cord-000018-amvlm09p	2008	Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNα/β, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. These results are consistent with data gained from previous experiments in Vero cells ( Figure 1E ) and indicate that neither induction of SOCS-3 mRNA nor inhibition of STAT phosphorylation is dependent on virus-induced type I IFN expression. To answer the question whether enhanced STAT phosphorylation in SOCS-3 deficient cells would also lead to enhanced expression of ISGs, total RNA was isolated at different time points p.i. from infected wild type and knock out cells and monitored for induction of SP110, IRF-1 and OAS1 ( Figure 7E, 7F and 7G ).
cord-000063-tex6bgab	2009	Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. It was further demonstrated that no siRNA-resistant viral mutation appeared in siM2 targeting sequence even after the virus was cultured in the shRNA expressing stable cell line for 40 passages. A recent report by Zhou et al [30] also showed that several siRNAs targeting NP and M genes exhibited effective inhibition against influenza A virus replication in cultured MDCK cells and in animal models. Taken together, all the findings about effective RNAi target, lentiviral vector delivery and the establishment of stable shRNA expressing cell lines in our study provide rational information for the development of siRNAs as prophylaxis and therapy for influenza virus infection in humans.
cord-000083-3p81yr4n	2009	R. China Background: The objective of this study was to evaluate the early virologic response for prediction of achievement of HBeAg seroconversion and hepatitis B virus (HBV) DNA negativity after two years of lamivudine treatment in chronic hepatitis B (CHB) patients. Methods: A total of 620 patients who tested positive for hepatitis B surface antigen and were referred to Chiba University Hospital between February 1985 and March 2008 were included in the study, and their following characteristics were analyzed: age, gender, the status of HBeAg, ALT, HBV-DNA level, and PLT. Methods: A total of 60 patients with chronic hepatitis B, 32 (53.3%) were HBeAg positive (group A) while 28(46.7%) were HBeAg negative (group B) were included in this study after meeting the following criteria: age 18 to 60 years, HBsAg positive for more than 6 months, serum HBV-DNA was >5 log(10) copies/mL and ALT more than two times the upper normal limit.
cord-000088-1xgjdhkx	2009	Using both strict and relaxed clock Bayesian phylogenetics we investigated 1) the substitutions rates of the structural P1 and capsid VP1 regions and 2) evolutionary timescale of currently circulating HPeV lineages. Their evolutionary history and population dynamics can be reconstructed by means of genealogy-based coalescent approaches using nucleotide sequences sampled over an epidemiological time frame in order to estimate timed viral ancestry as well as the rates of genetic change [27, 29] . We first identified the best-fitting substitution model for the HPeV sequences using the Modelgenerator package (GTR + Γ) [35] , and tested whether the evolution of the P1 and VP1 genetic regions was better described by a strict or relaxed lognormal molecular clock. The Bayesian analysis presented here first indicates that the structural P1 and the capsid VP1 region of this viral species evolve at a high rate of evolutionary change (~10 -3 substitutions per site per year).
cord-000113-d0eur1hq	2009	The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis. The advent of molecular biology and new technological initiatives that combine advances in biology with other disciplines will support the development of techniques capable of high throughput testing with a low turnaround time for rabies diagnosis. Another method for the detection of rabies virus antigen from postmortem samples is a recently developed rapid immunodiagwww.plosntds.org nostic test (RIDT) based on the principles of immunochromatography [13] . Development of RT-LAMP assays for use in diagnosis and surveillance is challenged by the considerable sequence variation observed within the rabies virus genome [44] that can frustrate specific primer design. Currently, high-throughput rabies virus molecular detection methods augment standard diagnostic tests or are in the process of development and refinement for use alone.
cord-000125-uvf5qzfd	2009	The interaction between a PAMP and a PRR triggers activation of the interferon (IFN) pathway in mammalian cells, which significantly changes the gene-expression profile in the cells and contributes to the well-documented off-target effect of RNAi. IFN induction is especially problematic in antiviral studies employing RNAi, where the antiviral effect of IFN must be distinguished from that of RNAi. Typical IFN-inducing structure patterns include dsRNA of certain length, single-stranded RNA (ssRNA) containing 5 0 -triphosphates (5 0 -ppp), the dsRNA analogue polyinosinic-polycytidylic acid (poly I:C), and certain dsDNA molecules. Mammalian expression plasmids encoding each of these proteins, as well as the dominant negative (DN) mutants of RIG-I and MDA5, were transfected into 293FT cells with shRNAs and an IFN-b promoter reporter construct.
cord-000128-t74b5j2j	2008	In this review, we will concentrate on peptide-mediated delivery of siRNAs and steric block oligonucleotides and discuss different methods for quantitative assessment of the amount of cargo taken up and how to correlate those numbers with biological effects by applying easy to handle reporter systems. The focus of this article is recent progress in the field of peptide-mediated cellular delivery of siRNA and steric block oligonucleotides in cell tissue culture as a starting point for further developments illustrated by own experimental data. In contrast to many noncovalent CPP-mediated siRNA delivery approaches, efficient splice correction was only achieved with conjugates of peptide and steric block oligonucleotide. As outlined above, our quantitative studies along with microscopic analyses of siRNAs and steric block oligonucleotides using either a peptide or a commercially availably cationic lipid as carrier clearly show that less than 0.1% -5% of molecules taken up are involved in a biological response, i.e. RNAimediated down regulation or splice correction-mediated up regulation of reporter gene activity.
cord-000143-2xvd5ogf	2009	In this process, following translation of an upstream open reading frame (ORF) and termination at the stop codon, a proportion of 40S subunits remain associated with the mRNA and reinitiate at the AUG of a downstream ORF, which is typically in close proximity. Recent studies of termination-reinitiation in the expression of the orthomyxovirus influenza BM2 protein have revealed a requirement for a shorter stretch of mRNA (45 nt) upstream of the stop-start window, but nevertheless, the RNA contains a similar TURBS Motif 1 [19] . Whilst in principle, reinitiation of translation of the MNV 49.7 VP2fluc ORF, following termination, could occur at the next available AUG, this is located 54 amino acids from the natural stop-start signal and initiation here would produce a substantially shorter product that would have been detectable by SDS-PAGE.
cord-000159-8y8ho2x5	2009	''Recoding'' is a term used to describe non-standard read-out of the genetic code, and encompasses such phenomena as programmed ribosomal frameshifting, stop codon readthrough, selenocysteine insertion and translational bypassing. It provides access to detailed information on genes known to utilize translational recoding and allows complex search queries, browsing of recoding data and enhanced visualization of annotated sequence elements. The term ''translational recoding'' describes the utilization of non-standard decoding during protein synthesis and encompasses such processes as ribosomal frameshifting, codon redefinition, translational bypassing and StopGo (1) (2) (3) (4) (5) (6) (7) . To facilitate further development of computational tools for the prediction of recoded genes in the ever faster growing body of sequence data, as well as to provide bench researchers with upto-date information on recoding, an efficient means of Recode database population and annotation are now required. RECODE: a database of frameshifting, bypassing and codon redefinition utilized for gene expression
cord-000238-om92cx5q	2010	We also draw attention to other infectious disease systems where robustness theory may prove useful for bridging evolutionary biology and biomedicine, especially the evolution of antibiotic resistance in bacteria, immune evasion by influenza, and malaria parasite infections. [14] [15] [16] In reviewing these results, we hope to highlight the importance of empirical work in RNA viruses for testing theory pertaining to robustness, as well as for better understanding the evolutionary biology and evolvability of infectious organisms in general. 1 However, theory and artificial-life data 9 support the idea that genetic robustness should be strongly favored when populations experience elevated mutation rates, suggesting that RNA viruses would be fruitful systems to explore how genetic robustness evolves. [33] [34] [35] Regardless, preliminary experiments showed that UVC exposure for periods up to 30 min greatly increased mortality in wild type phage 6 ͑Fig. 2͒, indicating that this environmental effect should produce strong selection for UVC resistance in populations of the virus.
cord-000248-zueoyesj	2010	These authors cite, for example, ''''mitochondrial dysfunction'''' [5, 6] (including, but not limited to ''''glucose avidity'''' [7] and ''''a shift in glucosemetabolism from oxidative phosphorylation to glycolysis'''' [6, 8] , ''''altered glycolysis'''' [9] , ''''altered bioenergetic function of mitochondria'''' [10] ), ''''dysregulation of cell cycle and defective genome-integrity checkpoints'''' [11] , ''''aberrant DNA methylation'''' [12] (''''promoter hypermethylation of hallmark cancer genes'''' [13] and ''''CpG island hypermethylation and global genomic hypomethylation'''' [14] ), ''''shift in cellular metabolism'''' [15, 16, 17] , ''''regional hypoxia'''' [18] , ''''microenviroment acidosis'''' [19] , ''''abnormal microRNA regulation'''' [20, 21] , ''''aneuploidy'''' and ''''chromosome aberrations'''' [22, 23, 24, 25, 26] , ''''disruption of cellular junctions'''' [27] , ''''avoidance of the immune response'''' [28] , ''''pre-existing chronic inflammatory conditions'''' [29, 30] , ''''cancerrelated inflammation'''' [29] , ''''disabled autophagy'''' [28] , ''''impaired cellular senescence'''' [31] , ''''altered NF-kappaB signalling'''' [32] , ''''altered growth patterns, not altered growth per se'''' [33] , ''''disregulated DNA methylation and histone modifications'''' [34] , ''''tissue dedifferentiation'''' [35, 36] , and ''''somatically heritable molecular alterations'''' [37] .
cord-000265-llilwq1u	2010	Autopsy studies have shown that human highly pathogenic avian influenza virus (H5N1) can infect multiple human organs other than just the lungs, and that possible causes of organ damage are either viral replication and/or dysregulation of cytokines and chemokines. Although H5N1 virus infection of humans is primarily one of the lower respiratory tract, more recent reports suggested that influenza A H5N1 may in rare, severe cases, disseminate beyond the lungs and infect brain [26, 27] , intestines [20, 27] and lymphoid tissues [27] , and result in extra-pulmonary clinical manifestations including encephalopathy or encephalitis [15, 28] . To better understand the pathogenesis of human H5N1 virus infection, and investigate the route of virus dissemination in vivo, we report on the use of different techniques to detect virus distribution and infection of 5 organ systems in a laboratory confirmed fatal human H5N1 virus infection, and analyze the relationship between viral load in tissues and host response.
cord-000269-v4jochbe	2010	cDNA libraries from five individuals and two pools of samples were prepared from retropharyngeal lymph node RNA enriched for polyadenylated RNA and sequenced using Roche-454 Life Sciences technology. Representatives of all bacterial phyla were detected in the seven libraries based on protein-coding transcripts indicating that viable microbiota were present in lymph nodes. Based on detection of both rRNA and protein-coding transcripts, we identified two new proteobacterial species; a Helicobacter closely related to Helicobacter cetorum in the Helicobacter pylori/Helicobacter acinonychis complex and an Acinetobacter related to Acinetobacter schindleri. The microbial community of mule deer lymph nodes Detection of protein-coding and ribosomal RNA transcripts provides strong support for the presence of viable and replicating microorganisms. As an alternative approach to identifying bacterial microorganisms present in lymph node tissue, we utilized amplicon DNA library sequencing technology.
cord-000293-pc4x5e24	2010	The requirements for À1 ribosomal frameshifting are the presence of a slippery heptanucleotide sequence X XXY YYZ (where X can be A, U, G or C; Y can be A or U; and Z does not equal Y; the spaces indicate the original reading frame) (8) followed by a downstream structural element, such as a pseudoknot, a hairpin or an antisense oligonucleotide duplex [for reviews, see (9) ]. We and others have demonstrated that small RNA oligonucleotides are able to mimic the function of frameshifter pseudoknots or hairpins by redirecting ribosomes into new reading frames (27, 28) . These results demonstrate that LNA modifications indeed enhance the antisense-induced frameshifting efficiency probably due to higher thermodynamic stability and RNA-like structural properties. In addition to RNA oligonucleotides, we demonstrated that LNA/DNA mix-mers are also capable of stimulating efficient À1 ribosomal frameshifting in contrast to DNA oligonucleotides. Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of À1 ribosomal frameshifting
cord-000295-ft5wl70x	2010	These short, single-stranded RNA molecules originate from larger precursor molecules that fold to produce hairpin structures, which are subsequently processed by ribonucleases Drosha/Pasha and Dicer to form mature miRNAs. MiRNAs play role in the posttranscriptional regulation of about one third of human genes, mainly via degradation of target mRNAs. Whereas the target mRNAs are often involved in the regulation of diverse physiological processes ranging from developmental timing to apoptosis, miRNAs have a strong potential to regulate fundamental biological processes also in the lung compartment. Small non-coding RNAs (miRNAs) play pivotal role in the posttranscriptional regulation of numerous human genes, mainly via degradation of target mRNAs. There is evidence that the lung has a very specific miRNA expression profile undergoing changes during the lung development.
cord-000322-8ctsa9sd	2011	Subsequently, T4 and MS2 ICs were evaluated in routine real-time PCR or RT-PCR virological diagnostic tests, using a series of 8,950 clinical samples (representing 36 distinct specimen types) sent to our laboratory for the detection of a variety of DNA and RNA viruses. It represents a valuable strategy for enhancing the quality of routine molecular diagnosis in laboratories that use in-house designed diagnostic systems, which can conveniently be associated to the use of specific synthetic ECs. The high rate of inhibitors observed in a variety of specimen types should stimulate the elaboration of improved technical protocols for the extraction and amplification of nucleic acids. Monitoring rt-PCR and rt-RT-PCR assays and validation of the results rely on the use of relevant external or internal controls (ECs or ICs) [1, 2] and commercial kits including such control systems are being increasingly improved for the molecular diagnosis of a number of pathogens such as HIV, hepatitis viruses, influenza viruses etc..
cord-000364-ikq38rm1	2011	Lung computer tomography (CT) on admission revealed pronounced diffuse bilateral interstitial infiltrates with pulmonary oedema, dependant atelectasis, and moderate pleural effusions (Fig. 1 ) which were later drained (>800 ml). Hantavirus infection was verified with the detection of PUUV RNA in plasma (630,000 copies/ml) on the day of admission, while IgM and IgG were negative. Consecutive plasma samples were analysed for PUUV RNA with declining viral copy numbers until negative 16 days post onset of Fig. 1 Chest CT-scans of two European patients with hantavirus pulmonary syndrome. Concerning the cases of European hantavirus infection in our present report, there was only mild or no renal impairment at the time of admission, whereas the respiratory involvement was early and severe, consistent with acute respiratory distress syndrome (ARDS), fulfilling criteria of HPS according to CDC case definition [19] .
cord-000372-wzwpyvll	2011	These findings are in agreement with the fact that host mRNA transcription takes place in 2A pro -expressing cells when both translation and RNA nuclear export are inhibited, upon cleavage of eIF4G and Nups [17] . In this regard, PV 2A pro has been very useful for examining the requirements for eIF4G to translate different cellular or viral mRNAs. In addition to this, in this paper, we have highlighted the role of PV 2A pro not only in the processing of the poliovirus polyprotein but also in the interaction with the host-cell. In particular, PV 2A pro cleaves cell proteins involved in transcription, pre-mRNA splicing, nucleus/cytoplasm transport and translation, in order to hijack those host functions and to concentrate the cellular resources on the production of the viral progeny.
cord-000435-2u49b7xo	2011	With respect to the non-structural polyprotein, SINV and Aura virus (AURAV) form a separate clade from VEEV, WEEV and EEEV but, again, the conservation analysis revealed striking tandem conservation peaks 3 0 of the RT site ( Figure 1B ) and, again, the conservation peaks corresponded to sequences with the potential to base pair to form an RNA structure-this time comprising an 11 bp stem with a 1 nt asymmetric 3 0 bulge, a 12 nt ''spacer'' from the RT stop codon, and a 154 nt ''loop'' region ( Figure 2 ). The potential to form an extended stemloop structure 3 0 -adjacent to a RT stop codon-phylogenetically conserved and supported by a pair of peaks in synonymous site conservation-was also found in a number of plant virus RT cases, for example, in the replicase gene in the genera (Figures 3 and 4) .
cord-000482-wifs97yy	2011	−1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base–base and base–sugar interactions between loop and stem nucleotides. In short, pDUAL-HIV(0) was digested by KpnI and BamHI, followed by insertion of complementary oligonucleotides to clone the SRV-1 gag-pro pseudoknot, various hairpins as shown in Figures 2C and 5, and a negative control (NC) which formed no apparent secondary structure downstream of the slippery sequence. These data support the notion that downstream structures serve as barriers to stall translating ribosomes to stimulate frameshifting, and demonstrate that there is a correlation between the thermodynamic stability of a hairpin and its frameshift inducing capacity. In the present study, a 12 bp hairpin derivative of the SRV-1 gag-pro pseudoknot with a calculated stability of À26.9 kcal/mol was capable of inducing 22% of frameshifting, which is only 1.4-fold .
cord-000532-e18licyc	2011	The results shown in Figure 2 revealed that a 1D SDS-PAGE assay could not firmly identify polypeptides originating from a À1 frameshifted ribosome stalled in the pseudoknot from the non-frameshifted product in a ''Downstream Stop'' construct. In order to quantify the amount of À1 frameshifted ribosomes stalled inside the pseudoknot, we performed a 2D SDS-PAGE separation of the radioactively labelled proteins originating from the ''Upstream Stop'' construct (Supplementary Figures S4 and S5) , which is the type of construct most commonly used throughout literature. In the ''Upstream Stop'' construct the non-frameshifting ribosomes will translate gene10 and terminate at a UAA stop codon in the spacer sequence and produce a 28 kDa polypeptide. In the following subsections ''Identification of transcripts from the T7gene10-PK-lacZ gene fusions'', ''Messenger RNA stability'' and ''Coupling between translation and transcription is required for full-length transcripts'', we will show that the observed proteins did indeed originate from stalled ribosomes and that they were not caused by other effects.
cord-000547-adfigzc1	2012	METHODOLOGY AND PRINCIPAL FINDINGS: We have investigated the structure of Ebola virus using a combination of cryo-electron microscopy, cryo-electron tomography, sub-tomogram averaging, and single particle image processing. Here we report the three-dimensional structure and architecture of Ebola virus and establish that multiple copies of the RNA genome can be packaged to produce polyploid virus particles, through an extreme degree of length polymorphism. From the same image data set, we combined extracted volumes from tomograms with 2-D single particle processing to determine the structure of the GP spikes ( Figure 5 ) to a resolution of 14 Å as measured by the Fourier Shell Correlation (FSC) 0.5 criterion. Analysis of 2090 distinct intact virions with a nucleocapsid from cryo-electron micrographs shows that the most common class length (53%) of virus particles is 982679 nm ( Figure 1A , Table S1 ).
cord-000556-uu1oz2ei	2012	Whole genome transcriptome analysis is a complementary method to identify "novel" genes, small RNAs, regulatory regions, and operon structures, thus improving the structural annotation in bacteria. Therefore, genome structural annotation or the identification and demarcation of boundaries of functional elements in a genome (e.g., genes, non-coding RNAs, proteins, and regulatory elements) are critical elements in infectious disease systems biology. Whole genome transcriptome studies (such as whole genome tiling arrays [13, 14, 15] and high throughput sequencing [16, 17] ) are complementary experimental approaches for bacterial genome annotation and can identify ''''novel'''' genes, gene boundaries, regulatory regions, intergenic regions, and operon structures. We compared the RNA-Seq based transcriptome map with the available genome annotation to identify expressed, novel, and intergenic regions in the genome. The single nucleotide resolution map helped uncover the structure and complexity of this pathogen''s transcriptome and led to the identification of novel, small RNAs and protein coding genes as well as gene co-expression.
cord-000578-jhetyd9t	2012	In this paper, we show that an essential translation factor, Ded1p DEAD-box RNA helicase of yeast, directly affects replication of Tomato bushy stunt virus (TBSV). In this paper, the authors show that the Ded1p DEAD-box helicase, which is an essential translation factor in yeast, is recruited by Tomato bushy stunt virus (TBSV) into its replicase complex. Interestingly, addition of purified Ded1p to the tombusvirus replicase assay containing the short RNA/DNA duplex ( Figure 7A Non-overlapping functions of Ded1p and GAPDH in promoting initiation by the tombusvirus replicase GAPDH (Tdh2p in yeast) RNA binding protein is also a host factor stimulating (+)RNA synthesis by the tombusvirus replicase [25, 50] . To test if Ded1p and GAPDH could play a complementary role during (+)RNA synthesis, we added the purified recombinant Ded1p and Tdh2p to the in vitro tombusvirus replicase assay based on the purified preparation ( Figure 8A ).
cord-000640-t0y0b0gb	2012	Following numerous failed attempts, hantavirus RNA was detected in ethanol-fixed liver tissue from two banana pipistrelles (Neoromicia nanus), captured near Mouyassué village in Côte d''Ivoire, West Africa, in June 2011. Phylogenetic analysis of partial L-segment sequences using maximum-likelihood and Bayesian methods revealed that the newfound hantavirus, designated Mouyassué virus (MOUV), was highly divergent and basal to all other rodentand soricomorph-borne hantaviruses, except for Nova virus in the European common mole (Talpa europaea). After innumerable failed attempts, hantavirus RNA was detected by RT-PCR in ethanol-fixed liver tissues from two of 12 banana pipistrelles (Neoromicia nanus Peters 1852), captured during June 2011 near Mouyassué village (5°22''07"N, 3°05''37"W) in Aboisso District, 130 km from Abidjan, in the extreme southeastern region of Côte d''Ivoire in West Africa ( Figure 1 ). The newfound hantavirus, designated Mouyassué virus (MOUV), exhibited low nucleotide and amino acid sequence similarity of less than 69% to all representative soricomorph-and rodent-associated hantaviruses, except for the 76.3% sequence similarity with Nova virus (NVAV), previously reported in the European common mole (Talpa europaea) [12] .
cord-000660-tsvzg0ax	2012	Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. In contrast, in acute infection by viruses that cause severe pathogenesis and death within a few days after infection, protection is primarily provided by the intrinsic antiviral actions of IFN-induced proteins encoded by the hundreds of IFN-stimulated genes (ISGs) [10] [11] [12] , several of which often contribute to the overall effect of IFN against a given virus. From the above observations, we conclude that after intranasal infection by VSV, Ifit2 protects mice from neuropathogenesis by suppressing replication or spread of the virus in brain neurons. In the complete absence of type I IFN action in the IFNAR 2/2 mice, intranasally infected VSV replicated vigorously not only in brains, but also in livers and lungs ( Figure 7A-C) .
cord-000715-zl1s82yi	2012	These samples were selected from among archived stool samples previously tested for enterovirus and MS2 after extraction by QIAamp Viral RNA Mini Kit. A sufficient number of samples with high, intermediate, and low levels of inhibitors were chosen for re-analysis to enable comparison between extraction procedures at each of these levels of inhibition. Analysis of variance ( Fig. 3 , part 2), indicated that there was no significant difference (paired t-test, P.0.05) between the inhibition of rRT-PCR of the MS2 external control and the added enterovirus (P.0.05) for protocols A, C, and D. Stool suspensions (N = 185) prepared for routine analysis of clinical stool samples sent to the Central Virology Laboratory (CVL) at Chaim Sheba Medical Center in Israel were used to evaluate the efficiency of four different RNA extraction systems in excluding inhibitors of rRT-PCR.
cord-000729-iq30z094	2012	The genome size (18,162 nt) and organization of CedPV is very similar to that of HeV and NiV; its nucleocapsid protein displays antigenic cross-reactivity with henipaviruses; and it uses the same receptor molecule (ephrinB2) for entry during infection. Preliminary challenge studies with CedPV in ferrets and guinea pigs, both susceptible to infection and disease with known henipaviruses, confirmed virus replication and production of neutralizing antibodies although clinical disease was not observed. As part of our on-going field studies on HeV genetic diversity and infection dynamics in the Queensland flying fox populations, urine samples were collected on a regular basis for PCR and virus isolation. CedPV is more closely related to HeV and NiV than henipavirus-like sequences detected in African bats [26, 32] as shown in a phylogenetic tree based on the only sequences available of a 550-nt L gene fragment (Fig. S5) .
cord-000736-6f8vyziv	2012	FINDINGS: We developed a real-time PCR array capable of simultaneously detecting eight human viral pathogens: human immunodeficiency virus types 1 and 2 (HIV-1 and -2), hepatitis B virus (HBV), hepatitis C virus (HCV), human T-cell leukemia virus-1 and -2 (HTLV-1 and -2), vaccinia virus (VACV) and West Nile virus (WNV). The analytical sensitivity of each primer set was determined in the single virus testing using FDA/CBER panels (kindly provided by Dr. Stephen Kerby, FDA/CBER) consisting of various amounts of the viruses (0-1,000 genome copies/ml) spiked into the ''''normal'''' human plasma. The results of sensitivity testing of the real-time PCR array primer sets specific for HIV-1, HIV-2, HBV, HCV, and WNV the with FDA/CBER analytical plasma panels. Tm and C(t) values obtained with primer sets specific for HIV-1, HCV, or HBV in testing of 17 human clinical samples in the format of PCR array targeting eight different viruses.
cord-000794-l565gha4	2012	Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Photoreactive ligand peptides for identification of interacting protein(s) of pre-S1 domain of L envelope protein To identify the pre-S1 interacting molecule(s), we employed a photo-cross-linking approach using a synthetic peptide derived from the native pre-S1 peptide with particular residues replaced by eLife digest Liver diseases related to the human hepatitis B virus (HBV) kill about 1 million people every year, and more than 350 million people around the world are infected with the virus. In this study, by employing a unique approach of tandem affinity purification combined with MS analysis against a Tupaia hepatocyte proteome database established by deep sequencing, we revealed that the liver bile acid transporter, NTCP, specifically interacts with a key region in the pre-S1 domain of the HBV envelope L protein.
cord-000804-0hlj6r10	2012	While the ectodomain, which is mainly formed by GP 1 , mediates binding to entry factors and receptors [87] [88] [89] [90] [91] [92] [93] [94] [95] , the transmembrane subunit GP 2 contains the fusion peptide and is presumed to mediate fusion of the viral and the cellular membrane based on similarity to EBOV GP 2 both at the amino acid and structural level [96, 97] . The IFN-inducible antiviral protein tetherin was shown to block the release of VP40-induced virus-like MARV and EBOV particles, suggesting that tetherin might act as a restriction factor for filovirus release [102, 103] . After the nucleocapsid is released into the cytoplasm of the infected cell, transcription and replication of the viral RNA genome takes place (Figure 7 ). Virus-like particles exhibit potential as a pan-filovirus vaccine for both Ebola and Marburg viral infections
cord-000822-iuglkdcp	2012	One strong point of the DotKnot method for single sequence pseudoknot prediction (Sperschneider and Datta, 2010; Sperschneider et al., 2011) is that the set of possible H-type pseudoknot candidates (and secondary structure elements) is explicitly computed and thus readily available for further investigation. (3) Use significant pairs to calculate the set of conserved structure elements and pseudoknots for the two sequences that maximizes a combined free energy and similarity score. The key point of the DotKnot-PW approach is how to score the similarity of stems, secondary structure elements and H-type pseudoknot candidates derived from sequences Seq x and Seq y . The optimal structure element alignment, which preserves the interval ordering includes structure elements p 1 , p 4 , p 7 in the first sequence and p 1 , p 4 , p 6 in the second sequence pairwise prediction with highest combined free energy and similarity score is taken, DotKnot-PW has an improved average MCC of 0.81.
cord-000830-jiy4cp4n	2012	The use of amplification techniques such as polymerase chain reaction, real-time polymerase chain reaction or nucleic acid sequence-based amplification for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range. The use of amplification techniques such as polymerase chain reaction (PCR), real-time PCR or nucleic acid sequence-based amplification (NASBA) [3] for virus detection, genotyping and quantification have some advantages like high sensitivity and reproducibility, as well as a broad dynamic range [4, 5] . NASBA assays could identify active infection by detecting viral messenger RNA (mRNA) but the most widely used tests in clinical virus diagnosis are quantitative real-time PCR techniques [8] . Some real-time PCR assays such as LightCycler parvovirus B19 quantitative assay (Roche Diagnostics, Indianapolis, IN) and ABI TaqMan (Applied Biosystems) have been developed for detecting B19 nucleic acids in association with infection during pregnancy or assessing the prevalence of the virus DNA in blood products [62, 63] .
cord-000866-dr2uow4m	2013	In the present study, we investigated the ability of MZP to directly inhibit the human RNA capping enzyme (HCE), a protein harboring both RNA 5′-triphosphatase and RNA guanylyltransferase activities. An RNA guanylyltransferase (GTase) then catalyzes a two-step reaction in which it initially utilizes GTP as a substrate to form a covalent enzyme-GMP (EpG) intermediate, with the concomitant release of pyrophosphate (PPi). Since ribavirin triphosphate and MZP share functional similarities, we investigated the attractive possibility that MZP could also inhibit the GTase activity of the human RNA capping enzyme (HCE). The enzyme-GMP complex formation assays were carried out for 3 min at 37uC in a buffer containing 1 mM [a-32 P]GTP, 4 mM HCE, 50 mM Tris-HCl, pH 7.5, 5 mM MgCl 2 , 500 mM DTT, 0.5 ng/ml pyrophosphatase (Roche), and various concentration of MZP (as indicated).
cord-000881-s90geszi	2012	In contrast to the relatively short lengths of previously described motifs, we found that most homomorphs are long, and each provides a structural connection between the template tunnel or NTP entry tunnel and the exterior of the protein. The structurally aligned sequences that comprised homomorph of Motif F (hmF) for RdRps and HIV are summarized in Figure 3A . Using T7 DNAP as a query (lowest segment of the figure) , only a small portion of the C-terminal edge of Motif D and a few species have similar structures. In the RdRps, the combined regions of structural homology represent $75% of the sequence from the start of homomorph of Motif G (hmG) through the end of hmE in each species ($375 residues). The tertiary position of each of the homomorphs includes at least one residue (and sometimes more) in contact with the exterior surface of the protein and one or more highly conserved functional residues located within or at the wall of the template tunnel.
cord-000895-z5rdf0mi	2013	A series of in silico shuffling algorithms were developed to account for these features and analyze the relative impact of mutational pressure components on codon usage bias in RNA viruses. A general rationale for analyzing impact of (di)nucleotide bias on ENC is randomizing or shuffling synonymous codons in the original genome sequence whilst preserving the factor under study [37] . Unfortunately, common statistical tests were poorly applicable to this approach, because all factors that affect codon preference (e.g. structural RNA elements) could not be accounted for, and therefore even a negligible difference in ENC between the original and generated sequences (e.g. 0.2 SD) passed a formal significance test upon increasing the number of replicates. Overall, dN 23 and dN 231 shuffling produced ENC values that differed from the original sequence by less than one in 25 of 29 viruses, indicating that we explored almost all types of pressure that affect overall codon usage.
cord-000937-8vk89i4h	2013	RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Seven RNA libraries (aihP01, hbvP02, hcvP02, hcvP03, hcvP05, nshP01, norP01) and seven DNA libraries (aihP01D, hbvP02D, hcvP02D, hcvP03D, hcvP05D, nshP01D, norP01D) were constructed from patients with autoimmune hepatitis (AIH), hepatitis B virus (HBV) chronic infection, hepatitis C virus (HCV) chronic infection, non-alcoholic steatohepatitis (NASH), and healthy subjects (NOR). Plasma samples were obtained from patients with autoimmune hepatitis (AIH), chronic hepatitis B virus (HBV) infection, chronic hepatitis C virus (HCV) infection, non-alcoholic steatohepatitis (NASH), and healthy subjects (NOR); each RNA library was named by diagnosis (e.g. aihP01) and a suffix ''D'' was added for each DNA library (e.g. aihP01D). To a lesser extent (about one read per million), we also detected sequences resembling RNA viruses in our DNA libraries (Supplemental Tables S15-S28 ). Assembly of viral sequences was also possible for all viruses shown in Figure 2 as the most abundant virus in each library (data not shown).
cord-000979-cav9n18w	2013	title: Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. Additionally, the structure and antigenicity of the proteins and epitopes were modelled to analyze the suitability of the identified sequences for future applications like diagnostic tools or vaccine development. For a summary of the predicted characteristics, see S9: Transmembrane and antigenic potential of three potential epitope sites for cj0920c.Further, specificity control assays revealed that these signals do not drop significantly when using antibodies to Salmonella enterica indicative of nonspecific binding to occur, see S10: Specific vs. jejuni and were able to determine the important residue involved in antibody binding as well as modelling the epitope''s accessibility within the full-length protein.
cord-000981-6vloa2w3	2013	The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca(2+)) homeostasis, gene expression and proliferation studies. The cells were stimulated with 100 mM histamine or 15 mM 2,5-Di-t-butyl-1,4-benzohydroquinone (BHQ: selective SERCA blocker) in the presence and absence of extracellular Ca 2+ , after incubation for 24 h with 25 mg/mL Poly I:C, 25 mg/mL dsRNA, 2.5 mg/mL L-DNA, 2.5 mg/mL total RNA or control solution. Double-stranded RNA (dsRNA) or its synthetic analogue (Poly I:C) significantly decreased the electric resistance and increased the permeability of the confluent human pulmonary artery endothelial (hPAEC) monolayer as shown on Figure 1 . Altogether, these data suggest that synthetic dsRNA treatment alters the function of SERCA, which inhibits cell proliferation by inducing G1 arrest in the hPAECs and contributing to endothelial dysfunction.
cord-001090-qg2r691d	2013	BACKGROUND: The ribosomal RNA content of a sample collected from a woman with bacterial vaginosis (BV) was analysed to determine the active microbial community, and to identify potential targets for further screening. Prevotella amnii was chosen as an example target, being the most metabolically active species present in the single patient with BV, and was found to be detected at a high concentration by qPCR in 31% of cohort with BV, with an association with both oral and penile-vaginal sex. An amplicon-based metagenomic library was generated from the extracted DNA using the universal bacterial PCR primers 27F and 338R that target the V1-V2 hypervariable regions of the 16S rRNA gene as previously described [15] . Comparison of cDNA to DNA from Nugent 10 patient All of the predominant bacteria with .1% total abundance identified in the cDNA library were also detected using the 16S rRNA gene amplicon-based approach on extracted DNA.
cord-001109-xs7df6a7	2013	We demonstrate that these defective viral genomes (DVGs) are generated naturally during respiratory infections in vivo even in mice lacking the type I IFN receptor, and their appearance coincides with the production of cytokines during infections with Sendai virus (SeV) or influenza virus. To further investigate the cellular mechanisms responsible for the efficient activation of the antiviral response by SeV DVGs, we evaluated the phosphorylation of transcription factors that are critical for the expression of type I IFNs in cells infected with equivalent amounts of infectious particles of a SeV strain Cantell stock containing high levels of copy-back DVGs (SeV Cantell HD) or with SeV Cantell depleted of DVGs (SeV Cantell LD). Based on the potent ability of SeV stocks containing a high content of copy-back DVGs to induce the host response to infection in vitro [23, 24, 25, 28] (Fig. 1 ) and on our prior reports of strong host responses to DVGs regardless of the presence of functional virus-encoded antagonists [23, 24] , we hypothesized that DVGs that arise in situ during viral infections provide essential stimuli to initiate an antiviral immune response.
cord-001152-v6uc0ijw	2013	Here, we provide evidence for the presence of discrete small RNAs of viral origin that are not associated with the RNA-induced silencing complex (RISC), that are highly expressed and detected by Northern blot analysis, and that accumulate as 21to 28-nucleotide (nt) species during infection. We report that the cellular antiviral endoribonuclease RNase L cleaves the viral genome, producing in turn the small RNAs. Surprisingly, we uncovered the presence of a modification on the 3′-end nucleotide of SINV-derived viral small RNAs (SvsRNAs) that might be at the origin of their stability. The identification of small RNAs from RNA viruses could be seen either as a result of a productive mechanism for the pathogen (as for virus-encoded miRNAs) or as the consequence of a host response to control the infection by degrading viral RNAs. SINV expresses four enzymes that play key functions during the viral replicative cycle.
cord-001228-4eh22ek7	2014	32 Thus, as far as we are aware, there are no reported examples of synthetic molecules able to alter HIV-1 frameshifting and interfere with viral infectivity via selective, high-affinity binding to the FSS RNA. Building on our laboratory''s longstanding interest in understanding the factors that drive affinity and sequence selectivity in small molecule recognition of RNA, 33 we previously reported the use of an 11,325-member resin-bound dynamic combinatorial library 34 (designed based on the structure of DNA-binding, bisintercalating peptide antibiotics) to identify a compound (1) able to bind the HIV-1 FSS upper stem-loop with moderate affinity (K D = 4.1 ± 2.4 μM immobilized on an surface plasmon resonance (SPR) chip via one of its amine groups; K D = 350 ± 110 nM in solution as measured by fluorescence 35 ) and good selectivity.
cord-001257-t21l6i3f	2014	Novel pro-viral function of AtRH2, AtRH5 and the yeast Dbp3p and Fal1p is to bind to the viral replication enhancer present in the tombusvirus minus-strand RNA To test if AtRH2, AtRH5 and the yeast Dbp3p and Fal1p play a comparable role with the previously analyzed yeast Ded1p (human DDX3-like) and Dbp2p (human p68-like) and the ortologous AtRH20 DEAD-box helicases [30, 49] , first we performed in vitro RNA binding experiments with affinity-purified recombinant helicase proteins. The replication process leads to the production of abundant (+)RNA progeny, while the (2)RNA templates are likely sequestered in dsRNA forms within the VRCs. The presented in vitro data based on the solubilized/purified tombusvirus replicase and the CFE assay containing the membrane-bound VRC indicate that the eIF4AIIIlike RNA helicases can mainly stimulate TBSV (+)-strand synthesis, while their effects on (2)RNA synthesis have not been observed (not shown).
cord-001397-nrq4ncdf	2014	Avenues for additional studies include determining if the multifunctional flavivirus protein NS5 has a role in viral persistence, the development of relevant animal models of viral persistence as well as investigating the host responses that allow vector borne flavivirus replication without detrimental effects on infected cells. The SL1 structure is essential for viral replication and acts as a promoter which is targeted initially by NS5 and then delivered to the 3 0 end via cyclization (Filomatori et al., 2006; Zhang et al., 2008; Lodeiro et al., 2009) Although there is low nucleotide conservation between flaviviruses and different CS homology (Hahn et al., 1987) , the 5 0 UTRs of TBFVs share the same genomic organization as the MBFVs (Kofler et al., 2006) Structural proteins Capsid (C) 11 kDa, 114 aa Cytosol/nucleus The capsid protein is a dimeric alpha-helical (Jones et al., 2003) protein and assembles into an icosahedral structure, measuring 30 nm in diameter, which initiates encapsidation of the associated genomic RNA in virus-induced membrane invaginations of the ER (Welsch et al., 2009 ).
cord-001453-l1r416w7	2014	title: Archaeal DnaG contains a conserved N-terminal RNA-binding domain and enables tailing of rRNA by the exosome Consistently, a fusion protein containing full-length Csl4 and NTD of DnaG led to enhanced degradation of A-rich RNA by the exosome. The RNase PH-domain containing subunits Rrp41 and Rrp42 are arranged in a catalytically active hexamer, on the top of which a trimeric cap composed of the RNA-binding proteins Rrp4 and Csl4 is bound ( Figure 1B ; 4, 5, [22] [23] [24] . The bacterial primase DnaG is composed of an NTD containing a Zn-finger motif involved in DNA binding, the central, catalytic TOPRIM domain and a CTD neces-sary for the interaction with the replicative helicase DnaB ( Figure 1A , refs. coli cell-free extract was easily detectable by pull-down assays with Strep-Tactin Sepharose beads (for an example see Figure 7A be-low), we conclude that the CTD of DnaG is important for the binding to the archaeal exosome.
cord-001521-l36f1gp7	2011	The IC 50 values determined in functional NI assays provide valuable information for detection of resistant viruses, but should not be used to draw direct correlations with drug concentrations needed to inhibit virus replication in the infected human host, as clinical data to support such inferences are inadequate. • Standardized reagents and protocols • Choice of detection technology • Simple instrumentation requirements • High sensitivity for use with low virus concentrations • Compatibility with batch-mode processing and largescale assay throughput • Broad specificity of influenza detection • Flexibility in assay format • Additional NA assay applications -cell-based viral assays, screening for new NIs, detection of NA from other organisms Functional neuraminidase inhibition assays enable detection of any resistance mutation and are extremely important in conjunction with sequence-based screening assays for global monitoring of virus isolates for NI resistance mutations, including known and new mutations. Such new assays need to include methods to measure local antibodies and virus-specific lymphocytes, especially in the case of live attenuated influenza vaccines, because of their potential to induce such broad-based immune responses.
cord-001541-5d64esp4	2015	We demonstrate that remarkable changes in genome size and complexity have occurred in rhabdoviruses in a clade-specific manner, primarily by extension and insertion of additional transcriptional units in the structural protein gene junctions, followed by occasional losses. All rhabdovirus genomes contained the five canonical structural protein genes (N, P, M, G and L); however, there was remarkable diversity in the number and location of other long ORFs. Across the data set, we identified 179 additional ORFs 180 nt in length of which 142 shared no detectable protein sequence similarity with any other protein in our data set or with those in public databases (S2 Table) . Interestingly, although substantial variation in the length of gene junctions was observed in several genera (including ephemeroviruses and lyssaviruses), most variation in genome size occurred as the result of the presence of new, non-canonical ORFs in the regions between the structural protein genes (Table 1) .
cord-001542-f089bs8r	2014	These may include monoclonal antibody (mAbs)-based therapies (e.g. ZMapp), anti-sense phosphorodiamidate morpholino oligomers (PMO AVI-6002), lipid nanoparticle small interfering RNA (LNP-siRNA: TKM-Ebola), and an EBOV glycoprotein-based vaccine using live-attenuated recombinant vesicular stomatitis virus (rVSV-EBOGP) or a chimpanzee adenovirus (rChAd-EBOGP)-based vector. The GP2 of the EBOV is able to counter the interferon (IFN)-inducible antiviral protein tetherin which restricts the VP40-dependent budding of the progeny viral particles from infected cells [16] [17] [18] . Currently available therapeutic agents that are effective in targeting the EBOV infection in cell or animal studies may include convalescent plasma, favipiravir, chloroquine, amiodarone, dronedarone, verapamil, clomiphene, toremifene, IFN-β, Na + /K + exchangers, Na + /K + -ATPase pump inhibitors, and antioxidants. The anti-EBOV activity of clomiphene and toremifene is dependent not on its estrogen receptor antagonistic action but upon the ability of both drugs to induce a Niemann-Pick C-like phenotype to inhibit viral entry at late endosome.
cord-001655-uqw74ra0	2015	The sets of viral genotypes ranged from that which would be expected for a straightforward co-infection by 2 virus strains in snake #45, to more complex combinations such as that in snake #33, which contained the sequences of 1 S and 10 distinct L segments. We applied homogenates from samples to cultures of boa constrictor-derived JK cells and monitored levels of virus RNA by qRT-PCR using genotype-discriminating primers. Thus, sequences of multiple viral genotypes Recombinant genome segments with unusual organizations. Virus populations replicate as stable ensembles in culture: (A) Liver homogenate from snake #38 was applied to cultures of JK cells and replication was monitored by measuring supernatant viral RNA levels using qRT-PCR and genotype-specific primers. Although we detected many instances of snake tissues containing multiple viral genotypes, our results do not prove that individual cells in these animals were multiply infected.
cord-001748-7e8px4vx	2015	The bicolored white-toothed shrew (Crocidura leucodon) has recently been identified as reservoir of the neurotropic Borna disease virus 1 (BoDV-1). In animals caught in 2013 (group 1: female #2, male #5, female #6), after an adaption phase of one month, samples of saliva, lacrimal fluid, skin surface, urine and excrements from the BoDV-1-infected shrews were taken weekly over a period of 4 weeks as necessary veterinary care. The five other shrews did not exhibit any evidence for BoDV-1-infection, neither infectious virus nor viral RNA was detected at any time point investigated. Current data from living shrews provide reliable evidence that natural BoDV-1-infection in these animals is indeed clinically inconspicuous over a long time period as already previously assumed [15, 18] despite persistent infection with shedding of infectious virus via various sites. Distribution of Borna Disease Virus Antigen and RNA in Tissues of naturally infected Bicolored White-Toothed Shrews, Crocidura leucodon, supporting their role as Reservoir Host Species
cord-001829-rwnbxmt4	2015	Genome-wide association studies performed on diverse patient populations have identified polymorphisms near the interferon-λ 3 (IFNL3; formerly IL28B) gene that predict the efficacy of interferon-based therapy for chronic infection. The differential effects on mRNA versus protein levels strongly suggest that the IFNL3 3′ UTR regulates gene expression by repressing the efficiency of mRNA translation rather than mRNA abundance in HeLa cells. We used polysome profiling to examine whether the variant IFNL3 reporter mRNAs were differentially associated with translating ribosomes in stable HeLa cell lines. Although rs4803217 is not independently associated with patient phenotypes in the cohort we analyzed, this SNP occurs in the 3′ UTR of IFNL3 and showed clear functional effects on reporter gene expression, suggesting a role for this variant in control of HCV infection.
cord-001835-0s7ok4uw	2015	Altogether, these results indicate that, although PHDs might be more selective for HIF as a substrate as it was initially thought, the enzymatic activity of the prolyl hydroxylases is possibly influenced by a number of other proteins that can directly bind to PHDs. Non-natural aminoacids via the MIO-enzyme toolkit Alina Filip 1 , Judith H Bartha-V ari 1 , Gergely B an oczy 2 , L aszl o Poppe 2 , Csaba Paizs 1 , Florin-Dan Irimie 1 1 Biocatalysis and Biotransformation Research Group, Department of Chemistry, UBB, 2 Department of Organic Chemistry and Technology An attractive enzymatic route to enantiomerically pure to the highly valuable a-or b-aromatic amino acids involves the use of aromatic ammonia lyases (ALs) and aminomutases (AMs). Continuing our studies of the effect of like-charged residues on protein-folding mechanisms, in this work, we investigated, by means of NMR spectroscopy and molecular-dynamics simulations, two short fragments of the human Pin1 WW domain [hPin1(14-24); hPin1(15-23)] and one single point mutation system derived from hPin1(14-24) in which the original charged residues were replaced with non-polar alanine residues.
cord-001963-4wjvykx7	2016	title: Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP''s RNA-binding affinity and hindered viral replication. As shown in Fig. 7A , upon H7 treatment at 15μM, the M1 viral RNA synthesis in the influenza virus-infected cells was reduced by 75% at T2. NP is the most abundant RNA-binding viral protein in influenza virus-infected cells and is responsible for recognizing RNA and forming a filamentous nucleocapsid 24 . Using fluorescence titration and an SPR assay of the NPs, we identified one potential natural compound, curcumin (H7), that targets Y148 of influenza virus NP and potently interferes with its RNA-binding activity.
cord-001964-iy6qzq58	2016	The wild boars persistently infected with CSFV were protected from superinfection by the virulent CSFV Margarita strain, as evidenced by the absence of clinical signs and the absence of Margarita RNA detection in serum, swabs and tissue samples. Additionally, in PBMCs, a well-known target for CSFV viral replication, only the primary infecting virus RNA (Cat01 strain) could be detected, even after the isolation in ST cells, demonstrating SIE at the tissue level in vivo. Interestingly, the RNA of the vaccinal C-strain was undetectable by specific RT-PCR [8] in any of the samples analysed after vaccination, including blood, nasal and rectal swabs, or organs throughout the experiment, suggesting a phenomenon of homologous interference, also known as superinfection exclusion (SIE), between the high viral load generated by the primary persistent infection and the CSFV vaccine strain.
cord-001985-iwfidoer	2016	This method revealed a previously unidentified viral RNA diversity of more than 20 complete RNA viral genomes including dsRNA and ssRNA viruses associated with an environmental diatom colony. We herein established a novel strategy to obtain full-length RNA virus sequences with extremely high efficiency by applying a short dsRNA full-length cloning method (8) for physically fragmented dsRNAs. The improved method, named FLDS (fragmented and loop primer ligated dsRNA sequencing), was applied to a diatom colony in a tide pool and revealed previously unidentified RNA viruses. These results indicated that FLDS effectively enriched dsRNA reads, thereby allowing the retrieval of complete genome sequences including terminal regions without the requirement for the additional rapid amplification of cDNA ends (RACE). A phylogenetic analysis of RdRp in DCASSRV-2 suggested that the RNA virus was classified into the genus Mitovirus, which has a non-segmented ssRNA genome, infects the mitochondria of fungi, and lacks viral particles (Fig. S4E) .
cord-002006-pwlybr2h	2016	AIM: To investigate the antiviral effects of vectors expressing specific short hairpin RNAs (shRNAs) against Hantaan virus (HTNV) infection in vitro and in vivo. In mice infected with lethal doses of HTNV, intraperitoneal injection of pSilencer-S or pSilencer-M (30 μg) considerably increased the survival rates and mean time to death, and significantly reduced the mean virus yields and viral RNA level, and alleviated virus-induced pathological lesions in lungs, brains and kidneys. Based on these results and the viral antigen expression results detected by IFA (data not shown), we concluded that the shRNAs that targeted the S and M segments of the HTNV gene were able to inhibit RNA transcript and virus production in the HTNV-infected cells and that shRNA-S1 and shRNA-M2 exhibited a stronger inhibitory effect against HTNV. The RNAi pSilencer-S and pSilencer-M plasmids were constructed, and their antiviral effects were further evaluated by detecting the viral protein synthesis and RNA transcript and progeny virus titers in the HTNV-infected cells.
cord-002015-s3tdllby	2016	2, 3 It is therefore a remarkable fact that the statistical incidence of topological entanglement in biomolecules such as proteins and DNA filaments, which are both long and compact, is substantially smaller than expected for general models of equilibrated polymers with equivalent length and packing conditions. It is accordingly plausible, as remarked by Levinthal in more general contexts, 7,8 that protein sequences have evolved to encode not only the thermodynamically-stable native fold, but also the sequence of steps leading to the native structure formation so to minimize the incidence of misfolded states, including knotted ones which would be very challenging to backtrack. It is possible that the sequence of naturally-occurring RNAs, some of which need to be efficiently translocated through biological pores, have evolved to harness folding kinetics and thermodynamics so as to minimize the incidence of various forms of selfentanglement, including knots, in their native structures.
cord-002045-m44fic4g	2016	Endogenous bornavirus-like L (EBLL) elements are inheritable sequences derived from ancient bornavirus L genes that encode a viral RNA-dependent RNA polymerase (RdRp) in many eukaryotic genomes. Furthermore, we showed that the EBLLs evolved under purifying selection and still possess functional motifs conserved among the Mononegavirales RdRps. These results strongly suggest that the EBLL elements encode functional proteins that may be RdRps. ORF screening for these elements in several mammalian species and found an EBLL element in the bat species Eptesicus fuscus (designated efEBLL-1; accession number ALEH01013293). Notably, this element (eEBLL-1) contains a 1,718-amino acid ORF that was conserved for more than 11.8 MY and included almost all of the sequence motifs essential for the enzymatic activity of a RNA virus RdRp. To the best of our knowledge, eEBLL-1 is the first example of an RNA virus-derived RdRp encoded by the mammalian genome.
cord-002102-0zbp3uqf	2016	title: Hepatitis E Virus Infection in Dromedaries, North and East Africa, United Arab Emirates, and Pakistan, 1983–2015 A new hepatitis E virus (HEV-7) was recently found in dromedaries and 1 human from the United Arab Emirates. Recently, HEV sequences were reported from 3 dromedaries sampled in the United Arab Emirates (UAE) in 2013 and were classified as a new orthohepevirus A genotype, HEV-7 (2, 3) . To determine the geographic distribution of HEV-7, we conducted a geographically comprehensive study of HEV-7 prevalence in dromedaries by testing 2,438 specimens sampled in 6 countries during the past 3 decades. Serum and fecal samples were collected from dromedary camels in the UAE, Somalia, Sudan, Egypt, Kenya, and Pakistan during 1983-2015 (5-7). Detections of HEV-7 RNA in feces in this and a previous study (2) point at feces or feces-contaminated camel products, such as milk, as putative additional sources of human infection.
cord-002179-v8lpw4r7	2016	title: Identification of RNA Binding Proteins Associated with Dengue Virus RNA in Infected Cells Reveals Temporally Distinct Host Factor Requirements Previously, we have described a high-throughput mass spectrometry method termed TUX-MS (thiouracil cross-linking mass spectrometry) to identify host factors that interact with viral RNA during a live infection in cell culture [15] . Development of a quantitative thiouridine cross-linking mass spectrometry (qTUX-MS) method for identification of proteins associated with the DENV RNA TUX-MS can be used to identify host factors by incorporating 4-thiouridine (4sU), a zero-distance cross-linker, into the viral RNA (vRNA) to enable cross-linking of proteins bound to vRNA during a live infection in cell culture [15] . Since some of hnRNPs are known to modulate cellular gene expression in response to dengue infection [60, 61] , we can not rule out that some of the observed effects on virus titers derive from their roles in regulating host mRNAs. Among the numerous qTUX-MS identified factors of interest, our study is the first to demonstrate the involvement of HMCES (or C3orf37) in viral infection.
cord-002180-gsdk5x3e	2016	Amino acid substitutions of NS5(Y26A) and NS5(F123A) that inhibit the ability for NS5 to attach to RNA and recruit host eukaryotic translation initiation factors, respectively, retained the ability to induce an accumulation of cells in the G(0)/G(1) phase as identified for wild-type NS5. Several RNA viruses, including murine norovirus 1 (MNV-1) have been characterized to manipulate cell cycle progression at the G 1 /S restriction point, often creating favorable conditions for viral replication [14] [15] [16] [17] [18] [19] [20] [21] . The effect of NS5 on the host cell cycle was therefore determined by transfection of RAW-Blue cells with RNA transcripts, encoding individual viral genes, NS1-2 from MNV-1 was included as a negative control (Fig 1A) . Furthermore, the NS5(F123A) variant decreased cyclin A protein expression by 67% when compared to the mocktransfected population in a synonymous manner to NS5, strongly implying that the host eukaryotic initiation factor binding domain of NS5 does not play a role in its cell cycle manipulation (Fig 3D) .
cord-002222-rgqwm3vb	2016	By screening a large number of predominantly fecal samples (n = 631) obtained from five carnivore species in the Serengeti ecosystem, East Africa, sapovirus RNA was detected in the spotted hyena (Crocuta crocuta, family Hyaenidae), African lion (Panthera leo, family Felidae), and bat-eared fox (Otocyon megalotis, family Canidae), but not in golden or silver-backed jackals (Canis aureus and C. Long-term monitoring of sapovirus in a population of individually known spotted hyenas from 2001 to 2012 revealed: i) a relatively high overall infection prevalence (34.8%); ii) the circulation of several genetically diverse variants; iii) large fluctuations in infection prevalence across years, indicative of outbreaks; iv) no significant difference in the likelihood of infection between animals in different age categories. A total of 20 partial RdRp gene sequences (16 from spotted hyenas, 3 from African lions and 1 from bat-eared foxes) were obtained and used for the phylogenetic analysis, together with publically available sequence data from 25 representatives of all sapovirus genogroups, divergent unclassified sapoviruses, and other genera in the Caliciviridae family, including Norovirus and Vesivirus.
cord-002238-fyztb8d9	2016	We have previously shown that IFIT1 is primarily responsible for the antiviral action of interferon (IFN) alpha/beta against parainfluenza virus type 5 (PIV5), selectively inhibiting the translation of PIV5 mRNAs. Here we report that while PIV2, PIV5, and mumps virus (MuV) are sensitive to IFIT1, nonrubulavirus members of the paramyxoviridae such as PIV3, Sendai virus (SeV), and canine distemper virus (CDV) are resistant. Despite their limited genetic information, the majority of paramyxoviruses encode small multifunctional accessory proteins that function to aid virus multiplication and block cellular antiviral defense mechanisms; typically, these proteins can block both the production of, and the signaling response to, interferons (IFNs) (for reviews, see references 3, 4, 5, 6, and 7). These results therefore show that MuV Enders is sensitive to IFIT1 but SeV and CDV are not, the weak inhibition of SeV and CDV protein synthesis observed in A549 and A549/shIFIT1 cells pretreated with IFN presumably being due to the action of other ISGs induced by IFN.
cord-002312-jyk7f8hz	2016	Massively parallel (deep) sequencing (RNAseq) of total RNA permitted analysis of all RNA sequences in MS (n = 6) and nonMS (n = 6) white matter samples, revealing that bacterial RNA (ribosomal and non-ribosomal) sequences were detected in all nonMS and MS brain specimens, including MS patients with relapsing-remitting disease (receiving disease modifying therapy) (RR-MS, n = 3) and progressive (untreated) MS (P-MS; n = 3) ( Fig. 2A) . The current study shows the presence of bacterial RNA and DNA sequences and proteins in human brain which are disrupted in conjunction with inflammatory demyelination in patients with MS. The present studies revealed the ratio of bacterium-encoded 16s rDNA to rRNA in matched brain samples to be ~1:2 in both white matter (and cortex, data not shown) with bacterial numbers of 1200-1400 genomes/cm 3 suggesting both bacterial burden and replication were low compared to active pathogenic infections in other tissues.
cord-002320-m99amd4y	2016	Systematic analysis of all CHIKV proteins using a Sf21 RNAi sensor cell line based assay revealed that non-structural proteins nsP2 and nsP3 exhibited RNAi suppressor activity. Using cell lysate of the transfected cell expressing nsP2 and nsP3, we evaluated their ability to bind double stranded RNA by using [γ 32P] labeled shRNA of GFP and running the bound complex in a 6% polyacrylamide gel (Fig. 3e) . Having confirmed that both nsP2 and nsP3 exhibit RNAi suppressor activities, efforts were taken to characterise the proteins better and to identify those specific domains that were and NS4B (dengue virus) were taken as positive controls and empty vector was used as negative control. Taken together, the current study has identified CHIKV proteins nsP2 and nsP3 to exhibit RNAi suppressor activity in the in-vitro system that was further demonstrated in its natural hosts, namely, an Aedes and mammalian cell line.
cord-002376-970934vm	2016	The quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay is nowadays considered as the gold standard method for detection and quantification of enteric RNA viruses such as hepatitis A virus (HAV), hepatitis E virus (HEV) or human noroviruses (NoV) (Mattison et al., 2009; Blaise-Boisseau et al., 2010; Di Pasquale et al., 2010; Vasickova et al., 2012; Hennechart-Collette et al., 2014) . The present article is a followup study of a previously published theoretical concept (Mikel et al., 2015) and describes a method of preparation of MS2 PLP carrying a specific control sequence and their use as a PCV in RT-qPCR detection and quantification of enteric RNA viruses in swab, liver tissue, serum, feces, and vegetable samples. MS2 PLP were added in the amount of 5 × 10 6 particles to the different types of matrices (swabs, liver tissue, serum, feces, and leafy green vegetables) to reveal their ability to serve as PCV in the RT-qPCR detection of enteric RNA viruses.
cord-002398-0a3okta0	2017	Here, we studied the structure of the 2H family member LigT from Escherichia coli both in the apo form and complexed with different active-site ligands, including ATP, 2′-AMP, 3′-AMP, phosphate, and NADP(+). coli LigT bound to tRNA in solution, and provide a model for RNA binding by LigT, involving flexible loops lining the active site cavity. The crystal structure of LigT with 2 0 -AMP was superimposed on the corresponding complex of CNPase, in order to distinguish common and divergent ligand binding determinants in 2H enzymes. Structural data [29] from these enzymes incidate that the 2 0 ,5 0 -adenosine bisphophate substrate binds along the opposite side of the active site, compared to LigT and CNPase (Fig 7A) . As the different structures predicted an RNA-binding surface on LigT, we further modeled a 3-residue RNA molecule with a terminal 2 0 -phosphate into the active site.
cord-002413-795wuqz5	2017	title: IRAV (FLJ11286), an Interferon-Stimulated Gene with Antiviral Activity against Dengue Virus, Interacts with MOV10 IRAV is an RNA binding protein and localizes to cytoplasmic processing bodies (P bodies) in uninfected cells, where it interacts with the MOV10 RISC complex RNA helicase, suggesting a role for IRAV in the processing of viral RNA. FLJ11286, which we refer to here as IRAV (interferon-regulated antiviral gene) (also annotated as C19orf66, UPF0515, or RyDEN), encodes a protein 291 amino acids (aa) in length with a calculated molecular mass of 33.1 kDa. Analysis of published microarray data suggests that IRAV (FLJ11286) is upregulated in response to type I and type II IFNs (6, 20, (24) (25) (26) . IRAV also associates with the host RNA binding proteins UPF1 and HuR (ELAV1) and interacts with MOV10 (a RISC complex RNA helicase), suggesting a role for IRAV in processing or stability of RNA.
cord-002423-1u44tdrj	2017	While this method does not explicitly model host-switching events, it does provide a simple means to compare multiple topologies of virus-host pairs, and accounts for differences in sample size and the fact that several viruses from a specific family can infect a single host species. Across the data set as a whole we found that all virus families displayed relatively large tree topological distances with nPH85 values of !0.6, suggesting that cross-species transmission is widespread, at least at the family-level (Fig 2; S3 Table) . As with the analysis of topological distances, this revealed that cross-species transmission was the most common evolutionary event in all virus families studied here, with co-divergence consistently less frequent (with the possible exception of the Hepadnaviridae-see below), and lineage duplication and extinction playing a much more minor role. To investigate the comparative prevalence of cross-species transmission among viruses we measured the congruence between virus and host phylogenetic trees using a normalized tree topological distance-based approach (nPH85, [14] ).
cord-002439-wesyiymn	2017	For this current report, optical tweezers (OT), a type of single molecule force spectroscopy, and steered molecular dynamic simulations (SMD) were used to examine the folding/unfolding pathways of the TSS. One possibility for why the TSS-only fragment was not stable was the omission of the upstream 5A, as it was subsequently shown that adjacent doublet mutations in 5A (positions 8 and 9 in Figure 1A ) or single mutations disrupting É 3 caused identical enhancements in flexibility of residues throughout the 5A/H4a/É 3 region as assayed Hairpins H4a, H4b and H5 and tertiary interactions É 2 and É 3 comprise the TSS. Initial explicit solvent SMD simulations performed on fragment TSS108 (C 5 through A 112 ) at pulling speeds of 1.0 Å /ps and 0.5 Å /ps terminated with H5 and H4b helices remaining partly basepaired, indicating that the RNA chain was ahead of its approximate equilibrium state.
cord-002482-2t09zqqi	2017	Here, we review the tools utilized by positive-sense single-stranded (+ss) RNA plant viruses to initiate non-canonical translation, focusing on cis-acting sequences present in viral mRNAs. We highlight how these elements may interact with host translation factors and speculate on their contribution for achieving translational control. In this review, we describe current knowledge on the mechanisms used by positive-sense single-stranded (+ss) RNA plant viruses to initiate translation, focusing on cis-acting sequences present in viral mRNAs. We also describe other protein translation strategies used by plant viruses to optimize the usage of the coding capacity of their very compact genomes, including leaky scanning initiation, ribosomal frameshifting and stop-codon readthrough.
cord-002542-f7l4ty2j	2017	Using a combination of longand short-read Next-Generation Sequencing, we have characterized the formation of DI-RNAs during serial passaging of Flock House virus (FHV) in cell-culture over a period of 30 days in order to elucidate the pathways and potential mechanisms of DI-RNA emergence and evolution. By combining these data, we aimed to determine the correlation of recombination events within single RNA virus genomes, characterize the distribution of defective RNA genomes, and determine the exact make-up of DI-RNAs during serial passaging of FHV in cell culture. This observation indicates the protection of cells from infection and/or CPE when supplied with a large dose of viral particles that contain a large proportion of DI-RNAs. In this manuscript, we sought to provide a thorough and comprehensive analysis of the frequency and identity of recombination events present during the serial passaging of Flock House virus in cell culture in order to elucidate the pathways and mechanism of DI-RNA emergence and evolution.
cord-002608-zn7tm1ww	2017	A CLIP-seq approach was used to screen for candidate sites of interaction between the viral Capsid protein and genomic RNA of Sindbis virus (SINV), a model alphavirus. The data presented in this report indicates that the SINV capsid protein binds to specific viral RNA sequences in the cytoplasm of infected cells, but its interaction with genomic RNA in mature extracellular viral particles is largely non-specific in terms of nucleotide sequence. This report details our efforts to identify and characterize the sites of interaction between the viral capsid protein and the genomic RNA using the model alphavirus Sindbis virus (SINV). C) The infectivity of the individual SINV C:R interaction site mutants and parental wild type virus as reported as the ratio of total particles per infectious unit as determined using BHK-21 cells.
cord-002651-9r384oxd	2017	Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. Consistent with the importance of epistatic interactions in the influenza virus polymerase, our data suggest that nucleoside analog resistance and replication fidelity are strain dependent. To this end, we identified variants within the polymerase complex of influenza virus that are able to tolerate drug-mediated increases in viral mutation rates. In most cases, mutagen-resistant variants encode polymerases that exhibit increased replication fidelity, and characterization of these mutants has elucidated the molecular mechanisms governing mutation rate. We tested each of the variant polymerases for reduced nucleoside sensitivity by comparing titers after replication of the corresponding virus populations in mock-or drug-treated cell cultures 24 h postinfection (Fig. 1A) . We used a serial passage competition assay (15) to measure the replicative fitness of each mutant virus relative to the WT PR8 and also performed growth curves to quantify RNA genome production.
cord-002673-5a1rfi6k	2017	This study uses statistical approaches and assessment of "characteristic attributes" (i.e. nucleotide positions that discriminate among strains) to assess whether published and new NNV RNA2 cds sequences show genetic differentiation over geography, host species and years. Within the red spotted grouper nervous necrosis virus (RGNNV) strain, to which all Asian grouper NNV belong, however, no one so far has reported evidence of genetic subgrouping by region, species or year in a formal statistical manner, especially when we restrict hosts just to Asian grouper. The goal of this report was to collate the most comprehensive data set to date on NNV RNA2 sequences for warm water Asian marine finfish, whether published and/or lodged in Genbank over the last 20 years, including some sequence data produced by our group for Vietnamese and Taiwanese grouper, to statistically test the data for evidence of NNV strain variation that associates with geography, host species and year and also to determine whether there are "characteristic attributes" that indicate regional (or host, year) specific differences among the strains.
cord-002676-zwkl1ywk	2017	However, the mechanisms of EBOV lifecycle in host cells, including viral entry, membrane fusion, RNP formation, GP-tetherin interaction, and VP40-inner leaflet association remain poorly understood. Then, the Tyro3 protein kinase (TAM) family, including Axl, Dtk, and Mer, which widely expressed in many cell types, span the plasma membrane and contain intracellular tyrosine kinase domains, are facilitate EBOV GP-dependent attachment [39, 40] . In addition, folate receptor-α (FR-α) was reported to be a co-receptor in binding cells that expressed MARV or EBOV GP, mediated syncytia formation triggered by GP, and facilitated cellular entry of the virus [28] . After the virus has been internalized into the endosome, fusion induced by cleaved GP is fundamentally independent of pH, which indicated an unidentified host cell factor critical for filovirus entry is sensitive to an acidic pH [56] . Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus
cord-002687-ql6zo8ka	2017	Neutralizing antibodies (nAbs), in addition to their capacity to specifically block viral entry via Fab (fragment of antigen binding) recognition of virus, have been found to exert a variety of immunological ''effector'' functions, including clearance of circulatory viruses as well as by mediating cytotoxic killing or phagocytosis of infected cells (DiLillo et al., 2014; Corti et al., 2011; Bournazos et al., 2014; Bruel et al., 2016; Lu et al., 2016; Hessell et al., 2007) , or even possibly triggering sustained host immune responses in vivo Pelegrin et al., 2015) . In the present study, we took advantage of a large non-immune phage display human antibody library and our human NTCP-enabled HBV cell culture system (Yan et al., 2012; Sun et al., 2016) to identify a panel of nAbs that specifically target the preS1 domain.
cord-002720-lrkscs71	2017	title: Development and evaluation of a rapid molecular diagnostic test for Zika virus infection by reverse transcription loop-mediated isothermal amplification The assay detected viral RNA at 14.5 TCID(50)/mL in virus-spiked serum or urine samples within 15 min, although it was slightly less sensitive than reference real time RT-PCR assay. We then evaluated the utility of this assay as a molecular diagnostic test using 90 plasma or serum samples and 99 urine samples collected from 120 suspected cases of arbovirus infection in the states of Paraíba and Pernambuco, Brazil in 2016. Therefore, it is difficult to detect ZIKV in blood samples from patients after the acute phase of infection, even with sensitive molecular diagnostic methods, such as reverse transcription-polymerase chain reaction (RT-PCR) 14, 18, 19 . These results suggested that the RT-LAMP assay could be used as a rapid, sensitive diagnostic test for ZIKV, the Tp value (i.e., less than 15 min) can be used as an indicator of the number of RNA copies in each reaction.
cord-002746-qn34eyul	2017	Codon modification of nucleotides (nt) 22-261 or 22-378 in gag inhibited viral replication by decreasing genomic RNA (gRNA) abundance, gRNA stability, Gag expression, virion production and infectivity. To determine if they are necessary for inhibition of HIV-1 replication, codons introducing CpG dinucleotides were mutated back to the wild type codon, which restored efficient Gag expression and infectious virion production. Gag expression and virion production were similar for wild type HIV-1 and HIV-1 ∆22-261 (Fig. 4c) , indicating that RNA or protein sequences in this region are not necessary for these steps of the viral life cycle. When only 11 CpG dinucleotides are inserted into gag in the context of the codon modified sequence (HIV-1 CM22-165), there is a large decrease in infectivity without a substantial loss of gRNA abundance, Gag expression or virion production (Figs. [100] reported that introducing CpG dinucleotides into env inhibited HIV-1 replication by decreasing the abundance of cytoplasmic gRNA, Gag expression, Env expression and infectious virus production.
cord-002763-n952re94	2017	title: Transcriptome analysis of avian reovirus-mediated changes in gene expression of normal chicken fibroblast DF-1 cells In this study, RNA-Seq technology was applied to investigate the transcriptome-wide changes of DF-1 cells upon ARV infection at the middle stage. CONCLUSIONS: Overall, the differential expression profile presented in this study can be used to expand our understanding of the comprehensive interactions between ARV and the host cells, and may be helpful for us to reveal the pathogenic mechanism on the molecular level. In this study, we tried to build a complete expression profile of ARV-mediated changes at the transcriptional level using RNA-Seq to unveil the complex interactions between ARV and host cells. The results show a similar pattern of ARV-mediated changes as was seen in the DEG analysis of RNA-Seq data (Fig. 4 ).
cord-002764-px0cz6qn	2017	These were based on fusions of an IDR derived from the RNA granule protein FUS (fused in sarcoma) to a multivalent poly-Src homology 3 (SH3) domain protein that phase-separates when mixed with a poly-proline–rich-motif (polyPRM) ligand. These were based on fusions of an IDR derived from the RNA granule protein FUS (fused in sarcoma) to a multivalent poly-Src homology 3 (SH3) domain protein that phase-separates when mixed with a poly-proline-rich-motif (polyPRM) ligand. Tyrosine mutations also block recruitment of IDRs into hydrogels and/or phase-separated liquids formed by the RNA-binding protein hnRNPA2 (47) and FUS (29) , as well as into RNA granules in cells (29) . We found that, when tethered to a polySH3 domain protein that phase-separates when mixed with a cognate poly-proline-rich motif (polyPRM) ligand, wildtype FUS decreases the threshold concentration for LLPS by 8-fold.
cord-002937-7xauocti	2018	We aimed to investigate whether primary cultures of human respiratory tract epithelial cells are helpful to understand H7N9 virus pathogenesis and tissue tropism, and to evaluate how patient-related characteristics can affect the host''s response to infection. In this scenario, primary cultures of human respiratory tract epithelial cells would be invaluable to understand H7N9 virus tissue tropism and pathogenesis, as well as to evaluate how patient-related characteristics can modulate the host''s response to infection. With regard to virus tropism, viral RNA quantities were significantly higher in epithelial cells obtained from the upper anatomical locations than from the lower anatomical locations, without adjustment (P = 0.030); however, the difference lost significance after adjustment for age, sex, medical comorbidities, and obesity (P = 0.490; Figure 2B ).
cord-002990-7flusgus	2018	The application of a cell-based fluorescence resonance energy transfer (FRET) assay for protease activity demonstrated that these substitutions were involved in the enhanced resistance to rupintrivir. Furthermore, we validated the effect of these mutations using reverse genetics in murine norovirus (MNV), demonstrating that a recombinant MNV strain with a single I109V substitution in the protease also showed reduced susceptibility to rupintrivir. In the present study, we isolated replicon cells with reduced susceptibility to rupintrivir after several passages in the presence of rupintrivir and identified two amino acid substitutions of alanine 105 to valine (A105V) and isoleucine 109 to valine (I109V) in the viral protease. To confirm the previously reported inhibitory effects of rupintrivir on human norovirus replication (10), we generated a human gastric adenocarcinoma cell line, HGT-NV, which stably maintained a Norwalk virus replicon encoding the neomycin resistance gene in place of the major capsid protein.
cord-002994-1zjrunzc	2018	Based on these alignments, we investigated the genetic properties of these different isolates circulating in West Africa, such as genome length and location of main conserved amino acid motifs previously described in mosquito-borne flaviviruses (MBFVs) with sometimes mutations which include no physicochemical properties changes [3] . The RNAz method [21] implemented in the Vienna RNA Websuite (http://rna.tbi.univie.ac.at/) [22] was used to detect thermodynamically stable and evolutionarily conserved structural RNA domains on complete non-coding regions of the 11 West African BAGV isolates characterized in this study and the isolates from Spain and CAR, because complete non-coding sequences are not currently available for the isolate from India. Here, we described location of main conserved amino acid motifs on BAGV proteins using in silico analysis of complete genome sequences of the 11 West African BAGV isolates characterized in this study and sequences from India, CAR and Spain.
cord-003006-lk2ny1wd	2018	These newly discovered roles are revealing new mechanisms of virus replication and pathogenicity, whilst enhancing our understanding of the broad functions of each ebolavirus viral protein (VP). Lastly, VP35 is a suppressor of RNA silencing, functionally equivalent to the human immunodeficiency virus (HIV-1) Trans-activator of transcription (Tat) protein and important for viral evasion of the innate immune response [34] . The authors propose that this mutation is likely a result of EBOV adaptation to the human host, as several viral variants have been seen to increase human cell infectivity while decreasing virus entry in nonhuman primates [83] [84] [85] . In addition, many ebolavirus proteins are seen to interact with immune cells, causing cell activation and/or cell death and facilitating both viral replication and spread (e.g., by recruiting monocytes to infected cells or by increasing vascular leakage) as well as enabling immune evasion (e.g., antibody neutralisation by sGP and by cleaved GP), roles that were previously solely attributed to VP24 and VP35.
cord-003044-9uqa39j9	2018	As viral fitness is reduced, interactions are less optimal and, consequently, the gene expression profile of the plant will be increasingly different from that resulting from the infection with the WT virus. Figure 2A shows the clustering (unweighted average distance method; UPGMA) of average expression data for those genes that significantly changed expression (62-fold) among plants infected with the seven viral genotypes (1-way ANOVAs with false discovery rate (FDR) correction; overall P < 0.05) relative to the mock-inoculated plants. tabacum into a novel, poorly susceptible one, Arabidopsis thaliana, have shown that adaptation of TEV to the novel host (i.e., concomitant to large increases in fitness) was associated with a profound change in the way the ancestral and evolved viruses interacted with the plant''s transcriptome, with genes involved in the response to biotic stresses, including signal transduction and innate immunity pathways, being significantly underexpressed in plants infected with the evolved virus than in plants infected with the ancestral one (Agudelo-Romero et al.
cord-003045-r707jl16	2018	The pipeline includes 4 major modules: The first module aligns and filter out human RNA sequences; the second module maps and count (remaining un-aligned) reads against reference genomes of all known and sequenced human viruses; the third module quantifies read counts at the individual viral-gene level thus allowing for downstream differential expression analysis of viral genes between case and controls groups. Available online at: https:// www.fda.gov/biologicsbloodvaccines/bloodbloodproducts/approvedproducts/ licensedproductsblas/blooddonorscreening/infectiousdisease/ucm080466.htm Abbreviations: HBV, Hepatitis B virus; HCV, Hepatitis C Virus; HERV K113, Human Endogenous Retrovirus K113; TCGA, The Cancer Genome Atlas; HCC, Hepatocellular carcinoma; NAFLD, nonalcoholic fatty liver disease; Hep B, Hepatitis B; Hep C, Hepatitis C; HepB + HepC, coinfected with both Hepatitis B and C virus; HBsAg, Hepatitis B surface antigen; HBeAg, Hepatitis B type e antigen; NGS, next-generation sequencing; RNA-seq, whole transcriptome sequencing; BAM, Binary version of Sequence alignment/map format; CDS, coding sequence; Cox PH, Cox Proportional Hazard; HBx, viral gene X; STS, Sequence-tagged sites; NCBI, National Center for Biotechnology Information; GFF, general-feature-format.
cord-003050-n25wnmq5	2018	The 4.2-kb plus-strand sequence of this virus encompasses four main open reading frames (ORFs), as expected for barnaviruses, including ORFs for a protease-containing polyprotein, an RNA-dependent RNA polymerase whose translation appears to rely on − 1 ribosomal frameshifting, and a capsid protein that is likely to be translated from a subgenomic RNA. Based on similar genome sizes, coding organizations, and P2-and P3-region sequence similarities, both MBV and RsBV1 appear to be most closely related to plant viruses in the unassigned genus Sobemovirus [12, 16] . As a result, the consensus sequence for the apparent new barnavirus reported here (GenBank MG686618) is 4206 nt long, appears to be coding complete for ORFs 1-4 ( Fig. 1) , and was newly assembled from a total of 821 individual reads (reads per position: mean, 19; range, 2-35).
cord-003070-6oca1mrm	2018	On the non-redundant benchmark test sets extracted from the PRIDB, the RPiRLS method outperformed RPI-Pred and IPMiner in terms of accuracy, specificity and sensitivity. The computational results showed that the RPiRLS classifier outperformed the RPiRLS-7G classifier in terms of various performance measurements, indicating that the diversity of amino acids at a sequence is important for the prediction of RPIs. The performance of predicting RPIs was evaluated by using 10-fold stratified cross-validation on the RPI2662 data set. For the RPI369 data set as shown in Table 4 , the performance of the RPiRLS method was 0.85, 0.92, 0.84 and 0.86 for predictive accuracy, AUC, specificity and sensitivity, respectively. The work presented here provided a computational method, called RPiRLS, to classify RNA-protein pairs as interacting or non-interacting by integrating a sequence-based derived kernel with regularized least squares.
cord-003104-9cx1gdze	2018	Here, we present data for an alternative model whereby RNA viruses evolve high mutation rates as a byproduct of selection for increased replicative speed. However, the observed attenuation of antimutator RNA viruses in vivo has led many to argue for the adaptive benefit of high mutation rates, as genetic diversity provides a rich substrate for a virus''s evolution in the face of varying intrahost environments [7, 10, [34] [35] [36] [37] [38] . This is a moderate fitness defect, falling in the 64th percentile in a dataset of 8,970 fitness values obtained for point mutants of poliovirus under similar conditions [16] (e.g., human epithelial cells [HeLa] multiplicity of infection [MOI] 0.1, 8-hour infection cycle, and 6 passages; Fig 1B) . The adaptability of WT and high-fidelity viruses have generally been compared using assays that measure the acquisition of drug resistance, the reversion of an attenuating point mutation, or escape from microRNA in a limited number of replication cycles [5] [6] [7] 34, 36] .
cord-003122-a3f4l6iu	2018	The segmentation of the influenza genome makes these additional trafficking requirements especially challenging, as each viral RNA (vRNA) gene segment must navigate the network of cellular membrane barriers during the processes of entry and assembly. To accomplish this goal, influenza A viruses (IAVs) utilize a combination of viral and cellular mechanisms to coordinate the transport of their proteins and the eight vRNA gene segments in and out of the cell. Influenza A viruses (IAVs) and type B viruses (IBVs) contain 8, negative-sense, single-stranded viral RNA (vRNA) gene segments ( Figure 1A ) (3, 4) , which encode transcripts for 10 essential viral proteins, as well as several strain-dependent accessory proteins ( Figure 1B) . In contrast to the early steps in IAV entry, vRNP trafficking to the nucleus following the fusion event is highly dependent on the host cell machinery and transport pathways [reviewed in Ref.
cord-003130-p2h8p5bm	2018	There are more than 70 viruses in the genus flavivirus, and they are transmitted by arthropods such as mosquitoes (dengue virus (DENV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV) and ticks (tick-borne encephalitis virus (TBEV), Langat virus (LGTV), Kyasanur forest disease virus (KFDV), Omsk hemorrhagic fever virus (OHFV), Powassan virus (POWV), and Louping-ill virus (LIV)) [1] [2] [3] [4] [5] . Two other ISGs which have been shown to be antivirally active against TBEV are virus inhibitory protein endoplasmic reticulum associated interferon inducible (viperin) and tripartite motif-79α (TRIM79α). The role of interferon in tick-borne encephalitis virus-infected l cells. A functional toll-like receptor 3 gene (tlr3) may be a risk factor for tick-borne encephalitis virus (tbev) infection Analysis of tick-borne encephalitis virus-induced host responses in human cells of neuronal origin and interferon-mediated protection
cord-003158-mhlqnj52	2018	Previous studies have demonstrated that the HCV JFH1 NS5A C-terminal is a flexible region which is capable of accommodating foreign gene inserts (such as EGFP, 720bp and Rennilla luciferase [Rluc] , 930bp) and still permit HCV replication and viral production (18, 19, (24) (25) (26) (27) (28) (29) . In this study, we used the JFH1-AM120 as a vector to explore if infectious reporter virus would be produced following insertion of LacZ gene that was three time larger than Rluc, into the NS5A C-terminus. This result provided evidence that fusion protein of NS5A and β-galactosidase can be co-expressed in Huh7.5 cells after transfection of JFH1-AM120-LacZ RNA. Our results demonstrate that the LacZ reporter gene can be inserted into the NS5A C-terminus of HCV JFH1-AM120 and will express the predicted NS5A-LacZ fusion protein, which can be detected by western blotting three days after RNA transfection of cells.
cord-003187-qdbcdn2j	2018	Although the two viruses are inhibited by the same three drugs, ZIKV is relatively more susceptive to serial passage in the presence of purine analogues (favipiravir and ribavirin), while USUV replication is suppressed more efficiently by 5-fluorouracil. We observe that ribavirin, favipiravir, and 5-fluorouracil are all inhibitors of both ZIKV and USUV, and that consecutive passage of virus in the presence of these drugs can lead to the complete extinction of infectivity. To investigate whether ZIKV replication could be affected by increased mutagenesis, we tested four different compounds known to be mutagenic for diverse viruses, 5-fluorouracil, ribavirin, favipiravir, and decitabine (25, 32, 35, (37) (38) (39) . To investigate whether the antiviral activities observed during treatment with favipiravir, ribavirin, and 5-fluorouracil are connected to their predicted mutagenic activity, we analyzed the mutation frequencies of both ZIKV and USUV rescued after 5 passages in the presence of these compounds.
cord-003254-yiqdsf9z	2018	Herein, we present a new statistical method for detecting overlapping genes in different reading frames that relies on only a single nucleotide sequence of a gene or genome. For the synonymous mutation test (C), codons that preserve the original amino acid sequence are randomly generated and the length of ORFs on alternative reading frames subsequently measured (note that codon replacement is not restricted to the example mutations shown in the figure, all of which occur in the third nucleotide positions, and that codon replacement with the original codon is also possible). Accordingly, whole genome sequences were downloaded from 2548 reference linear RNA viruses available on GenBank; this produced a total of 6408 coding regions that were used to estimate the sensitivity and false discovery rate of each test. where C is the empirical cumulative distribution function of the theoretical distribution of lengths calculated by permuting codons in the original coding sequence: that is, C(L) is the Simple Method to Detect Candidate Overlapping Genes .
cord-003284-hjx2d5rq	2018	Furthermore, using this infectious clone we have generated a mutant ZIKV containing a single amino acid substitution (A175V) in the NS2A protein that presented reduced viral RNA synthesis in cell cultures, was highly attenuated in vivo and induced fully protection against a lethal challenge with ZIKV wild-type. To analyze the genetic stability of the recombinant ZIKV harboring the point mutation A175V in the coding region of the NS2A protein (rZIKV-RGN-mNS2A), total RNA was purified from Vero cells infected with viruses from passage 1 (P1) to passage 5 (P5) using the RNeasy minikit (Qiagen), according to the manufacturer''s specifications. To investigate whether the reduced RNA synthesis of rZIKV-RGN-mNS2A in Vero cells could result in viral attenuation in vivo, the ability of the mutant virus to induce pathogenesis was analyzed in A129 mice and compared with that of the parental rZIKV-RGN ( Figure 6 ).
cord-003305-ya0siivm	2018	The intra-molecular NTD interactions with the RdRP palm that controls the active site closure, together with the observation of the NTD deletion mutant N-91 exhibited higher level of misincorporation in the de novo RNA synthesis, suggesting a unique mode of fidelity modulation. In order to quantitatively assess whether the fidelity levels are modulated by the intra-molecular NTD-RdRP interface interactions, we established the 32 P-radioactivity based NTP misincorporation assays using the T30/P2 construct utilized in the aforementioned de novo-mode synthesis assessment ( Figure 1B and C) . The effect of E472A mutation was highly consistent with AA mutation ( Figure 4B and Supplementary Figure S3A Since polymerase misincorporation level can be affected by the type of misincorporation and the sequence context of the misincorporation site, we established the second type of regular NTP misincorporation assays using the same T30/P2 construct for a more adequate assessment of the RdRP fidelity modulation brought by the NTD.
cord-003382-v3w1wi5c	2018	We examined the effect of human primary bronchial epithelial cells (PBECs) on PDC functions by performing RNA-sequencing of PDCs after co-culture with air liquid interface differentiated PBECs. Functional analysis revealed that PDCs co-cultured with PBECs displayed upregulation of type I IFN production and response genes. In keeping with the RNA-seq data, we observe increased secretion of type I IFN and other cytokines in response to influenza in PDCs co-cultured with PBECs. The PDCs also primed Th1 responses in T cells. PDCs exposed to PBECs secreted significantly higher levels of type I IFNs (p = 0.0001) on stimulation with influenza as compared to PDCs cultured alone (Fig. 3a) , which is in keeping with the RNA-seq data. In keeping with the activation genes in RNA-seq, we also observed increased secretion of type I IFN from influenza stimulated PDCs co-cultured with PBECs. The expression of TLR9 and ZBP1 was upregulated.
cord-003435-ke0az7nf	2018	Recombinant technologies further expanded the available therapies based on mAbs. In vitro antibody selection technologies like phage or ribosome display were developed to enable the generation of highly specific human mAbs out of libraries that may even be naïve for the specific antigens [31] [32] [33] [34] [35] [36] 1 Schematic illustration comparing active immunization and passive antibody immunotherapies. While T cell engineering for adoptive transfer inevitably requires the use of nucleic acids encoding a target-specific receptor, antibody immunotherapy can deploy recombinant proteins. However, the effects of mRNA modifications on translation, immunostimulation and resulting protein expression appear to be variable, for instance dependent on the type of target cells. In comparison to a single transfer of cells with retroviral CAR expression, an mRNA-encoded receptor required three consecutive lymphocyte infusions to obtain a comparable antitumor effect [262] .
cord-003482-f1uvohf0	2019	Quantitative probe-based reverse transcription PCR (qRT-PCR) was performed on seruminoculated Vero cell supernatants, serum, brain, lung, liver, spleen, kidney, urinary bladder, prostate and testes from bats from both studies. Brain and testicular tissues stained with both goat polyclonal goat anti-Iba1 (green) and monoclonal 4G-2 flavivirus E specific antibodies (red) showed co-localization (yellow) of ZIKV antigen in cytoplasm of activated microglial cells with their characteristic morphology in the cerebral cortex of infected bats 10 dpi in the time course study and 28 day dpi in the pilot study (Fig 9) . Two bat infection experiments were conducted in this investigation; 1) a pilot study to determine susceptibility of Jamaican fruit bats to ZIKV infection, and 2) a time course study to better understand pathophysiology and chronology of events pertaining to the dynamics of viremia, viral tropism, replication and shedding of the virus in a New World bat species.
cord-003505-qr6ukfti	2019	In this work, we have developed a novel colorimetric molecular assay that integrates nucleic acid analysis by dynamic chemistry (ChemNAT) with reverse dot-blot hybridization in an array format for a rapid and easy discrimination of Leishmania major and Trypanosoma cruzi. Once the abasic PNA probes hybridize with target sequences, the SMART-C-Biotin dynamic incorporation takes place, enabling the unequivocal identification of the parasite present in the amplicon sample, because of the unique colorimetric pattern that each Trypanosomatid amplicon generates. DNA amplicon products were denatured and then together with the dynamic chemistry reaction reagents added directly into the internal column of the Spin-Tube that supports the nylon membrane for the color-development assay (Fig. 4) . 20 ng of human gDNA were used as PCR template for its amplification with the set of primers described in our study and neither colorimetric signals nor bands were detected using the Spin-Tube and capillary electrophoresis analysis respectively (Fig. 5 , column 2: 2A and 2B), hence probing the specificity when human gDNA is present.
cord-003596-6dg7i06i	2018	The rise of mRNA nanomedicines is rapidly advancing their applications in a wide range of biomedical fields, such as vaccination, protein-replacement therapy, gene editing, and cell reprogramming and engineering. The four major biomedical applications of mRNA nanomedicine include: 1) nanovaccines derived from antigen-encoded mRNA for the activation of the immune system; 2) proteinreplacement therapy for the treatment of genetic disorder diseases and cancer due to the mutation or loss of protein expression; 3) gene-editing achieved by the co-delivery of Cas9-encoded mRNA and gRNA; and 4) cell programming and engineering through the introduction of mRNA encoding for transcript factors or other functional molecules. In other studies, SAM vaccines encoding influenza antigens were successfully delivered to DCs by chitosan-nanoparticles and PEI-based polyplexes, which were also reported to successfully induce humoral and cellular immune responses in mice [145, 146] . Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo
cord-003597-zj3w9ptj	2019	We combined nasal virus PCR, nasal and peripheral blood cell differentials, and nasal and blood RNA-seq for serial sample collections at baseline and longitudinally during episodes when participants reported cold symptoms to determine the key molecular pathways associated with subsequent asthma exacerbations. The primary aim of the study was to identify patterns of gene expression induced during events that progressed to asthma exacerbations (exacerbation, Ex + ), defined by clinical symptoms that resulted in systemic corticosteroid use within 10 d of cold Fig. 1 | Study design and primary and secondary endpoints. From nasal and blood samples, we combined measured cell percentages with RNA-seq data and used cell deconvolution and WGCNA to construct and validate a repertoire of 94 distinct gene expression modules representing a diverse array of biological functions (Supplementary Fig. 1 and Supplementary Table 1 ).
cord-003676-kr4o8hoc	2019	title: Serological evidence and experimental infection of cynomolgus macaques with pteropine orthoreovirus reveal monkeys as potential hosts for transmission to humans In this study, we screened 517 cynomolgus macaques caught in Singapore for evidence of exposure to PRV3M (also known as Melaka virus), which was first isolated from human patients in Melaka, Malaysia. We provide evidence that PRV exposure in the macaque population in Singapore occurs at a relatively high prevalence and this study suggests that cynomolgus macaques may be an intermediate or reservoir host for PRVs. Pteropine orthoreoviruses (PRVs) are a group of emerging bat-borne viruses, belonging to the genus Orthoreovirus within the family Reoviridae. Given the cross-species transmission of PRVs and frequent human-monkey interaction in Singapore and surrounding regions, we initiated this study to determine the seroprevalence of PRV3M in wild cynomolgus macaques and to examine the susceptibility of cynomolgus macaques to experimental infection with PRV3M and their potential role in acting as hosts facilitating cross-species transmission.
cord-003707-fbe47bgi	2019	I. scapularis NIRVS are enriched in bunyaand orthomyxo-like sequences, reflecting that ticks are a dominant host for these virus groups. NIRVS arise from the reverse transcription of viral RNA into cDNA and its integration into the genome of a host germ cell, followed by vertical transmission to offspring (Katzourakis and Gifford 2010) . Yet recent evidence has highlighted the abundance of NIRVS in some arthropod genomes-e.g., Ae. aegypti and Ae. albopictus contain >100 NIRVS from over eight RNA virus families encompassing þssRNA, ÀssRNA, and dsRNA viral groups (Palatini et al. Using the well-studied genomes of Aedes mosquitoes to validate our analysis, we employed a bioinformatic pipeline to characterise NIRVS in seven non-Aedes arbovirus vectors that have representative genomic sequences (Table 1 and Fig. 1 ). scapularis lacked NIRVS from positive-sense RNA viruses, which comprise about one tenth of NIRVS in Aedes mosquitoes ( Fig. 3; Supplementary Table S2 ).
cord-003711-l3brhmzq	2019	ADP-ribosylation is a reversible chemical modification catalysed by ADP-ribosyltransferases such as PARPs that utilize nicotinamide adenine dinucleotide (NAD(+)) as a cofactor to transfer monomer or polymers of ADP-ribose nucleotide onto macromolecular targets such as proteins and DNA. We further reveal that ADP-ribosylation of RNA mediated by PARP10 and TRPT1 can be efficiently reversed by several cellular ADP-ribosylhydrolases (PARG, TARG1, MACROD1, MACROD2 and ARH3), as well as by MACROD-like hydrolases from VEEV and SARS viruses. Importantly, PARP3 could only ADP-ribosylate DNA ends and did not have any activity on RNA oligos, while PARP10 specifically modified phosphorylated ssRNA oligo in the conditions tested ( Figure 1A and B). Since PARP10 can ADP-ribosylate both 5 and 3 phosphorylated ends of RNA, we tested both of these modified oligos as substrates for well characterized human ADP-ribosylhydrolases: PARG, TARG1, MACROD1, MACROD2 and ARH1-3.
cord-003792-v48xeqdz	2019	We characterized natural vertical transmission of Zika virus in pools of Aedes aegypti larvae hatched from eggs collected in Jojutla, Morelos, Mexico. We characterized natural vertical transmission of Zika virus in pools of Aedes aegypti larvae hatched from eggs collected in Jojutla, Morelos, Mexico. Several studies carried out under laboratory conditions have demonstrated that Zika virus can infect many different Aedes mosquito species (3) ; still, the key species for the transmission of Zika virus to humans are Ae. aegypti and Ae. albopictus (4) (5) (6) . In this study, we sought to demonstrate natural vertical transmission in Ae. aegypti mosquitoes by detecting viral RNA and isolating infectious Zika virus from larvae hatched from field-collected eggs. In this work, we were also able to demonstrate the natural vertical transmission of Zika virus in Ae. aegypti mosquitoes by the successful isolation of infectious Zika virus (31N) from larvae raised from field-collected eggs.
cord-003898-y6zpvw84	2019	title: RNA Sequencing of H3N2 Influenza Virus-Infected Human Nasal Epithelial Cells from Multiple Subjects Reveals Molecular Pathways Associated with Tissue Injury and Complications The aim of this study was to utilize RNA sequencing (RNAseq) technology to not only reveal the hNEC responses (from multiple individuals) against influenza infection, but also to identify those genes with high magnitude changes to serve as potential reference markers of the innate responses of influenza infection. After deriving the transcriptomes by RNAseq, we then further investigated whether the changes in expression of genes resulted in alterations in secretory cytokines and chemokines early in the infection of hNECs. Initially, we detected significant reductions in multiple cytokines at 8 hpi, with the exception of IL-15 which was increased ( Figure S2 ). In conclusion, RNAseq technology allowed us to accurately quantify the magnitude of gene expression changes, as well as the relevant enriched pathways during H3N2 influenza virus infection of hNECs, which can serve as a baseline for future clinical studies.
cord-004274-cot05vx7	2020	"STATE-OF-THE-ART" mRNA CONSTRUCTS AND DELIVERY TECHNOLOGIES The core principle behind mRNA as a technology for vaccination is to deliver the transcript of interest, encoding one or more immunogen(s), into the host cell cytoplasm where expression generates translated protein(s) to be within the membrane, secreted or intracellularly located. Perspective #2: Translational sciences will inform preclinical and clinical studies to promote rapid downselection of constructs and formulations A key aspect of vaccine development efforts is the goal of making early informed decisions, based on objective data that favor or disfavor a particular candidate. 64 The current focus from a clinical perspective is to optimize the benefit (immunogenicity and efficacy) while reducing the risk (safety) profile of a candidate mRNA vaccine by optimizing the quality attributes that dictate expression and/or augmenting delivery. Thus, early-phase clinical trials need to be designed in a way to appropriately capture the inflammatory component intrinsic to all mRNA vaccines, given that several intracellular innate immune response sensors are activated by RNA.
cord-004280-c470nlie	2020	In this study, we evaluated the use of a bioaerosol sampling method to noninvasively detect and quantify airborne influenza A virus (IAV) densities in a public elementary school. Significantly different (p = 0.049) airborne IAV densities were detected between all three indoor locations (i.e., gymnasium, classroom, and corridor) and all positive samples were collected during the last two weeks of 66 , and a 20-30% relative humidity level; Descriptive of an average elementary school student in the USA weighing ~23-32 kg with an assumed tidal volume (V T ) of 7 mL per kg of body mass. Given the high airborne IAV densities detected in the school corridor, along with elevated student contact rates, it is plausible to conclude that the school corridor is a "hotspot" for influenza virus transmission.
cord-004507-ezuyjcxs	2020	The study provided complete genome sequences for nine Letea virus strains and new information about orbivirus diversity, host range, ecology and evolution. The phylogenetic clustering of Orbivirus members results in clades indicating their putative or potential arthropod vectors: Culicoidesor sand fly-borne (C/SBOV), mosquito-borne (MBOV) and tick-borne orbiviruses (TBOV) [9] . Evolutionary relationship of LEAV with representative members of the Orbivirus genus were analyzed by inferring phylogenetic trees with amino acid and nucleotide ORF sequences of conserved genes encoding the polymerase (VP1), the subcore shell protein T2 (VP2/VP3) and the major core surface protein T13 (VP7) [5, 6] . Inclusion and demarcation of species within the Orbivirus genus considers several criteria, such as sequence identity of segments encoding the polymerase (VP1) and major subcore shell protein T2, gene reassortment between close strains, high levels of serological cross-reactivity against conserved antigens like the T13 protein, conservation of UTR terminal nucleotides, range of hosts and vectors or the clinical signs associated with orbivirus infection [1] .
cord-004509-jkzqmkz6	2020	In conclusion, Lyoph-P&P holds several advantages over extemporaneously preparer liquid formulation that merit to be considered when a novel real-time molecular assay is implemented in a laboratory in charge of routine diagnostic activity. Selected assays target two emerging viruses that are listed on the blueprint of the WHO as to be considered for preparedness and response actions [5] : chikungunya virus (CHIKV), a single-stranded positive-sense RNA alphavirus, and Rift Valley fever phlebovirus (RVFV), a tri-segmented, single-stranded negative-sense RNA phlebovirus. Detection of CHIKV RNA using Lyoph-P&P provides results in clinical samples that are at least equal and often better than those obtained with the extemporaneously prepared liquid formulation used as reference. Indeed, at laboratory level, it is likely that one or two different formats (number of tests per vial) will be either prepared or ordered; as a consequence, the time during which rehydrated material can be stored without affecting the expected performances of the assay is a key factor.
cord-004534-jqm1hxps	2009	HIV-1 to efficiently complete a replication cycle has to integrate its genome into the host cellular DNA.After HIV-1 enters target cells,neosynthesized viral DNA forms along with other proteins the pre-integration complex (PIC).PICs are then transported into the nucleus where integration,catalyzed by the viral integrase,takes place.HIV-1 viral particles engineered to incorporate integrase fused to EGFP have proven effective to study PICs within nuclei of infected cells.In this study we report the live imaging analysis of nuclear PIC dynamics obtained by time-lapse microscopy.Intranuclear trajectories of IN-EGFP-labeled PIC were collected in three dimensions and examined by both mean squared displacement (MSD) and cage diameter (CD) analysis.In CD the maximum distances measured between two positions occupied by a PIC in a time window of 2 minutes were calculated while in our MSD analysis 5-minute long trajectory segments were considered.Remarkably,MSD revealed the presence of an underlying active transport mechanism.To test the possible role of actin filaments,PIC nuclear trafficking was analyzed in cells treated with latrunculin B (actin polymerization inhibitor).Preliminary results suggest that the disruption of actin function impairs the active nuclear movement of PICs. Second harmonic generation microscopy reveals sarcomere contractile dynamics of cardiomyocytes N.
cord-004584-bcw90f5b	2011	Our goals are two-fold: (1) to monitor conformational changes in each domain upon its binding to specific ligands and then to correlate the observed changes with structural differences between the CRDs and (2) to investigate the interaction between the CRDs and lipid model membranes. Cholesterol-assisted lipid and protein interactions such as the integration into lipid nanodomains are considered to play a functional part in a whole range of membrane-associated processes, but their direct and non-invasive observation in living cells is impeded by the resolution limit of [200nm of a conventional far-field optical microscope. Therefore, to investigate the dynamic and complex membrane lateral organization in living cells, we have developed an original approach based on molecule diffusion measurements performed by fluorescence correlation spectroscopy at different spatial scales (spot variable FCS, svFCS) (1).
cord-004656-n4h295e5	1990	In an effort to determine if this phenomenon is specific to the rat or applicable to other species, we compared the ontogenic changes in hepatic and renal angiotensinogen mRNA expression in fetal (60, 90, 118, and 138 d of gestation, term being 145 d), newborn (7 d postnatal), and adult sheep. Angiotensinogen mRNA sequences were detected in all fetal liver samples and appeared to increase 3-fold from 60 to 138 d gestation and then to decrease after birth. In an effort to determine if this phenomenon is specific to the rat or applicable to other species, we compared the ontogenic Received January 18, 1990 ; accepted April 16, 1990 changes in hepatic and renal angiotensinogen gene expression during the last trimester of gestation in fetal sheep. Northern blot analysis of liver and kidney RNA from fetal sheep (1 18 and 138 d gestational age) 1 and newborn lamb are shown in Figure 3 .
cord-004719-3stcx0dd	1993	In several groups of positive-strand RNA viruses, the movement function is provided by the proteins encoded by the so-called triple gene block including two putative small membrane-associated proteins and a putative RNA helicase. It is concluded that classification of movement proteins based on comparison of their amino acid sequences does not correlate with the type of genome nucleic acid or with grouping of viruses based on phylogenetic analysis of replicative proteins or with the virus host range. Limited sequence similarities were observed between i) movement proteins of dianthoviruses and the MIP family of cellular integral membrane proteins, and ii) between movement proteins of bromoviruses and cucumoviruses and M1 protein of influenza viruses which is involved in nuclear export of viral ribonucleoproteins. With representative sequences of plant virus genomes from many groups available, efforts were made to identify regions of similarity between known movement proteins and to predict movement functions for uncharacterized ORFs. Early observations made by this approach have been reviewed previously (e.g.
cord-004827-bnf3mvaf	2005	Phylogenetic relationships have been found useful for virus identification, work on origin, speed and mechanisms of evolution, taxonomy, and the elucidation of transmission pathways (e.g. the transmission of HIV from a source to a victim [16] ). In vitro, a single round of replication of HIV-1 in T lymphocytes generated on average 9 recombination events per virus [14] . Approximately 50% of sequence changes are consistent with evolution by point mutations; other changes are due to multiple recombination events. After an introduction in which basic genetic terms were defined (mutation and mutation rate, hypermutation, recombination, reassortment, segmentation etc), the quasispecies concept was presented according to which any sample of an RNA virus represents a ''swarm'' of closely related mutants. ''To maintain RNA structure, evolution selects against better alternatives elsewhere in the genome''. The aim of the talk was to show the significance of RNA structural considerations for the evolution of viruses. Plant RNA virus evolution
cord-004848-2cfphi88	1990	In this way these workers have identified three intracellular RNAs (4.8, 4.2, and 2.4 kb) as well as the genome (8.2 kb) and shown that these form a nested set of 3'' co-terminal molecules similar to that observed in coronavirus infected cells. We have used this to probe FCV-infected cells for the synthesis of virus specific RNA and confirm and extend the observations of Neill and Mengeling. Subcloning of the virus 3'' end into a single stranded vector has allowed us to examine the occurrence of positive and negative sense RNA separately. Finally this inference was confirmed by subcloning the terminal EcoRI/PstI fragment into M13 and determining its sequence (Fig. 3a) , This showed a poly A stretch preceded by a short run ofpoly C which is exactly that structure expected for cDNA clones derived by this method from an mRNA 3'' end. However, the negative strand-specific probe also showed hybridization to subgenomic messages 2-4, but this was not observed until later in infection than the detection of the positive forms of these molecules.
cord-004851-h9ppa064	1991	The viral proteins have not been identified, but on the basis of the predicted amino acid sequences, hydrophobicity plots, location of potential glycosylation sites and similarities of these properties to those of pestiand flaviviruses, the following genome organization has been predicted. A recent study of stored blood samples from prospective studies of 15 U.S.A. patients with transfusion-associated chronic hepatitis documented by liver biopsies indicated that the time course of changes in plasma ALT activity and of the formation of anti-HepCV antibodies can vary considerably between patients [4] . The putative nucleocapsid protein sequence derived from PCR products of RNA extracted from healthy Japanese carrier plasma exhibited a 97.4% amino acid identity with the sequence determined for the original chimpanzee HepCV isolate (HepCV-1) but only a 90.5 % identity at the nucleotide level [45] [46] [47] .
cord-004879-pgyzluwp	1994	Furthermore kinetic experiments after complementation of HIV=RT p66 with KIV-RT pSl indicated that HIV-RT pSl can restore rate and extent of strand displacement activity by HIV-RT p66 compared to the HIV-RT heterodimer D66/D51, suggesting a function of the 51 kDa polypeptide, The mouse mammary tumor virus proviral DNA contains an open reading frame in the 3'' long terminal repeat which can code for a 36 kDa polypeptide with a putative transmembrane sequence and five N-linked glycosylation sites. To this end we used constructs encoding the c-fos (and c-jun) genes fused to the hormone-binding domain of the human estrogen receptor, designated c-FosER (and c-JunER), We could show that short-term activation (30 mins.) of c-FosER by estradiole (E2) led to the disruption of epithelial cell polarity within 24 hours, as characterized by the expression of apical and basolateral marker proteins.
cord-005010-xg2bv9gy	2015	title: Mechanism of Preferential Packaging of Negative Sense Genomic RNA by Viral Nucleoproteins in Crimean-Congo Hemorrhagic Fever Virus In the present work, by analyzing genomic sequences of RNA viruses either with negative or positive sense, performing different docking experiments and carrying out molecular dynamic (MD) simulations, we undertook to study the mechanism conferring different affinities to CCHFV nucleoprotein for negative and positive sense RNAs''. Figure 1 , also, shows that irrespective of their senses, long RNAs have comparatively higher affinities to nucleoprotein than short RNAs. Based on the results of aforementioned docking experiments, we then selected CCHFV nucleoproteins-RNA complexes of maximum binding energies for positive and negative sense RNAs (both short and long) to carry out MD simulations.
cord-005060-n901y2d4	2001	The largest ORF 2 encodes a polyprotein of 947 amino acids (103.6 kDa), which codes for a serine protease and an RNA-dependent RNA polymerase. The genome sequence of sobernoviruses has been determined in Southern bean mosaic virus (SBMV)''2,24), CfMV8315), Rice yellow mottle virus (RYMV)") and Lucerne transient streak virus (LTSV, accession number U31286). However, the con-served sequence, WAG + E/D rich sequence is detected in the region, and putative E/S cleavage sites are present on both sides of the region : proteolytic cleavage would result in a protein of 9 kDa. Possibly, the VPg of RGMoV is located between the protease and the RNA-dependent RNA polymerase domains in the same order as in the SBMV ORF 222) (Fig. 3) . In the RGMoV RNA sequence, no ORF corresponds to the second largest product of 68 kDa. The putative replicase of CfMV is translated as part of a single polyprotein by -1 ribosomal frameshifting between two overlapping ORFs having a coding capacity for 60.9 kDa and 56.3 kDa proteins7J8).
cord-005147-mvoq9vln	2017	Using whole-exome sequencing and trio-based de novo analysis, we identified a novel heterozygous de novo frameshift variant in the leukemia inhibitory factor receptor (LIFR) gene causing instability of the mRNA in a patient presenting with bilateral CAKUT and requiring kidney transplantation at one year of age. Loss of cdkl5 associated with deficient mammalian target of rapamycin (mTOR) signaling in mice and human cells We and other groups have shown that mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene cause a severe neurodevelopmental disorder with clinical features including intellectual disability, early-onset intractable seizures and autism, that are closely related to those present in Rett syndrome (RTT) patients. Functional characterization of novel GNB1 mutations as a rare cause of global developmental delay Over the past years, prioritization strategies that combined the molecular predictors of sequence variants from exomes and genomes of patients with rare Mendelian disorders with computer-readable phenotype information became a highly effective method for detecting disease-causing mutations.
cord-005281-wy0zk9p8	2017	In the human genome, this capacity is determined by the portion of chromosomal DNA, which does not contain species-specific protein-encoding sequences and, thus, can basically make a place for novel information that will be modified to reach a new balance. In fact, the scope of the described phenomena is not limited to retroviruses as such, since the ubiquity of retroviral elements in animal genomes, their activity in germline cells [31] , along with the fact that viral replication depends significantly on RNA expression, allow retroviruses to contribute in different ways to the insertion of nonretroviral genes into animal germline cells. Finally, the ability to incorporate parts of the viral genome into the chromosomal DNA of host germline cells can vary strongly among different taxonomic groups of viruses, i.e., orders, families, genera, and even species If insertions of viral sequences remain functionally active in the host cell genome, they can give rise to either proteins that function in a new environment or untranslated RNAs of different sizes.
cord-005377-36io7zsm	2012	The main isothermal methods reviewed here include loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), and helicase-dependent amplification (HDA). Development and evaluation of a novel loop mediated isothermal amplification (LAMP) method for rapid detection of SARS Corona virus Rapid detection of norovirus from fecal specimens by real-time reverse transcription-loop-mediated isothermal amplification assay Rapid detection and quantification of Japanese encephalitis virus by real-time reverse transcription loop-mediated isothermal amplification. Rapid and real-time detection of chikungunya virus by reverse transcription loop mediated isothermal amplification assay Development and evaluation of reverse transcription Loop mediated isothermal amplification assay for rapid and real-time detection of Japanese encephalitis virus Development of a quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes Development and evaluation of a real-time nucleic acid sequence based amplification assay for rapid detection of influenza A
cord-005378-u2bbgn8k	2013	Porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus that causes significant losses in the pig industry, is one of the most important animal pathogens of global significance. Similarly, mutagenesis studies have shown that EAV NSP1 (which contains a tandem of the PCPα and PCPβ domains, with PCPα having lost its enzymatic activity) is involved in regulating the accumulation of minusstrand templates to control the relative abundance of viral mRNAs, thereby coordinating genome replication, subgenomic mRNA synthesis, and virus production (Tijms et al., 2001 (Tijms et al., , 2007 Nedialkova et al., 2010) . Structure and cleavage specificity of the chymotrypsin-like serine protease (3CLSP/nsp4) of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Identification of the CD163 protein domains involved in infection of the porcine reproductive and respiratory syndrome virus
cord-005392-0pgcfk6b	2011	title: Development of a Quantitative Real-Time Nucleic Acid Sequence-Based Amplification Assay with an Internal Control Using Molecular Beacon Probes for Selective and Sensitive Detection of Human Rhinovirus Serotypes In this study, we developed the first quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes. Aim of this study was to develop the first quantitative real-time nucleic acid sequence-based amplification assay internally controlled using molecular beacon for selective and sensitive detection of HRV serotypes. To estimate the dynamic range of the real-time NASBA assay (range of concentrations over which the method performs in a linear manner with an acceptable level of trueness and precision), we used HRV standard dilutions from 10 8 copies/ll to 1 copy/ll. Evaluation of a real-time nucleic acid sequence-based amplification assay using molecular beacons for detection of human immunodeficiency virus type 1
cord-005654-n9u2em10	1984	41 The repeated mini-exon sequence that encodes the first 35 base pairs of all variant surface antigen mRNAs of Trypanosoma brucei directs the synthesis of a discrete I37-nucleotide transcript. 41 The repeated mini-exon sequence that encodes the first 35 base pairs of all variant surface antigen mRNAs of Trypanosoma brucei directs the synthesis of a discrete I37-nucleotide transcript. This result could be interpreted as protection by a short RNA of 137 nucleotides derived from the mini-exon repeats and/or by a longer transcript with a discontinuity at this position in the RNA/DNA duplex. The second method to detect and size transcripts from the mini-exon repeat used Northern blot analysis 31 • For this procedure, total trypanosome RNA was resolved by polyacrylamide gel electrophoresis (PAGE) in denaturing conditions, transferred to GeneScreen membrane 32 and hybridized with a [5/ -32 P]_labelled, synthetic oligonucleotide complementary to positions + 117 to +133.
cord-005865-7lohh5ty	2007	Here we present a microfluidic platform that can detect the highly pathogenic avian influenza virus H5N1 in a throat swab sample by using magnetic forces to manipulate a free droplet containing superparamagnetic particles. Here we present a microfluidic platform that can detect the highly pathogenic avian influenza virus H5N1 in a throat swab sample by using magnetic forces to manipulate a free droplet containing superparamagnetic particles. Starting from a throat swab sample, we show how HPAI H5N1 can be detected by using magnetic forces to manipulate a free droplet containing superparamagnetic particles. (h) After SPE, the immobilized viral RNA is magnetically pulled out of the raw sample solution, washed four times to remove residual contaminants, and desorbed into an RT-PCR solution positioned on top of a miniaturized real-time thermocycler (see also Supplementary Fig. 1 ).
