; pharmacology ; China ; DNA, Viral ; genetics ; metabolism ; Drug Resistance, Viral ; genetics ; Female ; HIV Infections ; drug therapy ; HIV-1 ; drug effects ; genetics ; metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; Middle Aged ; Mutation ; Proviruses ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; RNA-Directed DNA Polymerase