cord-006049-sw1hki4r	2010	Nucleic acid aptamers can be selected from pools of random-sequence oligonucleotides to bind a wide range of biomedically relevant proteins with affinities and specificities that are comparable to antibodies. Although this is true for biological nucleic acids [1] [2] [3] [4] , it was only recently that a series of technological advances allowed the development of in vitro evolutionary methods for the discovery of additional, non-biological oligonucleotides that can bind to protein targets. Since the invention of the SELEX process around 1990 (REfS 5, 6) , researchers have identified high-affinity aptamers that target a broad cross-section of protein families including cytokines, proteases, kinases, cell-surface receptors and cell-adhesion molecules (TABLE 1) . In particular, the site-specific placement of functional groups for conjugation means that the modification of aptamers after the solid-phase step (for example with high molecular mass PEG 46 ) leads to products with discrete stoichiometries and defined chemical structures. Selection of a RNA aptamer that binds to human activated protein C and inhibits its protease function
cord-006068-w3if1hns	2006	This Review summarizes recent in vitro and in vivo studies that point to an important connection between DNA-and RNA-containing immune complexes, the production of type I interferons (IFNs; that is, IFNα and IFNβ), the activation of TLRs and subsequent events in the development and/or the progression of systemic autoimmune diseases. . b | IFNα upregulates expression of Toll-like receptor 7 (TLR7) by B cells, promotes cell death and increased release of certain RNA autoantigens, and primes pDCs to respond more effectively to immune complexes. A limited number of studies have now provided data consistent with the idea that TLR7 and TLR9 have key roles in the production of pathogenic autoantibodies and/or in the development of clinical features of autoimmunity in experimental animals ( Mice that are homozygous for the lpr mutation do not express a functional form of the death receptor CD95 (also known as FAS), and they develop a lymphoproliferative disease that is similar to Canale-Smith syndrome in humans.
cord-006129-5rog0s98	2012	[12] Answering back, certain host miRNAs alter the cell gene expression to defend the cells against the viral infection by interfering with viral proteins or other cellular factors as a type of immune response against these particular viruses. [40] These virus-encoded miRNAs play important roles in the establishment of latent infection, as well as the pathogenesis of virally induced diseases. According to the most recent studies, herpesviruses utilize their encoded miRNAs in a wide range of biologic functions, such as inhibition of apoptosis, immune evasion, control of cellular proliferation, and regulation of viral replication. [58] Downregulation of UL114 protein, using miR-UL112-1, results in inhibition of viral DNA replication and subsequently triggers the latent phase of infection, making the virus able to evade the host immune system.
cord-006331-s2qf98lj	2010	After 16 rounds of selection from the 2'' amino modified RNA library the isolated aptamers formed a complex with FVIIa characterized by the K d value of 11.3 ± 1.3 nM. performed RNA selection to FIXa; after eight rounds of selection they found an aptamer, which bound to FIXa with the K d value of 0.65 ± 0.2 nM and exhibited 5000 fold higher affinity to FIXa compared with FVIIa, FXa, FXIa and activated protein C [20] . These aptamers formed a complex with ΔNS3 with the K d value of 10 nM, caused 90% inhibition of protease activity of the ΔNS3 peptide and full sized NS3 bound to a maltose binding protein (MBP NS3). Four teen rounds of selection yielded the RNA aptamer, exhibiting high affinity binding to p50 and inhibition of NF kB binding to DNA by preventing protein dimerization [91] .
cord-006452-mmdk2xom	2018	Nucleic acid-based therapeutics present huge potential in the treatment of pulmonary diseases ranging from lung cancer to asthma and chronic pulmonary diseases, which are often fatal and widely prevalent. In this review, we provide a comprehensive overview of the nucleic acid application for pulmonary diseases, covering action mechanism of the nucleic acid drugs, the novel delivery systems, and the current formulation for the administration to lungs. To overcome these biological barriers, strategies like chemical modification, conjugation, vector encapsulation, and selection of administration route have been utilized to improve the delivery of nucleic acids to lungs. One direction for developing new drugs to treat asthma is to target central pathways to the pathogenesis of the disease, and nucleic acid-mediated therapies silencing the specific effector or the upstream regulator can be a potential approach. Nucleic acid drugs hold great promises as new classes of therapeutic agents for pulmonary diseases, and some candidates have entered into clinical trials (Table III) .
cord-006826-vnmg0zid	2017	In this work, we wanted to determine how the length of the cargo impacts the stability and structure of the assembled CLPs. We hypothesized that cargo neutralizes the basic region of the alphavirus capsid protein and if the cargo is long enough, it will also act to scaffold the CP monomers together. Modest differences in particle size on DLS and EM suggest that changes in light scattering intensity indicate that similar amounts of CLPs were formed using 48hp, 48lin, and 90mer oligos than with the shorter 27mer ( figure 1(B) ). From our ionic strength stability results (figure 1(B)), we postulated that longer cargo that functioned to both neutralize charge and scaffold CP would favor the formation of complete, closed, spherical cores. This model provides an explanation for the structural defects observed in the cryo-EM classification, but does not account for the possible role of longer cargo in scaffolding or bridging between CP monomers to promote CLP assembly.
cord-006935-wzz5t3sv	1996	Only one conserved cysteine residue, thought to be the catalytic nucleophile, was identified in 3C p~° sequences of different origin [41, 47] and its indispensability for the PV 3C pr° proteolytic activity was proven by site-directed mutagenesis [48] . All RNA viral cysteine proteinases are synthesized as a domain in a polyprotein [17] and three major trends were discovered with respect to the position occupied by these enzymes. However, in an N-terminally extended precursor of 3C pr°, the Nterminal region of the proteinase could adopt a different structure that would fit the substrate binding pocket and might be processed in cis [5] . The $5-$2 subsites of the substrate-binding pocket, which interact with the less conserved P5-P2 positions of the cleavage sites, were recognized in the enzyme-substrate model of the HRV-14 3C pr° as well [5] .
cord-006951-tj22dh5o	2018	While viral RNAs are found to be compact and highly branched because of long distance base-pairing between nucleotides, recent experiments reveal that in a head-to-head competition between an ssRNA with no secondary or higher order structure and a viral RNA, the capsid proteins preferentially encapsulate the linear polymer! While branching makes the genome more compact, RNA base-pairing increases the effective Kuhn length of the RNA molecule, which could result in an increase of the free energy of RNA confinement, that is, the work required to encapsidate RNA, and thus less efficient packaging. In this paper, we vary the degree of branching as well as the effective Kuhn length and linear charge density of a model RNA, and study their impact on the optimal length of encapsulated genome by capsid proteins.
cord-006960-9pho3hk6	2013	In this work, we have utilized electro-actuation based DMF technology, integrated with suitably tailored resistive micro-heaters and temperature sensors, to achieve chip based real-time, quantitative PCR (qRT-PCR). Performance of the integrated DMF device was analyzed in real-time chip based qRT-PCR detection of in-vitro synthesized influenza A and C virus RNAs during 30-35 PCR cycles. Among the contact temperature control methods, the micro-fabricated resistive heaters/RTDs have smaller power requirement, faster thermal response and higher heating ramp rates and are therefore preferred for the proposed qRT-PCR micro-device. Mixing of the influenza C RNA sample and the off-chip prepared PCR reagents, followed by the RT reaction and thermal cycling over Micro-electrode 1, is shown in Figure 7 . This article demonstrates the design and micro-fabrication of integrated droplet microfluidic device, with nano-textured superhydrophobic top surface, that is capable of electro-handling of PCR samples/reagents, facilitating chip based mixing/sample preparation and chip based qRT-PCR amplification and detection of influenza viruses.
cord-007009-4wbvdg1r	2014	Severe fever with thrombocytopenia syndrome (SFTS), an infectious disease with a high case-fatality rate, is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic to central and northeastern parts of China [1, 2] . Vero cells were inoculated with RT-PCR-positive patient sera for virus isolation, cultured for 4-7 days, and examined for SFTSV antigen detection by indirect immunofluorescence assay (IFA) with a polyclonal antibody raised against SFTSV recombinant NP (rNP; rabbit anti-SFTSV rNP serum), which was produced as follows. Physicians were asked to volunteer information if they had treated patients who satisfied the following case definition: (1) fever of >38°C; (2) gastrointestinal tract symptoms, such as nausea, vomiting, abdominal pain, diarrhea, and melena; (3) thrombocytopenia, with <100 × 10 9 platelets/L; (4) leukopenia, with <4 × 10 9 white blood cells/L; (5) elevated levels of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase; (6) absence of other causes; and (7) death or admission to an intensive care unit because of the severity symptoms. Detection of severe fever with thrombocytopenia syndrome virus (SFTSV) RNA in the right cervical lymph node by the in situ hybridization ATtailing method.
cord-007041-rloey02j	2019	We sequenced virus populations in parallel using both MinION and Illumina, allowing us to corroborate the inferences of AssociVar. This then allowed us to directly infer relationships between mutations and to deduce the entire genome sequences of viral strains in the population. We then determined the population frequency of each mutation at passage 1 and passage 15 through whole genome deep sequencing as described below, using Illumina and MinION. After applying AssociVar to the data, we were able to identify five out of the six mutations appearing at a frequency above 10% in the Illumina results in p15A, and all eight positions within the p15B sample (Figure 4 , Supplementary Table S2 ). We applied AssociVar to sequencing data from an evolved population of phages where Illumina sequencing was available, allowing us to corroborate whether mutations we found based on analysis of the MinION data alone were indeed real.
cord-007066-zn10rnrm	2006	RNA can enter the oral cavity through various routes, including saliva secretions from the 3 major salivary glands (the parotid, submandibular, and sublingual glands) and minor glands, gingival crevice fluid (GCF), and desquamated oral epithelial cells. It is therefore possible that the RPS9 PCR products in panels B and C of Fig. 1 are produced from both RPS9 and RPS9P2 mRNAs. Previous expression-based microarray analysis showed that 185 different transcripts were consistently detected in the supernatants of 10 healthy human saliva samples (6 ) . Although individual variations exist, we detected ␤-actin mRNA in all 3 major salivary glands, GCF, and desquamated oral epithelial cells ( Fig. 2A) , suggesting that RNA enters the oral cavity from different sites. In addition to the saliva samples, we also measured RNA-macromolecule associations in serum and found that ϳ8% and Ͻ1% of ␤-actin mRNA could be detected in serum filtered through 0.45 and 0.22 m pores, respectively (see Fig. 1c in the online Data Supplement).
cord-007181-qpahuqld	1977	Sedimentation Analysis-PH]Virion RNA dissolved in 0.1 ml NET was layered on a 15-30% sucrose gradient in NET containing 0.3% SDS, then sedimented at 48,000 rpm for 90 min at room temperature in an SW 50.1 rotor. Isolation of Poly(A)-fHJAdenosine-or P''P]labeled MH virion RNA containing 50 (ig of carrier tRNA was digested with a combination of ribonuclease A (2//g) and Tl (50 units The hydrolysate was neutralized with HC1O, at 0°C and the precipitate was removed by centrifugation. We therefore extracted RNA from [*H]uridine-labeled MH virions with phenol/chloroform as well as 1 % SDS, and RNA''s obtained by both methods were compared by sucrose gradient centrifugation. To isolate poly(A), [''HJadenosine-labeled total virion RNA was digested with a combination of ribonuclease A and Tl. The digest was adjusted to 0.3 M NaCl-0.001 M EDTA-0.02 M Tris-HCl (pH 7.5)-7 M urea and applied to a DEAE-Sephadex column equilibrated with the same buffer.
cord-007208-wnkjdg6y	2005	Here we report the purification and identification of an androgen-stimulated 36-kDa glycoprotein, a minor protein component of mouse SVS that is able to enhance sperm motility in vitro. The following materials were obtained from commercial sources: DEAE-Sephacel (Amersham Pharmacia Biotech, Uppsala, Sweden); Protein PAK SP 5PW column (Waters, Milford, MA); Vydac 218TP54 C 18 column (Separations Group, Hesperia, CA); AminoLink coupling gel, bicinchoninic protein assay kit (Pierce, Rockford, IL); testosterone propionate, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), PMSF, periodic acid Schiff reagent, and silanated glass slides (Sigma Chemical Co., St Louis, MO); cDNA integrity kit, alkaline phosphataseconjugated streptavidin, and biotin-conjugated goat anti-rabbit immunoglobulin G (IgG; Kirkegaard & Perry Laboratories, Gaithersburg, MD); rhodamine-conjugated goat anti-rabbit IgG (Zymed Laboratories, San Francisco, CA); Nuclear Fast Red (Vector Laboratories, Burlingame, CA); enhanced chemiluminescent substrate and [␣32 A) Fractionation of soluble mouse SVS proteins by ion exchange chromatography on a DEAE-Sephacel column. A novel heat-labile phospholipid-binding protein, SVS VII, in mouse seminal vesicle as a sperm motility enhancer
cord-007236-8hiymqyb	2011	title: Inhibition of hepatitis C virus replication by Monascus pigment derivatives that interfere with viral RNA polymerase activity and the mevalonate biosynthesis pathway We demonstrated that a group of Monascus orange pigment (MOP) derivatives effectively inhibited NS5B RdRp activity and interfered with the mevalonate synthesis pathway, thereby suppressing HCV replication in cells harbouring an HCV genotype 1b subgenomic replicon and in cells infected with genotype 2a HCV. As shown in Figure 2 (a), among the selected group of MOP AADs that inhibited NS5B RdRp activity in vitro, Lys, Phe, Val, Leu and Ile derivatives also inhibited HCV replication in Huh7 cells, which harbour a HCV subgenomic replicon RNA. Together, these results suggest that in addition to a direct inhibition of NS5B RdRp activity, these MOP AAD compounds also inhibit HCV replication by interfering with the cholesterol biosynthetic pathway downstream of the HMG-CoA reductase step.
cord-007382-5kb16qb7	2016	With the additions of RNAi and the CRISPR/Cas system, specific nucleases including the restriction-modification (R-M) systems, and antiviral effector proteins partially discovered only recently in the context of rare hereditary inflammatory diseases, the new concept of nucleic acid immunity evolves. While innate nucleic acid immune-sensing receptors elicit antiviral signaling pathways, a number of nucleic acid-detecting effector proteins (viral restriction factors, e.g., PKR, ADAR1, IFIT1) directly detect and restrict nucleic acid function and replication. Another member of the RIG-I-like helicase family of receptors is MDA5 which was found to be responsible for the long sought after type I IFN-inducing activity of cytosolic long double-stranded RNA including poly(I:C) . Extrinsic effects inside the same cell include degradation of the nucleic acid (e.g., RNase L activated by 2 0 -5 0 -OA generated by OAS1 upon binding of long double-stranded RNA).
cord-007463-8g0zklzy	2002	The 3′ terminal labelling method for the analysis of genome profiles used in this study required only 1 ng of viral RNA, an increase of 1000-fold in sensitivity over ethidium bromide staining for detecting all rotavirus genome segments. To investigate the latter, mixtures containing known amounts of ds-RNA from two rotavirus isolates with disparate genome profiles (UK strain and a C7 type) were used to simulate the type of samples that might be recovered from calves concurrently infected with these two viruses. After 3'' terminal labelling the RNA segments were analysed by electrophoresis (Fig. 3) profiles, and meant that in a faecal sample containing two rotaviruses if one virus was present at a concentration 10-fold less than the other, i.e. 1 log10 dilution, it would not be detected. Rotaviruses isolated from 43 sub-clinically infected calves were characterised by genome profile analysis.
cord-007474-ckqghr3b	2002	Recombinant complementary DNA (cDNA) representing gene 9 (the gene encoding the neutralization antigens in VP7 glycoprotein) from OSU (porcine rotavirus serotype 1) and Gottfried (porcine rotavirus serotype 2) strains were used to determine the optimal hybridization conditions which allow specific detection of group A porcine rotaviruses. In the present study, a dot blot hybridization assay is described for the detection and serotypic differentiation of porcine rotavirus utilizing hybridization probes prepared from recombinant cDNA representing gene 9 from OSU and Gottfried strains (porcine rotavirus serotypes 1 and 2, respectively). Five subfragments of gene 9 from OSU and Gottfried (Table 1 ) were 32p-labeled and hybridized to heterologous rotavirus RNA under both high and low stringency conditions. They reported that rotavirus serotypes 2 and 3 could be differentiated with the gene 9 cDNA probe by using conditions of low stringency (56 ° C), and comparison with the results obtained with hybridization at 56°C, 13% formamide.
cord-007621-rapinodd	2002	Previous data from this laboratory had shown that the cytokine tumor necrosis factor-α (TNF-α) enhances IFN-γ-mediated class II antigen expression on astrocytes. To determine the steady-state level of mRNA for class II, Northern blot analysis was performed using a eDNA probe for murine class Ii genes (E-a), with total RNA isolated from cultured astrocytes. The duration of protein synthesis required to allow expression of the class II MHC gene in astrocytes was examined in cells that were pretreated with IFN-y or IFN-7/TNF-a for different lengths of time prior to the addition of CHX. In this study we have shown that primary neonatal rat astrocytes, upon stimulation with IFN-~,, express mRNA transcripts for class II MHC genes, and that TNF-a enhances the expression of IFN-~,-induced class II mRNA. The expression of class II mRNA was completely inhibited when CHX was included with IFN-~, and IFN-''t/TNF-~ treatment, indicating that newly synthesized protein is required for astrocyte class II MHC gene expression.
cord-007689-0vpp3xdl	2007	Since the discovery of type I IFNs in 1957, long double-stranded RNA formed during replication of many viruses was thought to be responsible for type I IFN induction, and for decades double-stranded RNA-activated protein kinase (PKR) was thought to be the receptor. It now became evident that not PKR but two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, and two cytosolic helicases, RIG-I and MDA-5, are responsible for the majority of type I IFNs induced upon recognition of viral nucleic acids. Based on the recent progress in the field, we now know that TLR7, TLR9, and RIG-I do not require long double-stranded RNA for type I IFN induction. These two cytosolic receptors are then responsible for the second and prolonged wave of type I IFN production and for the induction of apoptosis of virally infected cells. Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3
cord-007714-n3omlvfl	2008	In recent years, molecular and proteomic approaches have begun to dissect the pathways of ribosomal subunit assembly and transport from the nucleolus and examine the composition of protein complexes and RNPs involved in these processes (Grandi et al. In plants, the best-characterised nuclear bodies are the nucleolus and CBs. The plant nucleolus differs to some extent in organisation and structure from the animal nucleolus, although its major function in rRNA and ribosomal subunit production remains the same Shaw and Brown 2004) . Finally, the Arabidopsis nucleolus contains exon junction complex proteins, involved in linking transcription and splicing to translation, export and mRNA surveillance (Pendle et al. As with other large RNP complexes such as the spliceosome, correct assembly occurs in a regulated and co-ordinated step-wise pathway involving non-ribosome factors and ribosomal proteins required for correct rRNA folding, protein association and ultimately export of the ribosomal subunits through the nuclear pore complex to the cytoplasm (Venema and Tollervey 1999; Fatica and Tollervey 2002; Grannemann and Baserga 2004) .
cord-007819-51h2jrsy	2006	Use of the infectious clone also allows us to begin with a single DNA sequence, providing a well-defined starting point for studying PRRSV evolution. 8 The ORF3 protein has the greatest percentage of amino acid changes between the modified live vaccine (Ingelvac) and its parent strain, VR-2332, the isolate from which the infectious clone used in this study was derived. These sequences were then compared to a low passage VR-2332 cell culture propagated stock to determine if the use of an infectious clone was able to decrease the quasispecies variation as compared to a viral stock. The master sequence for each sample derived from the infectious clone was the same as the original plasmid for all genetic regions investigated. Generation of an infectious clone of VR-2332, a highly virulent North American-type isolate of porcine reproductive and respiratory syndrome virus
cord-007890-bie1veti	2002	Effects of Interferon alpha plus ribavirine therapy on frequencies of HCV, HIV and CMV specific CD4-T-cell responses in peripheral blood of HIV/HCV coinfected patients after 6 months of treatment SoA9.5 Methods: Two groups of patients with chronic HCV infection were studied: 26 HIV coinfected progressors with antiretroviral therapy and 13 HIV-negative controls. In order to assess the local temporal trend of antibiotic sensitivity of the most common urinary tract bacterial pathogen, all urine-cultured Escherichia coli isolates were reviewed as to susceptibility profile, and specimen source (community-versus hospital-acquired infection). Methods: A total of 87 penicillin resistant clinical strains isolated from patients at Hacettepe Children''s Hospital, Ankara, Turkey between 1999 and 2001 were tested for their in vitro susceptibility to various antibiotics that are commonly used in the treatment of respiratory tract infections.
cord-007920-mh3tesdc	2004	The three most detrimental shrimp viruses are white spot syndrome virus (WSSV), yellowhead virus (YHV), and Taura syndrome virus (TSV), all of which have caused serious epizootics in various regions of Asia and are considered notifiable by the Office International de Epizooties (OIE, 2002) . The RNA helicase consensus sequence, Gx 4 GK, is present at ORF1 amino acid positions 752 to 758, and the TSV helicase domain shows significant similarity with the cognate domain of insect picorna-like viruses (Drosophila C virus, DCV; Rhophalosiphum padi virus, RhPV; Plautia stali intestinal virus, PSIV; black queen cell virus, BQCV; Triatoma virus of the fungus Triatoma infestans, TrV; and Himetobi P virus, HiPV). In addition to helicase, protease, and RdRp motifs, the TSV genome contains a short aa sequence at the N-terminal end of ORF1 (positions 166 to 230) that shows significant similarity with the inhibition of apoptosis (IAP) proteins found in mammals, yeast, insects, and some DNA viruses (Mari et al., 2002) .
cord-008407-jbp8bxjz	1995	During serial undiluted passage of rubella virus (RUB) in Vero cells, two species of defective-interfering (DI) RNAs of approximately 7000 and 800 nucleotides (nts) in length were generated (Frey, T. Finally, to determine whether the majority of DI RNAs present in P12 RNA contained a deletion in the SP-ORF, an oligonucleotide probe which is complementary to a sequence located within the E1 protein coding region (nts 8472 to 8488, eligonucleotide 83, Fig. 3E ) that was deleted in all three cDNA clones was hybridized to P12 RNA. Analysis of the 5'' terminus of DI RNA generated during serial passage As shown in Figure 3A , oligonucleotide probes complementary to the exact 5'' terminal sequences of the RUB genome hybridized to the large DI RNAs. To confirm that these DI RNAs contained the authentic 5'' end of the genome, primer extension was performed on intraceilular RNA from standard RUB-infected cells and P12 RNA.
cord-008426-ktn8c0zx	1995	From a comparison of the predicted sequences of the ORFs of these three sobemoviruses and of the noncoding regions, it is suggested that the two SBMV strains differ from one another as much as they do from RYMV and that they should be considered as different viruses. Southern bean mosaic virus (SBMV) is the type member of the sobemovirus group of small icosahedral positive-strand RNA viruses (for reviews see Sehgal, 1981 ; Tremaine and Hamilton, 1983; Hull, 1988) . The recently published nucleotide sequence (4450 nt) of rice yellow mottle virus (RYMV), another sobemovirus (Ngon A Yassi et aL, 1994) , shows that it has a similar genome organization to SBMV-C (Fig. 1) . Amino acid sequence of southern bean mosaic virus coat protein and its relation to the three dimensional structure of the virus
cord-008541-0u2fatbg	2008	The idea was to develop an infectious RNA3 molecule stable in infection with a possibility of inserting sequences of interest and studying their recombinational activity. This indicated that progeny recombinants easily accumulated, because they easily outcompeted their parental RNAs. Figure 3 shows that all legitimate crossover events occurred within the long (197-to 220-nt) 3'' region of M4 or DM4 (homologous among three BMV RNA components) and the corresponding part of either wt RNAl or wt RNA2. The recombination activity between pairs of these mutants was determined by coinoculation rearrangements was tested by inserting long palindromic sequences into RNA3 molecules (Section IV,D). The sequence of the inserted region and the presence of a marker mutation a t the 3'' end indicated that B x 4 might have been formed through rearrangements between two mutant B RNA3 molecules.
cord-008556-oetrdm8g	2008	One consequence of the scanning mechanism is that deleting the "ribosome binding site" (i.e., the normal initiator codon and flanking sequences) will not abolish translation; ribosomes will simply use the next AUG codon downstream, which, in some cases, has been shown to direct the synthesis of a biologically active, truncated protein (Downey et al., 1984; Halpern and Smiley, 1984; Katinka and Yaniv, 1982) . The best evidence for this is the ability of both EMC and SFV 26 S mRNA to be translated in EMC virus-infected cells, in which host translation is drastically inhibited by a mechanism that has not been difined, but that clearly does not involve cap binding protein (Mosenkis et al., 1985) . In wild-type adenovirus-infected cells, in which host protein synthesis is drastically reduced, both adenovirus and influenza virus mRNAs are translated efficiently.
cord-008575-bbpmlo3c	2004	Although no reconstitution system is yet available for any of the reoviruses, studies conducted using baculovirusexpressed recombinant rotavirus-like particles containing the viral RNA polymerase coexpressed with the inner capsid protein have begun to clarify the mechanism by which the dsRNA genome is synthesized from the mRNA templates in rotavirus Patton et al., 1996; Zeng et al., 1996) . One of the more interesting observations about the life cycle of dsRNA viruses is that genome transcription occurs only within structurally intact TCPs. In rotavirus, the transcriptionally competent form of the virus has a double-layered capsid consisting of the structural proteins VP2 and VP6 surrounding the dsRNA genome segments and the enzymatic machinery in the core. Although observation of the actively transcribing rotavirus particle by electron cryomicroscopy has not provided a clear definition of the pathway of mRNA translocation through the inner capsid layer, atomic resolution structural studies of the bluetongue virus TCP have suggested a possible route (Grimes et al., 1998) .
cord-008588-4eu9v5d3	2008	The structures of the tRNA anticodon loop and the UUCG loop suggest that the specific loop sequences adopt conformations that are more stable because they contain more hydrogen bonding and stacking interactions-particularly interactions with the sugar-phosphate backbone. Determining the conformation of these loops in RNA is important for understanding how these nucleotides interact with other elements of secondary structure to form tertiary interactions and for understanding how proteins bind to bulge loops. In general, this could occur between any of the secondary structure regions containing unpaired nucleotides (single-stranded regions, hairpin loops, bulge loops, internal loops, and junction loops). There are several examples of RNA molecules containing tertiary contacts between nucleotides that are in loop regions of secondary structure. The high frequency with which known RNA structures contain tertiary pairing between nucleotides that are unpaired in the secondary structure stresses the importance of learning more about such interactions.
cord-008613-tysyq6o4	1988	Simian virus 5 (SV5), a prototype paramyxovirus, has a single-stranded, negative sense genomic RNA (vRNA) approximately 15,000 nucleotides in chain length that is transcribed in infected cells by the virion-associated RNA transcriptase to yield virus-specific mRNAs. The SV5 "P" gene has been shown to encode both the P protein (Mr = 44,000), and protein V (Mr = 24,000) by the arrest of translation in vitro of both P and V using a cDNA clone derived from SV5-specific mRNAs (Paterson et al., 1984) . Antigenic Region Mapping of the P and V Monoclonal Antibodies on the P and V Gene Using T7 RNA Polymerase Runoff Transcripts, In Vitro Translation, and Immunoprecipitation The full-length P203-1 DNA insert cloned using Xbal linkers (XX) and a Hindlll to Xbal fragment (HX) containing the 3'' two-thirds of the P203-1 DNA were placed under the control of the T7 promoter in the plasmid pGEM-2.
cord-009376-a35a92gh	2002	Applications of Q-PCR within biotechnology are discussed with particular emphasis on the following areas of biosafety and genetic stability testing: (a) determination of the biodistribution of gene therapy vectors in animals; (b) quantification of the residual DNA in final product therapeutics; (c) detection of viral and bacterial nucleic acid in contaminated cell banks and final products; (d) quantification of the level of virus removal in process validation viral clearance studies; (e) specific detection of retroviral RT activity in vaccines with high sensitivity; and (f) transgene copy number determination for monitoring genetic stability during production. We are developing Q-PCR assays for these repeat regions for rodent and primate residual DNA to obtain sensitivity in the fg range, however, one advantage of using a gene such as ␤-actin is gene stability, as this target should not undergo copy number changes within a stable cell line.
cord-009662-ntjngiem	2007	When delivering Morpholinos to cell cultures using Endo-Porter, starting with a 10 µM Morpholino concentration for both fluorescent delivery assays and functional experiments increases the chances that the fluorescence will be visible in the cytosol and that the first functional experiment will produce measurable results. However, assaying only for a phenotypic effect becomes problematic if the expected change in phenotype does not occur; if antisense activity is not separately assessed at the level of protein concentration or mRNA mass, the experimenter will not be able to discern whether (1) the oligo failed to reach and interact with its target mRNA to produce the knockdown or splice-block, or (2) the knockdown or spliceblock was successful but did not cause the expected phenotypic change. The negative control shows that the effects observed during the antisense experiment are due to the sequence of the targeting oligo and not to the backbone chemistry of the Morpholino or the cytosolic delivery method used.
cord-009943-fzynh14x	2005	Further, evidence is provided in this study that indicates that it would be feasible to use cDNA probes to detect human rhinoviruses in nasal washings. In a preliminary study we found that this probe does indeed cross-hybridize with a number of human rhinoviruses indicating the feasibility of cDNA:RNA hybridization for rhinovirus detection [Al-Nakib et al, 19861 . We have now extended these studies to include a larger series of human rhinoviruses (totalling some 56 viruses) and looked in more detail at the molecular relationship between these viruses, the limits of detection, and the feasibility of applying these procedures for the direct detection of viral RNA in nasal washings. However, in addition to detecting virus at lower titres, the strength of the hybridization signals obtained with 20 x SSC-37 % formaldehyde were particularly strong at all virus dilutions in nasal washings compared with phenol/chloroform extraction, indicating that more viral RNA has been immobilised onto the nitrocellulose filters.
cord-010045-eqzs01au	2006	Yeast cells containing recombinant plasmids, with the nucleoprotein gene in the correct orientation, produced a polypeptide of M, 47000, identical to the viral product, that reacted with a specific monoclonal antibody. Three recombinant plasmids, shown to contain cDNA inserts of 1.5 kbp and designated pTS13-2, pTS15-1 and pTSi 5-2, were labelled with p^S]-dATP by nick translation and hybridized to glyoxylated TGEV mRNA species separated on 1 % agarose gels as described in Experimentat procedures. The plasmid also contained the smaller 3'' ORF which initiates, in a different reading frame, 6 bp 3'' from the end of the nucleoprotein gene and terminates 166 bp 5'' to the Drall Sma\ combined site between the TGEV cDNA insert and the pUC9 DNA. A recombinant plasmid, pYNGI, was found to contain the TGEV nucleoprotein gene in the correct orientation for expression under the control of the yeast GAL1 promoter.
cord-010092-uftc8inx	2019	Prospective testing of blood donations in endemic areas of the U.S. revealed 0.38% of donors were positive for Babesia DNA or antibodies (Moritz, NEJM, 2016) Aims: -To report results of ongoing Babesia clinical trial -To explain significance of Babesia as a TT infection Methods: In cobas â Babesia for use on the cobas â 6800/8800 Systems, is a qualitative polymerase chain reaction nucleic acid amplification test, developed to detect in whole blood (WB) donor samples the 4 Babesia species that cause human disease: B. In sensitivity analyses, there were two discrepant results for HIV testing, three for HCV, and five for anti-HBc. Summary/Conclusions: Elecsys â infectious disease parameters on the cobas e 801 analyser demonstrate high specificity/sensitivity for screening first-time blood donor samples, with similar clinical performance to other commercially available assays.
cord-010120-mqvm9zn4	2004	Ribosomal frameshifting is different from frameshift suppression in that these events are directed by specific mRNA sequences and structures, rather than being a consequence of mutations in host gene products, e.g. tRNAs containing four base anticodon loops. In eukaryotes, frameshifting in the +1, or 3'' direction has been observed in the Ty retrotransposable elements in yeast (for review, see reference 25), and in the ornithine decarboxylase antizyme gene in mammalian ~e l l s .~~,~~ In Tyl and Ty3, +1 they splice their RNA.4 I: s6 All of these classes ribosomal frameshifting between the T Y A and T Y B genes in Tyl and the GAG3 and POL3 genes in Ty3 also results in the production of Gag-Pol fusion proteins. A longer ribosomal pause over the slippery site would follow, yielding increased efficiencies of -1 ribosomal frame~hifting.~.''~ A weak point of this model is that, in some mRNA pseudoknots, stem 1 is only five or six base pairs in slippery site, eliminating frameshifting.
cord-010161-bcuec2fz	2004	With the description of statistically significant phylogenetic clades within CV genera, data were available to recognize strains that might be natural recombinants within CVs. Two examples are the well-characterized Argentine strain 320 (Arg320) and Snow Mountain virus (SMV), one of the prototype CVs, recognized to be recombinants when the RNA polymerase and capsid regions of these strains were characterized (Hardy et al., 1997; Jiang et al., 1999) (Fig. 2) . While SMV was likely also to be a recombinant virus, the capsid and RNA polymerase region amplicons of SMV were generated separately and that fact did not exclude the possibility of different sources of strains. Infection of single cells simultaneously by two CVs implies absence of immune or molecular and of 40 nt near the 5'' end of that strain''s capsid gene (ID="B" sequence for this Fig.) . The sequence data indicated that recombination in strain Arg320 occurred at the ORF1/capsid gene junction where high sequence identity exists between the putative parent clades.
cord-010188-884d196k	2004	Sindbis virus and Semliki Forest vires are best known as valuable models for molecular and cell biology, and it is these two viruses that are presently being developed as vectors for the expression of heterologous genes. The basic strategy for using alphaviruses as vectors for the expression of heterologous genes has been to construct cDNAs of the alphavirus genome, in which the heterologous gene is placed downstream from the promoter used to transcribe a subgenomic RNA 13 (Fig. 2a) . A second potential problem is recombination between the packaging helper virus RNA and vector RNAs. The two Sindbis RNAs can undergo recombination to produce a single molecule of RNA containing the genes that encode both the nonstructural and structural proteins m. The initial studies with Sindbis and Semliki Forest virus suggest that both viruses are promising as vectors for heterologous gene expression.
cord-010273-0c56x9f5	2001	1,2 The identification of HCV led to the development of diagnostic assays for infection, based either on detection of antibody to recombinant polypeptides expressed from cloned HCV sequences or direct detection of virus ribonucleic acid (RNA) sequences by polymerase chain reaction (PCR) using primers complimentary to the HCV genome. 6 ''13 Remarkably, a series of plant viruses that are structurally distinct from each of the mammalian virus groups, and with different genome organizations, have RNA-dependent RNA polymerase amino acid sequences that are perhaps more similar to those of HCV than are the flaviviruses. In contrast to the highly restricted sequence diversity of the 5''NCR and adjacent core region, the two putative envelope genes are highly divergent between different variants of HCV (Table III) 111-114 and show a three-to-four-times higher rate of sequence change with time in persistently infected patients, ll5 Because these proteins are likely to lie on the outside of the virus, they would be the principal targets of the humoral immune response to HCV elicited on infection.
cord-010374-z9ygudv6	2008	The main characteristics of the member viruses are: (i) Morphological: Enveloped pleomorphic particles typically 100 nm in diameter (range 60-220 nm), bearing about 20 nm long club-shaped surface projections, (ii) Structural: A single-stranded infectious molecule of genomic RNA of about (5-7) × 10(6) molecular weight. (iv) Antigenic: 3 major antigens, each corresponding to one class of virion protein, (v) Biological: Predominantly restricted to infection of natural vertebrate hosts by horizontal transmission via the fecal/oral route. Recently, there have been reports of virus-specific RNA poly merases in coronavirus-infected cells, but the components of the enzyme have not been iden tified. UV inactivation studies in dicate that coronavirus mRNAs are not pro duced by the processing of a larger RNA, although extensive sequence homologies have been detected at the 5'' ends of ail murine hepatitis virus-specific subgenomic RNAs. For murine hepatitis virus, the mRNA function of each of the subgenomic viral RNAs has been demonstrated in vitro, and the mRNAs encod ing each of the virion proteins, or its precur sors, have been identified ( fig.
cord-010680-lc1onm53	2020	Some of these hurdles may be overcome through transient in vivo gene delivery platforms, such as non-viral synthetic plasmid DNA and messenger RNA vectors that are engineered to encode optimized mAb genes. In this review, we focus on nucleic acid delivery of antibody employing synthetic plasmid DNA vector platforms, and RNA delivery, these being important approaches that are advancing simple, rapid, in vivo expression and having an impact in animal models of infectious diseases and cancer, among others. The original studies surrounding in vivo antibody gene delivery focused primarily on gene delivery using recombinant viral vectors such as AAV and adenovirus (Ad), which were advanced clinically, building on work in the traditional gene therapy-based field. Additional studies to help evaluate fully human pDNA-mAbs in mouse models would be highly informative for both non-viral and viral delivery and provide an important path forward for preclinical evaluation of in vivo-delivered antibodies [109] .
cord-010762-c01wgg4v	2020	Previously reported NS5 structures represented by those from the Japanese encephalitis virus (JEV) and Dengue virus serotype 3 (DENV3) exhibit two apparently different global conformations, defining two sets of intra-molecular MTase-RdRP interactions. Data from in vitro polymerase assays further demonstrate that perturbing the JEV-mode but not the DENV3-mode intra-molecular interactions inhibits catalysis only at initiation, while the cell-based virological analysis suggests that both modes of interactions are important for virus proliferation. NS5 constructs bearing mutations specifically probing two modes of MTase-RdRP intra-molecular interfaces were tested in in vitro polymerase assays, and only the JEV-mode interface related mutants inhibited polymerase initiation primarily through a three-fold reduction in the Michaelis constant of the initiating NTP (K M, NTP ), while polymerase EC properties were not much affected by mutations probing both modes of interactions.
cord-010977-fwz7chzf	2020	In this review, we describe some of the approaches being taken to apply translational genomics to the study of diseases commonly encountered in the neurocritical care setting, including hemorrhagic and ischemic stroke, traumatic brain injury, subarachnoid hemorrhage, and status epilepticus, utilizing both forward and reverse genomic translational techniques. Termed "reverse translation," this approach starts with humans as the model system, utilizing genomic associations to derive new information about biological mechanisms that can be in turn studied further in vitro and in animal models for target refinement (Fig. 1) . These results highlight the value of reverse genomic translation in first identifying human-relevant genetic risk factors for disease, and using model systems to understand the pathways impacted by their introduction to select rationally-informed modalities for potential treatment. These observations provide vital information about cellular mechanisms impacted by human disease-associated genetic risk factors without requiring the expense and time investment of creating, validating, and studying animal models.
cord-011794-ejoufvvj	2020	Additionally, glycoprotein precursor (GPC)-derived virus-like particles of a German PUUV sequence allowed the generation of monoclonal antibodies that allowed the reliable detection of the isolated PUUV strain in the immunofluorescence assay. Finally, the reactivity of the isolates was determined with novel monoclonal antibodies raised against PUUV GPC VLPs. Bank voles were trapped in spring 2019 in the PUUV endemic region around Osnabrück following a standard snap trapping protocol [25, 26] . Dissection on site and inoculation of VeroE6 and bank vole MGN-2-R cells with homogenized lung samples resulted after three blind passages in four potential isolates that were detected by a novel PUUV RT-qPCR (Table S1 , Fig. 1) . Reactivity of novel PUUV GPC-specific monoclonal antibodies with hantavirus-infected VeroE6 cells in immunofluorescence assay (IFA). In conclusion, the PUUV isolate described here replicates in a bank vole cell line and its N and GPC proteins can be detected by specific monoclonal antibodies.
cord-011803-9122f1zc	2020	Collectively, our study describes an intricate relationship between cellular targets of an RNA quality control pathway and KSHV lytic gene expression, and demonstrates that NMD can function as a cell intrinsic restriction mechanism acting upon DNA viruses. EJCs are deposited at the exon-exon junction, thus we determined whether the EJC component eukaryotic translation initiation factor 4A3 (eIF4A3) is associated with spliced KSHV RNAs. To test EJC association with KSHV transcripts we performed eIF4A3 formaldehyde crosslinking RNA immunoprecipitation (fRIP) coupled to reverse transcription quantitative PCR (RT-qPCR) on lytic iSLK.219 and TREx-BCBL-RTA cells (Fig. 1g, h) . Leveraging p-UPF1 fRIP-seq we identified NMD targets transcriptome-wide in both latent and lytic PEL cells and targets include both host and viral RNAs. Remarkably, the mRNA encoding RTA, the master transcription factor governing KSHV reactivation, is targeted by NMD via its 3′UTR and silencing of UPF1 is sufficient to potentiate RTA-mediated transactivation.
cord-012032-zolowuhj	2020	To further examine the antiviral effects, another murine macrophage cell line, J774A.1, was used, and a similar inhibitory effect of 2''-FdC on MNV-1 replication was observed, with decreased viral RNA and NS1/2 protein expression ( Supplementary Fig. 1 ). As shown in Fig. 3D and E, the viral NS1/2 protein expression and viral titers were further decreased when the antivirals were used in combination, supporting the synergistic antiviral effects of 2''-FdC with MPA, ribavirin, or T705 against MNV replication. The combined effect of 2''-FdC and ribavirin on viral replication was analyzed using a qRT-PCR assay (n = 2-4) and mathematical modeling using MacSynergy. RAW264.7 cells were infected with MNV-1 at an MOI of 1 for 1 h and then left untreated or treated with 2''-FdC and MPA, ribavirin, or T705 at the indicated concentrations for 20 h, alone or in combination.
cord-012420-llh22iq2	2020	Interestingly, non-pathogenic OW Mopeia virus (MOPV), a close genetic relative to LASV, induces a strong initial IFN and cytokine response in infected moDCs and macrophages leading to a sustained T cell activation and immune response [67, 68] . Similar to NP, interaction of arenavirus Z proteins with RIG-I prevents further binding to MAVS and therefore inhibits the production of IFN-β and reduces antiviral host immune responses. Although the inhibition of RIG-I signalling by highly pathogenic arenaviruses in vitro suggests that there should be limited IFN1 response, it is notable that in vivo infection with some of these viruses (LCMV, JUNV) induces high levels of IFN1, ISGs and cytokines suggesting that this strategy is not completely effective or is widely varied in cell type and differs from host to host [134, 135, 186] .
cord-012484-c9ajmbw2	2020	The blood transcriptome of the children (n = 63) was investigated at time of FN diagnosed as viral, bacterial, co-infection or unknown etiology, respectively, and compared to control samples derived from 12 of the patients following the FN episode. In the present study of children during cancer treatment, the blood transcriptome was not suitable for determining the etiology of FN because of too few circulating immune cells for reliable gene expression analysis. To determine whether it is possible to detect pathogen-specific immune responses on the basis of gene expression in immunosuppressed children, we compared the blood transcriptomes of samples with viral infection, bacterial infection, co-infection, or unknown etiology with those of the control samples. To determine whether it is possible to detect pathogen-specific immune responses on the basis of gene expression in immunosuppressed children, we compared the blood transcriptomes of samples with viral infection, bacterial infection, co-infection, or unknown etiology with those of the control samples.
cord-012552-porty653	2020	Single-cell RNA-sequencing (scRNA-seq) analysis demonstrated considerable heterogeneity of olfactory sensory neuron (OSN) cell populations in wild-type (WT) mice, and revealed that UPF3B loss influences specific subsets of these cell populations. RNA-seq and Ribotag analyses identified classes of mRNAs expressed and translated at different levels in WT and Upf3b-null mOSNs. Integrating multiple computational approaches, UPF3B-dependent NMD target transcripts that are candidates to mediate the functions of NMD in mOSNs were identified in vivo. We identified UPF3B-regulated genes in mOSNs by performing RNA-seq analysis on FACSpurified mOSNs (YFP+ cells) from R26-eYFP; Omp-Cre mice (Figure 1-figure supplement 1B) . Among the antimicrobial genes expressed and upregulated in Upf3b-null OSN precursors and OSNs was Camp (also known as ''Cramp''), which encodes a member of the cathelicidin family of antimicrobial peptides that has an important role in the defense against microbial infections, and functions in cell chemotaxis, immune mediator induction, and inflammatory response regulation (Zhang and Gallo, 2016) .
cord-012784-c74jr4ga	2020	We previously reported that nagilactone E (NLE), a dinorditerpenoid isolated from Podocarpus nagi, possessed anticancer effects against lung cancer cells in vitro. To better understand the potential biological processes associated with the effects of NLE in lung cancer A549 cells, GO analysis was performed using the online DAVID 6.8 bioinformatics resource. To clarify the mechanisms underlying the anticancer effect of NLE in A549 lung cancer cells, we further analyzed the DEGs using the CMap dataset. As shown in Fig. 4a , the mRNA levels of NRF2, p21, STAT3, and ATF4 were upregulated after NLE treatment, which was consistent with the results obtained by RNA-seq analysis. Thereafter, the inhibitory effect of NLE on de novo protein synthesis in A549 cells was further confirmed using the Click-iT assay. CMap dataset analysis supported NLE as a protein synthesis inhibitor, which was further confirmed by the Click-iT assay.
cord-012909-o6t2srim	2020	Both virulent and live-attenuated porcine reproductive and respiratory syndrome virus (PRRSV) strains can establish persistent infection in lymphoid tissues of pigs. In this study, expression of several important chemokine ligands (CCL19, CCL21, CCL24, CCL22, CX3CL1, and CCL14) and chemokine receptors (CCR6 and CCR10) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (APCs) to the lymphoid tissues was down regulated in ILN of CON90-infected pigs (Figure 6a ). In this study, expression of several important chemokine ligands (CCL19, CCL21, CCL24, CCL22, CX3CL1, and CCL14) and chemokine receptors (CCR6 and CCR10) which play an essential role in migration and localization of lymphocytes and antigen-presenting cells (APCs) to the lymphoid tissues was down regulated in ILN of CON90-infected pigs (Figure 6a ). GO terms and KEGG analysis revealed that genes involved in the innate immune (complement and TLR) pathways, apoptosis, cytokine-chemokine signaling, T-cell exhaustion, and humoral responses are highly differentially expressed in PRRSV-infected animals compared to control.
cord-013171-wgn529rc	2020	title: STAU1 selectively regulates the expression of inflammatory and immune response genes and alternative splicing of the nerve growth factor receptor signaling pathway They extended this analysis to multiple cell types of diverse origin, including human embryonic STAU1 selectively regulates the expression of inflammatory and immune response genes and alternative splicing of the nerve growth factor receptor signaling pathway carcinoma stem cells (NT2), mouse neural progenitor cells (N2A), human retinal epithelial cells (ARPE19), and primary mouse embryonic fibroblasts (MEFs). RASEs detected by qPCR were consistent with those by RNA-seq, which demonstrated that STAU1 may play a significant regulatory role in the AS of ''nerve growth factor receptor signaling pathway''. In addition, the AS of multiple genes was also regulated by STAU1, and the main enriched pathways not only include ''retrograde transport'' and ''muscle cell differentiation'', but also the ''nerve growth factor receptor signaling pathway''.
cord-013176-6ckuya1w	2020	Quercetin, extracted from Embelia ribes (Mirsinaceae), exhibited antiviral effects against HCV, exerted through activity inhibition of the viral protease Non-Structural protein 3 (NS3), leading to a decrease in HCV replication [36] . The natural extract of Tetrastigma hemsleyanum (Vitaceae) contains many flavonoids, including vitexin, vitexin-2-O-rhamnoside, isorhamnetin, rutin, kaempferol, astragalin, quercitrin, quercetin and iso-quercetin, which were shown to be able to exert anti-influenza virus activity, with different efficiency, through the reduction of the number of plaques induced by the influenza virus in infected Madin-Darby Canine Kidney (MDCK) cells [21] . In future perspective, this approach could be considered in order to possibly improve the antiviral activity of some flavonoids, like baicalin, that was able, like fludarabine [65] , to act against HIV-1 chronic infection of human monocytes and macrophages, inhibiting the fusion of HIV virus envelope proteins with these cells [73] .
cord-013177-whd0znan	2020	Novel posa-like viral genomes were first identified in swine fecal samples using metagenomics and were designated as unclassified viruses in the order Picornavirales. These posa-like viruses have undergone a complex evolutionary process, and have a wide geographic distribution, complex host spectrum, deep phylogenetic divergence, and diverse genomic organizations. Novel posa-like virus genomes were first identified in swine fecal samples using metagenomics and were assigned as unclassified viruses to the order Picornavirales [12] . Further reports of novel posaviruses with low amino acid sequence identity revealed novel genomic organization features and phylogenetic characteristics of posa-like viruses [15, 16] . Due to the low genomic sequence similarity between the husavirus and unclassified posa-like viruses in Picornavirales, it was difficult to perform a phylogenetic analysis at the full-length genome level. Posa-like viruses identified in previous reports and in the present study formed a single group and clustered with the genomes of family Marnaviridae (Figure 4) .
cord-013243-1hj5clsw	2020	title: Editorial for "Methods to characterize virus small RNAs and RNA structures" One of the advantages of this enzymatic approach is that it minimizes problems that can arise upon annealing two complementary RNA strands, e.g., secondary structure within a ssRNA due to selfannealing; and low yields of long dsRNAs. These RNAs can be subsequently modified (e.g., Here, the small molecule dimethyl sulfate (DMS) preferentially methylates unpaired or dynamic adenosine and cytosine residues within a viral RNA genome. Methods for detection and study of virus derived small RNAs produced from the intramolecular base-pairing region of the picornavirus genome A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells Functional analyses of mammalian virus 5''UTR-derived, small RNAs that regulate virus translation From current knowledge to best practice: A primer on Viral diagnostics using deep sequencing of virus-derived small interfering RNAs (vsiRNAs) in infected plants
cord-013280-kczj24se	2020	Viral capsid protein VP1 and leading protein L pro can inhibit the production of interferon (IFN) and innate immune response by interacting with soluble resistance-related calcium-binding protein (sorcin) or host transcription factor ADNP [12, 13] . Viral capsid protein VP1 and leading protein L pro can inhibit the production of interferon (IFN) and innate immune response by interacting with soluble resistance-related calcium-binding protein (sorcin) or host transcription factor ADNP [12, 13] . FMDV VP1 interacts with host ribosomal protein SA (RPSA) to continually activate the MAPK signal pathway and promote virus replication by inhibiting the RPSA-mediated function [59] (Figure 2 , Table 1 ). It interacts with the VISA protein to inhibit the formation of VISA-regulated complex, thereby inhibiting the dimerization and phosphorylation of IRF3, inhibiting the expression of antiviral genes induced by IFN-β, and promoting FMDV replication [60] (Figure 2 , Table 1 ).
cord-013412-gj443yei	2020	The purpose of this review paper is to summarize the main approaches developed to date in the chemical and photodynamic inactivation of human and animal viruses using porphyrins and their analogues and to analyze and discuss the information on viral targets and antiviral activity of porphyrins, chlorins, of their conjugates with organic/inorganic compounds obtained in the last 10–15 years in order to identify the most promising areas. A cationic substituent is necessary to increase the solubility of porphyrins and their analogues in aqueous media, and three nitro groups provide linking of the gp120 protein with the glycoprotein ( Figure 3 ) and thereby inhibit fusion between the virus and the cell membrane. A cationic substituent is necessary to increase the solubility of porphyrins and their analogues in aqueous media, and three nitro groups provide linking of the gp120 protein with the glycoprotein ( Figure 3 ) and thereby inhibit fusion between the virus and the cell membrane.
cord-013784-zhgjmt2j	2020	To move beyond serum-free sphere culture-based models, we utilized a DLP-based rapid 3D bioprinting system to generate 3D tri-culture or tetra-culture glioblastoma tissue models, with a background "normal brain" made up of NPCs and astrocytes and a tumor mass generated by GSCs, with or without macrophage, using brain-specific extracellular matrix (ECM) materials (Fig. 1a ). 35 While patient-derived glioblastoma cells grown under serum-free conditions enrich for stem-like tumor cells (GSCs) that form spheres and more closely replicate transcriptional profiles and invasive potential than standard culture conditions, we previously demonstrated that spheres display differential transcriptional profiles and cellular dependencies in an RNA interference screen compared to in vivo xenografts. [49] [50] [51] g Therapeutic efficacy prediction of drugs in all cancer cells in the CTRP dataset based on differentially expressed genes between the 3D tetra-culture model and GSCs grown in sphere culture as defined by RNA-seq.
cord-013854-wadpugbj	2020	Since whole-exon deletions or duplications are the predominant type of pathogenic variant in the DMD gene (~78%; Table 1 ), an initial screen which detects the majority of these copy number variations (CNVs) should be the first diagnostic test offered (refer to Genetic testing strategy section and Fig. 1 ). In patients with an ascertained clinical diagnosis of dystrophinopathy but no CNVs or small variants identified, RNA-based methods offer a valuable tool with a high likelihood of being able to detect variants that escape detection using level 1 and 2 DNA approaches, such as complex rearrangements or deep intronic variants leading to pseudo-exon insertion or cryptic splice site recognition in the mature transcripts. If a pathogenic DMD variant is not identified by analysis for whole-exon deletions and duplications or after DMD gene sequencing, then in some cases alternative diagnoses should be considered, depending on the available clinical evidence and test results.
cord-014397-7b88ycv8	1996	Thus introduction of new mechanisms of disease resistance in livestock by gene transfer may be viewed as a logical continuation of the creative influence of humans on the evolution of farm animals and birds that could benefit mankind by improvements in food safety and production efficiency. As background for the discussion of the subject, the article deals briefly with coevolution of hosts and parasites and principal elements of virus-host interactions, and reviews past improvement of disease resistance in plants and livestock by conventional breeding and genetic engineering, as well as the potential ''biological cost'' of genetic manipulation. Basic understanding of the parallel evolution of viruses and their hosts provides a useful starting point for the consideration of strategies for genetic engineering of new mechanisms of resistance. Genetic engineering strategies that prevent entry of viruses into host cells would be effective against all three types of viral infection.
cord-014462-11ggaqf1	2011	Molecular diagnosis based on reverse transcription (RT)-PCR s.a. one step or nested PCR, nucleic acid sequence based amplification (NASBA), or real time RT-PCR, has gradually replaced the virus isolation method as the new standard for the detection of dengue virus in acute phase serum samples. Non-genetic methods of management of these diseases include quarantine measures, eradication of infected plants and weed hosts, crop rotation, use of certified virus-free seed or planting stock and use of pesticides to control insect vector populations implicated in transmission of viruses. The results of this study indicate that NS1 antigen based ELISA test can be an useful tool to detect the dengue virus infection in patients during the early acute phase of disease since appearance of IgM antibodies usually occur after fifth day of the infection. The studies showed high level of expression in case of constructed vector as compared to infected virus for the specific protein.
cord-014685-ihh30q6f	2005	This study has attempted to analyse the structural properties of membrane peptides and proteins through the use of model systems that have been designed to mimic their natural counterparts: Podlubnaya 2 1 Institute of Theoretical and Experimental Biophysics RAS, 2 Pushchino State University Amyloid brils are formed by proteins or their peptides in the result of a conformational transition from alpha helix into beta-sheet structure. Analysis of the results of such studies indicate that folding of SNase fragments is dominated by developing the local and non-local nucleation sites from native-like secondary structures and by intensifying the longrange interactions of residues at nucleation sites with residues further removed in sequence. The results show that at different pH values the aggregation processes of both proteins follow different pathways determined by the variations in the native structure and by the details of the involved conformational changes.
cord-014908-jys1y0k9	2010	Unluckily, the PCR-based tools do not persistently amplify nucleic acids if viruses are found in infected food or environmental samples at critically low level. Another successful innovative biosensor with combined microfluidics and biosensing capabilities, furnish real time and automated affinity bioanalysis (e.g. for antigen-antibody assays) through surface plasmon resonance (SPR)-based original optical transduction mechanism. Since molecular recognition trait is central in the biosensing systems, all the structural components can be targeted to device a biosensor for detection of the specific virion particles present in food and environment samples. Molecular nanotechnology-based new nanostructures/nanomaterials such as aptamers are capable for developing highly specific biosensor for target elements detection. DNA-based biosensors have great applications in food and environmental analysis including determination of the pathogenic bacteria , and virus DNA sequence such as that of SARS virus (Abad-Valle et al. Quartz crystal microbalance (QCM)-based piezoelectric sensors can detect the hybridized viral DNA and also the capsid protein-ligand interactions.
cord-015237-8cxfa8wf	2005	The solution structures of the four RNA-binding domains (RBDs) of polypyrimidine-tract-binding protein-1 (PTB1) in complex with RNA have now been solved by Oberstrass et al., leading to new models for the function of PTB1 as a repressor of alternative splicing. used NMR to look at the structure of RBD1-4 in complex with a 5′-CUCUCU-3′ oligonucleotide, which is a common feature of intronic regulatory sequences. Each RBD binds independently to one RNA molecule and recognizes a different consensus sequence within the oligonucleotide. The nucleotides interact with the flat β-sheet surface of each RBD but, unlike other RBD-RNA structures, the third β-strand is only weakly involved in RNA binding. A single PTB1 molecule can therefore bring two distant pyrimidine tracts into close proximity and induce RNA looping -a feature that has led to the proposal of various models for the function of this protein in alternative splicing. Structure of PTB bound to RNA: specific binding and implications for splicing regulation
cord-015376-z739ifu5	2006	These include: viral entry (inhibited by chloroquine and peptides); viral RNA (targeted by antisense approaches/RNAi); the main protease 3CLpro (inhibited by peptidic molecules such as HIV-1 protease inhibitors and miscellaneous compounds); the accessory protease(s) PLpro(s) (inhibited by zinc ions); RNA-dependent RNA polymerase (inhibited by aurintricarboxylic acid and antisense approaches); and helicase (inhibited by bananins). Chloroquine and HIV-1 protease inhibitors (with well-known toxicity profiles) should be considered for clinical tests if severe acute respiratory syndrome (SARS) re-emerges; however, there are other attractive compounds. The potential usefulness of 3CLpro as a drug target is supported by: i) its fundamental role in coronavirus replication; ii) its well defined 3D structure; and iii) preliminary clinical observation indicating that drugs cross-targeting this enzyme, that is, the HIV-1 protease inhibitors (HIV-1 PIs; 2 -6) produced some clinical benefits in patients treated with IFNs and ribavirin.
cord-015394-uj7fe5y6	2008	Studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein-1 (AP-1) transcription factor, cFos compared to theca cells, where cFos expression is virtually absent. Following acute hypoxia (0.5% O2) for one to six hours, RhoA mRNA, total protein and activation (RhoA-GTP) levels were analysed, using semi-quantitative PCRs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. Since there is an urgent need for non-invasive methods for determination of fetal (F) and placental (P) function, this study was designed to evaluate the genes differently and commonly expressed in P tissue and leukocytes in maternal (M) and F circulation.Material and Methods. The current study: 1) localized IL-6 mRNA levels in preeclamptic versus normal decidual sections; 2) evaluated mechanisms regulating IL-6 synthesis by targeting intracellular signaling pathways with specific inhibitors; 3) identified potential IL-6 targets by immunolocalizing the IL-6 receptor (IL-6R) to specific cell types in placental bed biopsies.
cord-015527-ph576eji	2019	Although we performed mappings, read countings, and normalization for all samples, bat genome assemblies and all six data sets ( Table 2 ; overall 1568 mappings), we only selected one comparison per data set to exemplarily show novel and significantly differential expressed ncRNAs (Supplementary Files S2.1-S2.15; divided by data set and input annotation). To give a better estimation of transcribed and potentially functional ncRNAs, we used six Illumina short-read RNA-Seq data sets derived from four bat species (Table 2) to estimate the expression levels of our novel annotations. To this end, we used the RNA-Seq data sets Field-2015 , Field-2018 , Hölzer-2019 and Weber-2019 (Table 2 ) as a basis to identify DE ncRNAs that were newly discovered in this study and were not part of the current NCBI or Ensembl genome annotations for this bat species.
cord-015606-h9bbvpzd	2006	When RNA extracted from IBV-infected and uninfected chick embryo kidney cells was added to the reticulocyte system there was a stimulation of methionine incorporation (Table 1 ) and a whole spectrum of polypeptides could be found in the product (Fig. la) . It has a molecular weight of 55 000, co-migrates with one of the bands formed by translation of the virion RNA and is also present in the IBV capsid. When the products formed in the wheat germ synthesis by IBV infected cellular RNA were compared with those formed by uninfected cellular RNA, two new bands could be identified, with molecular weights Lomniczi [4] have demonstrated that the RNA is infectious and that the virion does not contain any transcriptase activity (Lomniczi, unpublished results) . In the reticulocyte system one of the products formed by the virion RNA appears to have the same molecular weight as one of the virion proteins.
cord-015642-p46abodr	2013	On a more technical level, the problem to compute the partition function over RNA secondary structures with given end-to-end distance d, usually measured as the number of external bases (plus possibly the number of structural domains) arises for instance when predicting nucleic acid secondary structure in the presence of single-stranded binding proteins [9] or in models of RNA subjected to pulling forces (e.g. in atom force microscopy or export through a small pore) [10, 23, 11] . can be split as follows, This gives the recursion [10] , the investigate of loop entropy dependence [7] , the analysis of FRET signals in the presence of single-stranded binding proteins [9] , as well as in mathematical studies of RNA panhandle-like structures [2, 13] . As the graph-distance for a pair of nucleotides in a given secondary structure can be computed in O(n log n) time, even large samples can be evaluated efficiently 2 . The equilibrium partition function and base pair binding probabilities for RNA secondary structure
cord-015673-rz74sh32	2006	RNA interference (RNAi) is a technology developed after the recent discovery of well-conserved cellular processes that induce posttranscriptional gene silencing triggered by small fragments of double-stranded RNA. An ancient process for defense against viral infections and transposons, and in higher developed organisms an endogenous process that regulates gene expression, triggered by double-stranded RNA (dsRNA) was recently revealed (for reviews see Carrington and Ambros, 2003; Hammond et al., 2001; Sharp, 2001) . Furthermore, longer fragments seem to be more effective than short RNA particles, because they are more efficiently processed into more different siRNAs. The convenient method of introducing small dsRNA fragments into the cell by hairpin-expressing plasmids (Fig. 2) can overcome these disadvantages (Kawasaki and Taira, 2003; Yu et al., 2002) . Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter significantly induce RNAi-mediated gene silencing in the cytoplasm of human cells
cord-015850-ef6svn8f	2013	General overviews of eukaryote genomes are first discussed, including organelle genomes, introns, and junk DNAs. We then discuss the evolutionary features of eukaryote genomes, such as genome duplication, C-value paradox, and the relationship between genome size and mutation rates. Most of the protein coding genes of melon mitochondrial DNAs are highly similar to those of its congeneric species, which are watermelon and squash whose mitochondrial genome sizes are 119 kb and 125 kb, respectively. There are various genomic features that are specifi c to eukaryotes other than existence of introns and junk DNAs, such as genome duplication, RNA editing, C-value paradox, and the relationship between genome size and mutation rates. The Perigord black truffl e ( Tuber melanosporum ), shown as A i n Fig. 8.9 , has the largest genome size (~125 Mb) among the 88 fungi species whose genome sequences were so far determined, yet the number of genes is only ~7,500 [ 81 ] .
cord-015871-1tuf4zxi	2007	In contrast, a dose of ribavirin at least nine times greater was required to induce a comparable inhibitory effect on the yields of Rift Valley fever virus, for which the drug has been shown to inhibit replication in monkeys and rodents [104] . However hemorrhagic fever virus infections can be approached by the following different therapeutic strategies [6] : (i) administration of high-titered neutralizing antibodies and/or (ii) treatment with antiviral drugs. In recent times, several groups have studied the antiviral activities of interferons against hemorrhagic fever viruses. Human MxA protein inhibits the replication of Crimean-Congo hemorrhagic fever virus Type I interferon inhibits Crimean-Congo hemorrhagic fever virus in human target cells Genotoxic effect of ribavirin in patients with Crimean-Congo hemorrhagic fever Ribavirin efficacy in an in vivo model of Crimean-Congo hemorrhagic fever virus (CCHF) infection Inhibition of Crimean-Congo hemorrhagic fever viral infectivity yields in vitro by ribavirin
cord-015933-x5cq4k4x	2011	Virussen vormen een geheel aparte groep biologische agentia die ziekte bij de mens kunnen veroorzaken; hun meest karakteristieke eigenschap is dat zij voor hun vermeerdering afhankelijk zijn van levende gastheercellen. Dit duidt er dus tevens op dat een micro-organisme over een complex geheel van genetische eigenschappen moet beschikken, wil het pathogene betekenis voor de mens krijgen; dergelijke eigenschappen worden ook wel virulentiefactoren genoemd. Een aantal virussen staat bekend als tumorvirus omdat zij een permanente maligne transformatie in hun gastheercel kunnen teweegbrengen; het virus beïnvloedt dan de transcriptie van specifieke cellulaire oncogenen of andere gastheergenen, genen die betrokken zijn bij de regulatie van celdeling of spontane celdood (apoptose). Hoewel sommige micro-organismen (stafylokokken) zich direct aan dergelijke kunststoffen kunnen hechten, is het in de praktijk zo dat het oppervlak ervan snel door neerslag met bovengenoemde matrixeiwitten als fibronectine wordt bedekt, een proces dat ook wel conditionering van het biomateriaal wordt genoemd.
cord-016095-jop2rx61	2010	Instead of setting out to discover unknown mechanisms by analyzing effects that are dependent on specific causes, with some uncertainty as to the possible success of the enterprise being undertaken, which is the foundation stone of the Bernardian paradigm of the experimental method, many current research projects give themselves achievable and programmable objectives that depend upon the means available to them: sequencing of genomes with a view to comparing them, recognition of sequence similarities in proteins coded for by genes belonging to different species, with the aim of putting together phylogenetic trees, synthesis of interesting proteins in transgenic animals and plants, analysis of the three-dimensional structure of proteins, in order to find sites that are likely to fix medicinal substances, and synthesis of molecular species able to recognize pathogenic targets.
cord-016108-jlono0x7	2015	In this chapter, we describe a method to deep genome sequence porcine coronavirus on the Illumina MiSeq, avoiding the number of contaminating reads associated with the host and other microorganisms. (e) Remap the reads to the contig to verify accurate generation of the viral genome and suffi cient coverage. (a) Open the SeqManNGen program, select reference-based assembly, and load the Susscrofa genome and the correlating paired fastq fi les to the sample. However, the concentration by RT-PCR may not indicate successful generation of the complete viral genome since total RNA was used in the library preparation. If libraries have limited host contamination and have an acceptable concentration of viral RNA (Ct value <25), a 1 million read output per sample should be suffi cient for assembly. Since the MiSeq generates reads from total RNA, host reads need to be removed to facilitate de novo assembly, which can be done by fi rst mapping the reads to the swine genome and saving the unmapped reads.
cord-016144-280kwlev	2018	Further, modifications in PCR-based molecular detection techniques have generated a vast array of fast, reliable and specific assays which have widespread applications in veterinary diagnostics. The sensitivity of any genome detection-based method can be enhanced to a very high degree by manipulating any of the three pillars of the assay, i.e. by amplification of target, signal and probe. Common real-time PCRs include (1) SYBR green method where the fluorescent dye SYBR green binds to random dsDNA and can also give nonspecific amplification and (2) dual dyelabelled probe method which involves the use of sequence-specific DNA probes that are labelled with a fluorescent reporter, permitting specific detection after hybridization of the probe with its complementary sequence. To overcome these limitations and to increase efficiency comparable to symmetric PCRs, linear after the exponential (LATE)-PCR was developed based on primer pairs purposely designed for use at unequal concentrations to yield specific single-stranded DNA products in a robust way (Pierce et al.
cord-016179-4i1n9j4x	2015	title: Real-Time Reverse Transcription-Polymerase Chain Reaction for Detection and Quantitation of Turkey Coronavirus RNA in Feces and Intestine Tissues A specific one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay using TCoV-specific primers and dual-labeled fluorescent probe for detection and quantitation of TCoV in feces and intestine tissues is described in this chapter. In this chapter, the protocol for one-step real-time RT-PCR to detect, differentiate, and quantitative TCoV RNA in the feces and intestinal tissue is presented. In step 3, the extracted RNA was subjected to one-step real-time RT-PCR for detection and quantitation of TCoV in feces or intestine tissues. Specific real time reverse transcription polymerase chain reaction for detection and quantitation of turkey coronavirus RNA in tissues and feces from turkeys infected with turkey coronavirus The protocol "Real-time reverse transcription-polymerase chain reaction for detection and quantitation of turkey coronavirus RNA in feces and intestine tissues" outlined in this chapter had been successfully carried out in the authors'' studies on molecular diagnostics, molecular virology, immunology, and/or vaccinology of turkey coronaviral enteritis.
cord-016209-6p9btua0	2008	The use of highly specific siRNAs targeting distinct regions of the viral genome ( Fig. 1) as well as host genes that are relevant for virus entry and maturation represents a novel therapeutic strategy to cure or attenuate in particular coxsackievirus-mediated diseases. Several laboratories obtained significant inhibition of the HIV-1 replication applying both synthetic and vector-derived siRNAs/shRNAs directed against the viral genome and HIV-encoded RNAs, such as the TAR element, tat, rev, gag, env, vif, nef and reverse transcriptase (Boden et al. In our previous work, the application of the most effective siRNA directed against the RNA dependent RNA polymerase 3D resulted in an approximately fourfold prolonged survival of coxsackievirus-infected cells and an inhibition of viral replication by more than 10 5 -fold compared to control siRNAs (Merl and Wessely 2007) . Even though previous studies reported efficient suppression of hepatitis C virus replication by siRNAs targeting single-stranded regions inbetween two stem-loop motifs of the viral 5′ UTR (Yokota et al.
cord-016261-jms7hrmp	2005	Profiling models based solely on sequence content such as Hidden Markov Model (HMM) [12] may miss structural homologies when directly used to search genomes for noncoding RNAs containing complex secondary structures. ERPIN searches genomes by sequentially looking for single stem loop motifs contained in the noncoding RNA gene, and reports a hit when significant alignment scores are observed for all the motifs at their corresponding locations. In this paper, we propose a new method to search for RNA pseudoknot structures using a model of multiple CMs. Unlike the model of Brown and Wilson, we use independent CM components to profile the interleaving stems in a pseudoknot. Finally, in order to test the ability of our program to cope with noncoding RNA genes with complex pseudoknot structures, we carried out an experiment where the complete DNA genomes of two bacteria were searched to find the locations of the tmRNA genes.
cord-016293-pyb00pt5	2006	In the course of the project, especially in the early years, the plan stated that "much new technology will be developed that will facilitate biomedical and a broad range of biological research, bring down the cost of many experiments (mapping and sequencing), and finding applications in numerous other fields." The plan built upon the 1988 reports of the Office of Technology Assessment and the National Research Council on mapping and sequencing the human genome. These DNA chips have broad commercial applications and are now used in many areas of basic and clinical research including the detection of drug resistance mutations in infectious organisms, direct DNA sequence comparison of large segments of the human genome, the monitoring of multiple human genes for disease associated mutations, the quantitative and parallel measurement of mRNA expression for thousands of human genes, and the physical and genetic mapping of genomes.
cord-016309-6mw8okmt	2019	Those included usage restricted to a single virus and specific animal species, problems with high spectrum activity and low cytotoxicity, high costs of development of new chemical compounds and absence of rapid diagnostic techniques allowing prompt use of a specific antiviral agent in the course of an acute infection (Rollinson 1992a, b) . Nevertheless, several licensed human antiviral agents are being used with cascade principle for treatment of animal diseases (e.g. acyclovir, idoxuridine and trifluridine against feline herpesvirus-1 ocular infection in cats) (Thiry et al. The discovery of PAA (Fig. 22.4) as an antiviral drug gave rise to intense research on its biological activities, which demonstrated PAA and its derivatives'' ability to inhibit the replication of a number of viruses such as immunodeficiency, hepatitis and herpes viruses. To conclude with, equine herpesvirus type 1 (EHV-1) infection causes outbreak of respiratory and various neurological diseases in horses, against which acyclovir and valacyclovir are the most common drugs, but also IFN targeting IFNGR complex as a key mediator of virus-specific cellular immunity (Poelaert et al.
cord-016313-n4ewq0pt	2012	The field of possible application of mesenchymal stem cells in medicine and research expanded tremendously with the advent of improved Lentiviral-vectors capable of inserting stable copies of genes of interest and expressing proteins or biologically active RNA species ad libitum, performing delicate gene editing or active gene silencing or serving as advanced drug delivery systems utilized in ex vivo cell therapy. Implantation of Lentiviral vector-transduced human bone marrow mesenchymal cells using collagen scaffolds into immunode fi cient mice resulted in ef fi cient engraftment of gene-engineered cells and provided sites for transgene-expression in vivo. Moreover, it did not alter the differentiation potential of either HSCs or MSCs. In addition, the therapeutic potential of CD133+ and MSC progenitor cells transduced ex vivo with Lentiviral vector encoding the mature form of vascular endothelial growth factor D (VEGF-D ) or the enhanced green fl uorescent protein (eGFP) marker gene achieved permanent gene expression.
cord-016343-wc3i54fc	2008	RdRp, RNA-dependent RNA polymerase; IRES, internal ribosome entry site; 5BSL3.2, stem loop 3.2 within the coding sequence of NS5B normally result in the production of type I IFNs and the subsequent expression of IFN-induced effector proteins, which in turn would establish an antiviral state in the IFN-producing cell itself and in neighboring cells. Since MxA has the ability to efficiently inhibit a variety of different RNA viruses, MxA was the first IFN-induced effector protein to be analyzed for its antiviral activity in the HCV replicon system (Frese et al., 2001) . Rather indirect evidence that the OAS/RNase L pathway may indeed target HCV replication/translation was recently provided by Taguchi and co-workers, who reported that the N-terminal portion of NS5A (amino acids 1 to 148), which lacks the so-called PKR-binding domain, binds to OAS proteins and there by counteract the antiviral activity of IFN-α (Taguchi et al., 2004) .
cord-016419-v1f6dx3e	2016	Usage of recombinant DNA technology for large-scale production of proteins requires ample amount of time, labor, and resources, but it also offers many opportunities for economic growth. The recombinant proteins approved by FDA are obtained either from Escherichia coli or other prokaryotes; from Saccharomyces cerevisiae or other fungal species; from insect cells , mammalian cells , or human cells ; or from transgenic plants or animals. Their advantages are low cost, high mass production, scale-up, lack of human pathogens , and addition of eukaryotic PTMs. The fi rst recombinant protein obtained in 1986 from tobacco plants was human growth hormone . The vector can be modifi ed to express genes for insulin (tomato) or Hep-B surface antigen (HBsAg) for recombinant therapeutics providing a human glycosylation pattern have increased attention, and the efforts are being made for the development of novel glycoengineered cell lines for production of fully glycosylated protein therapeutic.
cord-016499-5iqpl23p	2014	A convenience population of 15 healthy children (1-9 years old) without asthma were followed during at least three seasons, and picornaviruses were detected in 5 % of 740 specimens (21 % of infections) not associated with symptoms, The impact of HRV typing and of sampling based only on symptoms. Clinical features and complete genome characterization of a distinct human rhinovirus genetic cluster, probably representing a previously undetected HRV species, HRV-C, associated with acute respiratory illness in children Comparison of results of detection of rhinovirus by PCR and viral culture in human nasal wash specimens from subjects with and without clinical symptoms of respiratory illness Detection of human rhinovirus C viral genome in blood among children with severe respiratory infections in the Philippines
cord-016538-4og05fuo	2017	Although in theory any of the grapevine-infecting viruses can be engineered into transient gene expression or VIGS vector, in practice, only one of them, the filamentous Grapevine leafroll-associated virus-2 (GLRaV-2) from the genus Closterovirus (family Closteroviridae), was demonstrated to fulfill these roles (Dolja and Koonin 2013; Kurth et al. Among these viruses, only GLRaV-2, a closterovirus, has been so far engineered into a vector capable of systemic infection of grapevine that either produces recombinant protein or elicits VIGS response (Kurth et al. The more recently developed CTV-based gene expression vectors were shown to be not only capable of systemic infection in the natural citrus hosts but also exhibited remark-able genetic stability in regard to retention of the inserted recombinant gene, as well as VIGS capability (Dawson et al. Another candidate to be developed as a vector for protein expression and VIGS is GRSPaV, which is the only grapevine-infecting member of the genus Foveavirus that was recently characterized (Meng et al.
cord-016652-x8t3lf1x	2011	This process is crucial for virus biology because if the viral proteins that are required for cytoplasmic functions such as RNA synthesis and encapsidation are sequestered in the nucleolus or nucleus, then progeny virus production will be affected as has been revealed by inhibitor and genetic studies (Lee et al. Viruses may interact with the nucleolus to usurp host cell functions and recruit nucleolar proteins to facilitate virus replication. Initial studies utilising the prototype g-2 herpesvirus, herpes virus saimiri (HVS), demonstrated that the HVS nucleolar trafficking ORF57 protein induces nucleolar redistribution of the host cell human TREX proteins, which are involved in mRNA nuclear export (Boyne and Whitehouse 2006) . The localisation of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence Sequence requirements for nucleolar localization of human T cell leukemia virus type I pX protein, which regulates viral RNA processing
cord-016755-ye37z5h9	2019	Sequence from a novel species of phlebovirus was identified by sequence independent single primer amplification (SISPA) from the serum of a patient with SFTS. The virus was isolated in Vero cell culture and its complete genome sequence was determined, only distantly related to other known phleboviruses. Phylogenetic trees based on complete viral genomic sequence of L, M and S segments from strains (HB29, HN6, AN12, LN2, JS3 and SD4) from 6 provinces in China in comparison with other known phleboviruses showed that SFTS virus was related to prototypic viruses of Phlebovirus. The novel phlebovirus was isolated in cultured Vero cells inoculated with acute-phase serum of patient HB29 from Shuizhou area, Hubei province. Specificity, sensitivity and cross reactivity of the methods were verified with serum samples collected from patients with SFTS confirmed by RT-PCR and sera collected from healthy donors from the areas without reported SFTS cases.
cord-016796-g4kqqpy1	2019	As a part of modern research on immunotechniques, a diagnostic approach for chronic hepatitis C infection (CHC), detects specific antibody to HCV (anti-HCV) (indirect tests) and assays that can detect, quantify, or characterize components of HCV viral particles, viz. Nonetheless, recent studies on respiratory syncytial virus (RSV) developed Luciferase Immunoprecipitation Systems (LIPS) assay to detect IgG Antibodies against Human RSV G-Glycoprotein. Sensitive and specific detection of Crimean-Congo hemorrhagic fever virus (CCHFV) was developed employing specific IgM and IgG antibodies in human sera using recombinant CCHFV nucleoprotein as antigen in μ-capture and IgG immune complex (IC) ELISA tests (Emmerich et al. A rapid diagnostic platform for colorimetric differential detection of DENV and CHIKV viral infections was recently developed with a possibility to alter clinical diagnosis of acute febrile illnesses in resource-limited settings. This novel antibody demonstrates noteworthy specificity to identify H7N9 virus compared to homemade target-captured ELISA, qRT-PCR, and rapid influenza diagnostic test (RIDT) with high sensitivity (Chang et al.
cord-016808-gy8d8285	2008	Modern hypotheses of viral origin are based on two major developments of the molecular biology: discovery of ribozymes (RNA-based enzymes) and formulation of the "RNA World" theory (RNA had been "invented" before proteins and DNA), on the one hand, and achievements of genomics (determination of the nucleotide sequences of a great number of cellular and viral genomes), on the other. Three distinct DNA viruses, which had infected RNA genome-containing cells, gave rise to the three distinct domains of life, bacteria, archea, and eukarya (Forterre, 2006) . To infect a human, an avian flu virus should change its receptor specificity, which depends on the interaction of viral hemagglutinin (HA) with a cellular membrane glycoprotein receptor. Such a change in the host range may be achieved by either mutations in the avian HA or acquisition by an avian virus of the HA gene from human influenza virus as a result of genetic exchange (reassortment) between these viruses during mixed infections. Three RNA cells for ribosomal lineages and three DNA viruses to replicate their genomes: A hypothesis for the origin of cellular domain
cord-017167-8cdbcrh7	2016	The nonstructural proteins (nsPs) of chikungunya virus (CHIKV) are expressed as one or two polyprotein precursors, which are translated directly from the viral genomic RNA. Similar to other alphaviruses, CHIKV nsPs not only perform virus RNA replication but are also crucial for other activities essential for virus infection and pathogenesis. However, recent studies of SFV P1234 processing reveal that a second mechanism, the presentation of cleavage sequences via long-range interactions between different domains of the polyprotein, Processing of CHIKV ns polyprotein P1234 and RNA synthesis. The main interaction appears to be mediated by a membrane-binding peptide located in the central part of the protein (approximately residues 244-263 in CHIKV nsP1; Fig. 2 ), which forms an amphipathic alpha helix, as characterized for the corresponding peptide from SFV (Ahola et al. However, the effects of mutations introduced into the NTPase/helicase active site were different for these viruses: in SINV such a mutation strongly reduced the nsP2-dependent degradation of Rpb1 whereas CHIKV nsP2 mostly retained its ability to block host gene expression.
cord-017181-ywz6w2po	2008	As this regulating process depends largely on structure formation, modelling of RNA folding would be a big step in the right direction for reflecting attenuation dynamics. Additionally, modelling interactions between mRNA and RNAP as well as mRNA and the ribosome are needed because both influence the kinetic folding process and RNA termination structures break up gene transcription. If observed structures are present during folding simulation, the macro level model can signalise this information and thus trigger dynamics to other components by adding new ports to itself (representing docking sites) or send messages over existing ports. Simulating the RNA folding with Kinfold [12] results in a five times higher amount of the 2-helix conformation than the single hairpin, but their total sum is about 60% of all molecules and thus less misfolded structures can be observed. The pattern observation function of the RNA folding model, which is realised at macro level, allows us to look for an intrinsic transcription termination structure [2] during simulation.
cord-017297-q3qtgrfc	2008	In a recent study on HSV-1 UL9 helicase, mutational analysis of the residues in the Ia motif implicated in DNA binding resulted in moderate to severe defects in single-stranded nucleic-acids binding and ssNA stimulated ATPase activity, while retaining the intrinsic ATPase activity similar to that of wildtype enzyme (Marintcheva and Weller 2003) . Thus, in order to understand the mechanism of helicase catalyzed unwinding reactions, it is important to understand all the individual steps to it, namely: nucleic-acid binding, NTP binding and hydrolysis, single-stranded translocation, and then finally the strand-separation function. Two early genes of bacteriophage T5 encode proteins containing an NTP-binding sequence motif and probably involved in DNA replication, recombination and repair Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases A functional interaction of ICP8, the herpes simplex virus single-stranded DNA-binding protein, and the helicase-primase complex that is dependent on the presence of the UL8 subunit
cord-017732-1pwa6zsk	2009	As in animal viruses, the −1 ribosomal frameshift site in the viral mRNA consists of a canonical shifty heptanucleotide followed by a highly structured frameshift stimulatory element, and the gene translated as a result of frameshifting usually encodes the RNA-dependent RNA polymerase. We suggest that these experiments may not have revealed all of the sequence requirements for frameshifting on the full-length viral RNA because the frameshifting constructs tested contained a very short (10 codon) first (zero frame) ORF from the start codon through the shifty site which is followed immediately by a stop codon. The 25-kDa protein that could be generated by a frameshift followed by cleavage at the HC-Pro/P3 cleavage site is indicated (Chung et al., 2008) anticipate results of future research and structural analysis to determine how these diverse plant viral RNAs induce ribosomes to change reading frames by what may be novel mechanisms.
cord-017764-h1w9gbxk	2018	A groundbreaking clinical trial that combined daclatasvir (1) with the protease inhibitor asunaprevir (52) established that a chronic HCV infection could be cured with small molecule therapy in the absence of immune stimulation, setting the stage for approval of this regimen for the treatment of GT-1b-infected subjects by the Japanese health authorities on July 4, 2014. The discovery of the hepatitis C virus (HCV) nonstructural 5A (NS5A) replication complex inhibitor daclatasvir (1) began with the development of a genotype 1b (GT-1b) replicon that was implemented as a phenotypic screen using a design that conferred a stringent triaging of hit molecules [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . A screen of compounds selected from the library of HCV NS5A inhibitors assessed in the presence of 1 using the Tyr93Asn GT-1aresistant replicon, followed by SAR optimization, identified Syn-395 (52) as a molecule capable of restoring the sensitivity of resistant virus to the inhibitory effects of 1. Discovery of daclatasvir, a pan-genotypic hepatitis C virus NS5A replication complex inhibitor with potent clinical effect
cord-017968-17d37a2z	2018	Here, we discuss recent progress to obtain a systems-level understanding of in vivo RNA–protein interactions in the reference plant Arabidopsis thaliana using protein-centric and RNA-centric methods as well as combined protein binding site and structure probing. Among the RBPs present in the Arabidopsis genome are 197 proteins with an RNA recognition motif (RRM), the most abundant type of RNA-binding domain, and 28 K homology (KH) domain proteins first identified in mammalian heterogeneous nuclear protein hnRNP K (Silverman et al. Functional characterization of a glycine-rich RNA-binding protein 2 in Arabidopsis thaliana under abiotic stress conditions Glycine-rich RNA-binding proteins are functionally conserved in Arabidopsis thaliana and Oryza sativa during cold adaptation process The circadian clock regulated RNA-binding protein AtGRP7 autoregulates its expression by influencing alternative splicing of its own pre-mRNA An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with target transcripts in Arabidopsis thaliana
cord-018017-c8myq6bi	2018	Numerous emerging infections caused by viral agents have imposed high impact on human survival (Table 3 .3). The apparent success of these viruses is that as they move from reservoir hosts to humans and as humans become immune to the initial infection, the population of diverse genomes offers multiple chances to adapt by finding a "fit" genome version which can propagate until the next transition requiring adaption. Human T-cell Lymphotropic Virus (HTLV-1) HTLV-1 is a single-stranded RNA retrovirus, defined by their use of reverse transcriptase, a polymerase, that makes a DNA copy of the RNA 7 kb viral genome. If we combine cardiovascular events and neoplasia caused by infection, then infectious disease is the most significant threat to human life and qualifies as the area of greatest impact. Adeno-associated Virus (AAV) is a single stranded DNA virus that infects humans but are not known to cause disease. is a 5229 base double-stranded DNA virus infecting less than 5 percent of the human population.
cord-018164-h5k1zsyg	2014	Studies of viral replication indicate that most viruses self-assemble as a result of interactions between the viral proteins to form a viral capsid that interacts with the nucleic acid to form the whole. The viral proteins are produced in one part of the cell, the replicated nucleic acid in another, and somehow they find each other, interact, and form virus particles that are expelled from the cell. Indirect contact spread includes cases where mucus from a runny nose may get onto the hands, or virus may be left on a surface such as a doorknob, telephone, or countertops, and is picked up by a second individual, who then touches his eyes or nose, resulting in infection. Vector transmission is a very common means of transmission; the best studied cases include yellow fever, dengue virus, and West Nile fever-viruses all transmitted by mosquitoes. As many as 400 million people are infected annually by dengue virus, which is caused by any one of four related viruses transmitted by mosquitoes.
cord-018437-yjvwa1ot	2013	Classifi cation is based on the genomic nucleic acid used by the virus (DNA or RNA), strandedness (single or double stranded), and method of replication. The nucleocapsids of some viruses are surrounded by envelopes composed of lipid bilayers and host-or viral-encoded proteins. The sequence of negative-sense ssRNA is complementary to the coding sequence for translation, so mRNA must be synthesized by RNA polymerase, typically carried within the virion, before translation into viral proteins. Among the families of viruses able to infect humans and other vertebrate hosts, there are many species that target and cause disease in the lung. The nucleocapsid is surrounded by an envelope derived from host-cell membrane and viral envelope proteins, including hepatitis B surface antigen. The genome of human parainfl uenza viruses is ~15 kb in length with an organization and six reading frames (N, P, M, F, HN, L) typical of the Paramyxoviridae (Karron and Collins 2007 ) .
cord-018564-3igg5s57	2013	Driven by the energy of ATP hydrolysis, this movement allows the protein to displace complementary strands of DNA or RNA [13] ; <38> the DEAD-box protein DED1 has the ability to balance RNA unwinding with a profound strand annealing activity in a highly dynamic fashion [11] ; <10,20> RNA helicase activity [2, 4] ; <12> multifunctional enzyme possessing serine protease, NTPase, and RNA unwinding activities [42] ; <12> NTPase activity analyzed, ambiguous helicase activity, enzyme capable for unwinding RNA and DNA [38] ; <39> RNA-stimulated ATPase activities determined, interaction between the replicative component nonstructural protein 3 (NS3) with the nonstructural protein 4A (NS4A) [44] ; <12> the Arg-rich amino acid motif HCV1487-1500, a fragment of domain 2 NS3 of Hepatitis C virus, as well as the complete domain 2, and domain 2 lacking the flexible loop localized between Val1458 and Thr1476, mediate competitive inhibition of diverse protein kinase C functions, inhibition of rat brain PKC, overview [39] ; <17> the West Nile virus RNA helicase uses the energy derived from the hydrolysis of nucleotides to separate complementary strands of RNA [62] ; <13> translation of HIV-1 gag mRNA is reliant on the ATP-dependent helicase activity of RNA helicase A [61] ) (Reversibility: ?) [2, 4, 5, 6, 11, 12, 13, 21, 22, 28, 30, 31, 32, 37, 38, 39, 41, 42, 43, 44, 45, 46, 61, 62 ] P ADP + phosphate S RNA + H 2 O <2,5,10,42> (<5> helicase/unwinding activity [43] ; <42> helicase/unwinding activity, either ATP or dATP is required for the unwinding activity [32] ; <2> RNA unwinding activity, the enzyme contains two RecA-like domains, opening and closing of the interdomain cleft during RNA unwinding [45] ) (Reversibility: ?) [32, 41, 43, 45 ] P ?
cord-018724-ss8x2g3b	2016	The variation we see within a single plant host has profound effects on the how the virus responds to selective pressures associated with new hosts, and factors such as the bottleneck events associated with cell-to-cell movement or vectoring. However, several forms of virus variation, such as the high mutation rates of RNA and some DNA viruses, recombination, and reassortment lead to resistance breaking (Duffy and Holmes 2008; McDonald and Linde 2002; Harrison 2002) . For example, genetic diversity (heterosis) induced tolerance to Turnip mosaic virus in wild cress (Lepidium sp.) hybrids, while plants that were selfed were more susceptable to disease, suggesting that small populations with low genetic diversity could lead to increased disease symptoms, and infection rates (Houliston et al. Genetic bottlenecks during systemic movement of Cucumber mosaic virus vary in different host plants Role of recombination in the evolution of natural populations of Cucumber mosaic virus, a tripartite RNA plant virus
cord-018798-yzxy9ogf	2018	This chapter also reviews the rich history of RNAi research, gene regulation by small RNAs across different organisms, and application potential of RNAi for generating transgenic plants resistant to major pests(.) But, there are some limitations too which restrict wider applications of this technology to its full potential. Different delivery methods of dsRNA that have been used for successful RNAi in insects and nematodes include microinjection, feeding on either artificial diet (Table 4 .2), and/or host-mediated delivery through transgenic plants (Fig. 4.1) . RNAi mechanism partly occurs in the host itself and partly in nematodes feeding on the transgenic host plant expressing dsRNA for the target gene. despite the absence of Sid-1 and Sid-2 genes exhibit systemic RNAi when subjected to silencing technology indicating a presence of similar receptor-mediated endocytic process for dsRNA uptake as reported in insects ).
cord-018804-wj35q88f	2007	High error prone replication, together with the short replication times and large population sizes typical of RNA viruses, instead of being a handicap for survival provides an extraordinary evolutionary advantage by permitting the generation of a wide reservoir of mutants with different phenotypic properties [7] . However, the fact that DNA organisms, which usually live in constant environments, have evolved corrector activities, whereas RNA viruses have not, suggests that replication with high error rates is a selected character that strongly favours viral adaptation to fast changing conditions. Quasi-species replicating during a long time in a near-constant environment in the absence of large population size fluctuations can present a low rate of fixation of mutations in the consensus sequence, despite the continuous occurrence of mutants that is characteristic of the underlying dynamics of the population. The infection of a new host constitutes a sudden change in the environment in which viral replication takes place, usually with the consequence of a drastic decrease in the average fitness of the virus population, which prevents further transmission.
cord-018944-du42ho11	2018	[6, 7] as follows: (1) extraction should be simple and rapid and should show high sensitivity and specificity; (2) it is preferred that there be no requirements for specialized equipment or special knowledge and skills; (3) the final nucleic acid should be pure and easy to modify for various amplification techniques; (4) the reagents and their product should be harmless; and (5) the process of preparation should resist contamination with other specimens. Although the phenolchloroform method is relatively easy compared with the CsCl/EtBr gradient and is very useful for the extraction of nucleic acids, it also is problematic for the clinical microbiology laboratory because phenol has important limitations due to its being toxic, caustic, and flammable [5, 15, 16] . In recent years, it has become possible to extract viral nucleic acid from clinical specimens having cellular components, and there have been trials of these commercially available kits to detect various clinically important viruses [30] [31] [32] .
cord-019051-gtruu1op	2009	Viruses with an established role in common cold are rhinoviruses, adenoviruses, parainfluenza viruses, coronaviruses and the respiratory syncytial virus, and these are reviewed in greater detail here. Therefore, the viral etiology and the role of viruses in the pathogenesis of common cold is complex and it is safe to say, not fully understood for each and every virus that is linked to respiratory tract infection. RSV infection is assumed to be frequently misdiagnosed, particularly in adults [56] , because the symptoms are similar to those caused by other respiratory viruses like influenza. Human parainfluenza viruses (HPIV) are important causes of respiratory diseases in infants and children. HMPV is thought to be the second or third cause of severe acute respiratory tract infection in children, just ranking behind RSV and influenza virus [146, 148] . Retinoic acid-inducible gene I mediates early Antiviral Response and Toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells
cord-019076-4qu9j953	2009	In this chapter, we review our current understanding of the expression and functions of key replicative enzymes, such as RNA polymerases, helicase, ribonucleases, ribose-2′-O-methyltransferase and other replicase gene-encoded proteins involved in genome expression, virus–host interactions and other processes. The RTC includes the key replicative proteins of the virus, such as RNA-dependent RNA polymerase (RdRp) and helicase activities, as well as enzymes that are thought to be involved in the processing and modification of viral and/or cellular RNAs, such as primase, endoribonuclease, exoribonuclease and ribose-2 0 -O-methyltransferase activities (for recent reviews, see Masters 2006; Ziebuhr 2005 Ziebuhr , 2008 . SARS-CoV pp1a and pp1ab are co-and post-translationally processed by two proteases, a papain-like protease (PL pro ) and the main protease (M pro , nsp5), resulting in 16 mature products called nonstructural proteins (nsps) 1-16 The 5 0 -terminal ORF(s) expressed from specific RNAs is/are shown as boxes.
cord-020010-q58x6xb0	2006	In the present study we reported the antiviral activity of neuraminidase inhibitor oseltamivir against lethal H5N1 influenza virus infection in ferrets, an appropriate animal model that closely resembles clinical signs of human influenza. Earl Kern 1 , Kathy Keith 2 , Robert Jordan 2 , Dennis Hruby 2 , Debra Quenelle 2 1 Department of Pediatrics, University of Alabama School of Medicine, Birmingham, AL, USA; 2 SIGA Technologies, Inc., Corvallis, OR, USA Although cidofovir (CDV) has been approved as an investigational new drug for emergency treatment of smallpox, its lack of oral activity and dose limiting toxicity dictates a need for continued development of better therapeutic agents for this potential bioterror disease. The in vitro antiviral activity of one of the most selective compounds, i.e. CHI-033, was assessed by (i) MTS-based cytopathic effect assays, (ii) virus yield reduction assays, (iii) real-time quantitative PCR (RT-QPCR) and (iv) by monitoring viral antigen expression.
cord-020235-stcrozdw	2012	Hepatitis A virus (HAV) was isolated directly from human stool in diploid human fibroplasrs, Viral antigen was expressed only after 210 days of incubation of the infected cultures. Univ., 0-8700 Wiirzburg Effect of Measles (SSPE) Antiserum on Viral Surface Proteins and Hormone Receptor Activity in C6/SSPE Persistently Infected Cells BARRETT, P. Inst, of Genetics, Univ., 0-5000 Co logne Virus-Cell DNA Recombinants in Human Cells Lytically Infected by Ad2 NEUMANN, R., WEYER, U., and DOERFLER, W. In vitro, however, the gag-specific peptide sequences are cleaved off upon addition of the purified viral piS protease; in the case of the replication-defective, transforming avian sarcoma virus PRC II the cleaved nongag part has a ryrosine-phosphorylaring kinase activity similar to that described for the RSV src-gene product pp60 src . f. Virologie, Univ., D·6300 Giefen, 3 A protein of a molecular weight of about 38.000 d has been found to be phosphorylated 1 h after the onset of cell transformation by Rous sarcoma virus (RSV).
cord-020969-lh2ergpm	2012	Together with methods for cloning and manipulating viral genomes, this information has made possible the use of viruses as vectors to express foreign genes. The use of minus-strand RNA viruses as vectors was delayed because the virion RNA itself is not infectious, but recent developments has made it possible to rescue virus from cloned DNA by using coexpression of the appropriate viral proteins in a transfected cell. It is also possible to transfect cells with the E1 or E3 expression cassette together with DNA clones encoding the rest of the adenovirus genome, in which case homologous recombination results in the production of virus. Because the poliovirus replicon lacks a full complement of the structural genes (it is a suicide vector), packaging to produce particles requires infection of a cell that expresses the polioviral structural proteins by some mechanism.
cord-021115-2fkghukw	2013	It is significant for predicting the structure and function of RNA that learning about the stability and the process of RNA pseudoknot folding and unfolding. The structural features of mouse mammary tumor virus (MMTV) RNA pseudoknot in different ion concentration, the unfolding process of the RNA pseudoknot, and the two hairpin helices that constitute the RNA pseudoknot were studied with all atom molecule dynamics simulation method in this paper. To study the factors that affect the stability and the unfolding pathways of the pseudoknots, the MMTV RNA was studied by all-atom molecule dynamics simulation methods under different ion concentrations and temperatures. The structural features of MMTV RNA pseudoknot in different ion concentration, the unfolding process of RNA pseudoknot, and two hairpin helices that constituted the RNA pseudoknot were studied with all atom molecular dynamics simulation method in this paper.
cord-021481-tvs1pnib	2018	Introns FIGURE 7.1 Critical events in HIV-1 replication are supported by RNA helicases Early steps in HIV-1 replication involve reverse transcription in the cytosol of capsid-associated viral RNA (n = 2) to double-stranded cDNA that transits the nuclear pore and integrates into the host chromosome (blue lines) to form a provirus (red line surrounded by black dotted line) with the involvement of at least two RHs (DHX9, DDX19A). The study of CTE/TAP activity significantly expanded knowledge of nucleocytoplasmic transport of cell mRNAs and regulation by many virus RNAs. Ten years ago and in context RSV, a genetically simple avian retrovirus, DDX19B/yeast DBP5 was determined to facilitate nuclear export of genome-length unspliced RNA (LeBlanc et al., 2007) . Influenza A virus polymerase recruits the RNA helicase DDX19 to promote the nuclear export of viral mRNAs RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs
cord-021568-tdfn6up8	2012	The conserved domains highlighted in the figure are thought to be important for the replication of the viroid (i.e., to form promoters recognized by RNA polymerase II) and for its cleavage to produce unit-length molecules. GSS, most FFI, and some cases of CJD occur as dominant inherited diseases, associated with mutations in the gene for the prion protein. The pattern of symptoms associated with a particular TSE may vary, however, depending in part on how the disease was contracted; on the source of the infecting agent; and on the nature of mutations in the prion protein. Studies in mice and other animals, as well as the finding that mutations in the prion protein are associated with inherited TSEs in humans, have made clear that the prion protein, abbreviated PrP, is intimately involved in the transmission of TSE and in the disease process.
cord-022084-hap7flng	2009	The Centers for Disease Control and Prevention (CDC) recommends the immunization of persons aged 50 years and older; residents of nursing homes; children and adults with chronic cardiovascular or pulmonary disease, including asthma; persons chronically ill with diabetes mellitus, renal dysfunction, or hemoglobinopathies; immunosuppressed patients including those with HIV infection; children and adolescents on chronic aspirin therapy who may develop postinfluenza Reye'' s syndrome; women who will be pregnant during the influenza season; children aged 6 to 23 months; those who can transmit influenza to persons at high risk, such as health-care workers and household contacts of those at high risk including children 0 to 23 months of age; crew members of cruise ships; providers of essential services; and unimmunized travelers to areas where influenza may be circulating, including the tropics, the southern hemisphere between April and September, and those traveling in large organized tourist groups.
cord-022128-r8el8nqm	2019	In the case of viral genomes, mutations can result from different mechanisms: (i) template miscopying (direct incorporation of an incorrect nucleotide); (ii) primer-template misalignments that include miscoding followed by realignment, and misalignment of the template relative to the growing chain (polymerase "slippage" or "stuttering"); (iii) activity of cellular enzymes (i.e., deaminases), or (iv) chemical damage to the viral nucleic acids (deamination, depurination, depyrimidination, reactions with oxygen radicals, direct and indirect effects of ionizing radiation, photochemical reactions, etc.) (Naegeli, 1997; Bloomfield et al., 2000; Friedberg et al., 2006) . In addition to the general environmental and sequence context consequences for templatecopying fidelity that may affect any genome type, mutation rates for DNA viruses will also be influenced by: (i) whether the DNA polymerase that catalyzes viral DNA synthesis includes or lacks a functional proofreading-repair activity.
cord-022196-1tionxun	2013	With most isometric particles and in all complex virions, the capsid encloses another protein structure containing the viral genome, called the core. All animal viruses with tubular nucleocapsids are enveloped, and in these the lipid layer from which glycoprotein peplomers project is probably applied to a protein shell (the membrane protein; see Fig. 1 -1), which may be relatively rigid, as in Rhabdovirus, or readily distorted (as in the myxoviruses) so that in negatively stained electron micrographs the virions appear to be pleomorphic. The RNA viruses that have the largest (single-stranded) genomes, those of the Leukovirus genus, also have a highly complex structure with an envelope enclosing an icosahedral capsid that, in turn, surrounds a tubular nucleocapsid. The conventional physicochemical criteria [(a) nucleic acid: type, strandedness, fragmentation, and molecular weight; (b) virion: shape, size, and symmetry] are suitable for classification at this level of family/genus, perhaps assisted by the serological cross-reactivity of "group" antigens where these have been recognized.
cord-022262-ck2lhojz	2007	The following viruses have been recognized as picornaviruses on the basis of their genome sequences and physico-chemical properties as well as the result of comparative sequence analyses (see the section on Evolution): equine rhinovirus types I and 2, Aichi virus, porcine enterovirus, avian encephalomyelitis virus, infectious flacherie virus of silkworm Clusters of enteroviruses refer to groups of enteroviruses arranged predominantly according to genotypic kinship (Hyypia et al., 1997) . Briefly, when expression vectors ( Figure 12 .6E) consisting of a gag gene (encoding p17-p24; 1161 nt) of human immunodeficiency virus that was fused to the N-terminus of the poliovirus polyprotein (Andino et al., 1994; Mueller and Wimmer, 1998) were analysed after transfection into HeLa cells, the genomes were not only found to be severely impaired in viral replication but they were also genetically unstable (Mueller and Wimmer, 1997) .
cord-022290-p0l1kv6n	2007	The primary function of the picornaviral proteinases is the cotranslational, specific cleavage of the viral polyprotein into the structural and nonstructural proteins. This is true even for some families of + RNA viruses which have developed additional strategies to generate individual gene products from a single RNA genome, e.g., subgenomic RNAs or multiple ORFs. Proteolytic cleavage as a covalent modification of the precursors of viral structural proteins is even more common and occurs even in DNA viruses. The sequence of the residues which form the last turn of this helix is highly conserved throughout the picornaviral 3C genes (K/RR/KNL/I), It is interesting that the structural and functional details of the proteolytic active site of the 3C proteinases are not the most conserved part of the 3C structure (Gorbalenya et al., 1988) . While the details of the specific enzyme substrate interactions gleaned from the crystal structures of 3C proteinases provide valuable information for the design of effective inhibitors, there is little experimental evidence for the mechanism of the chymotrypsin-like cysteine proteinases.
cord-022336-zqnczjpp	2007	The chapter also outlines the significance of work on viroid-like pathogens, circular RNA replication, and their potential relation to early RNA; and relates the early emergence of RNA mosaics to developments leading to today''s DNA-based systems of viral gene expression. Recent work to be reviewed below shows that there is one class of primitive life forms-the viroid-like pathogens -whose properties today could help us to understand how primitive RNA-based self-replication may have been compatible with expansion to produce more complex RNAs. In summary, the causative agent for human hepatitis delta contains two specialized domains, one concerned with replication and the other encoding a single protein (Branch et al., 1989; Purcell and Gerin, 1996; Taylor, 1996) . It is evident that if RNA conjunction leading to delta-like RNA mosaics with both replicating and functionally translatable protein-coding domains has taken place, we need to consider the consequences both for the evolution of primitive RNA systems, yielding today''s DNA-based cellular information system, and for presentday RNA-level events.
cord-022348-w7z97wir	2007	An analysis of proteins derived from complete potyvirus genomes, positive-stranded RNA viruses, yielded highly significant linear relationships. Under the rubric replication, a virus could vary to increase its fitness, exploit different target cells or evade adaptive immune responses. For a given virus, different protein sequence sets were compared to a given reference such as RT in the case of HIV/SIV. Although these data were derived from completely sequenced primate immunodeficiency viral genomes, analyses on larger data sets, such as p17 Gag/p24 Gag or gp120/gp41, yielded relative values that differed from those given in Table 6 .1 by at most 14%. An analysis of proteins derived from complete potyvirus genomes, positive-stranded RNA viruses, yielded highly significant linear relationships (Table 6 .1). In the clear cases where genetic variation is exploited by RNA viruses, it is used to overcome barriers to transmission set up by the host population, e.g. herd immunity.
cord-022888-dnsdg04n	2009	Methods: Phospho-specific Western blot analyses were performed to verify the functionality of the different IFN-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and HLA class I cell surface antigens, quantitative real time-PCR experiments to confirm the absence of JAK2 and presence of pathway relevant molecules as well as, genomic PCR and chromosome typing technique to prove the deletion of JAK2. In order to accomplish these objectives we induced priming or tolerance of ovalbumin (OVA 323-339 peptide)-specific T cells from DO11.10 TCR transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with E3 ubiquitin-protein ligases expression and the ubiquitination status of the TCR signalling machinery.
cord-022889-lv6fy6e6	2019	This report suggests that some plant ncRNAs (e.g miRNAs and siRNAs) show higher stability as compared to other ncRNAs due to peculiar chemical characteristics (2''‐O‐methylation at 3'' end).However, ingested or administered ncRNA must overcome many extracellular and cellular barriers to reach the intended target tissue or functional location in sufficient amount to exert any biological effect. Finally, the publications reporting the outcome of two EFSA procurements aiming respectively at investigating and summarising the state of knowledge on the mode-of-action of dsRNA and miRNA pathways, the potential for non-target gene regulation by dsRNA-derived siRNAs or miRNAs, the determination of siRNA pools in plant tissues and the importance of individual siRNAs for silencing 6 ; and reviewing relevant scientific information on RNA interference that could serve as baseline information for the environmental risk assessment of RNAi-based GM plants ) 7 were also used.
cord-022940-atbjwpo5	2016	We have studied the effect of inhibition of IRE1 (inositol requiring enzyme 1), which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in U87 glioma cells. Transient inhibition of Akt and mTOR protein kinase activation in tumor cells followed by reactivation of signaling pathway did not result in a time-dependent difference on EGFR, HER2 and HER3 expression levels. In our study we aimed to determine cytotoxic effect of RES in K562 human CML cell line and to evaluate the expressions of miRNAs that are associated with genetics of leukemia after treatment with RES; to investigate target genes of miRNAs which show significant expression alterations and molecular mechanisms of RES treatment.
cord-023017-k6edtg58	2006	14/55 (25%) patients in AC who did not discontinue by week 24 received ribavirin dose reduction in comparison to 31/108 ( The clinical outcome in response to combination therapy for treatment of chronic hepatitis C virus (HCV) infection appears to be different for Caucasian versus African American patients. Over the period of combination therapy, most patients in which serum virus titers were reduced to non detectable levels had significant increases in T cell responses to HCV proteins. CHRONIC Background: Recent large prospective trials demonstrated that the combination therapy of interferon (1FN)-alphalribavirin significantly increased the ratio of a sustained virological response in patients with chronic hepatitis C in comparison with IFN monotherapy, especially in patients with high HCV-RNA titer and genotype lb. Results: Patients with chronic HCV infection showed higher MxA gene expression levels than healthy controls, indicating that hepatitis C virus induces IFN production.
cord-023120-jcgf2401	2004	We suggest that, (1) c regions contain promotors for viral RNA synthesis, (2) cx contains a more efficient promotor than c" and (3) non-acute disease foliows the formation of critical viral-cell recombinants in which ( 2 ) The r e s u l t i n g CDNA i s f r a c t i o n a t e d and a l l fragments having a length g r e a t e r than 300 nucleotides are i s o l a t e d and c u t with a r e s t r i c t i o n endonuclease capable of d i g e s t i n g single-stranded DNA. Studies of the synthesis and processing of viral proteins in simian sarcoma associated virus (SSAV)infected and SSV(SSAV)transformed marmoset and human cell lines has demonstrated polyprotein precursors precipitable by anti-SSAV p30 and anti-SSAV gp70.
cord-023208-w99gc5nx	2006	In order to develop a synthetic protocol by an automated instrumentation, increasing yield, purity of the crude, and reaction time, a microwave-assisted solid phase peptide synthesis was validated comparing the use of the new generation of Triazine-Based Coupling Reagents (TBCRs) with a series of commonly used ones. Ubiquitinium is a well known mechanism in protein degredation of Eukaryotic cells ,in which many obsolte and corrupted three dimentional structure protein ,become marked by covalent attachment of ubuquitin through a multi-step enzymatic pathway.Ubiquitin is a small ,8.5 kDa peptide of 76 amino acid residues that targets such substrtes for proteolysis in proteasome .Recnt studies showed that an extra cellular ubiquitination process also taking place in the epididymes of humans and other animals marks protein on the surface of the defective sperm .it appears that structurally and functionally defective sperm become surface ubiquitinated by epididymal epithelial cells. This head-to-tailcyclized 14-amino-acid peptide contains one disulfide bridge and a lysine residue (Lys5) present in the P1 position, which is responsible for inhibitor specificity.As was reported by us and other groups, SFTI-1 analogues with one cycle only retain trypsin inhibitory activity.
cord-023346-8sqbqjm1	2005	• enhancement of automation/computerisation; • process control to provide an ''error-free pathway''; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody ''combi'' assays (e.g. for HIV, and recently, for HCV); • awareness of HBsAg vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or Chagas'' disease infection (for retrieval of otherwise wasted blood); • European Union''s in vitro diagnostics directive: this has caused some problems and reduced flexibility.
cord-023354-f2ciho6o	2005	• enhancement of automation/computerisation; • process control to provide an ''error-free pathway''; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody ''combi'' assays (e.g. for HIV, and recently, for HCV); • awareness of HBsAg vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or Chagas'' disease infection (for retrieval of otherwise wasted blood); • European Union''s in vitro diagnostics directive: this has caused some problems and reduced flexibility.
cord-023364-ut56gczm	2005	• enhancement of automation/computerisation; • process control to provide an ''error-free pathway''; • (national) surveillance and trend analysis of results, preferably based on national working standards; • significantly increased sensitivity, especially from development of antigen/antibody ''combi'' assays (e.g. for HIV, and recently, for HCV); • awareness of HBsAg vaccine-escape mutants and design of assays to cope with this; • extension of range of agents and markers tested for (varies in different countries); • increasing range of assays available for testing donors with a relevant history of exposure to malaria or Chagas'' disease infection (for retrieval of otherwise wasted blood); • European Union''s in vitro diagnostics directive: this has caused some problems and reduced flexibility.
cord-023608-w2g7v7g1	2017	ICAR retains its flavor and personality, providing an interdisciplinary forum at which investigators involved in basic, translational, and clinical research worldwide meet to review recent developments in all areas of antiviral research, drug and vaccine development. Additionally, satellite activities such as the Women in Science Roundtable, the Career Development Panel and the New Member and First Time Attendee luncheon (The Happy Hour) provided an opportunity to discuss other issues of relevance. With so many different competing conferences and meetings to attend and a long economic crisis of which scientific research did not escape, ISAR has gone through great financial efforts to continue supporting the participation of students, postdocs and young investigators. The TCFF Awards support the professional development of women with potential to make significant contributions to the field of Antiviral Research by providing funds to attend a conference, visit another laboratory, take a course, or acquire specialized training.
cord-023705-3q9yr6np	2014	Many important biochemical phenomena such as the splicing and other types of posttranscriptional processing of RNA, the posttranslational cleavage and glycosylation of proteins, the replication of RNA, reverse transcription, integration, and the transposition of viral genes and cellular oncogenes were first elucidated by virologists and have general application in cell biology. Many important biochemical phenomena, such as splicing and other types of posttranscriptional processing of RNA, posttranslational cleavage and glycosylation of proteins, replica tion of RNA, reverse transcription, integration, and transposition of viral genes and cellular oncogenes, were first elucidated by virologists and have general application in cell biology. The proteins translated from the early transcripts of DNA viruses include enzymes and other proteins required for the replication of viral nucleic acid, as well as proteins that suppress host cell RNA and protein synthesis.
cord-023724-5at0rhqk	2015	The problems plant viruses face in initiating infections of host cells have already been described (Chapter 4), as has the fact that no known plant virus employs a specific cellular receptor of the types that animal and bacterial viruses use to attach to cells. There are probably many different mechanisms involved in systemic resistance, but in general terms there is a tendency of these processes to increase local necrosis when substances such as proteases and peroxidases are produced by the plant to destroy the virus and to prevent its spread and subsequent systemic infection. Virus-resistant plants have been created by the production of transgenic plants expressing recombinant virus proteins or nucleic acids which interfere with virus replication without producing the pathogenic consequences of infection, for example: I Virus coat proteins, which have a variety of complex effects, including inhibition of virus uncoating and interference of expression of the virus at the level of RNA ("gene silencing" by "untranslatable" RNAs), I Intact or partial virus replicases which interfere with genome replication, I Antisense RNAs, I Defective virus genomes, I Satellite sequences (see Chapter 8), I Catalytic RNA sequences (ribozymes), I Modified movement proteins.
cord-023726-2fduzqyb	2012	Also shown for each family is the presence or absence of an envelope in the virion, the triangulation number (defined later) if the virus is icosahedral, the morphology of the nucleocapsid or core, and figure numbers where the structures of members of a family are illustrated. Structural studies of viruses have shown that the capsid proteins that form the virions of many plant and animal icosahedral viruses have a common fold. The largest particle is the nucleocapsid of herpes simplex virus, which is 1250 Å in diameter and has T=16 symmetry (the virion is enveloped but only the nucleocapsid is regular FIGURE 2.5 Gallery of three-dimensional reconstructions of icosahedral viruses from cryoelectron micrographs. For most RNA viruses, nucleocapsids can be recognized as distinct structures within the infected cell and can be isolated from virions by treatment with detergents that dissolve the envelope.
cord-023766-qx0qdjmt	2018	Differences observed in the secondary structure of these polymerases reflect not only the substrate diversity but also divergent mechanisms for initiation of RNA synthesis (primer dependent for HRV and RHDV but primer independent for HCV and bacteriophage ϕ6). Conserved aspartic acid residues in the polymerase palm domains coordinate the two magnesium ions needed for the catalytic polymerization reaction of the enzyme, with one metal activating the primer 3 0 OH for the attack of the nucleotide α-phosphate, and the other metal serving to stabilize the triphosphate moiety ( Fig. 11 .1). The EC of PV polymerase provided a required view about how the template and the RNA strand interact as they thread through the active site and showed that viral RdRPs use a unique palm-based structural change to close their active site for catalysis (Gong and Peersen, 2010) .
cord-023770-ymxapsv6	2011	In general, capsid proteins and their homologs (CPm) show a significant degree of sequence conservation and the duplicate copies probably retain the general spatial folding and some crucial properties of the CPs. Notable exception are a group of ampeloviruses with the smallest genomes in the family [e.g. grapevine leafrollassociated virus 4 (GLRaV-4), GLRaV-5, GLRaV-6, GLRaV-9, pineapple mealybug wilt-associated virus 1(PMWaV-1) and PMWaV-3] which do not appear to possess CPm. The genome expression strategy is based on: (i) proteolytic processing of the polyprotein encoded by ORF1a; (ii) 1 Pos. ssRNA ribosomal frameshift for the expression of the RdRp domain encoded by ORF1b, a mechanism not found in other ()RNA plant viruses; (iii) expression of the downstream ORFs via the formation of a nested set of 3 co-terminal sub-genomic RNAs (sgRNAs).
cord-023865-6rafp3x3	2009	Towards the end of the article, we will also discuss some recent reports regarding the possible clinically relevant use of the nucleocapsid protein, as a candidate diagnostic tool and vaccine against SARS-CoV infection. Interestingly, biochemically mediated inhibition of GSK3 activity in SARS-CoV infected cells also leads to around 80% reduction in viral titer and subsequent induction of a virus-induced cytopathic effect. Further, S-phase specific gene products like cyclin E and CDK2 were found to be downregulated in SARS-CoV infected cell lysate, which suggested that the observed phenomenon may be relevant in vivo. Based on this observation, Palese''s laboratory has studied the IFN inhibitory property of different SARS-CoV proteins, which revealed that ORF3, ORF6 as well as the N-protein have the ability to independently inhibit IFN production through different mechanisms. Intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells
cord-024282-t5wl0bih	2020	title: BOAssembler: A Bayesian Optimization Framework to Improve RNA-Seq Assembly Performance For example, RNA-Seq assembly tools typically require hyper-parameter tuning to achieve good performance for particular datasets. Results: Here we propose BOAssembler, a framework that enables end-to-end automatic tuning of RNA-Seq assemblers, based on Bayesian Optimization principles. The reference-based assembler together with its performance evaluation can be represented as an abstract function f (D, θ), where D includes both the read alignments used for assembly and the reference transcriptome (a set of ground truth RNA transcripts) used for evaluation, and θ refers to the parameters of f . After read alignments are assembled with given parameter θ, the assembly output (a set of RNA transcripts) will be compared with the reference transcriptome, and the quality of assembly is measured by scalar metrics such as precision p and sensitivity s.
cord-025181-eg108wcd	2020	In this study, with biologically cloned viruses from a single clinical isolate, we have established two mouse models of DENV infection, one is severe lethal infection in immunocompromised mice, and the other resembles self-limited disease manifestations in Balb/c mice with transient blockage of type I IFN responses. Further, we compared the infectivity of these two viral variants in a self-limited infection model, in which type I IFN receptor of wild-type Balb/c mice had been transiently blocked before infection, and found only the virus strain exhibiting larger plaque size caused infectious viral particles in sera. We have recently developed a ZIKV infection model in Balb/c mice with transient blockage of type I IFN Fig. 2 Phylogenetic analysis of DENV-2 1D4-5-SP and DENV-2 8H2-7-LP with representative serotype-2 dengue viruses of different genotypes isolated from different geographical regions.
cord-025948-6dsx7pey	2020	Direct massively parallel sequencing of SARS-CoV-2 genome was undertaken from nasopharyngeal and oropharyngeal swab samples of infected individuals in Eastern India. We have initiated a study on sequencing of SARS-CoV-2 genome from swab samples obtained from infected individuals from different regions of West Bengal in Eastern India and report here the first nine sequences and the results of analysis of the sequence data with respect to other sequences reported from the country until date. The A2a clade is characterized by the signature nonsynonymous mutations leading to amino acid changes of P323L in the RdRp which is involved in replication of the viral genome and the change of D614G in the Spike glycoprotein which is essential for the entry of the virus in the host cell by binding to the ACE2 receptor. We have also detected emergence of mutations in the important regions of the viral genome including Spike, RdRP and nucleocapsid coding genes.
cord-026641-eemp6b5j	2020	In the absence of NS1 apoptosis appears to be induced through the viral-RNA-mediated induction of retinoic acidinducible gene I (RIG-I) and interferon (IFN) signaling including protein kinase R (PKR) and eukaryotic initiation factor 2 alpha (eIF2α) activation and subsequent block of translation [39] [40] [41] . Another study screened a variety of wild-type influenza A viruses for their infectivity in pancreatic carcinoma cell lines and showed oncolytic effectiveness in a mouse model of human pancreatic cancer 67 . expressed a recombinant humanized cytotoxic T-lymphocyte-associated protein 4 (CTLA4) immune checkpoint inhibiting antibody from two different RNA fragments of the influenza A virus genome in order to enhance its anti-cancer effectiveness in a murine B16 melanoma model 96 . Further effects of oncolytic influenza A viruses on the cancer-immune microenvironment shown in murine models include activation of NK-cells and macrophage polarization towards immuno-stimulatory M1 phenotypes 66, 114 .
cord-027865-p1epjn51	2020	ISH is a method of localizing and detecting specific mRNA sequences in preserved tissue sections or cell preparations by hybridizing the complementary strand of a nucleotide probe to the sequence of interest. (See Data Sheet Kappa and Lambda Probe ISH Kit, Novacastra.) (Fig. 21.3.) Chromogenic in situ hybridization (CISH) is a method ''that enables the detection of gene expression in the nucleus using a conventional histochemical reaction'' (White 2005) ; it is used for the detection of abnormal genes and to identify a gene therapy treatment direction. However, FISH still has an advantage over chromogenic methods for labeling specific nucleic acid sequences in cells and tissues. This may be done by using the normal detection procedure for the ISH method on dots of labeled nucleic acid sequence and a labeled control applied at matching descending concentrations on a positively charged nylon membrane.
cord-027975-77544sed	2020	Several of the studied ssRNA characteristics, such as coat protein–RNA interactions and the ability to readily form virus-like particles in recombinant expression systems, have fueled many practical applications such as RNA labeling and tracking systems and vaccine development. Bacteriophages belonging to the family Leviviridae are among the simplest known viruses, exhibiting positive-sense single-stranded RNA (ssRNA) genomes of just 3.5-4.5 kilobases, typically encoding only 4 proteins. Due to their simplicity, ssRNA phages have been used as models to study various processes in molecular biology and virology, including translation repression, RNA-protein interactions and virus evolution. For quite some time, the only available structural information about protein-RNA interactions in ssRNA phage particles came from the studies of CP dimers in complex with a 19 nucleotide-long stem-loop fragment known as TR (translation repression) from the genome region located around the replicase start codon in bacteriophage MS2 ).
cord-029779-9b6zs1sb	2020	miR-181a inhibition increased neurite length in vitro in two different types of cell culture-the SH-SY5Y cell line and primary cultures of E14 rat midbrain-and increased expression of BMPR2 in differentiating SH-SY5Ys. Therefore, this miRNA may be a potential therapeutic target with neuroprotective effects. Analyses of previously performed microarray data showed altered expression levels in 70 miRNAs in umbilical cord whole blood in infants with moderate and severe HIE compared with controls and this data have been previously reported by our group [5] . The predicted downstream targets of the six miRNAs were predicted to exert neuroprotective effects, and functional examination of one of the associated pathways-the BMP signalling pathway (a member of the TGFβ superfamily [31] )-in differentiating SH-SY5Y cells revealed inhibition of miR-181a increases expression of the type II receptor BMPR2.
cord-030028-s6sxi8uj	2020	This technique has been used to differentiate isolates of some plant viruses, such as prunus necrotic ringspot virus (PNRSV), TYLCV and CTV (Gillings et al., 1993; Hammond et al., 1998; Font et al., 2007) ; iii) Single-strand conformation polymorphism (SSCP) analysis is based on electrophoresis of denatured dsDNA in non-denaturing gels so migration of single-stranded DNA depends on its conformation determined by its nucleotide sequence and the electrophoretic conditions. This technique followed by sequencing of the different haplotypes detected has been used to evaluate the genetic variation of some plant viruses, such as cucumber mosaic virus (CMV), citrus psorosis virus (CPsV) and CTV (Rubio et al., 1996; Rubio et al., 1999; Vives et al., 2002; Lin et al., 2003; Martıń et al., 2006) . Procedures to detect and identify various viruses or virus strains in a single assay simultaneously reduce time and cost of the analysis (see Pallaś et al., 2018 for a comprehensive review), and are especially suitable for evaluating mixed infections in individual plants.
cord-030654-8yxa1r1c	2020	This structure was revealed to be a horseshoe-like tetramer, which may play an essential role in nsp9 oligomerization and in the regulation of viral nucleic acid binding during the replication of the virus. The initial structure solved by molecular replacement showed that six SARS-CoV-2 nsp9 protomers form an OB-fold cluster in an asymmetric unit ( Supplementary Fig. 1a ). To obtain more information about the protein interfaces and the likely biological assemblies of the OB-fold cluster, we calculated the structure of SARS-CoV-2 nsp9 using PDBe-PISA [27] . These three contact surfaces in interface I b/c contribute a hydrophobic base with eight hydrogen bonds and one salt bridge, making the SARS-CoV-2 nsp9 tetramer extremely stable in the crystal structure. In this present study, we observed the nucleic acid-binding ability of SARS-CoV-2 nsp9, using the electrophoretic mobility The molecules in these two interfaces are shown as cartoons and colored and labeled as in Fig. 2a .
cord-031907-ilhr3iu5	2020	L.M., and the National Institutes of Health (R35GM119623) to T.R.G. The addition of a size exclusion chromatography step to various urinary extracellular vesicle concentrating methods reveals differences in the small RNA profile Introduction: Urinary extracellular vesicles (EVs) and their RNA cargo are a novel source of biomarkers for various diseases, however non-vesicular RNA (e.g. associated with proteins) is also present within urine. We then evaluated efficiency of heart targeting for eAAV9 or eAAV6 and standard AAV9 or AAV6 encoding for EGFP, mCherry or firefly luciferase in different human cell lines in vitro, in black mouse and in passive immunity nude mouse model in vivo using flow cytometry, confocal microscopy, Langendorff perfusion system and Methods: HLHS patients (n = 3) after Glenn procedure and swine (n = 3) after PAB were given RV injections of allogeneic/xenogeneic MSCs. Donor-specific, HLA-I+, exosomes were isolated from plasma.
cord-033692-txfuuu7d	2020	In this study, we investigated the molecular mechanisms of PRRSV infection by integrating the differences in the expression of various genes in tissues responsible for respiration (lungs) and immunity (bronchial lymph nodes [BLNs] , and tonsils) using RNA-Seq data at serial time points. To validate the results of specific groups, GSEA based on the KEGG database were performed using log 2 -normalised TMM counts at each time point for each tissue to identify the maximum gene expression changes for each group, and the GSEA results were consistent with the KEGG enrichment analyses of DEGs. GSEA using the 3-dpi data for all tissues revealed many significant pathways related to viral infection, among which influenza A showed the highest NES ( Figure 5A ). Significant KEGG terms related to viral infections and immune signalling were identified through KEGG enrichment analyses performed for each up-and down-regulated genes (343 and 287 genes, respectively), based on the time point for each tissue with a maximum FC value in the GCN, which was constructed using serial DEGs ( Figure 4A ).
cord-034648-vfqth54o	2020	To determine if gene expression changes were associated with changes in cellular response, transwell assays were performed to assess HTR-8 cell migration and invasion after exposure to 2.5% O 2 or 21% O 2 for 6hrs and 24hrs. Outcome parameters monitored were estradiol levels, follicle morphology and survival throughout the in vitro culture interval (IVC), germinal vesicle breakdown (GVBD), oocyte maturation to metaphase II stage, fertilization and development to blastocyst. Cell number in blastocysts was determined by Hoechst staining RESULTS: Pre-antral follicles measured 121.9 AE 40.9 mM with oocyte diameters of 61.1AE 4.1 mM at time of seeding. RESULTS: Based on scRNAseq data in non-human primate ovarian tissue, ACE2 and TMPRSS2 co-localize in a sub-population of oocytes in antral follicles (62% of cells, Pearson correlation¼0.37), but to a lesser extent in less mature oocytes and not at all in ovarian somatic cells.
cord-035067-ic843wr9	2020	Those infected may be asymptomatic, present typical symptoms (fever, dry cough and dyspnea), gastrointestinal symptoms (diarrhea, nausea, vomiting and abdominal pain) and viral RNA in stools. Information on country of origin, mean age, different comorbidities, typical symptoms (fever, cough, and dyspnea, among others), gastrointestinal symptoms (diarrhea, nausea, vomiting, and abdominal pain), and the presence of viral RNA in feces, when cited, were included in this study for analysis. (19) According to the descriptive, cross-sectional, multicenter study (three hospitals in Hubei, China) by Pan et al., with 204 patients, in which 107 were male, mean age of 52.91±15.98 years, 103 (50.5%) reported some gastrointestinal symptom, such as lack of appetite (81; 78.6%), diarrhea (35; 34.0%), vomiting (4; 3.9%), and abdominal pain (2; 1.9%). (26) Cipriano et al., conducted a systematic review with six studies of patients from China, which points to the possibility of SARS-CoV-2 infection in the gastrointestinal tract and fecal-oral transmission.
cord-035110-5lkzhjfh	2020	Another study reported that triple-negative breast cancer (TNBC) cells can activate stromal cells by releasing exosomes containing unshielded RNAs that mimic viral components to co-opt anti-viral immune responses, thereby promoting tumor growth [115] . Furthermore, CAF-derived exosomes contain intact metabolites, including amino acids, lipids, and TCA-cycle intermediates, which are internalized by prostate cancer cells to promote tumor growth [122] . EGFR carried in exosomes secreted from gastric cancer cells can be delivered to the liver and integrated into the plasma membrane of liver stromal cells, thus favoring the development of a liver-like microenvironment and promoting liver-specific metastasis [147] . In addition, abundant studies have demonstrated that tumor-derived exosomes can modulate the cell biology of MDSCs, including increasing their expansion, promoting their activation, and enhancing their immunosuppressive function [162] . Tumor-associated macrophages-derived exosomes promote the migration of gastric cancer cells by transfer of functional Apolipoprotein E
cord-048198-zjufx4fo	2001	Here we present results of a comprehensive co-variation mutagenesis study of equine arteritis virus TRSs, demonstrating that discontinuous RNA synthesis depends not only on base pairing between sense leader TRS and antisense body TRS, but also on the primary sequence of the body TRS. Synthesis of sg mRNAs initially was proposed to be primed by free leader transcripts, which would base-pair to the complementary TRS regions in the full-length minus strand, and would be extended subsequently to make sg plus strands ( Figure 1B ; Baric et al., 1983 Baric et al., , 1985 . 7220±7228, 2001 Using site-directed mutagenesis of TRSs of the arterivirus equine arteritis virus (EAV), we have shown previously that base pairing between the sense leader TRS and antisense body TRSs is crucial for sg mRNA synthesis (van Marle et al., 1999a) .
cord-048204-6lvn10f4	2000	In order to demonstrate the involvement of hnRNP A1 in MHV RNA replication and transcription, we established several DBT cell lines stably expressing either the wildtype (wt) hnRNP A1 or a C-terminus-truncated mutant lacking the M9 sequence and part of the glycine-rich domain. We showed that the mutant hnRNP A1, which was localized predominantly in the cytoplasm, exhibited dominant-negative effects on viral genomic RNA replication and subgenomic mRNA transcription. Cells infected with P0 viruses did not yield detectable amounts of DIssE, but contained the naturally occurring A59 DI RNA, whose replication was inhibited more strongly than the synthesis of MHV genomic and subgenomic RNAs in DBT-A1DC cells ( Figures 5B, lanes 8±10 and 6B, lanes 1± 3). In the present study, we established that MHV RNA transcription and replication were enhanced by overexpression of the wt hnRNP A1 protein, but inhibited by expression of a dominant-negative hnRNP A1 mutant in DBT cell lines.
cord-048222-1pq6dkl5	2005	With that prospect in mind, and with the aim of anticipating future standards by pre-normative research, we identified and tested two software packages recently developed to gauge the integrity of RNA samples with a user-independent strategy: one open source, the degradometer software for calculation of the degradation factor and ''true'' 28S:18S ratio based on peak heights (24) and the freely available RIN algorithm of the Agilent 2100 expert software, based on computation of a ''RNA Integrity Number'' (RIN) (25) . A RIN number is computed for each RNA profile (see Supplementary Table 4 online) resulting in the classification of RNA samples in 10 numerically predefined categories of integrity. The values of the mean fold changes, calculated according to the 2 ÀDDCt quantification method (see Materials and Methods), were found lower than 1.0, corresponding to the expression level (1·) in the sample exhibiting the highest RNA quality (Table 2 and Figure 5 ).
cord-048322-5eqdrd52	2006	The antitumorigenic effects of PEI/siRNA-mediated in vivo gene-targeting of tumor-relevant proteins like in mouse tumor xenograft models are described. The efficient protection against enzymatic or nonenzymatic degradation is particularly important for RNA molecules including siRNAs. In fact, while the therapeutic potential of siRNAs for the treatment of various diseases is in principle very promising, limitations of transfer vectors may turn out to be rate-limiting in the development of RNAi-based therapeutic strategies. Their ultimate success, however, will Figure 4 : Systemic treatment of mice with PEI-complexed HER-2-specific siRNAs leads to reduced growth of subcutaneous SKOV-3 tumor xenografts due to decreased HER-2 expression. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo Atelocollagenmediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo
cord-048327-xgwbl8em	2006	The ability of cis-acting RNA structures or trans-acting 2 0 -O-Methyl antisense oligonucleotides to induce ribosomal frameshifting was determined by in vitro transcription and translation of a dual luciferase reporter vector, p2Luc. p2Luc contains the Renilla and firefly luciferase genes on either side of a multiple cloning site, and can be transcribed using the T7 promoter located upstream of the Renilla luciferase gene (45) . As the AZ1A antisense oligonucleotide was designed to anneal directly adjacent to the UGA codon of the shift site, it was of interest to determine whether the wild-type antizyme pseudoknot could induce À1 frameshifting when located in the equivalent position. The ability of spermidine to stimulate antisense oligonucleotide induced ribosome frameshifting to the +1 reading frame at the UCC UGA shift site in the absence of the natural 3 0 stimulator demonstrates that this cis-acting element is not required for polyamine responsiveness.
cord-048471-7jszm1nd	2008	Although slightly larger than predicted the N-term protein was detected in a processed form in vivo, thus not only demonstrating initial translation of the ORF1 polyprotein but also activity of the viral protease. Previous studies of norovirus polyprotein processing have yielded three major products following in vitro transcription and translation, representing the uncleaved 3ABCD, N-term and 2C proteins. In addition, this important observation also confirms for the first time that the JV 3C protease was active in cells transfected with capped RNA as the size of the V5/N-term indicated successful cleavage of the protein from the ORF1 polyprotein. Unlike similarly studied human noroviruses JV initially did not appear to express the N-terminal protein, presenting the possibility that the encoding RNA sequence had a regulatory function itself, most likely involved in translation initiation in an IRES-like manner.
cord-048478-ftlb5b95	2008	One striking characteristic, which accompanies apoptosis in both vertebrates and yeast, is a fragmentation of cellular DNA and mammalian apoptosis is often associated with degradation of different RNAs. We show that in yeast exposed to stimuli known to induce apoptosis, such as hydrogen peroxide, acetic acid, hyperosmotic stress and ageing, two large subunit ribosomal RNAs, 25S and 5.8S, became extensively degraded with accumulation of specific intermediates that differ slightly depending on cell death conditions. For example, in metazoans the programmed cell death (PCD) called apoptosis, in addition to irreversible DNA damage, which is considered an apoptotic hallmark (3) , also involves specific cleavage of several RNA species, including 28S rRNA, U1 snRNA or Ro RNP-associated Y RNAs (4) . Together, this strongly suggests that rRNA degradation observed in apoptotic and oxidative stress conditions is not simply a result of cell death but is produced in the process that requires enzymatic activity and functional cellular machinery.
cord-048485-b8xb1f12	2008	RNA pools prepared from two infected piglets were used to compare whole mucosal gene expression at 12 and 18 hpi to expression in uninfected germ-free piglets (n = 3) using a porcine intestinal cDNA microarray. However, the fluorescence intensity was not higher than the background threshold for any of these probes, indicating that mRNA concentrations of these cytokines (including IFN-c) in infected and uninfected mucosal scrapings were too low to detect by microarray analysis, probably due to the relatively low percentage of cytokine-producing (immune) cells present in intestinal mucosa [6] . Using microarray analysis, we detected a set of genes that are differently expressed in rotavirus-infected jejunal mucosa compared to uninfected mucosa. Perhaps, enhanced expression of a PLA2-inh in our study may be a countermeasure of specific intestinal epithelial cells to normalize and/or inhibit PLA2 enzyme activity in response to extra-and intracellular changes in [Ca 2+ ] evoked by rotavirus replication, either to protect specific cells from extra-and intracellular membrane-damage or to negatively regulate A-acid production.
cord-102336-ex3zlq38	2020	Cryo-EM structures of transcription pre-initiation complex (PIC) and initiation complex (IC) of yeast mitochondrial RNA polymerase show fully resolved transcription bubbles and explain promoter melting, template alignment, DNA scrunching, transition into elongation, and abortive synthesis. Hence, the structural basis for promoter melting, DNA scrunching, and transcription initiation remains largely unknown for the mtRNAPs. RPO41 (∆N100) and MTF1 were assembled on a pre-melted promoter (-21 to +12, 15S yeast mtDNA promoter; Fig. 1A ) to generate the yeast mitochondrial transcription preinitiation complex (y-mtPIC) (Fig. S1 ). The 3.7 Å density map of IC locates many previously uncharacterized structural elements, including the C-tail of MTF1 and the scrunched DNA ( Fig. 2B-C) , and reveals the mechanism of template alignment and RNA synthesis during initiation. During the transition, the upstream DNA from position -1 onward is locked in the MTF1 NT-groove while the template strand undergoes a large conformational switching to align with the 2-mer RNA and the incoming UTPaS at the active site ( Fig. 2C; Movie S2).
cord-102412-cnlvyey4	2020	Results Here we outline several Galaxy workflows and learning resources for scRNA-seq, with the aim of providing a comprehensive analysis environment paired with a thorough user learning experience that bridges the knowledge gap between the computational methods and the underlying cell biology. The analysis of scRNA-seq within Galaxy was a two-pronged e ort concentrated on bringing high quality single-cell tools into Galaxy, and providing the necessary work ows and training to accompany them. The training pictographically guides users through the concepts of extracting cell barcodes from the protocol, explains the signi cance of UMIs in the process of read deduplication with illustrative examples, and instructs the user in the process of performing further quality controls on their data during the post-mapping process via RNA STAR and other tools that are native to Galaxy. A Galaxy-based training resource for single-cell RNA-sequencing quality control and analyses
cord-102547-nxut8ov1	2020	DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% to 99.6 % of the best reference genomes. Taxonomic classification of viral reads (Oxford Nanopore) and contigs (Illumina MiSeq) using MEGAN based on BLASTN matches against NCBI''s nucleotide (nt) database at the species and genotype levels. In order to confirm the genotype identification and to obtain a highly accurate whole genome sequence of the enterovirus genome, we subsequently subjected the sample E590 also to cDNA sequencing using Illumina MiSeq. In this case, the cDNA was produced using genotype-specific primers given that low number of reads was obtained by DRS and MiSeq . Illumina sequencing of the cDNA from sample E026 produced using oligo-dT primers and random hexamers showed similar distributions on the domain level: The majority of contigs belonged to bacterial species (98.6% and 98.9%, respectively for oligo-dT and random hexamer approaches), with a minority of reads mapping to eukaryotes (0.49%, 0.50%) and viruses (0.87%, 0.56%).
cord-102766-n6mpdhyu	2020	title: Short k-mer Abundance Profiles Yield Robust Machine Learning Features and Accurate Classifiers for RNA Viruses Machine Learning methods are becoming more reliable for characterizing sequence data, but virus genomes are more variable than all forms of life and viruses with RNA-based genomes have gone overlooked in previous machine learning attempts. We designed a novel short k-mer based scoring criteria whereby a large number of highly robust numerical feature sets can be derived from sequence data. Here, we present a novel short k-mer based sequence 28 scoring method that generates robust sequence information for training machine learning 29 classifiers. Here, we present a novel short k-mer based sequence 28 scoring method that generates robust sequence information for training machine learning 29 classifiers. VirFinder: a novel k-mer based tool for identifying viral sequences from 558 assembled metagenomic data.
cord-102866-40s64455	2020	To verify this activity and to determine whether Taq polymerasemediated reverse transcription might be leveraged for single enzyme RNA detection, we carried out the CDC-approved SARS-CoV-2-specific N1 TaqMan RT-qPCR assay using only Taq DNA polymerase and its accompanying commercial reaction buffer, ThermoPol, (New England Biolabs) seeded with different copies of N gene armored RNA (Asuragen), a commercial template preparation that is devoid of DNA. Even though the only polymerase present in these reactions was Taq DNA polymerase (NEB), and no dedicated reverse transcriptase was added, amplification curves were generated in response to 3 x 10 5 , 3 x 10 4 , and 3 x 10 3 copies of the SARS-CoV-2 N gene armored RNA templates (Figure 1 ). Similar to our results with armored N gene RNA, the NEB Taq DNA polymerase was able to perform TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA with all three CDC assays (Figure 2 ).
cord-102892-nt1zoktv	2020	Consensus tRNA primary 213 sequence with 2D structure for each isotype of each taxonomic domain was generated based 214 on the tRNA alignments used for building the isotype-specific covariance models in tRNAscan-215 SE 2.0 16 . 270 R2DT templates model the conserved core of most structured RNAs We classified each nucleotide in the resulting diagrams according to whether it matched a 282 template and found that 90.6% of nucleotides were displayed using the nucleotide locations 283 encoded in the templates, while 6.0% of nucleotides represented insertions compared to the 284 templates, and 3.4% of nucleotides matched the templates but required automatic repositioning 285 by the Traveler software (Table 2) . In addition, R2DT will benefit from the ongoing development Isotype-specific consensus tRNA sequences and 2D structures were generated using R-scape 52 389 from the alignments that were used to train and build the corresponding covariance models in 390 tRNAscan-SE 16 .
cord-102898-eyyd7ent	2020	Using ribosome profiling, we identify multiple mechanisms including frameshifting, tRNA dysregulation and alternate translation initiation sites that regulate viral protein synthesis. downstream polyprotein, 2) Significant modulation in levels of a distinct subset of ribosome-bound tRNAs 48 that cannot be explained by virtue of codon usage and 3) Translation from an upstream ORF (uORF) using 49 a non-canonical initiation codon in the 5 UTR region of JEV. However, these sites do not represent commonly associated Studies on RNA viruses have suggested adaptations in codon usage of viral genes to the host translation [30] . Interestingly, JEV infection appears to stimulate 251 expression from UUG start site by almost 67% suggesting viral or virus-induced host trans-regulatory factors 252 promoting uORF translation (Fig.4D) . We also identify a subset of ribosome associated 286 tRNAs whose levels are modulated globally upon JEV infection (Fig.3) .
cord-102931-vxkbctiz	2020	Here, we report that mitochondrial dysfunction in Caenorhabditis elegans activates RNAi-directed silencing via induction of a pathway homologous to the mammalian RIG-I helicase viral response pathway. elegans compared to most animals, and surprisingly, loss of function mutations in some of those genes cause an increase in the response to siRNAs: mutations in the RdRp RRF-3, the specialized Argonaute ERGO-1, the RNA helicase ERI-6/7, or the exoribonuclease ERI-1 enhance silencing by siRNAs (Fischer et al., 2008; Kennedy et al., 2004) . We find that reduction of function mutations in a wide range of mitochondrial components robustly enhanced RNA interference-mediated silencing of endogenous genes as well as a variety of reporters of RNAi. These antiviral responses to mitochondrial dysfunction are homologous to the RIG-I-based mitochondrial response in mammals because they depend on the RIG-I homologue, the DRH-1 RNA helicase. Our analysis of the eol-1 and drh-1 pathway from mitochondrial dysfunction to enhanced RNA interference and antiviral activity is a key output from mitochondria for anti-aging.
cord-102934-7e2mqooe	2020	Here, we established 60 total small RNA-sequencing profiles from 17 aggressive prostate cancer (PCa) patients tumor and adjacent normal tissue, and EVs isolated from urine, serum, and cancer cell culture media. We interrogated the key satellite alteration in tumor-derived EVs and found that resection of tumor prostate tissue leads to differential expression of reactive oxygen species (ROS), P53 pathways, inflammatory/cytokines, oncogenes, and tumor suppressor genes in the EV nanosatellites. We have conducted the total small RNA sequencing (n = 60) of aggressive prostate cancer patients (n = 17) tumor and adjacent normal tissue and EVs from urine, serum, and 22RV1 PCa cell line. We interrogated the key satellite alteration in tumor-derived EVs and found that resection of tumor prostate cancer tissue leads to differential expression of novel RNA biomarkers, ROS, P53 pathways, inflammatory/cytokines, major oncogenes, and tumor suppressors in the EV nanosatellites.
cord-102967-dx0tg077	2020	Coupled to PRO-seq in human blood samples, we propose to use PRO-seq as a single package method to detect (+)ssRNA virus RdRp activity and its interaction with host immune response through transcriptome-wide profiling of leukocyte gene expressions at once. This is different from Drosophila RNA Polymerase II, which appears to have comparable substrate specificity for all 4 biotin-NTPs. The ORF-proximal accumulation coincides with quadruplet rich regions of the template-strand of the DAV genome ( Figure 1D ). From this data, we did not detect significant PRO-seq sequences from (+)ssRNA viral genomes, indicating that none of the individuals had direct viral infections in the blood immune cells. Our PRO-seq data show expression levels of immune-response related genes from human peripheral blood leukocytes. PRO-seq density on DAV genome and base-quadruplet counts.The read count is indicated along the left side of each graph. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq)
cord-102968-mhawyect	2018	Results Here, we developed SilentMutations (SIM), an easy-to-use tool to analyze the effect of multiple point mutations on the secondary structures of two interacting viral RNAs. The tool can simulate destructive and compensatory mutants of two interacting single-stranded RNAs. This will facilitate a fast and accurate assessment of key regions, possibly involved in functional long-range RNA-RNA interactions and finally help virologists to design appropriate experiments. In this study, we present a tool called SilentMutations (SIM) that effectively simulates synonymous (silent) compensatory mutations in two single-stranded viral RNAs and is therefore appropriate for the in vitro assessment of predicted LRIs. Here, we present a command-line tool, called SilentMutations (SIM), that can simulate synonymous structure-destroying and structurepreserving mutation pairs within coding regions for long-range RNA-RNA interaction experiments. Figure 1 : Overall workflow of the SilentMutations tool, exemplary shown for two sequences from a negative single-stranded RNA virus genome (ssRNA-) (a) The first step will extract the defined range in each sequence and possibly increase the range to preserve codons in the given reading frame.
cord-103015-3dxwbmd2	2017	Molecular-docking simulations revealed a putative site of PIP binding to poliovirus (PV) 3C protein that was validated using NMR spectroscopy. 93 Validation of PIP-binding sites by NMR 94 In order to test the validity of our docking observations, we titrated 15 N-labeled 3C protein 95 with soluble dibutyl-PI4P to observe potential NMR chemical shift perturbations (CSPs), which 96 would indicate chemical environment changes in the presence of PI4P (Figure 2) . Out of the three 97 basic residues of the major cluster that were predicted to interact with the PI4P, R13 showed the 98 largest CSP (Figure 2A ). Titration of PI4P into a solution containing 3C caused CSPs that were consistent 249 with the major PI4P-binding site observed computationally (Figure 2A) .
cord-103163-0rreoh4o	2020	We determined the titers and RNA segment profiles of reovirus (rsT1L and rsT3D I ) and rotavirus (rsSA11) laboratory strains that had been rescued by reverse genetics then serially passaged ten times, each in triplicate lineages, in cultured cells. The two reoviruses accumulated non-canonical RNAs that retain 5′ and 3′ termini and feature one or more large internal deletions, while the rotavirus rarely accumulated such DVGs. Analyses of next-generation RNA-sequencing data sets from purified rsT1L reovirus RNA revealed many junctions, with hot spots for recombination in specific viral gene segments. Taken together, lin1 RNA profiles suggest non-canonical RNA species that differ in length but have identical termini to the parental segments accumulate variably during reovirus and rotavirus serial passage in cultured cells.
cord-103377-j1mmx7k7	2020	Since it was reported that RNase L activation increased translation of 3''UTR regions downstream of stop codons by interfering with factors that promote translation termination (eRF3) or ribosome recycling (ABCE1) (Le Roy et al., 2005) , we assessed the level of ribosome footprints in 3''UTRs. This level was assayed by computing the ratio of footprints in every 3''UTR relative to its respective main ORF within the coding sequence (density ratio, 3''UTR:ORF) for each transcript . The similarity in the effects suggests that translation of non-canonical regions occurs when RNase L is activated via naturally produced 2-5A from broad activation of the antiviral response by double-stranded RNAs. It should be noted that poly I:C treatment did result in slightly elevated 3''UTR ribosome footprints on some genes in a RNase L KO cell line (Supplemental Figure 4A ).
cord-103430-x6zzuu7v	2020	Among the many identified cellular interactors of SFV proteins, the enrichment of factors involved in translation and nonsense-mediated mRNA decay (NMD) was striking, reflecting the virus'' hijacking of the translation machinery and indicating viral countermeasures for escaping NMD by inhibiting NMD at later time points during the infectious cycle. Gene ontology (GO) enrichment analyses of protein complexes that could 10 form between the identified host interactors revealed highly significant GO terms related to 11 translation and Nonsense-mediated mRNA Decay (NMD). In addition to ribosomal proteins, many other RNA binding proteins were also 37 identified as having a potential role in SFV replication (Figure 3a (nsP1, nsP2, nsP3-Z, nsP4, capsid and Env) as well as for the merged list (All baits) was also applied.
cord-103511-31njndob	2020	Accordingly, sorted lung resident dendritic cells express 192 high levels of IFN-λ transcript after 5 days of poly (I:C) treatment, in contrast to epithelial cells, 193 alveolar macrophages and monocytes (Fig. 4A) , which, instead, express inflammatory cytokines (Fig. S8A, B) . Moreover, diphtheria toxin (DT)-mediated depletion of 195 CD11c + cells in CD11c-DT receptor (DTR) mice was sufficient to completely abolish IFN-λ 9 transcript and protein upregulation upon 6 days of poly (I:C) treatment (Fig. 4B, C) , while production remained unaltered (Fig. S8C with the response measured in vivo, TLR7 stimulation did not induce IFN production while it 207 induced upregulation of pro-inflammatory cytokines, and intracellular delivery of poly (I:C) induced 208 high levels of IFN-I but not IFN-λ (Fig.4D, Fig. S9A , B). Dendritic cells sorted from Ticam1 -/mice treated with poly (I:C) for six 222 days did not express appreciable levels of IFN-λ transcripts while still produced type I interferons 223 ( Fig. 4E, F) .
cord-103638-n5kpvsvg	2020	By extending the 3''or 5''-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discovered a new self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs were incorporated in a paper-based lateral flow assay that could detect the target with up to 23-fold higher sensitivity within 40-60 minutes. However, the ENHANCE showed 5.4-fold and 3.4-fold and higher trans-cleavage activity compared to the wild-type crRNA for targeting the methylated dsDNA and ssDNA, respectively (Fig. 3e, Supplementary Fig. 20a) . While no clinical samples were tested, the results indicated the 3''DNA7-modified crRNA consistently demonstrated higher sensitivity for detecting CoV-2 dsDNA within 30 minutes as compared to the wild-type crCoV-2 ( Supplementary Figs.
cord-103735-nil1vv6h	2020	Using a curated set of RNA oligonucleotides based on the ViroChip microarray assay [7] as baits in a hybridization capture system, multiple mammalian RNA and DNA viruses were detected from both eDNA and iDNA samples. Highlights Environmental DNA (water and blood-sucking leeches) provided a non-invasive method of screening wildlife for viruses A comprehensive viral RNA oligonucleotide bait set was developed to capture known and unknown mammalian virus diversity Leech blood meal host determination and viruses identified were congruent Viruses determined from water correlated with known and observed species visiting the water sources In brief Alfano, Dayaram, et al. In filtered water and sediment samples collected from the same waterhole, only one virus per sample was generally identified and in one location (WM20 and SM20) contigs from different viral families were isolated based on sample type. We provide evidence that environmental and invertebrate-derived DNA samples including waterhole water, sediment and wild haematophagous terrestrial leeches can be used to survey known and unknown viruses.
cord-103739-mmkrwj8t	2020	Metabolic labelling of newly-synthesized viral RNA followed by quantitative EM autoradiography revealed abundant viral RNA synthesis associated with DMVs in cells infected with the beta-CoVs MERS-CoV and SARS-CoV, and the gamma-CoV infectious bronchitis virus. In infected cells, the CoV RNA-23 synthesizing machinery associates with modified endoplasmic reticulum membranes that are 24 transformed into the viral replication organelle (RO). In infected cells, the CoV RNA-23 synthesizing machinery associates with modified endoplasmic reticulum membranes that are 24 transformed into the viral replication organelle (RO). 106 double-membrane spherules (DMSs) 107 We first set out to analyse the ultrastructure of MERS-CoV-infected Huh7 cells under sample 108 preparation conditions favourable for autoradiography (see Materials and Methods) (Fig 1, S1 109 Video). Association of polioviral proteins of the P2 89 genomic region with the viral replication complex and virus-induced membrane synthesis as 90 visualized by electron microscopic immunocytochemistry and autoradiography
cord-103787-qhftb6d7	2005	Scalable transcriptional analysis routine (STAR) represents a novel integration of reverse transcriptase-polymerase chain reaction and capillary electrophoresis that allows detection of dozens of gene transcripts in a multiplexed format using amplicon size as an identifier for each target. Scalable transcriptional analysis routine (STAR) represents a novel integration of reverse transcriptase-polymerase chain reaction and capillary electrophoresis that allows detection of dozens of gene transcripts in a multiplexed format using amplicon size as an identifier for each target. We have developed STAR (scalable transcription analysis routine), a gene expression analysis platform that represents an innovative integration of real-time multiplex PCR and capillary electrophoresis (CE), allowing the simultaneous quantitative measurement of multiple targets in a single sample with high sensitivity. In a typical STAR experiment (diagrammatically shown in Figure 1A ), a PCR reaction is set up in a single tube containing the analyte, common PCR reagents (eg, DNA polymerase, dNTPs), and, for each target to be amplified, gene-specific primers where at least one of each pair is labeled with a fluorophore.
cord-103807-x4hrwhkz	2020	Patients often present to the intensive care unit with broad phenotypes, including multiple organ dysfunction syndrome (MODS) resulting from infection, trauma, or other disease processes. Using multi-time point whole blood (cellular/acellular) total transcriptomics in 27 patients, we highlight the promise of simultaneously mapping viral/bacterial load, cell composition, tissue damage biomarkers, balance between syndromic biology vs. The power of RNA generated data likely is in its potential to effect therapeutic changes in individual patients with diverse clinical presentations, such as Multiple Organ Dysfunction Syndrome (MODS). Additional insights from the top mapped bacteria include normal flora elevation of Polynucleobacter necessarius and Bordetella parapertussis in patient 24 suggested to have issues in antigen processing and presentation (case study presented below for RNASEH2B), multiple Streptococcus strains (including pyogenes) identified in patients 11 and 5 that were culture positive, Pandoraea faecigallinarum in patient 10, and Cryobacterium arcticum in patient 10 day 0.
cord-103823-3rchp9yy	2008	Despite the computing power of supercomputers, computationally predicting secondary structures with thermodynamic methods is still not feasible when the RNA molecules have long nucleotide sequences and include complex motifs such as pseudoknots. Of course, the scientist who uses such an approach of sampling and rebuilding from segments to predict longer secondary structures has to benefit from the computing capabilities of such a framework without being required to cut and paste results from one code output to another, redirecting or reformatting output files (e.g., from FASTA to EMBL format) before forwarding them to the next step in the analysis, or dealing with distributed computer systems. RNAVLab addresses the challenges above by combining sampling of nucleotide sequences, predictions based on different codes and supported by grid computing technology, and analysis of large sets of secondary structures with different scientific scopes.
cord-103853-ar09nzmw	2020	20 In this work, we evaluate the performance of commonly-used packages capable of making thermodynamic predictions in two tasks that have been crowdsourced on Eterna and are emerging as central to RNA characterization and design: 1) predicting chemical reactivity data through calculating probabilities that nucleotides are unpaired, and 2) predicting relative stabilities of multiple structural states that underlie the functions of riboswitch molecules, a task that involves predicting affinities of both small molecules and proteins of interest. We evaluated commonly used secondary structure modeling packages in their ability to make thermodynamic predictions on a compilation of large datasets of diverse synthetic molecules from Eterna, which we termed EternaBench ( Figure 1A ). We trained models with a variety of combinations of data types to explore interactions in multitask training (Table 1) and evaluated performance on held-out test sets for single-structure prediction accuracy, chemical mapping prediction accuracy, and riboswitch fold change prediction.
cord-103899-6tqm99g1	2020	Hence, analyzing the role of these types of nucleotides in antiviral immune responses and the characterization of miRNA target genes might contribute to understanding the mechanisms of the interplay between the host and viruses, and in the future, potentially result in discovering therapeutic strategies for the prevention and treatment of acute COVID-19 infection. This review will summarize the recent discoveries associated with miRNAs in various respiratory infections caused by viruses, especially coronavirus, and address all feasible therapeutic options to mitigate the burden of VRIs. The humoral immunity is immunologically categorized as an acquired immune response in which T helper cells collaborate with B cells to differentiate these types of cells to plasma cells [17] [18] [19] . The immune responses against VRIs, such as IV, hRV, human coronavirus (HcoV), hMPV, and RSV, are correlated with the aberrant expression of several miRNAs in epithelial cells and participate in the pathogenesis of chronic and acute forms of respiratory disorders (Table 1 ) [16] .
cord-103914-ppgx7mci	2020	Here, we perform bulk RNA sequencing studies in laser-capture microdissected whole epithelium, FACS-sorted basal cells and cultured basal cells, as well as in vitro cell proliferation experiments, to address the intrinsic molecular differences between paediatric and adult airway basal cells. We found no significant differences in the proportion of cells in these three cellular compartments in paediatric and adult biopsies either by immunohistochemistry ( Figure 1A /1B), or by assessing basal, mucosecretory or ciliated cellassociated gene expression (Table S2 ) in bulk RNA sequencing in which we had laser-capture microdissected the whole epithelium ( Figure 1C ; Figure S1 ). Analysing this laser-capture microdissected whole epithelium RNA sequencing dataset using DESeq2 (Love et al., 2014) with a false discovery rate (FDR) of 1% and log2 fold change threshold of 1.2, we identified 37 genes with significant differential expression between paediatric and adult donors of which 17 were upregulated in adults and 20 were expressed at higher levels in children ( Figure 2A ; Table S3 ).
cord-103925-i73ymrov	2020	Finally, we use metabolic labelling and ribosome profiling to study 2A-mediated frameshifting and translation of the TMEV genome at sub-codon resolution in infected cells. A meta-analysis of the inferred P site positions of ribosomes relative to host mRNA start and stop codons reveals that RPFs map to coding sequences with a triplet periodicity reflective of the length of a codon ( Figure S3D ). Moving on to look specifically at the frameshift site, a single-nucleotide resolution plot of reads mapping to this region reveals a peak on the SS mutant genome corresponding to a ribosome paused with the GUU codon of the slippery sequence in the P site ( Figure 5G , Figure S4C ). These read lengths were selected for analysis as potential "disome-protected fragments", and their density plotted on the viral genome at the inferred P site position of the upstream, colliding ribosome ( Figure 6C and D).
cord-104162-fe51v2pt	2020	Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. While the regions are often also protein-encoding (e.g. NSP3, ORF3a), we suggest that their nucleic acid level functions – such as the ribosomal frameshifting element (FSE) that facilitates differential expression of 1a and 1ab polyproteins – can be considered potential "Achilles heels" for SARS-CoV-2, perhaps susceptible to therapies like those envisaged for AIDS. Assays of the base order-dependent component of the folding energy have shown that a highly conserved region, in otherwise rapidly mutating HIV-1 genomes, associates with an RNA structure corresponding, not to a protein-encoding function, but to an RNA packaging signal. This high GC% value can obscure the contribution of the base order-dependent component of the folding energy, which provides a sensitive indicator of local intraspecies pressures for the conservation of function within a population (i.e. a mutated organism is eliminated by natural selection so no longer can be assayed for function in the population).
cord-104186-fyw1xfgi	1996	Cells harboring the above hybrid UPFJ gene had the same three phenotypes as the mof4-1 strain, including: (i) elevated abundance of CYH2 precursor and frameshift reporter LacZ mRNA (Figures 2B and IC); (ii) inability to propagate the M1 killer virus (Table IV, Table IV , #5). A upflA strain (YGC106) containing the lacZ frameshift reporter construct in the zero or -1 frame relative to the translation start site (p315-JD86-ter or p315-JD85-ter) was transformed with a single copy plasmid harboring either the wild-type UPFJ gene, the mof4-1 allele or the vector alone. Thus, the higher expression level of the lacZ gene product in the -1 reading frame in mof4-1 cells as compared Hybrid genes between the wild-type UPFI and the mof4-1 alleles schematically represented in (A) were constructed, transformed into a upflA strain (Y52-) and CYH2 precursor abundance was determined by RNA blotting analysis as described in Figure 1 .
cord-122092-gdyt02er	2020	Comparison of different scenarios is based on tissue damage and viral load, highlighting the impact(s) of antibodies and adaptive cell-mediated immune response on infection dynamics. Surprisingly, our model also suggests that early treatment by either therapy alone can actually increase the duration of infection compared with a later therapy start, likely because suppressing virus production results in a reduced immune response. The model also includes non-structural proteins that are important for the viral life cycle, such as the replicase-transcriptase complex (RTC), and keeps track of the numbers of gRNAs (and subgenomic sgRNAs) at different stages of the replication process. We have included additional reactions into the model that describe remdesivir binding to the RTC complexes on the gRNAs and sgRNAs to capture this (see SI for details), and track the effect of a given, fixed number of remdesivir molecules per cell on the release of viral particles from an infected host cell.
cord-146406-85usg3uh	2011	Using next-generation sequencing (NGS) performed on a Genome Analyzer platform (Illumina), this study compared the viral populations within two bovine epithelial samples (foot lesions) from a single animal with the Inoculum used to initiate experimental infection. Some of the higher frequency polymorphisms identified encoded changes within codons associated with heparan sulphate binding and were present in both feet lesions revealing intermediate stages in the evolution of a tissue-culture adapted virus replicating within a mammalian host. The sequence diversity of viral populations within individual hosts is the starting 24 material for selection and subsequent evolution of RNA viruses such as foot--and--mouth 25 disease virus (FMDV). The sequence diversity of viral populations within individual hosts is the starting 24 material for selection and subsequent evolution of RNA viruses such as foot--and--mouth 25 disease virus (FMDV). Validation and analysis of sequence diversity in the samples 148 The frequency of site--specific polymorphisms was estimated from the frequency of 149 mismatches of the aligned reads to the reference genome.
cord-184744-oyc2djxk	2020	The present study evaluated the possibility of plant originated approved 117 therapeutics against the main protease protein (MPP), RNA-dependent RNA polymerase (RdRp) and spike protein (S) of SARS-CoV-2 including drug surface analysis by using molecular docking through drug repurposing approaches. The molecular interaction study revealed that Rifampin (-16.3 kcal/mol) were topmost inhibitor of MPP where Azobechalcone were found most potent plant therapeutics for blocking the RdRp (-15.9 kcal /mol) and S (-14.4 kcal/mol) protein of SARS-CoV-2. The main protease proteins, RNA-dependent RNA polymerase and spike protein of SARS-CoV-2 were employed to molecular docking study with the repurposed drug candidates from plant origin for find out the better drug option towards the COVID-19 pandemic. In the present study, five plantr based therapeutics such as Azobechalcone, Rifampin, Isolophirachalcone, Tetrandrine and Fangchinoline were suggested for potential inhibitors for the Main Protease protein, RNA dependent RNA polymerase and Spike protein of SARS-CoV-2 by using molecular docking based virtual screening study.
cord-243806-26n22jbx	2020	Here, we performed sequence and structural alignments among 62 SARS-CoV-2 strains and identified the conservation of specific elements in the spike S region, which provides clues on the evolution of domains involved in the binding to ACE2 and sialic acid. As highly structured regions of RNA molecules have strong propensity to form stable contacts with proteins 14 and promote assembly of specific complexes 15, 16 , SARS-CoV-2 domains enriched in double-stranded content are expected to establish interactions within host cells that are important to replicate the virus 17 . Analysis of functional annotations carried out with GeneMania 46 revealed that proteins interacting with the 5'' of SARS-CoV-2 RNA are associated with regulatory pathways involving NOTCH2, MYC and MAX that have been previously connected to viral infection processes ( Fig. 4E) 47, 48 .
cord-252268-o63ep08b	2014	As this equation relies on consistency between samples in the RNA quantity assayed, and the optimum reaction efficiency, as well as minimal fluctuation in the expression of housekeeping genes (endogenous controls) (Peters et al., 2007; Mane et al., 2008; Bruder et al., 2010) , these parameters were optimized using RNA extracted from cultured ferret lymph node cells stimulated with mitogens or with influenza virus. To test the ability of ferret leukocytes to produce mRNA cytokines and chemokines, lymph node cells from naïve ferrets were cultured with various mitogens known to activate T and B lymphocyte and macrophage/monocyte responses (Fig. 5) and with live or heat inactivated influenza virus (Fig. 6 ) and the cytokine and chemokine expression profiles were determined (summarized in (ConA) or Phytohaemagglutinin (PHA), which act by cross linking T cell receptors via sugars on the surface of human T lymphocytes (Chilson and Kelly-Chilson, 1989) , induced similar cytokine profiles, increasing expression of IL2, IL4, IL6, IL10, IL17, Granzyme A, TNF␣ and IFN␥, most with high fold changes, consistent with effective stimulation of T lymphocytes (Fig. 5) .
cord-252433-0e9lonq4	2009	As a result, HIV-1 mutants lacking a functional rev gene are able to express the proteins encoded by fully spliced viral mRNAs, that is, Tat, Nef, and the defective Rev protein itself, but cannot express any of the proteins encoded by incompletely spliced viral mRNAs, including Gag, Pol, and Env. The transcripts encoding these viral structural proteins, however, can be detected in the nucleus of cells infected by Rev-deficient viruses, where they are either degraded or eventually fully spliced and then exported (Cullen, 2003) . MPMV has a simpler genomic organization than HIV-1 and only encodes the three structural proteins Picornaviruses and some flaviviruses Recruits cellular translation factors and ribosomal subunits to viral translation initiation codons in the absence of an mRNA cap Gag, Pol, and Env. Nevertheless, MPMV expresses both a genome-length Gag/ Pol mRNA and a spliced Env mRNA.
cord-252466-usrpodjx	2012	Apparently, failure to develop the cellular immune response that would control dissemination of LASV, which is indicated by high serum virus titers, combined with disseminated replication in tissues and absence of neutralizing antibodies, leads to the development of fatal Lassa fever [64] . Downregulation of immune responses caused by LASV infection demonstrated in vitro is also in agreement with the results of clinical observations showing that fatal outcome of Lassa fever correlates with low levels or absence of interleukin (IL) 8 and IFN inducible protein 10 (IP-10) in circulation [70] . These data indicate that T cells are essential for rapid resolution of LASV infection; however, if the host fails to control virus replication due to inadequate activation of the immune system, T lymphocytes may play a key role in Lassa fever pathogenesis.
cord-252485-cxi3cr15	2015	We and other groups have recently reported that recombinant viruses of Sendai virus (SeV), a prototype of the family Paramyxoviridae, in which the C proteins are knocked-out or mutated, generate dsRNA in infected cells at levels similar to the production of IFN-β (Takeuchi et al., 2008; Irie et al., 2010) . These unusual RNAs exhibited distinct properties in infected cells in terms of encapsidation with the viral N protein and subcellular distribution with SG marker proteins and RLRs. Our results suggest that RNA-typedependent mechanisms recognize and accumulate virus-derived, IFN-β-inducible, unusual RNAs into specific compartment to trigger the production of IFN-β, and that SeV may evade detection by the host innate immune system by preventing the production of these RNA species. Since the naked cbDI genomes have been reported readily to form an ideal structure as the RIG-I ligands of 5 -triphosphated, blunt-ended dsRNA (Kolakofsky, 1976) , these results indicated that the major IFN-β-inducing viral RNA species produced in the cells infected with CNT was encapsidated cbDI genomes, whereas those for SeV-4C(-) and NDV were not.
cord-252871-qfrpuy3t	2020	We propose a new definition of viruses that is not restricted to the presence or absence of any genetic or physical feature, detail a scenario for how viruses likely originated from ancient cells, and explain technical and conceptual biases that limit our understanding of virus evolution. We propose a new definition of viruses that is not restricted to the presence or absence of any genetic or physical feature, detail a scenario for how viruses likely originated from ancient cells, and explain technical and conceptual biases that limit our understanding of virus evolution. In turn, the origin of archaeoviruses from Archaea, bacterioviruses from Bacteria, and eukaryoviruses from Eukarya also seems less likely as these viruses share several conserved protein folds involved in virion synthesis and other functions, indicating that they may have evolved prior to the diversification of LUCA into modern cells.
cord-253024-b393ea2u	1992	Six RNA+ mutants that reside within a single complementation group mapping within the S glycoprotein gene of MHV-A59 were isolated which did not cause syncytium at the restrictive temperature. Since these mutants do not produce syncytium at the restrictive temperature ( Fig. 1) and sequence analysis of RNA recombinant viruses suggested that the defect in LA7 mapped within the first 1 .l kb of the S glycoprotein gene (Banner et al., 1990; Keck eta/., 1987 Keck eta/., , 1988a Makino eta/., 1986b Makino eta/., , 1987 , these data provided an anchor for mapping the location of the remaining group F RNA+ mutants. Since the distance between the group Fl and F2 mutants would have required sizable deletions (>lOO bp) to result in ts+ virus (Fig. 4) these data suggested that large deletions were rare events in these crosses, and did not contribute to an increase in the recombination frequency within the S glycoprotein gene.
cord-253115-ekgdsv4f	2019	Commonly used drug delivery systems for respiratory diseases are polymer-based, lipid-based and peptide-based, and among these three, the lipid-based carriers are the most commonly used vectors for delivering RNAi. They include solid lipid nanoparticles, cationic liposomes, lipidoids, solid nanostructured lipid carriers and pH-responsive lipids [26] . The effective delivery of the drug and siRNA induced cell death of lung tumor cells by targeted gene silencing [56] . Glud et al., investigated pulmonary gene silencing effect of small interfering locked nucleic acid (siLNAs), targeting enhanced-greenfluorescent-protein (EGFP) in lung bronchoepithelium upon intravenous delivery of naked siLNAs and intranasal delivery of naked siLNA or chitosan based siLNA mucoadhesive nanoparticles. This study demonstrated that SAMiRNA nanoparticle is a stable siRNA silencing platform with less toxicity for effective in vivo targeting of genes involved in the pathogenesis of respiratory diseases [96] . Overcoming cisplatin resistance in non-small cell lung cancer with Mad2 silencing siRNA delivered systemically using EGFR-targeted chitosan nanoparticles
cord-253282-zwl0safn	2013	In previous studies, differences in the amount of genomic and subgenomic RNA produced by coronaviruses with mutations in the programmed ribosomal frameshift signal of ORF1a/b were observed. Here, analyses using synonymous protein coding mutations demonstrate that the region of the genome that harbors the frameshift signal affects the regulation of genomic and subgenomic RNA production without altering protein sequence. Here we describe deletion and mutagenesis experiments with a dual luciferase reporter to show that the effect the sequence between stems 1 and 2 has on frameshifting efficiency is due to structural changes those mutations cause in the pseudoknot. Similar to previously described viruses containing mutations in the slippery site of the frameshift signal [7] , here we show that mutations to the SARS-CoV frameshift stimulating mRNA pseudoknot can also affect the production of viral genomic RNA.
cord-253307-4bpdfgau	2014	Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut‐off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Therefore, in SINV nsP2 (P726G)-replicating BHK cells, a correlation exists between the inhibition of viral RNA replication and the release of nuclear proteins, in addition to the failure to completely block cellular mRNA translation. Thus, in agreement with previous results, these findings indicate that the shutoff of host translation after SINV infection is restricted when viral RNA synthesis is blocked and nuclear proteins are not released to the cytoplasm. Indeed, in cells co-transfected with rep C+luc(ΔnsPs) and EMCV(IRES)-nsP1-4 mRNA, there is low-level RNA replication, no release of nuclear proteins and the synthesis of cellular proteins is not blocked.
cord-253466-7gpije5d	2007	Significantly, Poliovirus infection causes enrichment of GEFs in membranes containing replicase proteins, and this would provide a mechanism for increasing levels of Arf1-GTP at sites of virus replication. There is evidence that Tobacco mosaic virus also uses the ER as a site of replication because the replicase enzyme and viral RNA are located on the ER of infected cells, and infection causes major changes in ER morphology (Reichel and Beachy, 1998) , including ER aggregation and formation of lamella structures. Even though these viruses infect a diverse range of hosts from different phyla, including vertebrates [poxviruses, African swine fever virus (ASFV)], arthropods (entomopox, ASFV, chloriridoviruses), amphibians and fish (Ranavirus, Megalocytivirus, and Lymphocystivirus genera of the Iridoviridae family), marine algae (phycodnaviruses), and protozoa (mimivirus), they all generate cytoplasmic factories as major sites of virus assembly and replication (illustrated in Fig. 4 ). Formation of DNA replication structures in herpes virus-infected cells requires a viral DNA binding protein
cord-253480-qchrw337	2016	Here, we found that the production of infectious DENV particles was significantly decreased by CBX treatment in DENV‐permissive cells, while the viral RNA and viral protein synthesis were not affected. Moreover, results from time-of-addition study showed that the inhibitory effect of CBX on DENV was exhibited by targeting the virus itself, not the host cells. In this study, we investigated whether CBX treatment inhibits dengue infection by measuring the production of progeny virions and viral RNA as well as viral protein expression. To determine whether CBX inhibits the production of infectious progeny DENV in other dengue-permissive cells, TCID 50 assay was performed to measure the virus titer in CBX-treated THP-1 and HUVEC cells, both of which are widely used in DENV studies [Halstead, 1988; Wu et al., 2000] . CBX treatment did not inhibit DENV-1 or DENV-2 RNA synthesis and protein expression in one replication cycle, but markedly reduced progeny virus The total RNA was isolated from the infected cells and analyzed by quantitative RT-PCR.
cord-253501-hkxlq3os	2018	[29] [30] [31] [32] Ribavirin inhibits host inosine monophosphate dehydrogenase, thereby depleting cellular GTP pools and blocking viral replication during HEV infection. Sofosbuvir, a prodrug of a uridine nucleoside analogue that acts as a direct-acting antiviral against hepatitis C virus (HCV) RNA-dependent RNA polymerase in its active form, was reported by Dao Thi et al. 63, 64 Zinc salts were shown to block the replication of both genotype 1 and genotype 3 HEV by inhibiting the activity of viral RNA-dependent RNA polymerase in cultured human hepatoma cells. Zinc directly inhibits HEV RNA-dependent RNA polymerase activity in vitro and displays moderate cooperativity with ribavirin in inhibiting viral replication in mammalian cell culture models of HEV infection. Ribavirin therapy inhibits viral replication on patients with chronic hepatitis e virus infection Zinc salts block hepatitis E virus replication by inhibiting the activity of viral RNA-dependent RNA polymerase
cord-253539-0kcujnfa	1996	It has been suggested that the genes for CP homologues arose by gene duplication that probably occurred in a common closterovirus ancestor; it is noteworthy that, despite significant divergence, the CP homologues of BYV and CTV have retained the profile of conserved amino acid residues that are believed to ensure the characteristic fold of the filamentous plant virus CPs (Boyko et al., 1992 , Dolja et al., 1991 . L I W RNA-2 contains genes for the small hydrophobic protein, the HSP7O homologue, the 59-kDa protein distantly related to the B W 64-kDa and CTV 61-kDa products, the 9-kDa protein, the 28-kDa CP, the 52-kDa protein whose C-terminal domain is homologous to the CP, and the 26-kDa protein The monopartite genome of another whitefly-transmissible closterovirus, cucumber chlorotic spot virus (CCSW, has a size of approximately 15.5 kb (Woudt et al., 1993a,b) .
cord-253616-7jyui5ca	2020	To investigate the anti-ZIKV activity of harringtonine, we assessed the inhibition of virus infection in Vero cells with MOI = 0.01 under different concentrations of harringtonine for 48 h. Intracellular viral RNA levels, protein expression levels and virus progeny in supernatants were respectively determined by RT-qPCR, western blotting and fluorescent focus assay (FFA). The dose-dependent anti-ZIKV activities of harringtonine were observed to decrease viral RNA/protein production and progeny yield ( Figure 1B-D) , indicating that virus propagation was suppressed. Harringtonine (625 nM) was administered at different stages of ZIKV infection with MOI = 0.1, and then the levels of ZIKV RNA in cells, as well as virus titers in supernatants, were determined after 24 h treatment. Harringtonine (625 nM) was administered at different stages of ZIKV infection with MOI = 0.1, and then the levels of ZIKV RNA in cells, as well as virus titers in supernatants, were determined after 24 h treatment.
cord-253862-jl1zhg13	2020	Although this novel virus is less severe than the first SARS-CoV outbreak, human-to-human transmission remains very high and the number of cases continues to rise exponentially in major urban areas, highlighting the urgent need to develop new containment, diagnostic, and treatment protocols. In the case of SARS-CoV-2, viral evasion of the innate immune system leads to an increase in cytokine production and late CD4+/CD8+ response, which then leads to pathogenic inflammation in patients with high viral loads. (ChiCTR2000029308), involving severe SARS-CoV-2 cases, compared lopinavir/ritonavir treatment with standard care alone, and they showed that the antivirals yielded no clinical benefits. In an open-label control study conducted by Cai et al., the antiviral activity of favipiravir + IFN-α was compared to that of lopinavir/ritonavir + IFN-α in patients with confirmed SARS-CoV-2 infection.
cord-254070-v9gabn1a	2015	For retroviruses, some base analog-resistant HIV strains were reported with reverse transcriptase mutations (e.g., M184V) that altered the fidelity of this RNA-dependent DNA polymerase. Several observations supported this last mechanism: G64S exhibited the same replication as wild-type virus in single-cycle infections; G64S generated fewer escape mutants to antiviral compounds; and G64S was also resistant to base analogs of different structure (Pfeiffer and Kirkegaard 2003; Vignuzzi et al. Finally, a high-fidelity polymerase variant was found for foot-and-mouth disease virus (FMDV, also in the Picornaviridae family), selected by serial passage in the RNA mutagen, 5-fluorouracil (5-FU). As with picornavirus high-fidelity variants, this variant was resistant to multiple RNA mutagens and generated populations with more restricted genetic diversity than wild-type virus. A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity
cord-254100-u6x5zd4i	2010	An increasing number of reports reveal that similar to the proteins of animal viruses, many plant virus proteins localize in the nucleolus to divert host nucleolar proteins from their natural functions in order to exert novel role(s) in the virus infection cycle. An increasing number of reports reveal that similar to the proteins of animal viruses, many plant virus proteins localize in the nucleolus to divert host nucleolar proteins from their natural functions in order to exert novel role(s) in the virus infection cycle. As their name suggests, MPs are involved in virus spread in infected plants, and the potential role of fibrillarin in this process will be discussed in Section IV.B. The multifunctional PVA (potato virus A)-encoded viral genomelinked protein (VPg) is also able to interact with fibrillarin (Rajamäki and Bonfiglioli et al.
cord-254192-86ksgl5t	2019	Here, to resolve the mechanism responsible for the disparity between IFN-λ3 and type I IFN in anti-mucosal virus infection, we compared the transcription profiles induced by the two IFNs in porcine intestinal epithelial (IPEC-J2) cells by RNA-Seq. Our results showed that the pretreatment of IPEC-J2 cells with IFN-λ3 resulted in the differential expression of 983 genes. Here, to resolve the mechanism responsible for the disparity between IFN-λ3 and type I IFN in anti-mucosal virus infection, we compared the transcription profiles induced by the two IFNs in porcine intestinal epithelial (IPEC-J2) cells by RNA-Seq. Our results showed that the pretreatment of IPEC-J2 cells with IFN-λ3 resulted in the differential expression of 983 genes. In this study, we comprehensively compared the transcriptional profiling of IFN-λ3-and IFN-α-induced genes in a porcine intestinal epithelial cell line (IPEC-J2) and verified the RNA-Seq results by reverse transcriptase quantitative PCR (RT-qPCR) in vitro, and further confirmed the transcriptional profile difference in crypt-derived porcine enteroids.
cord-254210-3mi2aop5	2011	title: Silencing of HTLV-1 gag and env genes by small interfering RNAs in HEK 293 cells Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. In the present study, siRNAs were used for inhibition of the expression of the HTLV-1 structural genes gag and env. Forty-eight hours after transfection, the expression of EGFP-target (gag or env) fusion proteins was observed directly under an inverted fluorescence microscope. HEK 293 cells were observed 48 h posttransfection under a fluorescence microscope to determine the silencing effect of siRNAs on Gag and Env protein expression in cultured cells (Fig. 2B and C) . Based on the fluorescence data, flow cytometry and real time quantitative PCR, two siRNAs targeted to the HTLV-1 gag gene reduced efficiently gene expression by different amounts compared to the negative siRNA, scrambled siRNAs and controls without siRNA.
cord-254250-l0v602x9	2020	title: A Novel RNA Virus, Macrobrachium rosenbergii Golda Virus (MrGV), Linked to Mass Mortalities of the Larval Giant Freshwater Prawn in Bangladesh De novo virus assembly revealed a 29 kb single-stranded positive-sense RNA virus with similarities in key protein motif sequences to yellow head virus (YHV), an RNA virus that causes mass mortalities in marine shrimp aquaculture, and other viruses in the Nidovirales order. rnaSPAdes assembly of combined libraries produced 38,826 contigs; 23 contigs, of average length 4560 bp, had similarity in protein sequence to YHV or gill-associated virus (GAV), but when the trimmed reads were aligned against the YHV genome (accession number GCA_003972805.1), no alignment was seen. rosenbergii were negative: MrNV and XSV, the causative agents of white tail disease [9, 10] ; MrTV, a virus associated with mass larval mortalities in China [15] , Spiroplasma eriocheiris [8] , and WSSV-shown to be able to infect M.
cord-254592-wa5il5go	2019	To quantify the effects of the most informative risk factors, averaged partial dependence was extracted from the random forests, describing the marginal predicted probabilities of severe virulence associated with each virus trait (Fig 4, S2 Table) . Predicted probability of classifying virulence as ''severe'' for each of the most informative risk factors in random forest models applied to all known human RNA viruses and zoonotic viruses only (primary tissue tropism, any known neural tropism, any known renal tropism, level of human-to-human transmissibility, primary transmission route, and any known vector-borne transmission). In both classification tree and random forest models, viruses were more likely to be predicted to cause severe disease if they caused systemic infections, had neural or renal tropism, transmitted via direct contact or respiratory routes, or had limited capability to transmit between humans (0 < R 0 � 1).
cord-254596-wsmnlnlk	2020	In this proof-of-principle study, we assessed direct RNA sequencing (DRS) using nanopore sequencing technology for fast whole-genome sequencing of viruses directly from clinical samples. DRS of total RNA extracted from three different enterovirus-positive stool samples produced long RNA fragments, covering between 59% and 99.6% of the most similar reference genome sequences. Here, we show that nanopore DRS can be used to correctly identify enterovirus genotypes from patient stool samples with high viral load and that the approach also provides rich metatranscriptomic information on sample composition for all life domains. In order to confirm genotype identification obtained via DRS and to obtain a highly accurate whole-genome enterovirus sequence, we also subjected sample E590 to cDNA sequencing using Illumina MiSeq. MiSeq sequencing library preparation was performed with stool material and following the routine diagnostic pre-treatment (as opposed to chloroform/bead treatment) given that low amount of patient stool sample was available (Figure 1 ).
cord-254713-ghcwfcx2	2015	RESULTS: From 351 frugivorous bats, we detected 14 coronaviruses from two endemic bats species, of which 13 viruses were identified from Pteropus rufus and one from Eidolon dupreanum, giving an overall prevalence of 4.5%. Studies which aimed to identify potential reservoirs of emerging human CoVs have revealed that the Betacoronavirus SARS-CoV was closely related to CoVs detected in bats, specifically members of the genus (Rhinolophus), which brought the hypothesis of a spillover of this virus to several animal species (including civet cats and raccoons) sold in Chinese markets as bushmeat for human consumption [9] [10] [11] . A total of 351 bats belonging to 3 endemic bat species of the family Pteropodidae were captured and sampled: Rousettus madagascariensis (n = 179), Pteropus rufus (n = 76) and Eidolon dupreanum (n = 96) ( Table 1) . In the context of this study, we detected 14 coronaviruses forming nine genetically distinct strains in two endemic Malagasy frugivorous bat species.
cord-254747-vox5xsgd	2018	Overall, current evidence indicates that the EndoU activity of CoV nsp15 is dispensable for viral RNA synthesis and virus replication in cell culture. It was first discovered that the EndoU activity of nsp15 mediates the evasion of host recognition of viral dsRNA by infecting primary macrophages with EndoU-deficient CoVs (Deng et al., 2017; Kindler et al., 2017) . Moreover, treatment with the PKR inhibitor did not affect IFN induction or RNase L-mediated ribosomal RNA degradation in the EndoU-deficient CoV infected-macrophages (Deng et al., unpublished data) . Macrophages infected by the EndoU-deficient CoVs exhibited an early, RNase L-mediated degradation of ribosomal RNA, demonstrating that the OAS-RNase L system was activated (Deng et al., 2017; Kindler et al., 2017) . Lack of MDA5 expression or treatment with the PKR inhibitor did not affect virus-induced RNA degradation (Deng et al., 2017; Kindler et al., 2017) , suggesting that the nsp15-mediated blockage of OAS-RNase L activation is independent of the MDA5-IFN and PKR pathways.
cord-254895-ym0jsir5	2008	Dendritic cell subpopulations express specific nucleic acid recognition receptors belonging to the Toll-like receptor family (TLR3, 7, 8, 9) and the cytosolic RNA helicase family (RIG-I, MDA5, LGP2). Apart from activating the NFkB and MAPK signaling pathways leading to inflammatory cytokine and chemokine production as well as costimulatory molecule expression, the intracellularly localized nucleic acid recognition receptors TLR3, 7, 8 and 9 specifically trigger type I interferon production via MyD88-and TRIF-dependent signaling pathways. Thus, TRAF6 seems to be required for NFkB activation but not IFN-b induction downstream of IPS-1 which is mainly mediated by TBK1/IKKe. In vitro studies performed with primary cells obtained from RIG-I knockout mice confirmed that RIG-I plays an essential role in eliciting immune responses against specific negative strand and positive strand RNA viruses such as NDV, SeV, VSV, Japanese encephalitis virus (JEV) and Influenza virus in various cell types with the exception of pDCs .
cord-254903-g9ropt9c	2020	RESULTS: In the present study, we used small RNA sequencing (sRNA-seq) to detect viruses in ticks and discovered a new MGTV strain in Amblyomma testudinarium ticks collected in China''s Yunnan Province in 2016. In the present study, we identified a new MGTV strain Yunnan2016 detected in Amblyomma testudinarium ticks [17] and aimed to achieve the following research goals: (1) establish a method to generate the full-length genome sequence of an RNA virus using sRNA-seq data; (2) determine the feasibility of using the sRNA-seq based method in the detection of viruses in a small tick; and (3) provide a high-quality and well-curated reference genome for future studies on MGTV, JMTV, KITV and GXTV.
cord-254916-y1rw9q11	2020	The overall level of amino acid sequence identity of viral proteins ranges from about 65 % in the least conserved parts of the S protein to about 95 % in the most conserved replicative enzyme domains, prompting the coronavirus study group of the International Committee on the Taxonomy of Viruses to classify the new agent within the species Severe acute respiratory syndrome-related coronavirus, which also includes the 2003 SARS-CoV [1] . In this report, we describe a comparative study of the basic replication features of SARS-CoV and SARS-CoV-2 in Vero E6 cells, including growth kinetics, virus titres, plaque phenotype and an analysis of intracellular viral RNA and protein synthesis. One of them is the rapid evolution -during virus passaging in Vero cells -of a specific region of the SARS-CoV-2 S protein that contains the so-called furin-like cleavage site.
cord-254963-cnvxlv6h	2019	In order to test this newly expanded probe panel and to specifically assess the effect of hybridization-based viral enrichment on the sensitivity of HTS for detection of a single virus within a complex environmental sample, commercial bat guano was spiked with increasing concentrations of Influenza virus (IFV). As expected, a dose-dependent effect in the proportion of sequencing reads derived from IFV was observed as the number of spiked genome copies increased ( Fig. 1a and Additional file 1: Table S1 ), in both the unbiased shotgun sequence data as well as the virus enriched sequence data. Such limitations have been of particular concern for U.S. Department of Defense (DoD) laboratories tasked with biosurveillance and biodefense activities in regions with limited material resources and human We demonstrate here that hybridization-based viral target enrichment yields robust coverage of small genomes from clinical samples, even yielding full-length, deeply covered genomes at concentrations whereby Fig. 3 Detection of close relative viruses irrespective of extensive multiplexing.
cord-255090-2gpsu1y4	2009	In addition, variations within multiple regions of the quasispecies of FMDV were retrospectively revealed by sequencing of FMDV genes, and a single nucleotide substitution was identified as the main factor in resistance to RNAi. Our data demonstrated that the three siRNA molecules synthesized with T7 RNA polymerase could have transient inhibitory effects on the replication of FMDV. Recently, several laboratories used RNAi to inhibit foot-and-mouth virus infection in cell culture or in animals taking forms of chemically synthesized siRNA, plasmid encoding shRNA, and adenovirus encoding shRNA (Chen et al., 2004 (Chen et al., , 2006 de los Santos et al., 2005; Kahana et al., 2004; Mohapatra et al., 2005) , showing rapid decrease of FMDV replication. Furthermore, among the five siRNAs candidates, three siRNAs were confirmed efficacious to transiently inhibit the replication of FMDV in BHK-21 cells by determining the relative viral amount within infected cells and the virus titers in supernatants as well as the observation of the emergence of CPE.
cord-255252-md0avnqg	2007	SARS‐CoV viral load and SARS‐CoV/GAPDH RNA ratio for each organ type were related to four time durations: onset of illness to death, death to post‐mortem tissue sampling, and total durations of treatment with ribavirin and hydrocortisone. Post-mortem tissues were collected with great care from the major organs including heart, kidney, liver, spleen, lung, small bowel, psoas (skeletal) muscle, and bone marrow. Figures 1-6 show the results, using semi-log plots, for each organ: heart, kidney, liver, spleen, lung, and small bowel, respectively, for SARS-CoV, GAPDH and the SARS-CoV/GAPDH RNA ratio. In the organspecific viral load results, the overall picture made up from the data points from the seven different patients with different durations of SARS illness, generally, the SARS-CoV/GAPDH RNA ratio never reached above one in heart, kidney, liver, and spleen tissue for all x-axis parameters analyzed.
cord-255495-xnoppq3y	2020	It is possible that this "Trojan horse" strategy represents possible explanation for the re-appearance of the viral RNA in the recovered COVID-19 patients 7–14 day post discharge, suggesting that viral material was hidden within such exosomes or extracellular vesicles during this "silence" time period and then started to re-spread again. The fact that SARS-CoV-2 can be present within the vacuoles or double membrane vesicles (DMVs) within the host cells was proven by the careful post-mortem histopathological analysis of the renal samples of patients with COVID-19 by light microscopy, electron microscopic examination, and immunostaining (Farkash et al., 2020; Su et al., 2020) . Is this "Trojan horse" strategy of the release of the SARS-CoV-2-loaded exosomes or EDMVs represent a reasonable explanation for the appearance of the viral RNA in the recovered COVID-19 patients 7-14 day post discharge?
cord-255499-31xmue1g	2008	In general, homologous recombination events occur much more often and they are most commonly known as genetic crossing-over that happens in every DNA-based organism during meiosis. The mechanism may involve either homologous crossing-over events or copy-choice processes that rely on template switching by DNA replicase. Aberrant homologous recombination involves crossovers between related RNAs, but the crosses occur at not-corresponding sites leading to sequences insertions or deletions. A double-stranded RNA Pseudomonas phage Phi6 was hypothesized to recombine its RNA based on a copy-choice template switching mechanism, where the crossovers would have occurred inside the virus capsid structures at regions with almost no sequence similarity. In some cases, the base pairing between a partial nascent strand and the acceptor template can lead to the appearance of the rearranged regions in DI RNAs. In addition to rearranged DI RNAs, some RNA viruses accumulate defective RNAs due to a single internal deletion in the genomic RNA of the helper virus.
cord-255545-nycdhdsd	1999	In theory, sense-specific measurement of viral RNAs may be achieved by reverse transcription polymerase chain reaction (RT-PCR) assays which utilize primers of defined polarity during the RT step. Key RT-PCR parameters which were optimized include the design of tagged primers, DNase treatment of in vitro transcribed RNA standards, specification of temperature differences between RT and PCR annealing steps, and use of competitive RNA templates for quantitative assays. In fact, several studies have demonstrated that RT-PCR assays based on specific RNA template recognition by RT primers of defined polarity will not reliably distinguish between viral RNAs of positive or negative sense. Despite the findings above, many published research reports are based on conventional RT-PCR assays, relying on the polarity of primers added to the RT step in putative sense-specific measurements of viral RNAs; rarely are control reactions performed to rigorously show that this method is in fact sense-specific.
cord-255576-738khdwv	2012	We have previously shown that Tax interacts directly with the cellular Rb (Retinoblastoma) protein and targets Rb for degradation via the proteasome pathway, resulting in a decrease in Rb protein expression in HTLV-1 infected cells and a dysregulation of the cell cycle [47] . Collectively, these data indicate that the Drosha in Tax-containing and HTLV-1 infected cells is mostly functionally inactive and the functional suppression of Drosha is dependent on its interaction with a small region of the N-terminus of Tax. We have shown above that Drosha is downregulated, degraded, and mostly inactive in HTLV-1 infected cells, however, it was not clear what effect this dysregulation of Drosha would have on viral replication. Collectively, these data indicate that proteins, such as IKK-b, among others, may directly be regulated by the Tax/Drosha interaction in HTLV infected cells.
cord-255607-dbexsugq	2020	Our preliminary results revealed that endogenous TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), the key components in the IFN signaling pathway were downregulated in PEDV infected IPEC-J2 cells by iTRAQ analysis. Moreover, PEDV nsp15 can directly degrade the RNA levels of TBK1 and IRF3 dependent on its EndoU activity to suppress IFN production and constrain the induction of IFN stimulated genes (ISGs), by which PEDV antagonizes the host innate response to facilitate its replication. Previous studies suggest that PEDV can restrain host innate immune response by different strategies, such as by degradation or cleavage of key factors essential the IFN signaling pathway [25, 26] , competitive interaction between viral encoded proteins and modulators for IFN production [27, 28] , or localization changes of antiviral components [29] .
cord-255619-5h3l6nh6	2013	title: Evolution of infectious bronchitis virus in Taiwan: Positively selected sites in the nucleocapsid protein and their effects on RNA-binding activity This study illustrates that the N protein of the current TW IBV variant has been shaped by both RNA recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the RNA-binding capacity of the N protein. This study illustrates that the N protein of the current TW IBV variant has been shaped by both RNA recombination and positive selection and that the latter may promote viral survival and fitness, potentially by increasing the RNAbinding capacity of the N protein. In this study, we showed that substituting either of the positively selected residues with the amino acid present in most CH IBVs dramatically reduced the binding capacity of the N protein for synthetic TRS repeats (Fig. 4) .
cord-255738-r8zfdsix	2007	Sequence analysis of the replicon RNA purified from SCR-1 cells soon after selection in blasticidin (passage number 6) found no sequence differences compared with the published sequence of SARS-CoV strain SIN2774 (GenBank Accession Number AY283798). Baric''s group constructed a transmissible gastroenteritis virus (TGEV) replicon for the expression of heterologous GFP gene (Curtis et al., 2002) and Thiel''s group generated a non-cytopathic, selectable replicon RNA (based on HCoV 229E) for the identification of coronavirus replicase inhibitors (Hertzig et al., 2004) . Compared to anti-viral agent identification systems based on purified proteins or nucleic acids, our SARS-CoV replicon cell line has two advantages: first, if a candidate inhibitor can inhibit replication of our replicon RNA, which occurs intracellularly, it thus demonstrates that this agent can permeate the cell. To obtain the complete sequence of the SARS-CoV replicon persisting in the SCR-1 cells, the total cellular RNAs isolated from SRC-1 cells at passage number 6 and 40 were used as the templates.
cord-255795-su7f5ges	2020	Here, testing a pooling approach for the standard RT-qPCR test, we find that a single positive sample can be detected even in pools of up to 32 samples, with an estimated false negative rate of 10%. We hope that such implementation of a pool test for COVID-19 would allow expanding current screening capacities thereby enabling the expansion of detection in the community, as well as in close integral groups, such as hospital departments, army units, or factory shifts. Here, we test the ability of the standard RT-qPCR test for detecting a single positive sample within a pool of negative samples. Pooling clinical RNA samples, we tested previously confirmed positive samples alone and combined with an increasing number of previously confirmed negative samples and found that positive samples can still be well observed in pools of up to 32 samples, and possibly even 64 with additional PCR cycles. We found that a single clinical sample with SARS-CoV-2 RNA can be consistently detected in a pool of up to 32 samples.
cord-255883-mz6nyisw	2020	Essential oils (EOs) have long been known to have anti-inflammatory, immunomodulatory, bronchodilatory, and antiviral properties and are being proposed to have activity against SARC-CoV-2 virus. An in vitro study conducted by Hoffmann and colleagues revealed that SARC-CoV-2 depends on cellular serine protease (TMPRSS2) for S proteins priming which are known to interact with human ACE2 receptors in the lungs and facilitate entry into the cells. The authors opted the following keywords to find relevant studies: "essential oils", "antiviral", "COVID-19", "SARC-CoV-2", "bronchodilation", "immunomodulatory'''', "anti-inflammatory'''', "corona virus''''. Thus, on the basis of these docking and in vitro studies, it is proposed that garlic essential oils and their isolated constituents, especially DAS, have potential to prevent the entry of virus into host cells as well as to activate molecular antioxidant pathways that decrease the secretions of culprit pro-inflammatory cytokines. Essential oils have long been known to have anti-inflammatory, antioxidant, immunomodulatory, and antiviral properties and are being proposed to have activity against SARC-CoV-2.
cord-256036-gd53s4dv	2013	In contrast to human hepatocytes, murine cells do not support HCV entry thereby creating a first and important barrier for a broader host tropism of the virus. Utilizing blocking antibodies specific to CD81 or the viral envelope protein E2, expression of entry factor mutants and mice with a targeted disruption of the SCARB1 gene validated uptake of HCV into murine hepatocytes in an HCV glycoprotein-mediated fashion. Taking advantage of the high mutational plasticity of HCV, three adaptive mutations in the viral glycoproteins E1 and E2 were identified that allowed the virus to enter cells expressing human SCARB1, CLDN1, OCLN and mouse CD81. Recently, a genetically humanized mouse model was constructed utilizing cell culture produced recombinant hepatitis C virus to activate a cellular encoded reporter (Dorner et al., 2011, in press ). Human occludin is a hepatitis C virus entry factor required for infection of mouse cells A humanized mouse model to study Hepatitis C virus infection, immune response, and liver disease
cord-256325-q70rky3r	2017	title: A Functional Genomics Approach to Henipavirus Research: The Role of Nuclear Proteins, MicroRNAs and Immune Regulators in Infection and Disease Here we largely focus on findings from two recent RNAi screens to identify protein-coding genes and host-encoded microRNAs impacting the henipavirus infection cycle in human cells. X-ray data have suggested that the methylation of rRNA requires the formation of this complex with involvement of four fibrillarin molecules interacting with different regions of the target rRNAs. The yeast equivalent of fibrillarin, NOP1, has been more extensively studied than the human counterpart but fibrillarin is a well-conserved protein in most organisms, reinforcing the notion that all post-transcriptional processes involving fibrillarin such as chemical modification (methylation) of rRNA, pre-rRNA cleavage and ribosome assembly are essential for proper cellular functioning (Rodriguez-Corona et al. Deffrasnes and colleagues showed that siRNA-mediated knockdown of fibrillarin expression dramatically reduced HeV protein production and viral genome replication but did not impact viral fusion, and that fibrillarin catalytic activity was essential to henipavirus infection.
cord-256370-cz88t29n	2016	This is the first report on isolation of an orthoreovirus from an arthropod host associated with bats, and phylogenetic and sequence data suggests that MAHLV constitutes a new species within the Orthoreovirus genus. Maximum Likelihood trees were prepared using amino acid sequences of all open reading frames from all segments, showing the placement of Mahlapitsi virus (MAHLV) in the Orthoreovirus genus relative to other viruses in this genus for which sequence is available on Genbank. A Maximum Likelihood tree, constructed with nucleic acid sequence data for the RNA-dependent RNA polymerase (RdRp) encoding segments of representative viruses from the different genera within Reoviridae (Figure 7) shows the placement of both isolates amongst other orthoreoviruses in the family. Maximum Likelihood trees were prepared using the deduced amino acid sequences from the open reading frames (ORF''s) of all the virus'' segments and those of other viruses in the Orthoreovirus genus (Figures 8-10) .
cord-256444-grw5s2pf	1997	Publisher Summary This chapter discusses the manipulation of clones of coronavirus and of complementary DNAs (cDNAs) of defective-interfering (DI) RNAs to study coronavirus RNA replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. This decade has seen the manipulation of these clones, and of complementary DNAs (cDNAs) of defective-interfering (DI) RNAs, to study coronavirus RNA replication, transcription, recombination, processing and transport of proteins, virion assembly, identification of cell receptors for coronaviruses, and processing of the polymerase. The species and tissue specificity of a coronavirus infection is a t least partially dictated by the nature and distribution of cellular receptors and other related molecules that regulate virus entry, as evidenced by the viral replication that results when viral RNA is directly introduced into cell types of other animal species.
cord-256508-ce59ovan	2020	To date, with the exception of intravenous Remdesivir and dexamethasone, which have modest effects in moderate to severe COVID-19, no strong clinical evidence supports the efficacy and safety of any other drugs against SARS-CoV-2. The current diagnostic strategy to identify patients with COVID-19 is to test samples taken from the respiratory tract to assess for the presence of SARS-CoV-2 specific nucleic acid targets [47] . The neutralization assay is a laboratory-based test that uses live virus and cell culture methods to determine if patient antibodies can prevent viral infection in vitro [72] . A randomized, controlled, openlabel trial involving hospitalized adult patients with confirmed SARS-CoV-2 infection and severe respiratory illness COVID-19 was performed [126] . Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals
cord-256510-orr2roxz	2012	For example, transmission electron microscopy (TEM) of cells infected with coxsackievirus showed intracellular organized lattices (Fig. 1E) , very similar to those assembled by the viral RNA polymerase in vitro (Kemball et al., 2010) close relationship between self-interaction and replication activity is reported for viral polymerases of other viruses such as FHV (Dye et al., 2005) , hepatitis C virus (Qin et al., 2002) and RUBV (Risco et al., 2012) . For viral genome replication, the virus first assembles cytoplasmic mini-nuclei with attached mitochondria (Tolonen et al., 2001) ; virus morphogenesis then starts an aggresome-like structure (Risco et al., 2002) , where immature viruses assemble using an atypical membrane remodelling mechanism that has been characterized by ET (Chlanda et al., 2009) . Although we are beginning to understand how replication organelles are assembled, information is still limited about how cell organelles are recruited, about the mechanisms of macromolecular transport between compartments, and about the signals that regulate the major structural changes in the factory during distinct stages in the virus life cycle.
cord-256561-fnh2do4z	2014	Based on these results, we propose the following consensus for designing intranasal antiviral siRNAs: (a) modified 19–27 nt-long double-stranded siRNAs are functional in the lung, (b) excessive 2′-OMe and 2′-F modifications in either or both strands of these siRNAs reduce efficacy, (c) limited modifications in the sense strand are beneficial, although their precise efficacy may be position-dependent, (d) cocktail of multiple siRNAs can be highly effective against multiple viral strains and subtypes. Therefore, enhancement of the intracellular and extracellular stability of synthetic siRNAs while increasing (or without compromising) their RNAi activity is a continuing goal for therapeutic translation of RNAi. A variety of chemical modifi cations, including terminal and internal ones, have been added to the fi rst-generation siRNA sequences to improve stability and delivery, leading to what we call "second-generation" siRNAs. Advantage has been taken of the free 2′-OH group of the ribose moiety of RNA (in contrast to DNA that lacks this OH group), to which various substituents were added.
cord-256615-gvq8uyfk	2014	RNA viruses, with their high potential for mutation and epidemic spread, are the most common class of pathogens found as new causes of human illness. An analysis of virus discovery indicates that the small number of novel viruses discovered annually is an artifact of inadequate surveillance in tropical and subtropical countries, where even established endemic pathogens are often misdiagnosed. Many of the emerging viruses of the future are already infecting humans but remain to be uncovered by a strategy of disease surveillance in selected populations. Despite the differences in clinical presentation and geographical location, these three pathogens share three characteristics: all were unknown before found infecting humans, all are RNA viruses, and all have proven or putative non-human, animal sources. A single subtropical bat species hardly represents all mammal species and indeed many viruses are known to infect more than one species; they tested for only 9 of the 25 virus families pathogenic to humans.
cord-256918-mauzesor	1998	Objectives: To discuss indeterminations inherent to a quasispecies structure and to the analytical procedures to define it, biological implications of quasispecies, and the need to take into account this type of population structure, in order to design effective strategies to prevent and control diseases caused by highly variable viruses. Results: Quasispecies have many biological implications, extending from viral pathogenesis to the emergence of new pathogens, rapid antigenic variation, and alterations in cell tropism, virulence, host range and viral gene expression. Since adaptability of RNA viruses is key to viral pathogenesis and strategies for disease control, it would seem obvious that quasispecies should have been regarded as highly relevant to these questions (Domingo, 1989 (Domingo, , 1996 Domingo and Holland, 1992; Duarte et al., 1994; Novella et al., 1995) . The evolution of an RNA virus may be rapid or slow, but mutant swarms are always hidden in their replicating genome populations.
cord-256940-yuja99jg	2020	title: Long-term positive severe acute respiratory syndrome coronavirus 2 ribonucleic acid and therapeutic effect of antivirals in patients with coronavirus disease: Case reports Despite treatment with recombinant human interferon, convalescent plasma from COVID-19 patients, arbidol, etc., nucleic acid results were still positive for SARS-CoV-2. After treatment with ritonavir-boosted danoprevir (DNVr, 100/100 mg, once daily), all four patients showed two to three consecutive negative SARS-CoV-2 RNA and were thus discharged from hospital. Therefore, DNVr may be a potentially effective antiviral for COVID-19 patients with long-term positive SARS-CoV-2 RNA. However, some COVID-19 patients have been reported to have long-term positivity for SARS-CoV-2 ribonucleic acid (RNA). On April 5, after three consecutive negative nucleic acid test results, he was discharged and transferred to another hospital for further treatment of comorbidities. Thus, DNVr may be a potential antiviral for COVID-19 patients with long-term positive SARS-CoV-2 RNA.
cord-257456-15bm9psj	2020	Using COVID-19 clinical specimens, we have collected evidence that the RT-qPCR assay can feasibly be performed directly on patient sample material in virus transport medium (VTM) without an RNA extraction step, while still producing sensitive test results. Using COVID-19 positive clinical specimens, we demonstrated that RT-PCR assays can be performed in as little as 12 min using untreated samples, heat-inactivated samples, or extracted RNA templates with our low-cost water bath setup. To further improve the speed of a diagnostic assay, we and others tested using untreated or heat-inactivated samples added directly to one-step RT-PCR master mixes without an RNA extraction step [6, [13] [14] [15] [16] [17] [18] [19] . Prior to COVID-19 emerged as a global pandemic, we have tested the feasibility of circumventing the sample preparation steps by adding a few microliters of the unprocessed sample (in VTM) directly into the RT-qPCR assay master mix targeting InfA, InfB, and RSV.
cord-257569-36qx1sy9	2004	title: A Large Variation in the Rates of Synonymous Substitution for RNA Viruses and Its Relationship to a Diversity of Viral Infection and Transmission Modes In conclusion, the variation of mutation rates for RNA viruses is caused by different replication frequencies, which are affected strongly by the infection and transmission modes. First, we estimated the rates of synonymous substitution for 46 different species of RNA viruses except Puumala virus, human T-lymphotropic virus 1 (HTLV-1) and GB virus C/hepatitis G virus (HGV), using the time-serial sample data. This indicated that the transmission mode affected the replication frequency and that differences in the replication frequencies contributed to the variation of the rate of synonymous substitution for RNA viruses. Moreover, in the present study, we proved that the variation in the synonymous substitution rates among RNA viruses was caused by variation of the replication frequency, and that differences in the infection and transmission modes affected the variation of replication frequencies.
cord-257652-ndt8f812	2018	In addition, despite frequent cross-species transmission, the RNA viruses in vertebrates generally follow the evolutionary history of their hosts, which began in the oceans and then moved to terrestrial habitats over timescales covering hundreds of millions of years. In addition, despite frequent cross-species transmission, the RNA viruses in vertebrates generally follow the evolutionary history of their hosts, which began in the oceans and then moved to terrestrial habitats over timescales covering hundreds of millions of years. However, following the extensive use of PCR and the Sanger sequencing methods for virus identification over the past decade, the number of RNA viruses sampled from lower vertebrates has steadily increased [28] , with notable examples being arenavirus and paramyxoviruses in reptiles [29] , and novirhabdoviruses and other RNA viruses from fish [30] , although these numbers were still very limited compared to the viruses described in birds and mammals.
cord-257693-rnchfjbe	2018	Observational studies have reported that prolonged influenza viral shedding in the respiratory tract is associated with severe outcomes of patients with seasonal influenza [10] and that early initiation of antiviral treatment with neuraminidase inhibitors (NAIs) for patients infected with 2009 pandemic influenza A(H1N1) virus (A[H1N1] pdm09) has a survival benefit [11] [12] [13] [14] [15] [16] [17] . We conducted a retrospective, multicenter study of 478 hospitalized patients with laboratory-confirmed A(H7N9) infection who were identified during April 2013-March 2017 to assess the impact of different NAI treatment strategies and corticosteroid treatment on the duration of A(H7N9) shedding. In a multivariable model that included available data from 478 patients, the time from illness onset to antiviral treatment (HR, 0.90 [95% CI, .91-.96]) and systemic corticosteroid administration (HR, 0.62 [95% CI, .50-.77]) were independent factors associated with the duration of A(H7N9) RNA shedding (Table 2 and Figure 4 ).
cord-258035-2tk7maqk	2003	Given that virus replication involves use and manipulation of multiple host proteins and molecular processes, cellular pathways related to transcription and translation, signal transduction, metabolism, host defense and cell cycle control are Review commonly altered in response to, or as a result of, infection with diverse virus types. A special case of virus modulation of host cell gene expression in which functional genomics will reveal novel targets and treatments is viral oncogenesis. The most recent method for analyzing gene inhibition studies has been RNAi or siRNA, where small 21 -23-nucleotide (nt) RNA duplexes interfere with the transcription program by directing degradation of homologous mRNA [34] [35] [36] [37] . Both new-generation antisense oligomers and siRNA can be used to knockdown expression of genes that were found by DNA microarrays to be upregulated in virally infected cells or organisms. RNA interference directed against viral and cellular targets inhibits human immunodeficiency virus type 1 replication
cord-258172-p54j4zzo	2020	Single cell RNA-Seq data from trachea indicated positive signals along the respiratory tract in key protective cell types including club, goblet, proliferating, and ciliary epithelial cells; while in lung the ratio of ACE2-expressing cells was low in all cell types (<2.6%), but was highest in vascular endothelial and goblet cells. Analysis of ACE2 promoter regions was performed using the TFBSfootprinter tool (https:// github.com/thirtysix/TFBS_footprinting) which uses transcription-relevant data from several major databases to enhance prediction of putative TFBSs, including: all cell types aggregated and merged human ATAC-Seq data from ENCODE [43] , transcription start sites and expression data from FANTOM5 [44] , expression quantitative trail loci from GTEx [39] , TFBS metacluster data from GTRD [45] , TFBS binding profile data from JASPAR [46] , and sequence and conservation data from Ensembl [47] .
cord-258286-lodjcj8c	1997	Previous studies have shown that immunocoma large plaque variant derived from the parental JHM petent mice infected with MHV exhibited increased exstrain (Stohlman et al., 1982) ; the small plaque variant pression of a number of cytokines, including IL-1, IL-6, DS (Stohlman et al., 1982) ; the neutralization-escape mu-TNF-a, and IFN-g, in the CNS at the time of viral cleartant 2.2-V-1 (Fleming et al., 1987; Wang et al., 1992) , and ance (Pearce et al., 1994) . The expressed IFNg exhibited antiviral activity, prevented virus spread in layers of DBT cells grown at approximately 70% confluence in 60-mm petri dishes were infected with MHV at vitro, and altered viral pathogenesis in mice. Expression of IFN-g using an MHV DI RNA vector cell metabolism prior to infection or it may be that interferon acts at an early stage of viral replication.
cord-258547-47cyyetb	2013	In the serum the acute phase proteins (haptoglobin and serum amyloid A), pro-inflammatory cytokines (interferon-γ and tumor necrosis factor-α), and serum sialic acid (total, TSA; lipid-bound, LBSA; and protein-bound, PBSA) concentrations were measured using validated standard procedures. Based on this evidence, the present study was undertaken to evaluate alteration in concentrations of the acute phase proteins [haptoglobin (Hp) and serum amyloid A (SAA)], pro-inflammatory cytokines (TNF-α and IFN-γ), and the serum level of total sialic acid (TSA), lipid-bound sialic acid (LBSA), and protein-bound sialic acid (PBSA) in experimentally infected chicks by IBV isolate IRFIBV32 compared with healthy chicks. The serum concentrations of IFN-γ, TNF-α, Hp, SAA, TSA, LBSA, and PBSA were increased significantly 2.50, 1.98, 1.82, 3.33, 2.25, 2.44, and 1.77 times, respectively, during experimental infection with IBV in chickens.
cord-258595-bk35vxlr	2020	Inoculation of differentiated Caco-2 cells for ten days with purified and concentrated wastewater (P2, P5, P11, and P12) did not result in the production of infectious SARS-CoV-2 particles (data not shown), which suggests that treated sewage appears to be non-infectious even though viral RNA fragments can be detected. Inter-comparing these nine catchment areas, we plotted the estimated cumulative and the acute prevalence against the measured SARS-CoV-2 load (Figure 8 ), the latter calculated from RT-qPCR measured M-gene copy concentration ( Figure 4 ) and the actual wastewater flow Q actual on the day of sampling (Table 2) . In contrast, plotting the incidence against SARS-CoV-2 concentration did not yield a conclusive correlation (not shown), likely because the precision of the qPCR employed was not sufficient to discriminate relatively minor differences in the incidence prevailing in the studied catchment areas at the time of sampling, ranging from 30 to 174 cases per 100,000 residents (less than an order of magnitude, Figure 8C and D).
cord-258678-0atfsivf	2013	Here, we report the development of a small and simple protein tag that complements the therapeutic and targeting functionalities of chimera with two functional domains: a dsRNA binding domain (dsRBD) for siRNA docking and a pH-dependent polyhistidine to disrupt endosomal membrane. Therefore, it is of critical importance to design a delivery system that is simple for potential regulatory approval and mass production, universal for all siRNAaptamer chimera, neutral and siRNA-binding specific to ensure aptamer targeting, and small to avoid major alteration of chimera''s biodistribution profile. To assess their dsRNA binding activity, siRNA-aptamer chimera labeled with fluorophore FAM were incubated with the protein tags and probed with gel electrophoresis (1% agarose). To evaluate the targeting specificity of the aptamer block, PSMA-positive LNCaP cells and PSMA-negative PC3 cells were treated with complex of chimera and dsRBD-His 18 (chimera/protein tag molar ratio at 152, 100 nM chimera) in serum free medium for 2 hours, followed by incubation in complete medium for another 12 h.
cord-258696-01wj76es	2008	The dogs, three 2.5-month-old and two 6-month-old pups, were successfully infected, shedding viral RNA with their faeces for the entire observation period (21 days) and displaying systemic clinical signs resembling those observed during the course of natural infection. At postmortem examination, remarkable lesions were observed in the internal organs of the euthanized pups, that tested positive for CCoV by real-time RT-PCR and virus isolation on cell cultures. Unexpectedly, CCoV type II RNA was detected at very high titres in the internal organs of the dead pups and the virus (strain CB/05) was isolated on canine cell cultures. (last day of observation) reaching the maximal mean value of 6.79 Â 10 5 RNA copy numbers/ml of template at day 10 p.i. Surprisingly, CCoV RNA was never detected in the blood of the 6-month-old pups, as well as in the euthanized animals, in whose organs remarkable viral RNA titres were found.
cord-259152-pwvcwlh8	2020	The current outbreak of viral pneumonia in the city of Wuhan, China, was caused by a novel coronavirus designated 2019‐nCoV by the World Health Organization, as determined by sequencing the viral RNA genome. To investigate possible virus reservoir, we have carried out comprehensive sequence analysis and comparison in conjunction with relative synonymous codon usage (RSCU) bias among different animal species based on the 2019‐nCoV sequence. 4 On 10 January, it was reported that a novel coronavirus designated 2019-nCoV by the World Health Organization (WHO) 5 was identified by high-throughput sequencing of the viral RNA genome, which was released through virological.org. Highthroughput sequencing of viral RNA from patients'' samples has identified a novel coronavirus designated 2019-nCoV by the World Health Organization. 10, 11 Results from our analysis suggest that 2019-nCoV has most similar genetic information with bat coronovirus and has most similar codon usage bias with snake.
cord-259233-smmhhroe	2016	Several RNA viruses induce the formation of these autophagosome-like vesicles (also referred to as DMVs) to enhance viral replication and non-lytic egression, such as poliovirus and CVB3, HIV-1 and HCV. Indeed, autophagosome-like vesicles may represent a trafficking pathway for these viruses, connecting to multivesicular bodies (MVBs), and assuring virus assembly and budding at the cell surface while protecting them from intrinsic antiviral factors and immune responses. Trogocytosis involves the exchange of cell surface membrane patches that may contain receptor clusters associated to viral particles, while exosomes are vesicles formed from MVBs that could participate in viral infection and spreading between cells of the Alphavirus group of this family [12] , couple their RNA synthesis to endosome and lysosome membranes modified by the association of virus specific components. It remains unclear how proteins from distinct viruses and host cells use the same intracellular membrane compartments or events (e.g. autophagy) to achieve viral replication, without affecting important cellular processes.
cord-259246-azt5sr9w	2020	This structure provides a missing snapshot for visualizing the catalysis dynamics of coronavirus polymerase, and reveals an unexpected base-pairing pattern between Favipiravir and pyrimidine residues which may explain its capacity for mimicking both adenine and guanine nucleotides. Recently, we and other groups have determined the structures of SARS-CoV-2 core polymerase complex in both apo and RNA-bound states 26-30 , providing important information for structure-based antiviral drug design. Similar observations were also reported recently that SARS-CoV-2 polymerase is more tolerant for mismatches between template and product residues than other viral RdRps 5 , which further highlights the requirement for the proofreading nuclease nsp14 to maintain the integrity of viral genome. Interestingly, Favipiravir could be incorporated into the RNA product with similar efficiencies to those of ATP or GTP substrates guided by U or C template residues, respectively (Fig. 1c) .
cord-259311-ccx61owl	2020	In order to provide better understanding of the Quality and performance of COVID-19 RNA detection kits on the market, we designed a system to evaluate the specificity (quantitation), sensitivity (LOD) and robustness of the kits using positive RNA and pseudovirus controls based on COVID-19 genomic sequence. At the time of submission, 23 diagnostic kits have been approved in China, of which 8 are based on quantitative Polymerase Chain Reaction (qPCR) using COVID-19 viral RNA sequence as templates and fluorescence detection. Our study aims at providing objective evaluation and comparison of the quality and performance characteristics of 8 of the currently marketed COVID-19 nucleic acid detection kits in China based on qPCR and fluorescence detection. Our study provides an elegant design to define the most important performance characteristics of the RNA detection kits for COVID-19, which are specificity (quantitative), sensitivity (LOD), and robustness.
cord-259500-ndjbrtrv	2003	title: Frameshift mutations in infectious cDNA clones of Citrus tristeza virus: a strategy to minimize the toxicity of viral sequences to Escherichia coli These data demonstrate that, when hosts are sufficiently susceptible for infection by transcripts of reduced specific infectivity, introduction of frameshift mutations at "slippery sequences" near toxic regions of viral cDNAs can be used as an additional strategy to clone recalcitrant viral sequences in high-copy-number plasmids for reverse genetics. A similar frameshift mutation was also found in the derivative replicon pCTV-⌬Cla. However, the sequence of the progeny virion RNA of CTV9 from infected protoplasts or citrus plants did not contain the additional nt at position 3732 that occurred in the cDNA clones, suggesting that the frameshift mutation present in the original cDNA clone was repaired either during the in vitro transcription or during replication in the protoplasts.
cord-259593-shrd1s7r	2007	title: siRNAs targeting terminal sequences of the SARS-associated coronavirus membrane gene inhibit M protein expression through degradation of M mRNA To study M protein function, three candidate small interfering RNAs (siRNAs) corresponding to M gene sequences were designed, transcribed in vitro, and then tested for their ability to silence M protein expression. The results showed that the mean green fluorescence intensity and M RNA transcripts were significantly reduced, and that the expression of M glycoprotein was strongly inhibited in those cells co-transfected with M-specific siRNAs. These findings demonstrated that the three M-specific siRNAs were able to specifically and effectively inhibit M glycoprotein expression in cultured cells by blocking the accumulation of mRNA, which provides an approach for studies on the functions of M protein and for the development of novel prophylactic or therapeutic agents for SCoV infection.
cord-259603-bh198xgl	2016	Reverse-genetics studies targeting specific residues in SARS-CoV nsp7 confirmed the protein''s importance for virus replication (Subissi et al., 2014b) , although the impact of single point mutations was smaller than anticipated on the basis of the biochemical characterization of the RNA-binding properties of nsp7-containing protein complexes in vitro (see later). The large number of viral subunits in these complexes (Subissi et al., 2014a) , the likely requirement for host factors (van Hemert et al., 2008) , and the concept of RNA synthesis occurring in a dedicated microenvironment in the infected cell (Knoops et al., 2008; V''Kovski et al., 2015) complicate the straightforward characterization of the CoV RdRp. To reconstitute the enzyme''s activities in vitro, purified recombinant nsp12 is a key reagent but, for many years, such studies were hampered by poor nsp12 expression in Escherichia coli.
cord-259671-7de21oaq	2014	Overall, the conservation pattern identified for 5′ and 3′-terminal RNA structural elements in the genomes of alphaand betacoronaviruses is in agreement with the widely used replicase polyprotein-based classification of the Coronavirinae, suggesting co-evolution of the coronavirus replication machinery with cognate cis-acting RNA elements. Other internal cis-acting elements include specific RNA signals required for genome packing, which have been characterized in a small number of coronaviruses Escors et al., 2003; Morales et al., 2013; Penzes et al., 1994) , and a complex RNA pseudoknot structure located in the ORF1a-ORF1b overlap region that mediates a (−1) ribosomal frameshift event and thus controls the expression of the second large ORF on the coronavirus genome RNA (ORF1b) (Brierley et al., 1987 (Brierley et al., , 1989 de Haan et al., 2002; Namy et al., 2006) .
cord-259710-qrht9tq3	2020	title: Molecular-based cross-species evaluation of bovine coronavirus infection in cattle, sheep and goats in Ghana Conclusions: Given that Ghana predominantly practices the extensive and semi-intensive systems of animal rearing, our study highlights the potential for spillover of BCoV to small ruminants in settings with mixed husbandry and limited separation between species. In this study, we employed a molecular-based detection method to investigate the presence of BCoVs in rectal samples of cattle, sheep and goats from four major regions in Ghana. In a prior study, we reported a high seroprevalence, cross-species infection and serological determinants of BCoV in cattle, sheep and goats in Ghana [19] . In this study, we sought to investigate the presence of BCoVs in rectal samples of cattle, sheep and goats from four major regions in Ghana using molecular-based detection method.
cord-259916-gr6v098c	2016	Like all positive-sense RNA viruses, hepatitis C virus (HCV) induces host membrane alterations for its replication termed the membranous web (MW). This review will summarize the recent studies that have led to our current knowledge of the role of viral and host factors in the biogenesis of the MWs and discuss how HCV uses this specialized membrane structure for its replication. A later study of cells containing a subgenomic HCV replicon reported that the membrane alterations induced by HCV replication consisted in part of double-membrane vesicles (DMVs; Figure 1 ) with a diameter around 200 nm that were also positive on immunoelectron microscopy with antibodies against NS5A and double-stranded RNA (dsRNA) [15] . [10] first showed that expression of viral nonstructural proteins resulted in membrane alterations that morphologically resemble those observed in replicon cells, demonstrating that viral RNA replication is not required for membranous web formation. Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex
cord-259927-xh9cw9ao	2017	When considering prevention or treatment of viral respiratory tract infections, potential targets include the causative pathogens themselves but also the immune response, disease transmission, or even just the symptoms. Here we provide an overview of the options and highlight some of the most promising approaches in vRTI treatment, including symptomatic medication, immunomodulatory drugs, antiviral agents, and natural products, as well as in vRTI prevention, ranging from vaccines to immunostimulators and public health policies. Early in vivo evidence suggested that azithromycin has anti-inflammatory and antiviral effects through induction of interferon-stimulated gene mRNA expression and reduced viral replication and release in patients with asthma and chronic obstructive lung disease. mAb therapies to viral infections, such as EBV (rituximab) or RSV (palivizumab), provide passive immunization and are licensed, whereas similar agents targeting influenza and other viruses are in preclinical development.
cord-260042-cs0wp99n	2019	The present study aimed to: a) determine mitochondrial counts in the cells of oviduct segments in laying hens at different time-points of egg formation in relation to the requirement of energy for egg production during IBV challenge; b) to determine the expression of nuclear DNA encoded genes in the shell gland to gain insights into their responses to IBV infection and time-points of egg-shell formation. Based on the lack of significant differences in mitochondrial count in the cells between the challenged and control groups for the magnum and isthmus, we focused further on shell gland tissue and studied the expression level of genes involved in mitochondrial density, biogenesis and fission. The lack of any significant difference in the relative expression levels of all of the genes except SDHA, between the control and IBV T challenged groups, may indicate that mitochondrial function may have been enhanced and thus overall egg quality may not have been affected by fewer mitochondria in the shell gland cells of IBV T infected hens.
cord-260168-rb7j94dh	2007	Negative controls also included an unrelated antisense probe against the fragment of the polymerase gene (R1AB) of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV), 20 as well as H5N1 in-situ hybridisation probes to tissues (including lung and tracheal) obtained from seven adults who died from infectious lung diseases other than H5N1 infl uenza (four, SARS; one, purulent bronchitis; two, pneumonia), one adult who died from a non-infectious disease (gastric ulcer), one pregnant woman who died from an amniotic embolism, and one aborted fetus. Presence of viral sequences and antigens in the CNS is consistent with the recent isolation of H5N1 virus from cerebrospinal fl uid of a boy who died from encephalitis 6 with neurological symptoms commonly seen in patients with H5N1 infl uenza (Gao Zh, unpublished), including the two cases in this study.
cord-260225-bc1hr0fr	2020	Integrating evolutionary, structural, and interaction data with human proteins, we present how the SARS-CoV-2 proteome interacts with human disorders and risk factors ranging from cytokine storm, hyperferritinemic septic, coagulopathic, cardiac, immune, and rare disease-based genetics. The most noteworthy human genetic potential of SARS-CoV-2 is that of the nucleocapsid protein, where it is known to contribute to the inhibition of the biological process known as nonsense-mediated decay. As we understand more of the dynamic and complex biological pathways that the proteome of SARS-CoV-2 utilizes for entry into cells, for replication, and for release from human cells, we can understand more risk factors for severe/lethal outcomes in patients and novel pharmaceutical interventions that may mitigate future pandemics. Additional SARS-CoV-2 proteins with mentions include nsp12 (RNA-directed RNA polymerase, 20/71), nucleocapsid (N, 17/71), membrane (M, 5/48), envelope (E, 4/31), nsp5 (3CLPro/Mpro, 7/26), nsp8 (3/19), nsp16 (2′-O-methyltransferase, 3/14), ORF8 (1/10), nsp10 (3/9), nsp14 (guanine-N7 methyltransferase, 1/8), nsp3 (papain-like protease, 16/6), and nsp15 (uridylate-specific endoribonuclease, 16/4).
cord-260250-t48y27wg	2004	A TaqMan(®) fluorogenic reverse transcriptase-polymerase chain reaction (RT-PCR) assay was developed for the detection and quantitation of canine coronavirus (CCoV) RNA in the faeces of naturally or experimentally infected dogs. The CCoV fluorogenic RT-PCR assay, which targeted the ORF5 (M gene), was more sensitive than a conventional RT-PCR assay targeting the same gene, showing a detection limit of 10 copies of CCoV standard RNA, and was linear from 10 to 10(8) copies, allowing quantitation of samples with a wide range of CCoV RNA loads. As shown in Table 2 , the detec-tion limit of the TaqMan RT-PCR was 1-2 log higher than that of conventional RT-PCR, ranging around 10 1 copies/l and 10 −1.50 TCID 50 /50 l for standard RNA and CCoV strain, respectively, with a detection rate of 100% for each positive dilution. The results of the conventional amplification and real-time analysis carried out on the faecal samples of the CCoV experimentally infected dog are summarized in Fig. 3 .
cord-260345-ugd8kkor	1992	203, sodium dodecyl-sulfate.; ted blood-cells; phosphoenolpyruvate carboxylase activity; nucleotide-linked dehydrogenases; alkaline-phosphatase isoenzymes; pathogenesis-related proteins; cr-L-fucosidase; polyactylamide-gel; produce phosphate; general-method. low-density~l&protein; high-performance liquid; chrcmatogmphy mass spectmmetry; chicken vitellogam gene; thin-layer chmmatography; apolipoprotein-VLDL-II; fatty-acid composition; laying turkey hens; egg-yolk; plasma-lipoproteins. human-skin fibroblests; IGF binding-protein; messenger ribonucleic-acid; cooh-teminal truncation; monoclonal-antibody; endothelial-cells; factor receptoc amniotic-fluid; DNA-synthesis; rat-heart. factor-binding-protein; erythroid colony formation; cultured human-tibroblasts; factor messenger-RNA, N-terminal sequence; fetal bovine serum; IGF-I; somatomedin C, clinical-applicstians; stimulating factors. shon-hved protein; dependent pmteolytic system; ubiquitin-conjugating enzyme; repair gene ra&, Escherichia coli; transfer RNA; Sacclurroqces cercvisiue; endoplasmic-reticulum; cell-cycle; amino-acid. polymense chain-reaction; fragment length polymorphisms; dependent diabetes-mellitus; sickle-cell anemia; factor-M gene; enzymatic AMPlificatiat; genomic DNA; mutations; sequence; diagnosis; predisposition; genetics. T-cell receptor; messenger-RNA degradation; gamma-delta; stress proteins; antigen-receptor, lymphocytes-T; ~ycobactcrium fuberc&arir. Wiskott-Aldrich syndrome; heat-shock proteins; receptor-delta; ataxia telangiectasia; transgenic mice; lymphocytes T; bearing cells; recognition; expression; incmase. human granulocyte-macmphage; protein kinase-c; recombinant human interleukin-3; cell growth-factor, murine bone-marrow; express functional receptors; acute lymphocytic-leukemia; GTPase-activating pm&n; factor-independent growth.
cord-260422-z22t57ju	2012	Today''s view is that RNA chaperones are nucleic acid binding proteins present in all living organisms, including viruses, where they Abbreviations: HIV-1, human immunodeficiency virus-type I; NCp7, nucleocapsid protein of HIV-1; ZF, zinc finger; NA, nucleic acid; TAR, transactivation response element; PBS, primer binding site; AA, amino acids. The substantial nucleic acid chaperone properties exhibited by Tat may account for its ability to promote the annealing of the primer tRNA to the viral RNA (Kameoka et al., 2002) and intervene in the first strand transfer (Boudier et al., 2010) and by this way, to stimulate RTion as does NCp7 (Harrich et al., 1997; Ulich et al., 1999; Apolloni et al., 2007) . Human immunodeficiency virus Type 1 nucleocapsid protein (NCp7) directs specific initiation of minusstrand DNA synthesis primed by human tRNA(Lys3) in vitro: studies of viral RNA molecules mutated in regions that flank the primer binding site
cord-260452-js4nr4d8	2017	Both the pathogen-associated molecule pattern derived from virions and intracellular stress molecules involved in the process of viral infection lead to activation of the NLRP3 inflammasome, which in turn triggers inflammatory responses for antiviral defense and tissue healing. IL-1β and IL-18 serve to activate myriad downstream cell responses, and orchestrate innate and adaptive immunity through MyD88/IRAK4/TRAF6-mediated NF-κB signaling and the JNK/p38 mitogen-activated protein kinase pathways (60-63), which may represent key events for the NLRP3 inflammasome-dependent antiviral defense. In BV-2 mouse microglia cells infected by Japanese encephalitis virus, the NLRP3 inflammasome induces production of IL-1β and IL-18 rapidly (within 3 h of exposure) and of TNF-α, CCL2, and IL-6 later (within 6 h after exposure) (40) ; the findings suggest that the NLRP3dependent protective inflammatory response is a very early phase innate immune response against RNA viral infection.
cord-260647-7bjhobg7	2016	A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The aim of this study was to develop real time RT-PCR assays for detection of a total of 19 human enteric viruses (including 3 genogroupes of norovirus and 4 coronaviruses) and two control process viruses (mengovirus and murine norovirus) generally used for monitoring the recovery of viral foodstuff extraction methods. The sensitivity of conventional qPCR assays targeting 21 viral genomes was compared to the quantitative digital RT-PCR array and to the qualitative nanofluidic real-time PCR array performed on Fluidigm''s BioMark System. Similarly, by testing genomes from viruses in stools and RNA from virus production in cells, the limit of detection (LOD) as determined by RT-dPCR was respectively 1.5 to 3.4 log 10 and 1.6 to 2.1 log 10 lower than the expected copy numbers calculated via the standard curve by RT-qPCR.
cord-260695-qwepi0we	2017	Of the host-encoded lncRNAs reported to be differentially expressed upon HIV-1 infection, only nuclear enriched abundant transcript 1 (NEAT1) and noncoding repressor of nuclear factor of activated T cells (NRON) have been functionally characterized (Lazar et al., 2016) . In this report, we describe a meta-analysis of two independent RNA-seq studies of HIV-1-infected cells and show that, unexpectedly, only three lncRNAs are differentially expressed in both of these datasets (Chang et al., 2011; Mohammadi et al., 2013) . To obtain an unbiased understanding of any changes in lncRNA expression during HIV-1 infection, we performed a meta-analysis of two independent RNA-seq studies conducted by two different groups using two different virus strains ( Supplementary Fig. S1 ) (Chang et al., 2011; Mohammadi et al., 2013) . To validate the results of the in silico analysis of lnc173 expression, we isolated RNA from 4 human cell lines and probed for the presence of both transcript variants by quantitative RT-PCR (qRT-PCR; Fig. 3A -D).
cord-260705-huyyw5z6	2012	During infection, many viruses induce cellular remodeling, resulting in the formation of insoluble aggregates/inclusions, usually containing viral structural proteins. The aggregates are utilized by viruses to house a large complex of proteins of both viral and host origin to promote virus replication, translation, intraand intercellular transportation. In plant cells, both RNA and DNA viruses are associated with large inclusions detected in the cytoplasm and nucleus, however, their role in virus propagation or oppositely in virus restraint is less investigated than in infected mammalian cells. In general, mammalian and plant viruses make use of aggregates as scaffolds for anchoring the replication complex, increasing the local concentration of viral and host components required for replication and assembly, and shielding the process of replication from host defense.
cord-260708-l9w5jhsw	2013	The nairoviruses are a rapidly emerging group of tick-borne bunyaviruses that includes pathogens of humans (Crimean-Congo hemorrhagic fever virus [CCHFV]) and livestock (Nairobi sheep disease virus [NSDV], also known as Ganjam virus), as well as a large number of viruses for which the normal vertebrate host has not been established. As several potential cysteine-protease-like cleavage sites have been identified in the L protein sequence of nairoviruses [94] and some viral proteins containing an OTU-like protease domain have also been shown to undergo autoproteolytic cleavage to generate multiple mature proteins, e.g., the replicase of BlScV [98] , it has been suggested that the L proteins of nairoviruses may also be autoproteolytically cleaved into an active RNA polymerase and protein(s) with additional function [85] . Role of actin filaments in targeting of Crimean Congo hemorrhagic fever virus nucleocapsid protein to perinuclear regions of mammalian cells Structural analysis of a viral ovarian tumor domain protease from the Crimean-Congo hemorrhagic fever virus in complex with covalently bonded ubiquitin Induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever virus infection
cord-260782-1lm8tzbc	2018	title: Viral RNA load and histological changes in tissues following experimental infection with an arterivirus of possums (wobbly possum disease virus) The aim of the current study was to describe histological lesions and viral RNA levels in tissues from possum experimentally infected with WPDV. Histology of brain, kidney and liver tissue from wobbly possum disease virus (WPDV)-infected possums. Whilst RNA levels in selected tissues (liver, spleen, brain and kidney) from WPD-infected possums have been previously reported (Dunowska et al., 2013) , we have expanded both the number of possums and the tissue types tested to provide a greater understanding of the tissues targeted by the virus in-vivo. Detection of moderate to high levels of viral RNA from the sera of all but one infected possum in this study also supports the use of blood or serum samples for detection of WPDV.
cord-261110-cnj0e0s9	2011	This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1–16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. We present X-ray diffraction data from these SARS-CoV nsp10-nsp16 crystals. The purified SARS-CoV nsp10-nsp16 complex was analyzed by 12% SDS-PAGE and stained using Coomassie Blue. Lane MK, molecular-weight markers; lane 1, 2 mg nsp10-nsp16 protein complex eluted from the Strep-Tactin column. The nsp10-nsp16 complex eluted from the Strep-Tactin column was analyzed on a 16/60 S200 gel-filtration column and the elution of protein and nucleic acid was followed by measuring the absorption at 280 nm (blue) and 260 nm (orange), respectively. The purified SARS-CoV nsp10-nsp16 complex was loaded onto a 4-12% NuPAGE gel and stained using Coomassie Blue. We have crystallized a complex of the SARS-CoV nsp10 and nsp16 proteins.
cord-261160-g92zhv19	2003	title: Lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero Even though PRRSV-specific antibody appears as early as 5 days post-infection and is followed by serum neutralizing activity and cell-mediated immunity Molitor, 1997, 1999; Rowland et al., 1999 Rowland et al., , 2001 persistently infected pigs can continue to shed virus. The purpose of this study was to further understand persistent infection in congenitally infected pigs by characterizing the course of clinical disease, sites of virus replication and the capacity to transmit virus in a group of pigs exposed to PRRSV in utero. Analysis of virus and antibody in blood and/or umbilical cords from the 28 live neonates showed that at least 20 or 74% were virus isolation (VI) or RT-PCR positive for PRRSV at the time of farrowing indicating that in utero infection was successful.
cord-261279-6mef38eo	2020	RESULTS: Using RNA extracted from cells infected by SARS coronavirus as a positive control, these assays were shown to have a dynamic range of at least seven orders of magnitude (2x10(−4)-2000 TCID(50)/reaction). In this study, we report the development of RT-PCR assays to detect this novel virus in human clinical specimens. Two monoplex real-time RT-PCR assays targeting the ORF1b and N gene regions of 2019-nCoV were designed based on the first publicly available sequence in Genbank (Accession number: MN908947). Viral RNA from cells infected by SARS coronavirus or DNA plasmids containing the target sequences were positive in the assays as expected. In addition, the N gene RT-PCR assay was found to be more sensitive in detecting 2019-nCoV RNA in the studied clinical samples.
cord-261417-4pf5nsw2	2017	Why the virus prefers to use these snRNAs as targets has yet to be experimentally established, but it has been proposed that the selective de-capping of U1 and U2 RNAs in combination with the binding of the viral NS1 protein to U6 snRNA may serve to inhibit host pre-mRNA splicing [66] . The folding of the TAR hairpin is key to the regulation of HIV-1 transcription [94] as it is used as a scaffold to recruit essential transcription factors, including the 86-101 amino acid (aa) viral trans-activator protein (Tat) [83] ( Figure 3C ). Interestingly, the human T-lymphotropic virus type 1 transcriptional activator Tax also utilizes P-TEFb for viral transcription and displaces P-TEFb from 7SK snRNP through binding CycT1 [101, 102] , suggesting that P-TEFb liberation from 7SK snRNP could be a common theme developed by different viruses to support their replication in host cells.
cord-261532-q923xxn2	2012	Microbial components and the endogenous molecules released from damaged cells can stimulate germ-line-encoded pattern recognition receptors (PRRs) to transduce signals to the hub of the innate immune signaling network-the adaptor proteins MyD88/TRIF/MAVS/STING/Caspase-1, where integrated signals relay to the relevant transcription factors IRF3/IRF7/NF-κB/ AP-1 and the signal transducer and activator of transcription 6 (STAT6) to trigger the expression of type I interferons and inflammatory cytokines or the assembly of inflammasomes. These receptors can recruit specific adaptor proteins, like myeloid differentiation primary response gene 88 (MyD88) or Toll/interleukin-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) in the TLR pathway, mitochondrial antiviral signaling protein (MAVS) downstream of RLRs, stimulator of interferon genes (STING) in the cytosolic DNA response pathway and, cysteine aspartic protease 1 (Caspase-1) as part of the inflammasome, all of which orchestrate the host innate responses, through activation of transcriptional factors such as nuclear factor κB (NF-κB), activator protein 1 (AP-1) and interferon regulatory factors (IRFs), to trigger the production of type І interferons (IFNs), inflammatory cytokines and chemokines.
cord-261735-03hvi4el	2011	A one-step real time quantitative RT-PCR (qRT-PCR) assay was developed to detect all published Dugbe virus (DUGV) genomes of the Nairovirus genus. Frequently detected in tick-borne virus surveys in Africa (Guilherme et al., 1996) , DUGV is a tri-segmented single-stranded negative RNA enveloped virus and is considered endemic in arid regions (Burt et al., 1996) . The aim of this study was to develop a sensitive, specific and rapid one-step quantitative real time RT-PCR assay (qRT-PCR) to detect DUGV in infected cell supernatants, ticks or serum samples. The specificity was evaluated by using RNA extracted from supernatants from CCHFV, Hazara virus, Coronavirus and Influenza A virus infected cells. Among the 498 captured ticks, one Dugbe virus RNA was detected in one tick using the qRT-PCR assay (0.2%). In conclusion, a sensitive and specific qRT-PCR assay was developed to detect and quantify DUGV RNAs in infected cell supernatants, extracts from ticks and potentially sera.
cord-262076-b5u5hp2r	2008	We show that the expression of individual miRNAs is greatly enhanced in multiplex hairpin transcripts that are properly processed into functional miRNAs. HIV-1 replication can be potently inhibited by simultaneous expression of four antiviral miRNAs. These combined results indicate that the multiplex miRNA strategy is a promising therapeutic approach against escape-prone viral pathogens. By repeating this procedure we obtained constructs expressing different combinations of 1, 2, 3, 4 and 6 pri-miRNAs. The RNA structures formed by the transcripts were predicted with the Mfold program (47) at http://frontend.bioinfo.rpi.edu/ applications/mfold/ and found to be similar to the predicted conformation of the wild-type pri-miRNAs. The firefly luciferase (FL) reporters containing HIV-1 target sequences pol47 (Luc-A pol47 ), pol1 (Luc-B pol1 ), gag5 (Luc-C gag5 ), r/t5 (Luc-D r/t5 ), ldr9 (Luc-E ldr9 ) and the anti-HIV shRNAs have been described previously (32) .
cord-262282-9xh51cd1	2020	Without going into details concerning live vaccine production via eukaryotic viruses, I think it reasonable to assume that eukaryotic virus production is more difficult, more expensive and less rapid than the production of phages. However, current efforts to human-engineer improved antigens for anti-RNA virus vaccines have shown that neutralizing antibodies typically react with viral proteins that are in states that are context dependent and unstable [12, 13, 15, 20] . I take the liberty of responding here to the obvious objection that no membrane-covered, single-stranded RNA phage has ever been isolated [21] and that the pandemic viruses include influenza, Zika-type and coronaviruses, all in this category. A non-specialist observer reasonably concludes that DNA and RNA vaccines, when viewed in the context of our overall objective, are examples of type 2 strategy options. Given that eukaryotic viruses have doubling times much greater than those of phages (2-5 min for typical coliphages), meeting this objective implies that a live virus vaccine has to be already present in the environment.
cord-262318-qpztmdnw	2020	In this work we have crystallised the protease in its native form with an unperturbed catalytic triad and have conducted crystal-based fragment screening of 844 compounds with the aim of discovering novel inhibitory functional groups which have the potential to be developed as therapeutic agents, either on their own or through chemical coupling. Interestingly, the β-hairpin formed by β9 and β10, which is involved in binding the N-terminal side of the substrate peptide, adopts an appreciably different conformation from that observed in an earlier inhibitor-complexed structure ( The SV3CP enzyme has approximately 90 % sequence identity with other GI noroviral 3C proteases and an identity of the order of 68 % with the enzyme from the GII genotype. The X-ray structure of the Southampton virus 3CL pro has been determined at 1.3 Å resolution in a crystal form that has allowed fragment-screening for novel inhibitors to be undertaken at similar resolutions.
cord-262347-ejhz9rra	2015	The nidovirus order constitutes a group of singlestranded positive-sense RNA viruses which share a hallmark replication/transcription strategy, similar genomic organization, and a defining set of genetic elements, but differ in host species and range, disease phenotype, virion morphology, cellular tropism, genomic size and encoded content . It has been shown that the EAV replicase proteins (ORF1a/b) are sufficient to support viral replication (Molenkamp et al., 2000b) , but that infectivity is dependent on the presence of the structural genes encoded at the 3 0 -end of the arterivirus genome Molenkamp et al., 2000b; Verheije et al., 2002) . Leader-body junction sequence of the viral subgenomic mRNAs of porcine reproductive and respiratory syndrome virus isolated in Taiwan Molecular cloning and nucleotide sequencing of the 3 0 -terminal genomic RNA of the porcine reproductive and respiratory syndrome virus Comparison of the 5 0 leader sequences of North American isolates of reference and field strains of porcine reproductive and respiratory syndrome virus (PRRSV)
cord-262511-96xp1v0r	2007	Post-transcriptional regulation contributes significantly to this rapid transit by several mechanisms, including mRNA stability modulation and translational control; collectively, they aim to control the expression of key gene products involved in the immune response. The stabilization of cytokine mRNA and other immune response gene products can occur by the activity of mRNA stabilization-promoting proteins such as human antigen (HuR) protein or by inactivation of RNA decay-promoting proteins such as the zinc finger protein, tristetraprolin (TTP). Traditionally, post-transcriptional regulation in innate immunity has been studied in response to the bacterial endotoxin, LPS, which binds CD14 in a complex with TLR4 on the surface of neutrophils and macrophages and initiates a cascade of signals that causes cell activation, the inflammatory response, and phagocytosis [35] . With the coordinated kinetics model, stabilizing RNA-binding proteins such as HuR can occur initially following immune cell activation, allowing rapid and early response of cytokine production.
cord-262592-0rdiosxd	2016	This revealed the presence of low-frequency sequences carrying large numbers of U-to-C or A-to-G base transitions, suggesting a role for hyper-mutation in NoV diversity. Based on the sequence context of the observed changes, we propose that NoV hypermutation might be driven by ADAR-mediated editing of the viral genomic RNA of either polarity during replication. We used 16 stool samples from patients acutely infected with NoV GII.4 to amplify by RT-PCR a 386-base region encompassing nucleotides 1 to 386 of the VP1 gene (reference sequence: GenBank JX459908; Fig. 1A ). After 48 h incubation, total RNA was extracted from cells, residual DNA was removed with DNAse I, a specific primer annealing to the minus-strand of the VP1 capsid gene was used for reverse transcription, and high-fidelity PCR amplification of a region encompassing positions 19 to 323 of the VP1 gene (305 bases, although only the 266 bases excluding primer regions Fig. 1 .
cord-262609-cssgzvus	1999	title: RNA sequence and secondary structural determinants in a minimal viral promoter that directs replicase recognition and initiation of genomic plus-strand RNA synthesis In contrast, we find that position-specific changes in the RNA sequence will affect replicase recognition, modulate the polymerization process, and contribute to the differential accumulation of viral RNAs. These functional results are in agreement with the phylogenetic analysis of BMV and related viral sequences and suggest that a similar mechanism of RNA synthesis takes place for members of the alphavirus superfamily. Results herein demonstrate that the endscript directing genomic plus-strand synthesis also interacts with the viral replicase via a mechanism that does not depend on a speci®c preformed RNA structure. An interesting conclusion from our analysis of the BMV genomic minus-strand RNA endscript is that template sequence may regulate the amount of RNA synthesis in a manner independent of replicase-promoter interaction.
cord-262753-jld1ygxt	2019	Analysis of sequence reads in the different fractions shows >60% of total cytoplasmic and polysome-associated reads map to the 5 viral genes by 6 hours post-infection, a time point at which robust host cell translational shut-off is observed. The cellular mRNAs that remain most polysome-associated following infection had longer half-lives, were typically larger, and were more AU rich, features that are shared with the viral mRNAs. Several of those mRNAs encode proteins known to positively affect viral replication, and using chemical inhibition and siRNA depletion we confirm that the host chaperone heat shock protein 90 (hsp90) and eukaryotic translation initiation factor 3A (eIF3A)—encoded by 2 such mRNAs—support viral replication. In the present study, we interrogate global mRNA translation in VSV infected cells using RNAseq analysis of the cytoplasmic mRNA transcriptome, and parallel sequencing of polysome-associated mRNAs. We obtain support for the model that an overabundance of viral mRNA contributes to host shut-off by leading to a re-distribution of cellular ribosomes onto viral mRNA.
cord-262776-6k7tcgfs	2004	Analysis of production parameters indicate that acid pH treatments and caprylic acid precipitations, which have been validated for the manufacture of some human IgG products, appear to provide the best potential for viral inactivation of antivenoms. Among those, caprylic acid and low pH treatments, both of which are commonly used also for the purification of antivenom IgG, have been shown to contribute to the viral safety of human plasma IgG products as described below. It should be kept in mind that treatment of whole plasma or crude fractions, as is the case for equine antivenoms production, may lead to lower rate and kinetics of viral inactivation, due to the high endogenous lipid content, as found in a study that evaluated the virucidal effect of sodium oleate [85] . However, a comparison with validated manufacturing processes used for human IgG clearly indicates that at least two widely used antivenom production steps, caprylic acid treatment and low-pH incubation, are likely to contribute in a robust manner to viral safety, at least against enveloped viruses.
cord-262841-nr42rs8f	2003	Study design: Peripheral blood mononuclear cells collected from SARS cases infected by the same infectious source were tested for both negative-stranded RNA (minus-RNA, "replicative intermediates") and positive-stranded RNA (genomic RNA) of SARS-CoV during the course of hospitalization by reverse transcription-polymerase chain reaction (RT-PCR). Although the virus has been identified Abbreviations: BNIBernhard-Nocht Institute for Tropical Medicine, Hamburg, Germany; BSL3biosafety level 3; CoVcoronavirus; MHVmouse hepatitis virus; PCRpolymerase chain reaction; minus -RNAreplicative negative-stranded RNA; plus -RNApositive-stranded genomic RNA; RTreverse transcription; SARSsevere acute respiratory syndrome; SCAsodium citrate anticoagulant. In order to evaluate (i) whether SARS-CoV can infect peripheral blood mononuclear cells (PBMCs) of infected persons, (ii) whether the virus can replicate in their PBMCs, and (iii) to reveal any dynamic changes to the virus during the course of the disease, we carried out follow-up investigations on the plusand minus-RNA forms in SARS patients.
cord-262844-qeheeqe3	2020	The zinc finger antiviral protein (ZAP, known as ZC3HAV1 in mammals or hZAP in human), a key component in mammalian interferon-mediated immune response, binds specifically to CpG dinucleotides in viral RNA genomes via its RNA-binding domain (Meagher et al., 2019) . If a coronavirus infects a different host tissue with different ZAP abundance, then its RNA genome will experience different selection pressure against its CpG. The most striking pattern in Fig. 1 is an isolated but dramatic shift in the lineage leading to BatCoV RaTG13 which was reported (Zhou et al., 2020) (Theys et al., 2018) , but also in experimentally CpG dinucleotide-enriched viral genomes (Antzin-Anduetza et al., 2017; Burns et al., 2009; Fros et al., 2017; Trus et al., 2019; Tulloch et al., 2014; Wasson et al., 2017) . To search for a mammalian host with the potential to select viral lineages with low Poder, 2011; Pratelli, 2006) , have genomic ICpG and GC% values similar to those observed in SARS-CoV-2 and BatCoV RaTG13 (Fig. 3A) .
cord-262923-kgzbd6w3	2018	Here, we report the development of an improved molecular diagnostics tool that utilizes CRISPR/dCas9-mediated biosensor that couples a nuclease inactivated Cas9 (dCas9) and single microring resonator biosensor, enables label-free and real-time detection of pathogenic DNA and RNA. In this study, we developed an improved diagnostic tool by combining a CRISPR/dCas9 and an isothermal diagnostic approach based on SMR biosensor for simultaneous nucleic acid (RNA and DNA) amplification and detection with speed as well as high sensitivity and specificity. For simultaneous amplification and detection of nucleic acid, sequence specific primer of target was immobilized to the surface of the SMR biosensor and dCas9 RNP was in reaction chamber with single temperature for isothermal reaction with RPA. To achieve sensitive detection with dCas9 RNP on SMR biosensor, we constructed guide RNAs (gRNAs) targeting two tick-borne pathogens that have substantially overlapping clinical presentations: Orientia tsutsugamushi, the causative agent of scrub typhus (ST), and bunyavirus, the causative agent of severe fever with thrombocytopenia syndrome (SFTS) (Fig. 1B) .
cord-263033-4790dhc5	2015	The review considers posttranscriptional modification of eukaryotic mRNA, focusing on the major modified nucleotides, the role they play in the cell, the methods to detect them, and the enzymes responsible for modification. Regions distant from the mRNA ends may contain N6 methyladenosine (m 6 A), 5 methylcytidine (m 5 C), pseudouridine (Ψ), and inosine (I), which were believed to play only a minor role because their pro portion in cell RNA is extremely low as compared with the standard nucleotides. In the case of eukaryotic mRNAs, the method is suitable for probing the adenosine methylation status in a particular site of a particular RNA in various cell growth conditions. A method known as site specific cleavage and radioactive labeling followed by ligation assisted extrac tion and thin layer chromatography (SCARLET) [23] makes it possible to establish whether adenosine is methylated in a given position of a given molecule and to estimate the proportion of modified and unmodi fied nucleotides (Fig. 2) .
cord-263157-8jin6oru	2008	Recent advances regarding the utility of RNA-mediated interference (RNAi) to specifically inhibit HIV-1 replication have opened new possibilities for the development of gene-based therapies against HIV-1 infection. Importantly, this study made the extraordinary demonstration that cell transfection of synthetic 21 base pairs (bp) short interfering RNA (siRNA) duplexes can mediate RNAi in a sequence-specific manner; this finding enabled the specific regulation of gene expression in a variety of biological systems, including diseased cells. Soon after the demonstration that synthetic siRNAs were able to induce the RNAi mechanism in mammalian cells (15) , several studies reported that HIV-1 gene expression and replication ex vivo could be inhibited by virus-specific synthetic siR-NAs (16 22) or expressed siRNAs (16, 18) that were targeted to early or late phases of virus replication. To counteract this strategic weakness, co-expression of multiple siRNAs or shRNAs that target conserved RNA sequences could reduce the emergence of single siRNA-resistant virus with a comparable effect to that achieved by the multiple anti-HIV drug combination approach employed by HAART.
cord-263239-andje0wu	2015	Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. To corroborate that PI4KA activity is required for the step of viral genome replication, we performed a time-of-addition experiment in which AL-9 was added to the cells at different time points after infection with RLuc-EMCV. The Picornavirus EMCV Converges on the Host Lipid Pathway Used by HCV localized throughout the cytoplasm and at the Golgi, OSBP was mainly found at ROs in infected cells, where it largely colocalized with 3AB ( Fig 6D, Pearson''s correlation coefficient = 0.71). Finally, data are presented suggesting that the OSBP-mediated exchange of PI4P and cholesterol at RO-MCSs is critical for EMCV genome replication and the global organization of ROs. Membrane alterations in the cytoplasm of cardiovirus-infected cells were already observed decades ago by electron microscopy [37, 38, 63] .
cord-263302-z5uhrta5	2000	Transfection of the reporter RNA into MHV-infected cells resulted in synthesis of a CAT-specific subgenomic mRNA detected by reverse transcription-polymerase chain reaction (RT-PCR). Further sequencing on cDNA clones derived from JHM2c mRNAs by reserve transcription-polymerase chain reaction (RT-PCR) has shown that the leader-body joining sites in subgenomic mRNA2-1 are more heterogeneous . When it was placed in front of the chloramphenicol acetyl-transferase (CAT) gene in the defective-interfering (DI) RNA-CAT reporter plasmid, the IG5-1, which is devoid of any known IRES sequence, can direct the synthesis of a subgenomic CAT-containing mRNA and expression of the CAT activity, thus confirming that the IG5-1 serves as a promoter for transcription of a subgenomic mRNA. To identify the minimal sequence required for subgenomic mRNA transcription, three deletions within the 140-nt sequence were made by PCR, and the deletion fragments were cloned into the DI RNA-CAT reporter vector in place of the wild-type, full-length (140-nt) se, and MHV-1 (F), respectively.
cord-263315-g7os15m1	2018	title: Identification of Secreted Proteins Involved in Nonspecific dsRNA-Mediated Lutzomyia longipalpis LL5 Cell Antiviral Response The two most abundant secreted peptides at 24 h in the dsRNA-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (FKBP), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. In human and mouse plasmacytoid dendritic cells (pDCs), which are professional interferon-producing cells specialized in recognizing viral RNA and DNA through the endosomal Toll-like receptors (TLRs) TLR7 and TLR9, respectively, PLSCR1 was described as a TLR9-binding protein that plays a significant role in type-1 interferon responses in pDCs by regulating TLR9 expression and trafficking [57] . Binding of FKBP51 to TRAF proteins facilitates the type-I interferon response induced by dsRNA transfection or Newcastle disease virus (NDV) infection in murine fibroblasts. Binding of FKBP51 to TRAF proteins facilitates the type-I interferon response induced by dsRNA transfection or Newcastle disease virus (NDV) infection in murine fibroblasts.
cord-263334-wwkdum94	2014	BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI¼ 0.01) for 48 h, Viral titers determined by plaque formation assay at 48 hpi. (F) BHK-21 cells transfected with different amounts of DDX3 shRNA plasmid were infected with JEV (MOI ¼0.01), 48 h later, the amount of virus released into the medium was determined by plaque formation assay. In order to determine whether cellular DDX3 is involved in virus assembly or release, the BHK-21 cells were transfected with DDX3 shRNA plasmid before being infected with JEV (MOI ¼0.01). The virus titers were detected 2 days later by plaque formation assay, the results showed that overexpression of DDX3r-K230E, DDX3r-S382L and the control plasmid pcDNA3.1 after DDX3 knockdown resulted in the reduction of JEV replication for 13-fold (p o0.01), 12-fold (p o0.01) and 15-fold (p o0.01). The DEAD-box RNA helicase DDX5 acts as a positive regulator of Japanese encephalitis virus replication by binding to viral 3′ UTR
cord-263357-krvei97r	2013	Sequencing and bioinformatic analysis of the viral genomic RNA showed that it is a novel virus not previously detected in any other species and that its closest relatives are two Asian bat coronaviruses. demonstrated how state-of-the-art sequencing technology and bioinformatic analysis can quickly provide critical insight into the viral genome sequence, phylogeny, replication strategy, and potential drug and vaccine targets and generate tools to evaluate the possible epidemic risk associated with this novel human virus. In June 2012, a novel coronavirus, tentatively named HCoV-EMC (for Erasmus Medical Center where the initial genome sequence was done), was isolated in a monkey kidney cell line from a sputum specimen from a 60-year-old man from the Kingdom of Saudi Arabia who died of acute severe pneumonia and renal failure. The genomic analysis suggests that HCoV-EMC may be a zoonotic virus that spilled over to infect the 9 laboratory confirmed patients, either directly or indirectly, from an unknown reservoir, possibly a bat.
cord-263433-oldy0gta	2015	As the ISGs described to date are coding genes, we sought to determine whether IFN also regulates the expression of long non-coding ISGs. To this aim, we used RNA sequencing to analyze the transcriptome of control and HuH7 cells treated with IFNα2. To address the issue of whether IFN could also regulate expression of lncRNAs, which may play key roles in the antiviral response, we analyzed the transcriptome of cells treated or not with IFNα2 by RNA sequencing (RNASeq). The results showed that at later times post-infection with the influenza virus lacking NS1, there was increased expression of lncISG15, lncBST2/BISPR, and their neighboring coding transcripts (Figure 3A) . To discriminate whether lncISG15 and lncBST2/BISPR are induced directly by the JAK/STAT signaling pathway or by a secondary wave of the IFN response, we evaluated the expression of these lncRNAs and their coding neighboring genes in HuH7 or A549 cells incubated or not with the JAK/STAT inhibitor ruxolitinib.
cord-263468-996kl9jz	1988	Abstract We assessed the alterations of viral gene expression occurring during persistent infections by cloning full-length transcripts of measles virus (MV) genes from brain autopsies of two subacute sclerosing panencephalitis patients and one measles inclusion body encephalitis (MIBE) patient. One of these nucleotide substitutions and one deletion resulted in alteration of the reading frames of two fusion genes, as confirmed by in vitro translation of synthetic mRNAs. One cluster of mutations was exceptional; in the matrix gene of the MIBE case, 50% of the U residues were changed to C, which might result from a highly biased copying event exclusively affecting this gene. most likely based on one hand on the low fidelity of RNA To assess the variability in strains of lytic viruses, and replication (Domingo et al., 1978; for review see Steinto estimate the number of mutations introduced during hauer and Holland, 1987) and on the other hand on the persistence, we counted the differences of lytic and perlow selective pressure exerted on viral genomes in nonsistent viruses from a consensus as defined above.
cord-263580-zxnmylkw	2011	New studies have revealed molecular mechanisms for coupling between ATP hydrolysis and unwinding, the physical basis for regulatory control by cofactors, and novel functions for RNA remodeling proteins. For DEAD-box proteins where the mechanochemical cycle has been studied, ATP hydrolysis (specifically, at the stage of Pi release) stimulates the dissociation of bound RNA molecules [46 ,47] , allowing recycling of these ''single-use'' proteins [7] . Like many DEAD-box proteins, Mss116 is capable of unwinding short RNA duplexes [46 ,52,53] , however, studies of Mss116 mutants in vivo and in vitro show that its role in splicing is not necessarily dependent on helicase activity [52, 54 ] . This structure reveals the Dbp5 export motor imbedded within the complex containing other proteins involved in its function, revealing roles for small molecule activators and a conserved mechanism for ATP hydrolysis in RNA release
cord-263645-wupre5uj	2018	One important example is the development of chemical probes, which has greatly progressed the study of proteins and related diseases (11, 12) but has been challenging for non-ribosomal RNAs. This powerful chemical tool requires small molecules with well-defined biological activity, cell permeability, and selectivity to accurately and reliably probe specific mechanistic and phenotypic questions (11, 12) . While ligands that bind non-ribosomal RNA in vitro have been reported for decades, the development of chemical probes with evidence of specific small molecule:RNA engagement in cell or animal models has dramatically increased in the last four years. Recent studies report several drug-like small molecules that target a range of RNAs in animal models, including riboswitches (15) , miRNAs, (16, 17) splice sites (18) , and mature mRNAs (19) , at least one of which is currently in clinical trials (NCT02268552).
cord-263699-gosqpg3k	2020	GBF1 has been shown not only to facilitate the intracellular traffic of different viral and cellular elements during infection, but also to modulate the replication of viral RNA, the formation and maturation of viral replication complexes, and the processing of viral proteins through mechanisms that do not depend on its canonical role in intracellular transport. In addition to the role of GBF1 in the transport of cellular and viral proteins relevant for RNA replication and viral assembly, it has also been observed that this factor can regulate the lipid composition of the membranous web induced by HCV. The role of GBF1 has been found, principally, to depend on its ability to activate distinct Arf proteins, to regulate different transport pathways that permit the communication between the viral replication organelles and cellular organelles (ER and LDs) involved in virus infection.
cord-263987-ff6kor0c	2017	BACKGROUND: Despite the long-anticipated possibility of putting sequence alignment on the same footing as statistical phylogenetics, theorists have struggled to develop time-dependent evolutionary models for indels that are as tractable as the analogous models for substitution events. MAIN TEXT: This paper discusses progress in the area of insertion-deletion models, in view of recent work by Ezawa (BMC Bioinformatics 17:304, 2016); (BMC Bioinformatics 17:397, 2016); (BMC Bioinformatics 17:457, 2016) on the calculation of time-dependent gap length distributions in pairwise alignments, and current approaches for extending these approaches from ancestor-descendant pairs to phylogenetic trees. CONCLUSIONS: While approximations that use finite-state machines (Pair HMMs and transducers) currently represent the most practical approach to problems such as sequence alignment and phylogeny, more rigorous approaches that work directly with the matrix exponential of the underlying continuous-time Markov chain also show promise, especially in view of recent advances.
cord-264051-ps0x2es1	2020	
cord-264159-e9071tyv	2020	In pathophysiological studies of infectious disease, single-cell omics offer excellent spatial-temporal resolution that help to not only reconstruct the uneven subcellular distribution of pathogen across the entire host cell population, but also reveal the sequence of immune events accompanied by the change of immune cell profiles. Moreover, viral mutation can be correlated with host gene expression status at the single-cell level to further investigate their potential mutual effect on one another throughout the course of infection and reveal the dynamic host responses and pathogen adaptations in the progression of infection [32] . Technologies that enable the simultaneous measurement of multiple parameters facilitate high-resolution characterization of transcripts and protein at the single-cell level and boost our understanding of how host immune responses are initiated and orchestrated against infection. As covered earlier in this review, the main applications of scRNA-seq in infectious disease study comprise of the following: (1) studying effect of host cell heterogeneity on infection, (2) identifying host immune responses, and (3) antibody discovery.
cord-264488-989t9ld1	2014	In the present study, the potential antiviral activity of RNase L against hepatitis B virus (HBV) was explored utilizing the recently reported infection protocol based on human hepatoma HepG2 cells stably complemented with the virus entry factor NTCP. These results suggest that HBV replication can be regulated through interferon-mediated RNA decay pathways and that activation of these host antiviral factors may represent a novel therapeutic strategy for HBV infection. Our data indicate that ligand-mediated activation of these enzymes results in a marked inhibition of HBV replication in both infected and transfected cells. With the newly established HepG2-NTCP cell culture system, which permits HBV infection, we showed that viral gene expression and replication can be profoundly reduced by 2-5Aor poly(I:C)-mediated activation of RNase L. In HBV1.2-transfected Huh-7 cells, it was further confirmed that this type of inhibition occurred mostly through viral RNA degradation via the ribonuclease function of RNase L.
cord-264678-wt0lvhfl	2009	To develop the IRES search system, it will be necessary to screen the database of virus sequences by the prediction of secondary structure to identify the candidate IRES element in the virus genome, especially those positive strand viruses with 5'' untranslated regions. Enterovirus IRES domain IV [25] , was first selected as a target to compare with the whole genome sequences from four different viruses (Enterovirus 71 (U22521), Bovine Enterovirus (NC_001859), Human Rhinovirus (NC_001617) and Hepatitis C virus (NC_004102) [26] [27] [28] ) were downloaded into IRSS and ran UTR2SQ.pl program to proceed RNA secondary structure prediction (see Figure 1 ). First, the IRSS search capability is evaluated while virus genomes sequences were substituted for the entire UTRdb. The known IRES element which was used for RNA comparison was selected such as HCV IRES domain III structure for example.
cord-264746-gfn312aa	2012	The success of this project (it came in almost 3 years ahead of time and 10% under budget, while at the same time providing more data than originally planned) depended on innovations in a variety of areas: breakthroughs in basic molecular biology to allow manipulation of DNA and other compounds; improved engineering and manufacturing technology to produce equipment for reading the sequences of DNA; advances in robotics and laboratory automation; development of statistical methods to interpret data from sequencing projects; and the creation of specialized computing hardware and software systems to circumvent massive computational barriers that faced genome scientists. Although the list of important biotechnologies changes on an almost daily basis, there are three prominent data types in today''s environment: (1) genome sequences provide the starting point that allows scientists to begin understanding the genetic underpinnings of an organism; (2) measurements of gene expression levels facilitate studies of gene regulation, which, among other things, help us to understand how an organism''s genome interacts with its environment; and (3) genetic polymorphisms are variations from individual to individual within species, and understanding how these variations correlate with phenotypes such as disease susceptibility is a crucial element of modern biomedical research.
cord-264884-ydkigome	2008	For example, common structural motifs from phage to eukaryotic DNA viruses (T4 and herpesvirus) suggest very ancient links in virus evolution that span all domains of life (see below). On an evolutionary time-scale, the majority of viral lineages tend to exist as species-specifi c persistent (aka temperate, latent, and chronic) infections in which individual hosts will be colonized by mostly silent (asymptomatic) viruses for the duration of their life . It has distinct genetic, fi tness, and evolutionary characteristics that require intimate, host (tissue)-specifi c viral strategies and precise gene functions to attain stable maintenance in the presence of immunity and to allow biologically controlled reactivation. Thus, the phycodnaviruses appear to represent a basal but diverse viral lineage that has both acute and persistent lifestyle and have some clear relationships to most large eukaryotic DNA viruses and many phage.
cord-264944-7xj27r98	1993	The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. The method was compared to existing extraction techniques by studying the quality of the templates for rcvcrsetranscriptase polymerase chain reaction amplification (RT-PCR), using virusinfected and mock-infected paraffin-embedded cell pellets as a model. In the work reported here we used paraffin-embedded torovirus-infected cell pellets to compare the effect of different fixatives and RNA extraction methods on RT-PCR amplification of tissue extracts.
cord-265095-lf5j4ic7	1990	
cord-265139-x7g3jcjm	2020	There is also growing evidence that circRNAs are closely linked to non-alcoholic fatty liver disease (NAFLD), a disorder that is caused by a plethora of factors including hepatic lipid accumulation, adipose tissue and mitochondrial dysfunction, a high-fat diet, obesity, a chronic inflammatory state, insulin resistance (IR), and genetic and epigenetic factors [48, 55] . In addition to classical epigenetic modifications, a variety of ncRNAs have been uncovered in different cells and organs including adipose tissues, many of which are involved in the regulation of adipogenesis and other metabolic processes implying their role in the etiology of obesity [69] . Emerging evidence from in vitro and in vivo animal studies suggest that circRNAs are expressed in adipose tissues and may modulate adipogenesis and lipid metabolism. Collectively, the results from the above studies demonstrate that several circRNAs are differentially expressed in adipose tissue and support a significant role of these RNA species in the regulatory networks of adipogenesis.
cord-265173-70wyecwj	2020	title: Host cell p53 associates with the feline calicivirus major viral capsid protein VP1, the protease-polymerase NS6/7, and the double-stranded RNA playing a role in virus replication Viruses modulate p53 functions in different manners: single-stranded RNA viruses such as the human coronavirus NL63 (HCoV-NL63) induces its degradation of p53 (Yuan et al., 2015) to ensure viral growth in infected cells (Ma-Lauer et al., 2016) , Zika (ZIKV) and West Nile (WNV) activate p53 to facilitate their replication (El Ghouzzi et al., 2016) (Teng et al., 2017) (Yang et al., 2008) while Adenovirus Even though p53 is implicated in the induction of apoptosis and in many viral infections, its role during calicivirus infection has not been studied. Here we found that p53 interacts with FCV dsRNA, the protease-polymerase NS6/7, and VP1 in the RC; moreover, knockdown of p53 resulted in a significant reduction of the NS viral protein synthesis and virus production, indicating its role for efficient viral replication.
cord-265381-ppjohov8	2003	6 -13 Cis-acting elements required for minus-strand RNA accumulation in vivo or RNA synthesis in vitro have been well studied for the plant plus-strand RNA viruses that contain tRNA-like structures in the 3 0 NTR 2 such as brome mosaic virus (BMV), 14 -16 cucumber mosaic virus (CMV), 17 tobacco mosaic virus (TMV) 18 and turnip yellow mosaic virus (TYMV), 19, 20 and for several other viruses such as alfalfa mosaic virus (AlMV), 21, 22 barley stripe mosaic virus (BSMV), 23 cymbidium ringspot virus (CymRSV), 24 red clover necrotic mosaic virus (RCNMV) 25 and turnip crinkle virus (TCV). The role of the 3 0 NTR cis-acting sequences and/ or structures in PVX RNA accumulation was studied by solution structure probing and by introducing wild-type (w.t.) and mutant transcripts into tobacco protoplasts for analysis of minus and plusstrand RNA accumulation by S 1 nuclease protection assays.
cord-265410-khwzdi79	2020	Life is defined as the instance of lyfe that we are familiar with on Earth, one that uses a specific organometallic molecular toolbox to record information about its environment and achieve dynamical order by dissipating certain planetary disequilibria. Hence, in the search for extraterrestrial life, we must consider that: (1) Life exactly as we know it may be rare in the universe, but a more general class of phenomena with life-like characteristics may be far more common; (2) There may be systems, yet to be discovered or even imagined, that more successfully satisfy the living criteria than even earthly life does; (3) By loosening our constraints on the definition of life, we open ourselves up to exploring the full parameter space of physical and chemical interactions that may create life. Lyfe is any hypothetical phenomenon that maintains a low-entropy state via dissipation and disequilibria conversions, utilizes autocatalytic networks to achieve nonlinear growth and proliferation, employs homeostatic regulatory mechanisms to maintain stability and mitigate external perturbations, and acquires and processes functional information about its environment.
cord-265461-hj2b1wc4	2017	Some virus families code for two different MTase domains carrying a cap-binding site (e.g., poxviruses [11] , coronaviruses [ Structure of inhibitors targeting enzymes involved in viral RNA capping pathways. Cap analogues exemplified here with m7 GTP, and several inhibitors of cap-binding protein have been identified through X-ray structure analysis of the influenza virus PB2-CBD in complex with the corresponding ligands. The X-ray structure of influenza A or B virus PB2 in complex with m7 GTP [49, 50] reveals a conserved cap-recognition mechanism in which the methylated guanosine is stacked between two aromatic residues similar to its binding mode with the eukaryotic initiation factor (eIF4E). However, the past research decade has a contrario unveiled that RNA capping is essential for virus replication, and is in fact a most interesting target for the design of potent antivirals due to two main reasons: (i) incomplete/inhibited RNA capping triggers a potent host immune response adding up to direct inhibition of viral gene expression, and (ii) structural and functional studies of viral capping enzymes have revealed a profound uniqueness of the viral enzymes involved, which shows promises to achieve high drug selectivity.
cord-265508-t1nfyzf5	1984	authors: Boursnell, M.E.G.; Brown, T.D.K. title: Sequencing of coronavirus IBV genomic RNA: a 195-base open reading frame encoded by mRNA B Abstract DNA sequencing of genomic cDNA clones of avian infectious bronchitis virus (IBV) has been carried out. The organisation of the messenger RNAs and in vitro translation studies have led to the hypothesis that the 5''-most sequences of each mRNA, which are not present in the next smallest mRNA, contain the complete coding sequences for the major protein product produced by that messenger species (Stern and Kennedy, 1980b; Lai et al., 1981) . In this paper we report the nucleotide sequence of a cloned cDNA copy of IBV genomic RNA in the region corresponding to the 5'' end of mRNA B. The region of the IBV sequence presented in this paper contains the 5'' ends, on the viral genome, of mRNAs A and B.
cord-265855-zf52vl11	2020	Zinc deficiency may increase ACE-2 receptor activity on type 2 pneumocytes and other cells that are infected by SARS-COV-2, mainly in the lower respiratory tract. Although there are no specific data regarding zinc in this pathway for SARS-CoV-2, zinc may limit infection through upregulation of IFN-alpha production and an increase in its antiviral activity (77, 78) . Thus, patients with IL-6-174 GG polymorphism (C-carriers) may be susceptible to developing a severe infection due to SARS-CoV-2, leading to an increase in IL-6 levels that produce a cytokine storm related to impaired zinc homeostasis. We believe there is enough evidence to further investigate how zinc status or homeostasis is involved in the pathogenesis of severe illness produced by SARS-CoV-2 infection, and its potential role as an active treatment should be assessed in clinical trials.
cord-265895-ck7eto16	1987	These data, coupled with the high frequency of RNA recombination during MHV infection, suggest that the viral polymerase may pause in or around regions of secondary structure, thereby generating pools of free leader-containing RNA intermediates which can reassociate with the template, acting as primers for the synthesis of full-length or recombinant RNAs. These data suggest that MHV transcription uses a discontinuous and nonprocessive mechanism in which RNA polymerase allows the partial RNA products to be dissociated from the template temporarily during the process of transcription. These data, coupled with the presence of discrete large leader-containing RNAs which range from 84 to 1000 nucleotides in length in MHV-infected cells (Baric et a/., 1985) suggest that discontinuous RNA intermediates may be dissociated and reassert between viral RNA templates to generate recombinant viruses by a copy-choice mechanism (Makino eta/., 1986a). The leader-containing RNAs larger than 1 10 nucleotides in length detected by the leader-specific cDNA probe were more heterogeneous (Fig. 2) (Baric et al., 1985) suggesting that multiple RNA species in this size range were present.
cord-266127-phv08xe2	2019	Consistent to our previous results, Rbx1 expression was successfully knocked down in response to Rbx1 siRNA in RV-SA11-infected cells lysed at 9 hpi as well as in mock-infected control but not in RV-SA11-infected cells harvested FIGURE 1 Host RNA interference is blocked during early hours of RV-SA11 infection. Together, the data suggest that actively replicating RV-SA11 triggers attenuation in protein levels of AGO2 leading to functional blocking of RNAi during early time points (2-6 hpi) of infection. Sensitivity of ectopic GFP (pEGFP-N1) expression to siGFP was also reduced in RV-NSP1overexpressing cells ( Figure S4A ), indicating that RV-NSP1 might FIGURE 3 Rotaviral nonstructural protein 1 triggers ubiquitination and proteasomal degradation of AGO2. Rotavirus nonstructural protein 1 suppresses virus-induced cellular apoptosis to facilitate viral growth by activating the cell survival pathways during early stages of infection
cord-266464-wuf3s8m0	2016	title: Viral RNA in Blood as Indicator of Severe Outcome in Middle East Respiratory Syndrome Coronavirus Infection We evaluated the diagnostic and clinical usefulness of blood specimens to detect Middle East respiratory syndrome coronavirus infection in 21 patients from the 2015 outbreak in South Korea. Respiratory specimens are preferred for viral RNA detection and confirmatory diagnosis of MERS-CoV infection in humans (5) . Our study aimed to evaluate the diagnostic utility of blood specimens for MERS-CoV infection by using large numbers of patients with a single viral origin and to determine the relationship between blood viral detection and clinical characteristics. Between the blood viral RNA-positive and -negative groups, we found no differences in age, duration from symptom onset to diagnosis of MERS-CoV infection, or an invasive procedure before the specimens were obtained (online Technical Appendix Table 3 ). Clinical features and viral diagnosis of two cases of infection with Middle East respiratory syndrome coronavirus: a report of nosocomial transmission
cord-266520-n439dwcx	2020	The antiviral efficacy of the 2''-F-siRNA swarms and the elicited cellular innate responses did not correlate suggesting that innate immunity pathways do not significantly contribute to the observed enhanced antiviral activity of the modified siRNAs. The results support further applications of enzymatically produced siRNA molecules with incorporated adenosine nucleotides, carrying fluoro-modification on ribose C2'' position, for further antiviral studies in vitro and in vivo. We have previously generated three antiviral siRNA swarms against herpes simplex virus type 1 (HSV-1) mRNAs encoding essential viral proteins, including glycoprotein B (UL27), the infected cell protein 8 (ICP8; UL29), and ICP27 (UL54) (Romanovskaya et al., 2012; Paavilainen et al., 2016) . The siRNA swarm targeting mRNA of HSV single-stranded DNA binding protein ICP8, encoded by the UL29 gene, harbors a most pronounced antiviral effect (Paavilainen et al., 2016) and induces only minimal non-specific cellular responses (Romanovskaya et al., 2012) .
cord-266521-vovas81d	2019	In this report, recent advances in synthetic riboswitches that function in mammalian cells are reviewed focusing on the regulatory mechanisms they exploit such as mRNA degradation, microRNA processing, and programmed ribosomal frameshifting. In this report, recent advances in synthetic riboswitches that function in mammalian cells are reviewed focusing on the regulatory mechanisms they exploit such as mRNA degradation, microRNA processing, and programmed ribosomal frameshifting. While the ribozyme was not specifically regulated by a small molecule via an aptamer, this work paved the way for the subsequent riboswitches that employ allosterically regulated ribozymes (aptazymes) embedded in the 5 0 and/or 3 0 UTR to chemically regulate gene expression in mammalian cells (Figure 1a ) [13] [14] [15] [16] . A new mode of engineered RNA-based gene regulation in mammalian cells was demonstrated by controlling the accessibility of a miRNA target site by aptamer-ligand interaction
cord-266585-jfjrk9gy	2007	During construction of an infectious clone from a Vero cell-adapted coronavirus infectious bronchitis virus (IBV), we found that a G–C point mutation at nucleotide position 15526, causing Arg-to-Pro mutation at amino acid position 132 of the helicase protein, is lethal to the infectivity of IBV on Vero cells. Further characterization of the in vitro-synthesized full-length transcripts containing the G15526C mutation demonstrated that this mutation blocks the transcription of subgenomic RNAs. Substitution mutation of the Arg132 residue to a positively charged amino acid (Lys) affected neither the infectivity of the in vitro-synthesized transcripts nor the growth properties of the rescued virus. To further demonstrate that the failure to rescue infectious virus from the G15526C mutant transcripts is due to a defect in subgenomic RNA transcription, the full-length clones with and without the G15526C mutation were used to generate recombinant IBV expressing the enhanced green fluorescent protein (EGFP) by replacing the 5a gene with EGFP.
cord-266634-bww62vx8	2015	In the present work, using partially purified recombinant RNA polymerase complex of rinderpest virus expressed in insect cells, we demonstrate the in vitro methylation of capped mRNA. Considering the presence of both KDKE tetrad, responsible for 2 0 -O-methyl transferase as well as GXGXG motif for SAM substrate binding, domain III could likely represent the methyl transfer module (both N 7 -guanine and 2 0 -Omethyl) of RPV L protein. Consensus sequence between the viral mRNAs is given in bold the capped RNA with the partially purified L-P complex from insect cells resulted in a concentration dependent N 7 guanine methylation of 6 bp substrate (Fig. 2b , marked as rL) which was not detected in a mock-purified high-density fraction from insect cells infected with non-recombinant baculovirus (Fig. 2b, mock) . Further, to functionally validate the methyl transferase activity of domain III (aa 1717-2183, LD3) of RPV L protein, LD3 was purified from insect cells using metal chelate affinity chromatography as described earlier [8] .
cord-266921-x9q7dwc4	2006	The exoribonuclease RNase II is representative of an extensive enzyme family found in all three domains of life, whose members play roles in the maturation, turn-Structure and mechanism of ribonucleases Worrall and Luisi 131 Whereas DEAD-box helicases use the free energy of ATP binding and hydrolysis to disrupt secondary structure, RNase R transduces the favourable free energy of RNA backbone hydrolysis into mechanical work that translates the single-stranded substrate further into the catalytic pocket, much like a ratchet. Crystal structures of the core of the exosomes from the archaea Sulfolobus solfataricus and Archaeoglobus fulgidus have recently become available, revealing a hexameric ring comprising two types of RNase-PH-like subunits, known as Rrp41 and Rrp42 (rRNA-processing proteins 41 and 42; see Figure 4a ) [36 ,37 ,38 ] . Structure of Escherichia coli RNase E catalytic domain and its implications for RNA turnover and processing
cord-266960-kyx6xhvj	2020	The sonification of codons derived from all three reading frames of the viral RNA sequence in combination with sonified metadata provide the framework for this display. CONCLUSION: The auditory display in combination with real-time animation of the process of translation and transcription provide a unique insight into the large body of evidence describing the metabolism of the RNA genome. Audio generated from each of these sequence motifs and metadata were combined to create a complex auditory display to represent either transcription or translation. High resolution analysis of gene expression in Coronavirus genomes has detected ribosome protected fragments which map to non-canonical ORF''s, these may be novel protein-coding ORFs and short regulatory uORFs. The tool highlights the occurrence of one such uORF of 30 nucleotides (including the stop codon) in the 5′ untranslated region downstream of TRS1 [35] that is not documented in the GenBank metadata. In the Additional file 4: supplementary example ''Sonification Sub-genomic RNA'' the auditory display represents the process of transcription.
cord-266985-9qwttt2y	2014	At present, the great strength of gene sequence data appears to be in giving information on the distribution and proportion of susceptible genotypes (for example due to the presence of the appropriate pathogen‐binding receptor) in the host population rather than in predicting specificities from the amino acid sequences concurrently obtained. The nature of the mutant spectrum in RNA viruses greatly complicates the application of omics approaches to the development of mechanistic dose–response models and prevents prediction of risks of disease progression (given infection has occurred) at the level of the individual host. The binding of NoV capsid protein to its HBGA receptor Table 1 Breakdown of the initial infection process into four steps for building a mechanistic dose-response relationship for RNA viruses through the oral route: information needs Host glycans play a central role in the pathogen infection process including binding of virus to specific receptors in steps 1 and 2 and also in the immune system.
cord-267027-diwm1940	1992	Abstract A combination of comparative sequence analysis and thermodynamic methods reveals the conservation of tertiary structure elements in the 5′ untranslated region (UTR) of human enteroviruses and rhinoviruses. Base pairings between highly conserved 17-nucleotide (nt) and 21-nt sequences in the 5′ UTR of human enteroviruses and rhinoviruses constitute a predicted pseudoknot that is significantly more stable than those that can be formed from a large set of randomly shuffled sequences. The predicted pseudoknots, K2 (tertiary interactions: between the region 497-501 and 550-554) and K3 (579-581 and 600-602) in the RLP of PV2L were found to be totally conserved in all 22 human enteroviruses and rhinoviruses. Based on the common RNA secondary structures (Le and Zuker, 1990 ) of the 5'' UTR in 18 human enteroviruses and rhinoviruses, two sequences complementary to the highly conserved polypyrimidine sequence in all picornaviruses were identified in human 18 S rRNA .
cord-267036-llngs3v5	2009	Numerous lines of evidence indicate that cellular SR proteins are important for regulation of viral RNA splicing and participate in other steps of post-transcriptional viral gene expression control. The E2 protein encoded by HPVs primarily regulates the transcription of early promoters by binding to a consensus element within the long control region of the viral genome, and also functions together with the E1 protein in viral DNA replication [16] . It has been shown that reduced activity of SR proteins resulting from viral infection can be recovered by overexpression of SR proteins or by their rephosphorylation in the host cells [74] , and that HIV expression can be greatly increased when SRp75 is phosphorylated by SRPK2 [69] . Cellular SR proteins and their cooperative or antagonistic factors may play a critical role in the life cycle control of viruses, which involves a series of alternative splicing events for expression of viral genome or proteins. HIV-1 infection induces changes in expression of cellular splicing factors that regulate alternative viral splcing and virus production in macrophages
cord-267115-6jqdi417	2020	Collectively, we established the first expandable human gastric organoid culture across fetal developmental stages, and we support the hypothesis that fetal tissue seems to be less susceptible to SARS-CoV-2 infection, especially in early stages of development. Principal component analysis (PCA) showed smaller heterogeneity in the organoid groups derived at different stages of fetal and pediatric development with respect to the primary tissues analyzed at the same stages, which may also include some heterogeneity from the surrounding cells as a result of the isolation procedure (Fig. 3a) . In order to validate both fetal and pediatric gastric organoids as functional in vitro models of SARS-CoV-2 infection and replication, we optimized the culture condition for viral infection in a 3D system (Fig. 4a) .
cord-267124-8efdzlc0	2020	In response to the pandemic spread of SARS-CoV-2, the authorities of the German federal state of Hamburg ordered mandatory autopsies in all patients dying with a diagnosis of COVID-19 confirmed by polymerase chain reaction (PCR). During autopsy, tissue samples for histology were taken from the following organs: heart, lungs, liver, kidneys, spleen, pancreas, brain, prostate and testes (in males), ovaries (in females), small bowel, saphenous vein, common carotid artery, pharynx, and muscle. In this autopsy study of 12 consecutive patients who died of COVID-19, we found a high incidence of deep venous thrombosis (58%). In studies that examined deceased patients with COVID-19 without relying on autopsy, no increased rates of pulmonary embolism were observed clinically. To our knowledge, only 3 case reports have been published on patients with COVID-19 who have undergone complete autopsy and a few more in which only lung tissue was examined (7, 8) .
cord-267326-355q6k6k	2018	This study shows that spatial factors (e.g., reservoirs/tributaries, land use) are the main drivers of the viral community structure in tropical freshwater ecosystems. However, up till now, studies of land use impacts on the virome community in freshwater ecosystems are still limited as they mainly rely on traditional methodology (culture-based method or qPCR/RT-qPCR), which focuses on limited human virus targets without considering the whole picture of the viral community in the water environment (Corsi et al., 2014; Lenaker et al., 2017) . Thus, the objectives of this study were to: 1) investigate the overall virome distribution and diversity in diverse freshwater ecosystems (reservoirs/tributaries) in a tropical environment, 2) compare the virome community based on the different land use patterns, 3) assess the extent of human-related pathogenic viruses in surface waters, especially emerging zoonotic and human-related viruses, which may have been undetected before.
cord-267377-wyhsxj6g	2014	The single, large ORF encodes a polyprotein with domains specifying methyltransferase (mtr), protease (pro), helicase/NTpase (hel), and polymerase (pol) activities fused to the sequence encoding the CPs. The major and minor coat proteins map to the same CP sequence and size differences between them are potentially determined by translation initiation at two different start codons (minor, AUG 5581 and major, AUG 5710 ). Oat protoplasts were inoculated with capped transcripts of wild type clone pOBDV, major CP mutants GH1-7 (premature termination mutant) and KL1-3 (initiation codon mutant), and AB15-25 (marafibox mutant) and incubated for 40 h. Accumulation of viral coat proteins (CPs) and RNA in oat protoplasts inoculated with premature termination and initiation codon mutants of the CP gene. Oat protoplasts were inoculated with capped transcripts of wild type clone pOBDV, premature termination mutants GH1-7 and EF2-2, and initiation codon mutants IJ4-7 (minor CP), and KL1-3 and KL2-5 (major CP) and incubated for 24 h.
cord-267475-6f4h3cck	2002	This depends on careful structural arrangements, however, which are rarely present in cellular mRNAs. Understanding the rules for initiation of translation enables understanding of human diseases in which the expression of a critical gene is reduced by mutations that add upstream AUG codons or change the context around the AUG(START) codon. (Whether the second AUG codon resides in a strong or weak context is not relevant; the ribosome reads the mRNA linearly and thus the decision to stop or to bypass the first AUG is not influenced by whether there is a better initiation site downstream.) The large number of genes that employ leaky scanning precludes discussion of the biological significance of the proteins thereby produced, but it merits noting that, for many of the viruses in Table 3 , replication requires production of both listed proteins.
cord-267532-5rnqd9mb	1998	HEor CAT-specific subgenomic mRNAs were detected in the brains at days 1 and 2 p.i. but not later, indicating that the genes in the DI vector were expressed only in the early stage of viral infection. In the present study, we used this DI RNA vector system to express the viral HE gene in the CNS of mice in the presence of an HE-deficient helper virus. To determine the basis for the reduction in mortality following A59-DE-HE infection compared to other viruses ( Fig. 2) , virus titers in the brains of mice infected with A59-DE-HE were initially compared to mice infected with the parental A59 or A59-DE-CAT at 6 days p.i. Based on the survival of most mice infected with A59-DE-HE, reduced viral replication in the CNS was anticipated. IL-6 mRNA was also increased in the CNS of mice infected with A59-DE-HE virus (Fig. 5B) ; however, no differences were detected in the liver between the two groups.
cord-267533-nmgtan4e	2020	By LASSO and multivariate Cox regression analyses, we observed that delayed hospital admission, subpleural lesion, and high-dose corticosteroid use were independent risk factors of prolonged SARS-CoV-2 RNA detection. The study of Xu and colleagues [5] estimated the risk factors of delayed viral shedding (≥ 15 days after illness onset) and found that male, delayed hospital admission, and invasive mechanical ventilation were positively associated with prolonged SARS-CoV-2 RNA detection duration. Delayed hospital admission, hypokalemia, and subpleural lesion were still the independent risk factors of long-term SARS-CoV-2 RNA detection in multivariate binomial logistic regression analysis with a generalized additive model. LASSO analysis with Cox regression model found six independent risk factors of prolonged SARS-CoV-2 RNA detection duration, including cough, dyspnea, delayed hospital admission, subpleural lesion, the use of methylprednisolone, and the use of thymosin.
cord-267588-ruuzr6l1	2020	This study aimed to examine the efficacy of six different swabs that are commonly found in hospital settings (PurFlock Ultra, FLOQSwab, Puritan Pur-Wraps cotton tipped applicators, Puritan polyester tipped applicators, MedPro 6" cotton tipped applicators, and HOLOGIC Aptima), along with more readily available alternative transport mediums (DMEM, PBS, 100% ethanol, 0.9% normal saline and VTM) for their use in molecular detection of SARS-CoV-2. Therefore, our results suggest that the cotton and wood For the portion of the study focusing on alternative transport media, we assessed the ability of DMEM, PBS, 0.9% Normal Saline, and 100% ethanol compared to VTM to be used as medium for the preservation and recovery of viral RNA to be quantified by molecular detection. Despite finding similar levels of viral RNA collected using different swabs and transport media, there is variation when evaluating different respiratory clinical samples while testing for SARS-CoV-2.
cord-267867-q52nvn0n	2016	Several siRNAs dramatically reduced or even abrogated the replication of selectable subgenomic HCV replicons upon cotransfection of human hepatoma cells with viral target and siRNAs, or upon transfection of cells supporting autonomous replication of HCV replicon with siRNAs. Importantly, three siRNAs also proved capable of strongly inhibiting virus production in cell culture. Several siRNAs dramatically reduced or even abrogated the replication of selectable subgenomic HCV replicons upon cotransfection of human hepatoma cells with viral target and siRNAs, or upon transfection of cells supporting autonomous replication of HCV replicon with siRNAs. Importantly, three siRNAs also proved capable of strongly inhibiting virus production in cell culture. We sought to determine whether the siRNAs si240-1a, si244, and si313, shown to be the most efficient in inhibiting the replication of HCV subgenomic replicons both transiently and under selective pressure, are also capable of efficiently blocking virus infection in cell culture.
cord-268071-ow2aijmj	2020	Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. Naturally occurring mutations in critical residues for drug efficacy can lead to drug resistance phenomena, with a significant loss in the binding affinity of these molecules to the RdRp. We focused our study on SARS-CoV-2 mutations in order to assess if new viral variants were spreading across the Countries. Among all mutation sites analyzed, RdRp mutant is particularly interesting given that the enzyme is directly involved in viral replication and its fidelity determines the mutagenic capabilities of SARS-CoV-2. In the present work we have compared the SARS-CoV-2 reference genome to those exported from the GISAID database with the aim of gaining important insights into virus mutations, their occurrence over time and within different geographic areas.
cord-268122-74nj66vb	2020	The NAD + decline during normal aging results in oxidative damage, metabolic disorder, circadian rhythm abnormalities, and mitochondrial dysfunction through regulating signaling pathways, such as p53, NF-κB, PGC-1α and HIF-1α, by sirtuins and PARPs. NAD + and its metabolites function as crucial regulators to maintain cellular redox homeostasis through replenishing the reducing power or modulating the activity of NAD + -consuming enzymes including sirtuins and PARPs. However, disequilibrium of NAD + metabolism could disturb physiological processes, including mitochondria function, circadian rhythm, inflammation, DNA repair and metabolism, leading to aging-associated dysfunction and cancer. c The deduced NAD + levels in kidney are attributed to the decreased expression of enzymes in NAD + de novo synthesis and increased consumption by DNA damage activated PARPs. NAD + depletion inhibits the SIRT1/PGC1α mediated mitochondrial quality control, ATP production and NAD + de novo biosynthesis.
cord-268139-tgpsu4qz	2005	title: Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication In the current study, a reverse genetic approach was used to define the role of the 28-kDa amino-terminal product (nsp1) of the gene 1 polyprotein during replication of the coronavirus murine hepatitis virus (MHV) in cell culture. To determine whether nsp1 is required for MHV replication and to identify residues critical for protein function, mutant viruses that contained deletions or point mutations within the nsp1-coding region were generated and assayed for defects in viral replication, viral protein expression, protein localization, and RNA synthesis. Mutagenesis of the murine hepatitis virus nsp1-coding region identifies residues important for protein processing, viral RNA synthesis, and viral replication Experiments were next performed to determine if viral gene expression occurred in cells electroporated with RNA for mutants that did not produce infectious virus (VUSB5, VUSB6, nsp1DFL, and nsp1DMid).
cord-268206-ino9srb6	2020	Recently, severe acute respiratory syndrome coronavirus 2 (SARS-COV-2), commonly known as coronavirus disease-2019 (COVID-19) has rapidly spread across China and around the world. In the current SARS-COV-2 pandemic, Wu and McGoogan (2020) showed that patients with chronic diseases, including diabetes, were at higher risk for severe COVID-19 infection and mortality. The former (S) is the wild type which is milder while the latter (L) is the novel one which resulted in high binding affinity between SARS-COV-2 virus with angiotensin-converting enzyme 2 receptor in human cells. The use of convalescent plasma was recommended before as an important treatment during outbreaks of Ebola virus, Middle East respiratory syndrome coronavirus, SARS-COV-1, H5N1 avian influenza, and H1N1 influenza (Zhou et al. In a study involving patients with pandemic influenza (H1N1) and SARS virus, treatment of severe infection with convalescent plasma was associated with reduced respiratory viral load, serum cytokine response, and mortality (Cheng et al.
cord-268337-o6lo55o8	2005	Human immunodeficiency virus-1 (HIV-1) infection of cells resulted in partial cleavages of eIF4GI that was mapped to three sites in two regions on either side of the eIF3-binding domain ( Fig. 1) (Ohlmann et al., 2002; Ventoso et al., 2001) . Translation assays based on luciferase reporter constructs in cells indicated that expression of HIV protease (HIV PR) primarily inhibited translation of capped mRNAs. Interestingly, comparison of translation function of eIF4GI C-terminal cleavage products produced by L pro and HIV PR revealed that the slightly shorter HIV PR-derived fragment was defective in supporting translation of the PV-IRES but not the EMCV IRES. Demonstration in vitro that eucaryotic initiation factor 3 is active but a cap-binding protein complex is inactive in poliovirus-infected HeLa cells Eukaryotic initiation factor 4GII (eIF4GII), but not eIF4GI, cleavage correlates with inhibition of host cell protein synthesis after human rhinovirus infection Cleavage of eukaryotic initiation factor eIF4G and inhibition of host-cell protein synthesis during feline calicivirus infection
cord-268467-btfz6ye8	1989	The 3′-noncoding region of the genome contains an 11-nucleotide sequence, which is relatively conserved throughout the Coronavirus family and lends support to the theory that this region is important for the replication of negative-strand RNA. This result suggested that the HCV229E subgenomic mRNAs possess a nested-set structure similar to other coronaviruses and that A34 represented a cDNA clone of either the 3''-end of the genomic RNA or the leader sequence. The 3''-noncoding region contains the sequence TGGAAGAGCCA, 75 nucleotides from the 3''-end (Fig. 4) which is relatively conserved among coronaviruses and is found at approximately the same location in all of these viral genomes (Kapke and Brian, 1986; Skinner and Siddell, 1984; Armstrong et a/., 1983; Lapps et al., 1987; Kamahora et a/., 1988; Boursnell et al., 1985) ( Table 1) . Three intergenic regions of coronavirus mouse hepatitis virus strain A59 genome RNA contain a common nucleotide sequence that is homologous to the 3''end of the viral mRNA leader sequence
cord-268565-2sg1tlrg	2006	However, because durable neutralizing antibodies are usually elicited against the VSV surface glycoprotein after a single vaccination with replication-competent rVSV vectors, glycoprotein exchange vectors were designed to allow more effective boosting of immune responses to target antigens. In these pioneering studies, rhesus macaques were immunized with a combination of two prototypic rVSV vectors expressing a HIV-1 89.6 Env gp160/VSV-G fusion polypeptide and simian immunodeficiency virus (SIV) Gag p55 protein. Vaccine vector administration by a combination of intramuscular and intranasal routes clearly demonstrated the ability of rVSV vectors to elicit potent antigen-specific cellmediated immune responses in NHPs. In addition, this study also clearly demonstrated for the first time the ability of rVSV vectors expressing Env and Gag proteins to provide highly significant protection from AIDS after an intravenous heterologous SIV/HIV (SHIV) 89 .6P challenge [89] . Immunogenicity of attenuated vesicular stomatitis virus vectors expressing HIV type 1 Env and SIV Gag proteins: comparison of intranasal and intramuscular vaccination routes
cord-268718-tt07cwrf	2020	54 Virus infectivity study has indicated that the SARS-CoV-2 is able to utilize ACE2 of human, Chinese horseshoe bats, civet, and pig but was not able to use mouse ACE2. The roles of ACE2 expression in SARS-CoV-2 pathogenesis and human COVID-19 susceptibility are largely unknown. B, ACE2 expression in lung cancer patients with different smoking histories analyzed using similar methods as described previously 106 other symptoms in addition to respiratory symptoms, suggesting that SARS-CoV-2 could perhaps infect other organs (Figure 3 ). 118 In addition to sputum, SARS-CoV-2 RNA has been detected in the stools of a COVID-19 patient, 119 F I G U R E 3 Tissue distribution of angiotensin-converting enzyme 2 (ACE2) expression and potential COVID-19 susceptibility. Expression of elevated levels of proinflammatory cytokines in SARS-CoV-infected ACE2 + cells in SARS patients: relation to the acute lung injury and pathogenesis of SARS
cord-268763-s16n7f17	2020	The sequences from these specimens was used for downstream analysis (Figure 1) Assessing Nasal Specimen Similarity 64 nasal brushing specimens from 15 PARDS and 10 control subjects collected on study days 1, 3, 7, and 14, were analyzed by DESeq2. In comparing Group A, B, and C subjects, only disease severity (PELOD2) was statistically significant (Supplemental Table 1 ), and for individual specimens, PARDS classification (None, Mild, Moderate, or Severe), and the presence of direct lung injury, a viral or combined viral/bacterial cause of ARDS were significantly different between groups A, B, and C (Supplemental Table 2 ). Interferon-γ and tumor necrosis factor-related signaling were notable in Group B ( in conjunction with our findings that the only clinical variables that differentiated specimens from groups B and C from A were severity of lung injury and a viral cause of ARDS, and that viruses were the most common trigger of ARDS in both groups B and C, these data demonstrate that nasal epithelial transcriptomics can identify three distinct endotypes in PARDS: Endotypes A, B, and C.
cord-268970-uz7q6z2f	2020	Most currently approved strategies for the collection of saliva for COVID-19 diagnostics require specialized tubes containing buffers promoted for the stabilization of SARS-CoV-2 RNA and virus inactivation. We found SARS-CoV-2 RNA in saliva from infected individuals is stable at 4°C, room temperature (~19°C), and 30°C for prolonged periods and found limited evidence for viral replication in stored saliva samples. To explore the viability of broadly deploying affordable saliva-based surveillance approaches 8 , we characterized SARS-CoV-2 RNA stability and virus infectivity from saliva samples stored in widely available, sterile, nuclease-free laboratory plastic (polypropylene) tubes. Following RNA extraction 9 and RT-qPCR 10 testing for SARS-CoV-2 on the day of saliva collection 2 , the remaining sample volumes (n=20) were aliquoted and stored at -80°C, room temperature (recorded as ~19°C) and 30°C. Moreover, SARS-CoV-2 RNA remained relatively stable in saliva samples left for up to 25 days at room temperature (~19°C; Ct increase of 0.027, 95% CI: -0.019, 0.071) ( Figure 1B) .
cord-269011-230p8rsf	2005	Biochemical and theoretical studies led to a topological model for the MHV M protein Rottier et al., 1984 Rottier et al., , 1986 , in which the polypeptide spans the lipid bilayer three times, leaving a small amino-terminal domain (15-35 residues) in the lumen of intracellular organelles (or outside the virus), whereas the carboxyterminal half of the protein is located on the cytoplasmic side of the membrane (or inside the virion). Using a mutational approach including deletions, insertions, and substitutions, both the transmembrane domain and the cysteine-rich region immediately downstream thereof, but not the carboxy-terminal part of the cytoplasmic tail, were shown to be important for MHV S protein-induced cell-cell fusion (Bos et al., 1995; Chang and Gombold, 2001; Chang et al., 2000) The cleavage requirements of the S proteins for the biological activities of the coronavirus spike remain enigmatic.
cord-269150-d1sgnxc0	2012	In a screen based on a yeast three-hybrid system using the 5′-untranslated region (5′-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. In this study, we describe the interaction of a cellular protein, MADP1 (zinc finger CCHC-type and RNA binding motif 1) with the 5 0 -UTR of IBV and SARS-CoV, using yeast-based three hybrid screen (34) and RNA-binding assays. Using indirect immunofluorescence, we confirmed that MADP1, despite being reported as a nuclear protein (35) , was detected in the cytoplasm of virus-infected cells and partially co-localized with the RTCs. Upon silencing of MADP1 using siRNA, viral RNA synthesis on general has been affected, resulting in a lower replication efficiency and infectivity.
cord-269193-a647hwu9	1991	Abstract The complete nucleotide sequence of the second largest RNA segment of Dhori/India/1313/61 virus was determined and the deduced amino acid sequence was compared with the polymerase (P) proteins of influenza A, B, and C viruses. The viral RNAs have been shown to encode information in the negative-sense (Clerx et al., 1983; Fuller et al., 1987; Freedman-Faulstich and Fuller, 1990) and it was previously shown that the Dhori nucleoprotein (encoded by RNA segment 5) shares conserved amino acid sequences with the influenza A, B, and C virus nucleoproteins (Fuller et al., 1987) . The PBl polymerase proteins are the most highly conserved among the proteins of the influenza A, B, and C viruses (Yamashita et a/,, 1989 ) and they are most likely required for nucleotide addition during viral RNA synthesis (Braam et al., 1983) .
cord-269194-b1wlr3t7	2015	Complementing serologic testing by detecting infections within the pre-seroconversion window period and infections with immunovariant viruses, real-time PCR provides a highly valuable tool for screening, diagnosing, or monitoring diseases, as well as evaluating medical and therapeutic decision points that allows for more timely predictions of therapeutic failures than traditional methods and, lastly, assessing cure rates following targeted therapies. Beyond this, quantitative real-time PCR facilitates advancements in the quality of diagnostics by driving consensus management guidelines following standardisation to improve patient outcomes, pushing for disease eradication with assays offering progressively lower limits of detection, and rapidly meeting medical needs in cases of emerging epidemic crises involving new pathogens that may result in significant health threats. With the development and administration of newer drugs that target specific biological processes of HIV, routine and clinical monitoring of viral loads using a real-time quantitative PCR assay continues to be critical to predict treatment failure and early emergence of drug resistance mutations, within a timeframe that would increase subsequent treatment success.
cord-269294-vx7xr80t	2005	Here, we discuss the role of these enzymes, factors affecting their potential as drug targets and progress in the development of agents that inhibit their activity using the hepatitis C virus-encoded helicase NS3 and the cellular helicase DDX3 adopted for use by HIV-1 as examples. The solution of the crystal structure of HCV helicase complexed with oligonucleotide, as well as mutagenesis studies, have identified key residues that are essential for enzyme activity or translocation of the RNA substrate 13, 72 . Theoretically, an unwinding assay should increase the odds of finding an inhibitor because inhibition of any of the potential mechanisms of action listed above, except those requiring a cellular environment (for example, turnover and replicase-complex formation), should result in ''hits'' (for example, low-potency chemical starting points for medicinal chemistry optimization). Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding
cord-269466-9hnal9ad	2013	In this study, we used a novel dual gene cassette construct that differentiated primary infected cells from cells infected after virus intercellular movement to show that the early as well as the late secretory pathway, but not endocytosis, was important for TuMV transport. By using a dual cassette of genes encoding fluorescent proteins that can differentiate between primary infected cells and cells infected after intercellular transport, we provide evidence that turnip mosaic virus (TuMV) needs a functional secretory pathway where pre-and post-Golgi trafficking and the actomyosin network are important for its movement. Fig. 2E shows that that there was no significant difference in the ratio of red over green fluorescence during BFA and CMA treatments compared with the no inhibitor treatment (TuMV alone) at 4 dpinf, indicating that viral protein production in the primary infected cells was not affected by the drug treatments.
cord-269496-tnw7sxlh	2020	Molecular dynamics of corresponding protein-drug complexes reveals that the drug bound state of RdRp with RNA has better structural stability than the Helicase NCB site and Importin-α, with MM/PBSA free energy of −187.3 kJ/mol, almost twice that of Helicase (−94.6 kJ/mol) and even lower than that of Importin-α (−156.7 kJ/mol). Together, being conserved and a necessary component for the replication of coronavirus, a multi-functional protein, Nsp13-helicase, is another vital SARS-COV-2 target (Jia et al., 2019) , which can be considered further for antiviral drug discovery provided a very small number of Nsp13 inhibitors reported to date . Molecular docking of Ivermectin with twelve SARS-COV-2''s targets along with Importin-a was carried out, followed by binding mechanism exploration and structural stability analysis using molecular dynamics (MD) simulation through the root-meansquare deviation (RMSD), root-mean-square fluctuation (RMSF), radius of gyration (R g ), and binding free energy of the complexes of Ivermectin with the best targets.
cord-269720-o81j3d1j	1990	Primer extension, of a synthetic oligonucleotide complementary to the 5′ end of the nucleoprotein gene of TGEV was used to produce a single-stranded DNA copy of the leader RNA from the nucleoprotein mRNA species from TGEV and PRCV, the sequences of which were determined by Maxam and Gilbert cleavage. Comparison of the leader RNAs of TGEV and PRCV with published leader RNAs of other coronaviruses was used to identify areas of conserved sequence and potential secondary structure that may be involved in the transcription of coronavirus subgenomic mRNA species. The site, ACTAAAC, is seven nucleotides upstream of the nucleoprotein initiation codon and has also been found to precede all the TGEV structural protein genes and two of the three potential genes shown to be at the 5'' end of mRNA species (15) (16) (17) . The two porcine coronavirus leader sequences were identical, indicating that the two viruses probably use the same RNA polymerase-leader complex binding site, ACTAAAC, for the synthesis of subgenomic mRNA species.
cord-269726-z0frgm7s	2020	Criteria for patients'' selection were diagnosis of SARS-CoV-2 infection [5] ; the subsequent meeting of criteria for hospital discharge (improvement of symptoms and two negative swabs collected at least 24 h apart) [4] ; and a positive respiratory sample collected after discharge. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Statement protocol [8] , a systematic review has been performed concerning the patients with a diagnosis of COVID-19 that, after clinical and virological recovery, presented a new positive respiratory sample (swab, sputum, saliva, tracheal aspirate, or BAL). The patient was discharged in good clinical conditions with indication to repeat quarantine and swab tests that came negative for SARS-CoV-2 (Allplex™ 2019-nCoV Assay) on April 27 and 28 (Fig. 1b) .
cord-269766-arjoemla	2020	title: Detection of Coronavirus in Tear Samples of Hospitalized Patients With Confirmed SARS-CoV-2 From Oropharyngeal Swabs This study was designed to detect CoV-RNA in the tears of polymerase chain reaction (PCR)-confirmed SARS-CoV-2 positive patients. METHODS: We performed a prospective case series study of hospitalized patients who have been confirmed SARS-CoV-2 positive by oropharyngeal swab within the previous 5 days. CONCLUSIONS: Using a tear fluid sampling technique similar to oropharyngeal lavage presents a higher percentage of SARS-CoV-2 positive tears in contrast to earlier reports that used a conjunctival swab. To clarify this, we tested the tear fluid of confirmed hospitalized SARS-CoV-2 patients by PCR using a method not previously used for the collection of tear samples. In this study, we could confirm SARS-CoV-2 RNA positive tear samples by PCR in as many as 28% of determined SARS-CoV-2 patients by oropharyngeal swabs. 13 In a more recent cross-sectional study, only 1 (1.38%) conjunctival swab of 72 confirmed SARS-CoV-2 cases was tested positive.
cord-269771-hffxb7bm	2020	title: Gastrointestinal Manifestations of SARS-CoV-2 Infection and Virus Load in Fecal Samples from the Hong Kong Cohort and Systematic Review and Meta-analysis We performed a systematic review and meta-analysis of published gastrointestinal symptoms and detection of virus in stool, and also summarized data from a cohort of patients with COVID-19 in Hong Kong. The proportion of patients with detectable stool viral RNA was higher among those with diarrhea than those without diarrhea Table 2 including the hospital admission period, places in which the patients were recruited, sample size, age, sex, disease severity, non-gastrointestinal symptoms (fever and respiratory symptoms) on presentation, and gastrointestinal symptoms (anorexia, nausea/vomiting, diarrhea and abdominal pain/discomfort). In this meta-analysis of 4,243 COVID-19 patients from six countries, the pooled prevalence of all gastrointestinal symptoms (including anorexia, nausea/vomiting, diarrhea or abdominal pain) was 17.6%. Clinical findings in a group of patients infected with the 2019 novel coronavirus (SARS-Cov-2) outside of Wuhan, China: retrospective case series
cord-269866-3tpyj04y	1992	We report here that two polypeptides with the sizes expected for the 5a and 5b products can be synthesised by in vitro translation of a single artificial mRNA containing both the 5a and 5b ORFs. To establish whether these polypeptides represent genuine virus gene products, both the 5a and 5b coding sequences were expressed as bacterial fusion proteins, and these were used to raise monospecific antisera. We report here that two polypeptides with the sizes expected for the 5a and 5b products can be synthesised by in vitro translation of a single artificial mRNA containing both the 5a and 5b ORFs. To establish whether these polypeptides represent genuine virus gene products, both the 5a and 5b coding sequences were expressed as bacterial fusion proteins, and these were used to raise monospecific antisera.
cord-269975-1ebmq7t8	2020	None of the filoviruses or henipaviruses has any FDA-approved therapeutics or vaccines available for prevention or treatment of human disease, and while ribavirin is sometimes used to treat Lassa fever, it is not a terribly effective drug against this viral infection [28] . Many of the therapeutics that are in different stages of either preclinical or clinical development for select biothreat pathogens include small molecule antivirals (Tables 7.3 and 7.4), antibody (or antibody cocktails) against viruses or bacteria/virulence factors (Table 7 .5), and combination drug therapy (Table 7 .6). Although no FDA-approved HDT therapies are yet available for treating infectious diseases, we have summarized in this section the antimicrobial Primary screening of small molecule chemical libraries in the phenotypic HCI assay will identify compounds that inhibit pathogen infection as well as those that may contribute to cellular toxicity.
cord-270143-muxrxvyo	2019	A high diversity of coronaand paramyxoviruses have been detected in different bat species at study sites worldwide, including Africa, however no biosurveillance studies from Rwanda have been reported. In this study, samples from bats collected from caves in Ruhengeri, Rwanda, were tested for the presence of coronaand paramyxoviral RNA using reverse transcription PCR assays. Although several surveillance studies have been implemented to detect potential zoonotic viruses in bats, including from countries in the Congo basin and East Africa, limited information is available for Rwanda. Confirmation of species identification of bats, in which viral RNA was detected, was performed by amplifying the cytochrome b (cyt b) or cytochrome oxidase one (COI) gene region and determining the DNA sequence. aegyptiacus-derived viral sequence (BatPV/Rou_aeg/UP438/RWA/2008) grouped within a Henipavirus-related clade and was near identical to a paramyxoviral sequence detected in the same host species previously reported from Kenya [36] .
cord-270243-moxleyjg	2018	Conclusions: In summary, this study has identified a highly divergent virus with in the Orthomyxoviridae family associated with Rhipicephalus ticks from Mozambique. Different studies have shown that viral pathogens, such as Thogoto viruses, Wad Medani virus, Nairobi sheep disease virus, Crimean-Congo hemorrhagic fever virus, African swine fever virus and Tick-borne encephalitis virus [1, [6] [7] [8] , can be found in Rhipicephalus ticks. Numerous studies have used metagenomics to explore viral communities in different arthropod species and have in these identified viruses associated with a broad range of animals, plants and insects. However, the identified ORFs exhibit high genetic diversity to known quaranjavirus genomes, with an amino acid identity of only 32-55%, indicating that these represent novel viral sequences belonging to the Quaranjavirus genus. The parvovirus sequences identified in the current study had closest similarity to non-structural protein 1 of different densoviruses, which were shown previously to integrate into tick genomes such as in Ixodes, Amblyomma and Rhipicephalus genera [28, 29] .
cord-270473-5tok4mqk	2003	We have previously shown that mitochondrial-aconitase binds specifically to the 3′ terminal 42 nucleotides of the Murine hepatitis virus (MHV) RNA along with three additional proteins of 70, 58 and 40 kDa to form a stable RNA-protein complex. The assays were done essentially as previously described [22] except that the partially purified post-mitochondrial lysates (approximately 3 µg total protein) were incubated at 22 • C with purified m-apo-aconitase (8-10 µg, graciously supplied by M.C. Kennedy, Gannon University, Erie, PA) and MHV-JHM 3 UTR RNA in ATPase buffer (20 mM Hepes, pH 7.4, 100 mM KCl, 5 mM MgCl 2 , 0.1% NP-40, 1 mM DTT, 1 mM ATP containing 2 µCi [γ-32 P]ATP plus cocktails of protease and phosphatase inhibitors). Gel supershift assays with anti-phosphotyrosine antibodies (Fig. 2B ) demonstrate that at least one of the proteins in the complex formed with MHV 3 (+)42 containing RNAs and m-aconitase, mtHSP70, HSP60, and HSP40 is tyrosine phosphorylated.
cord-270550-if748w2n	2020	To ascertain whether human pluripotent stem cell-derived cardiomyocytes (hPSC-derived 150 CMs) can serve as an appropriate model to study cardiac SARS-CoV-2 infection, we measured 151 ACE2 mRNA expression in hPSC-derived CMs. Quantitative RT-PCR revealed that hPSC-152 derived CMs abundantly expressed ACE2 mRNA. We identified numerous host genes that were differentially 226 regulated upon SARS-CoV-2 infection in each of the examined cell types and two-dimensional 227 tissues (Fig. 3c) . 236 GO pathway analysis revealed that infected hPSC-derived CMs and two-dimensional co-237 culture tissues showed upregulation of genes associated with immune cell activation, stress-238 induced transcription, and responses to pathogens including viruses. Consistent with the 343 possibility that disrupted sarcomere gene expression might contribute to reduced EHT 344 contractility, immunostaining of hPSC-derived CMs infected with SARS-CoV-2 revealed evidence 345 of sarcomere loss 3 days following infection (Fig. 6c) , a time point that preceded cell death.
cord-270594-62xotol3	2017	In this study, the viral structural protein 7 (VP7) polyclonal antibody was prepared with immunized mouse to explore the presence of small VP7 gene-encoded proteins in Bombyx mori cytoplasmic polyhedrosis virus. The replication of BmCPV genome in the cultured cells and midgut of silkworm was decreased by reducing the expression level of vp7 gene using RNA interference. In immunoprecipitation experiments, using a polyclonal antiserum directed against the VP7, one additional shorter band in BmCPV infected midguts was detected, and then the band was analyzed with mass spectrum (MS), the MS results showed thatone candidate interacted protein (VP7 voltage-dependent anion-selective channel-like isoform, VDAC) was identified from silkworm. A novel protein was detected from the overexpression of vp7 gene in the BmCPV infected cultured cells, with VP7 antibody. Total proteins from the BmCPV infected silkworm midguts (from the first day to the twelfth day) were also extracted, and detected with VP7 antibody.
cord-270604-u62437dh	2013	We present an empirical test of two theoretical models of preferential host switching, using observed phylogenetic distributions of host species for RNA viruses of three mammal orders (primates, carnivores, and ungulates). To overcome the above complications, this study takes an alternative approach, and reconstructs the dynamics of preferential host switching among 38 recorded "multihost" RNA viruses of mammals, on phylogenies of their primate, carnivore, and ungulate hosts. To achieve this, approximate Bayesian computation (ABC) is used to test the fit of the two models of preferential host switching to the observed distributions of multihost RNA viruses on the phylogenies of their mammal hosts (primates, carnivores, and terrestrial ungulates). This indicates that ABC model selection was effective with each of the three sample sizes used for calculation of the HSD summary statistics (which corresponded to the number of observed host-virus associations, of 22 for primates, 12 for carnivores, and 4 for ungulates).
cord-270670-cubh9jxc	2006	a Upon infection with an RNA virus (even with a single particle, as depicted here, enlarged about 10 6 times), viral replication leads to a mutant spectrum of related genomes, termed viral quasispecies. As further discussed in the text, in real infections multiple mutant spectra that can amount to a large number of replicating (or potentially replicating) genomes (up to 10 9 or even 10 12 per infected individual) provide highly dynamic mutant repertoire viral yields in cell culture, have been immensely powerful in characterizing the population dynamics of RNA viruses (see references in the reviews by Domingo and Holland 1997; Quiñones-Mateu and Arts 2002; Novella 2003; and the chapters by Quiñones-Mateu and Arts and Escarmís et al., this volume) . Despite these limitations, determination of nucleotide sequence heterogeneities in virus populations using correct reagents and adequate controls has consistently documented that most RNA viruses (and also some DNA viruses) consist of complex mutant spectra, with an average number of 1-100 mutations per genome (Sect.
cord-270892-ycc3csyh	2010	[79] [80] [81] [82] Taken together, the results of natural cold studies as well as of experimental infection in human volunteers clearly demonstrate that HRV are able to replicate in the upper as well as in the lower airways. Such an anti-HRV drug would have to be (i) with broad spectrum activity because of the high number of HRV serotypes, (ii) administered very early in infection to demonstrate a good antiviral effect because of the fast infection kinetics, (iii) very safe because of the broad application by millions of people, and (iv) directed against a highly conserved target with low risk of resistance development. The HRV-induced CPE, infectious virus titers, viral protein expression, and RNA synthesis can be chosen as parameters to evaluate the anti-HRV activity of compounds in cell-culture based assays. Due to the lack of a small-animal model for HRV infection until 2008, the experimental human challenge model has to be used to approve effects of potential antiviral drugs under controlled conditions in preclinical studies.
cord-270940-acwkh6ed	2005	Recently, during an outbreak in Finland in 2002, the causative agent of Pogosta disease was isolated for the first time in Europe from skin biopsies and a blood sample of patients [115] ; the virus strains were most closely related to SINV strains isolated from mosquitoes in Sweden and Russia 20 years previously. The genus Nairovirus (family Bunyaviridae) is composed of 34 predominantly tick-borne viruses that have been divided into seven serogroups [154] including several associated with severe human and livestock diseases (especially Crimean-Congo hemorrhagic fever virus (CCHFV) and Nairobi sheep disease virus). Rift Valley fever virus (RVFV), which is the type species of the genus and is transmitted by mosquitoes, causing an influenza-like disease that affects domestic animals and humans.
cord-271091-ffn59sgf	2007	These include the analysis of the function of individual proteins from important pathogenic viruses, the elucidation of key processes in viral replication through the development of systems that allow the replication of higher eukayotic viruses in yeast, and the use of yeast in antiviral drug development and vaccine production. Although the expression of viral proteins in yeast not always necessarily reflects their role in higher eukaryotes, here we selected some examples in which the analysis in yeast of their effect on highly conserved cellular processes such as cell cycle control, apoptosis or mRNA degradation have contributed to the current understanding of the pathogenesis of important viral pathogens such as HIV-1 and HCV. The identification of the host factors involved in viral RNA replication is a priority area of research in virology because it can provide new targets for antiviral drug development.
cord-271127-l9bxqtqs	2005	(2) Putative mutualistic virus: a reovirus present routinely in a wasp population in association with others viruses, but which does not appear to play a major role in the host/parasitoid relationship. DpAV-4 has been shown to be essential in the host/parasitoid relationship, as it is able to inhibit melanization, and to interfere with the cytolysis of host tissues in lepidopteran pupae parasitized by the wasp (Bigot et al., 1997b; Renault et al., 2002) . The occurrence of this domain in the protein coded by the DpRV-1 4.3 kb genomic fragment might be correlated with a possible role of this virus in its interaction with the ascovirus DpAV-4 in its lepidopteran and wasp hosts. Owing to the apparent absence of DpAV-1 virions or RNA within the egg, the transmission of DpRV-1 to the wasp larva during their development in host pupae was investigated.
cord-271130-6s79q1c1	2017	title: Putative progressive and abortive feline leukemia virus infection outcomes in captive jaguarundis (Puma yagouaroundi) Thus, the aim of this study was to perform additional serological and molecular tests and monitor the population of jaguarundis at FPZSP for FeLV infection and development of FeLV-related diseases for 5 years (2003) (2004) (2005) (2006) (2007) . Two captive-born male jaguarundis, the geriatric #1 and the mature adult #4, presented serological and molecular FeLV test results similar to the progressive FeLV infection outcome in domestic cats [25] . Moreover, consistent with findings in domestic cats with a progressive FeLV infection, no antibodies to FeLV antigens were detected in jaguarundis #1 and #4. Two captive-born jaguarundis, #2 and #22, presented test results similar to those reported for domestic cats with abortive FeLV infection and seroconversion as the only marker of FeLV exposure [28] .
cord-271188-ewlxy5po	2020	Abbreviations 311, 2-hydroxy-1-naphthylaldehyde benzoyl hydrazine; 3CL pro , 3C-like protease; ABCE1, ATP binding cassette subfamily E member 1; ACE, angiotensin-converting enzyme 2; ADK, aryl diketoacids; AIDS, acquired immunodeficiency syndrome; APN, aminopeptidase N; AT2, small population of type II alveolar cells; BMP, bone morphogenetic proteins; Bp4aT, 2-benzoylpyridine 4-allyl-3thiosemicarbazone; Bp4eT, 2-benzoylpyridine 4-ethyl-3thiosemicarbazone; COVID-19, novel coronavirus pneumonia; CoVs, coronaviruses; DFO, deferoxamine; DFP, deferiprone; DPP4, dipeptidyl-peptidase 4.; E, envelope; EPDTC, Nethyl-Nphenyldithiocarbamic acid zinc; ER, endoplasmic reticulum; HCMV, human cytomegalovirus; HFE, homeostatic iron regulator protein; HIV, human immunodeficiency virus; HSA, human serum albumin; IP10, interferon-inducible protein 10; M, membrane; MBD, metal-binding domain; MCP1, monocyte chemotactic protein 1; MERS, Middle East respiratory syndrome; N, nucleocapsid; PBMC, peripheral blood mononuclear cells; PL pro , papain-like protease; PMA, phenylmercuric acetate; PPY, phenyl-1-pyridin-2yl-ethanone; RdRp, RNA-dependent RNA polymerase; ROS, reactive oxygen species; S, spike; SARS, severe acute respiratory syndrome; SARS-CoV-2, the 2019 novel coronavirus; SCD, sickle cell disease; TDT, toluene-3,4-dithiolato zinc; TfR1, transferrin receptor1
cord-271241-w1q46y63	2020	Since the genomes of viruses are remarkably variable, high conservation rates strongly suggest a crucial role of G4s in the viral replication cycle and evolution, emphasizing the possibility of targeting viral G4s as a new pharmacological approach in antiviral therapy. 1 In the past few decades, pharmaceutical and biotechnological advance has succeeded in developing new therapeutics for the management of different viral diseases: for example, anti-retroviral therapy against the human immunodeficiency virus (HIV), or the pan-genotypic direct-acting antiviral drugs used for hepatitis C (HCV) management. In addition, such PQSs are highly conserved, despite the high recombination rates that characterize viruses, suggesting a crucial role for G4s in viral evolution and replication, and corroborating their targeting as a valid pharmacological approach for antiviral therapy.
cord-271419-v6dfel3l	2020	Among viruses, some coronaviruses (CoVs) are notorious for causing the severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). The said article has successfully predicted today''s COVID-19 outbreak by pointing out that novel pathogenic variants will readily emerge from very diversified severe acute respiratory syndrome-related coronaviruses (SARSr-CoVs) of the bat origin through their close coexistence and high genetic recombination ability (Figure 1) . Since RNA viruses are easy to mutate and coronaviruses have high potentials for recombination, we can easily see the track of mutations and evolutions of the viruses, especially for SARS-CoV and MERS-CoV. Thus, to consider the origin of new pathogens and the prevention of their transmission to humans, and control of the viruses, not only studies on SARS-CoV, MERS-CoV, and SARS-CoV-2, but also those on their relatives SARSr-CoVs and MERSr-CoVs are recommendable for bats tracked for the ecology and evolution.
cord-271434-30nh2gc7	2020	In this work, we develop an automated centrifugal microfluidic system (Figure 1 ) consisting of a microfluidic disc and a customized instrument for rapid sample-to-answer detection of SARS-CoV-2 armored RNA particles with high sensitivity and specificity. Virus lysis buffer, RT-LAMP reagents, and mineral oil were sequentially injected into the microfluidic disc for on-chip release of viral nucleic acids, amplification of target RNA, and sealing of reaction unit. To demonstrate the automated detection of viral nucleic acids by the centrifugal microfluidic system, we loaded different concentrations of SARS-CoV-2 armored RNA particles with N gene (0.5, 1, 10, 10 2 , 10 3 copies/μL) into five reaction units of the microfluidic disc, and used the instrument to carry out on-chip release of viral nuclei acids, RT-LAMP, and realtime fluorescence signal detection.
cord-271504-t3y1w9ef	2020	A confirmed case should have at least one of the following criteria: (i) a positive result for 2019-nCoV nucleic acid, using real-time PCR tests from respiratory or blood samples; (ii) a high homogeneity between viral gene sequencing from respiratory or blood samples and known 2019-nCoV; and (iii) serum samples positive for IgM or IgG to 2019-nCoV, or seroconversion in IgG, or a fourfold or more significant increase in IgG antibody titer to 2019-nCoV in the recovery phase than in the acute phase [25] . Using blood samples taken from alleged COVID-19 patients, the researchers detected antibodies targeting the spike protein that prevented the virus from killing cells in laboratory tests. showed a promising in vitro inhibitory effect of this serine protease inhibitor in SARS-CoV and 2019-nCoV on human lung cells, showing potential as a viable option for COVID-19 treatment [113] . Given that antiviral drugs have previously demonstrated reasonable inhibition of coronaviruses and therapeutic efficacy against coronavirus outbreaks, umifenovir, chloroquine, hydroxychloroquine, lopinavir-ritonavir, and ribavirin have been recommended in the latest guidelines for diagnosis and treatment of COVID-19, updated on 17 February 2020 [189] .
cord-271526-14nfqusv	1997	To determine the specificity of the nucleocapsid pro-RNA-protein complexes (Fig. 3, lanes 8-10 and 12) , tein-Ps180 RNA interaction in UV cross-linking assays, clearly indicating that the observed supershift was not competition experiments were performed. This interaction was studied by gel retardation and UV Lysates were prepared from the virus peak fractions (pcross-linking assays using an RNA probe containing the lysate) and from the gradient bottom fractions (b-lysate; 69-nt Ps and the N protein from MHV-A59 infected cell negative control). A structural model for coronaviruses was flanked by non-MHV sequences could confer specific proposed, in which a spherical core, composed of a combination of N and M proteins, was present in addition to encapsidation to a heterologous RNA in MHV infected INTERACTION BETWEEN NUCLEOCAPSID PROTEIN AND PACKAGING SIGNAL of a murine coronavirus in the absence of helper virus.
cord-271648-m2c5bvuj	2020	Coronaviruses (CoVs) are RNA viruses that have become a major public health concern since the Severe Acute Respiratory Syndrome-CoV (SARS-CoV) outbreak in 2002. However, unlike SARS-CoV, human-to-human transmission of MERS-CoV is not easy and has not been confirmed except in cases of very close contact with infected patients in health care settings [67] . Similar to the adaptation of SARS-CoV to human host, MERSr-CoVs that are circulating in bats had to undergo several amino acid changes in RBD of S protein to become capable of infecting camels and humans ( Figure 2 ) [74] . S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry Fully human monoclonal antibody directed to proteolytic cleavage site in severe acute respiratory syndrome (SARS) coronavirus S protein neutralizes the virus in a rhesus macaque SARS model
cord-271701-tx0lqgff	2011	Commonly, its core subunit is a single RNA-dependent RNA polymerase (RdRp) that drives the production of template strands for replication, new genome molecules, and-in many RNA virus groupsalso subgenomic (sg) mRNAs. This canonical RdRp is structurally conserved among RNA viruses and widely accepted to drive catalysis of phosphodiester bond formation via a well-established reaction mechanism involving two metal ions that are coordinated by aspartate residues in its motifs A and C (3) (4) (5) . Interestingly, both nsp8 and nsp(7+8) are able to extend the RNA primers beyond template length in the presence of heparin ( Figure 4D and Supplementary Figure S2B ), suggesting that these extensions result from terminal transferase activity and not from template switching, as was previously observed for poliovirus 3D pol (20) . Subsequent alanine substitution of the N-terminal D/ ExD/E motif, composed of D50 and D52 in SARS-CoV, greatly affected primer extension activity on the CU 10 template as shown in Figure 5C .
cord-271781-cfv0ta10	2020	To date, many studies have discussed that the rationale behind its transmission potential is that viral RNA has unexpectedly been detected in multiple bodily fluids, with some samples having remained positive for extended periods of time. In this evidence-based comprehensive review, we discuss various potential routes of transmission of SARS-CoV-2—respiratory/droplet, indirect, fecal-oral, vertical, sexual, and ocular. Additionally, studies have noted that its fecal-oral transmission potential may lie in the fact that prolonged viral shedding can occur in fecal matter-one case reported an asymptomatic COVID-19 patient experiencing viral detection in the stool for up to 42 days, while nasopharyngeal sampling was negative [31] . To oppose, in a retrospective review of nine COVID-19 pregnant mothers who underwent cesarean section, six patients had samples of amniotic fluid, cord blood, neonatal throat swab, and breastmilk samples tested for SARS-CoV-2, and all were negative [43] .
cord-271972-qhr6iir6	2010	[10] [11] [12] [13] [14] While the role of the Lsm proteins in eukaryotes is closely linked to mRNA degradation, several viruses use this complex instead to facilitate viral replication and, surprisingly, to enhance viral RNA stability. 25 It is notable that the virus-associated roles of the Lsm complex in mRNA stabilization, enhanced translation, and replication are at odds with its established cellular function in activating mRNA decapping to facilitate message degradation. However, the zinc-finger antiviral protein (ZAP), an important mediator of cellular response to retroviruses, 39 alphaviruses, 40 and filoviruses, 41 has been shown to bind the hRrp46 component of the exosome and to recruit the complex to viral mRNAs to promote their degradation. Nonetheless, given the widespread roles for the exosome in RNA quality control and turnover, viruses presumably activate this complex at least indirectly when triggering turnover of cellular mRNAs. For example, infection by select herpes and coronaviruses results in a global destruction of host messages.
cord-272050-0u62j7nj	2008	In contrast, cis-preferential function of the viral encoded proteins or a coupling between translation and replication has been reported for several viruses, including Alfalfa mosaic virus (AMV) (Neeleman and Bol, 1999; van Rossum et al., 1996) , Clover yellow mosaic virus (CYMV) (White et al., 1992) , Bovine coronavirus (Chang et al., 1994) , Cowpea mosaic virus (van Bokhoven et al., 1993) , Poliovirus (Hagino-Yamagishi and Nomoto, 1989; Johnson and Sarnow, 1991; Novak and Kirkegaard, 1994) , Turnip crinkle virus (TCV) (White et al., 1995) , Tobacco etch virus (Mahajan et al., 1996; Schaad et al., 1996) , Tobacco mosaic virus (TMV) (Lewandowski and Dawson, 2000) , Tomato bushy stunt virus (TBSV) (Oster et al., 1998) , Turnip yellow mosaic virus (TYMV) (Weiland and Dreher, 1993) , and Rubella virus (Liang and Gillam, 2001) . cis-Acting core RNA elements required for negative-strand RNA synthesis and cap-independent translation are separated in the 3¢-untranslated region of Red clover necrotic mosaic virus RNA1
cord-272268-8vrcwwll	2009	Cytoplasmic stress granules (SGs) and processing bodies (PBs) are dynamic structures that form in response to stress-induced translational arrest. Critical components of the ''''cell biology'''' of protein translation are mRNP granules known as processing bodies (PBs) and stress granules (SGs). These transient cytoplasmic ''''structures'''' are actively assembled from untranslated mRNA by a host of RNA-binding proteins, which determine whether specific transcripts will be reinitiated, degraded, or stored. In 1999, it was noted that stress-induced translational arrest causes untranslated mRNPs to assemble into large cytoplasmic ''''SGs,'''' whose formation is triggered by, and dependent upon, the phosphorylation of eIF2a. Virus infection also induces the assembly of SGs and PBs suggesting that RNA granules play a role in reprogramming mRNA translation/decay during viral infection. RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2a to the assembly of mammalian stress granules
cord-272378-umvi0veu	2019	MicroRNAs are single-stranded non-coding RNAs that are typically 18-25 nucleotides (nts) in length and are best known for their role in the post-transcriptional regulation of gene expression. However, it is possible that the frequency of MREs in the entire transcriptome of a given cell contributes to the dynamic gene regulatory process by acting as a sponge for mature miRNAs, thus regulating their functional availability. Thus, gene expression regulation is a complex process involving the dynamic interactions between miRNA-mRNA-lncRNA-circRNA. This Special Issue of Genes, entitled "MicroRNA Regulation in Health and Disease" consists of a series of articles spanning the clinical realm from colorectal cancer to pulmonary fibrosis. Somatostatin (SST) analogues were used to control the proliferation and symptoms of neuroendocrine tumors (NETs) in an article by Døssing et al., entitled "Somatostatin Analogue Treatment Primarily Induce miRNA Expression Changes and Up-Regulates Growth Inhibitory miR-7 and miR-148a in Neuroendocrine Cells" [15] .
cord-272573-wxqly479	2020	Here, we summarise community-driven approaches based on Free and Open Source scientific and medical Hardware (FOSH) as well as personal protective equipment (PPE) currently being developed and deployed to support the global response for COVID-19 prevention, patient treatment and diagnostics. Community and commercial open source efforts in diagnostic technology to date have focused on four areas: i) open platforms for scaling reactions as exemplified by Opentrons ( Fig 3A) [28] , an open source lab automation platform that has been working with BP Genomics and the Open Medicine Institute to automate up to 2,400 tests per day and achieve US FDA EUA approval and is now automating COVID-19 testing at the Biomedical Diagnostic Center (CBD) of Hospital Clinic of Barcelona; ii) trying to fill gaps where less attention is being paid by clinical diagnostics companies, such as Chia Bio''s Open qPCR (Fig 3B) environmental test kit for surveillance via surface swabs [111] ; iii) distributed reproduction of rapidly-published, lab-scale protocols, seen within the OpenCOVID initiative hosted by Just One Giant Lab [39] which involves many community labs worldwide; iv) initiatives such as the Open Enzyme Collection [93] , Free Genes [94] and Biomaker Challenge [112] which are investigating new approaches to foundational technologies such as reagents and instrumentation, with a view to building capacity and resources or global science and medicine to face a future pandemic.
cord-272576-ez731lif	2016	To face the world-wide threats caused by zoonotic RNA viruses, which suddenly cause serious outbreaks by invasion from nonhuman hosts and mutate rapidly, we must understand the molecular evolutionary changes in their genome sequences extensively by innovating various technologies, including those used in big data analyses, e.g., large-scale word count. The present time-series analysis on influenza A genomes showed that the 20-mers exhibiting the most evident directional changes observed in common for three different subtypes corresponded to several influenza A siRNAs, which were experimentally validated. The present study focused on time-series changes, but not on data variations caused largely by random mutations and sequencing uncertainty, and therefore, average characteristics of strains isolated in a similar period were analyzed, summing up the sequences isolated in one month and calculating an averaged mononucleotide composition for each month (Fig. 1b) .
cord-272579-aenuyht0	2005	A number of different viruses interact with the cell cycle in order to subvert host-cell function and increase the efficiency of virus replication; examples can be found from DNA, retro, and RNA viruses. The majority of studies have been conducted on DNA and retroviruses whose primary site of replication is the nucleus, but increasingly a number of researchers are demonstrating that RNA viruses, whose primary site of replication is normally the cytoplasm, also interfere with the cell cycle. Viral interference with the cell cycle can have a myriad of different effects to improve virus infection, for example to promote replication of viral DNA genomes, or to delay the cell cycle to allow sufficient time for RNA virus assembly. Increasingly we have found that proteomic approaches allow the rapid analysis of a whole plethora of cell cycle proteins that may be affected by virus infection.
cord-272666-3uidpr79	2018	In this study, membrane rearrangements induced when expressing viral non-structural proteins (nsps) from two different strains of IBV were compared. In contrast to previously studied coronaviruses, IBV nsp4 alone is necessary and sufficient to induce membrane pairing; however, expression of the transmembrane proteins together was not sufficient to fully recapitulate DMVs. This indicates that although nsp4 is able to singularly induce membrane pairing, further viral or host factors are required in order to fully assemble IBV replicative structures. In a subsequent study by others, it was shown that expression of only nsp3 and 4 from either MERS-CoV or SARS-CoV was able to induce DMV formation, and furthermore, addition of nsp6 made no difference to their shape or size, and did not induce the spherule-like structures seen following infection with whole virus [26] .
cord-272702-7uc4ozjy	2020	We therefore tested whether we could detect SARS-CoV-2 RNA by adding 1 μ l of each swab sample to 20 μ l TaqPath reactions containing the N1, N2, and RNase P (RP) probes ( Fig 2A) . Taken together, these results show that RT-qPCR with BEARmix can detect SARS-CoV-2 in clinical samples, either using purified RNA or by direct addition of swab samples, albeit with somewhat less sensitivity than commercial TaqPath master mix. To evaluate a complete protocol in which swab samples are collected into PK solution and then added directly to BEARmix RT-PCRs, we prepared contrived swab samples in which live virus was mixed with pathogenfree human nasal fluid prior to dilution into either DNA/RNA Shield, VCM containing 0.4 mg/ml proteinase K, or a solution of 0.4 mg/ml proteinase K in water (Fig 6) . Here we have developed simple, academic laboratory-derived methods for RNA extraction, direct sample addition, and RT-PCR detection that provide low-cost alternatives to the use of commercial kits (Fig 8) .
cord-272729-nbgdmavr	2012	Investigations into the mechanism of action of ribavirin against PRRSV and PEDV revealed that the addition of guanosine to the ribavirin treatment significantly reversed the antiviral effects, suggesting that depletion of the intracellular GTP pool by inhibiting IMP dehydrogenase may be essential for ribavirin activity. Further experiments revealed that suppression of ribavirin affects post-entry steps of the replication cycle of PRRSV and PEDV, including viral genomic and sg RNA synthesis, viral protein expression, and virus production. Several mechanisms of action for the antiviral activity of ribavirin have been suggested, including a reduction in cellular GTP pools via inosine monophosphate dehydrogenase (IMPDH) inhibition and increased mutation frequency on the virus genome leading to error catastrophe (Graci and Cameron, 2006) . Treatment of cells with ribavirin resulted in significant attenuation of postentry steps during the replication of porcine nidovirus, as determined by lower progeny production, diminished viral protein expression, and decreased synthesis of genomic RNA and sg mRNA.
cord-272871-gu9ptt9y	1991	Abstract A small group of 1.2-kb RNAs present on polyribosoes from clover yellow mosaic virus (CYMV)-infected tissue contains sequences from the genomic RNA (gRNA) of CYMV and is encapsidated by CYMV coat protein. Figure 2 shows Northern blots of polyribosomal RNA extracted from CYMV-infected tissue containing the 1.2-kb RNAs. The 7.0-kb gRNA is identified by all probes (Fig. 2 , lanes a, c, e, g, i, and k; probes 1 to 6), but the 1 .O-kb sgRNA encoding coat protein hybridizes only to probes corresponding to the 3'' region of the gRNA (Fig. 2 , lanes i and k; probes 5 and 6). The majority of the sequence of the prototype 1.2-kb RNA (Fig. 4) and of additional 1.2-kb RNAs is identical WHITE, BANCROFT, AND MACKIE with the corresponding region of CYMV gRNA reported by Sit et al.
cord-273019-hbpfz8rt	2018	Studies on the herpes simplex virus-1 (HSV-1) infection on Vero, BHK-21 and PtK 2 cells reported transportation of viral tegument-capsid by dynein to the cytoplasmic side of NPC [22, 23] . In order to construct these compartments, viruses alter host''s fatty acid metabolism, induce rearrangement of the membrane constituents and also recruit cellular machinery to produce proteins essential for its replication [59, 60] . Upregulation of mitophagy and degradation of the mitochondrial antiviral signalling protein (MAVS) in order to attenuate the antiviral immune response in non-small cell lung cancer (NSCLC) cells was reported upon measles virus infection [83] . The expression of matrix protein (M) of human parainfluenza virus type 3 (HPIV3) in HEK293T and HeLa cells was reported to induce mitophagy resulting in the suppression of type1 interferon response [84] . Many viruses or viral proteins are reported to localize to peroxisomes and/or exploit their functions to facilitate their replication in the host cells [108] .
cord-273326-gmw8gl2r	2018	In this line, and contrary to above mentioned report [73] , CQ, an FDA-approved anti-inflammatory 4-aminoquinoline and an autophagy inhibitor widely used as an anti-malaria drug that is administered to pregnant women at risk of exposure to Plasmodium parasites, was shown to have anti-ZIKV activity in different cell types (Vero cells, human brain microvascular endothelial cells (hBMECs), and human neural stem cells (NSCs)), affecting early stages of the viral life cycle, possibly by raising the endosomal pH and inhibiting the fusion of the envelope protein to the endosomal membrane [74, 75] . Similarly, by using a drug repurposing screening of over 6000 molecules, it was found that emricasan, a pan-caspase inhibitor that restrains ZIKV-induced increases in caspase-3 activity and is currently in phase 2 clinical trials in chronic hepatitis C virus (HCV)-infected patients, protected human cortical neural progenitor cells (NPC) in both monolayer and three-dimensional organoid cultures, showing neuroprotective activity without suppression of viral replication [82] .
cord-273366-xd84f8ct	2020	Interestingly, we found that IBV is able to inhibit multiple cellular stress granule signaling pathways, whilst at the same time, IBV replication also results in the induction of seemingly canonical stress granules in a proportion of infected cells. Moreover, IBV infection uncouples translational repression and stress granule formation and both processes are independent of eIF2α phosphorylation. Vero cells were infected with IBV and at the indicated time points, cells were fixed and labelled with anti-dsRNA to detect virus infection and with anti-G3BP1 to detect SG. Following identification of SG in IBV infected cells, the requirement for active virus replication in induction of granules was assessed. At 24 hpi, cells were fixed and labelled with anti-G3BP1 (red) to detect stress granules (SG) and IBV infected cells were detected with an anti-dsRNA antibody (green). Junin virus infection impairs stress-granule formation in Vero cells treated with arsenite via inhibition of eIF2alpha phosphorylation
cord-273367-gl266pvt	2020	Study participants (27 employees and 27 household members) consented to provide frequent nasal or oral swab samples that were analyzed by RT-qPCR for SARS-CoV-2 RNA using CDC protocols. While on study, the participant was SARS-CoV-2 RNA positive for at least 71 days and had elevated virus-specific antibody concentrations (medians: IgM, 9.83 ug mL-1; IgG, 11.5 ug mL-1; IgA, 1.29 ug mL-1) in serum samples collected at three timepoints. Conclusions Our clinical study met its primary objectives by using intense longitudinal testing to provide a safe work environment during the COVID-19 pandemic, and elucidating SARS-CoV-2 dynamics in recovering and asymptomatic participants. Subject 557 18, a self-quarantined employee who had just recovered from suspected COVID-19 (based on 558 symptomology) at the start of the study, repeatedly tested negative for SARS-CoV-2 RNA, but 559 tested positive for IgM antibodies that rapidly declined (τ1/2 = 8.8 d, Fig. 4A) .
cord-273379-w8vy5rl8	2000	RNase protection assays using the purified genome-length RI and two probes, which corresponded to the 5′ 300-nt region of mRNA 6 and to the same region of mRNA 7, showed the presence of nascent leader sequence-containing subgenomic mRNAs in the genome-length RI. We purified genome-length RI without contamination of any detectable level of MHV subgenomic mRNAs. We used RNase protection assays to look for nascent subgenomic mRNAs containing the leader sequence in the genome-length RI. If leader-sequence-containing subgenomic mRNA 6 elongates on the genome-length RI, then the RNase protection assay using probe 1 and purified radiolabeled genome-length RI should produce a radiolabeled 300-nt-long RNA fragment (300-nt fragment) that corresponds to the 5Ј-end 300 nt of nascent mRNA 6 (see Fig. 3A ). RNase A treatment of the gel-purified genome-length RI produced RF 1 (Fig. 2B) , and the RNase protection assay demonstrated the presence of nascent leader-sequence-containing subgenomic mRNAs in the genome-length RI.
cord-273487-nfgjz6f9	2012	Loss of DDX56 helicase activity did not affect expression of WNV capsid protein (Fig. 4A ) nor its secretion from infected cells in the form of virus particles (Fig. 5A ). Data in Fig. 5B show that virus particles isolated from infected cells expressing helicase dead mutants D166N and E167Q contained 3-4 times less genomic RNA than those isolated from non-silencing cells or cells expressing RNAi-resistant wild type DDX56. Data in Fig. 6 show that in transfected or infected cells, the WNV capsid does not bind to the DEAD box helicase-containing region of DDX56 (DDX56-NT-myc), but rather, the C-terminal part of the protein, which was produced in cells expressing DDX56-CT-myc. The helicase activity of DDX56 is not essential for replication or assembly of WNV virions per se but our data indicate that it is critical for infectivity of virus particles.
cord-273609-whm2ce4u	2012	title: Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment BACKGROUND: The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The most commonly used reference genes, including β-actin, cyclophilin, GAPDH, tubulin, and 18S and 28S ribosomal RNAs, have shown variable expression levels in different cells and tissues under different conditions, and therefore they are unsuitable for normalization purposes owing to large measurement error [6, . As seen in Table 7 , normalization of the RT-qPCR data against the reference genes suggested as optimal by the four software packages (hkgFinder, geNorm, BestKeeper, and NormFinder) or the 2 -ΔΔCT method, gave comparable relative expression levels of the target genes under fluconazole treatment in C.
cord-273711-bxijla09	2012	title: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV. These results suggest that the siRNA generated by in vitro transcription effectively and specifically inhibit the expression of GTPV ORF095 conserved regions in BHK-21 cells. To investigate whether or not knockout of ORF095 relieves cytopathic effect (CPE) induced by GTPV, Vero cells were transfected by plasmids expressing ORF095 protein-targeted shRNAs (p61/GFP, p70/GFP, p165/GFP and p296/GFP), respectively. Therefore, ORF095 gene is a good target to suppress GTPV replication by RNAi. In conclusion, this study demonstrates that vector-based shRNA methodology can effectively inhibit GTPV replication on Vero cells. RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells
cord-273723-srfypn7j	2020	title: Duration of SARS-CoV-2 RNA detection in COVID-19 patients in home isolation, Rhineland-Palatinate, Germany, 2020 – an interval-censored survival analysis As far as we are aware, there are currently no published data on the duration of RNA positivity in the upper respiratory of patients with mild COVID-19 that could inform a public health assessment of RT-qPCR as a tool for monitoring home isolation. At 14 days after onset, the earliest moment to discontinue home isolation currently recommended in Germany [18] , 53.5% of COVID-19 patients still had detectable SARS-CoV-2 RNA (Figure 2) . Duration of SARS-CoV-2-RNA positivity in COVID-19 patients in home isolation, Rhineland-Palatinate, Germany, 2020 (n = 537) For cases where laboratory monitoring is indispensable, knowledge of the RT-qPCR threshold cycle may improve our judgement on whether a positive result indicates infectiousness or not [20] .
cord-273910-fna7s9te	2007	Recent immunogenetic studies have associated polymorphisms of the genes encoding TLRs, NLRs, and key signal-transducing molecules, such as interleukin-1 receptor-associated kinase 4 (IRAK4), with increased susceptibility to, or outcome of, infectious diseases. With the availability of high-throughput genotyping techniques, it is becoming increasingly evident that analyses of genetic polymorphisms of innate immune genes will further improve our knowledge of the host antimicrobial defence response and help in identifying individuals who are at increased risk of life-threatening infections. Since the innate immune system senses only a limited number of highly conserved microbial-associated molecular patterns 23 via a limited number of receptors and signalling molecules, as anticipated, several polymorphisms have been found to confer an increased susceptibility to specifi c pathogens (table 2, table 3 , and fi gure 4). [36] [37] [38] Taken together, these data clearly show that mutations in the genes encoding TLRs and downstream signal-transducing molecules infl uence innate immune responses and increase susceptibility to many infectious diseases.
cord-274049-3gw65kpu	2017	© 2017 Wiley Periodicals, Inc. nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), have enabled the manipulation of genes by targeting DNA double-stranded breaks (DSBs) via non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways [Rudin et al., 1989; Rouet et al., 1994; Choulika et al., 1995; Bibikova et al., 2002; Moscou and Bogdanove, 2009 ]. Overall, these studies empower a broader range of disease modeling applications via CRISPR-Cas9-mediated genome engineering, allowing researchers to uncover fundamental mechanisms in disease initiation, maintenance and procession, and explore the therapeutic potential of the CRISPR-Cas9 system to correct disease-causing mutations. Owing to its ability to completely disrupt target genes and the simplicity of designing potent sgRNAs, the CRISPR-Cas9 system has been extended to large-scale loss-of-function (LOF) genome screens in human cells [Koike-Yusa et al., 2014; Shalem et al., 2014; Wang et al., 2014; Zhou et al., 2014] . Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease
cord-274080-884x48on	2018	For the majority of animal viruses, the activation of these fusion or penetration mechanisms occurs through conformational changes and structural rearrangements in viral surface proteins and/or the whole virion shell that may destabilize the capsid core. D: Three mechanisms (I.-III.) of DNA viruses replication are shown: (I): Following entry and uncoating, the DNA genome is transported to the nucleus; products of early genes (regulatory proteins, transcription factors) regulate the synthesis of viral enzymes (e.g. DNA polymerase) required for genome replication; expression of late genes encoding structural capsid proteins in the cytosol, they are then transported into nucleus where packaging and pre-assembly take place; preassembled procapsids exit the nucleus and leave the cell (e.g. Herpesviruses). Here, we provide an overview of in vitro methods, including cell-based assays, that may be suitable for screening of antivirotics that interfere with the key steps of viral life cycles and target either virus or cell-encoded proteins required for the infectivity.
cord-274097-11hvriqy	2020	The few studies of SARS-CoV-2 RNA in donors or of donors developing COVID-19 after giving blood are a mix of small series wherein prospectively test-positive units were quarantined and not transfused or involved units quarantined after donation to permit the donor time to get ill before units are distributed. In a lookback to recipients of 17 transfused components from seven South Korean donors who developed COVID-19 6 to 15 days after donation, there was no associated clinical morbidity in the recipients; however, archived samples tested by PCR after the donors reported their illnesses were negative. 21 Precise estimates of the prevalence of asymptomatic/presymptomatic infection and especially of whether RNA-emia or, more germane to this topic, viremia occur in the absence of illness (especially in healthy donors or the larger well population who might be qualified to donate) are among the key missing data needed to inform our debate about any risk of TTI and subsequent TTD. Severe acute respiratory syndrome coronavirus 2 RNA detected in blood donations
cord-274110-nyyunoha	2010	A cDNA comprising the complete genome of West Nile Virus (WNV) was generated by chemical synthesis using published sequence data, independent of any preformed viral components. The synthetic WNV, produced by transfection of in vitro transcribed RNA into cell culture, exhibited undistinguishable biological properties compared to the corresponding animal-derived wild-type virus. Taking advantage of the rapid progression of gene synthesis technology (for review [24] ), we intended to adopt such a synthetic approach to produce a flavivirus cDNA system for the generation of a synthetic WNV seed virus for use in vaccine development. The production and characterization of the resulting West Nile Virus, which fully matched the sequence of the in silico designed viral genome, confirms the feasibility and accuracy of the synthetic flavivirus reverse genetic system. Cover slips with fixed cells were dried, rehydrated with phosphate-buffered saline and treated with a polyclonal mouse anti-WNV serum (1:50 dilution) obtained after immunization of mice with a formalin-inactivated whole virus vaccine preparation.
cord-274353-tzlcpx7q	2020	In order to legally perform diagnostic work on human samples in the United States, a lab needs certification from the Centers for Medicare & Medicaid Services, through its Clinical Laboratory Improvement Amendments (CLIA) program (2) . On March 12, for example, California Governor Gavin Newsom issued an executive order suspending certification and licensure requirements for any researcher with relevant skills who meets CLIA requirements to run the COVID-19 test; such a person may now temporarily run the assays under the supervision of a medical laboratory scientist in a CLIA-certified lab (4). To get around its limitations, UC Berkeley is temporarily extending the clinical lab''s CLIA certificate into the larger Innovative Genomics Institute on campus, and will staff it with volunteer researchers who have the relevant skills to run the assay, supervised by a licensed medical laboratory scientist.
cord-274401-pjyvg53w	2020	An early wave featured injectable (i.e., intramuscular, subcutaneous) biodegradable polymeric microspheres to control drug release profiles for peptides and small molecules (e.g., Lupron Depot®, Risperdal Consta®). With these early successes for microspheres, research shifted to exploring systemic delivery by intravenous injection, which required smaller particle sizes and modified surface properties (e.g., PEGylation) to enable long circulation times. These new innovations resulted in the nanoparticle medicines Doxil® and Abraxane®, designed to improve the therapeutic index of cytotoxic cancer agents by decreasing systemic exposure and delivering more drug to tumors. In 2003, the FDA approved Risperdal Consta®, a controlled release form of risperidone encapsulated in PLG microspheres that enabled dosing once every 2 weeks by intramuscular injection. The many successes of controlled release microsphere-based medicines led innovators to explore the potential of drug delivery for systemic administration to reach specific sites of disease, with particular interest in targeting tumors for treating cancer.
cord-274463-0nvw2egm	2006	Viral RNA or DNA accumulation on root, stem, leaf, sepal, petal, stamen, pistil and ovary tissues of infected carnation or Saponaria vaccaria plants was analysed by non-isotopic molecular hybridisation. A negative hybridisation signal was also observed in root tissue of CVMV infected carnation plants (Fig. 1B) whereas the viral RNA accumulation was determined to be 625 ng g À1 of infected pistil tissue (dilution 5 À3 ) followed by petal, stamen and pistil with 125 ng (dilution 5 À2 ) and sepal with 25 ng (5 À1 ) ( Table 1 ). The viral distribution results showed two patterns for the six carnation viruses: high titres of CarMV, CRSV, CIRV, and CLV accumulated with small differences among the plant tissue analysed whereas there were low amounts of CERV and CVMV irregularly distributed over the infected plant.
cord-274536-fv7mltj7	2020	RESULTS: Of the enrolled 1008 severe patients, the nasopharyngeal swab specimens showed the highest positive rate of SARS-CoV-2 RNA (71.06%), followed by alveolar lavage fluid (66.67%), oropharyngeal swab (30.77%), sputum (28.53%), urine (16.30%), blood (12.5%), stool (12.21%), anal swab (11.22%) and corneal secretion (2.99%), and SARS-CoV-2 RNA couldn''t be detected in other types of specimen in this study. Firstly, we analyzed the possible sites of infection in hospitalized patients with COVID-19 by detecting viral RNA with 12 different types of specimens, including nasopharyngeal swab, oropharyngeal swab, sputum, bronchoalveolar lavage fluid (BALF), stool, anal swab, urine, peritoneal dialysis fluid (PDF), blood, sweat, cerebrospinal fluid (CSF) and corneal secretion. The 20 discharged cases of COVID-19, the criteria [12] for which was the SARS-CoV-2 virus RNA detection negative in two consecutive respiratory specimens (at least 1 day of time interval of sampling) for patients who have reached the standards of isolation period (14 days) after clinical cured, during the isolation period were selected to detect SARS-CoV-2 RNA with multiple specimens including nasopharyngeal swab, oropharyngeal swab, sputum, stool, anal swab, urine and blood.
cord-274567-xd37wxxf	2002	title: Application of a Real-Time Polymerase Chain Reaction with Internal Positive Control for Detection and Quantification of Enterovirus in Cerebrospinal Fluid A quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method based on TaqMan technology was developed to determine the presence and amount of enterovirus RNA. Amplification of the internal positive control was effective in all but two specimens, confirming the absence of PCR inhibitors and allowing the results of amplification to be validated. Detection of EVs by amplification of viral RNA from CSF using reverse transcriptase-polymerase chain reaction (RT-PCR) assay has already been reported [4, 5, 6 ]. The fluorogenic RT-PCR was applied to detection of EVs in the CSF of 104 patients presenting with signs of meningitis. Amplicor enterovirus polymerase chain reaction in patients with aseptic meningitis: a sensitive test limited by amplification inhibitors Comparison of use of cerebrospinal fluid, serum, and throat swab specimens in diagnosis of enteroviral acute neurological infection by a rapid RNA detection PCR assay
cord-274569-jh0dyyz7	2015	These routes require maturation of the EE into recycling endosomes or multivesicular bodies (MVBs), which can either fuse with lysosomes (L) to generate endolysosomes (EL) or with the plasma membrane to release intraluminal vesicles to the milieu as exosomes. These routes require maturation of the EE into recycling endosomes or multivesicular bodies (MVBs), which can either fuse with lysosomes (L) to generate endolysosomes (EL) or with the plasma membrane to release intraluminal vesicles to the milieu as exosomes. This mechanism was recently reported for HCV [113] , where exosomes derived from infected human hepatoma cells containing full-length viral RNA, along with core and envelope proteins [115] , were shown to be infectious and a major route of transmission. demonstrated that B lymphocytes infected with Epstein-Barr virus (EBV), a human gammaherpesvirus associated with a variety of lymphoblastoid and epithelial cancers, released exosomes containing MHC II molecules, and that these vesicles were capable of activating specific CD4 + T cell clones in vitro [122] .
cord-274663-zyzgk2z3	2011	Using NGS, we detected small but significant changes in host gene expression affecting T cell function that coincided with the initiation of viral RNA production at 12 h postinfection (hpi). In addition, by using NGS, we observed the dramatic expansion of viral mRNA expression and detected new viral splice events occurring during viral replication and differential expression of noncoding RNA species, including microRNA host genes. In our data set, we observed no change in the expression of the Crm1-encoding gene XPO1, but several members of the Ran signaling pathway were downregulated, suggesting that the overall export of unspliced viral RNA was suppressed (see Fig. S5B in the supplemental material; data not shown for XPO1). The regulation of these and transcription factors specific for other functions (e.g., EGR1, KLF13, and MYC) may explain how HIV initiated replication with minimal disruption to host gene expression at 12 hpi but elicited larger-scale changes later in infection.
cord-274773-3jhka8wl	2019	title: Pathogenicity of porcine deltacoronavirus (PDCoV) strain NH and immunization of pregnant sows with an inactivated PDCoV vaccine protects 5‐day‐old neonatal piglets from virulent challenge High levels of IgG antibodies and NA were also detected in the serum of neonatal piglets born to immunized sows, which suggests that the antibodies were successfully transferred through the colostrum and milk. The protective efficacy of passive immunity elicited by the inactivated PDCoV vaccine against challenge with a highly pathogenic virulent strain in neonatal piglets born to immunized sows was investigated. These results suggest that within the first week, IgG antibodies in colostrum and milk of immunized sows could provide protection for piglets against TGEV virulent challenge. Moreover, high levels of IgG antibodies and NA responses were detected in serum, which protected the piglets against virulent PDCoV challenge. Pathogenicity of porcine deltacoronavirus (PDCoV) strain NH and immunization of pregnant sows with an inactivated PDCoV vaccine protects 5-day-old neonatal piglets from virulent challenge
cord-274785-9jgg8ukr	2019	TIA/G3BP/PABP-specific stress granules (SG) and GW182/DCP-specific RNA processing bodies (PB) are two major distinguishable RNA granules in somatic cells and contain various ribosomal subunits, translation factors, scaffold proteins, RNA-binding proteins, RNA decay enzymes and helicases to exclude mRNAs from the cellular active translational pool. The process of SG formation can be artificially divided into the following steps ( Fig. 2) : (1) accumulation of stalled translation initiation complexes ) in response to various types of stress; (2) the RNA-binding proteins such as RAS-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and T cell-restricted intracellular antigen 1 (TIA1) bind mRNAs and aggregate to nucleate SG formation. SG proteins (eIF4G, eIF3, PABP) are selectively sequestered within Ebola virus inclusion bodies and co-localize with viral RNA to form inclusion body-bound granules, which are functionally and structurally different from canonical SG, probably leading to inhibit the antiviral role of SG (Nelson et al. Interaction of TIA-1/TIAR with West Nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly
cord-275225-fvq8hezk	2012	The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. The quantitative sg-mRNA detection method revealed more than 10-50,000 times increase of the M gene sg-mRNA in organ materials of feline infectious peritonitis cases, compared to those of the enteric FCoV variants present in the faeces of normal, healthy cats. These results indicate the applicability of the new P-sg-QPCR test as a powerful novel tool for the better detection and quantitation of FCoV and for the improved diagnosis of feline infectious peritonitis, this important disease of the Felidae, causing serious losses in the cat populations at a global scale. The detailed analysis of the feline infectious peritonitis positive cat samples by SYBR-Green QPCR method revealed that the following organs harbour FCoV most frequently: lungs 6/6 (100%), liver 6/6 (100%), kidney 10/11 (90.9%), mesenteric lymph node 18/20 (90%), spleen 16/20 (80%) and gut 15/10 (66.7%).
cord-275252-4e3cn50u	2020	In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineered viral attenuation and antigen presentation. A common primary focus of mutational analysis of emerging viruses is the alteration in amino acid sequence of viral proteins that may provide enhanced or new functions for virus replication, immune avoidance, or spread. However, the potential of these mutations to impact upon RNA structure and miRNA recognition provides a basis for ongoing monitoring of viral evolution at these sites in the SARS-CoV-2 genome.
cord-275307-d7htyfcl	2015	Development of a novel bioinformatics pipeline to detect highconfidence SOX cleavage sites across the transcriptome following PARE Prior analyses of individual mRNAs indicated that the KSHV RNase SOX cuts at specific locations within the RNA, in a manner dependent on the sequence surrounding the cleavage site [5] . This example shows the expected distribution for a cut site followed by exonucleolytic degradation due PARE libraries from two replicates of SOX-expressing or GFP control cells and extracted the 5'' end of each mapped read, which represents the cleavage site (S1 Table) . Indeed, the sequences flanking the set of reproducible SOX cut sites (identified with a confidence level of 99.99%) were a closer match to the motif compared to those surrounding GFP-specific fragment ends, as shown by the distribution of the log likelihood scores (Fig 6A) .
cord-275403-g4rohhtt	2002	To examine characteristics of putative nonstructural proteins (nsp) encoded in ORF1b, which have been identified by nucleotide similarity to domains of equine arteritis virus, defined genomic regions were cloned and expressed in the pRSET expression system. As initial proof of helicase-like activity, experiments were performed to test the ability of purified PRRSV-14 nsp10 protein to hydrolyze radiolabeled ribonucleotides in the presence or absence of polyribonucleotides. To further analyze the predicted helicase activity of the PRRSV nsp10 protein, experiments were performed using duplex substrates prepared with synthetic oligonucleotides containing single-strand regions at the 3Ј or 5Ј ends and short duplex regions (21 and 24 bp). The 5Ј-to-3Ј orientation of the unwinding activity of the PRRSV helicase was further confirmed in experiments using substrates prepared with in vitro transcribed RNA that contained singlestrand regions at the 5Ј end of both strands and duplex regions of 67 bp (5Ј5ЈDuplex #1) and 85 bp (5Ј5ЈDuplex #2), as shown in Fig. 9B .
cord-275565-xerr4vki	2020	For the first time, we present, i) an account of decay in the genetic material loading of SARS-CoV-2 during Upflow Anaerobic Sludge Blanket (UASB) treatment of wastewater, and ii) comparative evaluation of polyethylene glycol (PEG), and filtration as virus concentration methods from wastewater for the quantification of SARS-CoV-2 genes. Thus, there still remains questions pertaining to: i) capability of conventional WWTPs to reduce the abundance of SARS-CoV-2 RNA, ii) better understanding of the protocol, virus J o u r n a l P r e -p r o o f Journal Pre-proof precipitation through PEG and filtration which one is better methods for concentrating the samples before RNA isolation. Appraising the genetic loading reduction through Upflow Anaerobic Sludge Blanket (UASB) systems, and iii) Comparing the performances between PEG and filtration as virus concentration methods in terms of SARS-CoV-2 RNA sensitivity and inhibition removal.
cord-275602-cog4nma0	2018	SUMMARY: In addition to the aforementioned pathogens, the Severe Acute Respiratory Syndrome, Middle East Respiratory Syndrome, Nipah virus, New Delhi metallo-ß-lactamase-1 Enterobacteriaceae, Rift Valley Fever virus, and Crimean-Congo Hemorrhagic Fever virus are reviewed. In 1992, an expert committee that produced the Institute of Medicine report on emerging infections defined them as "new, reemerging, or drug-resistant infections whose incidence in humans has increased within the past two decades or whose incidence threatens to increase in the near future." Additionally, six major contributors to these diseases were presented and included changes in human demographics and behavior, advances in technology and changes in industry practices, economic development and changes in land-use patterns, dramatic increases in volume and speed of international travel and commerce, microbial adaptation and change, and breakdown of public health capacity [1] . The World Health Organization has prioritized a number of infectious diseases as requiring urgent need for research and development given the concern for potential of severe outbreaks.
cord-275683-1qj9ri18	2019	Against the background of an extensive viral diversity revealed by metagenomics across many environments, new sequence assembly approaches that reconstruct complete genome sequences from metagenomes have recently revealed surprisingly cosmopolitan viruses in specific ecological niches. However, these techniques can only detect previously known viruses, and often require Box 1 Use of complementary methods to target different types of viruses A number of approaches have been developed to specifically select and survey the genetic material contained by virus particles in a given sample. Virus sequences obtained from "bulk" metagenomes will typically reflect viruses infecting their host cell at the time of sampling, either actively replicating or not, while viromes enables a deeper and more focused exploration of the virus diversity in a specific site or sample. With viral metagenomics being applied to a larger set of samples and environments, and with bioinformatic analyses including genome assembly and interpretation constantly improving, novel groups of dominant and widespread viruses may thus be progressively revealed across many environments.
cord-275720-kf9m4zho	2012	At the early point of growth of an infected strain as compared to an uninfected strain, genes associated with protein synthesis, including ribosome assembly, nucleolus, and ribosomal RNA processing, were significantly up-regulated. This is the first report of a genome-wide fungal gene expression analysis during mycovirus infection using a 3′ tiling microarray, and our findings show global differences in host cellular pathways in F. For example, according to the qRT-PCR and microarray results, the transcript levels for three genes, including FGSG_01379, FGSG_03143, and FGSG_03911, were highly reduced at both 36 h and 120 h, whereas FGSG_03788, FGSG_00023, FGSG_07804, FGSG_07801, and FGSG_13222 were strongly induced regardless of the time point ( Figure 3A -C). graminearum harboring FgV1-DK21 in detail, samples were harvested at two different time points, thus providing lists of differentially expressed genes early and late in the host containing FgV1-DK21 as compared to an uninfected strain.
cord-275795-ee7qyw5h	2018	We focus on immunity generated against both natural infection and vaccination, where a steady shift in conferred vaccination immunogenicity is observed from quantifying activated and proliferating, long-lived effector memory T cell subsets, as the prominent biomarkers of long-term immunity against viruses and their associated disorders causing high morbidity and mortality rates. Since that time, the occurrence of epidemics and outbreaks are now at lower risk, following the introduction of massive vaccination programs able to induce immune system targeting of viruses causing severe disorders affecting distinct geographical locations, and with many epidemiological reports demonstrating long-term efficacy of viral control of non-naïve populations. This approach is being developed to use virus-infected cell-killing antibodies that produce an antiviral environment; these termed antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, which are predicted to link innate and adaptive immune responses, and is becoming possible due to new technologies for rapid isolation and characterization of monoclonal antibodies targeting conserved regions of influenza virus, reviewed in Jegaskanda et al.
cord-275859-ix8du1er	2012	In contrast, there is a strong correlation between frameshift efficiency and the local thermodynamic stability of the first 3–4 bp in the stem–loop, which are predicted to reside at the opening of the mRNA entrance channel when the ribosome is paused at the slippery site. Here, we investigate the role of the HIV-1 RNA structure in frameshifting, focusing on elucidating the relationships between frameshift efficiency and (i) the downstream RNA stem-loop thermodynamic stability, (ii) spacer length and (iii) surrounding genomic secondary structure. Our data further indicate that the base pairs important for frameshifting are located at a distance of 8 nt from the slippery site, which corresponds to the length of the spacer and is consistent with a structural model of the ribosome paused at the frameshift site. Instead, we observe a strong correlation (R 2 = 0.88) between frameshift efficiency and local stability of the first 3 bp at the base of the stem-loop using a one-phase exponential decay function ( Figure 3C and Supplementary Table S3 ).
cord-276006-mjjnkqv6	2020	Those anions show antiviral properties by affecting Larson studied modified PEI composed of N,N-Dodecylmethyl-PEI that exhibited antiviral effect on HSV-1 and HSV-2 viruses (see also Figure 6 ) [98] , influenza A virus [99] and on poliovirus and rotavirus [100] . Larson studied modified PEI composed of N,N-Dodecylmethyl-PEI that exhibited antiviral effect on HSV-1 and HSV-2 viruses (see also Figure 6 ) [98] , influenza A virus [99] and on poliovirus and rotavirus [100] . Xiao and Xue examined the antiviral effect of quaternary pyridinium containing co-polymers on several Influenza viruses (A, PR8, 8, 34) , as demonstrated in Figure 11 [35]. Xiao and Xue examined the antiviral effect of quaternary pyridinium containing co-polymers on several Influenza viruses (A, PR8, 8, 34) , as demonstrated in Figure 11 [35].
cord-276185-ysspkbj7	2018	In experiments in vitro, three human APOBEC3 proteins (A3C, A3F and A3H) inhibited HCoV-NL63 infection and limited production of progeny virus, but did not cause hypermutation of the coronaviral genome. Although the precise molecular mechanism of deaminase-dependent inhibition of coronavirus replication remains elusive, our results further our understanding of APOBEC-mediated restriction of RNA virus infections. HCoV-NL63 infection resulted in upregulation of expression of A3A, A3C, A3D and A3F and A3C, A3F and A3H all inhibited HCoV-NL63 replication, we tested whether this inhibition was the result of the catalytic activity of the APOBECs. Plasmids encoding variants of A3C, A3F and A3H with Glu → Gln substitutions in the catalytic site were prepared 31 and mRNAs encoding active or inactive proteins were transfected into LLC-Mk2 cells, which were infected with HCoV-NL63 and cultured prior to visualisation of viral proteins with specific antibodies.
cord-276198-psjua913	2015	Many nidovirus and all coronavirus TM1 proteins contain one or more ubiquitin-like domains which may help to anchor the viral RNA to the membranes where replication takes place (Hurst et al., 2013) . The maze-like paired-membrane structures that resulted from coexpression of SARS-CoV TM1 and TM2 have not ever been reported in coronavirus-infected cells, suggesting that this should be interpreted as a conditional, or perhaps partial phenotype, that is not observed when the full viral replicase polyprotein is expressed. Since DMVs are reminiscent of the double-membranes of autophagosomes, several lines of controversial evidence hypothesized a diversion of Atg (autophagyrelated) proteins and autophagosome function during coronavirus replication, as it is the case for other +RNA viruses (Prentice et al., 2004; Snijder et al., 2006; Zhao et al., 2007; Maier and Britton, 2012; Richards and Jackson, 2013) . Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex
cord-276364-zyw5aukk	2019	Over the past few decades, a growing body of research has defined the critical role of this pathway in facilitating infection by numerous +RNA RNA viruses, including poliovirus (PV) [7, 8] , Coxsackievirus B3 (CVB3) [9, 10] , CVB4 [11] , Enterovirus 71 (EV71) [12] , Human rhinovirus (HRV) [13] , Foot-and-mouth disease virus (FMDV) [14] , encephalomyocarditis virus (EMCV) [15] , Dengue virus (DENV) [16, 17] , Zika virus (ZIKV) [18, 19] , Hepatitis C virus (HCV) [20] , Mouse hepatitic virus (MHV), Newcastle disease virus (NDV) [21] , Severe and acute respiratory syndrome coronavirus (SARS-CoV) [22] , Chikungunya virus (ChikV) [23] , and Japanese encephalitis virus (JEV) [24] . Delineating the process of viral assembly from replication is technically challenging, especially since both processes would very likely Induces formation of autophagosome-like double-membrane liposomes [112] Summary of Interactions between proteins from positive strand RNA viruses and host autophagy machinery.
cord-276493-hoaxv5e0	2020	With increasing structural data of key proteins in both SARS-CoV-2 and the host, such as the spike glycoprotein (S), the main protease (M pro ), RNA-dependent RNA polymerase (RdRp), and human angiotensin-converting enzyme 2 (hACE2), the structure-based design of new drugs has emerged as the most promising antiviral strategy. Several structure-based drug discovery studies have investigated the interaction of inhibitors in the substrate-binding pockets of SARS-CoV-2 M pro ( Figure 3C ) (Dai et al., 2020; Jin et al., 2020; Zhang et al., 2020b) . Because most inhibitors occupy the substrate binding pocket of SARS-CoV-2 FIGURE 4 | CryoEM structure of RdRp in complex with cofactors (nsp7 and nsp8), RNA template, and remdesivir. In addition, we provided structural insights into the mechanism of action of well-characterized drugs targeting the interaction between hACE2 and the spike protein of SARS-CoV-2 for viral entry, as well as M pro and RdRp for viral replication.
cord-276541-u9ebql5a	2011	title: Inhibition of porcine hemagglutinating encephalomyelitis virus replication by short hairpin RNAs targeting of the nucleocapsid gene in a porcine kidney cell line Here, we constructed a single short hairpin RNA (shRNA) plasmid expression system that targeted the N gene and investigated whether shRNAmediated RNA interference could inhibit PHEV replication in PK-15 cells. To study the inhibitory effects of RNA interference on PHEV replication, the level of viral antigen produced in the PK-15 cells after shRNA transfection and viral infection was examined 48 h post-infection by an indirect immunofluorescence assay (IFA) using anti-PHEV serum. To quantify the effect of shRNA on viral replication at 48 h postviral infection, the viral genome copy number was determined by real-time PCR, using the serially diluted plasmid pT-N as a standard. It is clear from this study that the DNA vector-based shRNA approach, that is, the use of RNAi expression plasmids directed against the PHEV N gene, could effectively block expression of the viral target gene and inhibit viral replication.
cord-276575-jfug80yu	2007	From the mechanism, it becomes clear that small interfering RNAs (siRNAs) play a pivotal role in triggering RNAi. Thus, the efficient delivery of target gene-specific siRNAs is one major challenge in the establishment of therapeutic RNAi. Numerous studies, based on different modes of administration and various siRNA formulations and/or modifications, have already accumulated promising results. While the inhibition of the activity of (aberrant) gene products, e.g., through small molecule inhibitors or inhibitory antibodies is one major focus in therapy, much attention has now shifted to an earlier step, i.e., the initial knockdown of the specific gene of interest through RNAi. However, for the in vivo application of RNAi in mammals as a therapeutic approach for reversing a pathological condition as well as for the study of particular gene functions, sophisticated strategies for the induction of RNAi are needed. RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo
cord-276739-84vf5bts	2011	Using SHRT-PCR, 86 strains of influenza viruses isolated by the Tokyo Metropolitan Institute of Public Health were tested; the results showed 100% sensitivity and specificity for each influenza A and B virus, and swine-origin influenza virus. This method offers high sensitivity and selectivity, but generally requires approximately 2 h per run; therefore, qRT-PCR is not appropriate for rapid virus detection or subtyping in outbreaks of fast-spreading and/or highly pathogenic viruses at public health centers, hospitals, airports, and other public transportation hubs. The commercial reaction mixture was from the qRT-PCR kit, RNA-Direct TM SYBR ® Green Real-time PCR Master Mix (Toyobo, Osaka, Japan; http://www.toyobo.co.jp/e/). The SHRT-PCR system detects the highly conserved sequence of the corresponding viral genome, and the newly designed primer sets targeted for typing MP segments do not exhibit any cross reactions among other influenza viruses (Table 5 ).
cord-276908-9jthjf24	2020	In last two decades, entire world faced three major outbreaks of coronaviruses like Severe Acute respiratory syndrome (SARS), middle east respiratory syndrome (MERS) and novel coronavirus disease i.e., COVID-19. Previously, CoV causes an epidemic of SARS in humans and infected thousands viruses belong to family Coronaviridae, which shows crown-like appearances under an electron microscope. A recent study published, relied on this approach, using the predicted structure of all SARS-CoV-2 proteins based on their homology with other known coronavirus protein structures, and identified several compounds with potential antiviral activity. [39, 77] A biological preparation provides active acquired immunity against particular infectious disease like COVID19 [51, 68] 5 Shenzhen, China SARS-CoV, NL63, HKU1 The organosulfur in the essential garlic oil inhibit the ACE2 (host-receptor site of the virus) and main protease of the virus as well as to treat the infection due to SARS-CoV-2.
cord-276914-44ji0g78	2020	However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Patient 3 (Figure 3(A) ) was transferred to the ICU directly on illness day 11 because of his severe condition, the 2019-nCoV virus was laboratory detected both in pharyngeal (Ct = 30 + 30) and blood samples (Ct = 37 + 39) on day 12, And his infection was confirmed by CDC on day 13. His disease advanced pretty fast and became severe on day 7 and he was transferred to ICU after his blood sample was detected to be virus-positive (Ct = 32 + 37). For patient 1, a high concentration of viral RNA (Ct = 23 + 27, on day 13) was detected in anal swab but not in pharyngeal (the same day) and blood (1 d ahead).
cord-276988-bvsz5q6d	2020	Additionally, we will focus on the currently known concepts of post-transcriptional control mechanisms important for RNA fate within platelets with a special emphasis on RBPs. Blood platelets-the major players in hemostasis-are small anucleate cell fragments with a characteristic discoid shape and a diameter of 1 to 3 µm that originate from megakaryocytes (MKs). The m 7 G cap and poly(A) tail promote mRNA translation and stability, while the UTRs expose sequences for RNA-binding proteins (RBPs) and regulatory sites for microRNA (miRNA)-mediated translational and degradation control [45, [52] [53] [54] . Moreover, RNA-Seq analysis of platelet miRNAs in patients with myocardial infarction revealed nine differentially expressed platelet miRNAs compared to healthy controls, which were released upon platelet aggregation and taken up by endothelial cells via a vesicle-dependent mechanism [80] . Megakaryopoiesis, megakaryocyte maturation, as well as platelet formation, were shown to be highly complex processes that are regulated on multiple levels including epigenetic, transcriptional as well as post-transcriptional gene expression control mechanisms.
cord-276997-hbovh7s9	2020	However, the infectivity of airborne human noroviruses has not been possible to assess in real-world environments, nor in laboratory experiments, since there is no well-established cell culturing system for these viruses working at the low concentrations obtained in air samples 6 . SLAG: Sparging liquid aerosol generator; HEPA filter: high efficiency particulate air filter; nsRNA: negative sense RNA; psRNA: positive sense RNA; MNV: murine norovirus; qRT-PCR: quantitative reverse transcriptase polymerase chain reaction. Taken together, as compared to the atomizer, a smaller amount of SLAG aerosol is collected by the BioSampler (i.e., higher physical dilution), but these particles contain more MNV psRNA copies (lower viral dilution relative the physical dilution). The experimental setup described here highlights some difficulties in studies on aerosolized viruses: (1) the lack of standards in how to generate bioaerosol that results in significant differences in aerosol particle size and concentration, (2) the necessity to determine both physical and viral dilution factors, and (3) www.nature.com/scientificreports/ during airborne transport due to the low-solute solution and dilution in the setup.
cord-277306-r8jki3x4	2011	At two of the rural sampling sites, CoV RNAs were detected in big brown and long-legged bats during the three sequential summers of this study. Alphacoronavirus RNA was detected at a high prevalence in big brown bats in roosts in close proximity to human habitations (10%) and known to have direct contact with people (19%), suggesting that significant potential opportunities exist for cross-species transmission of these viruses. To increase the sensitivity of RNA detection, based on our previously published bat CoV sequences [17] and new data from this study, we designed specific primers within the amplicons of alphacoronaviruses from bats of several species in the genus Myotis and big brown bats (Table S1 ). Although the number of big brown bats sampled at site #5 was small (4 in 2008 and 14 in 2009), the prevalence of CoV RNA in these bats during these two summers was high (29% to 100%) ( Table 2) .
cord-277318-cwuls6xs	2016	13 A recent single-molecule experiment indicates that ribosome helicase action during the translational elongation cycle may be twofold: it destabilizes the helical junction at the mRNA entry site favoring an open conformation, and it appears to pull mRNA strands apart during the translocation step when relatively large structural rearrangements occur on the ribosome. 26 We will review methods and experiments that apply force to the single molecules to determine the mechanical properties of codon-anticodon interaction at the slippery site, the stability of downstream mRNA structure motifs that give rise to −1 PRF, and elastic properties of the ssRNA elasticity bridging those elements. 104, 105 Single-molecule assays that probe the codon-anticodon dynamics at the slippery sequence and investigate the force dependence of the translation elongational cycle (discussed later in the chapter; see Fig. 8 ) should be fit to answer these questions and allow unfolding of −1 PRF mRNA structure motifs held at the ribosome''s entry site.
cord-277355-si3g5dih	2020	Although current studies on flaviviruses have shown that the flaviviral assembly process does not exhibit a necessary step occurring in the cell nucleus, it has been well demonstrated that many mosquito-borne flavivirus CPs partially localize in the cell nucleus [44] [45] [46] [47] [48] ; in the cytoplasm, in addition to localizing in the ER, DENV CP and ZIKV CP have also been demonstrated to accumulate on LDs [13, 16] , but the link between the functional importance and the subcellular distribution of CPs is still unclear. Interestingly, a study showed that a CD61-71 mutant had abolished ZIKV infectious virion production that was then restored by adaptive mutations (prM-E21K and NS2B-E27G) only in BHK21 cells but not in other cell lines (indicate complex interactions that apparently occur between structural and non-structural proteins during virus replication and/or assembly), making this live virus function like a single-round infectious particle (SRIP) in vivo and safely inducing strong immunity protection against vertical transmission in mice [33] .
cord-277547-2vim1wno	2011	In the present study, antiviral activity of four types of bioflavonoid against dengue virus type -2 (DENV-2) in Vero cell was evaluated. Daidzein showed a weak anti-dengue activity with IC(50 )= 142.6 μg mL(-1 )when the DENV-2 infected cells were treated after virus adsorption. Although there was no significant direct virucidal activity against DENV-2 by quercetin, continuous treatment of cells from 5 h before virus infection up to 4 days post-infection exhibited anti-dengue activity with IC 50 = 28.9 μg mL -1 (Figure 3a) . There was no significant change in the antiviral activity of daidzein when cells were treated continuously from 5 h before virus infection up to 4 days post infection comparing to its anti-dengue activity for postadsorption treatment (Figure 1 ). To investigate which of the many flavonoids could affect DENV infection, in the present study, we examined the potential effects of quercetin, naringin, hesperetin and daidzein on dengue virus infection of Vero cells.
cord-277566-j3ehiwn9	2008	Here, we study the involvement of the secretory pathway in mouse hepatitis coronavirus (MHV) replication by using the drug brefeldin A (BFA), which is known to interfere with ER-Golgi membrane traffic by inhibiting the activation of ADP-ribosylation factor (ARF) small GTPases. Therefore, LR7 cells infected with MHV-A59 were treated with BFA for 30 minutes starting 5.5 h p.i. They were subsequently fixed and processed for immunofluorescence using antibodies both against nsp8, which served as a protein marker for the MHV replication sites [50, 51] , and against the viral structural protein M, known to reside in the Golgi [52] . In complete agreement with the luciferase expression data shown above, this result demonstrates that BFA inhibits, but does not completely block, the formation of RCs. To study the effects of BFA on the DMVs at an ultrastructural level, MHV-infected LR7 cells were fixed at 6 h p.i. and embedded in Epon resin in order to be analyzed by electron microscopy.
cord-277687-u3q36o3e	2019	title: VAPiD: a lightweight cross-platform viral annotation pipeline and identification tool to facilitate virus genome submissions to NCBI GenBank In order to accept submitted viral genomic data, NCBI GenBank requires 1) viral sequence complete with at least one protein annotation, 2) author/depositor metadata, and 3) viral sequence metadata, such as strain, collection date, collection location, and coverage. VAPiD handles batch submissions of multiple viruses of different types without prior knowledge of the viral species, correctly annotates RNA editing and ribosomal slippage, performs spellchecking on annotations, handles batch or individual submission of metadata, runs with a simple one-line command, and creates annotated viral sequence files for GenBank submission. This first example is the task that the authors originally wrote VAPiD for -annotating large numbers of genomes from different viral species, which mirrors the type of data that many clinical and public health laboratories may encounter.
cord-277830-6fsz9iy7	2005	The crystal structure of a conserved domain of nonstructural protein 3 (nsP3) from severe acute respiratory syndrome coronavirus (SARS-CoV) has been solved by single-wavelength anomalous dispersion to 1.4 Å resolution. Sequence and structure comparison of all known macro-H2A domains combined with available functional data suggests that proteins of this superfamily form an emerging group of nucleotide phosphatases that dephosphorylate Appr-1″-p. One of its sequence homologs, Poa1p (YBR022) from Saccharomyces cerevisiae, was recently functionally characterized as a highly specific phosphatase that removes the 1 00 phosphate group of ADP-ribose-1 00 -phosphate (Appr-1 00 -p) in the latter half of the tRNA splicing pathway in yeast (Shull et al., 2005) , hinting at a similar substrate specificity for SARS ADRP. A view of the proposed active site of SARS ADRP along with the superimposed structures of AF1521 and yeast Ymx7 are shown in Figure 4B , highlighting the interactions that are likely between residues of the protein with the ligand.
cord-277841-7sp8ftbc	2020	Molecular diagnostic tests target the detection of any of the following markers such as the specific region of the viral genome, certain enzyme, RNA-dependent RNA polymerase, the structural proteins such as surface spike glycoprotein, nucleocapsid protein, envelope protein, or membrane protein of SARS-CoV-2. COVID-19 is a contagious disease, caused by a novel severe acute respiratory syndrome Coronavirus (SARS-CoV-2). In this article, we evaluated literature for reports informing various diagnostic methods, potential antiviral chemical therapeutics, and effective treatment strategies towards clinical management of COVID-19 patients. Molecular diagnostic methods target to detect either specific regions of the viral genome or RNA-dependent RNA polymerase (RdRP) and/or structural proteins of SARS-CoV-2 (Table 1) . Like most immunological diagnostic protocols, Enzyme-Linked Immunosorbent Assay (ELISA) for COVID-19 detection uses IgM and IgG antibody against nucleocapsid (N) and receptor binding domain spike proteins (S) of SARS-CoV-2. Table 2: Primers and probes for targeting SARS-Cov-2 genes in an RT-PCR test for COVID-19 diagnosis.
cord-277874-cr53ycrm	2020	We believe MPAD based SARS-CoV-2 protein quantitation represents a promising epidemiological tool with a sensitivity sufficiently superior to viral RNA measurement that, in addition to enabling early detection and population tracking of COVID-19 load, will also open the way to effective infection surveillance of specific facilities, schools and residences. Primary sludge and PEG precipitated influent fractions, collected from the contiguous cities of Ottawa and Gatineau in April through June 2020, were analysed for the presence of four SARS-CoV-2 structural proteins, N (nucleocapsid), M (membrane), S (spike), and E (envelope), by western blot. Next, in order to assure specificity for detection of SARS-CoV-2 proteins, we used MPAD with an expanded panel to simultaneously measure three viral proteins, N, S and M, along with six fecal content control proteins in PEG precipitated "influent solids" samples drawn from the Ottawa WRRF during the study period ( Fig 5) .
cord-278055-v2ed3tei	2020	Previous study of SARS-CoV (Urbani strain) in 5-weeks-old golden Syrian hamsters showed robust viral replication with peak viral titers detected in the lungs on 2 dpi, followed by rapid viral clearance by 7 dpi, but without weight loss or evidence of disease in the inoculated animals 20 . Our results indicate that the golden Syrian hamster is a suitable experimental animal model for SARS-CoV-2, as there is apparent weight loss in the inoculated and naturally-infected hamsters and evidence of efficient viral replication in the nasal mucosa and lower respiratory epithelial cells. c, Transmission of SARS-CoV-2 to naïve hamsters (N=3) that were each co-housed with one inoculated donor on 1 dpi; infectious viral load and viral RNA copy numbers detected in the nasal washes of contact hamsters were shown. e, Transmission of SARS-CoV-2 to naïve hamsters (N=3) that were each co-housed with one donor on 6 dpi; infectious viral load and viral RNA copy numbers detected in the nasal washes of contact hamsters were shown.
cord-278081-tk7vn1v1	2017	Here is presented new details to the hypothesis, explaining how the disrupted chromatin can lead to subsequent disruption of the nucleolus, even nucleolar fragmentation, which results in ineffective nucleolar functioning, misfolded RNAs, misassembled or incompletely assembled ribonucleoprotein (RNP) complexes, and stabilization of nucleolar components in autoantigenic conformations. For now there is no direct connection between viruses and the "X chromosome-nucleolus nexus" hypothesis to the increased risk of cancers among autoimmune disease patients but we can consider the induction by viruses of increased polyamine levels and the possible reactivation of X-linked polyamine genes as means by which competition for the cellular methyl donor, SAM, could reduce DNA methylation and open oncogenes for overexpression in proliferation competent cells. A disrupted Barr body could generate an abundance of polyamines and Alu RNA from X-linked genes and elements that further stress and damage the nucleolus, making it very inefficient in its functions, even fragmenting it and possibly leading to cell death.
cord-278099-ypov9ha3	2017	The four dsRNAs eluted from the agarose gel were purified and have been used as templates for RT-PCR amplification employed in SISPA to generate fulllength cDNAs. It is of interest to examine if ArCV-1 RNA dependent RNA polymerase (RdRp) structurally resembles the known RdRp of the dsRNA bacteriophage Փ-6, reovirus, or with other viruses like calciviruses and picornaviruses [12] [13] [14] [15] [16] . We report here the results of elaborated computer-assisted analysis of ArCV-1 replicase which revealed the presence of conserved sequence motifs (A to G) present in the fingers and palm subdomains of the polymerase that are shared in most of the RdRps. Interestingly, ArCV-1 replicase has more structural resemblances with several members of ssRNA (+) mono-partite Picornaviruses (viral replication by primer-dependent initiation), than the de novo dsRNA bacteriophage Փ-6 and reovirus polymerases. Possible functions of the residues of the A to G motifs described for identical RdRps was conserved with respect to the ArCV-1 3Dpol structure and was discussed in structural analysis of ArCVTable 1 ) and the 3'' terminus contained the sequence "GCA CCCATATTC".
cord-278123-mq56em3z	2020	Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. Standard approach for detection of SARS-CoV-2 RNA from nasopharyngeal specimens in our laboratory involves extraction of total nucleic acids from specimens in an IVD-labeled, automated extraction platform followed by RT-qPCR, based on one of the assays (Table 1) suggested by World Health Organization (WHO) [11] . Based on these results, the optimal pre-treatment and reaction conditions for the direct approach were: i) transfer and dilute (4-fold) 10 μl of NPFS specimen in NFW; ii) incubate at 65˚C for 10 min; and iii) test 8 μl of heat lysed specimen in a 20 μl reaction using TaqPath™ 1-Step RT-qPCR Master Mix. The analytical sensitivity of the direct RT-qPCR assay using specimens prepared in this manner was determined by serially diluting a specimen positive for SARS-CoV-2 with a negative specimen as a diluent.
cord-278186-t3izmz6n	2020	ABBREVIATIONS: cDC, conventional dendritic cell; CMT, cytokine modulating treatment; CRC, colorectal carcinoma; CTL, cytotoxic T lymphocyte; DC, dendritic cell; dsRNA, double-stranded RNA; FLT3LG, fms-related receptor tyrosine kinase 3 ligand; HNSCC, head and neck squamous cell carcinoma; IFN, interferon; IL, interleukin; ISV, in situ vaccine; MUC1, mucin 1, cell surface associated; PD-1, programmed cell death 1; PD-L1, programmed death-ligand 1; polyA:U, polyadenylic:polyuridylic acid; polyI:C, polyriboinosinic:polyribocytidylic acid; TLR, Toll-like receptor Alongside, a pilot study on patients with metastatic HNSCC and melanoma who received intratumoral or intramuscular Hiltonol™ reported clinical benefits for at least one of the 8 individuals enrolled in this trial, coupled to moderate side effects (such as inflammation at the injection site and fatigue) as well as increased levels of CD4, CD8, PD-1, and PD-L1 in tumors, confirming the activation of systemic immunity.
cord-278250-dwok857k	2019	We performed metagenomic sequencing of fecal samples to detect the bacterial microbiome and virome composition of healthy one-year-old rhesus monkeys housed at the IMBCAMS (Fig. 1) . We comprehensively analyzed the interactions among the gut virome, bacterial microbiome and metabolomes based on the above results there were noticeable differences in bacterial β-diversity between control and experimental animals, as determined using principal component analysis (PCA), and the results showed good repeatability within a single group (Additional file 2: Figure S2F ). Briefly, the fecal virome composition was noticeably altered after depletion of the bacterial microbiome, and the abundances of many DNA viruses, bacteriophages and RNA viruses in the gut were clearly decreased. As expected, the whole gut bacterial microbiome, including gram-positive and gram-negative bacteria (Additional file 2: Figure S2E ), was depleted after treatment with the antibiotic cocktail, except for Escherichia-Shigella species belonging to Proteobacteria, which were resistant to the cocktail.
cord-278436-job4854r	2009	Recent studies suggest that some RNA-binding proteins facilitate the folding of non-cognate RNAs. Here, we report that bacteriophage MS2 coat protein (MS2 CP) bound and promoted the catalytic activity of Candida group I ribozyme. This mechanism is similar to that of the yeast mitochondrial tyrosyl-tRNA synthetase on other group I introns, suggesting that different RNA-binding proteins may use common mechanisms to support RNA structures. Interestingly, CYT 18 also stimulates the activity of several non-cognate group I introns [12] , questioning the specificity of the action of RNA-binding proteins. In this study, the protein-protected RNA cloning (PRC) method has been developed to successfully identify the MS2-binding site on the Candida group I ribozyme RNA. In summary, the finding of MS2 CP binding of the P5ab-P5 structure of the Candida group I ribozyme suggests that this phage protein can interact with a bulged RNA paired region connected to an asymmetric internal loop containing three adenines.
cord-278482-j5zlismf	2016	Summary The adenosine nucleoside analog BCX4430 is a direct-acting antiviral drug under investigation for the treatment of serious and life-threatening infections from highly pathogenic viruses, such as the Ebola virus. The adenosine nucleoside analog BCX4430 is a direct-acting antiviral drug under investigation for the treatment of serious and life-threatening infections from highly pathogenic viruses, such as the Ebola virus. In a study in cynomolgus macaques infected with MARV [5] , 0/6 controls survived compared to 17/18 animals treated with a 15 mg/kg BD dose of The maximum viral load, as measured by quantitation of viral RNA copies in the peripheral blood, was reduced by ∼600-fold (geometric mean 0.008 × 10 9 compared to 4.79 × 10 9 copies/mL, p < 0.0005). The slope of the relationship of the dose of BCX4430 to both antiviral effect and survival in nonclinical models of lethal viral infections is steep.
cord-278635-vwdxr1bl	2020	In the present study we compared the clinical outcome, mortality and transmission in chickens and turkeys infected with the same infectious doses of H7N7 low pathogenic avian influenza virus containing different levels of defective gene segments (95/95(DVG-high) and 95/95(DVG-low)). Virions containing highly deleted forms of genome segments (defective viral genes-DVGs) are able to replicate only in the presence and at the expense of fully infectious virus, hence the term "defective interfering particles" (DIPs) [4] . To evaluate the effect of DIPs on the course of infection with low pathogenic avian influenza virus (LPAIV), a comparison of pathogenicity of two virus stocks of H7N7 LPAIV with different levels of defective genomes was performed in turkeys and chickens. Infected birds received the same infectious dose of the virus but with different amount of DVGs. The semiquantitative analysis of defective particles was done by a combination of RT-PCR, real time RT-PCR and whole genome sequencing and indicated significantly higher amount of truncated gene segments in 95/95(DVG-high).
cord-278647-krh63hqp	2012	At the time of its disappearance in 2009, the human H1N1 lineage had accumulated over 1400 point mutations (more than 10% of the genome), including approximately 330 non-synonymous changes (7.4% of all codons). This process may play a role in natural pandemic cessation and has apparently contributed to the exponential decline in mortality rates over time, as seen in all major human influenza strains. Given this large body of data, it becomes feasible to test the attenuation model using mutation accumulation rates, non-synonymous amino acid changes, changing dN/dS ratios, changing transition/transversions ratios, and changes in codon specificity over time. Using the amended 1918 Brevig Mission virus as a reference and including all human and porcine viruses in the database, we calculated SNPs, indels, transitions, transversions, non-synonymous amino acid changes, dN/dS ratios, predicted protein lengths (for all 11 proteins), the normalized codon scores (NCS) and relative synonymous codon usage (RSCU) [51] score for each predicted protein of each genome.
cord-278684-txlvla0j	1998	The BDV paradigm is amenable to study virus–cell interactions in the CNS that can lead to neurodevelopmental abnormalities, immune-mediated damage, as well as alterations in cell differentiated functions that affect brain homeostasis. Evidence provided by epidemiological and clinical data, together with virological studies, have led to the hypothesis that chronic viral infections of the CNS contribute to human mental disorders of unknown etiology. Therefore, neuronal damage seen in BD appears to be mediated by the cytotoxic activity of CD8 ϩ T-cells present in the brain parenchyma of BDV-infected rats. Studies on PTI-NB rats may provide valuable information regarding the contribution of CNS resident cells to disturbances in cytokine gene expression caused by BDV. Borna disease virus replicates in astrocytes, Schwann cells and ependymal cells in persistently infected rats: Location of viral genomic and messenger RNAs by in situ hybridization Expression of tissue factor is increased in astrocytes within the central nervous system during persistent infection with Borna disease virus
cord-279070-cy049zbi	2011	DNA metaviromes were similar between healthy and diseased tissues and comprised of contiguous sequences (contigs) that matched primarily metazoan and bacterial proteins. Ten samples of sea fans (5 each of visually ''healthy'' and ''diseased'' tissues) were used to generate metagenomic libraries of the viral fraction following protocols for tissues in Thurber et al. Contigs [ 100 bp that did not match protein databases by BLASTx against nr at e-values \ 10 -5 were further characterized by tBLASTx analysis against all viral genomes in GenBank. The proportion of annotated sequence reads (BLASTx against nr databse) and contigs was highest for DNA virus libraries and least for the RNA viral reads (Fig. 1) . The majority of matches for both DNA and RNA viral contigs and reads were to bacterial and metazoan proteins. In contrast to the RNA metaviromes, DNA viruses were well annotated, where *50% of contigs [ 100 bp matched proteins in the nr database.
cord-279346-7del8d2p	2007	title: Heterologous viral RNA export elements improve expression of severe acute respiratory syndrome (SARS) coronavirus spike protein and protective efficacy of DNA vaccines against SARS Here, we demonstrated that efficient expression of S from the wild-type spike gene in cultured cells required the use of improved plasmid vectors containing donor and acceptor splice sites, as well as heterologous viral RNA export elements, such as the CTE of Mazon-Pfizer monkey virus or the PRE of Woodchuck hepatitis virus (WPRE). Upon immunization of mice with low doses (2 μg) of naked DNA, only intron and WPRE-containing vectors could induce neutralizing anti-S antibodies and provide protection against challenge with SARS-CoV. The influence of SS, MPMV-CTE or WPRE on expression of S protein in transfected cells and on induction of a protective SARS-CoV-specific immunity in mice after naked DNA immunization are compared.
cord-279404-u0fs6xcj	2008	The search for the animal reservoir of the severe acute respiratory syndrome coronavirus (SARS-CoV) led to extensive surveys of coronaviruses in wild and domestic animal populations in China, resulting in the detection of a wide variety of novel bat coronaviruses (Bt-CoVs) (2) (3) (4) (5) . Our detection of RNA from group 1 CoVs in Trinidadian bats shows that Bt-CoVs have a wider distribution than previously suspected and is added support for bats as the original host species for these viruses. Despite the geographic proximity of the bats from which the Trinidadian Bt-CoV sequences were derived-Couva and Fyzabad are 28 km (17 miles) apart, and Trinidad has an area of only 4,769 km 2 (1,864 square miles)-they are relatively highly divergent. Trinidadian bat coronavirus (Bt-CoV) sequences are highlighted in red and North American Bt-CoV in blue. Detection of group 1 coronaviruses in bats in North America
cord-279418-3r1ijafm	2020	We particularly examine the interplay between viral factories and the cellular innate immune response, of which several components also form membrane-less condensates in infected cells. With the rapid identification of cellular membraneless compartments and proteins that undergo LLPS in vitro, a major challenge in the field is to demonstrate unambiguously that a specific structure is indeed a phase-separated liquid body in the cellular context. Until now, only a few specific cellular factors, which directly interact with viral proteins such as the nucleoproteins and phosphoproteins of MNV, have been shown to concentrate in these structures. Upon Infection, Cellular WD Repeat-Containing Protein 5 (WDR5) Localizes to Cytoplasmic Inclusion Bodies and Enhances Measles Virus Replication The Ebola Virus Nucleoprotein Recruits the Nuclear RNA Export Factor NXF1 into Inclusion Bodies to Facilitate Viral Protein Expression The Cellular Protein CAD is Recruited into Ebola Virus Inclusion Bodies by the Nucleoprotein NP to Facilitate Genome Replication and Transcription
cord-279623-ezax8c1u	2012	(CCND1) promoter can produce low-copy transcript lncRNAs in human cell lines which can be combined to the 5 ′end regulatory region of the CCND1 that are induced in response to DNA damage signals for the recruitment of the translocated-in-liposarcoma protein to the CCND1 promoter to cause gene-specific Fig. 1 Paradigms for functions of lncRNAs expression inhibiting the CREB-binding protein and p300 histone acetyltransferase activities [59] . The results directly implicate long non-coding RNAmediated transcriptional regulation of gene expression as a fundamental control mechanism for physiologic processes, such as vascular development [32] . To sum up, intense investigation of the lncRNA transcription will likely expand our understanding of both the cell biology and functions of lncRNAs. Non-coding RNAs revealed during identification of genes involved in chicken immune responses Long antisense non-coding RNAs function to direct epigenetic complexes that regulate transcription in human cells
cord-279691-v5kpmk0b	2012	Upon infection, coronaviruses extensively rearrange cellular membranes into organelle-like replicative structures that consist of double-membrane vesicles and convoluted membranes to which the nonstructural proteins involved in RNA synthesis localize. This review will summarize the current knowledge on the biogenesis of the replicative structures, the membrane anchoring of the replication-transcription complexes, and the location of viral RNA synthesis, with particular focus on the dynamics of the coronavirus replicative structures and individual replication-associated proteins. A distinctive common feature of +RNA viruses is the replication of their genomes in the cytoplasm of the host cell in association with rearranged cellular membranes that are remodeled into organelle-like membranous structures to which the viral replication-transcription complexes (RTCs) localize. The first detectable membrane rearrangements in CoV-infected cells are 200 to 350 nm organelle-like structures that have been described for both MHV [47, 62] and the SARS-CoV [5, 63] and consist of spherical vesicles containing double lipid bilayers, termed DMVs ( Figure 2 ).
cord-279841-oq25o4qr	2005	Further insights into RNA virus vector design and optimization are emerging from recent advances on the function of viral RNA replication factors, the nature of the viral RNA replication complex as a membrane-bounded compartment sequestering replication components from competing processes and host defenses, and identification of surprisingly diverse host genes contributing to many virus replication steps. An unusual feature of BMV for identifying and characterizing host functions in viral replication is that BMV directs RNA replication, gene expression and virion formation in the genetic model yeast, Saccharomyces cerevisiae [13] . In recent years, classical yeast genetics have been used to identify host genes that function in controlling BMV translation [14, 15] , selecting BMV RNAs as replication templates [16] , activating the viral RNA replication complex [17] , maintaining a lipid composition required for membrane-associated RNA replication [18, 19] , and other steps.
cord-279985-de0b27nq	2008	Kunjin replicon VLP vaccines encoding HIV-1 gag have also been shown to induce CD8 T cell immunity comparable to that seen after immunisation with recombinant vaccinia [11] . Here we describe the behaviour of four different Kunjin replicon VLP vaccines encoding SIV gag and show that only the Gag-pol vaccine (i) induced good levels of both effector memory and central memory T cell responses 10 weeks post-vaccination, comparable to those induced by the previously described HIV-1 gag Kunjin replicon VLP vaccine [11] , (ii) showed good levels of protection against challenge with A20 cells expressing SIV Gag ≈9 months post-vaccination, and (iii) displayed high levels of insert stability. In summary we describe here a Kunjin replicon SIV Gag-pol VLP vaccine, which showed high insert stability, good induction of effector and central memory responses, and good protection against a model challenge.
cord-280001-y7pvj2l1	2020	If the test is positive though, the result is most likely correct, although stray viral RNA that makes its way into the testing process (for example, as the specimen is being collected or as a result of specimen cross-contamination or testing performed by a laboratory worker who is infected with SARS-CoV-2 [these are just some examples]) could conceivably result in a falsely positive result. Testing patients for SARS-CoV-2 helps identify those who are infected, which is useful for individual patient management, as well as for implementation of mitigation strategies to prevent spread in health care facilities and in the community alike (Fig. 1) . Given that SARS-CoV-2 can infect anyone and result in transmission prior to the onset of symptoms or even possibly without individuals ever developing symptoms, testing asymptomatic patients could even be considered. Finally, serologic testing can possibly be used diagnostically to test viral RNA-negative individuals presenting late in their illness.
cord-280003-ndpuezpo	2020	Serial sera of COVID-19 patients were collected and total antibody (Ab), IgM and IgG antibody against SARS-CoV-2 were detected. The first detectible serology marker is total antibody and followed by IgM and IgG, with a median seroconversion time of 15, 18 and 20 day post exposure (d.p.e) or 9, 10 and 12 days post onset, separately. In order to answer some of the questions, we investigated the characteristics of antibody responses in 80 Covid-19 patients during their hospitalization periods, through detecting total antibody, IgM and IgG using immunoassays. A total of 80 Covid-19 patients and 100 to 300 healthy people were tested for antibodies against SARS-CoV-2 using different immunoassays. The present data showed that the sensitivity of total antibody detection was higher than that of IgM and IgG (p<0.001) while the specificities are overall comparable when the same testing technic (ELISA, CLMA or LFIA) is used.
cord-280048-b4dz1lnn	2019	Research on quasispecies has proceeded through several theoretical and experimental avenues that include continuing studies on evolutionary optimization and the origin of life, RNA-RNA interactions and replicator networks, the error threshold in variable fitness landscapes, consideration of chemical mutagenesis and proofreading mechanisms, evolution of tumor cells, bacterial populations or stem cells, chromosomal instability, drug resistance, and conformation distributions in prions (a class of proteins with conformation-dependent pathogenic potential; in this case the quasispecies is defined by a distribution of conformations) [16, 20] . Adaptability of RNA viruses is linked to parameters that facilitate exploration of sequence space: genome size (1.8 to 33 Kb), population size (variable but that can attain an impressive 10 12 individual genomes in an infected host at a given time), replication rate, mutation rate, fecundity (yield of viral particles per cell), and number of mutations required for a phenotypic change (surprisingly low for several relevant traits) (see [49] ).
cord-280130-ewqe9edq	2007	In most nucleated body cells, viral infections activate transcription of the ''''classic'''' IFN-b gene [1] by a signaling chain which is initiated by the RNA sensors RIG-I and MDA-5, which in turn act trough the adaptor IPS-1 and the kinases TBK-1 and IKK-3 to activate the transcription factor IRF-3 (see reviews by P. Many RNA and DNA viruses therefore express proteins which bind this key molecule to avoid both IFN induction and activation of dsRNA-dependent antiviral enzymes [7, 8] . Binding of the influenza virus NS1 protein to double-stranded RNA inhibits the activation of the protein kinase that phosphorylates the elF-2 translation initiation factor Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling Double-stranded RNA binding of influenza B virus nonstructural NS1 protein inhibits protein kinase R but is not essential to antagonize production of alpha/beta interferon
cord-280272-mn596x1p	2020	We have tested the ability of magnetic Nanotrap® (NT) particles to improve stability and detection of Venezuelan equine encephalitis virus (VEEV), viral capsid protein, and viral genomic RNA in whole human blood at elevated temperature and prolonged storage conditions. We have tested the ability of magnetic Nanotrap ® (NT) particles to improve stability and detection of Venezuelan equine encephalitis virus (VEEV), viral capsid protein, and viral genomic RNA in whole human blood at elevated temperature and prolonged storage conditions. In this study, we sought to apply new magnetic NT particles that consist of NIPAm copolymers functionalized with reactive red 120 to evaluate the efficacy of preservation of infectious VEEV, viral RNA, and VEEV capsid protein in whole blood samples at ambient and elevated temperature as well as at low and high humidity conditions.
cord-280427-smqc23vr	2020	The various evidence from the past clearly suggest that the evolution of the virus in both reservoir and intermediate animal hosts needs to be explored to better evaluate the emergence of SARS-CoV-2 in humans. The qPCR and virus titration test conducted on the various isolated organs of the ferrets on day 4 post inoculation detected infectious virus in the nasal turbinate, soft palate and tonsils of ferrets indicating the possible replication of the virus in the upper respiratory tract of the ferrets while no infection was found in other organs such as trachea, lung, heart, spleen, kidneys, pancreas, small intestine, brain and liver of the ferrets (Kim et al. This study results stipulate ferret to have high susceptibility for the SARS-CoV-2 and this infectious virus sheds by multiple routes of body discharge specimens such as urine and faeces of the infected ferrets which serve as a potential source of viral transmission to close contact.
cord-280616-9mwr6a4x	2017	reported a mechanosensitive miRNA, hsa-miR-103a-3p, that exhibited negative effects on Runx2 during cyclic mechanical stretch (CMS)induced osteoblastogenesis, leading to decreased bone formation both in vitro and in vivo (Zuo et al., 2015) . To understand whether other effective paracrine pathways exist in the interaction between the two cell types, Li and colleagues conducted miRNA-mediated osteoclast-directed osteoblastic bone formation in ovariectomized (OVX) mice, indicating that inhibition of osteoclast-derived exosomal mmu-miR-214-3p induced significantly suppressed osteoclastogenesis (Li et al., 2016b) . In addition, Krzeszinski and colleagues found that mmu-miR-34a-5p knockout and heterozygous mice exhibited elevated bone resorption and reduced bone mass by targeting transforming growth factor-b-induced factor 2 (Tgif2), indicating that miR-34a-5p was a critical osteoclast suppressor and a potential therapeutic strategy to combat osteoporosis, and could exert both anti-catabolic and anabolic effects compared to current drugs that are solely anticatabolic (Krzeszinski et al., 2014) .
cord-280795-wtrt13ij	2007	As a step toward better understanding the relationship between activities and structures of the helicase-like domain of BaMV replicase, we set out to investigate the importance of each signature motif to its NTPase and RNA 5′-triphosphatase activities by mutational and kinetic analyses. The apparent K i value of AMPPNP in inhibiting RNA 5′-triphosphatase activity The enzymatic activity was determined at 20°C by enzyme-coupled assay in 1 ml solution that contained 10 pmol enzyme, 0.1 to 3 mM NTP and other buffer components as described under Materials and methods except that the amounts of pyruvate kinase were increased up to 50, 75 and 300 U for GTPase, UTPase and CTPase assay, respectively, to assure the rate of NTP hydrolysis being the limiting step within the coupling reaction. The helicase-like domain of plant potexvirus replicase participates in formation of RNA 5′ cap structure by exhibiting RNA 5′-triphosphatase activity
cord-280941-ds6x0yym	2018	The receptor-binding domain (RBD) of Middle East respiratory syndrome-coronavirus (MERS-CoV) was fused with the RNA-interaction domain (RID) and bacterioferritin, and expressed in Escherichia coli in a soluble form. The concentration of the ion Fe(2+), salt, and fusion linker also contributed to the assembly in vitro, and the stability of the NPs. The kinetic "pace-keeping" role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of NPs and virus-like particles as recombinant vaccines and for serological detection of viral infections. Taken together, the results confirmed the immunologically relevant conformation of the MERS-CoV RBD displayed on the hybrid ferritin particles, and the crucial role of RNA in controlling the kinetic pathway for the assembly of viral antigen monomers into stable NPs. To evaluate the immunogenicity of ferritin-based NPs, BALB/c mice (n = 5) were immunized with RBD, RBD-FR, and RBD-[SSG]-FR NPs antigens.
cord-280994-w8dtfjel	2020	Here, we describe the near-atomic resolution structure of its core polymerase complex, consisting of nsp12 catalytic subunit and nsp7-nsp8 cofactors. This structure highly resembles the counterpart of SARS-CoV with conserved motifs for all viral RNA-dependent RNA polymerases, and suggests the mechanism for activation by cofactors. Biochemical studies revealed reduced activity of the core polymerase complex and lower thermostability of individual subunits of COVID-19 virus as compared to that of SARS-CoV. Simultaneous 193 replacement of the nsp7 and nsp8 cofactors further enhanced the efficiency for RNA synthesis 194 to ~2.2 times of that for the SARS-CoV-2 homologous complex ( Figure 4B ). After 3 rounds of extensive 2D classification, ~924,000 particles 437 were selected for 3D classification with the density map of SARS-CoV nsp12-nsp7-nsp8 438 complex (EMDB-0520) as the reference which was low-pass filtered to 60 Å resolution. One severe acute respiratory syndrome 631 coronavirus protein complex integrates processive RNA polymerase and exonuclease activities
cord-281020-g1muealp	2012	The universal requirement of (+)RNA viruses for cellular membranes for genome replication, and the formation of membranous replication organelles with similar architecture, suggest that they target essential control mechanisms of membrane metabolism conserved among eukaryotes. These viruses universally require cellular membranes for assembly of their replication complexescontaining viral proteins, RNA, and host factorsto amplify their genome. Expression of poliovirus and HCV proteins in the presence of BFA resulted in the appearance of new membrane structures that are indistinguishable from those observed without the drug, indicating that GBF1 is more probably involved in the function of replication organelles than in their formation [41, 45] . For HCV, the absence of PI4KIIIa activity induced a dramatic change in the ultrastructural morphology of the membranous replication complex, suggesting a role of PI4P in recruiting specific membrane-shaping proteins [51 ] .
cord-281124-4nhy35xn	2011	To examine this domain in more detail, the 18 aa peptide (M(11)PVRRPLPPQPPRNARLI(29)) encompassing this sequence was synthesized and found to bind nucleic acids similarly to the full-length N protein in EMSAs. The data indicate a fundamental role for the GAV N protein proline/arginine-rich domain in nucleating genomic ssRNA to form nucleocapsids. In a preliminary attempt to identify an RNA packaging signal in the GAV genome, EMSAs were performed using ssRNAs synthesized to various genome regions including (i) an ORF1b gene 39-region spanning the relative position to the genome packaging signal identified in MHV [28] , (ii) a 39-terminal genome region corresponding in position to the region in the infectious bronchitis virus (IBV) genome reported to contain an RNA binding domain [29] and (iii) the 59-genomic RNA terminus which, in coronaviruses, has also been reported to interact specifically with N protein [30] .
cord-281174-3c1vue0y	2003	The application of a rapid nucleic acid sequence-based amplification (NASBA) assay for the detection of NLV RNA in stool is described using the NucliSens® Basic Kit. Primers and probes for the NLV Basic Kit assay were based on the RNA polymerase region of the prototype NLV, Norwalk virus (NV) genome and could consistently detect 10(4) RT-PCR detectable units of NV RNA in a stool filtrate. Despite the specificity of the NASBA primer/probe sequences for NV, other representatives from both NLV genogroups I and II could be detected by the Basic Kit assay in outbreak stool specimens, although the results were inconsistent. In this study, we report the application of a rapid NASBA assay for the detection of NV RNA in stool using the NucliSens † Basic Kit. This assay was compared directly with RT-PCR and used for the detection of NV and other NLVs in fecal specimens obtained from human challenge studies and NLV outbreaks.
cord-281254-x7ivjvti	2012	The most promising newly developed technology for intervention in SARS may be RNA interference, an endogenous cellular process for the inhibition of gene expression mediated by sequence-specific double-stranded RNAs. Numerous studies have reported the therapeutic potential of RNA interference for the treatment of various human diseases ranging from cancers to infectious diseases such as HIV and hepatitis. Since SARS-CoV rep-To address the issue related to delivery of siRNAs into cells or lication also requires certain host proteins, genes from host cells living organisms, researchers have used several approaches, ininvolved in viral replication can also be selected as targets. Therefore, RNA interference this coronavirus family), siRNAs targeting different genes of can be a tool for down-regulation of gene expression in cultured SARS-CoV were used by various groups to inhibit virus gene cells as well as in living organisms.
cord-281281-knelqmzx	2020	The use of bioinformatics and other computational tools in addition to molecular modeling has helped researchers from different areas in the search for strategies for diagnosing viral infection, in the development of vaccines for its prevention, as well as in the discovery of new anti-SARS-CoV-2 agents. In the context of COVID-19, this characteristic was important for a better understanding of the origin of SARS-CoV-2 from the comparative analysis of genomic data of the new virus with others from the same family, suggesting its origin from natural selection, with modifications in its spike protein, more specifically in the host receptor binding domain, which may have enhanced its interaction and recognition by the human cell [83, 91] . The contributions of bioinformatics and molecular modeling in elucidating essential targets for the planning and development of new drugs, and the analysis of already known compounds, support the search for safer and more effective treatments against SARS-CoV-2 infection.
cord-281285-5g1rw202	2020	Based on its MOA, repurposed drugs with anti-SARS-CoV-2 activity can be divided into substances that prevent viral entry into host cells (1-2) and inhibit viral proteases (3) and inhibitors of viral replicase (4). The disappointing clinical results might be related to sub-therapeutic levels for inhibition of SARS-COV-2 because application of 400/100 mg of lopinavir/ritonavir twice daily was shown to yield median serum concentrations of 7.2 mg/l (11.5 µM) in patients with HIV (van der Lugt et al, 2009), which is significantly lower than the observed EC 50 in the in vitro studies. In this comparative review, we focus on repurposed drugs with antiviral effects against SARS-CoV-2 in cell-based assays as those substances offer great opportunities for a treatment early in the course of COVID-19 by inhibition of viral replication and might be even suitable for preventive strategies as shown for neuraminidase inhibitors in case of influenza (Jefferson et al, 2014) .
cord-281385-oxohdfpu	2014	Crystal structures of almost all available flavivirus methyltransferases contain S-adenosyl-L-methionine (SAM), the methyl donor molecule that co-purifies with the enzymes. The SAM-depleted and SAM-containing MTases exhibited comparable enzymatic activities (N-7 MTase, 2 0 -O MTase, and covalent GMP-MTase complex formation the natural product Sinefungin, showing that this is a viable approach to identify novel small molecules that bind to the same pocket. This conclusion was supported by two complementary structural and functional approaches: (i) the crystal structure unequivocally shows that absence of SAM does not affect the overall conformation of DENV MTase, including the SAM-binding pocket; (ii) depletion of SAM from the recombinant MTase did not change the activities of cap methylations and GMP-MTase complex formation. Structural and functional analysis of methylation and 5 0 -RNA sequence requirements of short capped RNAs by the methyltransferase domain of dengue virus NS5
cord-281565-v8s2ski3	2020	These PQS and PiMS have been assessed according to their potential to form, uniqueness, frequency of appearance, conservation rates between 3297 different 2019-nCoV clinical cases, confirmed quadruplex-forming sequence presence and localization within the genome. Then, these PQS and PiMS were evaluated by their frequency of appearance in the corresponding genome, the presence of known-to-form quadruplex structures sequences within and their probability of quadruplex-formation score (as the mean of G4Hunter (52) and the adaptation of the PQSfinder algorithm (53) ). A flexible configuration of quadruplex definitions will detect larger amounts of candidates at the expense of requiring more computing power and accepting sequences that are more ambiguous in forming quadruplex structures in vitro (as determined by their score; with longer loops, smaller runs, more bulges and more complementary G/C %, Figure 1 , B). Expanding the search for common candidates to the entire virus realm, we matched one PQS and PiMS from the 2019-nCoV with the potential quadruplexes found in four viruses from
cord-281717-kzd9vvci	2020	CpG dinucleotides are under-represented in the genomes of single stranded RNA viruses, and coronaviruses, including SARS-CoV-2, are no exception to this. CpG suppression amongst coronaviruses does not significantly differ according to genera of virus, but does vary according to host species and primary replication site (a proxy for tissue tropism), supporting the hypothesis that viral CpG content may influence cross-species transmission. 79 SARS-CoV-2 was recently reported to have a CpG composition lower than other members of the 80 betacoronavirus genus, comparable to certain canine alphacoronaviruses; an observation used to draw 81 inferences over its origin and/or epizootic potential (Xia 2020 in GC content (from ~ 0.32 -0.47) was seen across the Coronaviridae, and as expected, all viruses 97 exhibited some degree of CpG suppression, with CpG O:E ratios ranging from 0.37 to 0.74 (Fig 2A) .
cord-282372-nmii30mc	2020	Here, we develop a feeder-free, long-term three-dimensional (3D) culture technique for human alveolar type 2 (hAT2) cells, and investigate infection response to SARS-CoV-2. By imaging-based analysis and single-cell transcriptome profiling, we reveal rapid viral replication and the increased expression of interferon-associated genes and pro-inflammatory genes in infected hAT2 cells, indicating robust endogenous innate immune response. Although basic molecular mechanisms in SARS-CoV-2 infection have been identified [5] [6] [7] [8] , most findings have been obtained from experiments using non-physiological cell lines 9 , model animals, such as transgenic mice expressing human angiotensin-converting enzyme 2 (ACE2) 10 , ferrets 11 and golden hamsters 12 , or from observation in clinical cohorts 13 and/or inference from in-silico computational methods [14] [15] [16] . Immunostaining for double-stranded viral RNA (dsRNA) and nucleocapsid protein (NP) of SARS-CoV-2 identified widespread viral infection in hAT2 cells co-expressing pro-SFTPC and ACE2 in hAOs ( Fig. 2a and 2b; Extended Data Fig. 3) .
cord-282618-tjvjlyn9	2010	To maintain a low risk of insertional mutagenesis-mediated gene activation, expression-augmenting sequences would ideally function to improve transgene expression from transiently transfected intact plasmid, but not from spurious genomically integrated vectors. A neomycin resistance gene (NeoR) without an upstream Kozak sequence was cloned downstream of an enhanced green fluorescent protein (EGFP) transgene in different configurations similar to that used with PREs. Quantifiable neoR translation products were present in all tested configurations, as was biologically active neoR protein after plasmid transfection into both HEK293 and CHO cell lines (Supplementary Table S1 ). The assay was in the same format as in (a), except for the fact that Pol III inhibitor-treated cells were transfected with EGFP plasmids containing the CMV-HTLV-I R promoter with or without VA1; (c) Inhibition of PKR, not of adenosine deaminase acting on RNA (ADAR) or RNA interference (RNAI), was required for VA1 expression enhancement effect.
cord-282742-eyukbot7	2013	Geiss, Pierson, and Diamond (2005) observed that siRNAs targeting the C gene had no effect on virus replication when transfected into cells 10 h after WNV infection using lipid-based reagents. In addition, no significant reduction in viral protein or RNA levels was seen in WNV replicon-expressing cells transfected with siRNAs targeting the NS3 gene using lipid-based reagents. Also, a recent report showed that siRNA toward the TNF-a gene reduced cytokine response in DENV-infected DCs, highlighting the potential of targeted RNAi-based approaches to simultaneously decrease viral replication and the detrimental host immune response (Subramanya et al., 2010) . In addition, it has been shown that WNV (Chotkowski et al., 2008) and DENV (Mukherjee & Hanley, 2010) infection (Mukherjee & Hanley, 2010) of Drosophila cell lines induce functional virus-specific siRNAs that promote a protective RNAi response. So far we have described the antiviral effect of the RNAi mechanism induced by exogenous delivery of siRNA or precursors, and how cellular miRNA can target sequences artificially introduced within the genome of flaviviruses.
cord-282764-d9x1wii6	2006	title: Influence of intron and exon splicing enhancers on mammalian cell expression of a truncated spike protein of SARS-CoV and its implication for subunit vaccine development A truncated S protein of the TW1 strain, S(TR2) (88 kDa), carrying three S fragments (S74–253, S294–739, and S1129–1255) was investigated to study the influences of intron and exon splicing enhancers to improve S(TR2) protein expression in mammalian cells. Therefore, several different strategies for improving S TR2 protein expression in mammalian cells were investigated in this report, including intron addition and the application of exon splicing enhancers. The intron-dependent enhancement of S TR2 protein expression in CHO/dhFr− cells was further investigated by measuring total RNA level, in vivo RNA stability, and RNA elongation rate in this study. The results indicated that the intron-dependent S TR2 protein expression in mammalian cells correlated with a higher level of total RNA accumulation as determined by quantitative RT-PCR (Fig. 4A) .
cord-283097-rlf5nv5q	2014	Newcastle disease virus-vectored vaccines expressing the hemagglutinin or neuraminidase protein of H5N1 highly pathogenic avian influenza virus protect against virus challenge in monkeys Quantitative basic residue requirements in the cleavage-activation site of the fusion glycoprotein as a determinant of virulence for Newcastle disease virus Role of C596 in the Cterminal extension of the haemagglutinin-neuraminidase protein in replication and pathogenicity of a highly virulent Indonesian strain of Newcastle disease virus Role of the cytoplasmic tail amino acid sequences of Newcastle disease virus hemagglutinin-neuraminidase protein in virion incorporation, cell fusion, and pathogenicity Evaluation of the Newcastle disease virus F and HN proteins in protective immunity by using a recombinant avian paramyxovirus type 3 vector in chickens Role of fusion protein cleavage site in the virulence of Newcastle disease virus A single amino acid change, Q114R, in the cleavage-site sequence of Newcastle disease virus fusion protein attenuates viral replication and pathogenicity
cord-283132-rfw8njpo	1993	List of abbreviations: ADE=antibody-dependent enhancement; BCV=bovine coronavirus; C'' =complement; C''-ADE=complement-mediated antibody-dependent enhancement; CCV=canine coronavirus; CNS=central nervous system; CR=complement receptor; CVLP=coronavirus-like particle; ds=double-stranded; DTH=delayed-type hypersensitivity; EAV=equine arteritis virus; FcR = Fc receptor; FECV = feline enteric coronavirus; FeLV = feline leukemia virus; FIP = feline infectious peritonitis; FIPV = feline infectious peritonitis virus; HCV-229E = human coronavirus 229E; HCV-OC43=human coronavirus OC43; HE=hemagglutinating esterase; HEV=hemagglutinating encephalomyelitis virus; HIV=human immunodeficiency virus; HRSV=human respiratory syncytial virus; IBV = infectious bronchitis virus; kB = kilobases; kDa = kilodaltons; LDHV = lactate dehydrogenase virus; M = membrane (protein); mAb = monoclonal antibody; MHC = major histocompatability; MHV=mouse hepatitis virus; mRNA=messenger RNA; N=nucleocapsid (protein); Nlinked = asparagine-linked (glycosylation); NS = nonstructural (protein); O-linked = serine-or th reonine-linked (glycosylation); ORF--open reading frame; Pol = polymerase (protein); PRCV = porcine respiratory coronavirus; RCV = rat coronavirus; RECV = rabbit enteric coronaivirus; RI = replicative intermediate; rHuIFN~ =recombinant human interferon alpha; S= spike (protein); SDAV = sialodacryoadenitis virus; SIV = simian immunodeficiency virus; SPF = specific-pathogen-free; TCIDs0=tissue culture infectious dose 50%; TCV=turkey coronavirus; TGEV=transmissible gastroenteritis virus; ts=temperature-sensitive; VN=virus neutralization (-izing).
cord-283168-kl1hoa1x	2011	Fecal specimens, including swabs and litter extracts, collected from chickens, domestic ducks, turkeys, and Canadian geese were tested using degenerate primers targeting regions encoding for conserved amino acid motifs (YGDD and DY(T/S)(R/K/G)WDST) in calicivirus RNA-dependent RNA polymerases. Analysis of 2.2–3.2 kb extended genome sequences including the partial P2 (2C) and complete P3 (3A, 3B (VPg), 3C(pro), and 3D(pol)) regions of selected strains indicated that viruses yielding the 280/283 bp amplicons represent a putative new genus of Picornaviridae. This study utilized a broadly reactive primer set targeting conserved amino acid motifs encoding regions present in calicivirus RNA-dependent RNA polymerases (RdRp) and are partially also present in other viral RdRps. As part of the study here we report the serendipitous detection of novel picornaviruses in chicken and turkey samples that included diagnostic cases with runting-stunting syndrome (RSS).
cord-283346-0v4b6do2	2006	CCL2 was the most strongly regulated gene at mRNA level and its serum concentration was significantly elevated in viremic compared with aviremic and HIV-1 seronegative controls, indicating a positive correlation between viremia and CCL2. For example, the C-C chemokines macrophage inflammatory protein (MIP)-1a/CCL3, MIP-1b/CCL4 and regulated upon activation, normal T cell expressed and secreted (RANTES)/ CCL5 inhibit M-tropic HIV-1 infection by competing with the virus for its binding to the co-receptor CCR5 (11) (12) (13) (14) (15) (16) (17) . To investigate the impact of HIV-1 viremia on the host inflammatory cytokine/chemokine network, more specifically on CCL2, we utilized DNA microarray approach on PBMC RNA derived from HIV-1-infected viremic and aviremic individuals. In this study, we report that HIV-1 viremic patients show an altered expression of key inflammatory cytokines and chemokines as compared with aviremic individuals. Furthermore, increased mRNA transcripts were again detected in CCL2, CXCL10 and IFN-c when more RNA samples derived from additional aviremic and viremic patients were analyzed individually.
cord-283411-40ojqv1y	2020	title: Detection and infectivity potential of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) environmental contamination in isolation units and quarantine facilities This study assessed the infectivity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) contamination on surfaces and objects in hospital isolation units and a quarantine hotel. Surfaces and air sampling was conducted at two COVID-19 isolation units and in a quarantine hotel. Viral RNA detected in 29/55 (52.7%) and 16/42 (38%) surface samples from the surrounding of symptomatic COVID-19 patients in isolation units of two hospitals and in a quarantine hotel for asymptomatic and very mild COVID-19 patients. Surface Environmental, and 263 Personal Protective Equipment Contamination by Severe Acute Respiratory Syndrome Coronavirus 2 264 (SARS-CoV-2) From a Symptomatic Patient Detection of Severe Acute 268 Respiratory Syndrome Coronavirus 2 RNA on Surfaces in Quarantine Rooms. Severe acute respiratory 294 syndrome coronavirus 2 RNA contamination of inanimate surfaces and virus viability in a health care 295 emergency unit.
cord-283439-hqdq2qrh	2020	The suggested treatments for COVID-19 are, but not limited to, the use of (i) convalescent plasma for COVID-19 treatment [63] [64] [65] ; (ii) ribavirin, a nucleoside analogue in combination with recombinant interferon showed inhibition of MERS-CoV replication [66] ; (iii) lopinavir/ritonavir-a combination of a protease inhibitor and a booster used for the treatment of human immunodeficiency virus infection [67] ; (iv) remdesivir, a nucleotide analogue that inhibit RNA polymerase with a broad spectrum of anti-viral activities; in inhibition of human and zoonotic coronavirus [15, 68, 69] ; (v) favipiravir (also known as T-705, Avigan or favilavir) is a pyrazinecarboxamide derivative known to inhibit RNA polymerase [70] . In the current pandemic of SARS-CoV-2, Zn supplement could play an important role to treat COVID-19 patients such as (i) added immune boosting effects with anti-viral drugs and (ii) stopping SARS-CoV-2 replication in infected cells, if combined with chloroquine.
cord-283590-xvnv17zy	2020	Since December 2019, SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2; previously known as 2019-nCoV) has generated over 70000 cases of COVID-19 (Corona Virus Disease 2019, formerly known as Novel Coronavirus Pneumonia, NCP) in China, including 1870 deaths, as of 17 February 2020 (National Health Commission of the People''s Republic of China, 2020). Currently, COVID-19 patients remain the primary source of infection (Chan et al., 2020 ; General Office of National Health Commission and General Office of National Administration of Traditional Chinese Medicine, 2020; Special Expert Group for Control of the Epidemic of Novel Coronavirus Pneumonia of the Chinese Preventive Medicine Association, 2020). According to the guideline in China, patients should be isolated until two consecutive SARS-CoV-2 RNA tests of respiratory tract specimens are both negative, with an interval of at least 24 h (General Office of National Health Commission and General Office of National Administration of Traditional Chinese Medicine, 2020).
cord-283880-lrrkuist	2017	Use of sequence dependent (i.e; generic PCR assays and microarray) and sequence independent (i.e; single primer amplification (SISPA) and random priming) approaches for nucleic acid amplification combined with Sanger sequencing or HTS allowed the rapid identification of new viruses after 1980 (Bishop-Lilly et al., 2010; Chang et al., 1994; Day et al., 2010; Grard et al., 2012; Kapoor et al., 2015; Ladner et al., 2016; Linnen et al., 1996; Matsui et al., 1991; Mokili et al., 2012; Muerhoff et al., 1997; Nichol et al., 1993; Qin et al., 2014; Quan et al., 2010; Simons et al., 1995b) (Fig. 1) . For the virome analysis of clinical samples with an abundance of host cells, like blood or tissues, pre-extraction based enrichment is not appropriate as the virus genome itself can be present in its non-capsidated or transcribed form. In positive selection methods, samples are enriched for viral nucleic acids directly using probes targeting the viruses like in PCR assays, microarray or virus capture (in solution based hybridization) approaches.
cord-283895-1p5uog38	2020	Indeed, several reports indicate that SARS-CoV-2 RNA was readily detected in wastewater, and it is proposed that such approach could anticipate the occurrence of novel COVID-19 outbreaks in low prevalence regions , La Rosa et al., 2020 , Medema et al., 2020 , Orive et al., 2020 , Randazzo et al., 2020 . First, we showed that the Ebola standard (Ebo Std) primer/probe set was not detecting RNA from SARS-CoV-2-infected Vero E6 cells (Table 1) . . https://doi.org/10.1101/2020.07.08.20148882 doi: medRxiv preprint Next, we measured the SARS-CoV-2 RNA levels using N1 and N3 primer/probe sets in wastewater collected upstream of the main WWTP of the Montpellier metropolitan area on May 7 th , 18 th , 26 th , June 4 th , 15 th and 25 th (Figure 2A ). This intriguing result is reminiscent of a recent Spanish study, in which the authors could detect SARS-CoV-2 RNA in wastewater weeks before the first COVID-19 cases were reported (Randazzo et al., 2020) .
cord-283998-whwksoxt	1977	Abstract A homogeneous RNA complex with a sedimentation coefficient of 70 S and an apparent molecular weight of approximately 6.1 × 106 was released from purified 32P-labeled, mouse-brain-derived OC-43 virus after treatment with 1% sodium dodecyl sulfate (SDS) for 15 min at 23°. This suggests that (a) extensive nicking of a large RNA molecule has occurred during viral growth, due to ribonucleases which are inactivated during phenol extractions; (b) heterogeneity for OC-43 RNA is not due to internal ribonuclease activity and fragments are held together by noncovalent linkages much weaker than those present in the 70 S retroviral RNA complex, or by small proteins; or, most probably, (c) a combination of extensive nicking and weak noncovalent linkages results in the heterogeneous denaturation products. The RNA of avian infectious bronchitis virus (IBV) was first described by Tannock (1973) , who obtained from purified virions a highly heterogeneous array of RNA fragments using a phenol-sodium dodecyl sulfate (SDS) extraction procedure.
cord-284076-087oltss	2007	Of 12 peptide-conjugated PMO (PPMO), four were found to be highly effective at inhibiting PRRSV replication in cell culture in a dose-dependant and sequence-specific manner. In a previous study (Zhang et al., 2006) , we tested six PPMO and found one of them (5UP1), designed to target the 5 terminal region of the PRRSV genome, to be a highly effective inhibitor of PRRSV replication in a sequence-specific and dose-dependent manner. Confirmation of the CPE observations and PRRSV yield titration was obtained by IFA on CRL11171 cells after virus inoculation and PPMO treatment (Fig. 2B ). demonstrated that PPMO generated little cytotoxicity in both CRL11171 and PAM cells, further indicating that the suppression of virus replication observed in the antiviral experiments above was due to sequence-specific effects. Treatment of cells with PPMO 5UP2 or 5HP led to suppression of PRRSV replication of all North American strains, producing virus yields not detectable (ND) in this assay.
cord-284118-z8zwjvbu	1984	Measles virus-specific mRNAs were present, but the amount of matrix (M) protein mRNA was greatly reduced in comparison to lytically infected cells and phospho-(P) protein mRNA was hardly detectable whereas the level of the corresponding intermediate-sized (is-) RNA was greatly increased. Measles virus-specific mRNAs were present, but the amount of matrix (M) protein mRNA was greatly reduced in comparison to lytically infected cells and phospho-(P) protein mRNA was hardly detectable whereas the level of the corresponding intermediate-sized (is-) RNA was greatly increased. This interpretation is further supported by studies of non-productive cell lines which, although persistently infected with SSPE viruses, do not express measles virus M protein (Lin and Thormar, 1980; Machamer et al., 1981; Carter et al., 1983a) . This failure could result from an alteration of this specific mRNA which would prevent correct function in translation reactions, as studies of non-productive cell lines persistently infected with SSPE virus indicate (Carter et al., 1983a) .
cord-284156-btb4oodz	2017	Retinoic acid-inducible gene-I (RIG-I) is critical in triggering antiviral and inflammatory responses for the control of viral replication in response to cytoplasmic virus-specific RNA structures. They function as cytoplasmic sensors for the recognition of a variety of RNA viruses and subsequent activation of downstream signaling to drive type I IFN production and antiviral gene expressions. (c) Interactions between RIG-I-TRIM25 complex and 14-3-3ϵ promote RIG-I translocation to mitochondrial mitochondrial antiviral signaling protein (MAVS) for downstream signaling, leading to interferon production. Protein purification and mass spectrometry analysis identified that phosphorylation of Thr170 in the CARDs antagonizes RIG-I signaling by inhibiting TRIM25-mediated Lys172 ubiquitination and MAVS binding (68) . Ebola virus VP35 protein binds double-stranded RNA and inhibits alpha/beta interferon production induced by RIG-I signaling Inhibition of dengue and chikungunya virus infections by RIG-I-mediated type I interferon-independent stimulation of the innate antiviral response
cord-284549-edliu3it	2019	In this study, we screened all the nonstructural proteins of HCV and found that HCV NS2 could suppress RNAi induced either by small hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) in mammalian cells. In this study, we uncovered that HCV nonstructural NS2 protein possessed a potent in vitro VSR activity that suppressed the RNAi induced by short hairpin RNA (shRNA) and siRNA in mammalian cells. Our results showed that the reversal effect of EGFP silencing could be observed at 48 hpt (Fig. 2C) , indicating that the VSR activity was dependent on the expression level of HCV NS2 protein. To investigate whether HCV NS2 can inhibit this step, small RNAs harvested from HEK293T cells co-expressing EGFP-specific shRNA together with NS2 were subjected to Northern blotting with a DIG-labeled RNA probe targeting EGFP siRNA produced from shRNA by Dicer.
cord-284609-1q75zw6b	1982	RNAase Tl fingerprints of virus RNA, prepared from representatives of each recombinant type, confirmed the approximate crossover sites that had been deduced from the inheritance of proteins. The possibility of genetic recombination in such viruses was first suggested many years ago by Hirst (1962) and Ledinko (1963) , who showed that infection of cells with a mixture of inhibitor-sensitive variants of poliovirus resulted in an enhanced yield of resistant progeny that were genetically stable. Second, we have obtained biochemical evidence of genetic recombination by crossing temperature-sensitive mutants of aphthovirus that possess second-site mutations affecting polypeptide charge. This paper describes a cross between two subtype strains of aphthovirus that has provided the first biochemical demonstration of genetic recombination at the level of RNA. Several spontaneous temperature-sensitive mutants of subtype OS were isolated and screened by electrofocusing polypeptides induced in virus-infected cells.
cord-284707-72vx11aq	1988	For mouse hepatitis virus (MHV), one of the most extensively studied coronaviruses, there are seven species of MHV-specific mRNA present in infected cells, the largest of which is indistinguishable from virion RNA (Leibowitz et a/., 1981; Lai et a/., 1981; Spaan et a/., 1981) . In this paper we report the characteristics of a permeabilized cell system and demonstrate that it incorporates ribonucle-otide triphosphates into RNA molecules which appear identical to the virus-specific mRNAs synthesized in MHV-infected cells. Protease inhibitors and RNase inhibitors have both been reported to increase the RNA-dependent RNA polymerase activity present in extracts of West Nile virus-infected cells (Grun and Brinton, 1986) . The kinetics of the development of the MHV-specific RNA polymerase activity over the course of infection was determined in permeabilized cells. In this work we report the development and characterization of a permeabilized cell system for assaying MHV-specific RNA polymerase activity.
cord-284933-flbibrcm	2017	title: Characterization of the Transcriptome and Gene Expression of Brain Tissue in Sevenband Grouper (Hyporthodus septemfasciatus) in Response to NNV Infection Ubiquitin-like protein 4a (UBL4a), C-C motif chemokine 2 (CCL2), lysozyme g (LYG_EPICO) and two novel genes (ID: SGU016297, SGU008676) were highly expressed in the NNV-infected group compared to that in the mock group. In this study, we performed a transcriptome analysis of the brain tissue of sevenband grouper infected with NNV compared to mock brain tissue using a RNA-Seq. The total number of unigenes and the average length of the unigenes were 104,348 and 845 bp, respectively. In this study, we performed a transcriptome analysis of the brain tissue of sevenband grouper infected with NNV compared to mock brain tissue using a RNA-Seq. The total number of unigenes and the average length of the unigenes were 104,348 and 845 bp, respectively. In this study, we identified innate immune response relevant genes of sevenband grouper involved in NNV infection.
cord-284941-wfn0pnev	2008	The members of this virus family are enveloped and have genomes consisting of a single segment of negative-sense RNA that contains 6–10 genes encoding up to 12 proteins. The family Paramyxoviridae contains a large number of viruses of animals (Table 1) , including a number of major animal pathogens (such as Newcastle disease virus (NDV), canine distemper virus, and rinderpest virus), zoonotic pathogens (such as Hendra and Nipah viruses), and a number of somewhat obscure viruses whose natural histories are poorly understood. A number of animal paramyxoviruses have been recovered from cDNAs using reverse genetics, including simian virus 5, NDV, bovine parainfluenza virus 3, Sendai virus, canine distemper virus, rinderpest virus, bovine respiratory syncytial virus, and avian metapneumovirus. Infection occurs by several different routes, including aerosols (NDV, bovine respiratory syncytial virus, avian metapneumovirus) and contaminated feed and water (Newcastle disease, canine distemper, and rinderpest viruses).
cord-285159-gytebbua	2020	Here, we report the use of a purified and highly active SARS-CoV replication/transcription complex (RTC) to set-up a high-throughput screening of Coronavirus RNA synthesis inhibitors. Principle of SARS-CoV RNA synthesis detection by a fluorescence-based high throughput screening assay Highlights A new SARS-CoV non radioactive RNA polymerase assay is described The robotized assay is suitable to identify RdRp inhibitors based on HTS -A new SARS-CoV non radioactive RNA polymerase assay is described -The robotized assay is suitable to identify RdRp inhibitors based on HTS the RdRp core nsp12 and shown to confer full activity and processivity to nsp12 (Subissi et al., 2014) . Picogreen kinetic assay was based on polymerase activity of SARS nsp12 in complex with nsp7L8, which catalyzed the reaction using a poly (A) template and uridine triphosphate (UTP).
cord-285262-690kpupt	2020	Apoptosis, necroptosis and pyroptosis represent three distinct types of regulated cell death forms, which play significant roles in response to viral and bacterial infections. Abstract Apoptosis, necroptosis and pyroptosis represent three distinct types of regulated cell death forms, which play significant roles in response to viral and bacterial infections. In this chapter, based on the current advances in the research, we give a detailed description about the key cell death modalities, including apoptosis, necroptosis and pyroptosis emerging in response to pathogenic insults, and we discuss how bacterial and viral infections can modulate these signaling pathways. The components of the bacterial T3SS trigger inflammasome formation and pyroptotic cell death in Shigella infected macrophages through the activation of the NLR family CARD domain containing protein 4 (NLRC4) (Fig. 3) . Macrophage activation redirects yersinia-infected host cell death from apoptosis to caspase-1-dependent pyroptosis
cord-285290-l7mnq4yb	2018	Here, we discuss principles for discovering small-molecule drugs that target RNA and argue that the overarching challenge is to identify appropriate target structures – namely in disease-causing RNAs that have high information content, and consequently appropriate ligand binding pockets. In our view, successful targeting of RNA in therapeutically useful ways with small molecules requires three distinct components: (1) identification of RNAs and RNA motifs with disease-related function, (2) use of screening approaches and libraries likely to identify drug-like molecules with appropriate pharmacological properties and (3) identification of and focus on RNA motifs with sufficient structural sophistication that make it likely that high affinity and specificity in small-molecule binding can be achieved. (1) a therapeutically compelling RNA target, (2) a screening approach that will identify drug-like lead molecules with appropriate pharmacological properties and (3) the identification of RNA motifs with sufficient information content such that high-specificity and high-potency binding to a high-quality pocket is achieved.
cord-285330-td4vr0zv	2015	title: Viral quantity and pathological changes in broilers experimentally infected by IRFIBV32 isolate of infectious bronchitis virus An Iranian isolate of avian infectious bronchitis virus IRFIBV32 was quantified in experimentally infected broilers using real-time reverse transcriptase polymerase chain reaction and histopathological changes was investigated. In this study, we identified IBV load in different tissues of experimentally infected broilers to clarify the replication strength of IRFIBV32 Isolate at intervals post challenge. Gross lesions were recorded and their trachea, lungs, kidneys, caecal tonsils, testes and ovaries were aseptically collected separately for the virus detection, titration and histopathological evaluations. We detected the viral RNA in the caecal tonsils of infected chicken from 1 to 20 dpi and the maximal loads of the virus were on 5 dpi. Pathogenesis and tissue distribution of avian infectious bronchitis virus isolate IRFIBV32 (793/b serotype) in experimentally infected broiler chickens Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens
cord-285505-8norumv6	2014	The focus of John''s presentation was on the research conducted in his own and his collaborators'' laboratories that ultimately led to the invention of three compounds which were discovered to have antiviral activity against human cytomegalovirus (HCMV) and which later entered clinical trials: BDCRB pyranoside (GW275175X) (Phase I), maribavir (Phases I, II and III) and cyclopropavir (Phase I). In monotherapy studies after oral dosing with TDF (300 mg) and TAF (25 mg), the plasma TFV AUC is reduced from 1920 to 268 ng.h/ml respectively whereas the reduction in HIV load from baseline is improved, from log 10 0.97 to log 10 1.46 copies/ml, respectively, reflecting the more efficient delivery of TAF to target cells and tissues. David Margolis, University of North Carolina, NC, USA In HIV-infected patients, there is a long-lasting reservoir of HIV in the form of integrated viral DNA in resting CD4+ memory cells of the host immune system.
cord-285676-4kgy20o9	1997	The overlapping ORFs 1a and b found at the 58 end of the nidoviral genome are frequently referred to as the ''''polymerase gene.'''' However, there is little doubt that the processing of the encoded polyproteins yields proteins required for RNA synthesis as well as a number of products involved in other aspects of virus replication. The sequence conservation between the more closely related corona-and toroviruses is clustered in six domains, four of which are also found in the arterivirus POL1b: the ''''classical'''' RNA-dependent RNA polymerase (RdRp) and helicase (H) domains, which are also present in the polymerases of most other viruses, a zinc finger motif (zf), and a short region of 80-100 residues, which has not yet been identified in other viral polymerases and was called the ''''coronavirus-like'''' (CVL) domain (3) (motif 2 in Fig. 1b) .
cord-285785-29ohzeug	2020	Expression analysis and bioinformatical functional annotation through RNA sequence by using line 6 3 and line 7 2 showed that lncRNAs were aberrantly expressed and were involved in immune-related pathways that indicate that lncRNAs participate in regulating MDV infection [70] . Although the underlying functional mechanism of ncRNAs has been revealed in many species, it is still beginning to emerge in response to avian disease, except for miRNAs. Researches on circRNAs and lncRNAs are basically performed on the analysis of their expression profile and associated pathways. For lncRNAs and circRNAs, they mainly exhibit their function through lncRNA/circRNA-miRNA-mRNA axis, however, in some cases they could also regulate the immune process by directly interacting with RNA binding proteins or virus gene sequence. Gene expression profile and long non-coding RNA analysis, using RNA-Seq, in chicken embryonic fibroblast cells infected by avian leukosis virus
cord-285868-fz5utxss	2014	In addition to regulation of cell death, it is reported that HCV induces the expression of CHOP at mRNA and protein levels and is correlated with autophagy induction; knockdown of CHOP not only increases HCV PAMP-mediated innate immune activation, but also elevates its inhibitory effect on virus replication (Ke and Chen, 2011) . Overexpression of HCV E1 and/or E2 induces the expression of CHOP in a PERK-dependent manner (Chan and Egan, 2005) ; while upon HCV infection, CHOP protein is upregulated by PERK, activating transcription factor (ATF6), and inositol-requiring transmembrane kinase/endonuclease 1 (IRE1) collectively. It was reported that rotavirus NSP4 viroporin initiates autophagy to transport viral proteins to sites of virus replication for assembly of mature particles, which involves an increase of cytoplasmic calcium and subsequent activation of the CaMKK-β-AMPK pathway. Regulated IRE1-dependent decay pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication
cord-285935-5rsk6g7l	2019	Cyclic peptides (CP) that had been developed to abrogate interaction of p6Gag and TSG101 and inhibited viral release of HIV Virus like particles (VLPs) [76] were tested for their activity against HEV [77] . The compounds RBV and mycophenolic acid (MPA), both of which target enzymes involved in nucleotide synthesis, are either already used as treatment against HEV or have been reported for their potential to inhibit the virus. So far, the antiviral activity against HEV of only four drugs (Sofosbuvir, pegIFN-α, Ribavirin and silvestrol) was approved in experimental settings beyond in vitro cell culture systems. Sofosbuvir Inhibits Hepatitis E Virus Replication In Vitro and Results in an Additive Effect When Combined With Ribavirin Sofosbuvir shows antiviral activity in a patient with chronic hepatitis E virus infection Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase The natural compound silvestrol inhibits hepatitis E virus (HEV) replication in vitro and in vivo
cord-286103-cgky6ar6	2006	In this study, we adopted RNA interference (RNAi) strategy and examined whether small interfering RNAs (siRNAs) can be used to inhibit replication of MeV and SSPE virus. We report here that siRNAs targeted against L mRNA of MeV, either synthetic siRNAs or those generated by pcPUR + U6i-based expression plasmids, effectively and specifically inhibited replication of both MeV and SSPE virus without exhibiting any cytotoxic effect. We report here that siRNAs targeted against L mRNA of MeV, either synthetic ones or those generated by pcPUR + U6i-based expression plasmids, effectively inhibit replication of both MeV and SSPE virus. Indeed, we demonstrated in the present study that siRNAs targeted against selected portions of L mRNA of MeV, especially MV-L2 -L4 and -L5, either synthetic siRNAs or those expressed by pcPUR + U6i-based plasmids, effectively inhibited replication of both MeV and SSPE virus (Figs.
cord-286149-awhnjwyc	2017	In previous investigations of MUO in dogs, only brain samples were tested for infectious agents; however, CSF is a common sample utilized in the clinical evaluation of neurologic disease for the detection of infectious agent nucleic acids, especially by PCR. Additionally, RNA was extracted from postmortem brain samples from a mule deer (Odocoileus hemionus), green tree python (Morelia viridis), American crow (Corvus brachyrhynchos), and American robin (Turdus migratorius), all of which had previously been tested by PCR, metagenomic sequencing, or both, and were found to be infected with specific known infectious agents. There are several possible biological and technical explanations for our study''s inability to identify a candidate etiologic agent for MUO, including the underlying pathogenesis of the disease, sample type and collection methods, case inclusion criteria, sensitivity of diagnostics, and database limitations.
cord-286232-jo24ia4s	2009	Derm cells by infectious virus recovery assay after viral internalization, suggesting that EHV-1 enters E. In HeLa and receptor-expressing CHO cells, infectious entry of HSV requires trafficking of the virus to an acidic intracellular compartment, phosphatidylinositol 3-kinase activity, glycoprotein D (gD) receptors, as well as viral gB, gD, and gH-gL (Nicola et al., 2003; Nicola and Straus, 2004) . With the use of ultrastructural analysis, we have previously suggested that EHV-1 enters equine brain microvascular endothelial cells (EBMECs) via endocytosis (Hasebe et al., 2006) . Localization of EHV-1 and caveolae during viral internalization Caveolar endocytosis has emerged as a route of entry for several viruses including simian virus 40 (SV40) (Anderson et al., 1996) , mouse polyomavirus (Richterová et al., 2001; Gilbert et al., 2003; Gilbert and Benjamin, 2004) , echovirus (Marjomäki et al., 2002) , human papillomavirus type 31 (Bousarghin et al., 2003; Smith et al., 2007) , human polyomavirus BK (BKV) (Eash et al., 2004) , and species C human adenovirus (Colin et al., 2005) .
cord-286243-ddpemgqt	2018	One such factor is the multifunctional enzyme human apurinic/apyrimidinic endonuclease 1 (APE1), known best for its DNA backbone cleavage activity at AP sites during base excision repair (BER). Abbreviations: 1-nt, 1-nucleotide; 5OHU, 5-hydroxy-2′-deoxyuridine; 8-oxoG, 8-oxoGuanine; AP, apurinic/apyrimidinic; APE1, human apurinic/apyrimidinic endonuclease 1; APE2, apurinic/apyrimidinic endonuclease 2; BER, base excision repair; DHT, 5,6-dihydrothymidine; DHU, 5,6-dihydro-2′-deoxyuridine; dRP, deoxyribonucleotide-phosphate; IR, ionizing radiation; NIR, nucleotide incision repair; pBQ-C, benzetheno exocyclic adduct of cytosine; PG, phosphoglycolate; PNK, polynucleotide kinase; PUA, α,β-unsaturated aldehyde; ROS, reactive oxygen species; THF, tetrahydrofuran; Tdp1, tyrosyl-DNA phosphodiesterase 1; XRCC1, X-ray repair cross-complementing protein 1 ⁎ Corresponding author. The BER pathway requires the coordinated activity of at least five enzymes including: (1) a DNA glycosylase capable of excising the modified base; (2) an AP-endonuclease, such as APE1, to generate a nick at the lesion site; (3) DNA polymerase β, which performs both lyase and DNA synthesis activities to remove the 5′ dRP (deoxyribonucleotide-phosphate) and fill the resulting gap; and, finally (4) DNA ligase III and (5) XRCC1 (X-ray repair cross-complementing protein 1) scaffolding to seal the nick and complete the repair, (Fig. 1) .
cord-286298-pn9nwl64	2020	Another group of researchers reported that the virus originated from bats based on the genome sequence of SARS-CoV-2, which is 96% identical to bat coronavirus RaTG13. These factors include, but are not limited to: (1) travel to or contact with individuals who have recently visited Wuhan, China, or other places experiencing an outbreak; (2) close contact with persons who are diagnosed positive for the disease, such as healthcare workers caring for patients with SARS-CoV-2; (3) contact with droplets and secretions (produced by sneezing or coughing) from an infected person and eating or handling wild animals native to China such as bats. These factors include, but are not limited to: (1) travel to or contact with individuals who have recently visited Wuhan, China, or other places experiencing an outbreak; (2) close contact with persons who are diagnosed positive for the disease, such as healthcare workers caring for patients with SARS-CoV-2; (3) contact with droplets and secretions (produced by sneezing or coughing) from an infected person and eating or handling wild animals native to China such as bats.
cord-286332-cdg4im5h	2017	Herein, the genomic part coding for the spike glycoprotein ectodomain was replaced by that of the coronavirus mouse hepatitis virus (MHV), allowing for the selection and propagation of recombinant mIBV in murine cells. This problem was solved by transfecting IBV genomic RNA into otherwise non-susceptible cells, exchanging the IBV spike gene by that of the mouse hepatitis virus (MHV) provided as part of a synthetic RNA, and by subsequently rescuing recombinant IBV from infected/ transfected cells in embryonated eggs (Fig. 1) . b Stage 1 in targeted RNA recombination: an interspecies chimeric murinized IBV with a MHV spike ectodomain (mIBV) is generated by a single recombination event of IBV genomic RNA with synthetic RNA transcribed from donor plasmid p-mIBV in the 3′-end region of the 1b gene (indicated by a black curved line). Upon transfection of synthetic rIBV donor RNA into mIBV-infected murine LR7 cells, subsequent infectious IBV virus particles could be rescued in ECE.
cord-286343-s8n1ldol	2020	We were able to detect co-circulating virus variants, some specifically prevalent in England, and to identify changes in viral RNA sequences with time consistent with the recently reported increasing global dominance of Spike protein G614 pandemic variant. We conclude that viral RNA sequences found in sewage closely resemble those from clinical samples and that environmental surveillance can be used to monitor SARS-CoV-2 transmission, tracing virus variants and detecting virus importations. However, it was clear that there was a large reduction of SARS-CoV-2 RNA concentration in sewage between 14th April and 12th May. Positive and negative results were independently confirmed using a second real-time PCR platform (Stratagene 3000P) in a different NIBSC laboratory. However, it was clear that there was a large reduction of SARS-CoV-2 RNA concentration in sewage between 14th April and 12th May. Positive and negative results were independently confirmed using a second real-time PCR platform (Stratagene 3000P) in a different NIBSC laboratory (data not shown).
cord-286352-uftl1mx5	2003	Since the region downstream of the stem-loop increases frameshifting in the presence of this stem-loop, as shown in Figure 3 , these results also support our suggestion that the frameshift stimulatory signal in MVP5180 is more complex than a simple stemloop and could correspond to a pseudoknot structure. The frameshift efficiencies of the constructs containing these mutations were assessed in vitro and in cultured cells and are presented in Figure 4 (b) (see also Table 1 To provide an independent support for the pseudoknot structure in subtype MVP5180 of HIV-1 group O frameshift stimulatory signal, we made a structural probing analysis of an RNA fragment encompassing the gag/pol frameshift region of this subtype. Our results show that the frameshift stimulatory signal of subtype MVP5180 of HIV-1 group O is a pseudoknot, where 8 nt of a 10-nt loop capping an 8-bp stem base-pair with a downstream complementary sequence.
cord-286416-8eu6wp9b	2012	If deadenylation (e.g., CCR4/Not1), destabilization (e.g., TTP/XRN1) and decapping (e.g., DCP1/DCP2) complex; and even RISC (Ago) complex are recruited to mRNA, these will be targeted to PBs. Conversely, if TIA-1/TIAR or proteins such as G3BP/USP10 are recruited to the stalled initiation complexes, these will be directed to SGs. Different pathways in SG assembly are described (in red): (i) phosphorylation of eIF2␣ induced by the exposure to different stress inducers (e.g., arsenite and thapsigargin) (Fig. 1) ; (ii) Hippuristanol and Pateamine A, drugs that inhibit the helicase activity of eIF4A altering ATP binding or ATPase activity; and (iii) the overexpression of SG markers, such as G3BP or TIA-1. West Nile virus infections suppress early viral RNA synthesis and avoid inducing the cell stress granule response Interaction of TIA-1/TIAR with West Nile and dengue virus products in infected cells interferes with stress granule formation and processing body assembly
cord-286603-4p3t0vre	2020	title: TMT-based quantitative proteomics analysis reveals the attenuated replication mechanism of Newcastle disease virus caused by nuclear localization signal mutation in viral matrix protein Therefore, the reduced viral RNA synthesis and transcription caused by M/NLS mutation might be one of the reasons responsible for the attenuated replication of rSS1GFP-M/NLSm. Virus-host protein interactions based on quantitative proteomics analysis have become important methods in understanding cellular proteins involved in virus replication and pathogenesis [22, 23, 41, 42] . Therefore, together with the above results, we speculated that the relatively decreased expression of DEPs involved in ribosome structure, protein posttranslational modification and trafficking due to the disrupted nuclear accumulation of M protein affected viral protein synthesis and budding, which might be the third reason responsible for the attenuated replication of rSS1GFP-M/NLSm. Inflammatory responses are important aspects of the innate immune system during virus infection.
cord-286711-nr6vnl9h	2003	Some of these viruses discussed in this chapter are toroviruses, picobirnaviruses, enteroviruses, human immunodeficiency virus (HIV), herpesviruses, and coronaviruses. Cytomegalovirus (CMV) and herpes simplex viruses, members of the Herpesviridae family, are found as the cause of colitis and esophagitis, mainly in HIV-infected patients. Besides the viruses producing the majority of human viral gastroenteritis (Sections II-V), other viruses infect more rarely, but are sometimes able to cause epidemics. HIV, the causative agent of the Acquired Immunodeficiency Syndrome (AIDS), is a member of the Lentivirus genus of the Retroviridae family. HIV, the causative agent of the Acquired Immunodeficiency Syndrome (AIDS), is a member of the Lentivirus genus of the Retroviridae family. Cytomegalovirus (CMV) and herpes simplex viruses, members of the Herpesviridae family, are found as the cause of colitis and oesophagitis, mainly in HIV-infected patients (Levinson and Bennets, 1985; Jacobson and Mills, 1988; Dieterich and Robinson, 1991; Theise et al. Enteric viruses and diarrhoea in HIV-infected patients
cord-286719-1xjmlwqr	2018	The developed AuNP-based detection techniques are reported for various groups of clinically relevant viruses with a special focus on the applied types of bio-AuNP hybrid structures, virus detection targets, and assay modalities and formats. These techniques represent the majority of molecular techniques applied in virus detection and include various types of target amplification techniques (e.g., PCR, loop-mediated isothermal amplification (LAMP), transcription-mediated amplification, and nucleic acid sequence-based amplification), signal amplification techniques (e.g., branched DNA and hybrid capture), and probe amplification techniques (e.g., ligase chain reaction and strand-displacement amplification). [70] developed an impedimetric electrochemical assay for the detection of AIV M gene sequences based on measuring changes in the impedimetric behavior of the electrode when the target DNA hybridizes with the capture DNA probes immobilized onto its surface and is subsequently labeled by AuNPs via streptavidin/ biotin interaction (Fig. 12C) .
cord-286842-04cuk2cn	2005	title: Recombinant Tula hantavirus shows reduced fitness but is able to survive in the presence of a parental virus: analysis of consecutive passages in a cell culture Tula hantavirus carrying recombinant S RNA segment (recTULV) grew in a cell culture to the same titers as the original cell adapted variant but presented no real match to the parental virus. Using the two specific RT-PCR conditions, the presence of V-type and REC-type S RNA was monitored on ten sequential passages of the mixture of TULV02 and RecTULV5 variants (Fig. 2 ). Although relatively short, the observed survival time of the recTULV in the presence of the original variant TUL02 seems to be sufficient for transmission of a recombinant virus, in a hypothetical in vivo situation, from one rodent to another. The data presented in this paper show that the recTULV presents no real match to the original cell adapted variant and that the lower fitness of the recombinant virus can not be increased by pre-passaging in cell culture.
cord-286877-0h5vgi5c	2012	When the pAlpha-CPV-VP2 was injected intradermal as DNA-launched replicon-based DNA vaccine in dogs, it induced CPV-specific humoral and cell mediated immune responses. To increase antigen production and immunogenicity with DNA vaccines, a new strategy has been developed to express the target heterologous antigen under the control of replicon from positive-strand RNA viruses with the promise of using the ability of these viruses to produce large amounts of viral proteins in infected cells. The replicon-based CPV DNA vaccine plasmid (pAlpha-CPV-VP2) encoding CPV-VP2 was constructed and evaluated to express CPV-VP2 protein in transfected cells in SDS-PAGE and Western blot (data not shown). To assess the protective efficacy of different CPV vaccines, sera collected from all immunized and control dogs were analyzed for presence of virus neutralizing (VN) antibody. CD8 + lymphocytes in dogs immunized with replicon-based CPV DNA vaccine compared to controls (Fig. 7) . There were over 24 thousand times more CPV-VP2 mRNA transcripts in pAlpha-CPV-VP2-transfected BHK-21 cells compared to respective non-replicating control indicating the self-replicating ability of replicon-based CPV-VP2 DNA vaccine.
cord-287018-g4y5kjju	2006	In order to solve this durability problem, we tested DNA constructs encoding virus-specific long-hairpin RNAs (lhRNAs) for their ability to inhibit HIV-1 production. The lhRNA constructs were compared with in vitro diced double-stranded RNA and a DNA construct encoding an effective nef-specific shRNA for their ability to inhibit HIV-1 production in cells. For this reason, the DNA constructs encoding lhRNAs (a single-hairpin molecule) and long dsRNAs (two complementary molecules that form a duplex) were designed to target tat, rev and nef sequences as indicated in Figure 1 . 21 nef2 dsRNA induced a decrease in luciferase expression, but an even more pronounced level of inhibition was Figure 1 Scheme of the human immunodeficiency virus type 1 (HIV-1) pLAI proviral genome and target sequences used for the design of long-hairpin RNAs (lhRNAs).
cord-287093-9mertwj7	2011	In this review, we discuss how three supergroups of (+)RNA viruses generate replication sites from membrane-bound organelles and highlight research on perinuclear factories induced by the nucleocytoplasmic large DNA viruses. In this review, we discuss how three supergroups of (+)strand RNA viruses generate replication sites from membrane-bound organelles and highlight research on perinuclear factories induced by the nucleocytoplasmic large DNA viruses (NCLDV). The RNA-dependent RNA polymerases (RdRp) of the (+)strand RNA viruses are targeted to the cytoplasmic face of membrane-bound organelles and subsequent assembly of the replicase complex induces membrane curvature and the formation of densely packed membrane vesicles (reviewed in [1, 2] ) ( Figure 1 ). This suggests that replication may take place on CM and that genomes are transferred to vesicular packets for envelopment and budding, while excess viral RNA may be stored in DMVs. Picornaviruses generate densely packed DMVs between 200 and 400 nm in diameter, a series of single membraned vesicles resulting from fragmentation of the Golgi, and autophagosomes possibly generated as a bystander response to infection [11 ,12-16] .
cord-287153-jbuuph6w	2016	The most prevalent viral diseases in Norwegian salmon aquaculture are heart and skeletal muscle inflammation (HSMI) caused by Piscine orthoreovirus (PRV), and pancreas disease (PD) caused by Salmonid alphavirus (SAV). The SAV RNA level in heart was significantly lower in the co-infected fish at 4 and 6 WPC-SAV compared to the SAV3 controls (p < 0.05). Using a non-parametric rank test of the ordinal histopathological score, changes in pancreas were found to be significantly lower at 4 and 6 WPC-SAV in both early and late co-infection compared to the SAV control groups (p < 0.05), except at 4 WPC-SAV in the PRV-SAV3-late group (Figures 6 and 7) . Microarray analysis was performed on hearts sampled at 4 and 6 WPC-SAV from the late co-infection and differences between the SAV3 control and PRV-SAV3-late group were analyzed (Table 2) . SAV RNA levels in heart were significantly lower 6 WPC-SAV in the early co-infected groups compared to the SAV controls.
cord-287228-0qm939ve	2020	In one study including 70 patients with COVID-19, 21% clinically recovered patients with two consecutive negative results of nucleic acid detection experienced a later positive testing for SARS-CoV-2, and the longest duration of viral RNA positivity in this study was 45 days following infection [4] . A total of 2860 COVID-19 patients were hospitalized and followed in this hospital since the epidemic, and those with persistent or intermittent viral RNA positivity in respiratory samples (including the nasopharyngeal, oropharyngeal and sputum samples) for at least 4 weeks were included in our study, regardless of the age and their clinical status. However, in most of these studies, PCR testing was used as a marker to indicate the existence of virus, and patients with positive viral RNA was considered infectious though they have been infected for months without further clinical symptoms.
cord-287275-vwyny1vt	2018	In this study, a Chinese PDCoV strain, designated CHN-HG-2017, was isolated from feces of a suckling piglet with severe watery diarrhea on a farm located in central China. Tissues of small intestines from CHN-HG-2017-infected piglets at 4 DPI displayed significant macroscopic and microscopic lesions with clear viral antigen expression. In addition, it has been reported that newborn piglets inoculated with two recent Chinese PDCoV isolates, named CHN-HN-2014 and CHN-GD-2016, developed clear clinical signs, including severe diarrhea, vomiting, and dehydration [21, 22] . Analysis of the genomic sequence suggested that CHN-HG-2017 is a potential recombinant virus derived from Chinese and Vietnam PDCoV strains. To confirm that virus propagation had occurred, the infectivity of the plaque-purified CHN-HG-2017 strain in LLC-PK1 cells was tested by IFA and western blot with an mAb against the PDCoV N protein. Isolation, genomic characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014
cord-287324-ecpicv5v	2017	In this study, we detected the viromes of RNA viruses of one mock sample, one pooled duck feces sample and one pooled mink feces sample on the Personal Genome Machine platform using the sequencing libraries prepared by three methods. In this study, we detected the viromes of RNA viruses of one mock sample and two pooled authentic samples, using the libraries prepared by the three methods on the Personal Genome Machine (PGM) platform, with the aim to generate data of significance for virome detection of RNA viruses and characterize the viromes of RNA viruses in ducks and minks. In the future, it is of significance to compare methods 2 and 3 in detecting viromes of RNA viruses in some clinical samples containing limited viral RNA. Detection of viromes of ducks and minks increases our understanding of the viral diversity in the animals, and provides novel clues for further studies regarding diagnosis of infectious diseases, identification of novel viruses and research of host-virus relationships.
cord-287349-1zcq7kzx	2020	title: Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex Here we present cryo-electron microscopic structures of the SARS-CoV-2 holo-RdRp with an RNA template-product in complex with two molecules of the nsp13 helicase. The structure places the nucleic acid-binding ATPase domains of the helicase directly in front of the replicating-transcribing holo-RdRp, constraining models for nsp13 function. The analogous structural 234 arrangement leads us to propose that the SARS-CoV-2 RdRp may backtrack, generating a single-235 stranded RNA segment at the 3''-end that would extrude out the RdRp secondary channel 236 Table S1 ; Video S1). This aspect of helicase function could provide the NTP-296 dependent motor activity necessary to backtrack the RdRp. In cellular organisms, DdRp 297 backtracking plays important roles in many processes, including the control of pausing during 298 transcription elongation, termination, DNA repair, and fidelity (Nudler, 2012) . Structural Basis for RNA Replication by the SARS-CoV-2 Polymerase
cord-287372-ya5uvoki	2020	This study provides a detailed description of the pathogenesis of a low-passage SARS-CoV-2 isolate in two macaque models and suggests that both species represent an equally good model in research for both COVID-19 prophylactic and therapeutic treatments. Rhesus macaques have also been applied 116 in COVID-19 pathogenesis studies [22, 24, 32, 33] , and to test the efficacy of remdesivir in the 117 treatment of SARS-CoV-2 infection [34] . we compared SARS-CoV-2 replication in rhesus and cynomolgus macaque species and 129 monitored signs of COVID-19-like disease symptoms for three weeks after infection. The animals from this study were not 342 euthanized to be able to perform re-infection studies or to monitor them for late clinical signs, 343 or co-morbidities related to We conclude that the course of SARS-CoV-2 infection of both macaque species is highly 345 similar, indicating that they are equally suitable models to test vaccines and antivirals in a 346 preclinical setting for safety and efficacy.
cord-287396-18p171nr	2014	Other meta-analysis studies, examining the immune response to Salmonella typhimurium, combine microarray information with data such as serum cytokine measurements or microbiota differences. typhimurium will be discussed in the section ''''Overall value of transcriptomics in important infectious swine diseases.'''' In addition, whole genome microarrays were used to study pig response to Haemophilus parasuis infection by Zhao et al. In 2010, Xiao and collaborators performed a 3'' tag digital gene expression (DGE) analysis of the porcine lung transcriptome on pigs infected with the PRRS virus (Xiao et al. Most pig immune studies conducted to identify host response to common porcine pathogens or to immune response stimulators such as LPS or PMA/ionomycin described in this review provide gene expression data from a single tissue or isolated cell type, and this at a limited number of times post-infection/stimulation. Recently, such a meta-analysis was performed by combining results of several microarray-based pig immune studies to find PRRS-specific responses (Badaoui et al.
cord-287466-ag5y781z	2016	As evidenced by the presence of genomic-length and sgmRNA-length replicativeintermediate double-stranded (ds)RNAs in shrimp cells infected with gill-associated virus (GAV) (Cowley et al., 2002a) , the type species okavirus (Cowley et al., 2012) , it is speculated that transcription termination of the antisense RNAs might occur at precise positions, resulting in common 3′-termini, and that these then act directly as promoters for transcription initiation of the genomic and sgmRNAs. In all other nidoviruses, however, and for the longest of the sgmRNAs transcribed by toroviruses, the (−) and (+) strand sgmRNAs are transcribed using a far more complex discontinuous process involving the splicing of a common "anti-leader" sequence derived from the genome 5′-terminus to each (−) strand sgmRNA that then acts as a universal promoter for transcribing each (+) strand sgmRNA (Pasternak et al., 2006; Sawicki et al., 2007; Smits et al., 2005; van Vliet et al., 2002) . Nidoviruses of aquatic species include the rod-shaped okaviruses GAV and yellow head virus (YHV) that primarily infect Penaeid shrimp (Longyant et al., 2005; Flegel, 2012; Flegel et al., 1997a; Cowley et al., 2000a Cowley et al., , 2002a Cowley and Walker, 2002; Sittidilokratna et al., 2008) and a morphologically similar virus with a ~22 kb ssRNA genome detected in diseased Chinese mitten crabs (Zhang and Bonami, 2007) .
cord-287487-qeltdch7	2017	Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. High-or low-fidelity variants are described for many RNA viruses infecting animals, including the coronaviruses (CoVs) murine hepatitis virus (MHV-A59) and severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (13) (14) (15) (16) (17) , as well as foot-and-mouth disease virus (18) (19) (20) (21) (22) , poliovirus (23) (24) (25) (26) (27) (28) (29) , Chikungunya virus (30, 31) , influenza virus (32) , coxsackievirus B3 (33, 34) , and human enterovirus 71 (35) (36) (37) . The evolved mutations in MHV-ExoN(-) nsp14 and nsp12, which encodes the RdRp, accounted for only part of the increased nucleoside analogue resistance of MHV-ExoN(-) P250, implicating multiple replicase proteins in adaptation for viral fitness.
cord-287488-h102xn29	2020	BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. METHODS: After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS: The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks viable (titre 2.11 × 10(6) TCID50/mL, titre 1.5 × 10(6) PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad.
cord-287501-7it4kh0e	2012	We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV) nucleocapsid (N) protein. Among the polyphenolic compounds examined, (−)-catechin gallate and (−)-gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. 33 In this study, we report a novel approach for the inhibitor screening of SARS-CoV N protein using a quantum dots (QDs)-conjugated oligonucleotide system with wide applicability for facile and sensitive imaging analysis on a biochip. To the best of our knowledge, this is the first report on the inhibition effects of (-)-catechin gallate and (-)-gallocatechin gallate on SARS-CoV N protein using an optical nanoparticle-based RNA oligonucleotide platform. Among the polyphenolic compounds screened, (-)-catechin gallate and (-)-gallocatechin gallate showed high anti-SARS-CoV N protein activity. At a concentration of 0.05 µg mL -1 , (-)-catechin gallate and (-)-gallocatechin gallate showed more than 40% inhibition activity on a QDs-RNA oligonucleotide biochip platform.
cord-287658-c2lljdi7	2020	The discovered sequences are first validated on samples from other repositories, and proven able to separate SARS-CoV-2 from different virus strains with near-perfect accuracy. The discovered sequences are validated on samples from NCBI and GISAID, and proven able to separate SARS-CoV-2 from different virus strains with near-perfect accuracy. For example, we can use this sequencing data with cDNA, resulting from the PCR of the original viral RNA; e,g, Real-Time PCR amplicons to identify the SARS-CoV-2 16 . The global impact of SARS-CoV-2 prompted researchers to apply effective alignment-free methods to the classification of the virus: For example, in 26 the authors propose the use of Machine Learning Digital Signal Processing for separating the virus from similar strains, with remarkable accuracy. We calculated the frequency of appearance of different primer sets'' sequences used in SARS-CoV-2 RT-PCR tests developed by WHO referral laboratories and compared it to our primer design in the dataset from the GISAID ( Table 2) repository.
cord-287748-co9j3uig	2018	Several recent studies have reported that various bat species harbor bat hepatitis E viruses (BatHEV) belonging to the family Hepeviridae, which also contains human hepatitis E virus (HEV). Here, we collected and screened 81 bat fecal samples from nine bat species in Japan to detect BatHEV RNA by RT-PCR using HEV-specific primers, and detected three positive samples. These data support the first detection of BatHEVs in Japanese microbats, indicating their wide geographical distribution among multiple bat species. BLAST analysis indicated that BtHEV-Ej1/-Ej2 showed the highest sequence identities to BatHEV/BS7, a German strain detected from the Serotine bat (Eptesicus serotinus), among strains previously reported in other countries. The closely related BatHEVs (BtHEV-Ej1/-Ej2 and Bat HEV/BS7) have been detected in different species of Eptesicus bats (E. PCR amplifications were performed using the KOD FX Neo (Toyobo) with consensus HEV primer sets (PanHEV F and R), which were designed in this study to amplify a 191-bp fragment of the RNA-dependent into Orthohepevirus D, in Japanese bats, suggesting wide geographical distribution of BatHEV among multiple bat species.
cord-287758-da11ypiy	2020	The increase in studies related to SARS-CoV-2 during the first semester in 2020 has allowed the rather speedy identification of promising therapeutic targets for both developing immunotherapies and producing/identifying antiviral drugs. 5, 64 So far, structural proteins and enzymes that participate actively in the process of viral replication are the most investigated targets for the development of molecules for anti-CoVs therapies (FIG. Based on results from previous studies as well, nelfinavir was considered a likely therapy for COVID-19 after its indication for clinical trials as a promising anti-SARS drug. 218 In addition to this well-known antitumor effect, imatinib has also shown in-vitro antiviral properties against several virus, such as infectious bronchitis virus (a viral model for studying the role of tyrosine kinase activity during CoV infection), by interfering with virus-cell fusion, 219 and other RNA viruses including coxsackie virus, 220 hepatitis C virus, 221 Ebola, 222 among others, mainly by blocking viral entry or egress from the host cell.
cord-287931-cxqzac4a	2013	RESULTS: Using multiple probes designed to specifically detect a microbial genus or species, EOPM can correctly identify known pathogens at the species or genus level in blinded testing. To facilitate the application of EOPM in multiple surveillance sites for infectious diseases, we designed software with a user-friendly interface, which is supported by a statistical analysis method based on a comprehensive microbial sequence identification database. Analysis of the enrichment results at the genus level revealed Cardiovirus as the number one match, showing significant enrichment ( The microarray raw data of other symptom-causing pathogens, such as streptococcus and mycoplasma, identified by EOPM in peripheral blood in infectious patients, were also submitted to the GEO database. These microarray platforms use long oligonucleotide probes (60-70-mer) and random PCR amplification, and have successfully identified unexpected pathogens in infectious disease outbreaks, even discovering novel viruses with homology to known species [8, 11] .
cord-288093-012ipcdr	2018	Moreover, high-throughput studies have identified additional non-coding RNAs that are likely processed by Dicer [9] as well as several pre-miRNA binding proteins that may regulate its cleavage activity [10] [11] [12] . The structure and activity of the purified Dicer were assessed by size-exclusion chromatography coupled to multi-angle light-scattering and refractive index (SEC-MALS/RI), negative stain transmission electron microscopy (TEM), binding assay and steady-state kinetics. Our kinetic analysis for Dicer cleavage of the pre-let-7a-1 substrate were performed under strict steady state conditions and reveals k cat and K M values that are much higher than previously reported. Moreover, SEC -MALS/RI analysis of a purified Dicer sample stored for 6 months at − 80°C in sucrose/DDM-containing buffer shows that the purified protein remains almost exclusively monomeric ( ≥ 94%), indicating that the protein is intact after long-term storage (Additional file 1: Figure S1 ).
cord-288167-976qxja2	2018	title: Replicative virus shedding in the respiratory tract of patients with Middle East respiratory syndrome coronavirus infection BACKGROUND: Information on the duration of replicative Middle East respiratory syndrome coronavirus (MERS-CoV) shedding is important for infection control. This study examined the duration for detecting MERS-CoV sub-genomic mRNA compared with genomic RNA in diverse respiratory specimens. In the present study, replicative MERS-CoV was detected in sputum or transtracheal aspirate for up to 4 weeks after symptom development in MERS-CoV-infected patients with severe pneumonia. In conclusion, replicative MERS-CoV was detected in lower respiratory tract specimens for up to 4 weeks after symptom development, which was well correlated with the detection of genomic RNA. In upper respiratory tract specimens, the detection of sub-genomic mRNA and genomic RNA did not correlate. Middle East respiratory syndrome coronavirus (MERS-CoV) genomic RNA (upE) titers in sputum and transtracheal aspirates with vs.
cord-288390-p1q3v1ie	2015	These sensors induce expression of cytokines, affect cellular functions required for virus replication and directly target viral nucleic acids through degradation or sequestration. These sensors induce expression of cytokines, affect cellular functions required for virus replication and directly target viral nucleic acids through degradation or sequestration. In this review we concentrate on intracellular nucleic acid sensors and effector proteins that evolved to mediate specialised tasks including, firstly, expression of cytokines such as type I interferons (IFN-a/b); secondly, modulation of cellular machineries required for virus replication and thirdly, direct inhibition of virus growth ( Figure 1 ). Among the best characterised cytoplasmic proteins involved in virus sensing are RIG-I-like receptors (RLRs), a family of DExD/H-box helicases which specifically identify viral RNAs and have the ability to stimulate expression of IFN-a/b and other cytokines (Figure 3 ) [4, 17] . Virus infection activates a restricted set of sensor and effector proteins that modulate cellular pathways and directly target viral nucleic acid, thereby shaping the innate immune response.
cord-288651-bgo8istm	2005	RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Specific inhibition of cellular mRNA by RNAi can be triggered in mammalian cells by the introduction of synthetic 21-to 23-nucleotide duplexes of RNA N genes of SARS-CoV and evaluated their effects on viral genes expression in Vero E6 cells. The results show that all siRNA duplexes specifically reduced SARS-CoV genes expression to different extents compared with the control (Fig. 1) . Kinetic study results (Fig. 2B ) revealed a continuous increase in the specific inhibition of SARS-CoV genes expression by No. 5, No. 6, and No. 16 siRNA from 24 to 72 h after transfection.
cord-288669-46tkedw7	2006	P129-ΔE-transfected cells however produced virion particles in the culture supernatant, and these particles contained viral genomic RNA, demonstrating that the E protein is essential for PRRSV infection but dispensable for virion assembly. Our study suggests that the PRRSV E protein may function as a viroporin in the virion envelope that facilitates uncoating of the virus in order to release the genomic RNA into the cytoplasm for subsequent replication. At 24 h postinoculation, Marc-145 cells were fixed, and virus infection was examined by immunofluorescent staining with N-specific MAb. Marc-145 cells inoculated with a culture fluid from cells cotransfected with P129-ΔE and E gene showed bright N-specific fluorescent signal, indicating that the P129-ΔE replication may be rescued by trans-complementation of the E protein (Fig. 2C ). Strand-specific RT-PCR experiments were carried out and demonstrated that PRRSV-infected Marc-145 cells produced reduced levels of positive-sense genomic RNA at 2 days post-infection in the presence of the drugs chloroquine, amantadine and verapamil (Fig. 5A, upper panel) .
cord-288701-nx9fg4yn	2015	The aim of the present study was to develop a multiplex real-time RT-PCR assay, based on the TaqMan technology, for the rapid and unambiguous characterisation of all bovine pestiviruses, including the emerging HoBi-like strains. Analysis of field samples tested positive for BVDV-1, BVDV-2 or HoBi-like virus by a nested PCR protocol revealed that the developed TaqMan assay had equal or higher sensitivity and was able to discriminate correctly the viral species in all tested samples, whereas a real-time RT-PCR assay previously developed for HoBi-like pestivirus detection showed cross-reactivity with few high-titre BVDV-2 samples. To overcome these limitations, we have developed a multiplex real-time RT-PCR assay for simultaneous detection of the different species of bovine pestiviruses, including the emerging HoBi-like group, allowing a rapid, sensitive and specific diagnosis of pestivirus infection and characterisation of the viral species.
cord-288879-rj03dsib	2018	While the mechanisms for the function and toxicity of extended polyQ segments (or the nucleic regions that encode them) in eukaryotic proteins continue to be actively studied [16] , there has been little exploration of their occurrence and possible roles, even in neurovirulent viruses. At the start of this work, the ViPR database [29] , which allows rapid access to the published sequences of over 75,000 viral genomes or genome segments, was used to determine which RNA and DNA viruses contain polyQ repeats. As discussed below, the longest repeats were found in DNA virus proteins that function in enhancing transmissibility (cowpox ATI) or contribute to viral latency (herpes viruses). Under growth conditions allowing the virus to resume lytic growth, where the enzyme activity is required to ensure efficient replication, the region Fig. 2 Soluble polyQ segments (of cell or viral origin) may prevent beclin-1-induced autophagy, which depends on the DNA binding ability of the polyQ segment of wt-ataxin-3 (based on [2, 67] ).
cord-288960-v6l6o5va	2017	Possibly via RIG-I -dependent RNA detection which may in turn induce STING [6] Vesicular stomatitis virus Negative strand ssRNA virus Unknown [6] Human immunodeficiency virus Negative strand ssRNA virus dsDNA reverse transcribed from viral RNA induces cGAS-STING pathway [28] Influenza A virus Negative strand ssRNA virus Possibly via membrane fusion or unknown mechanism independent of DNA recognition [29] Mycobacteria tuberculosis Bacteria producing c-di-GMP c-di-GMP [30, 31] Streptococcus pneumoniae Bacteria dsDNA Bacterial dsDNA [32] Streptococcus pyrogenes Bacteria dsDNA Bacterial dsDNA [33] Staphylococcus aureus Bacteria producing c-di-AMP c-di-AMP [34] Listeria monocytogenes Bacteria producing c-di-AMP c-di-AMP [35] Vibrio cholera Bacteria producing 3′-3′ cGAMP 3′-3′ cGAMP [36, 37] Abbreviations: dsDNA double-stranded DNA, ssRNA single-stranded RNA The type I interferon signal adaptor protein STING is responsible for mediating double-stranded DNA sensing responses and the detection of bacterial cyclic dinucleotides c-di-AMP, c-di-GMP, and 3′-3′ cGAMP.
cord-288962-jgtoehcr	2000	To define the role of enteroviruses and human rhinoviruses as etiological agents in childhood bronchiolitis, clinical aspirates from 84 infants admitted to hospital with symptoms of obstructive bronchiolitis were tested by picornavirus RT‐PCR assay, adenovirus PCR assay and classical immunofluorescence antigen detection of common respiratory viral agents. In summary, combination of molecular and classical detection assays of common viruses can be used to demonstrate enterovirus and human rhinovirus respiratory infection in childhood bronchiolitis, and provides an improved approach to obtain new insights into concomitant viral respiratory tract infection in infants. To investigate the role of enteroviruses and human rhinoviruses as etiological agent in bronchiolitis, 84 nasopharyngeal aspirates were tested by picornavirus RT-PCR assay, classical immunofluorescence antigens detection of respiratory syncytial viruses, influenza viruses, parainfluenza viruses, coronaviruses, and adenovirus PCR assay.
cord-289038-15yp9uqy	2020	The purpose of this study was to identify microRNA with predicted binding sites in the SARS-CoV-2 genome, compare these to their microRNA expression profiles in lung epithelial tissue and make inference towards possible roles for microRNA in mitigating coronavirus infection. Another recent study used a high-throughput reporter screen of miRNA from human and mouse respiratory epithelial cells to identify hsa-miR-127-3p, hsa-miR-486-5p, and hsa-miR-593-5p as contributors to the antiviral defence against influenza A virus by targeting the genomes of the H3N2 and attenuated PR8 (H1N1) viral strains [16] . Given the wealth of evidence supporting a role for miRNA in host cell antiviral defence mechanisms, we sought to identify human miRNA that have the potential to target the SARS-CoV-2 genome. DEA of Calu3 cells infected with SARS-CoV revealed that only hsa-miR-155-3p (upregulated) and hsa-let-7a-3p (downregulated) out of the 128 miRNA we identified in this study, were differentially expressed ( Figure 4B ).
cord-289093-si8btsab	2014	To explore these interactions a functional high throughput small interfering RNA (siRNA) screen targeting 6719 druggable cellular genes was undertaken to identify host factors (HF) influencing the replication and spread of an eGFP-tagged VACV. Multiple components of the AMPK complex were found to act as pro-viral HFs, while several septins, a group of highly conserved GTP binding proteins with a role in sequestering intracellular bacteria, were identified as strong anti-viral VACV HFs. This screen has identified novel and previously unexplored roles for cellular factors in poxvirus replication. The methodology in the previously published VACV screens varied considerably; Mercer et al [32] measured the growth of a thymidine-kinase-deficient VACV (strain Western Reserve) after only 8 h of infection, thereby identifying cellular proteins involved in the initial stages of virus replication but excluding analysis of viral spread.
cord-289192-1ecr16a3	2019	title: Development of a homogeneous time-resolved fluorescence assay for detection of viral double-stranded RNA Here, we describe a simple, rapid, cost-effective, high-throughput method using a homogeneous time-resolved fluorescence (HTRF) assay [2] focusing on viral dsRNA. The dsRNA-HTRF assay may be a useful method for screening of antiviral agents against (+) ssRNA viruses. Moreover, we determined whether the dsRNA-HTRF assay could detect the concentration-and time-dependent signals. Therefore, to evaluate whether the dsRNA-HTRF assay can detect (+) ssRNA viruses, we focused on infection by picornaviruses represented by HRV. Schematic diagram of the double-stranded RNA (dsRNA) -homogeneous time-resolved fluorescence (HTRF)-based assay. In addition, we examined whether the dsRNA-HTRF assay could detect a single cycle of viral replication. H1-HeLa cells were infected with HRV-B14 at MOI of 10 to achieve single-cycle growth, and viral dsRNA was detected at 6 h post-infection using the dsRNA-HTRF assay ( Supplementary Fig. 2) , indicating that this assay could evaluate the single-cycle growth of HRV-B14.
cord-289248-6mx4o0eb	2018	These two mutants grew to high titer in Vero cells, were genetically stable, and were significantly more attenuated in vitro and in vivo compared to the parental rMV-Hu191 vaccine strain. We next compared the replication kinetics of the rMV-Hu191 mutants and the parental virus in Vero cells in the time course of 120 h after infection (Fig. 5 ). These data suggest that rMVs carrying mutations in the SAM binding site were more attenuated in Vero cells than the parental MV vaccine strain. In this study, we successfully generated two recombinant measles viruses with amino acid substitutions in the SAM binding site of L protein and examined the effects of these mutations on viral replication, safety, and immunogenicity. We generated two recombinant MV-Hu191 carrying mutations in the SAM binding site, which not only grew to high titer in Vero cells and were genetically stable but also were significantly more attenuated and immunogenic compared to the currently used Chinese MV vaccine strain.
cord-289321-ahl46ql9	2018	Differential visualization of drug-resistant and -susceptible RNA genomes within cells revealed that resistant variants of NS3/4A protease and NS5A phosphoprotein are cis-dominant, ensuring their direct selection from complex environments. Our goal was to screen the HCV-encoded viral proteins that are current targets of antiviral compounds to determine the intracellular dominance relationships between drug-resistant and drug-susceptible genomes. To test whether susceptibility to NS5A inhibitors was dominant in the context of viral infections, we analyzed U, S, S + R and R cell populations by flow cytometry as previously performed for the NS3/4A inhibitor in Figure 3 . To test whether exogenously expressed drug-susceptible NS5A proteins could co-assemble with drug-resistant NS5A, we utilized the previously described HCV plasmid that expresses HA-tagged and GFP-tagged NS5A within the same polyprotein but does not support genome replication ( Figure 5A) . Failure of NS5A proteins to mix during infection is a likely explanation for the cis-dominance of drug resistance observed in cultured cells (Figure 4) .
cord-289407-8fje16z1	2020	Understanding how Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is spread within the hospital setting is essential if staff are to be adequately protected, effective infection control measures are to be implemented and nosocomial transmission is to be prevented. 6 Air samples taken during tracheostomy procedures, high flow nasal oxygen treatment, non-invasive ventilation and nebulisation have not contained SARS-CoV-2 RNA 7 and HCWs exposed to unrecognised COVID-19 patients undergoing similar high-risk AGPs have not become infected. . https://doi.org/10.1101/2020.09.24.20191411 doi: medRxiv preprint Several studies, utilising a range of air and surface sampling methods, have been carried out to determine the presence and prevalence of SARS-CoV-2 in the healthcare environment. [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] The detection of viral RNA in air samples differs with study with some reporting widespread airborne contamination 14, 18, 21 but many reporting low or non-detectable concentrations 13, 15, 16, 19 even in samples collected 10 cm from the face of positive patients. Detection of air and surface contamination by SARS-CoV-2 in hospital rooms of infected patients
cord-289535-srrfr1es	2020	One concern with vaccine development for SARS-CoV-2 is that the immune response can cause disease, often in the act of clearing the infection. Preclinical animal studies have demonstrated that DNA vaccines encoding the M, N, 3a or S proteins of the SARS-CoV-1 virus could elicit immune responses [180] [181] [182] . The S protein is the target of the only SARS-CoV-1 DNA vaccine to progress to Phase I clinical trial, delivered by bio-injector, and it was safe and induced neutralizing antibody responses [183] . T cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice Targets of T cell responses to SARS-CoV-2 coronavirus in humans with COVID-19 disease and unexposed individuals A SARS DNA vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a Phase I clinical trial
cord-289612-4x5t4c5u	2020	Laboratory-confirmed SARS-CoV-2 infection requires the detection of viral nucleic acid in respiratory tract samples by the use of real-time reverse-transcription polymerase chain reaction (rRT-PCR) assay. In the course of this phase, upper respiratory specimens were tested by RT-PCR for viral RNA and the majority of the patients showed positive results for SARS-CoV-2. These results contrast with another German smaller study by Wolfel et al., conducted on 9 COVID-19 patients, with no discernible difference in viral loads or detection rates when comparing nasal and throat swabs [38] . found that 66.67% of laboratory-confirmed COVID-19 patients were tested positive for SARS-CoV-2 RNA in stool specimens. enrolled a total of 173 confirmed cases of COVID-19 by the use of rRT-PCR on samples from the respiratory track reported that the seroconversion sequentially appeared for the total antibody (Ab), IgM and then IgG, with a median time of 11, 12 and 14 days, respectively. Correlation of chest CT and RT-PCR testing in coronavirus disease 2019 (COVID-19) in China: a report of 1014 cases
cord-289926-y1rjgbui	2014	Using sequence analysis methods we have discovered significant sequence similarity between this central domain of HDAg and the HMG (High Mobility Group) box of the SRY gene [40] , whose product binds to DNA and is essential for sex determination. The sequence similarity between the central domain of HDAg and the HMG (High Mobility Group) box of the SRY gene suggests that SRY may be a cellular cognate of HDAg. Another candidate for the ''captured'' cellular RNA was recently proposed: a 202 residue cellular protein referred to as DIPA (delta interacting protein A) was identified by co-immunoprecipitation with HDAg and by its in vivo ability to inhibit viral replication [5] . Statistically significant sequence similarities are limited to the functionally essential regions of both genes: in the SRY gene the similarity is confined to the HMG (High Mobility Group) box, a DNA binding motif found in the HMG protein superfamily [24, 31] , in HDAg the similarity is limited to RNA-binding domain.
cord-289965-qcezqpze	2015	The 39-terminal domain of the most conserved ORF1b in three of the four families of the order Nidovirales (except for the family Arteriviridae) encodes a (putative) 29-O-methyltransferase (29-O-MTase), known as non structural protein (nsp) 16 in the family Coronaviridae and implicated in methylation of the 59 cap structure of nidoviral mRNAs. As with coronavirus transcripts, arterivirus mRNAs are assumed to possess a 59 cap although no candidate MTases have been identified thus far. The 39-terminal domain of the most conserved ORF1b in three of the four families of the order Nidovirales (except for the family Arteriviridae) encodes a (putative) 29-O-methyltransferase (29-O-MTase), known as non structural protein (nsp) 16 in the family Coronaviridae and implicated in methylation of the 59 cap structure of nidoviral mRNAs. As with coronavirus transcripts, arterivirus mRNAs are assumed to possess a 59 cap although no candidate MTases have been identified thus far.
cord-290218-dvyeg5fk	2020	Interestingly, the structure of complexed nsp12 is almost identical to nsp12 in apo RdRp, with an RMSD of 0.5 Å [17] , coinciding with the high processivity of the viral RNA polymerase, which does not need to consume extra energy for conformation changes in the active site during the replication cycle (Fig. 3B ). These "sliding poles" are stabilized by interactions formed between the positively charged residues at the extended N-terminal of nsp8 and bases in RNA backbones ( Fig. 3E ) and reported to account for the known processivity of the RdRp, which is required for replicating the long coronavirus genomes [39] . Although the sequence identity of nsp12 across the RNA viruses is low, the polymerase active site is structurally highly conserved, suggesting that RdRp inhibitors may serve as a potential J o u r n a l P r e -p r o o f broad-spectrum antiviral drug against RNA viruses.
cord-290254-m9l8ntur	2020	In this work, we report the development of a rapid PoC diagnostic test (< 20 min) based on reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) and semiconductor technology for the detection of SARS-CoV-2 from extracted RNA samples. For validating the incorporation of the RT-LAMP assay onto our PoC platform (RT-eLAMP), a subset of samples was tested (n=40), showing average detection times of 12.89 {+/-} 2.59 min for positive samples (n=34), demonstrating a comparable performance to a benchtop commercial instrument. Currently, reverse transcriptase polymerase chain reaction using real-time benchtop platform (RT-qPCR) is considered the gold standard for COVID-19 diagnosis due to its capability to detect the presence of SARS-CoV-2 RNA close to the onset of symptomatic illness which is critical for isolation. In this paper, we combined LAMP with an in-house LoC device to develop a rapid PoC diagnostic test (< 20 min) for the detection of SARS-CoV-2 RNA from extracted samples.
cord-290472-w77cmljm	2010	These markers, such as protein (including autoantibodies, which are antibodies specific to self-antigens [43] ), hormonal markers (such as lack of insulin in Type I diabetic patients [89] ), and genetic/genomic markers (such as BRCA1 mutation in breast cancer patients [52] ), enable clinicians to diagnose the disease while it is still at early stages, to ensure appropriate surgical intervention, efficient drug treat-ment and monitoring, and to predict an individual''s risk of developing specific diseases before they experience symptoms. Scientists, such as the group led by Gil Mor at Yale University, recruited proteomics-based approaches using antibody-based protein microarrays to identify new serum biomarkers, which, in combination with CA-125, may enhance the early detection of ovarian cancer [48, 66, 110] . To date, no studies that attempt to identify novel breast cancer markers have been performed using high-density protein microarrays.
cord-290481-i2ppvsh5	2006	It was concluded that, at least in part, viral pathogenicity is due to interference of silencing suppressors with developmental function of plant small RNAs. Despite their mechanistic similarity, p21 and p19 appear to be structurally and evolutionarily unrelated and neither has detectable homologues outside the respective virus genera (Vargason et al., 2003; Ye and Patel, 2005) . Although Citrus tristeza virus (CTV) encodes p20, a p21like suppressor of RNA silencing, screening of the CTV genome revealed an additional suppressor, p23, that has no homologues in other closteroviruses (Fig. 2) (Lu et al., 2004) . A comparison of TMV and BYV, which both evolved from the alphavirus-like ancestors, shows that the large part of the ∼9 kb genomic surplus of BYV is dedicated to facilitating the synthesis of the virion RNA and multiple sgRNAs. The rest of the surplus was invested in the formation of the complex virion tail that empowers virus transport within and transmission between the host plants and in suppression of RNA silencing (Fig. 1) .
cord-290550-u8x9drva	2012	Following on from electron microscopy, cell culture and PCR, next-generation sequencing is one of these methodologies that is now changing the way that we understand viruses, particularly in the areas of genome sequencing, evolution, ecology, discovery and transcriptomics. In addition, improved methods for analysis will become available, opening yet further applications in virology including routine diagnostic work on individuals, and new understanding of the interaction between viral and host transcriptomes. Although this review has concentrated on the use of NGS to study viral sequences directly, it is clear that these approaches provide new opportunities to explore the interaction of replicating viruses with their host, particularly their transcriptome, in a non-targeted, hypothesis-generating mode. Viral transcriptome analysis of feline immunodeficiency virus infected cells using second generation sequencing technology
cord-290744-m0vpizuh	2016	Here, we will summarize the insights gathered so far on an important aspect of virulence and host adaptation, the interactions of SARS-CoV and MERS-CoV with antiviral interferon (IFN) responses of human cells. The broad antiviral activity of IFNs occurs on several levels, namely virus entry, viral polymerase function, host cell translation, RNA availability, RNA stability, particle budding, apoptosis, or general boosting of innate and adaptive immune responses. Crystal structure of the middle east respiratory syndrome coronavirus (MERS-CoV) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells Middle East respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response
cord-290801-dv6aak01	2012	Since the core protein of flaviviruses is also endowed with potent RNA chaperone activities, we decided to examine the effect of West Nile virus (WNV) core on 5′–3′ genomic RNA annealing in vitro. These results indicate that core protein – besides its function in viral particle formation – might be involved in the regulation of flavivirus genomic RNA cyclization, and thus virus replication. In this study, we examined the effect of WNV core protein chaperoning on viral 5 -3 UTR annealing, using an in vitro model system with separate 5 and 3 RNAs. We found that core protein binding greatly increases the rate of 5 -3 complex formation, and is required for the interaction when full-length 3 UTR RNAs are used (Fig. 2) .
cord-290802-761wqgbe	2020	title: Structural Insights into the Binding Modes of Viral RNA-Dependent RNA Polymerases Using a Function-Site Interaction Fingerprint Method for RNA Virus Drug Discovery To this end, we describe structural binding-site insights for facilitating COVID-19 drug design when targeting RNA-dependent RNA polymerase (RDRP), a common conserved component of RNA viruses. In summary, the binding characteristics determined here help rationalize RDRP-targeted drug discovery and provide insights into the specific binding mechanisms important for containing the SARS-CoV-2 virus. In sum, structurally, SARS-CoV-2 has high global/ core structural similarity to the RDRP catalytic domains of all other RNA viruses, which provides an opportunity for structure-based COVID-19 drug design and repurposing, noting that keys differences lie in the subtle details. According to the similarity of functionsite interaction fingerprints over all complexes, it was possible to divide the binding modes into four classes, where each class contains multiple PDB structures from different kinds of viruses (Table 1) .
cord-290813-6ylwj5je	2006	To date, based on the publicly released full genomic sequences of SARS-CoV, various molecular detection methods based on reverse-transcription polymerase chain reaction (RT-PCR) have been developed. Subsequently, together with the improvement of viral RNA extraction in which plasma or serum requires no ultracentrifugation, two real-time quantitative RT-PCR assays, one aimed toward the polymerase region and the other toward the nucleocapsid region of the virus genome ( Fig. 1) , were developed for measuring the concentration of SARS-CoV RNA in serum/plasma samples from SARS patients (13, 14) . With the use of the real-time quantitative RT-PCR assay, SARS-CoV RNA has recently been shown to be detectable in the plasma samples of pediatric patients during different stages of SARS (Fig. 4) (14) . Quantitative analysis and prognostic implication of SARS coronavirus RNA in the plasma and serum of patients with severe acute respiratory syndrome
cord-290948-cuu78cvl	2008	Using the combination of yeast two-hybrid screening and GST pull-down assays, we have now analyzed all potential interactions between SARS-Coronavirus nonstructural proteins, which may contribute to the structure and/or function of the viral replication/transcription complex. SARS-CoV nsp3 is a large multidomain protein of 1922 amino acids Thiel et al., 2003) that is thought to contain at least seven domains: (1) an N-terminal Glu-rich acidic domain (AD); (2) an X domain (XD) with poly(ADP-ribose) binding properties Saikatendu et al., 2005) ; (3) the SUD domain (for SARS-CoV Unique Domain, an insertion not found in any other coronavirus thus far) with a specific affinity for oligo(G)-strings (Tan et al., in press); (4) a papain-like protease (PLP2), recently shown to exhibit deubiquitinating activity (Barretto et al., 2005; Harcourt et al., 2004; Lindner et al., 2005; Ratia et al., 2006) ; (5) an unknown domain possibly extending the papain-like protease domain, termed PLnc for Papain-Like noncanonical (see below); (6) a transmembrane domain (Kanjanahaluethai et al., 2007) corresponding to the N-terminal of the Y domain; and (7) the remainder of the Y domain, the abbreviation "Y domain" will be used for this part in this study.
cord-291026-99cit4ig	2015	In this study, we describe validation of a new probe‐based insulated isothermal reverse transcriptase PCR (iiRT‐PCR) assay for rapid detection of classical swine fever virus (CSFV) on a compact, user‐friendly device (POCKIT (™) Nucleic Acid Analyzer) that does not need data interpretation by the user. Archived viral nucleic acid from 18 laboratory-amplified non-CSF viruses including eight other pestiviruses (BVDV 1-Hastings, Singer and NY1 strains; BVDV 2-Ames 125c, 890, 24515 strains; BDV-Coos Bay; HoBi atypical pestivirus), African swine fever virus-Lisbon, swine vesicular disease virus-ITL 19/92, porcine respiratory and reproductive syndrome virus-YNL, swine influenza virus (H3N2), porcine circovirus 1 (PCV1, derived from infectious clone based on GenBank accession no. The iiRT-PCR assay accurately detected all CSFV RNA samples which represented all three genotypes, and eight of 11 subgenotypes that were available for testing and gave negative results for three BVDV type 1 strains, three BVDV type 2 strains, BDV, HoBi atypical pestivirus, ASFV and nine other viruses that affect livestock.
cord-291029-oldket3n	2005	The QIAamp MinElute Virus kit (Qiagen Inc., Valencia, CA) was compared to the two existing methods currently used in our laboratory, IsoQuick (Orca Research Inc., Bothell, WA) for DNA extraction and RNAzol B (Leedo Laboratories Inc., Houston, TX) for RNA extraction, of viral nucleic acids. In this study, we have chosen both nasopharyngeal swab (NPS) and cerebrospinal fluid (CSF) specimens submitted for influenza A virus, enterovirus and herpesvirus testing to validate the MinElute nucleic acid extraction system. Nucleic acid recovery rates between the MinElute and RNAzol B were further determined by quantitating HIV-1 or CMV viral loads in extracted RNA or DNA specimens using a real-time TaqMan PCR. The MinElute kits possessed an equivalent sensitivity in nucleic acid recovery and detection, in comparison to the IsoQuick and RNAzol B, for both DNA and RNA extraction.
cord-291063-de7v4e5s	2009	The expressions of EBV-encoded miRNAs in clinical samples and computational analysis to predict putative targets were applied to unravel the biological functions of EBV miRNAs. These approaches showed that the miR-BARTs are abundantly expressed in latently infected epithelial cells, nasopharyngeal carcinomas, EBV-associated gastric carcinoma cell lines and tissues, Burkitt''s lymphomas latency type I, EBV positive primary effusion lymphomas, and diffuse large B-cell lymphomas, but at a significantly lower level in B cells. However, computational alignment of the potential HIV-1 miRNAs with specific human T-cell mRNAs identified potential cellular targets including genes encoding CD4, CD28 and interleukin-2, IL-3, and IL-12 [119] . The idea of targeting viral transcripts is not new, and RNA interference has been demonstrated to efficiently mediate inhibition of replication of human pathogenic viruses such as HIV-1, HCV, dengue virus, severe acute respiratory syndrome (SARS) coronavirus, poliovirus, human rhinovirus, influenza A virus, hepatitis D virus, HBV, HSV-1, HPV, JCV, EBV, and CMV in cell culture (reviewed in [12] ).
cord-291225-75ys908n	2018	title: A Transgenic Flock House Virus Replicon Reveals an RNAi Independent Antiviral Mechanism Acting in Drosophila Follicular Somatic Cells Here we describe transgenic Drosophila melanogaster lines encoding a Flock House Virus-derived replicon (FHV∆B2eGFP), expressing GFP as a reporter of viral replication. This replicon is efficiently controlled by the siRNA pathway in most somatic tissues, with GFP fluorescence providing a reliable marker for the activity of antiviral RNAi. Interestingly, in follicular somatic cells (FSC) of ovaries, this replicon is still partially repressed in an siRNA independent manner. We did not detect replicon derived Piwi-interacting RNAs in FSCs and identified 31 differentially expressed genes between restrictive and permissive FSCs. Altogether, our results uncovered a yet unidentified RNAi-independent mechanism controlling FHV replication in FSCs of ovaries and validate the FHV∆B2eGFP replicon as a tool to screen for novel tissue specific antiviral mechanisms.
cord-291513-vpehn6nx	2020	Conclusions: Compared to using a clinical-grade synthetic swab, detection of SARS-CoV-2 from environmental samples collected from ICU rooms of patients with COVID was similar using consumer grade swabs, stored in 95% ethanol. Here we characterize the suitability of detecting SARS-CoV-2 RNA in experimental conditions as well as COVID-19 patient and built-environment samples using viral-inactivating storage solutions and alternative medical-grade and consumer-grade swabs. In a subset of seven COVID-19 patient nares samples stored in 95% EtOH, we also detected signi cantly higher SARS-CoV-2 viral load in RNA extracted from the swab head versus eluent ( Fig. 1b ; one-tailed paired Student''s t-test p = 0.03). Based on the results from these initial experiments, we conducted a proof-of-concept study in the clinical setting by performing RT-qPCR for the SARS-CoV-2 N1 amplicon and human RNase P gene on RNA extracted from the swab head of nasal samples collected using TMI and/or CGp swabs alongside the recommended SYN swabs.
cord-291590-24psoaer	2020	In line with such a role, ExoN-knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously found to have a crippled but viable hypermutation phenotype. Remarkably, using an identical reverse genetics approach, an extensive mutagenesis study revealed the corresponding ExoN-knockout mutants of another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV), to be non-viable. Our study thus reveals an additional function for MERS-CoV nsp14 ExoN, which apparently is critical for primary viral RNA synthesis, thus differentiating it from the proofreading activity thought to boost long-term replication fidelity in MHV and SARS-CoV. Strikingly, we now established that the equivalent knockout mutants of MERS-CoV ExoN are non-viable and completely deficient in RNA synthesis, thus revealing an additional and more critical function of ExoN in coronavirus replication.
cord-291677-zcbyhsf1	2020	To investigate the 2′-O methyltransferase activity of SARS-CoV-2 Nsp10/16, we applied fixed-target serial synchrotron crystallography (SSX) which allows for physiological temperature data collection from thousands of crystals, significantly reducing the x-ray dose while maintaining a biologically relevant temperature. We conducted serial synchrotron crystallography (SSX) experiments at 297 K to test whether low radiation dose could help uncover the structure of Nsp10/16 in a complex with Cap-1. The SARS-CoV-2 Nsp10/16 2′-O MTase complex provides a molecular arrangement for binding of the mRNA Cap-0 and subsequent methylation of the first transcribed nucleotide. The further development of SSX and implementation of time-resolved SSX crystallography is an approach that could visualize chemical processes and protein molecular dynamics -such as of the transfer of the methyl group catalyzed by Nsp10/16 2′O-MTase from SARS-CoV-2. Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2′-o-methyltransferase nsp10/nsp16 complex
cord-291727-4wfhuvww	2012	There are two main mechanisms that produce out of frame peptides: changes in the genome sequence that result in insertions or deletions (indels) and programmed ribosomal frameshifting as a consequence of the ribosome either slipping back one nucleotide (−1 frameshifting) or skipping one nucleotide (+1 frameshifting) (Figure 1) . By contrast, programmed ribosomal frameshifting can result in dual-coding genes that produce alternative functional proteins, which form an integral part of the organism''s physiology. Although the slippery sequence and pseudoknot are the most common motifs for frameshifting identified thus far, there are alternative mechanisms that may result in the production of out-of-frame proteins. First, one has to identify in mRNAs the requirements for a productive frameshift peptide: One might argue that a slippery sequence and a pseudoknot are a good predictor of frameshifting events, but-as pointed out above-there are alternative mechanisms that result in frameshifting (see also Figure 2 ). searched the genome for slippery sequences and pseudoknot FIGURE 3 | Methods to detect frameshifting events and out-of-frame peptides.
cord-291749-revhbd0q	2019	Diagnosis can be upheld by observing the cause of disease under the microscope or detecting the presence of nucleic acid and proteins of the pathogens. Sequencing of PCR amplicons or whole pathogen genomic DNA can be applied to comprehensively screen for infecting agents and their drug resistance phenotype, if exists, at the same time. Sequencing ensures detection of DNA composition unique to an organism, the presence of antimicrobials-destroying genes, or mutation that can change the function or conformation of proteins related to drug resistance. Targeted sequencing with MinION is powerful and fast to detect pathogens in clinical samples. A novel diagnostic method for malaria using loopmediated isothermal amplification (LAMP) and MinION TM nanopore sequencer Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis MinION nanopore sequencing of multiple displacement amplified Mycobacteria DNA direct from sputum.
cord-291754-1zxztadu	2019	In this study, a pathogenic avian infectious bronchitis virus (IBV) QX-type strain YN was successfully rescued by vaccinia virus based reverse genetic technology. To compare the in vitro replication of the rescued virus rYN and its parental strain YN on CEK cells, 200 μl PBS containing 10 3.0 TCID 50 of rYN or YN virus were inoculated onto the CEK cells in 24-well plates, and 200 μl supernatants from three wells from each group were harvested at the time points of 6, 12, 24, 36, 48, and 60 hpi for a real-time PCR detection assay for IBV N gene as described above. Collectively, these results demonstrate the successful rescue of the pathogenic IBV strain YN from cloned cDNA by using electroporation of full-length IBV in vitro transcripts into N-protein expressing cells and subsequent virus amplification in the allantoic cavities of ECE.
cord-291765-97lk5qfo	2006	To test the requirements for nsp1 and nsp14 in replication and to probe their functions, deletions or mutations were engineered into the viral genome in nsp1 and nsp14 and mutant viruses were analyzed for virus viability, replication, protein expression, and RNA synthesis. When infectious genome RNA containing these changes was electroporated into permissive cells, only the carboxy-terminal nsp1 deletion allowed recovery of an infectious mutant virus (nsp1 124-242). Based on the above results, systematic mutagenesis of clustered-charged residues was performed, both within the putative essential amino-terminal two-thirds of nsp1 as well as the dispensable carboxy-terminal third of the nsp1 protein domain. Deletions of P1-Gln residues in the flanking cleavage sites between nsp13-14 (VUSS6) and nsp14-15 (VUSS17) were engineered in the infectious clone cDNA and used to generate full-length genome RNA for electroporation into permissive cells.
cord-291860-dw1sfzqx	2019	Herein, were studied the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. Herein, were studied the performance of an in-house mNGS protocol for routine diagnostics of viral respiratory infections with potential for automated pan-pathogen detection. Clinical sensitivity was analyzed using the optimized procedure, which in short consisted of total nucleic acid extraction, including internal controls (1:100 dilution); the adapted New England Biolabs Next library preparation protocol, including fragmentation with zinc, for combined RNA and DNA detection (see Library Preparation); and sequencing of 10 million reads (Illumina NextSeq 500). The Centrifuge default settings, with NCBI''s nucleotide database and assignment of sequence reads to a maximum of five labels per sequence, resulted in various spurious classifications ( Figure 4) [eg, Lassa virus ( Figure 5 ), evidently highly unlikely to be present in patient samples from the Netherlands with respiratory complaints].
cord-291916-5yqc3zcx	2020	
cord-291962-rp172ugk	2019	title: Nucleotide-binding oligomerization domain-like receptor X1 restricts porcine reproductive and respiratory syndrome virus-2 replication by interacting with viral Nsp9 To test this, increasing dose of NLRX1 was transfected into Marc-145 cells followed by PRRSV infection, qPCR was then performed to test the total viral RNA levels. On the other hand, recent literature indicated that the interaction of Nsp9 with SUMO E2 conjugating enzyme Ubc9 and cellular protein interleukin-2 enhancer binding factor 2 (ILF2) through its RdRp domain resulted in a significantly decrease of virus titers, indicating that cells utilize host antiviral factors as defense mechanisms to limit PRRSV infection Wen et al., 2017) . An intracellularly expressed Nsp9-specific nanobody in MARC-145 cells inhibits porcine reproductive and respiratory syndrome virus replication Porcine reproductive and respiratory syndrome virus nucleocapsid protein interacts with Nsp9 and cellular DHX9 to regulate viral RNA synthesis The DEADbox RNA helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral Nsp9 in vitro
cord-291965-9r9ll83m	2009	Studies conducted in China in the aftermath of the SARS epidemic have identified CoVs in bats (Chiroptera) and implicated this speciose mammalian order as the most likely reservoir of all known coronaviruses (3) (4) (5) (6) (7) . Bayesian phylogenetic inference with different substitution models and parallel analysis using Metropolis coupling now placed the virus reliably next to a common ancestor with the 2b group of CoV (SARS-like viruses, Figure 3 ). These fragments could be combined into contig*MRCA, most recent common ancestor; CI, confidence interval; HPD, high population density; SARS, severe acute respiratory syndrome; hCoV, human coronavirus; GTR + + I, general time reversible gamma-shaped rate distribution across sites and an invariant site assumption. One of our Hipposideros CoVs was in a basal phylogenetic relationship with the SARS-like clade (group 2b); their most recent common ancestors date back to ≈400 bc.
cord-292045-pnid9dmq	2020	While infectivity of SARS-CoV-2 through the excreted viral genetic material in the aquatic environment is still being debated, the presence and detection of genes in wastewater systems makes a strong case for the environmental surveillance of the COVID-19 pandemic. Consistency between abundance of SARS-CoV-2 genetic materials and number of confirmed cases was observed in the previous reports in Australia, France, Italy, Spain and Japan Further, referring to the limitations of the present study owing to lockdown scenario, we recommend that although based MPC analysis, the efficiency of RNA extraction and RT-PCR is considered high for all the wastewater samples collected for this study, the efficiency of PEG method could have been better established. The first proof of the capability of wastewater surveillance for COVID-19 in India through the detection of the genetic material of SARS-CoV-2
cord-292112-dejrksum	2020	This study provides a lncRNA catalog for future research concerning the mechanism of the transdifferentiation of cBMSCs into IPCs. Long noncoding RNAs (LncRNAs) are the rest of the protein-coding transcripts >200 nt in length that lack coding potential [1] . The expression levels were verified by qRT-PCR, and the trends were consistent with the RNA-Seq data; however, the expression level of the lncRNAs was much lower than that of the protein-coding transcripts (Figure 8 ). The top 20 signaling pathways enriched by the colocalized genes of differentially expressed lncRNAs from pairwise comparison "I vs. The top 20 signaling pathways enriched by the colocalized genes of differentially expressed lncRNAs from pairwise comparison "I vs. The top 20 signaling pathways enriched by the colocalized genes of differentially expressed lncRNAs from pairwise comparison "I vs. The top 20 signaling pathways enriched by the colocalized genes of differentially expressed lncRNAs from pairwise comparison "I vs.
cord-292347-d7xq7x5g	2020	375 While RT-PCR-based viral RNA detection has been widely 376 used in diagnosis of COVID-19, it cannot be used to monitor 377 the progress of the disease stages and cannot be applied to 378 broad identification of past infection and immunity. 46,47 410 The determination of SARS-CoV-2 exposure relies largely 411 on the detection of either IgM or IgG antibodies that are 412 specific for various viral antigens including, but not exclusively, 413 the spike glycoprotein (S1 and S2 subunits, receptor-binding 414 domain) and nucleocapsid protein. While RT-PCR has been 571 the dominant technique for detection of viral RNA, other 572 nucleic acid assays including isothermal amplification assays, 573 hybridization microarray assays, amplicon-based metagenomics 574 sequencing, and the cutting-edge CRISPR-related technologies 575 are also under development or have resulted in approved 576 tests.
cord-292353-z86rjwle	2011	Hantaviruses pose a serious threat to human health because their infection causes two highly fatal diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The sequences at both the 3 0 and 5 0 termini of each RNA segment are complementary forming ''''panhandle'''' structures that are specifically recognized by the N protein and were shown to be important for viral transcription and replication. Further studies revealed that cellular 5 0capped mRNA oligoribonucleotides are rescued by N in virus-infected cells and stored in P-bodies for the later use as primers by the viral RdRp during transcription initiation . The UTRs are encapsidated by nucleocapsid protein and associate with RdRp both in the host cells and in the virion, and only these nucleocapsids are believed to be functional templates for mRNA synthesis and RNA replication by the viral RdRp. c.
cord-292416-3hhi4wps	2020	Five years later, in 2020, when the World Health Organization declared the coronavirus disease 2019 (COVID-19)-caused by the newly emerging SARS-CoV-2 virus-to be a pandemic, this talk was widely acknowledged to be almost prophetic. 24, 25 All four reportedly mild pathogenic coronaviruses are associated with 10%-30% of cases of the common cold, 26 -28 yet they have the potential to cause severe lower respiratory tract infection in infants, in the elderly, and in patients with other underlying illness, 29 while hCoV-OC43, like SARS-CoV-2, has been associated with neurologic dysfunction as well. Development of animal models for SARS-CoV-2 infection is vital in providing comprehensive understanding of the pathogenic mechanisms involved but may also serve for screening anti-viral drugs and vaccines. Accordingly, transfusion of convalescent plasma is likely to be beneficial to SARS-CoV-2, 45 ,46 yet its effect on virus shedding and disease outcome must be evaluated when given to healthy individuals and patients at different stages and severity of the disease.
cord-292643-n6xp5mlz	2013	The relative abundance of a virus (or viral nucleic acid) in a sample, compared to that of other organisms such as bacteria or host cells (or their genomes), is a critical factor for the discovery of viruses when using metagenomics. A study on human liver tissue compared enrichment techniques of freeze-thaw, centrifugation and nuclease-treatment for the detection of Hepatitis C Virus using both Roche 454 and Illumina high-throughput sequencing platforms (Daly et al., 2011) . After an initial 10 min reverse transcription step at 45 • C and 10 min denaturation Table 1 Virus enrichment process prior to sequencing in metagenomic studies on human and animal samples. This artificial sample represents a starting point to evaluate simple and rapid viral enrichment methods for use in virus metagenomics studies that seek to detect a virus that is causing disease in humans or animals.
cord-292673-00s3wgem	2020	In the quest of an effective antiviral drug, the most specific target for an RNA virus is the RNA-dependent RNA-polymerase (RdRp) which shows significant differences between positive-sense and negative-sense RNA viruses. Journal of Translational Medicine *Correspondence: l.buonaguro@istitutotumori.na.it 1 Innovative Immunological Models, Istituto Nazionale per lo Studio e la Cura dei Tumori, "Fondazione Pascale"-IRCCS, Via Mariano Semmola, 52, 80131 Naples, Italy Full list of author information is available at the end of the article SARS-CoV-2 is a positive-sense RNA virus belonging to the Orthocoronavirinae (coronavirus, CoV) family and, in particular, to the genus beta (group 2) together with the other two new human coronaviruses SARS-CoV and MERS-CoV. However, all three of them have been developed for negative-sense RNA viruses which show a significant difference in the RdRp sequence and structure compared to the positive-sense SARS-CoV-2 RNA virus. In conclusion, as for all RNA viruses, the RdRp of the newly identified positive-sense human SARS-CoV-2 RNA virus represents the most optimal target for an antiviral drug.
cord-292751-tk1oggi9	2020	Novel coronavirus SARS-CoV-2, designated as COVID-19 by the World Health Organization (WHO) on the February 11, 2020, is one of the highly pathogenic β‐coronaviruses which infects human. The previously reported viral zoonotic pathogens include SARS-CoV (severe acute respiratory syndrome coronavirus) and MERS (Middle East respiratory syndrome coronavirus) [3, 4] , that can cause severe respiratory disease in human [5, 6] . SARS-CoV-2, a novel coronavirus (which causes COVID19) , has fast spread like a pandemic since its outbreak in Wuhan, China, in December 2019 [7] . Nowadays, Griffithsin, as an inhibitor of SARS and MERS spike, Remdesivir, favipiravir and ribavirin (nucleoside analogues), lopinavir/ritonavir (protease enzyme inhibitors) [61] , oseltamivir (neuraminidase inhibitors), anti-inflammatory drugs and EK1 peptide [62] , the clinical potential to be applied against the 2019-nCoV infection [67, 68] . Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan
cord-292831-oihcay6w	2013	The purpose of this study was to develop a one step mRT-PCR that could detect respiratory viruses including influenza A viruses, H1N1pdm09, seasonal H1N1, H3N2, influenza B viruses, respiratory syncytial virus (RSV) A and B, human metapneumovirus (HMPV), parainfluenza viruses (PIV) 1, 2, 3, 4, rhinovirus, enterovirus, corona viruses OC43, 229E, NL63 and HKU1 in three sets in human clinical samples and to compare it with rRT-PCR. The specificity of the multiplex PCR assay was evaluated by cross reaction tests with known viral isolates and different panels of sequence confirmed known clinical respiratory samples as reference material. Specificity of the mRT-PCR assay was evaluated by cross reaction tests against known respiratory virus isolates/positive samples and a WHO QA/QC panel for influenza viruses showed no cross reactivity amongst different viruses. For validation of set 1 of the multiplex assay, 114 respiratory clinical specimens previously positive for influenza viruses by rRT-PCR were used and results were 100% concordant.
cord-292983-msuluuuu	2020	In particular, self-amplifying RNA (saRNA) derived from alphavirus expression vectors has shown to be very efficient to induce humoral and cellular responses against many antigens in preclinical models, being superior to non-replicating mRNA and DNA. In particular, self-amplifying RNA (saRNA) derived from alphavirus expression vectors has shown to be very efficient to induce humoral and cellular responses against many antigens in preclinical models, being superior to non-replicating mRNA and DNA. This type of LNP-delivered saRNA vaccines, named SAM (for self-amplifying mRNA) platform, have shown great potential to generate immune responses against influenza virus [20] [21] [22] and Toxoplasma gondii [23] . Despite the fact that the ta-RNA system was able to induce good immune responses in vivo against influenza virus HA, it did not outperform vaccination with a single saRNA molecule expressing the same antigen.
cord-293038-pjjvfdnq	2008	We propose that this new structure, unique among enveloped viruses, assembles in association with the most stable component of Golgi stacks, the actin‐containing matrix scaffold, connecting viral replication and morphogenesis inside viral factories. Modified membranes harbouring viral replication complexes (RCs) frequently integrate into a complex structure known as the ''viral factory'' where the cytoskeleton participates, cell organelles are recruited and the different steps of the virus life cycle are sequentially connected. We propose that this new multifunctional structure associates with the actin-containing matrix of the Golgi stacks providing a stable scaffold for viral replication and early morphogenesis. LtA and CyD had very little or no effect, respectively, on viral replication and assembly as determined by measurement of infectious viral particles released to the culture supernatants at 6 and 10 h p.i. Complete tubes, virus budding profiles and viral particles assembled in Golgi stacks of cells treated with LtA, as observed in thin sections of treated cells studied by EM (not shown).
cord-293163-udcw1mx5	2005	The functional validation of angiogenic factors for their specific role has been greatly facilitated by the use of RNAi inhibitors, revealing a network involving the early activation of the VEGF pathway and interactions among MMPs and adhesion molecules, leading to the regulation of signal transduction pathways. MMP9 and MMP2 are important for the mobilization of sequestered VEGF and initiation of tumor angiogenesis [17] , whereas specific integrins mediate interactions between endothelial cells and the BM by activating the integrin receptor signaling that controls many key functions, such as proliferation [18] . The involvement, revealed by siRNA, of diacylglycerol kinase a (Dgka) in hepatocyte growth factor (HGF)-stimulated cell migration, which impaired angiogenesis in vitro, indicated its essential role in both proliferative and migratory response to VEGF, and suggested that it is a novel therapeutic target for controlling angiogenesis [43] .
cord-293215-6flf5ig0	2007	title: Generation of Recombinant Coronaviruses Using Vaccinia Virus as the Cloning Vector and Stable Cell Lines Containing Coronaviral Replicon RNAs Based on cloned coronaviral cDNA, we describe the generation of recombinant coronaviruses and stable cell lines containing coronaviral replicon RNAs. Initially, the vaccinia virus-based reverse genetic system was established for the generation of recombinant human coronavirus 229E. This section describes the preparation of purified vaccinia virus DNA that can be used: (i) for the in vitro ligation with the assembled coronavirus full-length cDNA (see Section 3.1.4), and (ii) as template for in vitro transcription reactions (see Section 3.3.1). First, a fulllength coronavirus RNA is produced using the genomic DNA of a vaccinia virus containing the full-length coronavirus cDNA insert as a template for in vitro transcription. 1. Based on a full-length coronavirus cDNA cloned in vaccinia virus, a replicon RNA-encoding cDNA can be generated using vaccinia virus-mediated homologous recombination as described in Section 3.2.
cord-293355-0v71xwqy	2016	Thus, the pattern of small RNAs generated in infected cells can be used as a molecular footprint to identify and characterize viruses independent on sequence homology searches against known references. Different RNAi mechanisms can generate vsRNAs during viral infection including the mammalian miRNA pathway, Drosophila small interfering RNA (siRNA) pathway, and mosquito piRNA pathway. 22, 23 In animals, there are at least three separate RNAi mechanisms that can generate vsRNAs during viral infection: the microRNA (miRNA), piwi-interacting RNA (piRNA), and small interfering RNA (siRNA) pathways ( Figure 1 ). Interestingly, EVEs seemed to favor the generation of small RNAs with molecular characteristics of piRNAs rather than siRNAs. Second, some viruses have developed viral suppressors of the siRNA pathway (VSRs) that can significantly affect the pattern of vsRNAs. VSRs allow for accumulation of viral RNA that can be degraded by other host mechanisms.
cord-293375-qcy56ui7	1992	coma-, nepo-, and potyviruses; coronaviruses; and flaviviruses (and their proposed relatives pestiviruses and hepatitis C virus.) Originally these domains were predicted to have proteolytic activity based on the presence of certain conserved amino acid residues and on the basis of protein-modeling studies (Bazan and Fletterick, 1989; Boege et a/., 1981; Gorbalenya et al., 1989; Hahn eta/., 1985) . Furthermore, we present data to show that none of the asparagine residues in the proteinase domain of Sindbis nsP2 that are conserved among alphaviruses are absolutely required for proteolytic activity, but that Trp-559, adjacent to His-558, is essential for function. We also examined the effect of changing Cys-525, one of the two remaining conserved cysteine residues in the C-terminal half of nsP2, to serine or arginine, as well as changing Ser-535, which is found in a domain of limited similarity to the active site serine of serine proteinases, to threonine.
cord-293417-oqusfhei	2010	The higher resolution provides more detailed structural information than previous reports, showing that the NTD structure from MHV shares a similar overall and topology structure with that of SARS-CoV and IBV, but varies in its potential surface, which indicates a possible difference in RNA-binding module. To gain insight into the precise mechanism of N protein, several crystallographic or NMR structural results were reported, including MHV N-terminal RNA binding domain (residues 60-195) (Grossoehme et al., 2009) , two proteaseresistant domains of the N protein from SARS-CoV (Huang et al., 2004; Luo et al., 2006; Yu et al., 2006; Chen et al., 2007; Saikatendu et al., 2007; Takeda et al., 2008) , and IBV (Beaudette strain and Gray strain) (Fan et al., 2005; Jayaram et al., 2006) . In overall ribbon posture, the high resolution structure of MHV-NTD determined using two forms of crystals with different packing modes is similar to previously reported SARS-CoV and IBV structures, with a remarkable difference in surface electrostatic distribution.
cord-293481-bmfj50fb	2020	Remdesivir or GS-5734 is a prodrug of a nucleoside analog with direct antiviral activity against several single-stranded RNA viruses, including SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). Recently, preliminary data from a randomized placebo-controlled clinical trial showed that remdesivir reduces the time to recovery in patients with COVID-19 (5) , leading to an emergency-use authorization (EUA) by the U.S. Food and Drug Administration (FDA) only 2 days after the first press release from the National Institute of Allergy and Infectious Diseases (NIAID) (6) . There were strong arguments for the antiviral effect of remdesivir against coronaviruses emerging from multiple cell-based in vitro models, including primary human airway epithelial (HAE) cell cultures (25) , and, for MERS-CoV, from a mouse model of pulmonary infection (28) . After the outbreak of SARS-CoV-2 in January 2020, remdesivir was rapidly tested in a Vero E6 cell-based model that made use of direct viral quantification by rtPCR along with the antimalaria and immune-modulating drug chloroquine and known antivirals such as ribavirin and penciclovir.
cord-293525-c7nwygl1	2019	An EriCoV-specific BRYT-Green(®) real-time reverse transcription PCR assay was used to test 351 samples of faeces or distal large intestinal tract contents collected from casualty or dead hedgehogs from a wide area across GB. Characterisation of these Erinaceus coronavirus (EriCoV) nucleotide sequences revealed high nucleotide identity to MERS-CoV [3] , the cause of an acute respiratory syndrome in humans with high case fatality rates [5, 6] . Many animal species seem to have the capacity for coronavirus infection in the absence of apparent disease, including bats [15] , aquatic birds [16] and rabbits when inoculated with MERS-CoV [17] . Whole genome sequencing was performed on RNA extracted from one faecal sample collected in 2014 (R618/14) which was identified as EriCoV-positive by real-time RT-PCR. The highest proportion of EriCoV-positive hedgehog samples were submitted from the South of England (34/217, 16%); however, BLR showed no significant association (P = 0.678) between EriCoV infection status and wider region when other factors including age and year were included.
cord-293646-d4qcckh1	2003	The first small molecule inhibitor of Hepatitis C Virus (HCV), the NS3 protease inhibitor BILN-2061, entered phase 2 clinical trials, producing a striking reduction in viral load in treated individuals. Inhibitors of Hepatitis B Virus (HBV) -Adefovir dipivoxil iHepsera''") (1) was approved in the US for the treatment of HBV on September 20'', 2002 and in the European Union on March 1 lth, 2003, providing a second small molecule antiviral to add to lamivudine (3TC) and the injectable protein IFNa as the only approved agents for treating HBV infection. Pyridinedicarboxamide S represents the first report of a non-nucleoside inhibitor of HBV reverse transcriptase enantiomer of q is active in cell culture and appears to prevent proper formation of the viral nucleocapsrd. A significant advance towards establishing a correlation between replicon inhibition and clinical efficacy was recently accomplished with the disclosure of preliminary clinical data for BILN-2061, a selective inhibitor of the NS3 serine protease of HCV that is structurally related to 13.
cord-293651-96cmduez	2006	We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. Molecular assays use the reverse transcriptase-polymerase chain reaction (RT-PCR) to detect viral RNA directly from a clinical sample or from virus isolated in a laboratory host system. Clinical samples consisting of 229 tracheal swabs from commercial chickens experiencing upper-respiratory disease, submitted as routine diagnostic cases to the Delaware (Georgetown, DE) and Maryland (Salisbury, MD) State Diagnostic Laboratories were tested by the real-time RT-PCR assay, as well as, by virus isolation at those laboratories (Gelb and Jackwood, 1998) . In this report we present the development and evaluation of a real-time Taqman ® -based RT-PCR assay for the detection and quantification of IBV genomic RNA directly from tracheal swabs.
cord-293747-ds8rhbkv	2015	Three compounds: silymarin, quercetin and kaempferol were evaluated for their in vitro antiviral activities against CHIKV using a CHIKV replicon cell line and clinical isolate of CHIKV of Central/East African genotype. A cytopathic effect inhibition assay was used to determine their activities on CHIKV viral replication and quantitative reverse transcription PCR was used to calculate virus yield. Different non-cytotoxic concentrations of silymarin, kaempferol and quercetin were tested on CHIKV-infected Vero cells to find the effective compound. To confirm the post-entry antiviral activity of silymarin against CHIKV a virus yield assay using qRT-PCR was used. In contrast, dose-dependent reduction of amounts of nsP1, nsP3 and E2 proteins was observed (Fig. 6) indicating that silymarin limited CHIKV replication and virus-encoded protein synthesis within the treated cells. However, as in virus expression and replicon cell lines the synthesis of viral RNAs and proteins are coupled further study is necessary to evaluate the direct effect of silymarin on inhibition of newly synthesized CHIKV proteins.
cord-293766-vpfda3pd	2020	authors: Ji, Jingjing; Zhang, Jinxia; Shao, Ziyun; Xie, Qifeng; Zhong, Li; Liu, Zhifeng Patients were diagnosed as mild type, general type, severe type, and critical type according to the Chinese Recommendations for Diagnosis and Treatment of Novel Coronavirus (SARS-CoV-2) Infection (Trial 7th version) [4] . The current multicenter cohort study demonstrates that GC therapy does not change viral clearance and peripheral lymphocyte counts in COVID-19 patients. Low-dose corticosteroid therapy does not delay viral clearance in patients with COVID-19 Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients Jinxia Zhang, Ziyun Shao, Qifeng Xie, and Li Zhong were responsible for collecting the data. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.Ethics approval and consent to participate
cord-293790-7hyelm88	2010	title: Autophagy protein ATG5 interacts transiently with the hepatitis C virus RNA polymerase (NS5B) early during infection To identify novel cellular factors that may play an essential role in HCV RNA replication, we have previously screened a human liver cDNA library for proteins interacting with the HCV NS5B RNAdependent RNA polymerase (RdRp). Here we report that ATG5, a protein required for the formation of DMV in embryonic stem cell (Mizushima et al., 2001) , specifically interacts with HCV NS5B. As a control, a panel of proteins (RAR-β, RAR-α, HCV core, and nonstructural protein: NS3 prot , NS3 hel , NS4A, and NS4B) cloned in the GAL4 DNA binding domain was tested for interaction with ATG5. A. Soluble yeast extracts containing NS5BΔ21 (N-terminal c-myc tag) and ATG5 (N-terminal HA tag) were incubated with different monoclonal antibodies and the immunoprecipitates were pulled down using protein A/G beads.
cord-293852-r72c6584	2020	Different studies found that the values of cardiac Troponins were increased in COVID-19 patients with more severe disease [4, 5, [68] [69] [70] , indicating an association of SARS-CoV-2 with myocardial damage. Moreover, the single-cell RNA-sequencing (scRNAseq) approach has been used to profile the SARS-CoV-2 host-response in the PBMCs of COVID-19 patients, and to comprehensively characterize the immunological changes [124] [125] [126] [127] [128] [129] [130] . However, SARS-CoV-2 infection of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) induced cytotoxic effects and RNA-seq findings highlighted significant transcriptional changes in gene pathways related to cellular metabolism and immune response [131] [132] [133] . This analysis also revealed several host-derived lncRNAs differentially expressed in COVID-19 patient-derived lung tissue, and in SARS-CoV-2 infected epithelial cells, including MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and NEAT1 (nuclear-enriched autosomal transcript 1) [151] (Fig. 5) .
cord-293913-frkb8iso	1996	Abstract The RNA polymerase gene of murine coronavirus MHV-JHM encodes a polyprotein of greater than 750 kDa. This polyprotein is proposed to be processed by two papain-like cysteine proteinases, PCP-1 and PCP-2, and a poliovirus 3C-like proteinase domain, 3C-pro, to generate protein products. To investigate which viral proteinase may be responsible for generating p72 and p65, we expressed the 5′-region of the MHV-JHM RNA polymerase gene including the two papain-like cysteine proteinase domains in an in vitro transcription/translation system and analyzed the translation products for proteolytic processing. To determine how p72 and p65 are generated from the polymerase polyprotein, we expressed cDNA clones encoding the 5''-end of ORF l a, including the 2 papain-like cysteine proteinase domains, and analyzed the translation reactions for the presence of proteolytic products.
cord-293988-f5gvwjyh	2020	The pandemic respiratory disease COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan in December 2019 and then spread throughout the world; Italy was the most affected European country. In this study, a domestic cat with clear clinical signs of pneumonia, confirmed by Rx imaging, was found to be infected by SARS-CoV-2 using quantitative RT–qPCR from a nasal swab. The World Health Organization (WHO) declared COVID-19 disease, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as a worldwide pandemic [1] . As the cat''s pathology evolved rapidly and harmfully (the animal died in as little as three days), with clinical signs and rate of disease progression similar to human COVID-19 patients, and because previously published papers reported different cases of feline infection [10, [13] [14] [15] [16] , a nasal swab was collected in order to verify a possible infection with SARS-CoV-2.
cord-294056-7e477y1x	1992	Previously, we have identified a transcription initiation site (for mRNA 2-1), which is more efficiently transcribed by viruses containing two copies of UCUAA sequence in the leader RNA than by those with three copies. To understand the mechanism of MHV mRNA transcription, we have attempted to determine whether the differential regulation of transcription initiation by leader RNA containing different UCUAA copy numbers is unique to mRNA 2-l. The analysis of several other recombinant viruses with different genome structures suggested that mRNA 3-l was synthesized only by viruses with a leader RNA containing three copies of UCUAA and gene 3 sequence derived from A59 but not JHM (10, 13). Using various primers representing sequences of different regions of gene 1 of the JHM strain of MHV (2) and a second primer representing the 5''-end of the leader RNA, we detected several PCR products which represented mRNAs initiated from various sites.
cord-294108-uvnh0s9r	2020	[2, [8] [9] [10] This article discusses SARS-CoV-2 nanostructure, the virus biology in connection to its epidemiology, clinical manifestations, and potential and future therapeutic options including repurposed drugs, vaccine/protein therapies, immune therapies, and nanotherapeutics. Mechanisms such as inhibition of viral enzymes (DNA and RNA polymerases, 3CL pro, TMPRSS2, reverse transcriptase, neuraminidase, endonucleases, and other proteases) or processes such as ACE2 cellular receptor inhibitors and endosomal acidification mediators prohibiting viral fusion; molecules interfering with glycosylation of the viral protein, viral assembly, new viral particle transport, and release, and immunomodulation of cytokine release can be potential targets in developing various antiviral drugs for the SARS-CoV-2. [85] A randomized, placebo-controlled, Phase IV clinical trial assessing the safety and efficacy of umifenovir as an adjuvant therapy to the combined therapeutic regimen of IFN 1a, lopinavir/ritonavir and hydroxychloroquine in moderate to severe COVID-19 patients (NCT04350684) is underway.
cord-294138-h7sfd1wa	2020	Both countries were involved in the United States Agency for International Development''s (USAID) Emerging Pandemic Threats PREDICT program, and surveillance of bats in wildlife markets in rural areas in Laos using family-level PCR assays revealed the presence of CoV RNA in 41 animals [5] . Considering the significant interactions of wildlife and especially rodents with humans in Laos, we were interested in investigating the presence of CoVs in these animals, which can be primary or intermediate hosts for CoVs with zoonotic potential. Since there are abundant contact opportunities for wildlife pathogens and humans in Laos, and considering that coronavirus-zoonotic events can involve intermediate hosts, as in the cases of SARS and MERS, we focused our screening on non-bat species potentially capable of playing that role. It is worth noting that we targeted rodents most likely to be in contact with humans and transmit virus and found fewer CoV-RNA-positive animals than in other studies of rodents in the region or elsewhere.
cord-294260-g410mavp	2011	Arrow, RNA3 start site at nt 2721 (Lindenbach et al., 2002) ; (D) Schematic of the RCNMV genome showing the relative positions of the in trans interacting RNA elements: the loop portion of a stem-loop in RNA2 (termed trans-activator or TA) and a complementary sequence in RNA1 (termed TA binding site or TABS) located just upstream from the initiation site for SG mRNA transcription (Guenther et al., 2004) ; (E) Representation of CLSV genome and the proposed AS2 and RS2 interaction during regulation of SG RNA2 synthesis (Xu and White, 2008) ; (F) Overview of the BYDV genome and the in cisinteraction between genomic 5′-UTR stem-loop (BCL) and the genomic 3′-BTE, and the in trans interaction between the genomic 3′ BTE and the 5′ UTR of SG RNA1 .
cord-294363-bv6xa8v8	2020	Several studies demonstrated angiotensin converting enzyme 2 (ACE2) as an important therapeutic target of SARS-CoV-2 entry and infection, and many potential targets were subsequently proposed, such as the spike (S) protein and transmembrane serine protease 2 (TMPRSS2). Therefore, accelerating research for potential therapeutic target confirmation, promising drug discovery, and clinical verification development will speed up efforts to combat SARS-CoV-2. In addition to inhibiting the virus directly, ASOs are also expected to target the disease-related proteins involved in the inflammatory cytokine storm process, which could be considered a promising therapeutic strategy for combating SARS-CoV-2 . Although many strategies have been used to block the attachment, entry, replication and release processes to inhibit SARS-CoV-2 infection, how to prevent viral evasion from host immune responses and virus-induced cytopathic effects is considered one of the most urgent problems that need to be solved in SARS-CoV-2-induced pneumonia-associated respiratory syndrome (PARS) patients.
cord-294483-mozabpcs	2020	Ten-fold serial dilutions of each transcribed RNA products were tested with respective gene primer probe sets for specific detection and limit of detection. Further, the IVT RNA of each gene was serially diluted 10-fold (10 1 to 10 10 ), and the performance was tested with genespecific primer probe by real-time RT-PCR. When the assay was first set up at the National Influenza Centre of ICMR-NIV, Pune, the IVT RNA for E and SARS coronavirus Frankfurt 1 strain were received from EVAg. The real-time PCR screening assay (E gene) was also established at the 13 VRDLs as part of ICMR''s efforts to expand testing to VRDLs closer to major airports 3 . This necessitated the development of an indigenous IVT RNA for E and RdRp. In addition, majority of the WHO screening protocols (5 of 6) are based on N gene targeting different nucleotide positions and require multiple specific positive controls 4 .
cord-294592-zwvr57a0	2020	title: Global cataloguing of variations in untranslated regions of viral genome and prediction of key host RNA binding protein-microRNA interactions modulating genome stability in SARS-CoV-2 Furthermore, we found that despite the variations in the UTR regions, binding of host RBP to them remains mostly unaltered, which further influenced the functioning of specific miRNAs. CONCLUSION: Our results, shows for the first time in SARS-Cov-2 infection, a possible cross-talk between host RBPs-miRNAs and viral UTR variants, which ultimately could explain the mechanism of escaping host RNA decay machinery by the virus. We have also looked at the possible regulation of viral genomic RNA through binding of host RNA binding proteins (RBPs) and miR-NAs in specific sequences of the viral UTRs. There are experimentally validated evidences of human RBPs binding to the regulated signals within the untranslated region of SARS-CoV RNA in order to control the viral RNA synthesis and turnover.
cord-294718-n3gx862b	2020	title: Detectable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human breast milk of a mildly symptomatic patient with coronavirus disease 2019 (COVID-19) Although RNA has been detected in various clinical samples, no reports to date have documented SARS-CoV-2 in human milk. This case report describes an actively breastfeeding patient with COVID-19 infection with detectable viral RNA in human milk. The first sampling of human milk occurred five days following maternal symptom onset with no episodes of breastfeeding in those five days prior to collection of the sample. An additional six samples of human milk were collected with one further sample demonstrating detectable SARS-COV-2 RNA (Figure 1 ). These samples continued to have detectable RNA sixty-six days following infant symptom onset (Figure 1 ). To our knowledge, this is the first case of detectable SARS-CoV-2 RNA from human milk in a patient with COVID-19.
cord-294764-v28wbrqp	2017	The in vitro assays and in vivo animal models that have been developed to identify and characterize inhibitors of norovirus RdRp are also summarized, followed by an update on the current antiviral research targeting different regions of norovirus RdRp. In the future, structure-based drug design and rational optimization of known nucleoside and non-nucleoside inhibitors of norovirus RdRp may pave the way towards the next generation of direct-acting antivirals. Despite this limitation, the in vitro effect of small molecules on HuNoV replication has been studied using a stable human hepatoma cell line expressing the part of the Norwalk virus RNA genome encoding the non-structural proteins and carrying the neomycin resistance gene (Chang et al., 2006) . Finally, since norovirus RdRp shares functional and structural features with proteins from other RNA viruses such as HCV, it may be possible to identify nucleo(s/t)ide analogs, like 2CM-C, which inhibits a range of viral polymerases.
cord-294800-akr4f5p8	2020	They also summarized that as viral load is quite high during the time of hospital admissions, use of potent antiviral agents at an early stage might prove Abbreviations: ACE2, angiotensin converting enzyme 2; AP, antigen presentation; APCs, antigen presentation cells; APN, aminopeptidase N, ARBs, angiotensin II receptor blockers; ARDS, acute respiratory distress syndrome; CDC, Centers for Disease Control; nCOVID-19, novel coronavirus disease 2019; CoVs, coronaviruses; DPP4, dipeptidyl peptidase 4; dsRNA, double-strand RNA; EC 50 , half maximal effective concentration; ED, emergency department; ELISA, enzymelinked immunosorbent assay; EUA, emergency use authorization; FDA, Food and Drug Administration; GGO, ground-glass opacity; HCV, hepatitis C virus; HIV, human immunodeficiency virus;, MHC, major histocompatibility complex; or HLA, human leukocyte antigen; ICU, intensive care unit; IL-6, interleukin 6; LPV/r, lopinavir/ritonavir; mAbs, monoclonal antibodies; MERS, Middle East respiratory syndrome; N7-MTase, N7-methyltransferase; NSAIDs, nonsteroidal anti-inflammatory drugs; PRRs, pattern recognition receptors; PUI, patient under investigation; RdRp, RNA-dependent RNA polymerase; RSV, respiratory syncytial virus; S protein, spike protein; SAM, S-adenosyl-methionine; SARS, severe acute respiratory syndrome; SARS-CoV-2, Severe acute respiratory syndrome coronavirus 2; TMPRSS2, transmembrane serine protease 2; WHO, World Health Organization.
cord-294842-aesiff1f	2014	Three-dimensional reconstructions of the WNV KUN replication sites revealed an intimate association of the rough ER (rER) with the bounding membrane of the VPs [20] (Figure 2B ), resembling the vesicles observed in DENV-infected cells. In cells infected with TBEV, one of the most important tick-transmitted viruses in Europe and Asia, virus particles and membrane-connected vesicles were also observed inside the ER [25] , similar to what was described for DENV and WNV KUN . Importantly, pulse-radiolabeling experiments localized sites of active RNA replication to the outer surface of single-membrane tubules [71] and isolation of the membranous replication factories and their subsequent visualization by EM revealed that they form rosette-like structures composed of virus-induced cytoplasmic vesicles [124] . Formation of plant RNA virus replication complexes on membranes: Role of an endoplasmic reticulum-targeted viral protein
cord-294890-93ldjyi5	2018	KEGG analysis revealed that most of the differentially expressed target genes (mRNAs) of the lncRNAs were enriched in the "plant-pathogen interaction" and "plant hormone signaling" pathways during early longan SE. CONCLUSION: Analyses of lncRNAs during early longan SE showed that differentially expressed lncRNAs were involved in expression regulation at each SE stage, and may form a regulatory network with miRNAs and mRNAs. These findings provide new insights into lncRNAs and lay a foundation for future functional analysis of lncRNAs during early longan SE. Additionally, 11 potential target genes (mRNAs) and five lncRNAs related to auxin response factors were verified by qPCR in the three stages of early longan SE. Based on the functions of miRNAs and mRNAs in longan and other plants, we speculate that some lncRNAs are involved in regulation of gene expression by acting as miRNA precursors during early longan SE.
cord-295019-8tf8ah6g	2011	Synthetic mammalian transcription circuits consisting of a chimeric small-molecule-responsive transcription factor and a cognate synthetic promoter were originally designed for future gene-based therapies, and the aim was to adjust therapeutic transgene expression in mammalian cells in response to a pharmacologically active substance 34, 47, 49, 91 . When mammalian cells that are transgenic for the screening circuit are exposed to a compound library, they detect and modulate reporter gene expression in the presence of a non-toxic, cellpermeable and bioavailable molecule that has a classspecific core structure and corresponding drug activity (for example, antibiotic activity) (FIG. The availability of compact RNA sensor-actuators that are easy to design and to alter and that control transgene expression in response to intracellular levels of key proteins may also improve the ability to link metabolic disease states with gene-based therapeutic interventions.
cord-295130-e7j7kac0	2020	In this study, we compared the RT-qPCR results from 253 paired samples obtained from saliva and swabs of ambulatory patients; the RNA in the swab samples was extracted using a commercial RNA purification kit, and the saliva samples were directly mixed with a lysis buffer, boiled, and used for the RT-qPCR protocol. To evaluate if saliva is a good source of viral RNA for the RT-qPCR, we determined the presence of the SARS-CoV-2 genome in paired saliva and swab samples from 253 ambulatory patients. Direct lysis of nasopharyngeal or oropharyngeal swab samples in viral transport medium using the QE buffer has been reported as a suitable method for direct RT-qPCR for SARS-CoV-2 detection, with rates similar to those of methods based on column purification (11, 15) .
cord-295217-z2erqkr9	2020	Majority of scRNA-seq approaches provide the steady state kinetics of mRNA (messenger RNA) expression without deeper insights into transcriptional dynamics of cells. However, a recent method called scSLAMseq (single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing) profiles the transcriptional activity at the singlecell resolution which can help in differentiating old and new RNA for thousands of genes 14 A very recent method SMART-Seq3 provides the allele and isoform resolution in scRNA-seq approach 17 . This results in a data set that is roughly symmetric and often roughly normal Mutual Nearest Neighbours: a pair of cells from each batch is contained in each other''s set of nearest neighbours Batch correction: scRNA-seq datasets generated across different conditions or from technologies that contain batch specific systematic bias leading to batch-effect. From single-cell transcriptomic data, Cell-PhoneDB calculates significant receptor-ligand pairs from cluster information and differentially expressed genes. A benchmark of batch-effect correction methods for single-cell RNA sequencing data
cord-295351-0zr2e8lh	2020	Following ZIKV infection, the accumulation of misfolded virus polyproteins in the ER lumen overwhelms the ER protein-folding capacity leading to ER stress and triggers the activation of the UPR (Fig. 2) [47] . Following the dissociation of GRP78 that unmasks the GLS, ATF6 translocate to the Golgi apparatus and undergoes sequential proteolytic processing by Fig. 2 ZIKV-induced ER stress initiates host cell unfolded protein response (UPR). ZIKV infection induces ER stress due to the increased amount of unfolded/misfolded viral (red strand) and host cell (grey strand) protein aggregates in the ER lumen. To summarize, ZIKV virus bypasses the UPR by inhibiting stress granules assembly and reticulophagy to ensure continuous viral protein translation and virion production while simultaneously protecting the virus from host cell defense mechanisms.
cord-295467-9fnis6ci	2014	title: The European race of Gremmeniella abietina hosts a single species of Gammapartitivirus showing a global distribution and possible recombinant events in its history Phylogenetic analysis based on 46 partial coat protein (CP) cDNA sequences divided the GaRV-MS1 population into two closely related clades, while RNA-dependent RNA polymerase (RdRp) sequences revealed only one clade. (2) to analyse their genetic diversity and population structure; and (3) to assess evolutionary processes, such as recombination and selection, to better understand possible host-virus coevolution. The Spanish isolate of Gremmeniella abietina H1-4 was chosen for determination of the full-length sequence of a putative new strain of GaRV-MS1 (GenBank accession numbers for the CP, RdRp, and the unknown protein III: KJ786411eKJ786413). abietina appears to be composed of a single species (GaRV-MS1) with low genetic variability, which is seemingly stable within the different populations of the fungal host.
cord-295733-f3rt1fyk	2020	When compared with previous study, more places that could be potentially contaminated by COVID-19 patients were sampled for viral RNA detection, such as the flush button of the toilet bowl, medical refuse transfer trolley, elevators, and the examination rooms for these patients. These areas could not be used for non-COVID-19 patients until all the environmental samples collected were negative for SARS-CoV-2 RNA detection. In this study, surface samples collected from the examination rooms were all negative for SARS-CoV-2 RNA detection, and the samples collected from isolation wards and other places were also negative for viral RNA detection, which indicated that the terminal disinfection was effective. Other researches had revealed the presence of SARS-CoV-2 RNA in aerosol, which indicated the air could be contaminated by the virus, and patients could be infected in the isolation wards [12, 28] . Detection of air and surface contamination by SARS-CoV-2 in hospital rooms of infected patients
cord-296250-7ln7p715	2020	
cord-296309-i1mpov7k	2017	We will also explore two areas in which viral WGS has recently proven its clinical utility: metagenomic sequencing to identify viruses that cause encephalitis (BOX 1) ; and the role of WGS in molecular epidemiology and public health management of the Pan-American Zika virus outbreak (BOX 2) . However, the increasing number of resistance genes that are located across viral genomes, together with decreasing costs of sequencing and the use of sequence data for transmission studies, are driving a reappraisal of the need for WGS. The numerous phylogenetically informative variant sites that can be obtained from full-length or near full-length genomes removes the need for high-quality sequences, which enabled the robust linking of cases of Ebola virus infection and public health interventions in real time during the 2015 epidemic 39 . There are several methods that are available to achieve WGS of viruses from clinical samples; amplicon sequencing, target enrichment or metagenomics.
cord-296847-r752bcsu	2007	In the present study, we quantified the hRSV RNA load (both subgroups A and B) in NPAs from hRSV-infected infants by quantitative RT-PCR to investigate the possibility of defining the etiologic role of hRSV in the current ARTI episode from a series of infants admitted to the hospital either with a single hRSV infection or with two or three simultaneous or sequential viral infections including hRSV. NPAs from a series of 253 infants aged less than 1 year and admitted to the hospital in the period November 2005-May 2006 with a diagnosis of ARTI were examined for hRSV as well as other respiratory viruses by DFA, CC, and qualitative RT-PCR, as reported (Rovida et al., 2005) . In addition, 14 infants with multiple simultaneous (coinfections) or sequential (within 30 days) infections in the respiratory tract (including hRSV) were examined prospectively in order to identify the virus responsible for the current ARTI episode.
cord-296977-yzhsdz9c	2020	; https://doi.org/10.1101/2020.11.04.20225888 doi: medRxiv preprint imaging camera and SYBR Green I derived fluorescence transduction by naked eye or smartphone camera 27 ; (3) Microfluidic cartridge combining immune-capture, lysis and LAMP to detect viable bacteria using a reader platform comprising two light sources for fluorometric and/or turbidimetric analysis resorting to a smartphone camera 30 ; (4) a hermetic container providing power-free chemical-based heating for LAMP amplification followed by detection using a smartphone flashlight and camera for fluorometric detection 32 ; (5) Centrifugal platform combining silica-based DNA extraction and integrated LFA strips to multiplex the detection of multiple LAMP products using anti-DIG antibodies and colorimetric detection 32 ; (6) Centrifugal platform with automated bead-beating lysis followed by direct RT-LAMP by continuous measurement of fluorescence with UVC illumination and a standard camera 22 ; and (7) Centrifugal platform incorporating non-contact heating of the disc and colorimetric detection of LAMP products using a white LED for illumination and filtered photodiodes for signal acquisition 24 .
cord-297039-vfuem6bk	2020	Recent findings propose that circular RNAs (circRNAs) may play a prominent role in regulating the patients'' immune system against different pathogens, including bacteria and viruses. Due to the role that circRNAs play in the modulation of different cytokines and immune proteins [62, 63] , altered states of alternative splicing in sepsis may alter the expression of circRNAs, which could partially explain the changes in the immune response of septic patients. However, circRNAs may play a key role in sepsis because of their ability to modulate different molecular mechanisms [10] , including inflammation [83] and immune response [62] , and to control multiple biological processes in metabolic organs (i.e., liver, pancreas [84] ) ( Figure 3 and Table 1 ). Interestingly, circRNAs may function as "molecular sponges", by controlling the expression of different types of non-coding RNAs, such as miRNAS, involved in regulating different processes in sepsis [105] [106] [107] [108] .
cord-297078-pxggjaby	2005	Leipe, Aravind, and Koonin (1999) suggest that LUCA possessed a hybrid DNA/RNA genome, thereby providing an explanation for the universal distribution of certain components of the DNA replication/repair apparatus. We argue from structural and functional data that the second of these, RNA polymerase-dependent proofreading and repair, is likely to have been a feature of LUCA rather than a recent innovation as part of selection for improved messenger RNA (mRNA) quality control, though this is no doubt the current function of this phenomenon. In this scenario, cleavage-stimulatory factors evolved twice independently (GreA/GreB in bacteria and transcription factor S/TFIIS in archaea/eukaryotes) with an initial function in RNA genome repair. This implies that the LUCA possessed an RNA genome, and in this scenario, bacterial RNA polymerase-associated cleavage-stimulatory factors (GreA/GreB) and their archaeal/eukaryotic equivalents (TFS/TFIIS) were originally involved in proofreading and repair of the genome, as indicated by [Gen] .
cord-297092-oq14cwka	2020	This study developed whole genome sequencing methods to facilitate the control of SVA and provide a reference for the timely detection and prevention of other emerging infectious diseases. For the sequencing of cell culture samples, raw pass reads were mapped to the SVA reference genome (GenBank: MN164664) using Minimap2 [34] , then analyzed using Qualimap [35] , generating raw error rates and coverage information which was then visualized using GraphPad prism software (GraphPad Software, San Diego, CA, USA). After determining the consensus generation strategies for both sequencing methods, optimization was performed in terms of total input sequencing yield and the pre-treatment of raw reads using the cell culture virus in which the whole genome sequence was already known. In order to evaluate and compare the general performance of DRS and PCS for viral whole genome recovery, two whole genome sequencing runs using a cell culture-grown virus with a known reference sequence were carried out for each method (Table 1 ).
cord-297323-l3f12hg4	2020	Like many viruses, SARS‐CoV‐2 has evolved strategies to circumvent innate immune detection including low CpG levels in the genome, glycosylation to shield essential elements including the receptor binding domain, RNA shielding and generation of viral proteins that actively impede anti‐viral interferon responses. These subsequently induce expression of type I IFNs (IFNα/β) and interferon stimulated genes (ISGs) [figure 2] many of which have potent antiviral activities, as well as other proinflammatory mediators e.g. cytokines, chemokines and antimicrobial peptides that are essential to initiate the host innate and adaptive immune response. Likewise, viral load, obesity, gender, race, blood groups and comorbidities have all been reported to influence the response to SARS-CoV-2 infection, [ Table 4 ; (101) (102) (103) (104) (105) (106) (107) (108) (109) (110) (111) (112) ] although few studies have fully examined the extent to which subversion and activation of innate immune components contribute to susceptibility in these cases. Toll-Like Receptor 3 Signaling via TRIF Contributes to a Protective Innate Immune Response to Severe Acute Respiratory Syndrome Coronavirus Infection
cord-297579-ohpm5ys0	2018	Despite the clinical significance of norovirus infection, antiviral studies have been hindered, because until recently, human norovirus could not be successfully propagated in cell culture. The cross-genotypic activity displayed by Nbs illustrates that these molecules have the potential to overcome the narrow antigenic spectrum typically displayed by conventional mAbs. However, despite these findings, mAb and Nb studies have been based mostly on VLP-binding and structural analysis of that binding (Table 1 ) and thus the effects of such compounds against norovirus in cell culture or in vivo need to be explored further before continued development toward clinical application. Most recently NTZ was shown to potently inhibit FCV replication in cell culture with an EC 50 of 0.6 µM, 189 and the GI norovirus replicon at a clinically relevant concentration (5 μg/mL), 190 which was later shown to result in a broad antiviral response. The viral polymerase inhibitor 2′-C-methylcytidine inhibits Norwalk virus replication and protects against norovirus-induced diarrhea and mortality in a mouse model
cord-297760-uzzuoy9v	2006	title: siVirus: web-based antiviral siRNA design software for highly divergent viral sequences siVirus () is a web-based online software system that provides efficient short interfering RNA (siRNA) design for antiviral RNA interference (RNAi). siVirus searches for functional, off-target minimized siRNAs targeting highly conserved regions of divergent viral sequences. siVirus will be a useful tool for designing optimal siRNAs targeting highly divergent pathogens, including human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus and SARS coronavirus, all of which pose enormous threats to global human health. Consequently, only a limited fraction of 21mers is suitable for use as antiviral siRNAs. In this study, we developed a novel web-based online software system, siVirus, which provides functional, off-target minimized siRNAs targeting highly conserved regions of divergent viral sequences. Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference siDirect: highly effective, target-specific siRNA design software for mammalian RNA interference
cord-297776-k38jssr0	2020	gene expression, we report significant transcriptional upregulation of multiple genes associated with activation of ER sensors IRE1, ATF6, and PERK, such as Edem1, Hspa5 (encoding BiP protein), and Ddit3 (encoding CHOP), in response to virus replication, with the most robust response detected in cells infected with the wild-type or DUBmut virus infection (Fig. 5B, C, and D) . Overall, these differential gene expression analyses in macrophages reveal similar host responses to the wild-type and DUBmut viruses that include activation of UPR pathways and proinflammatory genes, whereas a distinct transcriptional profile during infection with the EndoUmut virus is predominately defined by a focused, robust antiviral response. The results presented here, and from studies of the MERS-CoV dORF3-5 mutant virus (26) Upon infection of a BMDM with EndoUmut, host double-stranded RNA (dsRNA) sensors (including MDA5, PKR, and OAS) are activated, resulting in robust transcription of type I IFN genes and rapid induction of apoptosis, the latter of which precludes the development of a potent inflammatory response.
cord-297790-tpjxt0w5	2018	Among them are filoviruses (e.g., Marburg, Ebola), coronaviruses (e.g., SARS, MERS), henipaviruses (e.g., Hendra, Nipah) which share the common features that they are all RNA viruses, and that a dysregulated immune response is an important contributor to the tissue damage and hence pathogenicity that results from infection in humans. It is likely that differences in evolutionary history of pathogen exposure between bats and humans have led to distinct adaptations in anti-viral immune responses and the ability to tolerate certain infections without disease while being susceptible to others. We summarize this work below, but comparisons of observations made across species suggest that although a number of species appear to be capable of avoiding the pathological effects of RNA virus infection, each bat species may have achieved this through distinct pathways, possibly involving changes to both increase pathogen replication control and to mitigate any immunopathology through decreased inflammatory responses and hence increased disease tolerance.
cord-297834-me1ajoyb	2014	The immune response is energetically expensive for wild animals, thus the findings of experimental studies will be critical for understanding the ecoimmunology of reservoir hosts of hantaviruses [6, 7] , and experiments using wild rodents in natural or semi-natural environments [8, 9] will be required to validate laboratory findings. Currently, three laboratory infection systems have been developed to study hantavirus infections of reservoir hosts: Seoul virus (SEOV) infection of the Norway rat (Rattus norvegicus), Puumala virus (PUUV) infection of the bank vole (Myodes glareolus), and Sin Nombre virus (SNV) infection of the deer mouse (Peromyscus maniculatus) [12, 14, 16] . Experimental data have also shown that patterns of the expression of genes related to the immune response are different in infected males and females [32] , and it is likely these differences have important roles in hantavirus ecology.
cord-297880-jlnv90vn	2018	Using the prototype species equine torovirus (EToV), we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyze the relative expression levels of the known torovirus proteins and transcripts, chimeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle), and changes in host transcription and translation as a result of EToV infection. The genomes of members of the order Nidovirales are positive-sense, polycistronic RNAs. One of the hallmarks of this virus order is the utilization of an unusual transcription mechanism to express the genes encoding structural and accessory proteins, which reside downstream of the large replicase open reading frames (ORFs) 1a and 1b (Fig. 1) . RNA sequencing (RNA-seq) confirmed previous reports that EToV utilizes a unique combination of both discontinuous and nondiscontinuous RNA synthesis to generate its repertoire of sgRNAs. Strikingly, we also identified a small proportion of chimeric transcripts spanning from the leader to the body TRS of the N protein gene, indicating that discontinuous and nondiscontinuous mechanisms compete in this location.
cord-297974-sduz0j35	2020	Here we describe a method to detect SARS-CoV-2 RNA of a single infected individual within a bulk sample comprised of up to 26 individual patient samples by combining a hybridizationcapture-based RNA extraction approach with smartphone app-assisted colorimetric detection of RT-LAMP products, a procedure that can be performed in less than one hour ( Figure 1A) . To investigate whether it is possible to detect single infected individuals in pools of gargle lavage samples, we created eleven pools of 25 patient samples each, all of which had been tested negative in RT-qPCR assay and in the Cap-iLAMP assays for the Orf1a and the N gene ( Figure 2D ). All tested gargle lavages from single healthy individuals (n=6) and 11 pools of 25 healthy individuals (n=275) correctly tested negative for both the Orf1a gene and N gene in the Cap-iLAMP assay ( Figure 2C and E), indicating that false positive results which were sometimes observed when samples are added directly into the iLAMP reaction (Supp.
cord-298032-3zlu8g8y	2018	An earlier study showed that a 22mer PPMO targeting the translation start site region of EBOV VP35 positive-sense RNA exhibited sequence-specific, time-and dose-dependent inhibition of EBOV replication in cultured cells (Enterlein et al., 2006) . However, PPMO targeting conserved internal ribosome entry site (IRES) sequences have been shown to be highly effective in protecting cultured cells against infection by human rhinovirus type 14, coxsackievirus type B2, and poliovirus type 1 (PV1) (Stone et al., 2008) , with reduction of PV1 titers by up to 6 log10. In this study, virus replication in MDCK cells was significantly inhibited by three PPMO targeting either the translation start site region of PB1 or NP mRNA or the 3 -terminal region of NP viral RNA (vRNA). Inhibition of influenza virus infection in human airway cell cultures by an antisense peptide-conjugated morpholino oligomer targeting the hemagglutinin-activating protease TMPRSS2
cord-298036-2zurc60t	2020	Subsequently, granzyme-B induces mitochondrial apoptosis by performing cleavage of the BCL-2 homology domain-3 (BH3)-only protein, BH3 interacting domain death agonist (BID), which then leads to BAX/BAK-mediated MOMP and the initiation of the caspase-9-driven apoptotic pathway [16] . Still, the mechanism, by which IRF-3 triggers cell death signalling pathways is only partially understood and the studies indicate a strong cell type specificity in the apoptosis sensitivity in response to viral PAMPs Z-RNA and z-DNA fragments, which are distinct from the B-structure of eukaryotic RNA and DNA are recognized by z-DNA/RNA binding protein-1 (ZBP1; also: DAI). Necroptosis initiation takes place upon TNFR ligation, which, however, primarily leads to NFkB activation via the assembly of so called complex-I, including adaptor proteins TNFRSF1A associated via death domain (TRADD), TRAF2, cellular IAP (cIAP) and ubiquitinated receptor interacting serine/threonine kinase 1 (RIPK1) [10] .
cord-298078-uqrwq5qk	2011	The results suggest that ANXA2 is a cellular RBP that can modulate the frameshifting efficiency of viral RNA, enabling it to act as an anti-viral cellular protein, and hinting at roles in RNA metabolism for other cellular mRNAs. Ribosomal frameshifing is a recoding process of translation where a specific messenger RNA (mRNA)-mediated signal directs a ribosome to shift its reading frame and to continue in the new frame. To search for cellular proteins that directly interacted with IBV pseudoknot RNA, a RNA pull down assay was performed in the presence of cell extracts (Figure 2A ). Through the RNA-immunoprecipitation assay, we showed that ANXA2 specifically interacted with wild-type IBV pseudoknot RNA but not with mutant IBV RNA in LNCaP and HEK293T cells ( Figure 3C and 3D). To test how ANXA2 regulates the frameshifting efficiency of IBV pseudoknot RNA, we first overexpressed ANXA2 protein in the presence of the reporters and measured the luciferase activities.
cord-298233-qqhgmqrg	2016	
cord-298281-wkje5jyt	2020	Primary outcomes were the urological manifestations of COVID-19, and SARS-CoV-2 viral RNA detection in urine and stool samples. Primary outcomes of our study included urological manifestations of COVID-19, detection rates of SARS-CoV-2 viral RNA in urine and stool samples, and special considerations in urological conditions. For the urological manifestations and viral RNA detection rates, data were pool analysed using MetaXL and Microsoft Excel when there are two or more studies with at least four patients reporting the same outcome under the same definition. There were a total of 11 studies that reported the number of patients who had their urine tested for SARS-CoV-2 viral RNA. Our meta-analysis included 12 studies that reported the number of patients with stools tested for SARS-CoV-2 viral RNA. Our study showed that 5.74% of the COVID-19 patients had positive viral RNA in urine samples.
cord-298779-0mjizsoo	2016	The abnormal RNA disruption bands that occur upon chemotherapy drug exposure are smaller in molecular weight than the 28S and/or 18S rRNAs. To determine whether the abnormal bands originate from the 28S and/or 18S rRNAs, Northern blotting experiments were performed on total RNA prepared from A2780 cells after incubation in the absence or presence of docetaxel for up to 48 h (Fig. 4) . Previous studies have shown that a variety of apoptosisinducing agents with distinct mechanisms of action (glucocorticoids, okadaic acid, tumor necrosis factor (TNF), dexamethasone, a calcium ionophore and a tricothecene mycotoxin) were able to induce an ordered, apoptosis-associated rRNA degradation in several different cell types, including plant cells and the unicellular organism yeast [11-13, 15-17, 24] . However, this study is the first to report the ability of structurally distinct cytotoxic chemotherapy agents with different mechanisms of action to induce the formation of high molecular weight rRNA degradation fragments (Figs.
cord-298820-nogoqyxl	2016	Here, we profiled the expression of mRNAs and long noncoding RNAs (lncRNAs) in THP-1 cells using the emerging RNA-seq to investigate the transcriptional changes during HCMV latent infection. These studies identified a subset of differently expressed genes, which are involved in a variety of biological functions including innate immunity response, inflammation pathway, cell cycle regulation, cellular metabolism and cell adhesion, during lytic HCMV infection. The present study adopted RNA-seq to identify global changes of mRNAs and lncRNAs in host cell during HCMV experimental latent infection of THP-1 cells, in an attempt to derive insights into the mechanism underlying alterations in response to infection. In this study, we profiled the expression of mRNAs and lncRNAs in host cell using the emerging RNA-seq to investigate the transcriptional changes in HCMV latent infection.
cord-298847-szezd2vb	2003	Virus inoculation of 21-day postnatal C57BL/6 and BALB/c mice led to a generalized infection of the whole CNS, demonstrating HCoV-OC43 neuroinvasiveness and neurovirulence. Moreover, mice inoculated with supernatants from cell cultures infected with brain tissue from affected mice developed the same disease, demonstrating that the virus was responsible for pathology. Cells positive for viral antigens were first observed at HCoV-OC43-infected mice gained weight normally during the first 5 days after infection, after which they all lost weight during the acute phase of the disease. (A) 100% of brains from C57BL/6 mice inoculated ic with 10 TCID 50 of HCoV-OC43 were positive for viral RNA between 3 and 11 days postinfection. However, RT-PCR analysis revealed that viral RNA could not be detected after the second week postinfection, suggesting a nonpersistent infection of HCoV-OC43 virus in 21 DPN mice. Every 2 days postinfection, five animals from two groups of infected mice of each strain were sacrificed and processed for detection of viral RNA, viral proteins, and infectious virus.
cord-298905-c2uuvfm5	1987	Using coronaviruses as examples the changes in virulence have been traced back to single mutational events; recombination, however, is likely to be an alternative mechanism by which virus-host interactions (e.g. the cell-, organor animal species-spectrum) can dramatically change. Parainfluenzaviruses, for example, attach to neuraminic acid-containing receptors; since glycolipids and glycoproteins containing neuraminic acid abound in vertebrate cell membranes the adsorption/penetration process lacks the specificity required to explain the restrictions in host range and tissue tropism of paramyxoviruses 29. Also in influenza virus infection cap structures are essential: these are cannibalized from host cell nuclear RNA precursor molecules and used as primers for viral RNA replication and synthesis 28. Autoimmune phenomena involving both the humoral and cellular limbs of the immune response have been identified in neurological conditions following infections with e.g. canine distemper virus3; invasion of brain tissue is supposed to cause changes in the molecular constitution of myelin and membrane components, making them recognizable as ''nonself''.
cord-298934-vtrfqozl	1988	
cord-298938-xemarhlv	2004	title: Apoptosis induced by a cytopathic hepatitis A virus is dependent on caspase activation following ribosomal RNA degradation but occurs in the absence of 2′–5′ oligoadenylate synthetase We have presented previously evidence that the cytopathogenic 18f strain of hepatitis A virus (HAV) induced degradation of ribosomal RNA (rRNA) in infected cells [Arch. Based on the pattern of rRNA degradation in intact ribosomes, we suggested that the 18f virus activates the interferon (IFN) controlled 2 -5 oligoadenylic acid-dependent RNase L pathway (for recent reviews, see Stark et al., 1998; Barber, 2001; Sen, 2001) . First, the level of IFN, 2 -5 OAS and RNase L mRNAs were investigated by RT-PCR to determine if any of these RNAs were induced following virus infection (Fig. 8) . RT-PCR amplification of selected cellular and viral mRNAs. Two microgram of RNA isolated from virus infected, IFN-␤, or dsRNA treated cells were reverse transcribed in a volume of 20 l using oligo(dT) 15 as primer as described in Section 2.
cord-299509-7xjdryoq	2013	Infectious cDNA clones of viruses have become invaluable tools that allow reverse genetics studies to elucidate the contribution of specific amino acids or RNA structures to viraemia, virulence, antigenicity, replication kinetics, interactions with host factors, adaptation to new vectors, and many other aspects of the viral life cycle. To obtain a virus that -in terms of virulence, sensitivity to antiviral compounds, and CHIKV-host interactions -is expected to have the general characteristics of the E1-226V CHIKV strains that were circulating during the 2005-2009 outbreaks, we have constructed a completely synthetic CHIKV cDNA clone based on the consensus sequence of the aligned genomes of these recent isolates. Indirect immunofluorescence analysis of Vero E6 cells infected with CHIKV LS3, LS3-GFP, or ITA07-RA1 at various time points showed that the localization and expression kinetics of E2 and dsRNA were similar for the natural isolate and the synthetic viruses (Fig. 6) .
cord-299560-np6nfvf2	2020	
cord-299747-qovrstak	2014	title: Inhibition of viral RNA polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand RNA viruses beyond hepatitis C virus Inhibition of viral RNA polymerases by nucleoside and nucleotide analogs: therapeutic applications against positive-strand RNA viruses beyond hepatitis C virus Jerome Deval, Julian A Symons and Leo Beigelman A number of important human infections are caused by positive-strand RNA viruses, yet almost none can be treated with small molecule antiviral therapeutics. The initial major class of nucleoside analogs of therapeutic potential to demonstrate potent inhibition of HCV RNA polymerase activity were 2 0 C-methyl-ribonucleosides, The first 2 0 C-methyl ribonucleosides were originally synthesized in the 1960s [18] . 0 -azidocytidine) is a potent inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus replication in cell culture
cord-299754-tgexahwd	2017	Downstream of the initial pattern recognition, TRIMs also influence the recruitment and interaction of adaptor molecules (stimulator of IFN genes (STING), mitochondrial antiviral signaling protein (MAVS), TGF-β-activated kinase 1(TAK1)/MAP3K7-binding protein (TAB) 2, Myeloid differentiation primary response gene 88 (MyD88), TIR-domain-containing adapterinducing interferon-β (TRIF), NF-κB essential modulator (NEMO), nucleosome assembly protein (NAP-1), and tumor necrosis factor (TNF) receptor-associated factors (TRAF) family memberassociated NF-κB activator (TANK)) and enzymes (TRAF3, TRAF6, TAK1, inhibitor of NF-κB (IκB) kinase (IKK) α,β,ε, TANK binding kinase 1 (TBK1)) to signaling complexes in order to activate transcription factors. Downstream of the initial pattern recognition, TRIMs also influence the recruitment and interaction of adaptor molecules (stimulator of IFN genes (STING), mitochondrial antiviral signaling protein (MAVS), TGF-β-activated kinase 1(TAK1)/MAP3K7-binding protein (TAB) 2, Myeloid differentiation primary response gene 88 (MyD88), TIR-domain-containing adapter-inducing interferon-β (TRIF), NF-κB essential modulator (NEMO), nucleosome assembly protein (NAP-1), and tumor necrosis factor (TNF) receptor-associated factors (TRAF) family member-associated NF-κB activator (TANK)) and enzymes (TRAF3, TRAF6, TAK1, inhibitor of NF-κB (IκB) kinase (IKK) α,β,ε, TANK binding kinase 1 (TBK1)) to signaling complexes in order to activate transcription factors.
cord-299848-fft1brwz	2009	Using human rhinovirus as a model system, we have combined NMR contact information, small-angle X-ray scattering (SAXS) data, and previous mutagenesis results to determine the shape, position and relative orientation of the 3C(pro) and SLD components. Binding between SLD and 3C(pro) induces structural changes in the proteolytic active site that is positioned on the opposite side of the protease relative to the RNA/protein interface, suggesting that subtle conformational changes affecting catalytic activity are relayed through the protein. NMR and small-angle X-ray scattering data were combined with results from previous mutational analysis (Andino et al., 1993; Leong et al., 1993) (Fig. 1) to construct a structural model of the HRV-14 3C pro -SLD complex. The largest SLD-induced chemical shift perturbations on the 3C pro surface cluster primarily to a patch (dark red in Fig. 5A ) that includes D32 from the loop connecting b-strands 2-3, N80, F83 and F89 from the inter-domain linker and V179 from the C-terminal region.
cord-299943-wzkh04dv	2020	In a sensor detection scheme, ssDNA(s) specifically hybridizes with a target DNA sequence that is being employed as a probe(s): a capture probe used to attach the target DNA to the surface of materials and/or a reporter probe labeled with signaling molecules, e.g., redox-active molecules. To make a quantitative measurement, the DNA hybridization event is coupled with electrochemical reactions, in a way that a probe-target complex increases/decreases a coupled redox reaction at the electrode surface. DPV-Differential pulse voltammetry, CV-Cyclic Voltammetry, EIS-Electrochemical Impedance spectroscopy, CSD-Circular strand displacement, RCA-Rolling circle amplification, EXPAR-Isothermal exponential amplification, HCR-Hybridization chain reaction, HDA-Helicase dependent amplification, TMB-3,3 ,5,5 -tetramethylbenzidine, N,S-GQDs@AuNP-Nitrogen, sulfur codoped graphene quantum, CNT-PANI-Carbon nanotube-polyanilline, NA-Not applicable, * If limit of detection is not reported, lowest detected value is provided. Different methods have been employed in signal amplification approaches to detect a low copy number of target DNA on the electrode surface.
cord-300023-2dg7njki	2015	
cord-300399-21xozruq	2020	
cord-300470-vgd1ol2z	2016	
cord-300489-gzcb6uqw	1993	
cord-300685-bcjnujlj	2003	The detection of live virus (4 ) and the detection of high copy numbers of viral sequence from NPA samples in the current study clearly support that the concept that cough and sneeze droplets from SARS patients are a major route of spread of this infectious agent. Interestingly, two of four available stool samples from the SARS patients in this study were positive in the assay (data not shown). RNA samples from this study were subjected to nested reverse transcription-PCR (4 ), and no evidence of metapneumovirus infection was detected in any of the patients in this study (data not shown), suggesting that the novel coronavirus is the key player in the pathogenesis of SARS. The PCR products from all 23 positive cases in this study had the same melting point, strongly suggesting that there was no viral sequence variation in the target region of samples collected at the two Hong Kong hospitals during the 1-month period of patient accrual.
cord-300884-rqfxe0x1	2007	Analysis of the sequence identified 11 open reading frames which encode two replicase polyproteins, five structural proteins (hemagglutinin esterase, spike, envelope, membrane, and nucleocapsid) and four accessory proteins (NS2, p4.7, p12.7, and I). In contrast to the replicase proteins which are directly translated from the genomic RNA, coronavirus structural and accessory proteins are expressed from a nested set of 3′ coterminal subgenomic (sg) mRNAs that also possess a common 5′ leader sequence derived from the 5′ end of the genome (Pasternak et al., 2006; Sawicki et al., 2007) . Following multiple alignments with the S proteins of other group 2a coronaviruses, a potential cleavage recognition sequence (RRQRR) was identified at residues 764-768 which would predict a cleavage between amino acids 768 and 769, separating the ECoV S protein into S1 and S2 subunits (Fig. 1) .
cord-300963-1n1f8mf2	2020	Previous studies based on SARS-CoV-1 showed that the "cytokine storm" was strongly associated with viral sepsis, inflammation-induced lung injury, and acute respiratory distress syndrome (ARDS) [32, 34] . With regard to IBD-specific risk factors, it is speculated that patients on immunosuppressive agents, those with active IBD symptoms, malnutrition, and frequent visits to clinics or hospitals are at greater risk of acquiring SARS-CoV-2 infection [50] . The International Organization for the Study of Inflammatory Bowel Diseases (IOIBD) maintains a registry for reporting COVID-19 in IBD patients called SECURE-IBD registry. Hence, all the societies have recommended that patients continue their IBD medications to sustain remission, because the risk of disease flare-up outweighs the chance of contracting SARS-CoV-2 infection. The management strategy will depend on multiple factors, such as the patient''s age, the severity of the COVID-19 infection, the clinical status of the IBD, and the presence of other comorbid conditions.
cord-301115-sedfbjlw	2020	title: Assessing SARS-CoV-2 RNA levels and lymphocyte/T cell counts in COVID-19 patients revealed initial immune status as a major determinant of disease severity The results of our analysis demonstrated that the initial SARS-CoV-2 RNA loads varied in patients, but were comparable in different patient groups stratified by age, gender, comorbidities and disease severity. We compared the measured SARS-CoV-2 RNA levels in sputum specimens from COVID-19 patients at admission among groups divided according to age, sex, underlying diseases and disease severity (Fig. 2a) . a, b The measured SARS-CoV-2 RNAs levels in sputum (a) and throat swab (b) specimens from COVID-19 patients at admission were compared according to the age, sex, comorbidity, and the disease severity. In this study, we analyzed the clinical features including SARS-CoV-2 RNA load and immunological characteristics of peripheral blood in a patient cohort with COVID-19 from Anhui Province, China.
cord-301226-hmc2wmst	2020	Methods: Here, we have used RT-qPCR for SARS-CoV-2 detection in a series of longitudinal metropolitan wastewaters samples collected during the earliest stages of the epidemic in the Region of Valencia, Spain. Here, we show that SARS-CoV-2 can be reproducibly detected by RT-qPCR in longitudinal samples from sewage treatment plants that receive wastewaters from over one million inhabitants in the metropolitan area of Valencia, Spain. Following concentration of viral content by flocculation, a standard RT-qPCR procedure allowed us to detect SARS-CoV-2 RNA in 12/12 samples collected from March 9 to April 14, 2020, with Ct values ranging between 34•00 and 37•84, correspondingly revealing between 5•22 and 5•99 log 10 genomic copies (gc)/L (Table 1) . Interestingly, we consistently detected SARS-CoV-2 RNA in samples collected on March 9 and March 11, when only 50 and 76 cumulative cases were declared in the entire Region of Valencia.
cord-301233-nenw0f81	2020	Here we report the structure of favipiravir ribonucleoside triphosphate (favipiravir-RTP) in complex with the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) bound to a template:primer RNA duplex, determined by electron cryomicroscopy (cryoEM) to a resolution of 2.5 Å. The structure reveals an unusual, non-productive binding mode of favipiravir-RTP at the catalytic site of SARS-CoV-2 RdRp which explains its low rate of incorporation into the RNA primer strand. Here we report the structure of the SARS-CoV-2 RdRp, comprising subunits nsp7, nsp8 and nsp12, in complex with template:primer double-stranded RNA and favipiravir ribonucleoside triphosphate (favipiravir-RTP), determined by cryoEM at 2.5Å resolution. In this study, we determined the cryoEM structure of favipiravir-RTP at the catalytic site of the SARS-CoV-2 RdRp, in complex with template:primer dsRNA, and investigated the influence of this nucleotide analogue inhibitor on RNA synthesis in vitro.
cord-301285-p83ondy8	2018	To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus vaccine strain, TC-83. Due to the error-prone nature of the RNA-dependent RNApolymerase (RdRp), RNA virus replication is characterized by a high mutation rate that results in increased genetic diversity of progeny viruses (Domingo et al. When compared with unpassaged, wild-type (wt) viruses, fidelity mutants have similar growth kinetics in vitro, but are attenuated in vivo due to the alteration of diversity produced during replication, which hampers the ability of the virus to overcome bottlenecks in the host (Pfeiffer and Kirkegaard 2005; Vignuzzi et al. The 4x mutant, while exhibiting phenotypic similarities with other altered fidelity mutants, had no significant difference in virus diversity compared with the TC-83 parent after one cell culture passage.
cord-301362-f3lp10lm	2017	Recognition of viral double-strand RNA (dsRNA) molecules by intracellular Toll-like receptors (TLRs) or retinoic acid inducible gene I-like receptors (RLRs) is a central event which entails the early steps of the immune response elicited during viral infections. Despite several differences among host range, viral structure, genome organization or membrane-donor organelles from the cell, these analyses revealed that +sRNA viruses are able to induce two types of membranous modifications as replicative niches: invaginated vesicles or spherules or a double membrane vesicle type. Endogenous RNAs forming secondary double-stranded structures that are released after necrosis and tissue damage after viral infection represent another source of dsRNA molecules reaching the endosomes, inducing host-derived dsRNA-mediated inflammatory responses through TLR-3 recognition (Kawai and Akira, 2010) . Other +sRNA viruses such as the enterovirus Coxsackievirus (Kemball et al., 2010) , Hepatitis C virus (Flaviviridae family) (Sir et al., 2012) , or Coronavirus such as MVH (Reggiori et al., 2010) also usurp the autophagy pathway and induce remarkably alterations in intracellular membranous components to harbor the sites for viral RNA replication.
cord-301535-eui41zyg	2020	This assay uses the addition of a frame-shifted spike-in, a modified PCR master mix, and custom Sanger sequencing data analysis to detect and quantify SARS-CoV-2 RNA at a limit of detection comparable to existing qPCR-based assays, at 10-20 genome copy equivalents. Crucially, our assay was able to detect SARS-CoV-2 RNA from viral particles suspended in transport media that was directly added to the PCR master mix, suggesting that RNA extraction can be skipped entirely without any degradation of test performance. Quantitative analysis of the Sanger sequence chromatogram gives qSanger an extremely high sensitivity and specificity for all positive results with a limit of detection of 10-20 genome copy equivalents (GCE), equivalent to gold-standard qPCR methods. To further evaluate the feasibility of a direct VTM, extraction-free method for Sars-CoV-2 detection, we also examined the ability of qSanger to quantify the amount of viral particles in the Seracare positive control specimens (Fig. 3B ).
cord-301904-mjfbvl5n	2014	Astrovirus as a cause of hospital-acquired viral diarrhea in young children is second only to rotavirus and norovirus, occurring at rates of 4.5-6%, and, in some studies, surpasses rotavirus in rates of nosocomial infections. Recent studies reported that an HAstV-VA1-like strain was detected in a patient with new-onset celiac disease and associated with extra-intestinal dissemination (including neural tissue) in immunocompromised children. Given the susceptibility of different cell lines for HAstV infection (depending on the serotype and genotype) it is possible that astroviruses use a variety of attachment proteins or receptors including carbohydrate moieties. The immunological response to astrovirus infection is poorly defined; however, observations in humans and animal models suggest that both the adaptive and innate responses play important roles in controlling and eliminating the virus. While astrovirus antibodies protected individuals from symptoms associated with infection, virus was identified in the feces, suggesting that such antibodies do not necessarily prevent viral replication.
cord-301997-63160t7f	1987	We have shown that RNA transcripts from a translocated 11K and from the authentic 11K and 4b late promoters are extended by approximately 35 nucleotides beyond the "start site" determined by Sl mapping using vaccinia genomic DNA as a probe. We have shown that RNA transcripts from a translocated 11K and from the authentic 11K and 4b late promoters are extended by approximately 35 nucleotides beyond the "start site" determined by Sl mapping using vaccinia genomic DNA as a probe. As a negative control, we have performed primer extension with a dhfr primer and wild-type mRNA that does not contain dhfr sequences ( Figure 1, cDNA-RNA hybrids were incubated in the presence or absence of RNAase A at a final concentration of 10 Kg/ml and the hybrids were cap-selected by immunoprecipitation using a rabbit anti-m7G antiserum as described. After RNAase treatment-cap selection of the cDNA-RNA hybrids, we obtained the same results as for the wild-type translocated promoter-dhfr messengers (data not shown).
cord-302020-ypsh3rjv	2020	In addition to the canonical genomic and 9 subgenomic RNAs, SARS-CoV-2 produces transcripts encoding unknown ORFs with fusion, deletion, and/or frameshift. Functional investigation of the unknown transcripts and RNA modifications discovered in this study will open new directions to our understanding of the life cycle and pathogenicity of SARS-CoV-2. (A) Read counts from nanopore direct RNA sequencing of total RNA from Vero cells infected with SARS-CoV-2. We further discovered RNA modification sites and measured the poly(A) tail length of gRNAs and sgRNAs. To delineate the SARS-CoV-2 transcriptome, we first performed DRS runs on a MinION nanopore sequencer with total RNA extracted from Vero cells infected with SARS-CoV-2 (Be-taCoV/Korea/KCDC03/2020). To unambiguously investigate the modifications, we generated negative control RNAs by in vitro transcription of the viral sequences and performed a DRS run on these unmodified controls ( Figure S4A ).
cord-302047-vv5gpldi	2019	Viruses are widely used as vectors for heterologous gene expression in cultured cells or natural hosts, and therefore a large number of viruses with exogenous sequences inserted into their genomes have been engineered. Viruses genera covered in relevant studies Conclusions of this review All viruses • Inserted sequences are often unstable and rapidly lost upon passaging of an engineered virus • The position at which a sequence is integrated in the genome can be important for stability • Sequence stability is not an intrinsic property of genomes because demographic parameters, such as population size and bottleneck size, can have important effects on sequence stability • The multiplicity of cellular infection affects sequence stability, and can in some cases directly affect whether there is selection for deletion variants • Deletions are not the only class of mutations that can reduce the cost of inserted sequences, although they are the most common I: dsDNA
cord-302085-xyru2q9o	2016	IRMA provides segment level read sorting based on LABEL, a sequence classification tool ideal for segmented genomes [11] ; iteratively gathers reads and iteratively edits the reference templates to account for high population diversity and mutational rates; and provides redesigned variant calling (heuristics as well as statistical tests) and phasing to allow for the analysis of diverse viral sub-populations. We solve these referenced-based assembly complications by using iterative refinement-moving the reference template closer to the reads-to obtain quality assemblies with increased sensitivity to distant and novel reads BLAT* or LABEL match sort into segments rough align chimeric low quality high quality templates removed module consensuses [1] de-multiplexed sample [1] [2] [3] [4] [5] READ GATHERING: do steps [3] to [5] optimize assembly [6] insertion, deletion, substitution merge reads+ [7] call variants [8] phase variants [9] Perl scripts, samtools, R Steps in (b) are also labeled under the steps of (a) where they correspond genetic variants.
cord-302195-25gjbyi1	2020	This article aims to examine the latest investigations on SARS-CoV-2 plausible environmental transmission modes, employment of wastewater surveillance for early detection of COVID-19, and elucidating the role of solid waste, water, and atmospheric quality on viral infectivity. There is no conclusive evidence for aerosol or faecal-oral transmission of SARS-CoV-2 despite several researchers considering them as plausible routes that may explain the high infectivity and global spread of COVID-19 (Chen et al., 2020; van Doremalen et al., 2020; Wang et al., 2020a) . From the literature studied, concerns of COVID-19 infection through environmental contact pertain mainly to areas that lack proper sanitation and wastewater treatment, lack adequate solid waste management infrastructure, in areas where raw sewage is discharged directly into natural water bodies, and in cities where air pollution is problematic.  Robust evidence is needed to assess impact of air pollution, solid waste management, and sewage contamination of water bodies on COVID-19 spread and infectivity.
cord-302316-raf5rlkq	2020	US researchers studied the viral and cellular transcriptional response upon infection of cell cultures and in animal models with different respiratory viruses including influenza A virus and SARS-CoV-2. A French study randomizing 181 COVID-19 patients with pneumonia on hydroxychloroquine or placebo, observed, however, no significant effect of treatment on transfer to ICU, mortality, or in the prevention of development of acute respiratory distress syndrome (Mah evas et al., 2020). A total of 86 COVID-19 cases of patients from China with mild/moderate disease were randomized on the antiviral lopinavir (an inhibitor of HIV protease combined with ritonavir, which prolongs the presence of drugs in the body) or the antiviral arbidol (an influenza virus fusion inhibitor only registered in Russia) or in a control group in a 2:2:1 ratio. Effect of high vs low doses of chloroquine diphosphate as adjunctive therapy for patients hospitalized with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection: a randomized clinical trial
cord-302355-3se1wp8o	2018	Although XRN1 digestion of a 3''-terminal 800-nt RNA could stall at a position to generate the sfRNA in vitro, we found that knocking out XRN1 had no effect on the accumulation of sfRNA in Japanese encephalitis virus (JEV) infected cells. Furthermore, the minus-strand templates covering the putative promoter region used for an in vitro RdRp assay gave rise to synthetic products, suggesting that the JEV sfRNA could be initially transcribed from the antigenome and may be further trimmed by XRN1 or other unidentified exoribonucleases. Although efficient RNA replication is required for the detection of any flaviviral RNAs despite which mechanism used for the sfRNA formation, our results were clearly different from the observations from WNV that BHK-21 cells transfected with replicon constructs containing various deletions had no effect on the accumulation of sfRNA when compared to the WT [8] .
cord-302368-uhhtvdif	2016	Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure μs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Additionally, we show that the measurements of relaxation parameters using CPMG, R 1 , and CEST are possible for both small and large RNAs. Furthermore, we demonstrate substantial improvements in signalto-noise and line width for relaxation optimized spectroscopy (TROSY) experiments compared to the traditional heteronuclear single quantum coherence (HSQC) exNucleic Acids Research, 2016, Vol. 44, No. 6 e52 periments for isolated two-spin systems approximated by our purine and pyrimidine labeling schemes (30) (31) (66) (67) . Thus, RNAs synthesized with our selective site-specifically labeled NTPs should benefit from TROSY based NMR experiments that reduce the problems of crowding, fast signal decay, low resolution, and decreased S/N ratios (12, 34, 31, (66) (67) (80) (81) .
cord-302409-40ktyt5q	2020	OBJECTIVES: The aim of this paper was to monitor the presence of SARS-Cov-2 among hospital environment surfaces, sewage, and personal protective equipment (PPE) of staffs in isolation wards in the First Affiliated Hospital of Zhejiang University, China. The monitoring data in this study suggested that the strict disinfection and hand hygiene could decrease the hospital-associated COVID-19 infection risk of the staffs in isolation wards. Detection of SARS-CoV-2 RNA among health-care settings, sewage, and staffs'' PPE In routine cleaning and disinfection, the 36 samples of environmental surface in isolation wards including the clean area, the semi-contaminated area, and the contaminated area were all negative. With routine cleaning and disinfection, none of SARS-CoV-2 RNA was detected among object surfaces in isolation wards including the clean area, the semi-contaminated area, and the contaminated area. In conclusion, the SARS-CoV-2 RNA monitoring results of the hospital isolation wards demonstrated the routine disinfection measures of air, object surface and sewage in the hospital were sufficient and the hand hygiene of staffs was effective.
cord-302425-aaxvlktp	2019	In contrast, other RNA viruses including Kobuvirus, Astrovirus, Sapovirus, Sapelovirus, Teschovirus, and Torovirus, have been detected in pig faeces but its role as causative agents of neonatal diarrhoea has not so far been fully elucidated [10] [11] [12] [13] [14] . The results reported among the 47 diarrhoeic samples analysed include representatives of 12 virus species corresponding to 8 genera of RNA viruses (Additional file 1): Kobuvirus, Rotavirus (RVA, RVB and RVC), Sapovirus (SAV), Mamastrovirus (Porcine Astrovirus types 3 -AstV3 -, 4 -AstV4 -and 5 -AstV5 -), Alphacoronavirus (PEDV), Enterovirus (Enterovirus G, EntVG), Pasivirus (PasiV) and Posavirus (PosaV). Regarding KobuV, our results also agree with an increased prevalence of this agent observed in cases of diarrhoea in suckling piglets worldwide: Brazil [22] , Korea [29] and Vietnam [30] ; despite several (See figure on previous page.) Fig. 5 Neighbor-joining phylogenetic tree based on the p-distance among the nucleotide sequences of the VP7 segment for Rotavirus B.
cord-302830-5psqxxc8	2016	To characterize the torovirus RTCs, we generated specific sera for BEV M pro , Hel and RdRp, three key proteins in the formation and functioning of nidovirus RTCs, that were used in confocal and transmission electron microscopy (TEM) assays to analyse the location of viral components involved in BEV replication. It has been shown that in cells infected with different nidoviruses, virus-induced membrane reorganization produces characteristic DMVs, as well as other membranous structures, which have been related with the viral RNA replication and transcription processes. To investigate whether torovirus DMVs are the sites where replication and transcription occur, we obtained specific sera for the BEV M pro , Hel and RdRp, three key proteins in the formation and functioning of RTCs. All showed a high colocalization with each other and with newly synthesized RNAs. However, the dsRNA, widely used as a marker of RTC in several positive-strand RNA viruses, only showed a partial colocalization with the nsps.
cord-302895-471zei5o	2013	Biochemical studies using recombinant arterivirus and coronavirus helicases revealed similar enzymatic properties, including nucleic acid-stimulated ATPase and 5 0 -3 0 duplex unwinding activities on both RNA and DNA substrates containing 5 0 single-stranded regions (34, 35) . Amino acid substitutions in ZBD or the adjacent ''spacer'' that connects it to the downstream domain can profoundly affect EAV helicase activity and RNA synthesis, with most replacements of conserved Cys or His residues yielding replicationnegative virus phenotypes (36, 37) . Thus, our study not only highlights how nidovirus helicase activity depends on the extensive relay of interactions between the ZBD, accessory and HEL1 domains but also provides a framework to propose and explore a role for the enzyme in the posttranscriptional quality control of nidovirus RNAs. Nsp10 of the EAV-Bucyrus isolate (NCBI Reference Sequence NC_002532) is composed of amino acids 2371-2837 of replicase pp1ab, which will throughout this study be referred to as nsp10 residues 1-467.
cord-302980-2jlz4c58	1988	Summary Sequences encoding the N protein of the bovine enteritic coronavirus-F15 strain (BECV-F15) have been cloned in PBR322 plasmid using cDNA produced by priming with oligo-dT on purified viral genomic RNA. The 3′-non-coding end of the gene has an 8-nucleotide sequence in common with the homologous genome areas of MHV, TGE and IBV viruses. The location of the insert along the viral genome was determined by Northern blot analysis: full length or purified products of insert restriction cleavage were hybridized with poly(A) § RNA extracted from infected or non-infected cells. We have determined, by cDNA cloning of BECV-F15 genomic RNA using an oligo-dT primer, a sequence of 1,710 nucleotides. For every coronavirus so far studied, the gene coding for the N protein is located at the 3''-end of the viral genome. Recently [11] it was described for the US Mebus strain of the related bovine corona virus (BCV), that the N protein gene was at the 3''-end of the viral genome.
cord-303111-iv4lzpev	2014	Until recently, the study of CoV genetics was broadly restricted to the analysis of temperature-sensitive (ts) mutants Baric, 1992, 1994; Lai and Cavanagh, 1997; Schaad and Baric, 1994; Stalcup et al., 1998) , defective RNA templates which depend on replicase proteins provided in trans by a helper virus (Izeta et al., 1999; Narayanan and Makino, 2001; Repass and Makino, 1998; Williams et al., 1999) , and recombinant viruses generated by targeted recombination (Masters, 1999; Masters and Rottier, 2005 reverse genetic system devised for CoVs at a time when it was not clear whether the construction of full-length infectious cDNA clones would ever be technically feasible. These reverse genetic systems have been established using non-traditional approaches, which are based on the use of targeted recombination, BACs, in vitro ligation of CoV cDNA fragments, and vaccinia virus as a vector for the propagation of CoV genomic cDNAs. The availability of CoV full-length infectious clones and recombinant viruses expressing reporter genes constitute important tools for the study of CoV replication and transcription mechanisms, virus-host interaction and pathogenesis, and also for the rapid and rational development and testing of genetically defined vaccines.
cord-303153-z7bdiuvx	2010	This approach has allowed us to establish that MHV induces the formation of six membranous rearrangements in the following order: DMVs, CMs, virions, LVCVs, TBs and CMSs. Importantly, we were able to show that most membrane rearrangements (LVCVs, TBs, CMSs and possibly CMs) observed in addition to the key structures in the infection (DMVs and virions) actually appear to be the consequence of a massive synthesis of viral proteins. In order to be able to correlate our EM and IEM analyses with the progression of a CoV infection inside the host cells, we first measured important known parameters that reflect the CoV life cycle: viral RNA replication/transcription, viral protein synthesis and secretion of progeny virus. In the centre of the DMV clusters, we frequently observed a small network of membranes with a diameter varying from 200 to 600 nm, which have recently been described in SARS-CoV-infected cells and called CMs [ Fig. 2A and B, arrowheads; (Knoops et al., 2008) .
cord-303189-ktl4jw8v	2015	Acting in both autocrine and paracrine manner, IFN interferes with viral replication by inducing hundreds of different IFN-stimulated genes with both direct anti-pathogenic as well as immunomodulatory activities, therefore functioning as a bridge between innate and adaptive immunity. In these cells, the HCV-induced miR-21 has been recently reported to be involved in evasion of IFN-I production and stimulation of HCV replication, upon suppression of MyD88 and IRAK1 expression, that is required for the TLR7-mediated sensing of the virus [100] . Amongst RNA viruses that, as HCV, can establish a persistent infection, HIV-1, a lentivirus from the Retroviridae family, represents a paradigm for its ability to prevent or circumvent the innate immune response mediated by IFN-I. Overall, viruses as HCV and HIV-1 have evolved nifty strategies to dampen the host innate response in cells where a productive infection may take place, while they induce infection-independent mechanisms in non-permissive cells to facilitate the viral life cycle and promote a chronic inflammation.
cord-303265-v6ci69n0	2019	Topics covered include molecular mechanisms of genetic variation, with emphasis on high mutation rates, Darwinian principles acting on viruses, quasispecies dynamics and its implications, consequences for virus-host interactions, fitness as a relevant parameter, experimental model systems in cell culture, ex-vivo and in vivo, long-term virus evolution, the current situation of antiviral strategies to confront quasispecies swarms, and conceptual extensions of quasispecies to nonviral systems. With regard to the concepts of genome stability versus variation addressed in this book, it is helpful to divide viruses into four groups, depending on whether it is DNA or RNA the type of genetic material, which acts as a replicative intermediate in the infected cell (bottom gray shaded boxes in Fig. 1.1 ). They were selected for replicability, stability, and evolvability with trade-offs 1.4 Origin of life: a brief historical account and current views (acquisition of benefits for one of the three traits at some cost for another trait) likely play a role at this stage (see Chapter 4 for trade-offs in virus evolution).
cord-303319-v3iyur78	2019	The findings of these studies have suggested that antiviral responses are mediated by cytosolic or intracellular compartment sensors and their adaptor molecules (e.g., TLR, myeloid differentiation primary response 88, retinoic acid inducible gene‐I, IFN‐β promoter stimulator‐1, cyclic GMP‐AMP synthase and stimulator of IFN genes axis) for the primary sensing of virus‐derived nucleic acids, leading to production of type I IFNs, pro‐inflammatory cytokines and chemokines by the host cells. Thus far, many different immune evasion strategies employed by various viruses have been identified, including: (i) interference with the functions of the host innate immune response via physical interactions with viral antagonistic proteins targeted to sensors, adaptors, related intracellular kinases and transcription factors; (ii) inducing degradation or specific cleavage at the protein level; and (iii) sequestration of signal transduction molecules targeting the PTM systems.
cord-303377-lkewcf8a	2020	Our results show that RNA extracted using the AGPC method is fully comparable to modern automated systems regarding analytical sensitivity, specificity and accuracy with respect to detection of SARS-CoV-2 as evaluated by RT-qPCR. A total of 87 clinical sample specimens were chosen based on SARS-CoV-2 status from the Cobas ® 6800 system and used to evaluate the analytical sensitivity, specificity and accuracy of our in-house SARS-CoV-2 RT-qPCR assay after RNA purification using the Maxwell ® RSC 48 and AGPC methods. The AGPC method delivers high analytical sensitivity, specificity and accuracy for SARS-CoV-2 testing To evaluate whether conventional AGPC based extraction of RNA could serve as a viable alternative to automated systems with respect to reliability and accuracy, we isolated RNA using the AGPC method from 87 clinical specimens (oropharyngeal or nasopharyngeal swabs) with known SARS-CoV-2 status (57 positive and 30 negative), and performed a side-by-side comparison with the identical samples extracted on a Maxwell ® RSC 48 instrument.
cord-303403-9th2jiq6	2014	CypA is also known to play critical roles in the proliferation of a number of viruses, including human immunodeficiency virus type 1 (HIV-1), influenza virus, hepatitis C virus (HCV), vesicular stomatitis virus (VSV), vaccinia virus, severe acute respiratory syndrome coronavirus (SARS-CoV), rotavirus (RV) and human papillomavirus (HPV), by interacting with viral proteins or facilitating IFN-b production [2, 3] . CypA was further revealed to interact with extracellular CD147, which is the main receptor for CypA on the cell membrane of human leukocytes, and this interaction can induce the phosphorylation of HIV-1 matrix protein to regulate the liberation of the reverse transcriptase complex into cytoplasm during an early stage of HIV-1 infection or can function in HIV-1 attachment to host cells [8] . The results we report here demonstrate that the CypA host factor played a crucial role in the uncoating process during the entry step of EV71 infection, and the action site of CypA was mapped to the H-I loop of capsid protein VP1.
cord-303408-coesfldm	2007	BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) can be inhibited by means of RNA silencing or interference (RNAi) using synthetic short interfering RNAs (siRNAs) or gene constructs encoding short hairpin RNAs (shRNAs) or long hairpin RNAs (lhRNAs). Although these escape variants lost the ability to trans-inhibit HIV-1, they effectively outgrew the wild-type virus in competition experiments in SupT1 cells. In the current study, we constructed the HIV-lhNef variant, which contains a 300 bp extended hairpin structure at the 3'' genome position of the Nef gene of the otherwise wild-type HIV-1. HEK293T cells were transfected with the wild type HIV-1 construct or HIV-lhNef. We measured CA-p24 in the culture supernatant as a measure of virus production 2 days post-transfection. Compared to the effective HIV-lhNef inhibitor, all AS escape variants had lost the ability to actively inhibit wild-type virus production (Fig. 4B ).
cord-303533-6s01qplg	2014	This review takes a virus-centric look at the coronavirus replication transcription complex organelle in the context of the wider world of positive sense RNA viruses, examining how the mechanisms of protein expression and function act to produce the factories that power the viral replication cycle. This review takes a virus-centric look at the coronavirus replication transcription complex organelle in the context of the wider world of positive sense RNA viruses, examining how the mechanisms of protein expression and function act to produce the factories that power the viral replication cycle. Whatever their purpose, it is clear that the coronavirus organelle is dynamic [9] , closely tied to vesicular transport in the host cell [5, 10] , and consists mainly of paired membranes that form a variety of complex shapes including convoluted membranes and double-membrane vesicles (DMVs) [2, 11] . Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein
cord-303915-14yfs4pa	2013	The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. Using this clone, recombinant MERS-CoV (rMERS-CoV) deletion mutants were constructed lacking genes nonessential for virus replication. An infectious cDNA clone was assembled as a BAC under the control of the cytomegalovirus (CMV) immediate-early pro-moter, based on the genome sequence of the MERS-CoV-EMC12 strain (GenBank accession number JX869059) (15) . Based on published data showing that the deletion of CoV E protein resulted in either replication-competent, propagation-defective viruses (24) or attenuated viruses (25, 26, 31) , a cDNA clone with the E gene deleted (pBAC-MERS-⌬E) was constructed from pBAC-MERS FL .
cord-304014-k62mtr9j	2019	Many differentially expressed lncRNAs act as elements to competitively attach microRNAs (miRNAs) which target to messenger RNA (mRNAs) to mediate expression of genes that related to toll-like receptors (TLRs), NOD-like receptors (NLRs), tumor necrosis factor (TNF), and RIG-I-like receptors (RLRs) pathways. Meanwhile, we found that the differentially expressed lncRNA TCONS_00058367 might lead to a reduction of phosphorylation of transcription factor p65 (p-p65) in TGEV-infected IPEC-J2 cells by negatively regulating its antisense gene promyelocytic leukemia (PML). CONCLUSIONS: The data showed that differentially expressed lncRNAs might be involved in inflammatory response induced by TGEV through acting as miRNA sponges, regulating their up/down-stream genes, or directly binding proteins. The data showed that differentially expressed lncRNAs might be involved in inflammatory response induced by TGEV through acting as miRNA sponges, regulating their up/down-stream genes, or directly binding proteins.
cord-304044-i1ikf96b	2007	Here, we described specific silencing of five white spot syndrome virus (WSSV) genes in Litopenaeus vannamei in vivo with sequence-specific siRNAs. These genes included DNA polymerase (dnapol), ribonucleotide reductase small subunit (rr2), thymidine kinase and thymidylate kinase (tk-tmk), vp24 and vp28. control for interference action of siRNAs. Delivery of the selected sequence-specific siRNAs resulted in increase of survival rate of shrimp infected by WSSV, as compared to the virus injected controls (Fig. 1) . To examine if treatment of specific and mutant siRNAs resulted in a reduced expression level of the selected WSSV genes in shrimp in vivo, semiquantitative and highly sensitive RT-PCR analyses were used to compare the relative silencing effects. The results showed that sequence-specific siRNAs significantly inhibited replication of WSSV relative to that of positive controls and the mutant siRNA injected group at 24 h during experiment (Fig. 3) . Our data strongly indicated that sequence-specific siRNAs significantly inhibited expression of target genes and viral replication to protect shrimp from WSSV.
cord-304058-i8cywew0	2009	title: Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. In the context of viral host switching, it is interesting that several SARS-CoV proteins encoded on subgenomic (sg) RNAs seem to be dispensable for virus replication in cultured cells of primate or rodent origin, as well as in rodent models [17] [18] [19] . Both BACs were sequenced, confirming presence of all marker mutations and absence of any further mutations (refer to Influence on apoptosis and type I interferon induction by overexpression of ORF 7a, ORF 7b, and ORF 7b with the Frankfurt-1-specific deletion Interferon beta promoter-specific reporter gene expression (y-axis), shown as the factor of induction as compared to the mock-transfected, non-superinfected control (see below).
cord-304283-nv4ret1f	2006	Since only a readily available short synthetic DNA fragment is needed for constructing this novel reporter-based siRNA validation system, this system not only provides a powerful strategy for screening highly effective siRNAs but also implicates in the use of RNAi for studying novel gene function in mammals. Since only a readily available short synthetic DNA fragment is needed for constructing both the targeting reporter and triggering siRNA expression vectors, this novel system should not only greatly facilitate large-scale lossof-function genetic screens in mammalian cells but also provide the basis for an improved approach to screen and identify the most potent siRNA for the purpose of the therapeutics. As the results shown in Fig. 5A , the siLMP1-2 exhibited no inhibition effects on pEGFP-3UTR-siLMP1-2-induced EGFP and pLuc+-3UTR-siLMP1-2-induced firefly luciferase expression as compared with the two standard positive controls, siEGFP and siLuc, indicating that the selected RNAi targeting sequence siLMP1-2 was inactive and non-functional.
cord-304306-rxjahqwh	2020	The currently available antiviral option for hospitalized patients is remdesivir, which may inhibit the replication process by targeting the RdRp. Previously proposed treatments for hospitalized patients included hydroxychloroquine, which thought to disrupt virus endocytosis, and lopinavir/ritonavir, which thought to inhibit SARs-CoV-2 main protease (Astuti and Ysrafil, 2020; Magro, 2020) . Silibilin is predicted to have a dual activity against SARS-CoV-2 infection; silibilin can potentially reduce viral replication activity by targeting NSP12 as a remdesivir-like inhibitor, and modulate inflammatory responses by direct inhibition of STAT3 (BoschBarrera et al., 2020) . A recombinant form of the human ACE2 protein was synthesized as a therapeutic treatment for COVID-19, functioning as a decoy for SARS-CoV-2 and essentially preventing the virus from binding to the cell surface ACE2 (Schuster et al., 2010) . Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2): An overview of viral structure and host response
cord-304356-jyp9gjh9	2020	We performed single cell RNA-Seq in 5 bronchoalveolar lavage fluid samples collected from patients with severe COVID-19 within 48 hours of intubation. b. Sankey diagram illustrating relationship between number of BAL samples from participants with COVID-19, other viral pneumonia, non-viral pneumonia (other pneumonia) and non-pneumonia controls 1) enrolled in the SCRIPT study (534 samples), 2) analyzed via flow cytometry (344 samples), 3) bulk RNA-seq on flow-sorted alveolar macrophages (243 samples) and 4) single-cell RNA-seq (6 samples). To define the immune cell profile over the course of severe SARS-CoV-2-induced pneumonia, we analyzed 116 samples from 61 patients with confirmed COVID-19 in our cohort. As our analysis of transcriptomic data from alveolar macrophages suggested that SARS-CoV-2 pneumonia is uniquely associated with the activation of pathways induced by interferons, we looked for the expression of type I interferons in our single cell dataset.
cord-304424-048xo7jn	2018	That same year 36,789 colony sequences from DNase-treated RNA extracted from viral concentrates of human feces revealed a preponderance of pepper mild mottle virus and other plant viruses, but no new human viruses (Zhang et al., 2005) . While finding a new virus in the environment is not necessarily a problem in either DNA or RNA, the low fidelity of RNA polymerases and the sequence space they are capable of sampling, along with the possibility of recombination, lend themselves to new species and genera that are the trophies of metagenomic viral discovery. While metagenomics delivered on the promise of finding novel human viruses, viral discovery in humans has increasingly become a tragic story of patients interacting with the wrong squirrel or tick on the wrong day and most samples sequenced are frankly negative (Hoffmann et al., 2015; McMullan et al., 2012) . The greatest paradigm shifter in recent viral metagenomics work has been the sheer number and diversity of novel RNA viruses present in arthropods and invertebrates.
cord-304498-ty41xob0	2011	Genetic inactivation of exoN activity in engineered SArS-Cov and MHv genomes by alanine substitution at conserved De-D-D active site residues results in viable mutants that demonstrate 15-to 20-fold increases in mutation rates, up to 18 times greater than those tolerated for fidelity mutants of other rNA viruses. Genetic inactivation of exoN activity in engineered SArS-Cov and MHv genomes by alanine substitution at conserved De-D-D active site residues results in viable mutants that demonstrate 15-to 20-fold increases in mutation rates, up to 18 times greater than those tolerated for fidelity mutants of other rNA viruses. The high mutation rates of RNA viruses also render them particularly susceptible to repeated genetic bottleneck events during replication, transmission between hosts or spread within a host, resulting in progressive deviation from the consensus sequence associated with decreased viral fitness and sometimes extinction.
cord-304553-gbwb7fqi	2008	Nonspecific, broad-spectrum immune responses can be induced by double-stranded (ds)RNAs such as poly (ICLC), or oligonucleotides (ODNs) containing unmethylated deocycytidyl-deoxyguanosinyl (CpG) motifs. Poly (ICLC), a dsRNA, and CpG ODNs, molecular mimics for TLR3 and TLR9, respectively, have been reported to non-specifically stimulate the innate immune system and to provide protection against various bacterial and viral pathogens, including influenza. Mice treated with two doses of poly (ICLC), 48 h apart, up to 12 d prior to viral challenge were completely protected from infection, whereas survival rates decreased to 80, 40 and 0%, when pre-treatment was given 14, 16 or 20 d prior to virus challenge, respectively [32] . CpG ODN treatment of splenocytes from senescence-accelerated SAM-P1 strain of mice increased IFN-γ and administration in vivo generated virus-specific cytotoxic T lymphocyte responses, NK cell activation, virus-specific Ig isotype switch from IgG1 to IgG2a, increased viral clearance and survival following influenza viral challenge. Viral infection and Toll-like receptor agonists induce a differential expression of type I and lambda interferons in human plasmacytoid and monocyte-derived dendritic cells
cord-304794-z2kx314h	2014	Conversely, a G-quadruplex or G4 is formed by nucleic acid sequences (DNA or RNA) containing G-tracts or Gblocks (adjacent runs of guanines) and composed of various numbers of guanines. Short RNA templates from the central region of the HIV-1 genome contain G-rich sequences near the central polypurine tract (cPPT) at the 3 end of the pol gene (IN coding sequence); this is a region where one of the two primers used for synthesizing the (−) strand DNA is produced during reverse transcription. In addition, one could imagine alternative therapeutic strategies focused on targeting RNA structures within viral ORFs to interfere with the virus cycle as well as to promote antigen presentation and to stimulate the host immune response. Topology of a DNA G-quadruplex structure formed in the HIV-1 promoter: a potential target for anti-HIV drug development U3 Region in the HIV-1 genome adopts a G-quadruplex structure in its RNA and DNA sequence
cord-304873-ppb9k3zu	1998	Our S1, V1, and T1 endonuclease mappings, together with UV melting analysis, clearly indicate that this sequence element of the HIV-1 mRNA frameshift site forms a stem-loop structure, not a pseudoknot structure. The enhancer secondary RNA structure, either a stem-loop or a pseudoknot, downstream of the shift site induces pausing of the ribosome and stimulates w x slippage at the shift sequence 4-7 . Here, we determine the secondary structure of the HIV-1 mRNA frameshifting sequence elements including the shift site, the spacer, and the downstream enhancer sequence by using ultraviolet melting and enzymatic mapping analysis. Our results agree well with the mutational studies showing a simple stem-loop structure for the downstream enhancer sequence of the frameshifting region of the HIV-1 mRNA. Nucleotides A24-A27 were cleaved This cleavage pattern, as summarized in Fig. 4 , indicates that in the context of the frameshifting sequence elements of the HIV-1 RNA consisting of the spacer and the enhancer, the enhancer sequence adopts a stem-loop structure separated from the shift site by a spacer sequence.
cord-304876-txaoz7oh	2018	42 Viral polymerase: An important molecular target for antiviral therapy Nucleoside analogs represent one of the dominant classes of antiviral agents due to their widespread use against the common chronic infections caused by human immunodeficiency virus (HIV), hepatitis B virus, and herpesviruses. 43 After being metabolized by host kinases to their triphosphate form, antiviral nucleotides compete with natural nucleoside triphosphates (NTPs) to bind to the active site of viral polymerases and alter DNA or RNA synthesis. 122 However, the results summarized here indicate that nucleoside analogs targeting the viral RNA polymerase of rhinovirus, EV71, and other enteroviruses have the potential to be efficacious in preclinical animal models, providing a rationale to conduct human studies with safer molecules sharing the same mode of action. Structure and functional analysis of the RNA-and viral phosphoprotein-binding domain of respiratory syncytial virus M2-1 protein
cord-305290-xnjwv0d7	1990	A single base change in the mouse mammary tumor virus (MMTV) gag-pro shift site, from the normal A AAA AAC to A AAA AAG, surprisingly leads from ~2% to 50% -1 frameshifting at this sequence (a 25-fold increase); and the lo-fold decrease between A AAA AAG and A AAA AAA affords an interesting glimpse at how the nuances of codon-anticodon interaction can govern the efficiency of this type of shifting. These results should be interpreted with caution, as all the higher eukaryotic frameshifting studies with altered sequences have been done with a reticulocyte lysate cell-free translation system; there are likely to be differences in the number of ribosomes loaded per message, and perhaps in the tRNA balance, from less specialized cells in vivo.
cord-305336-wxiazglk	2014	In the present study, we showed that a plant-pathogenic RNA virus, tobacco ringspot virus (TRSV), could replicate and produce virions in honeybees, Apis mellifera, resulting in infections that were found throughout the entire body. While intracellular life cycle, species-level genetic variation, and pathogenesis of the virus in honeybee hosts remain to be determined, the increasing prevalence of TRSV in conjunction with other bee viruses from spring toward winter in infected colonies was associated with gradual decline of host populations and winter colony collapse, suggesting the negative impact of the virus on colony survival. Conventional RT-PCR was performed on RNA samples extracted from adult bees, Varroa mites, different tissues, and bee bread collected from the same colony for the presence and distribution of TRSV.
cord-305393-96mrxt8a	2011	Recombinant 1b hepatitis C virus polymerase was found to enhance TLR3 signaling in the lung epithelial BEAS-2B cells when added to the media along with either poly(I:C) or viral dsRNAs. The polymerase from the genotype 2a JFH-1 HCV was a poor enhancer of TLR3 signaling until it was mutated to favor a conformation that could bind better to a partially duplexed RNA. These results demonstrate that several viral RNA-binding proteins can enhance the dsRNA-dependent innate immune response initiated by TLR3. Transfection of two plasmids, one containing an interferon stimulated response element (ISRE) promoter-driven firefly luciferase and a second encoding a constitutively expressed Renilla luciferase allow the analysis of TLR3 activation by different RNAs. HEK 293T cells expressing WT TLR3 responded to poly(I:C) (1-2 mg/ml), better than viral dsRNAs (1-2 mg/ml) purified from Reovirus and BPEV (Fig. 1B) .
cord-305591-ir3wz6nr	2020	We have analyzed and identified 25 four contiguous GG runs (G(2)N(x)G(2)N(y)G(2)N(z)G(2)) in the SARS-CoV-2 RNA genome, suggesting putative G-quadruplex-forming sequences (PQSs). We confirm Gquadruplex structure forming in the top-ranked PQSs by multiple spectroscopic assays in vitro and characterize the crosstalk between G-quadruplexes and viral helicase by microscale thermophoresis (MST) and molecular docking. Our analysis of Gquadruplex-forming sequences in SARS-CoV-2 provides insights into the design of anti-viral treatment by targeting the viral helicase and G-quadruplex structures. Interestingly, PQSs at positions 13385 and 24268 with the highest G-scores indicating high probability to adopt G-quadruplex structures only share high sequence similarity to the bat CoVs (see Supplementary Information S1 available online at https://academic.oup.com/bib and Table 2 ). In comparison, our G-quadruplex search across the genome of SARS-CoV-2 also identified a number of GG PQSs. PQS at position 13385 was confirmed to adopt G-quadruplex structures, which also contains a [GAAAG] sequence in the middle ( Table 1 ).
cord-305737-bnzd7b25	2013	Targeting the viral Achilles'' heel: recognition of 5 0 -triphosphate RNA in innate anti-viral defence Jan Rehwinkel 1 and Caetano Reis e Sousa 2 Some RNA virus genomes bear 5 0 -triphosphates, which can be recognized in the cytoplasm of infected cells by host proteins that mediate anti-viral immunity. Both the innate sensor RIG-I and the interferon-induced IFIT proteins bind to 5 0 -triphosphate viral RNAs. RIG-I signals for induction of interferons during RNA virus infection while IFITs sequester viral RNAs to exert an antiviral effect. Recent work shows that the IFN system targets 5PPP RNAs during both phases: both RIG-I, a virus sensor that induces IFN expression, and IFITs, effector molecules that execute anti-viral activities, can specifically recognize 5PPP RNAs. As such, 5PPP RNAs appear to be Achilles'' heel of many RNA viruses in their interaction with the innate immune system (Figure 3a ).
cord-305811-987dhnf7	2013	Because both 59CC-WT and 13363-13520 constructs share 27 identical nucleotides upstream of their slippery sites, the attenuation activity difference is not likely to be caused by an E-site flanking sequences effect [12, 13] but rather by the disruption of the two potential AU base pairs. We noticed a potential to form four extra base pairs between 59and 39-flanking sequences (GACG and CGUU, respectively) of the 6BPGC hairpin stem (and other deletion mutants) due to the existence of a 59 SalI cloning site (Fig. S1A ). The results (Fig. S1C ) indicate that the two potential base pairs involving E-site sequences are not the main cause of observed attenuation activity in 293T cell cultures. Furthermore, mutating two nucleotides (27 nucleotides upstream of the E site) to disrupt Watson-Crick base pairs in the lower hairpin stem dramatically impairs attenuation activity (Fig. 2) , indicating that attenuation is not caused by primary sequencemediated flanking-sequences effects [12, 13] .
cord-305859-vt8vwo3y	2017	Fecal virus shedding, seroconversion and histopathology were evaluated in 3-7-year-old gnotobiotic calves orally inoculated with porcine deltacoronavirus (PDCoV) (9.0-9.6 log(10) genomic equivalents [GE] of OH-FD22-P5; n=4) or porcine epidemic diarrhea virus (PEDV) (10.2-12.5 log(10) GE of PC21A; n=3). In PDCoV-inoculated calves, an acute but persisting fecal viral RNA shedding and PDCoV-specific serum IgG antibody responses were observed, but without lesions or clinical disease. Our study demonstrated that Gn calves orally inoculated with the PDCoV strain OH-FD22 (ICs of cell-culture grown PDCoV from infected Gn pigs) develop an acute infection with persistent fecal PDCoV RNA shedding and PDCoVspecific serum IgG antibody responses, but show no signs of significant intestinal lesions or clinical disease [8] . In our study, there were no detectable fecal viral RNA shedding, virus-specific serum IgG antibody responses, histological lesions, and clinical disease in Gn calves orally inoculated with the PEDV strain PC21A (ICs of wild-type PEDV-infected Gn pigs) [6] .
cord-305871-w1quh4fx	2017	Furthermore, inoculation of inactivated plasma on Vero E6 cells did not result in any cytopathic effect (CPE) even after 7 days of incubation and three consecutive passages, nor the detection of MERS RNA compared to pretreatment samples which showed complete CPE within 2 to 3 days postinoculation and log viral RNA titer ranging from 9.48 to 10.22 copies/ mL in all three passages. Furthermore, inoculation of inactivated plasma on Vero E6 cells did not result in any cytopathic effect (CPE) even after 7 days of incubation and three consecutive passages, nor the detection of MERS RNA compared to pretreatment samples which showed complete CPE within 2 to 3 days postinoculation and log viral RNA titer ranging from 9.48 to 10.22 copies/ mL in all three passages. Similar to SARS-CoV, there is no proven evidence so far of transfusion-transmitted MERS-CoV infections, 25 but the presence of viral RNA in plasma and serum of acute patients raises this concern especially in endemic areas like Saudi Arabia.
cord-306076-ygfnkgqp	2013	Although the success in delivering siRNAs intranasally in rodents cannot be completely extrapolated to human use because of the significant differences in lung anatomy [37] , this approach has potential for the clinical application of siRNAs. Phase II clinical trials have been initiated for the treatment of respiratory syncytial virus (RSV) infection, making use of intranasal application of naked chemically modified siRNA molecules that target viral gene products [17, 38] (see Section 3.1.1. The simultaneously inhibition of several genes would also minimize the risk of drug resistance normally encountered with small molecule-based therapies, involving siRNAs and miRNAs. There have already been significant improvements in siRNAs for primary or metastatic lung cancer treatment by targeting oncogenes such as Akt1 [9] , Wilms tumor 1 (WT1) [12] , overexpressed genes such as the insulin-like growth factor receptor 1 (IGF-1R) [77] , NUPR1 [53] and EZH2 [78] .
cord-306288-w43wec48	2017	The aptamers and expression platforms were reassembled in a mix-and-match fashion to create chimeric riboswitches that retained their regulatory activities to turn off transcription upon ligand binding, and even combinations using artificial RNA aptamers were successful in regulating gene expression. Engineering of natural RNA-based regulation for dynamic control of gene expression requires detailed knowledge of each mechanism. Aptamers were placed upstream of IRES and several base-pairing sequences were designed to control the formation of critical structures (PK-III) depending on ligand binding (Figure 2b ). RNA molecules that regulate translation (IS10) or transcription (pT181) were combined with aptamers to control the formation of intramolecular structures depending on ligand binding [24] (Figure 2c ). Moreover, simultaneous utilization of high-throughput measurement of activity and next-generation sequencing analysis can not only screen and optimize dynamic RNA controllers, but also provide new design principles from sequence-structure-function relationships [14 ,15] .
cord-306424-gf0bglm0	2017	Thus, these data indicate that MAMs are critical locations for antiviral signaling and have an important role in expression of type I and III IFNs. Moreover, increasing evidence suggests that at least some +RNA viruses in fact occupy or hijack MAM-membranes during infection, as MAMs of HCV-infected cells were found to contain proteins involved in virus assembly and fully assembled virions [111] . At last, based on recent studies that demonstrated how IFN-g inducible GTPases are capable of disrupting PVs, we discussed the possibility of a general function of IFN-inducible GTPases in the targeting of viral ROs. In summary, upon infection, +RNA viruses hamper IFN and ISG induction at multiple levels to decelerate antiviral innate immune signaling. Antiviral innate immune response interferes with the formation of replication-associated membrane structures induced by a +RNA virus
cord-306535-j26eqmxt	2020	The majority of candidate genes identified in our screen that were testis-specific were already identified by the Human Protein Atlas [9] and/or our reanalysis of (See figure on previous page.) Fig. 1 Summary of the human and mouse RNA-seq samples used in the identification of novel male reproductive tract-specific drug targets. Additional file 14: Fig. S6 shows the complete list of male reproductive tract-specific human genes for which a previously generated mouse model shows male infertility phenotype, as identified in each of the respective cell and/or tissue datasets. Through the integration of hundreds of published and newly acquired human and mouse reproductive and non-reproductive tissue and cell RNA-seq datasets, we have generated a list of novel genes expressed predominantly or exclusively in the male reproductive tract that are worthy of consideration for functional validation in an animal model and potential targeting for a male contraceptive.
cord-306688-po4p1466	2008	Among other advances, BMV was used to define the first ribosome binding sites in eukaryotic mRNAs, and to produce the first template-selective eukaryotic viral RNA-dependent RNA polymerase extract, the first infectious transcripts from cloned RNA virus cDNA, the first engineered RNA virus expression vectors expressing foreign genes, the first definition of subgenomic mRNA synthesis pathways and determinants, and the first demonstration of higher eukaryotic viral replication in yeast. In parallel to in vitro systems, cultured plant protoplasts provided a valuable substrate for in vivo replication studies due to their ability to be infected or transfected with nearly 100% efficiency with virions, virion RNAs, or in vitro transcripts from cloned viral cDNAs. For BMV, barley protoplast systems developed and refined by the groups of Okuno and Furusawa, Hall and others in the late 1970s have allowed studies of all aspects of BMV RNA replication, subgenomic RNA synthesis, progeny RNA encapsidation, and the like.
cord-306754-qohrnpgq	2017	Our report provides advances in investigating several important aspects of TGC assay design, including (i) the range of fold enrichment possible for targeted nucleic acids, (ii) comparison of TGC pathogen detection with traditional methods currently used by diagnostic laboratories, (iii) documentation that a single assay design can be applied to more than one host species, (iv) the applicability of TGC to characterize pathogens in the context of veterinary medicine, and (v) the ability of TGC-NGS to characterize intrahost genetic diversity of viral pathogens (4) (5) (6) . Mean enrichment values of 250,000-fold and 9,100,000-fold with the DNA and RNA probes, respectively, and 147,000-fold for FIV contained in tissues clearly demonstrate the ability of the targeted capture probes to dramatically alter the relative abundance of specific nucleic acids across a range of sample types and diverse pathogen taxa.
cord-306921-3afgpunj	2019	A small siRNA screen targeting human membrane trafficking genes identified vasolin-containing protein (VCP-p97) as an important protein essential after PV viral replication and it interacts and colocalizes with 2 BC/2C as well as 3AB/3B in poliovirus infected cells [83] . Human host factors-viral protein studies identified nuclear factor; adenosine-uridine (AU)-rich element RNA binding factor 1 (AUF1) is targeted for cleavage by CV-B3 viral 3C protease upon translocation to the cytoplasm for enhanced stability of the IRES dependent viral RNA production [112] , similar antiviral observations were made for poliovirus, coxsackievirus and human rhinovirus [113] . A subsequent study by Mohamud and colleagues demonstrated that SQSTM1 and another host factor calcium binding and coiled-coil domain-containing protein 2/nuclear dot 10 protein 52 (CALCOCO2) regulate CV-B3 virus infection by targeting autophagy receptors; via their interaction with viral protein 1 [177] .
cord-306934-29ljbl7g	2009	Finally, molecular modeling investigations indicated that compounds of structure A–C, active against BVDV, could work targeting the viral RNA-dependent RNA-polymerase (RdRp), having been observed a good agreement between the trends of the estimated IC50 and the experimental EC50 values. First of all, the binding site identified by our procedure is very close to the putative binding site proposed for two allosteric inhibitors of BVDV RdRp, VP32947 and BPIP, 23 Table 5 Cytotoxicity against MT-4, MDBK, BHK and Vero-76 cell lines and YFV, Reo-1, CVB-2, RSV, HSV-1 and Sb-1 inhibitory activity of triazene derivatives of structure F and G Tables 3 and 4. In view of these considerations, molecular modeling investigations were performed to study wether the active compounds of structures A-C could target the BVDV RNA-dependent RNA-polymerase (RdRp), which shares some structural similarity with HCV RdRp. Indeed a good agreement was observed between the trend exhibited by the IC 50 (calculated from the estimated free energies of binding) and the corresponding biological activities determined for these compounds in BVDV infected MDBK cell line.
cord-306948-wkisfz1m	2014	Serologic marker candidates identified among B-cell linear epitopes of Nsp2 and structural proteins of a North American strain of porcine reproductive and respiratory syndrome virus A full-length cDNA infectious clone of North American type 1 porcine reproductive and respiratory syndrome virus: expression of green fluorescent protein in the Nsp2 region Identification of nonessential regions of the nsp2 replicase protein of porcine reproductive and respiratory syndrome virus strain VR-2332 for replication in cell culture Establishment of a DNA-launched infectious clone for a highly pneumovirulent strain of type 2 porcine reproductive and respiratory syndrome virus: identification and in vitro and in vivo characterization of a large spontaneous deletion in the nsp2 region Recovery of viable porcine reproductive and respiratory syndrome virus from an infectious clone containing a partial deletion within the Nsp2-encoding region Determination of the complete nucleotide sequence of a vaccine strain of porcine reproductive and respiratory syndrome virus and identification of the Nsp2 gene with a unique insertion
cord-307354-dkwcheu0	2015	Alpha-herpesviruses such as herpes simplex-1 (HSV-1) express a FEN1-like nuclease termed virion host shutoff protein (vhs) that is directed to mRNAs through interactions with the translation Fig. 1 . Quality control decay pathways such as NMD recognize aberrant mRNAs during translation, including the presence of premature termination codons (PTC), and induce endonucleolytic cleavage, whereupon the fragments are degraded by exonucleases. The RNAi pathway restricts gene expression by processing the long double stranded RNAs frequently generated during viral replication into short interfering RNAs (siR-NAs), which guide endonucleolytic cleavage of complementary target mRNAs. Although mammalian cells possess the RNAi machinery, in most cases RNAi does not appear to play a significant antiviral role, and has instead been supplanted by the protein-based interferon response (Cullen, 2014) . Small interfering RNAs that deplete the cellular translation factor eIF4H impede mRNA degradation by the virion host shutoff protein of herpes simplex virus
cord-307598-p54p7enk	2013	Similar to TLRs, RIG-I and MDA5 induce type I IFN and chemokines (but no IL12) upon activation by viral but also bacterial RNA. Since type I IFN induction by this RNA required RNase L, the authors concluded that RNase L recognizes and processes viral mRNA into a MDA5 activating structure. Before 5 triphosphate was identified as the crucial RNA modification to induce RIG-I activation, Marques and colleagues observed that synthetic blunt ended dsRNA oligonucleotides can stimulate RIG-I ( Fig. 3 ) (Marques et al. (2010b) confirmed the requirement of a base paired 5 -ppp end of dsRNA for RIG-I activation and suggested that some arenaviruses and bunyaviruses use a prime and realign mechanism for genome synthesis, leading to 5 overhangs in order to evade RIG-I recognition (Marq et al. pneumophilae did not induce type I IFN in HEK293 cells, thus excluding RIG-I-mediated recognition of RNA polymerase-III transcripts in the host cell, as previously suggested (Chiu et al.
cord-307603-uqr6r14u	2006	Several studies have demonstrated that LNA-modified oligonucleotides exhibit unprecedented thermal stability when hybridized with their DNA and RNA target molecules (Koshkin et al. 2005 ); (2) easy access-LNAs (fully modified or mixmers) are commercially available; (3) high-affinity and sequence-selective targeting of RNA molecules in vitro or in vivo Koshkin et al. Recently, it has been demonstrated that LNAzymes containing 3-4 LNA monomers at the ends of the binding arms cleave viral RNA structures that are resistant to hydrolysis by the corresponding unmodified DNAzyme, i.e., that efficient cleavage is correlated with improved binding affinity towards the target (Schubert et al. Efficient selection of polyadenylated mRNA from eukaryotic cells and tissues is an essential step for a wide selection of functional genomics applications, including full-length complementary (c)DNA library construction and sequencing, Northern and dot blot analyses, gene expression profiling by microarrays, and quantitative RT-PCR. LNA (locked nucleic acid): high affinity targeting of complementary RNA and DNA
cord-307817-2vy28i4m	2014	The prevalence of chronic viral infectious diseases, such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza virus; the emergence and re-emergence of new viral infections, such as picornaviruses and coronaviruses; and, particularly, resistance to currently used antiviral drugs have led to increased demand for new antiviral strategies and reagents. Based on the complex structure of the PA C-terminal domain (PA C ) and the first 25 amino acids of PB1 [99] , a subset of modifications on N-terminal peptide of PB1 was shown to diminish the binding affinity of PA and PB1, inhibit polymerase activity, and attenuate the replication of influenza virus [100] [101] [102] . Because both the polymerase complex and NP show significant conservation between different influenza viruses, these results demonstrated that targeting the formation of viral RNP is a valid approach to the development of small molecule therapies against serious antiviral resistance to currently available drugs, such as adamantanes or neuraminidase inhibitors.
cord-307860-iqk1yiw4	2020	Structures retrieved from PDB (August 12, 2020) were analyzed for relevant information on COVID-19 infection, synthesis of new inhibitors, SARS-CoV-2 interaction with host receptors, and the neutralizing antibodies interactions with spike glycoprotein. The first X-ray structure found (PDB ID 6LU7) belongs to the nonstructural protein 5 (3C-like protease) of the SARS-CoV-2 in complex with the Michael acceptor-based inhibitor N3 (PRD_002214). There is a cryo-EM crystal structure of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) complex (nsp12/nsp8/nsp7) with the antiviral drug remdesivir (PDB ID 7BV2) [37] . Previous studies on the crystal structures of SARS-CoV S glycoprotein mutants neutralized by 80R-specific antibodies have been considered a hope for the immunotherapeutic Fig. 8 The phylogenetic tree (cladogram) of the CoVs Spike (S) sequences of CoVs with different origin. Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites
cord-307893-mvl0wrsj	2014	Poliovirus was the first enteric virus to be widely recognized, causing foodborne disease outbreaks in the early 1900s associated with the consumption of contaminated raw milk. This method, as well as cultivation methods for the vaccine strain of poliovirus, eventually allowed for the quantification of infective virus plaque forming units and facilitated studies on detection and control of enteric viruses in water and foods, with a particular focus on molluscan shellfish. Although promising, the utility of these molecular amplification methods for virus detection in food and environmental samples was limited by low levels of contamination; high levels of matrix-associated inhibitory substances that interfered with nucleic acid amplification; and the lack of broadly reactive primers and probes for HuNoV. Recent epidemiological data continue to support the fact that viruses, particularly HuNoV, are the most common cause of foodborne disease of known etiology in USA. HEV is a positive-sense single-stranded RNA virus that is transmitted via the fecal-oral route, generally through the consumption of water and sometimes food that has become contaminated with human feces.
cord-307904-lnagg1uw	2003	One RNA, ␥KpnI/HpaI, provided a source of the ␥a replicase protein and also contained a 350-nt deletion (from positions 2112 to 2461 on RNA␥) to remove the majority of the ␥b ORF, and a second derivative designed to assess promoter activity contained deletions engineered within the putative sgRNA␥ promoter ( Fig. 2A) . This result implies that competition between the two promoters has a major role in regulating the differential rates of synthesis of the two sgRNAs. To initially define sequences flanking the sgRNA␤2 promoter, two large deletions were generated in the ␤1Ϫ34/ ϩ14 clone that eliminated RNA␤ positions 1705 to 2109 and 2287 to 2434. Analysis of four deletions extending from position Ϫ179 to positions Ϫ110, Ϫ76, Ϫ52, and Ϫ28 relative to the transcription start site revealed that the mutant RNAs were able to replicate in protoplasts, but only mutants containing the deletions Ϫ179/Ϫ110 or Ϫ179/Ϫ76 were able to direct synthesis of sgRNA␤2 (Fig. 4A ).
cord-307914-lgprrwee	2020	Indeed, the functionalization of CRISPR/Cas systems as a tool for genomic editing have revealed important differences in human and murine NA sensing, including distinct cell subset expression patterns of NA-sensing Toll-Like Receptors (TLRs) or species differences in the structural requirements for the detection of cyclic dinucleotide cGAMP by STING. As a long dsRNA with a 5 0 diphosphate terminus, polyI:C is known to activate the sensing receptors TLR3, MDA5, and RIG-I (Alexopoulou et al., 2001; Gitlin et al., 2006; Kato et al., 2008; Yoneyama et al., 2005) , accessory proteins such including LGP2 and members of the DDX and DHX families (reviewed in Oshiumi et al., 2016) as well as the restriction factors PKR and the OAS family (Farrell et al., 1978; Hovanessian et al., 1977; Zilberstein et al., 1978) .
cord-307934-84zfabti	2014	Further studies by cofractionation analysis and immunoelectron microscopy of the released particles showed that NS5A-Core complexes, but not NS4B, were present in the low-density fractions, but not in the high-density fractions, of the HCV RNA-containing virions and associated with the internal virion core. Overall, our results suggest that HCV NS5A is associated with the core of the low-density virus particles which exit the cell through a preexisting endosome/exosome pathway and may contribute to HCV natural infection. Both NS5A and Core proteins are found to be closely associated with and co-transported along the microtubules from the perinuclear region of cells via the LDs and endosomes to the plasma membrane. (A) The HCV-infected cells (at day 10 p.i.) were labeled with antibodies specific for Core protein (red) and NS5A (green) (upper row) or NS4B (green) (lower row).
cord-308034-9b219k0v	2014	We have employed gene-trap insertional mutagenesis to identify candidate genes whose disruption confer phenotypic resistance to lytic infection, in independent studies using 12 distinct viruses and several different cell lines. Expression of nine SNORA/Ds encoded within RCC1 (which also encodes the shorter non-coding SNHG3 gene), or SNHG1 were silenced with siRNAs for 2 days prior to infection with either cowpox virus (CPV), Dengue Fever virus (DFV), influenza A (FLU), human rhinovirus 16 (HRV16), herpes simplex virus 2 (HSV2), or respiratory syncytial virus (RSV). While a previous study showed that RCC1 supports HSV1 replication [21] , we did not observe that silencing RCC1 or the non-coding Small Nucleolar RNA Host Gene 3 (SNHG3) inhibits HSV2 (data not shown), whereas inhibiting expression of the RCC1encoded SNORA73A did. The discovery of these classes of non-coding genes prominently represented in the mutant clones selected in our virus surviving cell lines suggests an importance of SNORAs and SNORDs in facilitating viral replication.
cord-308216-s6rd8p41	2011	title: Small interfering RNA targeting for infected‐cell polypeptide 4 inhibits herpes simplex virus type 1 replication in retinal pigment epithelial cells Background: This study sought to inhibit herpes simplex virus type 1 replication using small interfering RNA which targeting infected‐cell polypeptide 4 genes to mediate transcription of early and late viral genes in herpes simplex virus type 1 lytic (productive) infection in retina epithelial cells. Compared with herpes simplex virus type 1 infection alone or transfection with negative control small interfering RNA, the viral titre and the retina epithelial cell cytopathic effect were significantly decreased in retina epithelial cells transfected with infected‐cell polypeptide 4‐targeting small interfering RNA (50 and 100 nM) (P < 0.05). The inhibition of infected‐cell polypeptide 4‐targeting small interfering RNA on infected‐cell polypeptide 4 protein expression was also verified by Western blot in herpes simplex virus type 1 infected human cornea epithelial cell, human trabecular meshwork cells and Vero cells.
cord-308331-55ge7kmr	2013	Using ViReMa, we demonstrate that by mapping the distribution and frequency of recombination events in the genome of flock house virus (FHV), we can discover de novo functional genomic motifs required for viral replication and encapsidation. Here, segments at the 5 0 and the 3 0 end of a complex recombination event have been mapped to nt 500-550 and nt 1040-1080 of FHV RNA 1, but there remain a small number of trimmed nucleotides in the middle. We generated 5 043 791 synthetic reads containing 99 033 unique recombination events and aligned these reads to the FHV genome with ViReMa using a seed length of 20 nt. Our analysis of FHV demonstrates that by isolating a small number of virus particles, deep sequencing the encapsidated RNA and mapping the positions of recombination events, functional RNA motifs can be discovered.
cord-308835-999kewdw	1981	Abstract Seven virus-specific, polyadenylated RNA species have been identified in mouse cells infected with the murine coronaviruses MHV-A59 (A59V) or MHV-JHM (JHMV). MHV-infected 17CL·1 cells were labeled with [32P]orthophosphate in the presence of actinomycin D and the cytoplasmic RNA was extracted and analyzed by agarose gel electrophoresis. The largest intracellular RNA species is identical to RNA isolated from purified virions, as determined by agarose gel electrophoresis and oligonucleotide fingerprint studies of ribonuclease T1 digests. Oligonucleotide fingerprints of the six subgenomic RNAS show that the sequences they contain are present in virion RNA, confirming their virus-specific nature. Identification of MHV-Specific RNA Species A59V, JHMV, and mock-infected cells were labeled with [32P]orthophosphate from 4 to 8 hpi in the presence of actinomycin D. To determine if there is temporal regulation of MHV-specific RNA synthesis, replicate cultures of A59V, JHMV, or mock-infected cells were pulse labeled for 1 hr at hourly intervals and the intracellular RNA was extracted and analyzed by gel electrophoresis.
cord-308884-erofmh39	2018	authors: Yang, Seung Won; Jang, Yo Han; Kwon, Soon Bin; Lee, Yoon Jae; Chae, Wonil; Byun, Young Ho; Kim, Paul; Park, Chan; Lee, Young Jae; Kim, Choon Kang; Kim, Young Seok; Choi, Seong Il; Seong, Baik Lin It should be noted that specificity of the antibody response to a reporter protein relative to the RID docking protein was not appreciably different regardless of the origin of an RID and the animal species to be immunized (compare Fig. 5A with 5D for eGFP and Fig. 5B with 5F for the HAgD), probably as a result of high homology (;80%) in an amino acid sequence between the murine and rabbit counterparts. D) ELISA data showing that according to the number of boosts, high-titer antibodies in serum samples from mice (n = 5) immunized with mRID-HAgD (20 mg/mouse) bound to the PR8 (H1N1) virus (10 4 PFU/well).
cord-309043-dlmx12vt	2007	The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. There are reports that a number of MHV and SARS-CoV replicase proteins colocalize and eventually interact in cytoplasmic membrane bound complexes, in which viral RNA synthesis occurs [18, 19] . We therefore cloned the SARS-CoV ORFeome by recombinatorial cloning (GATEWAY technology) and performed a genome-wide analysis for viral protein interactions by yeast-two-hybrid (Y2H) matrix screen. To systematically study the subcellular localization of viral proteins within eukaryotic HeLa cells the SARS-CoV ORFs were transfected in eukaryotic vectors with either N-or C-terminal Flag tags and detected with an anti-Flag antibody. In this study we report the cloning of the complete ORFeome of SARS-CoV and the results of a matrix-based yeast two-hybrid screen of pairwise viral protein-protein interactions.
cord-309048-emmtplv3	2018	(1999) displayed peptides of either 10 or 15 amino acids from the spike protein of the coronavirus murine hepatitis virus on the surface of assembled particles. coli-produced protein with a minimum of 20% of plant-made TMV CP, an approach that enabled efficient RNA-guided assembly of TMV-CP His6 into particles of the expected length (Eiben et al., 2014) . Assembly of the particle of tobacco mosaic virus from RNA and disks of protein β-Structure of the coat protein subunits in spherical particles generated by tobacco mosaic virus thermal denaturation Expression of tobacco mosaic virus coat protein and assembly of pseudovirus particles in Escherichia coli In vitro assembly of tobacco mosaic virus coat protein variants derived from fission yeast expression clones or plants Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications Display of peptides on the surface of tobacco mosaic virus particles Assembly of hybrid RNAs with tobacco mosaic virus coat protein.
cord-309469-2naxn580	2019	For example, polyadenylated nuclear (PAN) RNA encoded by Kaposi''s sarcoma-associated herpesvirus (Sun et al., 1996; Zhong and Ganem, 1997) , the~2-kb latency-associated transcript (LAT) expressed by Herpes simplex virus (Bloom, 2004 ), a con-served~5-kb intron expressed by Human cytomegalovirus (hCMV) (Kulesza and Shenk, 2004) , and a 7.2-kb RNA expressed by mouse CMV (Kulesza and Shenk, 2006) , U-rich RNAs (HSURs) produced by Herpesvirus saimiri (HVS), (Albrecht and Fleckenstein, 1992; Ensser and Fleckenstein, 2005) ; (3) Subgenomic ncRNAs from single-stranded RNA viruses by incomplete degradation of genomic RNA by the cellular 5-3′ exonuclease XRN1. The results confirmed previous report that IBV can synthesize sgRNA via template switch mediated by a noncanonical core sequence (Bentley et al., 2013) Notably, the mutant virus carrying four mutations (A27100U/A27111U/G27113C/G27114C) was also unable to produce ncRNA (Fig. 4) , suggesting these nucleotides are required for ncRNA production.
cord-309722-04pp3lv0	2016	Notes: AHR, airway hyperresponsiveness; ALI, Acute lung injury; BALF, bronchoalveolar lavage fluid; CD86, cluster of differentiation 86; C-kit, a stem cell factor receptor; DCs, dendritic cells; HMGB1A, high mobility group box-1 A peptide; IFU, infectious unit; LPS, lipopolysaccharide; Mpl, myeloproliferative leukemia virus oncogene; OVA, ovalbumin; R3V6, an arginine-rich peptide; Rip2, receptor-interacting protein 2; RSV, respiratory syncytial virus; S1Plyase, sphingosine-1-phosphate lyase, SOCS, Suppressors of cytokine signaling protein 3; STAT6, signal transducer and activator of transcription factor 6; Syk, spleen tyrosine kinase; Tf-PEI, transferrin polyethylenimine; T h 2, T helper 2 cells; TNF-α, tumor necrosis factor-α; VEGFR, Vascular endothelial growth factor. After siRNA targeting, SOCS3 was intranasally administered to the lungs of chronic asthmatic mouse model [12] , the silencing of SOCS3 down-regulated the expression of T h 2 cell associated cytokines, IL-4, IL-5 and IL-13, leading to substantial reduction of airway inflammation, AHR as well as IgE production.
cord-310086-9e4txeck	2020	Here, we report three confirmed cases of COVID‐19 whose IgM was negative and IgG was positive before the first discharge, while nasopharyngeal swab test of SARS‐CoV‐2 RNA turned positive again during hotel isolation. The SARS-CoV-2 RNA test of all three patients was positive before being admitted to hospital. The results of SARS-CoV-2 RNA test during the two periods of hospitalization were shown in Table 1 . Although no special discomfort was found, all patient''s nasopharyngeal swab test of SARS-CoV-2 RNA were positive during the isolation. The detoxification procedure does occur which will cause re-detectable positive SARS-CoV-2 RNA in recovered COVID-19 Accepted Article patients [6] . However, these patients re-detectable positive for SARS-CoV-2 RNA displayed high levels of IgG and negative IgM in the plasma during two hospitalization periods. Results of SARS-CoV-2 RNA and Antibody tests of re-admission patients Recurrence of positive SARS-CoV-2 RNA in COVID-19: A case report
cord-310141-2jofy8fo	2018	Short interfering RNA technology affords a potential tractable strategy to combat viral pathogenesis because siRNAs are specific, easy to design, and can be directed against multiple strains of a virus by targeting their conserved gene regions. For example, siRNAs directed against different genes of deadly viruses like human immunodeficiency virus (HIV), 9, 10 influenza virus (INFV), 11, 12 hepatitis B virus (HBV), 13 SARS coronavirus (SARS-CoV), 14, 15 human papillomavirus (HPV), 16 and West Nile virus (WNV) 17 in infected cells displayed encouraging results in inhibiting viral replication. 18, 19 Short interfering RNAs for various human viruses like respiratory syncytial virus (RSV), hepatitis C virus (HCV), HBV, and HIV are also appearing in clinical trials, which further elucidate their importance in inhibiting viral infections. Effective small interfering RNAs targeting matrix and nucleocapsid protein gene inhibit influenza A virus replication in cells and mice
cord-310192-8x37nx4s	2019	In this review, we will give a broad overview of different methods for structure determination and discuss some recent advancements in both sample preparation and data acquisition that have advanced the study of large RNAs by nuclear magnetic resonance (NMR) spectroscopy. Base-pairing, hydrogen bonding, nucleotide accessibility and other secondary structure-related characteristics, can be obtained by treating RNAs with specific chemical reagents (typically dimethyl sulfate [DMS] and/or one or more SHAPE reagents) and probing for the sites of modification by sequencing (Cordero, Kladwang, VanLang, & Das, 2012; Merino, Wilkinson, Coughlan, & Weeks, 2005; Tian & Das, 2016; Weeks, 2010) . Traditionally, these studies have been applied to relatively small RNAs. The ability to site-specifically incorporate 13 C labels within an RNA ribose and/or base makes NMR spectroscopy a powerful tool for dynamics studies of large RNAs, expanding the types of experiments that can be conducted and simplifying analysis (see data acquisition and analysis advancements below).
cord-310268-8q4tk6fd	2015	Nucleic acid aptamers are RNA and single-stranded (ss) DNA oligonucleotides with lengths typically ranging from 15 to 70 mers, which have the same level of target-binding affinity as monoclonal antibodies (the dissociation constant (K d ) usually ranges from 0.1 to 50 nM) [5, 6] . This SELEX is able to generate DNA aptamers that recognize the cell-surface or intracellular target protein in their native conformation, which shows great potential in cell-specific therapeutics and diagnostic applications [26] [27] [28] . Recently, modified cell-SELEX methods have been developed to select aptamers targeting specific cells like disease state cells and metastatic cells. In the era of personalized medicine, DNA aptamer-based therapeutics and diagnostics are believed to have great potential for extensive application because of their flexibility to specifically bind to any molecule targets.
cord-310371-pylrg91h	2008	The onset of acute enteritis is associated with infection by viruses that replicate at or near the site of entry into the intestinal mucosa, including caliciviruses, rotaviruses, adenoviruses, astroviruses, and coronaviruses. . viruses causing localized inflammation at any level of the intestinal tract, predominantly in small intestinal mucosa, resulting in acute gastroenteritis, for example, rotaviruses, caliciviruses, adenoviruses, astroviruses; . The family Caliciviridae contain small RNA viruses that cause enteric disease in a wide variety of hosts including cattle, pigs, rabbits, and humans. Caliciviruses causing enteric infections (in humans and other animals) are classified as belonging to the family Caliciviridae, which is divided into four genera. The recent demonstration that human noroviruses can infect and replicate in a three-dimensional cell culture model of human intestinal epithelium, should improve our understanding of the pathogenesis, and antigenic diversity of this important group of enteric viruses.
cord-310605-r63sg73c	2020	Here we report an aberrant immune response in fatal Covid-19, principally involving the lung and reticuloendothelial system, that is not clearly topologically associated with the virus, indicating tissue-specific tolerance of SARS-CoV-2. This supports prioritising pathogen tolerance as a therapeutic strategy in Covid-19, by better understanding non-injurious organ-specific viral tolerance mechanisms and targeting aberrant macrophage and plasma cell responses. As analysis of SARS-CoV-2 RNA confirmed presence in numerous organs, detailed histological analysis of multiple tissues was undertaken on every patient to determine the associated pathological consequences and inflammatory responses. The present study shows that fatal Covid-19 is associated with variable but widespread distribution of viral RNA and protein but with a discordant inflammatory response to local viral presence, both between and within tissues, demonstrating tissue-specific tolerance of SARS-CoV-2.
cord-310748-ao29zx1u	1991	Our results showed that within a 1-kb region of the peplomer gene, RNA recombination occurred at almost every potential crossover site. To study RNA recombination in the absence of selection pressure, we developed a polymer-ase chain reaction (PCR) assay using two primers specific for the potential recombinant viruses which have a crossover site between the two primers. Only recombinant RNAs which had a crossover between the two primers and contained A59-specific sequences on the 5''-side and JHM-DL-specific sequences on the 3''-side could be detected by this PCR approach. DNA sequence analysis of 35 cloned PCR products showed that the crossover sites were almost randomly distributed throughout the nearly 1-kb region of the peplomer gene studied ( Fig. 2A) . Analysis of 53 recombinant clones revealed that, similar to the intracellular recombinants, the crossover sites in the viral recombinant RNAs were almost randomly distributed over the 1 -kb region of the peplomer gene (Fig. 2B) .
cord-310771-tnwfp1je	2005	A method was developed for qualitative and quantitative detection of the seminal cell-associated PRRSV RNA in relation to endogenous and exogenous reference RNAs. As endogenous control for one-step real-time reverse transcription (RT)-PCR UBE2D2 mRNA was selected. Particularly for the analysis of persistent infections associated with low copy numbers of PRRSV RNA, UBE2D2 mRNA is an ideal control due to its low expression in seminal cells and its detection in all samples analysed (n = 36). For the development of one-step real-time RT-PCR for four endogenous reference RNAs (HPRT, UBE2D2, PPIA, and HMBS) appropriate target regions were selected and the assay conditions were optimised for amplification efficiency. One-step real-time RT-PCR assays for PRRSV-1 and -2 RNA allowed quantitation with optimal efficiency (Fig. 1a ; standard curves for two additional viremic pigs infected with PRRSV-1 (data not shown)) as achieved for endogenous and Fig. 1 .
cord-310861-9kb0b6rq	2017	In this study, we report an isothermal and rapid one-step RNA amplification/detection (iROAD) assay to simultaneously amplify and detect the viral RNA in a label-free and real-time manner. Furthermore, we demonstrated the clinical utility of the iROAD assay by detecting respiratory viral RNAs extracted from the 63 nasopharyngeal samples with either IFN-A/B or HCoV-OC43/229E or RSV-A/B. Subsequently, the iROAD assay detected the respiratory viral targets from the clinical sample by measuring the resonance wavelength shift within 15-20 min (Fig. 1) . In case of the iROAD assay with label-free and real-time capabilities, the wavelength shift was sequentially increased based on the concentrations (2.5×10 1 to 10 5 copies/reaction) of IFN-B within 20 min, as compared to the negative control. In the case of the real-time RT-PCR assay, the fluorescent SYBR Green signal was observed in RNA samples diluted up to 2.5×10 2 copies/reaction and showed good linearity (R 2 =0.9795) for different concentrations of the target (Fig. 3C ).
cord-310920-itqwhi6a	2020	Each of these techniques provide important vantage points to understand the complexities of virus-host interactions, but we attempt to make the case that by integrating these and similar methods, more vivid descriptions of how viruses reprogram the cellular environment emerges. Obtaining structural details of the UTRs and identifying functional binding sites of RBPs will be deeply insightful in elucidating how this virus replicates within host cells. Given the large number of RBPs known to interact with genomic and subgenomic viral RNAs to modulate translation, replication and the shift between these two stages, CLIP-seq can be employed to understand virology at the molecular level. Studying RNA structural interactions and the effects of viral-host RBPs on RNA structure and function are essential for understanding translation, replication, and transcription processes in order to better understand how viruses reprogram the cellular environment.
cord-310947-aqau2n7q	2008	In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. However, the viral protein interaction maps have been generated until now only for a limited number of viruses, including T7 bacteriophage [1] , vaccinia virus [2] , potato virus A [3] , pea seed-borne mosaic virus [3] , wheat steak mosaic virus [4] , hepatitis C virus [5, 6] , porcine teschovirus [7] , Kaposi sarcoma-associated herpesvirus [8] , and very recently severe acute respiratory syndrome coronavirus (SARS-CoV) [9, 10] .
cord-310967-15mv5yx7	1989	One of these variants, ATIIf cord virus, which induced a chronic demyelinating disease in 2or 10-day-old intracerebrally inoculated Wistar Furth rats, had a deletion in the coding region of the peplomer glycoprotein mRNA. In this report, we investigated viral variants that arose in the CNS of rats with a JHM-induced demyelinating disease and studied the effect of alterations in their mRNAs. When lo-day-old rats were inoculated with ATllf brain virus or ATlIe brain virus, the rats developed a rapid encephalitis instead of the more chronic demyelinating disease that has previously been seen with wild-type parental JHM virus (Sorensen et al., 1980; Jackson et al., 1984) and was observed with ATIIf cord virus-injected rats. In contrast, when 2-day-old rats were inoculated with ATllf cord virus, the more chronic CNS disease resulted instead of the rapid encephalitis that has been reported for wild-type JHM (Sorensen et a/., 1980; Parham et a/., 1986) and was observed with the ATIIf brain virus variant.
cord-311007-0i1abjfa	2016	High levels of infectious virus were produced following transfection of the plasmid bearing the wild-type MR766 ZIKV genome, but not one with a disruption to the viral nonstructural protein 5 (NS5) polymerase active site. Multicycle growth curve and plaque assay experiments indicated that the MR766 virus resulting from plasmid transfection exhibited growth characteristics that were more similar to its parental isolate than previously published 2010 Cambodia and 2015 Brazil cDNA-rescued ZIKV. Indeed, sequence analysis of bacterial plasmid clones of these RT-PCRs demonstrated that all products from wild-type plasmid-transfected cell RNA lacked the inserted intron (Fig. 3B) . In contrast to parental virus RT-PCR products (Fig. 3B) , these sequences carried a single silent G3127A mutation that was inserted during intron cloning, which indicated that these RNAs were generated from the MR766 plasmid. The addition of supernatants from wild-type plasmid-transfected or parental virus-infected 293T cells resulted in readily detectable levels of viral proteins.
cord-311625-d7iycdyh	2014	The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log(10) from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. The data analyzed by one-way ANOVA, Tukey post hoc test showed significant high viral RNA genome copy number of 4.03 × 10 14 for virus inoculated cells as compared to circular TFO1, TFO3, TFO4, and TFO5 treatments ( ≤ 0.05). In this study, circular Triple Helix Forming Oligonucleotide (TFO) RNAs, specifically targeting the short regions of viral genome for triplex formation, were designed and evaluated.
cord-311628-ep795pil	2016	Our objective here is to review the novel delivery platform based on Bacteriophage MS2 virus-like particles (VLPs), including introduction to their structure, their potential as a delivery platform, and their expected use in medicine and other fields. A series of research findings showed that an MS2 VLP-based vaccine can effectively induce innate and cognate immune responses and can be used as a specific preventive intervention in some diseases, such as foot-and-mouth disease (Bittle et al., 1982; Dong et al., 2015; Van Lierop et al., 1992; Wong et al., 2000) , prostate cancer (Li et al., 2014) , and illnesses caused by human papilloma virus (HPV) (Tumban et al., 2012) . These particles offer an effective and convenient way to package RNAs, DNAs, epitope peptides, and drugs into bacteriophage capsids, forming different kinds of VLPs. MS2 VLPs can not only deliver various kinds of agents with a good safety profile and strong immunogenicity but also ensure tissue-specific targeting, which is determined by the species of the virus.
cord-311982-wkg56xeq	2007	Comparisons of the enteric (jejunum) and non-enteric (liver) derived viral RNA sequences revealed 100% nucleotide identity, a finding that questions the well accepted ''internal mutation theory'' of FIPV pathogenicity. Comparisons of the enteric (jejunum) and non-enteric (liver) derived viral RNA sequences revealed 100% nucleotide identity, a finding that questions the well accepted ''internal mutation theory'' of FIPV pathogenicity. Furthermore, the structural and accessory gene regions of viral RNA isolated from the liver of the same cat (FCoV C1Li) were sequenced and the data derived from the enteric (jejunum) and non-enteric (liver) sources were compared. Sequence data previously generated for the laboratory strain FCoV, FIPV 79-1146, were used to design primers for conventional reverse transcriptase polymerase chain reaction (RT-PCR) amplification of short lengths (100e500 bases) of the field strain RNA. Analysis of the accessory gene 7 region of the FCoV C1Je genome identifies two ORFs, which have translation products sharing high amino acid identity with proteins 7a and 7b of FIPV 79-1146.
cord-312001-8p7scli8	2019	Consequently, animal cells have evolved devoted pathways which (1) sense and recognize pathogen-associated molecular patterns (PAMPs) and, more particularly, virus-associated molecular signatures; (2) initiate signaling cascades stemming from the site of detection, translocating the information to the nucleus; and (3) induce a transcriptional program that confers an antiviral state to the host ( Figure 1 ). While the cytosolic recognition of viral RNA is almost exclusively mediated by RLRs, several proteins have been proposed to play a role in DNA sensing and triggering innate immune responses, such as the DNA-dependent activator of IFN-regulatory factors (DAI), DDX41, RNA polymerase III, IFI16 and DNA-PK [62] [63] [64] [65] [66] [67] . Although the pathway leading to the transcriptional activation of Vago is still poorly understood in insects, these studies established that DExD/H-box helicase containing proteins, like Dicer and RLRs, may represent an evolutionarily conserved set of viral nucleic acid sensors that direct antiviral responses in animals [159] .
cord-312223-qgwzgazd	2013	RESULTS: Screening of NanoTrap particles indicated that one particle, NT53, was the most efficient at RVFV capture as demonstrated by both qRT-PCR and plaque assays. RVFV that was inactivated through either detergent or heat treatment was still found bound to NT53, indicating the ability to use NanoTrap particles for viral capture prior to transport to a BSL-2 environment. Our study demonstrates that NanoTrap particles are capable of capturing whole virus, and can be assayed with both qRT-PCR and plaque assays. A) Seven different types of NanoTrap particles were incubated with viral supernatants containing RVFV (1E+7 pfu/ml) for 30 minutes at room temperature and washed 4 times with water. In order to determine if the amplification observed in the qRT-PCR assay was due to the NanoTrap particle capturing intact viral particles or association of viral RNA (presumably due to lysed virus) with the particles, plaque assays were performed.
cord-312240-0k8y86pf	2017	Nasopharyngeal/oropharyngeal (NP/OP) swabs from 70 children <5 years with CAP of unknown etiology and 90 asymptomatic controls were tested by next-generation sequencing (RNA-seq) and pan viral group (PVG) PCR for 19 viral families. We first assessed the ability of RNA-seq and PVG PCR to detect known respiratory pathogens using specimens from children with CAP (n = 63) and asymptomatic control (n = 52) subjects in whom viral or atypical bacterial pathogens had been detected using the EPIC study protocol. We validated RNA-seq and PVG PCR methods to detect known respiratory pathogens by testing specimens using both methods from children with CAP (n = 63) and asymptomatic control subjects (n = 52) in whom viral or atypical bacterial pathogens had been detected using the EPIC study protocol. Using RNA-seq and PVG PCR, we identified additional viruses from upper respiratory tract specimens in >30% children hospitalized with clinical and radiographic pneumonia but in whom no pathogen was identified despite extensive testing by culture, molecular, and serologic methods.
cord-312332-rwmuucsp	2020	title: The importance of virion-incorporated cellular RNA-Binding Proteins in viral particle assembly and infectivity Different proteomic studies have identified hundreds of cellular factors within the particles of several RNA viruses [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] , many of which are RBPs. Here, we discuss the ''knowns'' and ''unknowns'' of the roles that virion-incorporated cellular RBPs could play in the assembly of viral particles and the early steps of infection in the new host cell. Many ivRBPs such as annexins, heat shock family proteins (HSP), peptidylprolyl isomerase A (PPIA -also cyclophilin A), eukaryotic translation elongation factors (EEF), heterogeneous nuclear ribonucleoproteins (HNRNP) or poly(rC) binding protein 1 (PCBP1), have been linked to infection in multiple ways (Fig. S2) , and here we show that they are incorporated in the particles of several viruses (Table S1B) .
cord-312392-8zxl48af	2006	We report an outbreak of fatal disease in puppies caused by a pathogenic variant of CCoV that was isolated from organs with severe lesions. NSP 3b (22 aa) was 49 aa shorter than expected because of a 38-nucleotide deletion and a frame shift mutation in the downstream sequence that introduced To confirm the pathogenic potential of strain CB/05, we experimentally infected two 6-month-old dogs (authorization no. Experimental infection of dogs with the virus isolate resulted in a severe systemic disease that mimicked the clinical symptoms observed in the outbreak. Epidemiologic studies are required to determine whether the pantropic CCoV strain is a new coronavirus variant emerging in canine populations or a widespread infectious agent of dogs that usually goes undetected. Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease Genotype-specific fluorogenic RT-PCR assays for the detection and quantitation of canine coronavirus type I and type II RNA in faecal samples of dogs
cord-312431-de7zhswl	2013	These results indicate the potential of viruses in the water samples especially from the lower catchment areas of the Umgeni River to infect human hosts throughout the year. It is well recognised that monitoring the presence of enteric viruses could be challenging due to the relatively low level of infectious viral particles towards the respective host species and small viral particle size existing in environmental waters, thus making it essential to start with a large water sample volume and concentrate it to several orders of magnitude [27] [28] [29] [30] [31] . The present study was conducted to optimise procedures to extract and enumerate indigenous virus-like particles (VLPs) and to determine the community structures and infectivity of these viruses from river water. Canonical correspondence analysis (CCA) was used to reveal the association amongst the bacteriophages, VLPs and the physical and chemical water quality variables, which were measured from the same sites and seasons in concurrent studies performed in this laboratory [46] , with a view to defining the significant variables accountable for the observed spatial and temporal distribution of the communities.
cord-312461-5qzpo6l1	2019	A substantial proportion of pandemic and biological threat preparedness activities have focused on list-based approaches that were in part based on pandemic influenzas of the past, historical biological weapon development programs, or recent outbreaks of emerging infectious diseases (e.g., SARS, MERS, Ebola) (Centers for Disease Control and Prevention 2017; Casadevall and Relman 2010) . Cultivating and maintaining expertise in the epidemiology, surveillance, and pathogenicity of all classes of microbes, with explicit incorporation of a One Health approach-which incorporates and integrates information from infectious diseases of plants, amphibians, and reptiles-will help foster the broad capacities needed for emerging pandemic and global catastrophic biological risks. Pathogen-based lists, both USA and global, based on influenza precedents, historical biological weapon programs, and emerging infectious diseases were responsible for galvanizing early activities in the field of pandemic preparedness and have helped drive many important contributions.
cord-312517-b24zlaqt	2020	title: The Brighton collaboration standardized template for collection of key information for benefit-risk assessment of nucleic acid (RNA and DNA) vaccines The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) has prepared a standardized template to describe the key considerations for the benefit-risk assessment of nucleic acid vaccines. The Brighton Collaboration formed the Viral Vector Vaccines Safety Working Group (V3SWG) in October 2008 to improve the ability of key stakeholders to anticipate potential safety issues and meaningfully assess or interpret safety data, thereby facilitating greater public acceptance when viral vector vaccines are licensed [2] . When completing information on adverse effects in Section 8, please provide as many details as possible based on the Brighton Collaboration Guidelines for collection, analysis and presentation of vaccine safety data in pre-and post-licensure clinical studies [13] .
cord-312544-vip4jtlv	2006	METHODS: A one-step reverse-transcription PCR assay was developed to detect the H5N1 avian influenza A virus. RESULTS: Validation on 145 field specimens from Vietnam and Malaysia showed that the assay was specific without cross reactivity to a number of other infuenza strains as well as human respiratory related pathogens. In this study, we describe the development of a nucleic acid detection test that is rapid, specific and sensitive, thus allowing greatly improved detection of the H5N1 avian influenza A virus. To establish the specificity of the assays for H5N1 subtype detection, we then tested the primers on several known strains of influenza A viruses derived from avian sources (H3N8, H5N3, H7N3 and H9N2). A total of 145 field samples comprising of known and suspect cases from chickens, ducks and muscovies isolated from Vietnam and Malaysia during the 2004 to 2005 outbreak were tested for H5N1 RNA (Table Detection of H5N1 avian influenza A virus by one-step RT-PCR 2).
cord-312688-12san3m7	2016	title: Filovirus proteins for antiviral drug discovery: A structure/function analysis of surface glycoproteins and virus entry After replication of the viral genome and RNA transcription, nascent viral particles are assembled in a process mediated by the matrix protein VP40, and virus budding occurs at the cell surface membrane in a process that involves hijacking the host ESCRT machinery (Hartlieb and Weissenhorn, 2006; Noda et al., 2006) . However, no direct interaction between both molecules has been demonstrated yet, and recent studies suggest that a 5 b 1 -integrin is not required for GP-mediated binding of internalization, but rather is a positive regulator of cathepsins, which play an important role in processing GP 1 into its fusion-competent form within the endosomes of infected cells (Schornberg et al., 2009) . After attachment mediated by interaction between the filovirus surface protein GP 1,2 (PDB: 3CSY) and various attachment factors, the complex is internalized and routed to the endosome, where GP 1,2 is processed to trigger fusion of viral and host membranes.
cord-312741-0au4nctt	2020	160, 161 Once the PAMPs from invaded viruses are detected, RIG-I and MDA5 interact with the mitochondrial antiviral signaling protein (MAVs) that is a mitochondrial membrane-bound F I G U R E 2 Escape mechanisms of innate immune response of SARS-CoV and MERS-CoV adaptor molecule, followed by the activation of several kinase complexes and multiple subsequent transcription factors (IRF3, IRF7, and NF-κB). Antiviral peptides analogous derived from these regions exhibited inhibition to the spike protein-mediated cell-cell fusion and viral entry in viruses such as SARS-CoV, MERS-CoV, as well as HCoV-229E. Receptor-binding domain of severe acute respiratory syndrome coronavirus spike protein contains multiple conformation-dependent epitopes that induce highly potent neutralizing antibodies Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response Inhibition of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infectivity by peptides analogous to the viral spike protein
cord-312886-o3ipzn05	2014	Viral infection and stress granules Viral invasion and replication are detected by innate immune sensors in cells, triggering downstream signaling pathways that can ultimately result in the activation of systemic immune responses. In some cases these bodies have been given different names in an attempt to distinguish them from SGs; in this review, however, we refer to virusinduced SG-like granules collectively as SGs. Many viruses induce SGs through the activation of the eukaryotic translation initiation factor (eIF)2a kinases PKR and, in some cases, general control non-depressible 2 (GCN2), which are both triggered by detection of RNA in the cytoplasm [28] ( Figure 2 ). In the stress-recovered condition, GADD34 protein is rapidly downregulated by an unknown mechanism and the phosphorylated form of eIF2a reaccumulates in the cells, resulting in an oscillating pattern of SGs. In cases where viral infection appears to not induce SGs, accumulating evidence suggest that these viruses inhibit SG formation.
cord-312892-p72zwmtb	2017	It in turn causes the multimerization of cytoplasmic TIR domains, which will recruit downstream adaptors TRIF or MyD88 through homotypic interaction, further forming signaling complex called signalosome and activating downstream transcription factors: one is NF-jB that induces proinflammtory cytokines, another is interferon regulatory factor (IRF) that induces anti-viral type I Interferon (IFN) (6) . The summary of cellular localizations and distributions, ligand recognitions, activation mechanisms, cell signaling, recognition of pathogens, and cross-talks for RNA PRRs (2) TLR3 (2) RIG-I (2) The understanding of RNA PRR immune biology including the ligand recognitions, cellular localizations, cell signaling pathways, mechanisms of activation, recognized pathogens and the interactions between different RNA PRRs will definitely be helpful to improve the anti-viral immune response. Third, TLR3, 7, 8 are primarily expressed by macrophages and DCs and recognize viral RNA within the endosomal compartment, whereas RLRs (RIG-I, MDA5) and NLRs (NLRP3, NOD2) are ubiquitously expressed and sense viral RNA within the cytoplasm of infected cells (Table 1 ).
cord-313138-y485ev30	2013	Whether cause or effect, the lack of TLR8 in avian monocytes/ macrophages likely does contribute to the susceptibility of birds to RNA viruses (West Nile virus, Newcastle disease virus, influenza virus and others) and intracellular bacterial infections, including mycobacteria. The gene encoding RIG-I, DDX58, is not annotated in the chicken genome sequence, and is missing in some fish species, but MDA5 homologues are present in all vertebrate families (Zou et 2009). Riplet/RNF135 is a cytoplasmic E3-ligase identified by yeast two-hybrid as one of the proteins binding RIG-I, and is essential for RIG-I activation in human cell lines upon infection with an RNA virus (Oshiumi et al., 2009; Oshiumi et al., 2010) . The upregulation of IFIT5 following viral infection of chicken cells expressing duck RIG-I ( Barber et al., 2013) or infection of ducks (Vanderven et al., 2012) suggests IFIT5 is an important antiviral effector in avian species.
cord-313161-07iwwsfz	2014	Furthermore, sequential immunization with SIN and VEE replicon particles expressing the type 1 HIV gp140 envelope (Env) and trimeric Env protein in MF59 adjuvant provided partial protection in macaques against intravenous challenges with high doses of simian-human immunodeficiency virus (SHIV) [61] . In another study, chimeric VEE/SIN replicon particles were applied for the expression of measles virus hemagglutinin (H) and fusion (F) proteins, which elicited high-titer neutralizing antibody and IFNɤ-producing T-cells in macaques after intradermal vaccination [48] . Furthermore, vaccination with recombinant particles expressing the P1A gene [105] and the human papilloma virus (HPV) E7 gene [89] from SFV and VEE vectors, respectively, provided protection against further tumor development in mice. When TRAMP mice were prophylactically immunized with a prostate stem cell antigen (PSCA) DNA plasmid followed by VEE-PSCA particle administration, a specific immune response and anti-tumor protection were observed in 90% of vaccinated animals [102] .
cord-313301-7mkadtp9	2007	In particular, the high pernucleotide mutation rates of RNA viruses (Drake 1993) provide extensive genetic variation that fuels evolution by natural selection, making the study of reproductive isolation and speciation especially feasible (Holmes 2004) . We tested the plausibility of the no-gene mechanism of speciation by examining the consequences of adaptation to a novel host in laboratory populations of the RNA phage 6, which infects a number of Pseudomonas species. The same microevolutionary processes of mutation and natural selection, which led to the adaptation of 6 populations to a novel host also resulted in a macroevolutionary event: the evolution of a new virus species that is reproductively isolated from the ancestral phage 6 wt . Beyond uniquely demonstrating the evolution of reproductive isolation in the laboratory, our study extends the literature describing the evolutionary genetics of narrowed host range when viruses adapt to a single host.
cord-313439-cadyykks	2019	Studies evaluating sensitivity and specificity of the detection of serum antibodies in comparison to either histopathology, a combination of diagnostic tests or clinical suspicion of feline infectious peritonitis (FIP). Recent studies evaluated the use of a quantitative RT-PCR (RT-qPCR) to detect FCoV RNA in FNA samples of the mesenteric lymph nodes and other abnormal tissues of clinical cases [119, 140] and hypothesized that this technique would be a useful tool to diagnose FIP for veterinary practitioners, especially in cats without effusion. Sensitivity and specificity from different studies evaluating the detection of feline coronavirus (FCoV) spike (S) gene mutations in tissue samples. RT-nPCR and subsequent S gene sequencing of serum and plasma samples from cats with FIP (diagnosed by histopathology ± IHC or by positive immunofluorescence in effusion) and control cats (diagnosed with another disease either ante or post mortem) revealed a sensitivity of only 7%, which confirms the very low virus load in blood.
cord-313541-fpqwzf9k	2020	The growing demand for commercial kits used for automated extraction of SARS-CoV-2 RNA, a key step before rRT-PCR diagnosis, could cause a shortage of stocks that hinders the rapid processing of samples. SARS-CoV-2 detection by direct rRT-PCR without RNA extraction and inactivating samples at 95 °C for 5 minutes, was showed from specimens placed in UTM and molecular water, but not from samples in Hanks medium and saline buffer (Merindol et al., 2020) . Thus, the four different media used in this study (UTM, PBS 1x solution, Hanks medium and DNA/RNA Shield TM ) did not affect analytical results, because all precipitated samples were able to be detected by rRT-PCR using the whole viral panel (Orf1ab, N and S genes) and the internal control gene. Here, a simple protocol to detect SARS-CoV-2 from NPSs using rRT-PCR after a heat inactivation and a precipitation/concentration step is proposed.
cord-313684-61hkogdh	2020	Coronavirus disease 2019 (COVID-19), an acute onset pneumonia caused by a novel Betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in the Wuhan City of China in December 2019 and evolved into a global pandemic. These include antivirals (remdesivir, lopinavir/ritonavir, umifenovir, and favipiravir), interferon, antimalarials (chloroquine/hydroxychloroquine), antiparasitic drugs (ivermectin and nitazoxanide), biologics (monoclonal antibodies and interleukin receptor antagonist), cellular therapies (mesenchymal stem cells and natural killer cells), convalescent plasma, and cytokine adsorber. Though several observational studies have claimed many of these agents to be effective based on their in vitro activities and extrapolated evidence from SARS and Middle East respiratory syndrome (MERS) epidemics, the currently available data remains inconclusive because of ill-defined patient selection criteria, small sample size, lack of concurrent controls, and use of intermediary outcomes instead of patient-relevant outcomes.
cord-314019-8n0jafsk	2014	We focus on two important and well-studied genera of picornaviruses, namely Enterovirus and Cardiovirus, and discuss how their RNAs are recognized by RLRs, and how they antagonize the IFN-a/b induction pathway and SG formation in infected cells. Picornavirus infections activate a cytosolic RNA sensor, MDA5, which in turn, induces a type I interferon response, a crucial component of antiviral immunity. Picornavirus infections activate a cytosolic RNA sensor, MDA5, which in turn, induces a type I interferon response, a crucial component of antiviral immunity. However, it is important to point out that the caspase-and proteasome-mediated MDA5 cleavage events were observed under conditions where MDA5 expression level was artificially upregulated prior to infection (either by poly(I:C) or viral RNA transfections) [39, 42] , which may sensitize cells for virus-induced apoptosis, thereby promote caspase-and proteasome-mediated protein degradations.
cord-314254-9ye8tfvz	2014	To date, there is no evidence for an animal reservoir of viruses closely related to hepatitis C virus which may have crossed the species barrier to cause disease in humans and resulted in the current pandemic. Recently, several studies discovered new viruses related to hepatitis C virus, belonging to the hepaciand pegivirus genera, in small wild mammals (rodents and bats) and domesticated animals which live in close contact with humans (dogs and horses). Non-primate hepaciviruses (NPHV) were initially discovered in domestic dogs and subsequently in horses 12, 13 and other diverse and widespread HCV-like viruses have been reported in wild populations of rodents and bats. Furthermore, liver function analyses revealed no indication for hepatic inflammation as c-glutamyl transferase and glutamate dehydrogenase values were within reference range, with the exception of a mildly elevated c-glutamyl transferase New HCV-like viruses in different mammalian hosts Pfaender et al 4 level in one horse.
cord-314316-hsspggp8	1997	Small intestinal sections from pigs experimentally and naturally infected with TGEV were hybridized for various times at 52°C and 70°C with a radiolabelled or a fluorescein-labelled RNA probe in a standard hybridization or a REH buffer. Small intestinal sections from pigs experimentally and naturally infected with TGEV were hybridized for various times at 52°C and 70°C with a radiolabelled or a fluorescein-labelled RNA probe in a standard hybridization or a REH buffer. With the fluorescein-labelled probe, viral RNA was detected in virus-infected cells of the intestines after 30 min of hybridization by using the REH buffer. With the fluorescein-labelled probe, viral RNA was detected in virus-infected cells of the intestines after 30 min of hybridization by using the REH buffer. Signal intensity was greater with the REH buffer than with the standard hybridization buffer when compared at each hybridization time and hybridization temperature using both radiolabelled and fluorescein-labelled probes.
cord-314369-o4nis91y	2020	Although overall CT values of TW were higher than that of contemporary swab tests from hospitalized cases, TW from symptomatic cases had comparable CTs. Conclusions: The study suggests that TW may be a valid alternative to the detection of SARS-CoV-2 RNA. During the initial months of the COVID-19 swabs and other collection methods were used by LHW in the institute to identify SARS-Cov-2 RNA in upper respiratory tract, but occasionally throat wash (TW) was alternatively used. We compared the CT obtained at this survey to results generated from contemporary swab collections, sent as routine testing at the institute, that provide SARS-CoV-2 rtPCR testing to clinical services. The study did not compare the rate of positivity in paired samples, and only one individual was documented that performed both a swab test (negative) and a positive throat wash collection at a same day. The study suggests that throat wash may be a valid alternative to the detection of SARS-CoV-2 RNA.
cord-314560-rswa5zdn	2006	RNA interference (RNAi), initially recognized as a natural antiviral mechanism in plants, has rapidly emerged as an invaluable tool to suppress gene expression in a sequence-specific manner in all organisms, including mammals. However, in recent years, a new type of genomic immunity mediated by RNA interference (RNAi) has emerged and has sparked intense interest as a potential treatment strategy for a variety of diseases, including viral infections, cancer and degenerative diseases [1] [2] [3] [4] . In RNAi, long double-stranded (ds) RNA generated during viral infection is cleaved by an enzyme termed Dicer into short, 21-23 nucleotide (nt) dsRNA molecules termed small interfering (si)RNAs that mediate sequence-specific gene silencing [5, 6] . A landmark development in the field occurred with the discovery that the introduction of 21-nt-long synthetic RNA resembling the Dicer-processed siRNA into mammalian cells induces sequence-specific gene silencing without evoking the interferon response [10] .
cord-314567-purplsjn	2018	HCV-core associated proteins are implicated in RNA processing and RNA virus infection as well as in functions previously shown to be altered in Hepatitis C virus core expressing CD4 + T cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. HCV-core associated proteins are implicated in RNA processing and RNA virus infection as well as in functions previously shown to be altered in Hepatitis C virus core expressing CD4 + T cells, such as cell cycle delay, decreased proliferation, and induction of a regulatory phenotype. As studies using the whole virus do not allow for the elucidation of the specific molecular mechanisms in which each protein is implicated, in this work, we focused on a single viral protein, showing that in CD4 + T cells, HCV core protein mostly localizes in the nucleus and specifically in the nucleolus where it is greatly enriched.
cord-314572-1pou702r	2016	Conformational and functional analyses indicate that the engineered theophylline-responsive RNA functions as a mammalian riboswitch with robust theophylline-dependent −1 PRF stimulation activity in a stable human 293T cell-line. In a first step to constructing a ligand-responsive −1 PRF stimulator, we designed Switch-0 RNA with a theophylline aptamer replacing the stem 3 of SARS-PK ( Figure 1A and C). We rationalized that such an engineered switch hairpin of reasonable stability (predicted free energy of −12.7 kcal/mole (37)) would be the dominant conformation that could interfere with the formation of pseudoknot stem 2 in the absence of theophylline (Supplementary Figure S2A) . To improve the dynamic range of ligand response and to see if theophylline aptamers can be functional while existing in both positive and negative regulators of −1 PRF, we fused previously designed theophylline-dependent upstream attenuator, theoOFF2 (24) with Switch-1 ( Figure 5A ) and examined theophylline-dependent −1 PRF activity in vitro.
cord-314753-xflhxb13	2017	The percentage of reads mapping to RNA virus genomes in the rRNA-depleted BBV Panel samples was between 40 and 150-fold higher than in corresponding untreated controls. The depth plots in Fig. 3 again show unbiased and even coverages across both genomes, and the percentages of reads mapping to viral targets was again much higher in the rRNA-depleted sample than in the untreated comparator (61-fold and 85-fold for HCV and HPgV respectively). By depleting host-derived nucleic acids and making modifications to an existing library preparation protocol to account for ultra-low RNA input quantities, we have been able to reconstruct effectively full-length genomes of HCV, HEV and HIV from plasma samples with viral loads of 10 4 IU/ml (copies/ml for HIV) and substantial fractions of complete genomes at 10 3 IU/ml. Additionally, our system was able to recover viral sequences from a panel of diverse RNA viruses diluted in human plasma, with a broad correlation between the genomic coverage and depth metrics and approximate concentration.
cord-314833-6fue84x6	2014	The nucleocapsid phosphoprotein of the severe acute respiratory syndrome coronavirus (SARS-CoV N protein) packages the viral genome into a helical ribonucleocapsid (RNP) and plays a fundamental role during viral self-assembly. proposed a structure-based domain arrangement for SARS-CoV N protein where the NTD and CTD are sandwiched between three IDRs. Sequence alignments suggested that other coronavirus N proteins might share the same structural organization based on intrinsic disorder predictor profiles and secondary structure predictions (Fig. 2) . noticed that effective binding to RNA by MHV N protein in host cells required the presence of both the NTD and CTD (Hurst et al., 2009) , suggesting that the NTD and CTD formed a single bipartite RNA interaction site, a feature to be reiterated in the final SARS-CoV RNP model. Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA
cord-314877-db7tze8j	2020	When viewed in the context of the RNA secondary structure model for the TBSV genome (48) , the DE/CE interaction corresponds to the closing stem of a sizable RNA domain, termed large domain 3 (LD3), which, along with formation of the adjacent LD2, acts to unite the AS2 and RS2 sequences ( Figure 1B) . Translational readthrough for the CIRV genome requires a long-distance RNA-RNA interaction (LDRI) between RTSL and the 3 UTR, involving the PRTE and DRTE partner sequences, respectively ( Figure 1A , B) (39) . The binding of RTSL-TL to SL59-5 was investigated functionally by introducing compensatory nucleotide substitutions into the candidate partner sequences and assessing the effects on sg mRNA1 accumulation following transfection of mutant viral RNA genomes into protoplasts pairing potential in mutants TC-6 and TC-7 diminished sg mRNA1 plus-and minus-strand levels below ∼10% of wt, while regenerating pairing capacity with alternate nucleotides in mutant TC-8 restored levels up to ∼50-62% of wt ( Figure 3B, C) .
cord-314891-brgtwxhe	2018	Using cell culture assays, PPNDS, quercetagetin and GC376 did not display antivirals effects, however, we identified nitazoxanide and 2′-C-methylcytidine (2CMC) as potent inhibitors of FCV replication, with EC(50) values in the low micromolar range (0.6 μM and 2.5 μM, respectively). The combined inhibitory effects of nitazoxanide (0 to 0.6 µM) and 2CMC (0 to 4 µM) were tested over a range of combinations against FCV in the cell culture using the plaque reduction assay. Of the six NNI compounds tested in the current study, PPNDS and quercetagetin showed an inhibition of FCV RdRp activity with IC 50 values in the low micromolar range (Figure 2 and Table 1 ). Finally, we reported the identification of two compounds (nitazoxanide and 2CMC) with antiviral activity against FCV in cell culture at low micromolar concentrations with a potential combinational therapeutic utility to treat FCV-infected cats.
cord-315054-kji2kfek	2020	In an effort to be economical with available resources, snap-frozen euthanized mice are the straightforward solution; however, this method increases the risk of temperature abuse during the thawing process at the beginning of the tissue collection. We found that prolonged immersion of snap frozen mouse carcass in 10% neutral buffered formalin at 4°C yielded minimal microscopic signs of ice crystallization and delivered tissues with histomorphology that is optimal for hematoxylin and eosin (H&E) staining and fixation on glass slides. An omics-pathology integration approach could be the most effective for phenometo-genome interpretation if omics assays are conducted using the particular tissue specimen, where injury signatures are informed by histopathology image analysis (Pathak and Dave, 2014; Yu et al., 2017) . These snap-frozen carcasses had to undergo onground freeze-thaw cycles before tissue collection, which is typically expected to compromise overall sample quality and make the histopathologic analysis challenging (Lyons et al., 1979; Pikal-Cleland et al., 2000) .
cord-315069-xo4mbxei	1991	Abstract Defective interfering (DI) RNAs were generated de novo in each of 12 independent isolates of tomato bushy stunt virus (TBSV) upon serial passage at high multiplicities of infection (m.o.i.) in plants, but not in any of 4 additional isolates after 11 serial passages at low m.o.i. The DI RNAs were detected in RNA isolated from virus particles and in 2.3 M LiCl-soluble RNA fractions isolated from inoculated leaves. On each host species, six isolates were passed at high m.o.i. and two control isolates were passed at low m.o.i. In these hosts, DI-free TBSV induces lethal necrosis within 7-l 0 days postinoculation (d.p.i.), except in the presence of DI RNAs which are associated with attenuation and the establishment of persistent infections (Hillman et a/., 1987) . The small RNAs which developed in each of the high m.o.i. isolates hybridized to both 5''-and 3''-proximal TBSV genomic sequences, but generally were larger (-600 bases) than the previously characterized DI 1 RNA species (-400 bases).
cord-315072-b28yikvj	2016	title: Chicken interferome: avian interferon-stimulated genes identified by microarray and RNA-seq of primary chick embryo fibroblasts treated with a chicken type I interferon (IFN-α) To generate a chicken ISG database we have compared data from three transcriptomic technology platforms: (i) the classical 3′-biased GeneChip Chicken Genome Array (32K; Affymetrix, High Wycombe, UK), (ii) the Chicken Gene 1.0 Sense Target (ST) whole transcriptome Array (Affymetrix) and (iii) Illumina (Little Chesterford, UK) RNA-seq. One of the most highly-expressed contigs was one which, when analysed by BLAST, proved to represent a homologue of STAT2, which is missing from the current ENSEMBL annotated reference chicken genome In (B) PYURF shows 24-fold suppression by IFN but the sequence surrounding PYURF shows 87-fold induction from the right-hand end of the unannotated, antisense LOC422513 and considerably higher upregulation from the left-hand end (due to its lower uninduced levels), consistent with these representing homologues of IFN-inducible human genes HERC6 and HERC5. Technologies identifying significant IRGs are listed as "1" RNA-seq (using Kal''s Z test); "2" Affymetrix 32K GeneChip Chicken Genome Array and "3" Chicken Gene 1.0 ST Array'' .
cord-315085-rucfowvv	2020	In this study we report postmortem findings and detection and sequencing of SARS-CoV-2 viral RNA from formalin-fixed paraffinembedded (FFPE) samples of multiple organs collected in 2 patients with antemortem detection of SARS-CoV-2. The patient''s medical history was otherwise notable for dementia, radiologic evidence of a left lung mass (managed with hospice care), coronary artery disease (status post coronary artery bypass grafting), atrial fibrillation (biventricular pacemaker implanted), congestive heart failure, peripheral artery disease (status post iliac stenting), diabetes mellitus, hypertension, dyslipidemia, chronic kidney disease, gout, smoking, cerebrovascular accidents, and urinary tract infections. On day 1 after admission, ❚Image 2❚ (Case 1) Postmortem microscopic examination of the lungs showed diffuse alveolar damage characterized by hyaline membrane formation (A, ×100) and scattered squamous metaplasia of distal airways (B, ×100) on a background of emphysematous changes.
cord-315384-eqiokrub	2005	The viral RNA was more prevalent in samples from outpatients (7.9%) than from hospitalised patients (3.2%, p = 0.003), and co-infection with either respiratory syncytial virus or parainfluenza virus 3 was observed frequently. The samples had been collected from hospitalised patients and outpatients from December 1999 to October 2001 in four different regions in Germany as part of the prospective population-based PRI.DE study and analysed for RNA from respiratory viruses. The samples had been collected from hospitalised patients and outpatients from December 1999 to October 2001 in four different regions in Germany as part of the prospective population-based PRI.DE study and analysed for RNA from respiratory viruses. To investigate the prevalence of HCoV-NL63 and its involvement in respiratory diseases, we now analysed 949 samples from the Paediatric Respiratory Infection in Germany (PRI.DE) study, a prospective population-based study on lower respiratory tract infections (LRTIs) in children under 3 y of age in Germany [8, 9] .
cord-315483-l6dm82pp	2018	To confirm the sequence of the cDNA and to identify the genomic structure of the identified IFIT gene, the transcript was amplified from RNA extracted from NDV-infected CEFs. Complete sequence analysis of the gene revealed an open reading frame (ORF) of 1440 bps (479 amino acids excluding stop codon) with high sequence identity (48% and 67%) with the human and duck IFIT5 proteins, respectively ( Supplementary Fig. 1B) . The levels of chIFN-β gene induction was proportional to the NDV replication (Fig. 2D) , therefore, it can be inferred that the virus-induced expression of chicken IFIT5 is IFN-dependent and that chIFIT5 is an early-ISG with capacity to modulate initial steps of virus life cycle. To compare anti-viral effects of chIFIT5 (only IFIT gene identified in chickens so far) with its orthologous and homologous human proteins, huIFIT1, huIFIT2, huIFIT3 and huIFIT5 were expressed in chicken cells and antiviral affect was monitored (Fig. 5A ).
cord-315611-xbj41ekc	2020	Using a combination of bioinformatics and computational tools, we modelled the 3D structure of the RdRp (RNA-dependent RNA polymerase) of SARS-CoV2 (severe acute respiratory syndrome coronavirus-2) and predicted its probable GTP binding pocket in the active site. 20−22 In this report, using computer-aided homology modeling, docking, and molecular simulations, we have predicted the protein structure and probable small-molecule inhibitors against SARS-CoV2 RdRp (CoV2-RdRp). Taking together the aforementioned interaction and comparison of the model and experimentally determined structures, we propose the probable initiation complex of CoV2-RdRp bound to RNA and GTP molecules in Figure 2D . Molecular Dynamics simulation studies of the native and ligand-bound complexes of CoV2-RdRp. MD (Molecular dynamics) simulations were performed for the modelled structure of the RdRp protein and docked complexes for the GTP, lead optimized, and FIH compounds for a 50 ns time period.
cord-315616-pvt0amth	2004	Ribonucleotide reductase itself, which synthesises deoxyribonucleotides from ribonucleotides, requires complex protein radical chemistry, and RNA world genomes may have reached their limits of coding capacity well before such complex enzymes had evolved. Even with this gap in the RNA world model, current knowledge suggests the RNA organism that evolved protein synthesis was likely to have been dangerously close to the Eigen limit for RNA, and probably emIn the ¢rst step, a radical is generated at some distance from the active site. By virtue of its non-speci¢c dsRNA methylating activity, this enzyme is hypothesised to have been recruited into methylation of genomic RNA, improving the stability of the genetic material prior to the origin of DNA. The predicted stabilising effect of 2P-O-methylation on genomic RNA argues that the coding capacity of the genome would have increased suf¢ciently for genuinely catalytic proteins such as RNA polymerase, and later ribonucleotide reductase, to arise, paving the way to the DNA world (Figure 4) .
cord-315909-vwugf0wp	2019	Here, we describe methods for in vitro serial passaging of Middle East respiratory syndrome coronavirus (MERS-CoV) to select for mutations which increase replication on semi-permissive cell lines as described in Letko et al., Cell Rep 24, 1730–1737, 2018. Forced adaptation experiments have been used to determine viral mutations that facilitate escape from drugs [4] [5] [6] , monoclonal antibodies [7, 8] , host restriction factors [9] [10] [11] , and species variation in host receptors [12] [13] [14] and to elucidate various viral mechanisms of infection and replication [15] [16] [17] . Below is the method employed to adapt MERS-CoV to a semi-permissive host receptor, Desmodus rotundus DPP4. To increase selective pressures on a viral population which is beginning to show signs of adaptation, one can apply a population bottleneck in the subsequent passage by reducing the amount of viral Evolution of MERS-CoV supernatant passaged to the next cell culture.
cord-316134-lkd2mj27	2020	Investigation of possible molecular interactions between components of PEDV, PDCoV and TGEV and their influence on replication of each virus would provide a crucial insight into comprehensive understanding of these CoVs. Of all viral proteins, we have chosen to start with the N protein, as it is among the most abundant and ubiquitous structural proteins in infected cells. For viral infection in the transient CoV N expression experiment, VeroE6 cells were transfected with 1 or 2 μg of pCAGGS-PEDV N-Myc, pCAGGS-PDCoV N-HA, pCAGGS-TGEV N-FLAG, or the empty pCAGGS vector and were incubated for 24 h to allow for protein expression. To investigate how this protein-protein interaction might affect PEDV replication, we transiently transfected VeroE6 cells with varying amounts of the pCAGGS plasmid expressing N proteins from either PDCoV or TGEV for 24 h before infection with PEDV-mCherry (MOI = 0.0001) and followed the course of viral replication for each condition.
cord-316179-kmdxltie	2020	Here we report the development of an amplification-free CRISPR-Cas13a-based mobile phone assay for direct detection of SARS-CoV-2 from nasal swab RNA extracts. When the SARS-CoV-2 sequence became public in January 2020, we set out to develop a Cas13-based direct-detection assay for viral RNA that would avoid the need for amplification and enable point-of-care testing. We tested the performance of the device for detecting SARS-CoV-2 RNA using the triple-crRNA Cas13a assay and a dilution series with full viral RNA isolated from supernatants of infected Vero CCL-81 cells . (B) RNPs made with crRNA 2 and crRNA 4 individually and in combination (50 nM total RNP concentration for each reaction) were tested against 2.9 x 10 5 copies/µL (480 fM) of SARS-CoV-2 IVT N gene RNA, and compared to fluorescence from no target RNA RNP alone controls ("RNP 2," "RNP 4," and "RNP 2+4").
cord-316503-wtmmewiz	2012	Phosphorodiamidate morpholino oligomers (PMOs) are synthetic antisense oligonucleotide analogs that are designed to interfere with translational processes by forming base-pair duplexes with specific RNA sequences. Peptide conjugated PMOs (PPMOs; Fig. 2 ), which include positively charged amino acid residues such as arginine, have been viewed as particularly promising transporters and have shown efficacy in multiple in vivo models of viral infection (Enterlein et al., 2006; Paessler et al., 2008; Stein, 2008; Swenson et al., 2009) . While PPMOs are efficacious in multiple in vivo models of viral infections, PPMOs are more poorly tolerated in vivo compared to neutral-charge PMOs. In mice, dose-dependent reductions in weight, behavioral alterations, and mild liver histopathology were observed following repeat administration of 200-300 lg doses of a PMO conjugated to an arginine-rich peptide (Deas et al., 2007) .
cord-317037-1qydcc5e	2020	Virus-infected cells secrete various lipid-bound vesicles, including endosome pathway-derived exosomes and microvesicles/microparticles that are released from the plasma membrane. HIV-infected U1 macrophages upon Cigarette smoke condensate (CSC) treatment enhanced the packaging of IL-6 in EVs; IL-8 served as a biomarker for HIV patients with altered immune function due to alcohol and tobacco abuse [20, 116, 117] Host protein APOBEC3G Inhibit replication of viral infectivity factor (vif) -deficient and wild-type HIV-1 in recipient cells [118] miRNA vmiR-88 and vmiR-99 Hepatocytes secreted exosomes participate in virus replication [142] Viral miRNAs HBV-miR-3 Represses viral protein production and HBV replication [143] HTLV-1 Viral proteins gp61, Tax, and HBZ Increase cell-to-cell contact and promote a potential increase in viral spread [144] Zika Viral genetic material and protein RNA and ZIKV-E EVs derived from Infected C6/36 cells promote infection and activation of monocytes with enhanced TNF-α mRNA expression.
cord-317244-4su5on6s	2014	In the present study, among 985 bats belonging to 6 species sampled in the Belinga caves of Gabon, RNA of an unclassified paramyxovirus (Belinga bat virus, BelPV) was discovered in 14 African sheath-tailed bats (Coleura afra), one of which exhibited several hemorrhagic lesions at necropsy, and viral sequence was obtained in two animals. To further investigate the presence of the virus in bat populations, a strain-specific real-time RT-PCR assay (primers: GB09-478-F, 59-GGCGGCTCTTAAAAGT-GAATG-39; GB09-478-R, 59-GCGGGGTCAAATTGGTCAT-39; probe: GB09-478-P, 59-TCCAGCACAAACATATCCGAGAAGGCTAG-39) was designed within the initial PCR fragment and was used to test total RNA extracted from mixed liver and spleen samples from each of all the other bat species. In order to determine the organ distribution of this virus in infected bats, total RNA was extracted from heart, liver, spleen, kidney, lung, intestine and brain samples from all 14 real-time RT-PCR-positive bats, as described previously, and screened, using the same strain-specific real-time RT-PCR assay shown above.
cord-317455-6qx0v28w	2018	Turkey coronavirus, originally identified in the USA in the 1970s as one of the agents responsible for an acute enteritis named bluecomb (Panigrahy, Naqi, & Hall, 1973; Ritchie, Deshmukh, Larsen, & Pomeroy, 1973) and since with a multifactorial disease known as poult enteritis complex of turkeys (PEC) , has now been detected in most areas where turkeys are farmed Cavanagh et al., 2001; Dea & Tijssen, 1988; Domańska-Blicharz, Seroka, Lisowska, Tomczyk, & Minta, 2010; Martin, Vinco, Cordioli, & Lavazza, 2002; Maurel et al., 2009; Teixeira et al., 2007) , although TCoVs isolated in Europe have been shown to have a different genetic lineage to those isolated in the USA (Brown et al., 2016; Maurel et al., 2011) . At 1-day post-inoculation (dpi), two SPF turkey contacts were introduced into groups 1-4 as sentinels to demonstrate horizontal transmission of infectious virus. They were housed in a negative pressure room, under the same rearing conditions as in Exp 2, with three 11-day-old SPF turkeys introduced as contact-birds at 1 dpi to demonstrate horizontal transmission.
cord-317537-wgu5cd0y	2009	All constructs were replication competent in protoplasts as assayed by northern blot hybridization (Fig. 4I) , and statistics analysis (ANOVA) of real-time RT-PCR quantification of average relative percentage (from 3 independent experiments) of CymMV RNA from CymMV clone-infected protoplasts at 24 h postinoculation revealed no significant difference in percentage between these clones (P = 0.91). All constructs were replication competent in protoplasts as assayed by northern blot hybridization (Fig. 6J) , and statistics analysis (ANOVA) of realtime RT-PCR quantification of average relative percentage (from 3 independent experiments) of CymMV RNA from CymMV cloneinfected protoplasts at 24 h post-inoculation revealed no significant difference in percentage between these clones (P = 0.83). Our results indicated that the important amino acids of the CP that allowed for the CymMV systemic infection are located within the previously predicted RNA binding domain ( Fig. 7A; 1) .
cord-317591-qa6oxy4j	2009	To develop an effective and specific medicine targeting the SARS-coronavirus (CoV), a chimeric DNA-RNA hammerhead ribozyme was designed and synthesized using a sequence homologous with the mouse hepatitis virus (MHV). The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited multiplication of MHV in DBT cells by about 60%. A chimeric DNA-RNA hammerhead ribozyme was designed and synthesized to target a common nucleotide sequence between SARS-CoV and mouse hepatitis virus (MHV), and its effectiveness on suppression of MHV and SARS-CoV RNA expression evaluated in vitro. Figure 4 shows effect of the chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV RNA on the multiplication of MHV in DBT cells. Therefore, the chimeric DNA-RNA hammerhead ribozyme was designed with complementarity to common regions of SARS-CoV and MHV that included the target GUC sequence. In the present study, inhibition on the multiplication of MVH was approximately 60%, with 60% transfection efficiency of the chimeric DNA-RNA ribozyme targeting SARS-CoV into DBT cells.
cord-317715-xtsi663k	2012	Reporter gene activation (FFL) is shown as percentage (%) of LCMV NP-NP wt interaction (pC-NP-VP16 and pC-NP-GAL4) after normalization of transfection efficiencies with the Renilla luciferase expression plasmid pRL SV40 (ii). Unexpectedly, the D471A substitution impeded both NP-NP and NP-Z interactions, as well as NP functions in replication and transcription of the LCMV MG and the ability of NP to counteract SeV-mediated induction of the host IFN-I response, suggesting that change D to A at position 471 might have affected the overall structure of NP without affecting its stability as reflected by its unchanged expression levels determined by WB using anti-VP16 ( Figures 6A and 6B ) or anti-HA ( Figures 6C and 6D) antibodies. (C) Replication and transcription activity: BHK-21 cells were co-transfected with the LCMV MG as described in Figure 3 together with expression plasmids for the viral polymerase (L) and wt or indicated mutant NPs, and pSV40-Cluc expression vector to normalize transfection efficiencies.
cord-317720-gbi11oxx	2020	Concerned that the efforts of state laboratories would be further impacted by lack of resources, we began to identify sources -including the WHO, the CDC, and commercial vendors -of the required primers and probes for the reverse transcriptase polymerase chain reaction (RT-PCR) detection of the virus and placed orders for test reagents from potential suppliers. The document provided guidance for high complexity testing laboratories developing SARS-CoV-2 tests for submission for Emergency Use Authorization (EUA) status with respect to required validation experiments and reporting to the FDA. Initially laboratories were required to spike RNA transcripts into previously extracted nucleic acid from negative samples for the CDC assay to determine the limit of detection but this had its own challenges of not representing extraction of true clinical samples and issues with degradation were identified. Our plan included the production of enough contrived clinical specimens and control material to proceed with validation or verification of the multiple (laboratory-developed and CDC EUA) tests that we were evaluating.
cord-317773-jdq1d98i	2011	RESULTS: In this study, the avian leukosis virus subgroup J (ALV-J) proviral genome, including the gag genes, were treated as targets for RNAi. Four pairs of miRNA sequences were designed and synthesized that targeted different regions of the gag gene. To avoid the generation of escape variants during virus infection, expression vectors of multi-target miRNAs were constructed using the multi-target serial strategy (against different regions of the gag, pol, and env genes). CONCLUSION: The strategy of multi-target miRNAs might be an effective method for inhibiting ALV replication and the acquisition of resistant mutations. In some studies, transfection of siRNA designed to target C virus (HCV) remarkably inhibited the expression of virus-specific proteins and protected cells against HCV RNA, in vitro [4, 5] . In another study, Hepatitis B virus (HBV) replication was successfully inhibited after plasmid expression of HBV siRNA transfected into mouse liver [6] .
cord-317851-lj07947c	2008	In this review, we will focus on recent studies that used plant viruses to address evolutionary questions of general interest, such as the rate and fitness effects of deleterious mutations and the role of neutrality as a source of mutational robustness, the evolution of generalist viruses, or the effect of vertical versus horizontal transmission on virulence. Despite mutation rate is still high compared to that of DNA-based microorganisms, a classic field observation is that natural plant virus populations generally exhibit limited genetic variation (García-Arenal et al., 2001) , which may imply either that purifying selection may be strong or that genome replication occurs mainly by Luria''s stamping machine model (Luria, 1951) rather than exponentially (French and Stenger, 2003) , the two hypotheses being nonexclusive.
cord-318164-6rqi17oz	2020	This study therefore aimed to analyze the safety of sperm cryopreservation for cancer patients after the onset of the pandemic in Italy, through assessment of the risk of SARS-CoV-2 exposure and viral RNA testing of semen samples. CONCLUSION: This preliminary assessment suggests that a thorough evaluation (especially in the setting of a multidisciplinary team) and molecular confirmation of the absence of SARS-CoV-2 in seminal fluid from asymptomatic cancer patients may assist in ensuring the safety of sperm cryopreservation. This study thus aimed to evaluate the safety of sperm cryopreservation of cancer patients referred to our sperm bank after the onset of the pandemic in Italy through the assessment of the risk of SARS-CoV-2 exposure and, in selected volunteers, viral RNA testing of semen samples. This was further confirmed by testing seminal fluid samples from 10 asymptomatic cancer patients for SARS-CoV-2 RNA.
cord-318276-so5jooj0	1987	RNA was isolated from noninfected or infected cells and then hybridized to 32P-labeled probes isolated from the 5'' end region, upstream of the 11K coding sequences of cDNA clones 3, 8, and 9 (see Figure 2 ). To confirm this, and to identify the regions from which they are transcribed, the cDNA-derived probes used in the previous experiment were hybridized to cloned DNA restriction fragments representing the entire vaccinia virus genome ( Figure 4 ). %P-labeled DNA probes (~3, ~9, p9, pllK) derived from the 5'' region of the corresponding cDNA clones or from genomic DNA (see Figure 2 ) were hybridized lo RNA isolated from uninfected (HeLa) or infected (Early, Late) cells, or to tRNA immobilized on a nitrocellulose membrane. Total late RNA from infected cells protected fragments of 127 and 128 nucleotides of the genomic probe, consistent with the previous experiments, which demonstrated that the sequence complementarity between the genomic DNA and 11K mRNA ends just upstream of the 11K ATG codon.
cord-318359-41h90h05	2018	Here, we report the first high resolution analysis of retrovirus gene expression through tandem ribosome profiling (RiboSeq) and RNA sequencing (RNASeq) of MuLV-infected cells. Translation may proceed contiguously from this codon through the gag ORF; thus, presumably, giving rise to an alternative Glyco-Gag isoform which is N-terminally extended by a further 12 amino acids (MELTSSEHPAAT, assuming GUG is decoded by Met-tRNA i ) relative to the canonical Glyco-Gag. The GUG codon is present in some but not all MuLV strains, and is not well conserved among other gammaretroviral lineages for which sequence data are currently available (Additional file 1: Fig. S3-highlighted in green) , although some sequences have alternative nearby potential non-AUG initiation sites. Our data indicate the existence of new translation initiation sites, new translated short ORFs and the first measurement of the gag-pol stop codon readthrough efficiency in the context of the full-length virus genome during infection.
cord-318478-fn0gcxbb	2020	Available models for the RNA structure of SARS-CoV-2 and related viruses are largely confined to short-distance base-pairing which result in local folding of important cis-acting elements (Andrews et al., 2020; Huston et al., 2020; Kelly et al., 2020; Lan et al., 2020; Manfredonia et al., 2020; Ryder, 2020; Sanders et al., 2020; Sun et al., 2020) . In addition to the canonical UTR structures, we provide here a direct in vivo evidence for genome cyclization in SARS-CoV-2, mediated by long-range base-pairing between the 5′ and 3′ UTRs ( Figures 5B and S4B ). The long-distance RNA structure map for SARS-CoV-2 provides a practical starting point to dissect the regulation of discontinuous transcription, as it identifies cis-acting elements that interact with each other to create genome topologies that favour the synthesis of the ensemble of sgmRNAs. RNA viruses evolve sophisticated mechanisms to enhance the functional capacity of their size-restricted genomes and to regulate the expression levels of their replicase components.
cord-318495-1w74wf02	2019	The demonstration of hotspots for the generation of copyback DVGs from respiratory syncytial virus (RSV) and the identification of specific nucleotides that determine where copy-back DVGs rejoin further demonstrate that the generation of copy-back DVGs is not completely random, but instead that specific sequences encoded in the viral genome direct or facilitate their formation 50 in some infections, DVG generation is not a completely stochastic process and, instead, virus-encoded sequences favour the production and/or amplification of predominant DVGs. It remains to be determined whether conservation is a property of certain DVG types and which specific sequences and/or RNA structures lead to DVG generation in these conditions. Persistent infection with infectious pancreatic necrosis virus mediated by defective-interfering (DI) virus particles in a cell line showing strong interference but little DI replication I Interferon-inducing defective-interfering particles as mediators of cell sparing: possible role in persistent infection by vesicular stomatitis virus
cord-318551-c1qr27lg	2005	(Adapted with permission from Pasternak et al., 2001.) Transcription of segmented (À) strand RNA viruses such as the Orthomyxoviridae, Arenaviridae, Bunyaviridae, and Tenuiviruses requires a primer to initiate synthesis of the mRNAs. This is achieved by cap-snatching in which the replicase complex, or a protein thereof, binds to the 5 0 region of cell mRNAs, cleaves off the cap together with generally 7-15 nucleotides from the 5 0 end of the cell mRNA, and uses this fragment as a primer to initiate synthesis of the viral mRNAs (Bouloy et al., 1978; Nguyen and Haenni, 2003) . (1996) PV, poliovirus; MHV, mouse hepatitis virus; WNV, West Nile virus; BVDV, bovine viral diarrhea virus; HPIV-3, human parainfluenza virus-3; IG (À), intergenic region in (À) RNA; UTR, untranslated region; Leader RNA (À), 3'' end of (À) RNA; Leader RNA (þ), 5'' end of (þ) RNA; HF, host factor; PCBP, poly(C)-binding protein; PABP, poly(A)-binding protein; hnRNP A1, heterogeneous nuclear ribonucleoprotein A1; PTB, polypyrimidine tract-binding protein; TIA-1, T-cell-activated intracellular antigen; TIAR, TIA-1-related; RHA, RNA helicase A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
cord-318576-dc5n6ni4	2020	From the previous section, we demonstrated that human genes in the GO terms of the cell cycle and the regulation of the cell cycle process adopt codon usage patterns similar to those of þssRNA (subgroups 6, 7), -ssRNA (subgroup 4), retrovirus (HIV-1), and ambisense (subgroup 4) viruses. The lists of upregulated proteins upon viral infection were submitted to GO-TermFinder (Supplementary File 3) , and the enriched GO terms were compared to the enriched GO terms of human genes with codon usage patterns similar to RNA viruses from every subgroup. Several enriched GO terms of upregulated protein profiles during viral infections were found to be identical to the GO terms of human genes with codon usage patterns similar to RNA viruses ( Figure 4 ). Several GO terms of human genes with codon usage patterns similar to RNA viruses have been found by previous studies to be identical to the GO terms of upregulated protein profiles in viral infections.
cord-318749-k91oku7h	2020	Recently reported studies have demonstrated that ribosome biogenesis factors (RBFs) and ribosomal proteins (RPs) act as multifaceted regulators in selective translation of viral transcripts. Similarly, ribosomes are required for the protein synthesis of host cells and viruses, but the biogenesis factor RBFs can also impact the proliferation of virus and cell-intrinsic immune responses. The multifunctional nucleolar phosphoprotein nucleophosmin (NPM) plays a lead role in ribosome biogenesis to stimulate RNA Pol I-dependent transcription (Li and Hann 2013) and regulates SARS-CoV, IBV, HIV-1, HCV Recruited by viral proteins to facilitate viral replication Chen et al. RPS25 deletion in yeast or mammalian cells has minimal effects on cellular protein synthesis, which implies that this ribosomal protein may be selectively required for viral IRES-mediated translation (Jack et al. Conservation of multifunctional ribosomal protein metallopanstimulin-1 (RPS27) through complex evolution demonstrates its key role in growth regulation in Archaea, eukaryotic cells, DNA repair, translation and viral replication
cord-318751-4v2tl0gi	2013	While the efficient culture of human noroviruses (HuNoVs) in immortalized cells has yet to be achieved [5] , the development of a norovirus replicon [6] , which allows the generation of cell lines stably replicating Norwalk virus RNA, has facilitated many small molecule inhibitors to be tested in vitro. The discovery of murine norovirus (MNV) [7] , which replicates efficiently in immortalized macrophage cells and has both reverse genetics systems and small animal models available [8] , has also enabled the examination of the immune responses to noroviruses as well as the efficacy of inhibitors in vitro and in vivo. However, the vast majority of such studies are conducted with norovirus surrogates such as feline calicivirus or MNV, as testing decontamination procedures for HuNoV is difficult owing to the lack of an available cell culture system to detect any remaining infectivity.
cord-318853-mxyxwkhx	2005	Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. Hence, as relative concentrations of wild-type and variant viral proteins vary, alteration of both processivity and fidelity of RNA pol results, permitting viruses to adaptively respond to environmental changes, including immune recognition and reaction to evolving cell receptors. As clear evidence exists for viral disruption of leptin function [106] and virus-associated weight gain in humans [107] and monkeys [108] , is it possible the global epidemics of type II diabetes mellitus, insulin resistance, hyperlipidaemia and obesity now prevalent [109] [110] [111] [112] [113] [114] [115] [116] , are just that; epidemics fundamentally caused by viruses that co-opt insulin or leptin or other associated receptors for cell access and generate protein quasispecies that disrupt receptor function?
cord-319100-3gdawhfn	2020	authors: Kirkland, P.D.; Frost, M.J. title: The impact of viral transport media on PCR assay results for the detection of nucleic acid from SARS-CoV-2 and other viruses Also of concern are recommendations (3, 4) to include foetal bovine serum (fbs) as a source of protein to enhance the stabilising properties of VTMs. This report documents observations of the adverse impact of certain VTMs on real time reverse transcription PCR (qRT-PCR) assays for the detection of SARS-CoV-2 virus as well as on a Type A influenza virus and a herpesvirus and discuss the broader implications of the inclusion of foetal bovine serum as a protein supplement to VTMs. During the initial investigation, purified RNA from an Australian isolate (WMD DC1) of SARS-CoV-2 was supplied to the Elizabeth Macarthur Agriculture Institute (EMAI) by the Institute of Clinical Pathology and Medical Research (ICPMR), Westmead, New South Wales (NSW).
cord-319116-2ts6zpdb	2018	Since the number of reports describing the presence of G4s in virus genomes has boomed in the past 2 years and treatment with several G4 ligands has shown potentially interesting therapeutic activity, we here aim at presenting, organizing and discussing an up-to-date close-up of the literature on G4s in viruses and the classes of molecules that have shown antiviral activity by viral G4 targeting. The research of G4s in the HIV-1 genome has been quite productive, concerning not only the two RNA viral genome copies, but also the integrated proviral genome, specifically For each virus the following information is shown: virion structure and dimension, genome size and organization; schematic representation of the G4 (red dots) location in the viral genomes or in the mRNA and G4 binding proteins; number of G4s assessed through bioinformatics analysis, according to the corresponding references; G4 ligands reported to date to display antiviral effect and corresponding references.
cord-319179-gqaxf7mz	1987	When infected cells and isolated virions were assayed for this protein by two-dimensional gel electrophoresis, p28 could be detected in infected cells labeled at late times after infection, but not at early times or in purified virions. When infected cells and isolated virions were assayed for this protein by twodimensional gel electrophoresis, p28 could be detected in infected cells labeled at late times after infection, but not at early times or in purified virions. To determine if p28 was a minor protein actually present in virions, infected 17CL-1 cells were labeled with [35S] methionine and virus was purified as described in Fig. 4 . A portion of the labeled virus preparation was analyzed directly by two-dimensional gel electrophoresis whereas a second part was mixed with the [35S] methionine-labeled cell-free products prior to analysis (Fig. 4) .
cord-319194-ukuia48s	2004	Tracing the history of molecular changes in coronaviruses using phylogenetic methods can provide powerful insights into the patterns of modification to sequences that underlie alteration to selective pressure and molecular function in the SARS-CoV (severe acute respiratory syndrome coronavirus) genome. Tracing the history of molecular changes in coronaviruses using phylogenetic methods can provide powerful insights into the patterns of modification to sequences that underlie alteration to selective pressure and molecular function in the SARS-CoV (severe acute respiratory syndrome coronavirus) genome. Figure 1 shows the maximum likelihood tree produced using a set of homologous replicases from five SARS-CoV strains, 12 other coronaviruses representing both groups 1 and 2 of the genus [2, 3] , one torovirus (Breda virus) and one okavirus [yellow head (YH) virus], which were determined to most closely represent the consensus coronavirus sequence by a PSI-Blast search [12] .
cord-319501-a2x1hvkk	2016	Particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against SARS and MERS coronavirus infection are discussed. This suggests SARS-CoV N may interfere with RNA recognition by host immune sensors such as RIG-I and MDA5 thus achieving suppressive role in IFN production. Our group demonstrated that MERS-CoV ORF4a interacts with PACT, a cellular dsRNA-binding protein that optimally activates RIG-Iand MDA5-induced type I IFN production, in an RNAdependent manner (Siu et al., 2014c) . Infection with SARS-CoV and MERS-CoV has been accompanied with suppression of innate immune response, most notably with the suppression of type I IFN production and signaling pathways. Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I interferon, in infected cells Middle East respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses pact-induced activation of RIG-I and MDA5 in the innate antiviral response
cord-319635-kh99n7q2	2014	Such cleaved small RNA fragments may be further degraded through an RNA interference pathway triggered by viral double-stranded RNA during replication in mosquito cells, resulting in a lower frequency of RNA recombination in mosquito cells compared to that which occurs in mammalian cells. Yates'' chi-square test was used to assess the frequency of RNA recombination in cells coinfected by two virus strains or transfected by viral RNA fragments. Two and one recombinant form(s) were, respectively, identified in selected samples from BHK-21 and C6/36 cells, when they were coinfected with the T1P1-S1 and CJN-S1 strains of the Japanese encephalitis virus. As in our previous report, different strains of the JEV can coinfect host cells derived from mosquitoes or mammals [25] , which actually generates recombinant forms of the virus [30] . In this study, we infected host cells with Nakayama strains of the JEV, followed by transfection of the (+)5 3 -UTR-I RNA fragment.
cord-319649-d6dqr03e	2013	Here, we expressed VP5 from type 5 Helicoverpa armigera cypovirus (HaCPV-5) in a eukaryotic system and determined that this VP5 possesses RNA chaperone-like activity, which destabilizes RNA helices and accelerates strand annealing independent of ATP. In this study, we expressed HaCPV-5 VP5 in a eukaryotic expression system and determined that this CPV VP5 possesses an RNA chaperone-like activity to ATP-independently destabilize RNA helices and accelerate strand annealing. Moreover, we found that HaCPV-5 VP5 could facilitate the transcription initiation of an alternative polymerase (i.e. reverse transcriptase) through a CPV panhandle-structured RNA template, thereby strongly suggesting a direct role of the RNA chaperone activity of VP5 in the initiation of cypoviral dsRNA replication. In the family Reoviridae, CPV VP5 may not be the only RNA chaperone, as rotavirus nonstructural protein 2 (NSP2), which is a multifunctional enzyme involved in rotaviral dsRNA replication, was previously shown to contain ATP-independent nucleic acid helix-destabilizing activity (45) .
cord-319664-gyktrd36	2020	Finally, to evaluate the performance of molecular assays a standard curve was generated by 10-fold dilutions of SARS-CoV-2 RNA, isolated and extracted at Istituto Superiore di Sanità in Rome, Italy, and quantified by a well-established copy number of RNA synthetic E gene (Wuhan coronavirus, EVAg, www. All specimens were also manually extracted and tested for the presence of SARS-CoV-2 by in-house rt-Realtime PCR and the 2019-nCoV TaqMan RT-PCR Kit. In particular, we investigated the RNA availability and virus detection using both the purified and thermal/non-extractive procedures also with this commercial kit because it is based on the same primers, probes and assays developed by the CDC and used in the inhouse molecular method. This study corroborates our results for in-house rt-Real Time PCR, showing a lower sensitivity of the heat treatment (range ΔCT value of 0.5-1.0) when compared with purified samples, but, dissimilar to our findings, a total inhibition was found by the commercial kit RealStar SARS-CoV-2 RT-PCR (Altona Diagnostics, Hamburg, Germany), where all positive samples failed in the detection of Sars-CoV-2 [14] .
cord-319681-kjet3e50	2012	The mRNA signal for À1 FS is composed of two elements, a slippery sequence with consensus X_XXY_ YYZ (underlines denote zero frame; X can be any base, Y is A or U, Z is not G in eukaryotic systems) where the ribosome changes frame, and a downstream stimulatory RNA structure, a stem-loop or pseudoknot (reviewed in 3, 4) . A version of the À1 FS reporter plasmid with the IBV slippery sequence (p2lucIBV-AON) was also Spacer-length dependence of programmed À1 or À2 ribosomal frameshifting on a U 6 A heptamer supports a role for mRNA tension in frameshifting Based on the published literature, including our own studies of 80S ribosomes stalled at the IBV frameshift-stimulatory pseudoknot (22, 37) , we proposed a mechanical model of frameshifting in which a failure of intrinsic ribosomal helicase activity (15, 28) to unwind efficiently the stimulatory RNA during the translocation step leads to the build up of tension in the mRNA and subsequently, breakage of codon:anticodon contacts and realignment of the tRNAs in the À1 reading frame.
cord-319729-6lzjhn8j	2018	title: Lab-Attenuated Rabies Virus Causes Abortive Infection and Induces Cytokine Expression in Astrocytes by Activating Mitochondrial Antiviral-Signaling Protein Signaling Pathway Activation of mitochondrial antiviral-signaling protein (MAVS), the common adaptor molecule for RIG-I and MDA5, results in the production of type I interferon (IFN) and the expression of hundreds of IFN-stimulated genes, which suppress RABV replication and spread in astrocytes. Activation of mitochondrial antiviral-signaling protein (MAVS), the common adaptor molecule for RIG-I and MDA5, results in the production of type I interferon (IFN) and the expression of hundreds of IFN-stimulated genes, which suppress RABV replication and spread in astrocytes. To assess innate immune responses in astrocytes, cells were infected with DRV or B2c at an MOI of 0.1 and the expression of several proteins involved in the MAVS signaling pathway, namely, RIG-I, p-IRF7, STAT1 and IFIT1 (ISG56), was measured by Western blot.
cord-319780-rfj9t99r	2020	Analysis of the co-crystal structure suggested that the SARS spike protein binds to the active site of angiotensin converting enzyme 2 (ACE2, Li et al., 2005) . A truncated version of human recombinant ACE2, lacking the transmembrane domain, mitigated against SARS-CoV infection of cells (Li et al., 2003) and has been used in animal models to reduce symptoms of severe acute lung failure , diabetic nephropathy (Oudit et al., 2010) and cardiac hypertrophy and fibrosis . A recent cryo-EM structure suggested that ACE2 and B 0 AT1/SLC6A19 form a heterodimer which pairs up through interfaces between the two ACE2 partners (Figure 1) , with the RBD of SARS-CoV-2 spike protein binding to the peptidase active site of ACE2 suggesting that B 0 AT1/SLC6A19 may facilitate entry of the novel coronavirus. Tumor necrosis factor- convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensin-converting enzyme-2 (ACE2)
cord-319781-6thdg2up	2020	To understand factors that may contribute to viral spread and address long-term health sequelae for survivors, it is important to review evidence regarding viral presence in semen, sexual transmission potential, and possible effects on fertility. We review evidence for the following viruses: Ebola, Zika, West Nile, pandemic influenza, severe acute respiratory syndrome (SARS), and SARS-corona virus-2 (SARS-CoV-2). Then, we present the state of current research regarding presence in semen, sexual transmission, and fertility effects for the Zika virus (ZIKV), Ebola virus (EBOV), West Nile virus (WNV), pandemic influenza, severe acute respiratory syndrome (SARS), and SARS-coronavirus-2 (SARS-CoV-2) ( Table 1) . In this article, we have reviewed the presence in semen, possibility of sexual transmission, and fertility implications of each of the major recent viral pandemics: Zika, Ebola, West Nile, pandemic influenza, SARS, and SARS-CoV-2.
cord-319821-ij34t1ae	2017	Here, we will review recent efforts to develop direct-acting antivirals as well as host factor-targeting inhibitors to treat enterovirus infections (Table 1) . 2C ATPase and 3D pol may be more promising targets for direct-acting antiviral drugs as they can be inhibited by small molecules, and several inhibitors of these factors were found to have broad-range anti-enteroviral activity. Since targeted drug discovery depends heavily on basic knowledge of virus replication, fundamental research on the role of viral enzymes as well as essential host factors for enterovirus replication remains needed for the development of broad-range antiviral drugs against these important pathogens. FDA-aroved drug to target fungal infections, is identified as a broad-spectrum enterovirus inhibitor and shown to target the lipid shuttling activity of oxysterol-binding protein that is essential for viral replication organell formation and/or function
cord-319842-4mnaicki	2005	Infection of human cells with poliovirus induces the proliferation of double-membraned cytoplasmic vesicles whose surfaces are used as the sites of viral RNA replication and whose origin is unknown. Here, we explore the role of several constituents of autophagosome machinery in poliovirus-and rhinovirusinfected cells by monitoring: the presence of autophagosomal protein LC3 in virally induced vesicles, the acquisition of colocalization of LC3 and LAMP1 in virally infected cells, the viral induction of punctate structures that stain with monodansylcadaverine (MDC), and the effects of perturbing the autophagosomal pathway pharmacologically and via RNA interference on intracellular and extracellular virus yield. To determine whether the autophagosome-like membranes induced during poliovirus infection perform an antiviral function or facilitate viral replication, we tested the effect on poliovirus yield of pretreating H1-HeLa cells with either tamoxifen or rapamycin, known inducers of autophagy.
cord-319906-s7kzp795	2011	When for a given reference structure a structure-based search is performed on a set of proteins from the Protein Data Bank (PDB), StralSV identifies all structurally similar fragments from that set, evaluates the calculated structure-based alignments between the query (reference) motif (designated "segment" in this work) and the detected structure fragments, and quantifies the observed sequence variability at each residue position on the query structure. (For an illustration of a "span", see additional file 1: StralSV-RdRp_Suppl_Figure 1.docx.) All residue-residue pairs that are contained within a span''s alignment are used to calculate the sequence variability data at the corresponding position in the query structure. The qualified hits (structure fragments from PDB with detected local similarities to the query structure) for six selected sequence positions (positional hits) of polio RdRp (positions identified in Figure 3 ) were categorized and quantified based on SCOP (Structure Classification of Proteins database; version 1.75, June 2009 release) identifiers [32] .
cord-320169-dtv7to3l	2020	The coronavirus was officially named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses based on phylogenetic analysis. The spike glycoprotein of SARS-CoV-2 binds to angiotensin-converting enzyme 2 (ACE2) in human and Chinese horseshoe bats, civet for cell entry, that is also dependent on S protein priming by the serine protease TMPRSS2. Domestic animals can suffer from disease as intermediate hosts that cause virus transmission from natural hosts to humans; for example, SARS-CoV and MERS-CoV crossed the species barriers into masked palm civets and camels, respectively [30, 31] [ Table 1 ]. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2 Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan.
cord-320212-fw51w4nm	2012	When the novel strains were compared to nine Levivirus genogroup I strains, they shared 95–100% similarity among the maturation, capsid and lysis proteins, but only 84–85% in the RNA-dependent RNA polymerase gene. In all analysis programs, the nucleotide or amino acid sequences were aligned to other strains and were therefore approximate positions on the replicase gene. Breakpoint nucleotides for strains DL52 and DL54 (when aligned to genogroup I strains) occurred between nt 84-592 and 84-401, respectively ( Figure 5 a, b) , corresponding to the approximate amino acid breakpoint positions of 133-197 within the replicase gene. Human Noroviruses, a positive sense ssRNA virus with a genome length of 7400-8300 nt, are considered to belong to a prototype strain if they share approximately 85% overall nucleotide sequence identity and a high amino acid sequence identity (>95%) in the polymerase gene [20] .
cord-320325-sjab8zsk	2018	Using purified KSHV SOX protein, we reconstituted the cleavage reaction in vitro and reveal that SOX displays robust, sequence-specific RNA binding to residues proximal to the cleavage site, which must be presented in a particular structural context. Using an RNA substrate that is efficiently cleaved by SOX in cells, we revealed that specific RNA sequences within and outside of the cleavage site significantly contribute to SOX binding efficiency and target processing. Given that both substrates contain the requisite unpaired bulge at the predicted cleavage site (see Figure 2A and Supplementary Figure S2 ), these observations suggest that additional sequence or structural features impact SOX targeting efficiency on individual RNAs. Two SOX point mutants, P176S and F179A, located in an unstructured region of the protein that bridges domains I and II have been shown to be selectively required for its endonucleolytic processing of RNA substrates (Supplementary Figure S3A and S3B) (8, 21) .
cord-320351-47d0nby0	2020	Recently conducted researches indicated that circRNAs can be used as a miRNA sponge to inhibit targeted mRNA functions, showing interaction to RNA-binding proteins (RBPs) and translating proteins [9] . In contrast to the sensitive strains, the expression of 2909 circRNAs in A549 / Taxol was noticeably enriched and 8372 circRNAs were noticeably declined, demonstrating that abnormal cir-cRNA is likely to alter the occurrence of paclitaxel resistance [33] .Circ-PVT1 was found to facilitate paclitaxel resistance of gastric cancer cells by controlling ZEB1 expressing via the sponging process for miR-124-3p [34] . Lymphoblastic lymphoma [52] -T cell structure and degradation of circRNAs regulating PKR Activation in innate immunity circ-CDR1as B cell serving as the miR-7 "sponge" to increase expression of PTEN and restrain SLE [66] Other immune-related diseases circ_ 0057980 Circular RNA circ-PVT1 contributes to paclitaxel resistance of gastric cancer cells through the regulation of ZEB1 expression by sponging miR-124-3p
cord-320501-xqgqq55q	2020	title: Emerging vaccine delivery systems for COVID-19: Functionalised silica nanoparticles offer a potentially safe and effective alternative delivery system for DNA/RNA vaccines and may be useful in the hunt for a COVID-19 vaccine Liposomal encapsulation of drugs with bioavailability issues was a proven approach in small molecule pharma, and it was apparent to biopharma R&D scientists that LNPs could meet the basic requirements of an RNA/DNA delivery system too, namely to protect nucleic acid from in vivo digestion as it travels to the target. As scientists looked to alternative carriers that could be adapted to encapsulate and protect nucleic acids, mesoporous silica nanoparticles (MSNs)silica-based nanostructured materials with excellent biocompatibility and chemical stability This surface simply and effectively traps and protects nucleic acids (such as mRNA/pDNA) from nuclease enzymes as it travels to the cells. Once inside the cell, the DNA/RNA is released and will result in synthesis of the foreign/target protein which will activate the immune system, leading to both a cellular and humoral immune response.
cord-320709-2pnqpljt	2016	The Mx1, ISG56 and RANTES gene expression in the lungs of Jamaican fruit bats was analyzed as an indicator of the induction of an innate immune response to MERS-CoV infection. The tissue tropism of MERS-CoV in Jamaican fruit bats is comparable to the respiratory tract tropism observed in dromedary camels and humans 49, 50 . MERS-CoV and related batCoV-HKU4 can inhibit innate immune signaling in a variety of human cell lines in vitro via the ORF4b-encoded accessory proteins 52 Lungs of Jamaican fruit bat 5 were stained with α -cytokeratin as an epithelial marker (purple) and with a polyclonal α -coronavirus antibody (brown-red) to demonstrate that viral antigen was located along the basement membrane of alveolar pneumocytes of bat 1 at 2 dpi (indicated by black arrows). Middle East respiratory syndrome coronavirus (MERS-CoV) in dromedary camels
cord-320713-b37c8aye	2009	6 The rate of translation initiation in mammalian cells is also controlled by sequence elements within the 5 0 -and 3 0 -UTRs of mRNAs which regulate this process by providing sites for interaction of regulatory proteins and RNAs. These include upstream open reading frames (uORFs), microRNA (miRNA) target sites, and polyadenylation elements. 5 It was suggested that alternative eIF4F complexes lacking PABP could selectively promote the synthesis of viral, but not host, proteins, so that KSHV-encoded mRNAs would compete more effectively for host translation machinery in infected cells. Picornavirus translation is directed by internal ribosome entry sites (IRESs) within the 5 0 -UTRs of the viral RNAs. The central one-third of eIF4G, containing the eIF3 and one eIF4A-binding domain, is sufficient to support translation initiation from these IRESs. 43 This allows picornavirus RNAs to compete effectively for the host translation machinery following infection, although the situation appears to be more complicated than this (see Section III).
cord-320921-eumuid3r	2019	Our data indicate that despite relatively high viral RNA levels produced, low levels of infectious virus are excreted in the upper respiratory tract of rabbits as compared to dromedary camels, thus resulting in a lack of viral transmission. Besides dromedary camels, other animal species, i.e. llamas, alpacas, and pigs have been shown to be susceptible and develop upper respiratory tract infection upon experimental intranasal MERS-CoV inoculation [9] [10] [11] . We found that rabbits inoculated with the MERS-CoV EMC strain and those with the Qatar15 strain developed an equally mild infection and shed similar levels of viral RNA in their nasal and throat swabs (Figure 3 ). We found that rabbits inoculated with the MERS-CoV EMC strain and those with the Qatar15 strain developed an equally mild infection and shed similar levels of viral RNA in their nasal and throat swabs (Figure 3 ).
cord-320935-3n157yl4	2020	This paper aims to collate information on recent developments on WBE in monitoring the trend of community-scale SARS-CoV-2 prevalence as well as models to predict virus spread and transmission among populations. While several studies have identified the presence of SARS-CoV-2 in the faecal matter of corona-infected patients [35, 36] , there is a growing concern on the transmission of the virus through water treatment plants (WTPs) and WWTPs. Several studies also detected the genetic material of the virus in raw wastewater across the globe [22, 26, 27] . These studies provided enough excellent reasons for modelling the spread of 2019-nCoV with the external environmental conditions, assuming that the cases of infection will decrease through secondary infection routes due to the inactivation of the virus on different surfaces; however, the possibility of transmission via direct contact remains unchanged.
cord-321013-8pkrg0mx	2014	The coronavirus nucleocapsid (N) is a structural protein that forms complexes with genomic RNA, interacts with the viral membrane protein during virion assembly and plays a critical role in enhancing the efficiency of virus transcription and assembly. The M protein is the main core shell component and a 16 amino acid domain (aa 237-252) on the CTD of M protein binds directly to N protein via an ionic interaction, leading to specific genome encapsidation in the budding viral particle [81] [82] [83] .The N protein therefore plays an essential structural role in the CoV virion through a network of interactions with (i) the genomic RNA; (ii) M protein and (iii) other N proteins. Amino acid residues critical for RNA-binding in the N-terminal domain of the nucleocapsid protein are essential determinants for the infectivity of coronavirus in cultured cells Structure of the SARS coronavirus nucleocapsid protein RNA-binding dimerization domain suggests a mechanism for helical packaging of viral RNA
cord-321053-lgae22f8	2013	Numerous in vitro studies in combination with a growing number of HCV sequencing data from patients undergoing DAA treatment underline that the virus can develop drug-resistance and fitness restoring compensatory mutations [11] . An emerging third group of antivirals, so called hosttargeting antivirals (HTA), may be part of such future combination therapies, in particular as HTAs hold the promise of overcoming some of the caveats of DAAs. HTAs are antibodies, RNAs or small molecules, which interfere with host factors needed for HCV propagation. On the one hand, this demonstrates that HCV can in theory evade HTA therapy by mutating the viral binding partner of the targeted host factor and in fact suggests a low genetic barrier to resistance. Targeting HCV RNA Replication: Phosphatidylinositol 4-kinase III alpha (PI4KIIIα) Genome wide RNA interference screens and in depth cell culture replication assays with HCV replicons and full length infectious virus have revealed numerous additional host dependency factors, that could in principle serve as antiviral targets [99] [100] [101] [102] [103] [104] [105] [106] [107] .
cord-321155-dty18esg	2020	We also found that the SUD-like sequence is retained in the SARS-CoV-2 genome, while some other coronaviruses that can infect humans are depleted. To get the potential G-quadruplexes in the SARS-CoV-2 genome, we took the strategy described as follows ( Fig. 2A) : (i) Predicting the PG4s with three software independently. To further characterize the potential canonical secondary structures competitive with Gquadruplexes, the landscape of thermodynamic stability of the SARS-CoV-2 genome was depicted by using ΔG°z-score [55] . The distributions of loop length between the SARS-CoV-2 PG4s and the human two-quartet Gquadruplexes did not show discrepancies (Fig. S1 , Wilcoxon test, p-value = 0.4552). Recent research revealed that the G-quadruplexes in human UTRs (Untranslated Regions) are under selective pressures [58] , and some coronaviruses on bats and pangolins are closely related to SARS-CoV-2. Thus, we started to explore whether the SARS-CoV-2 genome contains the protein-coding sequence similar to SUD and whether SARS-CoV-2 retains the ability to bind RNA G-quadruplexes.
cord-321505-m40s6uw9	2007	Background and Aim: We have reported previously that synthetic small interfering RNA (siRNA) and DNA‐based siRNA expression vectors efficiently and specifically suppress hepatitis C virus (HCV) replication in vitro. Intravenous delivery of the adenovirus expressing shRNA into transgenic mice that can be induced to express HCV structural proteins by the Cre/loxP switching system resulted in specific suppression of virus protein synthesis in the liver. Here, we report that HCV replication was suppressed in vitro by recombinant retrovirus and adenovirus vectors expressing short hairpin RNA (shRNA) and that the delivery of the adenovirus vector to mice in vivo specifically inhibited viral protein synthesis in the liver. These results indicated that the decrease in luciferase activities was due to specific suppressive effects of shRNA on expression of HCV genomic RNA and the viral proteins, and not due to non-specific effects caused by the delivery of shRNA or to toxicity of the adenovirus vectors.
cord-321607-3r736dnk	2016	The active nuclease then cleaves ssRNAs, both cellular and viral, leading to downregulation of their expression and the generation of small RNAs capable of activating retinoic acid-inducible gene-I (RIG-I)-like receptors or the nucleotide-binding oligomerization domain-like receptor 3 (NLRP3) inflammasome. Although cleavage of RNA virus genomes appeared as the most direct mechanism of action, other important pathways have become evident, such as the regulation of host gene expression, stimulation of IFNβ production, activation of the NACHT, LRR, and PYD-containing protein-3 (NLRP3) inflammasome, and maintenance of the cell''s structural barrier to infection [27, 55, 83, 84] . These small RNAs are capable of stimulating RIG-I and MDA5 (melanoma differentiation associated gene-5) to activate mitochondrial antiviral signaling protein (MAVS) and induce the subsequent translocation of interferon regulatory factor 3 (IRF3) to the nucleus to drive transcription of IFNβ.
cord-321773-5fw9abzl	2018	Given the crucial roles of DDX5 in RNA biology, several RNA viruses were found to interact with the protein to promote viral replication (Table 1) , including severe acute respiratory syndrome (SARS) coronavirus (CoV) [16] , human immunodeficiency virus 1 (HIV-1) [17] , hepatitis C virus (HCV) [18] , Japanese encephalitis virus (JEV) [19] , porcine reproductive and respiratory syndrome virus (PRRSV) [20] , and influenza virus [21] . There are several studies that focus on specific inhibitors or drugs of the host DEAD-box helicase to inhibit virus replication or treat cancers [65] [66] [67] , but it remains to be determined whether small molecular inhibitors of the interaction between DDX5 and Rev can be found. The DEAD-box RNA helicase DDX5 acts as a positive regulator of Japanese encephalitis virus replication by binding to viral 3 UTR The DEAD-box RNA helicase 5 positively regulates the replication of porcine reproductive and respiratory syndrome virus by interacting with viral Nsp9 in vitro
cord-321938-pda4a5n7	2020	We therefore checked whether a clinically developed aptamer, BC 007, which is currently in phase 2 of clinical testing for a different indication, would also be able to efficiently bind DNA-susceptible peptide structures from SARS-CoV-2-spreading crucial proteins, such as the receptor binding domain (RBD) of the spike protein and the RNA dependent RNA polymerase of SARS-CoV-2 (re-purposing). In the Spike protein of SARS-CoV-2, several sequences which are highly susceptible to interaction with DNA (multiple amino acids with positive charged side chains) were identified, in particular at the angiotensin I-converting enzyme 2 (ACE2)-receptor binding domain (RBD): YRLFRK (SARS-CoV-2 specific from protein data bank (PDB) data base entry PBD ID: 6VXX, source: [23] ), as well as NRKRISN (PBD ID: 6VXX) and KIKRMK (PDB ID: 5X5B source: [24] ). This enabled us to exploit NMR-spectroscopy to investigate whether the selected peptide-sequences from SARS-CoV-2 proteins bind to this clinically advanced aptamer (BC 007), forcing it into its well described quadruple structure just by molecular interaction.
cord-321957-ybtk9cp1	2020	title: Protein Kinase C subtype δ interacts with Venezuelan equine encephalitis virus capsid protein and regulates viral RNA binding through modulation of capsid phosphorylation A virus with capsid phosphorylation sites mutated to alanine (VEEV CPD) displayed a lower genomic copy to pfu ratio than the parental virus; suggesting more efficient viral assembly and more infectious particles being released. These data suggest that cells infected with VEEV CPD output more functional viral particles than VEEV TC-83, potentially due to more efficient viral packaging and this phenotype is due to loss of capsid phosphorylation. Loss of PKCδ through siRNA transfection also resulted in increased capsid viral RNA binding, further solidifying the link between VEEV CPD and PKCδ (Fig 7B) . To determine the impact of VEEV capsid phosphorylation on vRNA binding, we utilized quantitative RNA-IP experiments to probe the interaction between the capsid protein and the vRNA at sites identified as highly enriched, intermediately enriched, and non-enriched by our CLIP-seq studies.
cord-322062-nnefbeo6	1991	We now report on the molecular cloning and sequencing of the complete HEV (Burma; B) viral genome together with the deduced amino acid sequences of viral-encoded proteins General perspectives on the genetic organization of the virus, as deduced from sequence and open reading frame analyses, indicate that HEV bears some similarity to the caliciviridae but may represent a new class of nonenveloped RNA virus. Bife was chosen as the RNA source for cDNA synthesis because it contained relatively large numbers of virus particles when HEV cDNA clones were identified from libraries made from randomly primed cyno bile (solid square), or from cyno liver after priming by oligo-dT (solid circle), random sequence hexamers (open circle) and HEV-sequence specific oligonucleotides (open square). The presence of HEV-specific subgenomic RNAs localized to the 3'' one-third of the genome suggests that these may be the transcripts from which these 3'' end ORFs are expressed and is indicative of a unique expression strategy among nonenveloped positive-sense RNA viruses infecting humans.
cord-322084-gkg1059v	1996	Abstract In our studies of murine coronavirus transcription, we continue to use defective interfering (DI) RNAs of mouse hepatitis virus (MHV) in which we insert a transcription consensus sequence in order to mimic subgenomic RNA synthesis from the nondefective genome. By using PCR-based sequences flanking the same intergenic region between site-directed mutagenesis, a 12-nucleotide-long segenes 6 and 7 do not affect the efficiency of subgenomic quence, TCTAATCTAAAC, was inserted into DI cDNA DI RNA transcription (Makino and Joo, 1993) . For all of the constructs used in Deletion analysis of those MHV DI RNAs that contain this study we sequenced the inserts that were derived the intergenic sequence from gene 6-7 with its naturally from PCR products to confirm the presence of specific occurring flanking sequences showed that reducing the mutations and the absence of extraneous mutations.
cord-322206-roxa3ix6	2020	Herein, we used RNA-based metatranscriptomics associated with Ion Torrent deep sequencing to allow for the high-quality reconstitution of an outbreak-related Zika virus (ZIKV) genome (10,739 nt), with extended 5′-UTR and 3′-UTR regions, using a newly-implemented bioinformatics approach. Besides allowing for the assembly of one of the largest complete ZIKV genomes to date, our strategy also yielded high-quality complete genomes of two arthropod-infecting viruses co-infecting C6/36 cell lines, namely: Alphamesonivirus 1 strain Salvador (20,194 nt) and Aedes albopictus totivirus-like (4618 nt); the latter likely represents a new viral species. Altogether, our results demonstrate that our bioinformatics approach associated with Ion Torrent sequencing allows for the high-quality reconstruction of known and unknown viral genomes, overcoming the main limitation of RNA deep sequencing for virus identification. Here, we applied RNA-based metatranscriptomics associated with Ion Torrent deep sequencing and a newly developed Bioinformatics approach to the high-quality reconstitution of viral genomes.
cord-322234-1zyy536y	2009	To evaluate the applicability of the test as a diagnostic tool in the screening of field specimens from swine, 64 field isolates of North American swine, 5 equine and 48 avian influenza viruses collected during diagnostic investigations were analyzed retrospectively as well as samples collected during an experimental in vivo infection with two novel H1N1 isolates, A/California/04/2009 (H1N1)v virus and A/Mexico/4108/2009 (H1N1)v. Swine and equine influenza virus isolates and the clinical samples from pigs infected experimentally with 2009 (H1N1)v were subjected to the USDA-validated qRT-PCR procedure for the general detection of type A influenza virus RNA (matrix screening assay), following procedures described previously (Spackman and Suarez, 2008) . All endemic North American swine influenza virus isolates were negative for (H1N1) 2009 specific matrix gene RNA using the present qRT-PCR assay, whereas the (H1N1) 2009 strains used as positive control were positive.
cord-322240-z8zkl2xh	2008	To date, only 1 case of disseminated infection has been reported in a solid-organ (kidney) transplant recipient; the diagnosis was made by molecular identifi cation in isolates from blood and marrow cultures. As a fi rst step in investigating unidentifi ed pathogens in bats and to help forecast the potential threat of emerging infectious diseases, we tried to isolate and characterize viruses that persistently infect bats. However, no viral nucleic acid sequence was detected from an RNA sample in the RV1-infected BKT1 cells. In addition, our success in DNA virus isolation might have resulted from usage of the adult animal latently and persistently infected with DNA viruses such as adenovirus and herpesvirus. Although its pathogenicity for humans is still unknown, knowledge of RV1 will be useful in epidemiologic studies of infectious diseases emerging from bats because persistently infecting viruses might be isolated together with primary pathogens.
cord-322410-k23engcx	2017	title: New real time and conventional RT-PCRs for updated molecular diagnosis of infectious bronchitis virus infection (IBV) in chickens in Egypt associated with frequent co-infections with avian influenza and Newcastle Disease viruses Conventional RT-PCRs amplifying hypervariable regions (HVRs 1–2 and 3) of the IBV S1 gene were developed and amplificates used for nucleotide sequence-based typing of IBV field strains in Egyptian chickens directly from clinical samples. The specificity of RT-qPCR primer set was evaluated by examining different avian respiratory viruses circulating in poultry in Egypt including AIV H5N1, H9N2 and NDV as well as different coronaviruses including a panel of IBV reference strains as listed in the materials section. Within HVR 1, 2 sequences, however, the existence of two distinct Table 3 RT-qPCRs reveal frequent co-infections in Egyptian poultry samples with avian influenza (AIV), Newcastle Disease (NDV) and infectious bronchitis viruses (IBV).
cord-322756-ouvn71r9	2020	Studies investigating RNA therapeutics in pulmonary diseases have rapidly expanded and drug administration by inhalation allows the direct delivery of RNA therapeutics to the target site of action while minimizing systemic exposure. Interestingly, it has been known for over a decade that naked RNA, including both siRNA and mRNA, can be transfected in the lung following pulmonary delivery, as shown in many in vivo studies [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] . Both studies demonstrated a gene-silencing effect of the powder formulations in lung tissues following intratracheal administration in mouse models of lung cancer, taking these delivery systems one step closer to clinical application. To take advantage of this phenomenon, pulmonary surfactant and surfactant protein B-coated dextran-based nanoparticles were developed for siRNA delivery, with successful gene-silencing effects observed in healthy mice and in a model of acute lung injury (ALI), respectively, following pulmonary administration [29, 69] (Table 1) .
cord-323029-7hqp8xuq	2020	For aptamer selection against rIgG [6] and IgE [7] , homogenous processes were used and the separation of bound and unbound nucleic Several different SELEX methods have been used to generate immunoglobulin aptamers including homogeneous, heterogeneous, bead-based SELEX processes, CE-SELEX (capillary electrophoresis-SELEX), µFFE-SELEX (micro-free flow electrophoresis-SELEX) and fully integrated selection processes selection of aptamers with proper binding properties. Molecular Light Switch Complex 100-800 100 [128] Fluorescence enhancement using a DNA aptamer 92-37,000 57 [130] Amplification through allostery-triggered enzymatic recycling amplification NA 5 [131] Fluorescent oligonucleotide probe based on G-quadruplex scaffold for signal-on ultrasensitive protein assay 4.72-7560 0.095 [132] Fluorescence anisotropy 1000-60,000 350 [133] Fluorescence anisotropy assay NA 20 [134] Fluorescence protection assay 100-50,000 100 [136] Aptamer-barcode-based assay based on instantaneous derivatization chemiluminescence coupled to magnetic beads 4.88-20,000 4.6 [137] Competitive fluorescence quenching assay 350-35,000 170 [138] Rapid fluorescence detection of immunoglobulin E using an aptamer switch based on a binding-induced pyrene excimer NA 1600 [139] 6.1.
cord-323585-iv2dcpqj	2015	The NS5A protein of classical swine fever virus (CSFV) is involved in the RNA synthesis and viral replication. The GST or GST-NS5A fusion proteins expressed in Escherichia coli BL21(DE3) were purified with glutathione Sepharose 4B resin and incubated with the lysate of HEK293T cells overexpressing the Myc-tagged eEF1A. The immunoprecipitate was analyzed by Western blotting using the anti-Flag MAb (1:1000) and a rabbit anti-Myc PAb (1:500); (D) Co-IP analysis of eEF1A and NS5A by the anti-Myc MAb. HEK293T cells were cotransfected with the indicated plasmids (+) or empty vectors (''), the cell lysate was collected at 48 h post-transfection (hpt), incubated with the anti-Myc MAb and Protein G-Agarose. The bound proteins were analyzed by immunoblotting using the anti-Myc monoclonal antibody (MAb) (1:1000); (B) eEF1A interacts with the CSFV IRES in a dose-dependent manner. The bound proteins were analyzed by immunoblotting using the anti-Myc monoclonal antibody (MAb) (1:1000); (B) eEF1A interacts with the CSFV IRES in a dose-dependent manner.
cord-323668-evzzfu04	2013	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. These findings suggest the widespread differential expression of lncRNAs in response to 0006 virus infection and their involvement in regulating the host response, including innate immunity [19] . Further analysis resulted in 313 differentially expressed lncRNAs and nearby coding gene pairs (distance < 300 kb) for each comparison between mock-and EV71-infected cells (Table S7) . In the present study, using Arraystar microarray analysis, we identified the differentially expressed lncRNAs in RD cells after EV71 infection, together with nearby differentially expressed mRNA pairs. They also observed the down-regulation of several genes encoding proteins involved in host RNA synthesis in EV71-infected SF268 cells. [19] performed functional enrichment analysis on the nearby protein-coding genes of differentially expressed lncRNAs in SARS-CoV infected mouse.
cord-323691-5s5almd2	2001	Abstract The ''infectious DNA'' approach, which is based on in vivo transcription of (+)RNA virus genome cDNA cassettes from eukaryotic promoters in transfected cells, became a popular alternative to the classical scheme in the infectious clone methodology. Substantial difficulties, however, were encountered in design of flavivirus ''infectious DNA'', requiring either modification of the viral genome cassette (Yamshchikov et al., 2001) to prevent unwanted expression of viral genome segments encoding toxic for Escherichia coli products, or deletion of the structural protein region (Varnavski et al., 2000) . For this reason we sought to investigate if the stability of constructs containing an unmodified virus genome cassette can be improved by preventing its deleterious expression at the transcriptional level, i.e. by minimizing spurious transcription from eukaryotic promoters in E.
cord-323737-6ajqy0ch	2020	title: Structural analysis, virtual screening and molecular simulation to identify potential inhibitors targeting 2''-O-ribose methyltransferase of SARS-CoV-2 coronavirus In the present study, we employed structural analysis, virtual screening, and molecular simulation approaches to identify clinically investigated and approved drugs which can act as promising inhibitors against nsp16 2′-O-MTase of SARS-CoV-2. In the present study, we employed structural analysis, virtual screening, and molecular simulation approaches to identify potential inhibitors targeting 2 0 -O-MTase of SARS-CoV-2. To identify inhibitors targeting nsp16, we first performed comparative analysis of primary amino acid sequences and crystal structures of seven human CoVs. Supplementary Table 1 lists the detailed genome and protein information that were employed in this study. As seen from MM-PBSA results and docking studies, drugs including Hesperidin, Osi-027, Rimegepant, Sonedenoson, and Gs-9667 had higher binding affinities than SAM with the 2 0 -O-MTase of SARS-CoV-2.
cord-323756-atnrw9ew	2013	When Janeway formulated the theory of pattern recognition in 1989, he proposed that host cells could sense microbial infection owing to receptors able to recognize invariant molecular structures defined as pathogen-associated molecular patterns (PAMPs). They share a similar organization with three distinct domains: (i) a C-terminal repressor domain (RD) embedded within the C-terminal domain (CTD); (ii) a central ATPase containing DExD/H-box helicase domain able to bind RNA; and (iii) a N-terminal tandem CARD domain that mediates downstream signaling, and which is present in RIG-I and MDA5 but absent in LGP2. DDX60 has also been shown to enhance the IFN-I response to RNA and DNA stimulation through formation of complexes with Frontiers in Immunology | Molecular Innate Immunity RIG-I, MDA5, and LGP2 but not with MAVS. Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA Nonself RNA-sensing mechanism of RIG-I helicase and activation of antiviral immune responses
cord-323845-s78t5qxj	2006	title: Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS) A one step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of viral hemorrhagic septicaemia virus (VHS). Reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viral hemorrhagic septicaemia virus (VHS) The aim of the present study was to develop a onestep, single-tube, accelerated RT-LAMP reaction for rapid detection of different serotypes of VHS virus. The specificity of the VHS-LAMP primers and conditions to VHS virus was confirmed by its aptitude to amplify only RNA from VHS virus serotypes, with no amplification of IHNVor non-infected fish (Fig. 5) . Detection of viral hemorrhagic septicaemia virus (VHS) in rainbow trout (Oncorhynchus mykiss) by reverse transcription followed by polymerase chain reaction. A loop mediated isothermal amplification (LAMP) method for detection of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss)
cord-323973-wszo9s3d	2020	[9] The analysis of larger volumes of biological fluids with a low viral load is likely to reduce the risk of false-negative results, but microfluidic point-of-care (POC) devices are typically unable to handle mL-scale volumes in a short time due to the requirement of slow flow rates. [15] The PCR test on samples prepared from blood and urine specimens is capable of detecting the virus in the early stages of the disease, resulting in early diagnosis and subsequent isolation of infected patients to block transmission. The total reaction time depends on a series of parameters, such as the chip size, the PCR master mix volume determining the value of C, the thermal conductivity of the substrate (G), and the temperature cycling rate. A number of fully integrated systems have so far been developed, starting with the non-portable GenExpert from Cepheid, which was first utilized for anthrax detection by the United States Postal Service, [64] and then with different primers to perform HIV and tuberculosis diagnostics in South Africa.
cord-323987-gh1m05gi	2018	RIDTs with digital readout systems showed many similarities to conventional assays like small sample volume (less than 150 µL) and short analysis time (around 15 min) but exhibited much better sensitivities, even one order of magnitude lower limits of detection (LODs). Among methods mentioned, general diagnostic tests for influenza base on virus culture (conventional and shellvial), detection of viral nucleic acid (PCR) or antigens (by neuraminidase enzymatic activity, fluorescent antibody or enzyme/optical immunoassay) and serologic tests. Main trends for influenza virus detection are: (I) modifications of traditional ''gold star'' methods like PCR, RIDTs, ELISA what results in analysis time shortening, costs lowering, LOD and limit of quantification (LOQ) improvement, (II) conjugating of traditional methods and creating new platforms, micro-biochips and others, (III) introducing known solutions to new ones, like smartphone-based analysis control with results data insertion into Google Maps, (IV) reuse of the functions of known devices, like glucometer, smartphone cameras, (V) the most common used detection methods: spectral/optical, electrical, (VI) and entirely new approaches.
cord-324137-nau83mjv	2018	Several reports convincingly demonstrate antimicrobial activity of quadruplex-binding ligands against clinically challenging pathogens, including HIV-1, HCV, Ebola virus, Plasmodium falciparum, and Mycobacterium tuberculosis. An earlier study reports that, following concatemeric replication of HHV-1, the cleavage of unit length genomes and their encapsidation is achieved by the binding of virus proteins to a DNA secondary structure formed by a DNA packaging sequence (pac-1) [50] . G4s have been shown to inhibit the transcription or translation of structural and nonstructural proteins in viruses, deleteriously affecting the virus loads and their pathogenicity; the stabilization of these quadruplexes with ligands has been investigated as a potential mechanism for targeting viruses. Negative regulation of virus transcription, translation or replication by quadruplex motifs in virus genomes forms the basis of using G4-binding ligands as antiviral agents. G-quadruplex forming structural motifs in the genome of Deinococcus radiodurans and their regulatory roles in promoter functions
cord-324212-aqp73hi9	2006	We identified a leadzyme substrate target sequence in genomic tobacco mosaic virus RNA and designed a 16‐mer oligoribonucleotide capable of forming a specific leadzyme motif with a five‐nucleotide catalytic loop. We identified a leadzyme substrate target sequence in genomic tobacco mosaic virus RNA and designed a 16-mer oligoribonucleotide capable of forming a specific leadzyme motif with a five-nucleotide catalytic loop. To achieve this goal, we analyzed the inhibition of tobacco mosaic virus (TMV) as a model system for inhibiting viral infections caused by positive single-stranded RNA (+)ssRNA viruses. In this article, we show that exogenous ssRNA with sequence complementarity binds the target site in TMV RNA to form a leadzyme motif, and in the presence of a catalytic amount of Pb 2+ , cleaves viral (+)ssRNA. Control assays, with 16-nucleotide catalytic RNA only or Pb 2+ applied in the presence of TMV, were performed to demonstrate the specificity of leadzyme cleavage.
cord-324324-8ybfiz8f	2020	In addition, the close contact between human beings and different animal species sold at the wet markets of East Asia represents the optimal situation for the host species jump and adaptation to humans of potentially zoonotic agents like CoVs. It is not a coincidence that two of the most severe zoonoses of the last two decades (highly pathogenic H5N1 avian influenza and SARS) have emerged in the same Chinese province of Guangdong where the contact between humans and animals is closer (Lorusso et al., 2020) . All these viruses as well as analogous IBV-like CoVs detected in other birds including penguins, pigeons, peafowl, parrots, waterfowl, teal, quail, duck and whooper swan (Cavanagh et al., 2002; Circella et al., 2007; Domanska-Blicharz et al., 2014; Torres et al., 2013; Hughes et al., 2009; Liu et al., 2005; Wille et al., 2016; Jordan et al., 2015; Bande et al., 2016; Suryaman et al., 2019) have been assigned to the same viral species known as Avian coronavirus (ACoV) within the subgenus Igacovirus of genus Gammacoronavirus.
cord-324495-0pee1i3o	2015	Therefore, the present study was conducted to determine whether Sasa quelpaertensis Nakai extract (SQE) inhibits PRRSV infection in cultured porcine alveolar macrophages (PAMs). Further experiments revealed that SQE suppresses post-entry steps in the replication cycle of PRRSV, including viral genomic and sg RNA synthesis, viral protein expression, and virus production. The presence of SQE notably altered expression of cytokine genes in PRRSV-infected PAM cells, suggesting that SQE activity is involved in the modulation of inflammatory responses during viral infection. As shown in Fig. 5 , SQE treatment resulted in a maximal reduction in the synthesis of PRRSV genomic RNA and sg mRNA of 90 % and 80 %, respectively, at a concentration of 5 mg/ ml, when compared with untreated infected cells. Treatment of cells with SQE resulted in significant inhibition of post-entry steps during the replication of PRRSV, as demonstrated by reduced progeny production, diminished viral protein expression, and reduced synthesis of genomic RNA and sg mRNA.
cord-324638-gwd8qin6	2006	We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The main objective of this study was to compare the resultant analytical sensitivity and quantitative performance of the serum SARS-CoV RNA test when either the manual or automated extraction protocol was used. The modified large volume protocol with the external lysis step was further compared with the external lysis protocol of the total nucleic acid isolation kit using a transport medium mixture containing 10 6 copies/mL of inactivated SARS-CoV. Serially diluted inactivated SARS-CoV isolate in transport medium was extracted by both the column-based manual method and the MagNA Pure LC instrument using the modified large volume protocol with external lysis.
cord-324640-2zhaknbi	2010	Although RNA synthesis and virus assembly occur in the cytoplasm, HRSV is known to induce nuclear responses in the host cell as replication alters global gene expression. More recently, 2DE was used to compare the potential effect of several different negative strand RNA viruses, including HRSV, parainfluenza virus, human metap-neumovirus, measles virus, and influenza virus, on the host cell proteome with common changes in proteins involved with apoptosis and endoplasmic reticulum stress being highlighted (27) . Together, the indirect immunofluorescence confocal microscopy images supported the observations from the quantitative proteomic analysis that the abundance of mitochondrial proteins (particularly pore proteins) was altered in HRSV-infected cells. Potential Disruption of Proteins Involved in Nucleocytoplasmic Trafficking-Network pathway analysis indicated that the abundance of nuclear pore complex components and proteins involved in the nucleocytoplasmic trafficking of proteins and RNA differed between HRSV-infected and mock-infected cells (Fig. 4) .
cord-324697-c0dv1zmi	2020	Consequently, viruses have evolved an arsenal of strategies to target these RNA features and ultimately take control of the pathways they influence, and these strategies contribute to the global shutdown of the host gene expression machinery known as "Host Shutoff". Throughout this section we will discuss how each of these RNA features render mRNA susceptible toand in many cases directviral endonuclease cleavage or similar strategies aimed at degradation of the host transcriptome during viral infection. Nsp1 thus emerges as a thorough RNA decay trigger that uses diverse and non-overlapping strategies to widely target host mRNAs. How the viral transcripts escape nsp-1 mediated is still under investigation. Overall, SARS coronavirus nsp1 is an interesting regulator of RNA stability: currently, nsp1 does not appear to have any endonucleolytic activity of its own, and instead binds to the 40 s subunit exploiting the host''s RNA quality control pathways to trigger mRNA degradation. Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression
cord-324928-cpryxa6p	2020	The differences between outgroup viruses and those belonging to the SFV complex had an impact on the design of the template RNAs. The 5'' region of the CHIKV genome contains seven SL structures that affect genome replication in mammalian and/or mosquito cells [33] . Cross-utilisation of template RNAs by alphavirus replicases structures can be predicted for RNAs of other members of the SFV complex (S1 Fig), the 5'' ends of the template RNAs of SFV, MAYV, RRV and ONNV had an identical design to that of the previously constructed CHIKV template, i.e. the 5'' UTR was followed by 231nt encoding the N-terminus of nsP1 [43] . The levels of Fluc and Gluc expression in human cells, transfected with plasmids expressing template RNA and corresponding polymerase negative control replicase (P1234 GAA ), were similar for trans-replicases derived from different viruses and the same was observed for Ae albopictus cells.
cord-324944-ixh3ykrc	2018	This review gives an overview of diagnostic technologies featuring a platform based approach: (i) assay (nucleic acid amplification technologies are examined); (ii) cartridge (microfluidic technologies are presented); (iii) instrument (various detection technologies are discussed); and at the end proposes a way that such technologies can be interfaced with electronic clinical decision-making algorithms towards a broad and complete diagnostic ecosystem. In studies that have recorded the clinical presentation of patients (and not only their laboratory results), the causes of fever in outpatients could be classified into four main syndromes: 1) acute respiratory infections (ARI, of any type); 2) diarrhea (gastroenteritis); 3) fever with another clear focus (e.g. meningitis or skin infection); and 4) non-specific fevers [13] (each diagnostic platform described in Section 5 focuses on at least one of the aforementioned cases).
cord-324984-ojrpsdt9	2020	In addition, host proteins are not under the genetic control of viral genome, and hence HTAs possess much higher genetic barrier to drug resistance as compared with DAAs. In recent years, much progress has been made to the development of HTAs with the approval of chemokine receptor type 5 antagonist maraviroc for human immunodeficiency virus treatment and more in the pipeline for other viral infections. 3 Altogether, targeting host factors is a very promising strategy with possibility to address the critical challenges faced with the DAAs. In this review, we summarize the recent advances made in HTAs from a medicinal chemistry standpoint, and the host targets are generally classified into three different categories based on the development stage of their corresponding inhibitors/modulators, namely the ones which reached Food and Drug Administration (FDA) approval, that have entered clinical trials and those in preclinical studies.
cord-325043-vqjhiv7p	1989	title: An NTP-binding motif is the most conserved sequence in a highly diverged monophyletic group of proteins involved in positive strand RNA viral replication These observations, taken together with the results of comparative analysis of the positions occupied by respective proteins (domains) in viral multidomain proteins, suggest that all the NTP-motif-containing proteins of positive-strand RNA viruses are homologous, constituting a highly diverged monophyletic group. Preliminary comparative analysis of the amino acid sequences of the NTP-motif-eontaining proteins of positive-strand RNA viruses by use of the programs DIAGON and OPTAL (see Methods) revealed three distinct families and some additional proteins in whose close relatives the motif was not conserved. In the present study we demonstrate that in a highly diverged group including similar proteins of positive-strand RNA viruses, the consensus sequences of the NTP-motif constitute the most strictly conserved stretches, encompassing four of the five invariant amino acid residues.
cord-325113-sou8xyld	2020	The use of unprocessed swap samples is enabled by employing a heat-stable RNAand DNA-dependent DNA polymerase, which performs the double task of stringent reverse transcription of RNA at elevated temperatures as well as PCR amplification of a SARS-CoV-2 specific target gene. A RNA-and DNA-reading heat-stable polymerase reverse transcribes and amplifies viral RNA Evidence of an acute SARS-CoV-2 infection depends on the detection of viral RNA species in patient samples, which necessitates reverse transcription of RNA followed by PCR amplification of the resulting DNA. To evaluate the potential of the high-temperature RT-PCR protocol using Volvano3G for the detection of viral RNAs in patient material, we assessed the presence of SARS-CoV-2 in RNA isolated from a small cohort of COVID-19 suspected cases. Interestingly, for most positive samples detected by the high-temperature RT-PCR with Volcano3G, the cq-values were lower compared to the standard RT-PCR (Fig 3C and 3D) , indicating that the detection of SARS-CoV-2 from unprocessed patient material is not limited by the sensitivity of this direct approach.
cord-325137-6c6er06a	2016	Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Thus, in some instances, large cDNAs, double-stranded DNAs (dsDNAs), or clones containing at least two-thirds of the genome may also be regulated as SAs. Positive-sense RNA viruses span multiple virus families, and the infectious nature of these genomic RNAs coupled with SA/biosafety/biosecurity concerns inhibit rapid removal from BSL-3/4 containment (7) or transport and handling of RNA samples that are known to contain viral genomes from outbreak settings.
cord-325197-j1uo8qmf	2020	Viruses causing severe pulmonary illness can use epigenetic-regulated mechanisms during host–pathogen interaction to interfere with innate and adaptive immunity, adequacy of inflammatory response, and overall outcome of viral infections. In this article, we provide an update on epigenetic-sensitive mechanisms and repurposed drugs interfering with epigenetic pathways which may be clinically suitable for risk stratification and beneficial for treatment of patients affected by severe viral respiratory infections. The goal of the review was to provide an appropriate pathogenic scenario in which epigenetic-sensitive mechanisms and epidrugs may be clinically useful to stratify risk and treatment of patients in ICU affected by severe viral respiratory infections. Here, we give an update on clinical evidence about the usefulness of novel and FDA-approved drugs interfering with epigenetic pathways, which were applied to ICU patients affected by highly pathogenic strains of influenza virus and CoV, with a particular interest about the novel SARS-CoV-2 (Table 4 ).
cord-325230-3kg4oe4g	2010	These proteins include: capsid proteins; an RNA-dependent RNA polymerase (3D pol ); a protein (VPg, or 3B) that serves as a primer for the initiation of RNA synthesis; an ATPase with a conserved superfamily 3 helicase motif (2C ATPase ) and an essential but poorly defined role in viral RNA replication; a chymotrypsin-like protease (3C pro ), which, as the mature protein or as a precursor, is a major factor in polyprotein processing; two hydrophobic membrane-binding proteins (2B and 3A) that participate in the generation of a virus-friendly environment; and, flanking the precursor of the capsid proteins, one or two highly variable proteins (L and 2A), the structure and functions of which are the subject of this Review.
cord-325280-4whzcmqv	2017	In this review, we summarize antiviral microbial products discovered by Institute of Microbial Chemistry (IMC) and discuss antiviral compounds against influenza virus and HBV, because these two viruses threaten global human health now and antiviral drug against these two viruses have been developed. THE CONTRIBUTIONS OF IMC TO ANTIVIRAL RESEARCH Nucleos(t)ide analogs are a major class of approved antiviral drugs that exert therapeutic effects through incorporation into viral DNA and RNA to inhibit virus replication. Viral polymerase complex is a promising target for the development of anti-influenza drugs, because transcription and replication are critical steps for virus propagation. Potential applications of microbial products in anti-HBV drug development Approved nucleos(t)ide analogs competitively inhibit DNA elongation of viral polymerase after the conversion to triphosphate form by cellular enzymes.
cord-325326-2bbqz4o7	2010	We have developed a high-resolution genomic mapping technique that combines transposon-mediated insertional mutagenesis with either capillary electrophoresis or massively parallel sequencing to identify functionally important regions of the Venezuelan equine encephalitis virus (VEEV) genome. Toward this goal, we used transposon mutagenesis, reverse genetics, and fragment analysis by capillary electrophoresis to identify regions of the nsP3 gene that are important for replication and that result in temperature sensitive (ts) mutations. We used transposon mutagenesis to construct a cDNA library with small DNA fragments randomly inserted throughout the VEEV genome and then produced replication-competent virus through reverse genetics. Comparing transposon insertion sites in the resultant viruses to those in the starting library, we were able to produce a functional map of the entire genome of VEEV, and to identify several hundred potential ts mutations, including those we originally identified with the capillary electrophoresis method.
cord-325328-3l3jznkj	2005	Structural studies and comparative sequence analyses have suggested that biological RNAs are largely modular in nature, composed primarily of conserved structural building blocks or motifs [4] of secondary (helices, and internal, external and junction loops) and tertiary (coaxial stacks, kissing hairpin loops, ribose zippers, etc.) structure. Other structures include the specificity domains of both A[17] and B-type ribonuclease P [18 ] ; RNAs corresponding to a guanine-responsive riboswitch (xpt) complexed with guanine [19 ] or hypoxanthine [20] , and an adenosine-responsive riboswitch (add) complexed with adenosine [19 ] ; a highly conserved stem-loop motif found at the 3 0 end of the genome of SARS (severe acute respiratory syndrome) virus and other coronaviruses [21 ] ; the core encapsidation signal of MMLV [2 ] ; and complexes between a high-affinity RNA aptamer and the NF-kB p50 homodimer [22] , and between the archaeal RNAbinding protein L7Ae and an RNA K-turn derived from a H/ACA small RNA [23] .
cord-325479-2r4oomdp	2020	This study aims (1) to compare the whole process recovery of Pseudomonas phage φ6, a surrogate for enveloped viruses, among combinations of primary concentration [ultrafiltration (UF), electronegative membrane vortex (EMV), and polyethylene glycol precipitation (PEG)] and RNA extraction methods (spin column-based method using QIAamp Viral RNA Mini Kit and acid guanidinium thiocyanate–phenol–chloroform extraction using TRIzol reagent) for three types of raw sewage and (2) to test the applicability of the method providing the highest φ6 recovery to the detection of SARS-CoV-2 RNA. This study aims (1) to compare the combination of primary concentration (UF, EMV, and PEG) and RNA extraction (QIAamp Viral RNA Mini Kit and TRIzol) for the whole process recovery of nonenveloped and enveloped virus surrogates and (2) to test the applicability of the method providing the highest φ6 recovery to detect SARS-CoV-2
cord-325529-pid58g2r	2020	METHODS: We tested the efficiency and sensitivity of pooling strategies for RNA extraction and RT-PCR detection of SARS-CoV-2. Implementing the 8-sample Dorfman pooling to test 26,576 samples from asymptomatic individuals, we identified 31 (0.12%) SARS-CoV-2 positive samples, achieving a 7.3-fold increase in throughput. Some key constrains are (1) a limit on the number of stages due to the importance of delivering a test result quickly, exemplified by the urgent clinical context of COVID-19 diagnosis; (2) a limit on the ability to dilute samples and still safely identify a single positive sample in a pool; and (3) favorability of simple algorithms which may minimize human error in a laboratory setting. Specifically, we have demonstrated that pooling lysates from 5 or 8 nasopharyngeal swab samples retains sufficient sensitivity of viral RNA detection, allowing identification of SARS-CoV-2-positive individuals, while increasing throughput 5-fold to 7.5-fold.
cord-325624-6anybxnk	2009	The unique phenotype of infected RL(−/−) mice was rather manifested in earlier onset and increased severity of demyelination and axonal damage in brain stem and spinal cord without evidence for enhanced neuronal infection. RNase L specifically protected the brain stem from sustained infection and prevented spread of virus to microglia/macrophages located in spinal cord grey matter. The increased pathological changes in both brain stem and spinal cord thus correlated with the high mortality rates of RL 2/2 mice starting at day 9 p.i. The inability to directly link increased axonal damage with enhanced neuronal infection remains puzzling and supports dysregulation of oligodendrocyte function and/or neuroprotective functions of microglia as contributing factors to the severe pathological phenotype and clinical outcome. Numerous functions of RNase L, not directly associated with anti-viral activity, may contribute to the increased susceptibility of RL 2/2 mice to MHV-JHM infection.
cord-325736-gs9d8y55	2000	title: Persistence of Viruses in Upper Respiratory Tract of Children with Asthma Conclusions: The persistent presence of viruses in the upper respiratory tract of asthmatic children shows a possible connection between viral infections and asthma. 4 In this study nasopharyngeal swabs were taken from asthmatic children whose asthma was well controlled, and when they were at least 3 weeks free of any respiratory infections. The samples were examined for the presence of adenovirus DNA and rhinovirus and coronavirus RNA. Five microlitres of c-DNA product were subsequently amplified in 50µl PCR reaction mixture consisting of 10 reaction buffer, 25 mM MgCl 2 , 20 mM dNTP, 5 U/l of DNA Taq polymerase (all reagents provided by Perkin Elmer, New Jersey, USA) and 50 µM of each specific primer for rhinoviruses and coronaviruses (TIB MOLBIOL, Berlin, Germany) (Table I) . found that upper respiratory tract viruses were associated with over 80% of asthma exacerbations in children.
cord-325820-tnyzmrm8	2020	To discover new antiviral agents, we performed a compound screen in cell culture-based infection models and identified two carbocyclic adenosine analogues, 6′-β-fluoro-homoaristeromycin (FHA) and 6′-fluoro-homoneplanocin A (FHNA), that displayed potent activity against CHIKV and Semliki Forest virus (SFV) with 50% effective concentrations in the nanomolar range at nontoxic concentrations. Enzymatic assays with purified wild-type (wt) SFV nsP1 suggested that an oxidized (3′-keto) form, rather than FHNA itself, directly inhibited the MTase activity, while a mutant protein with the K231R and K299E substitutions was insensitive to the compound. Our combined data suggest that FHA and FHNA inhibit CHIKV and SFV replication by directly targeting the MTase activity of nsP1, rather than through an indirect effect on host SAH hydrolase. Since we also identified FHNA analogues that efficiently inhibit host SAH hydrolase in vitro without being active against CHIKV in cell-based assays (31), we reconsidered the possibility of a direct effect of the compound on nsP1 activity.
cord-325925-010xj69x	2019	Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture. Together, sequencing of dead or moribund aquaculture salmon and live-sampled wild salmon, in-situ hybridization, and epidemiological surveys revealed that previously unknown viruses, some of which are associated with disease, infect wild salmon from different populations. High-throughput RT-PCR screening of >6000 wild juvenile Chinook and sockeye salmon showed dissimilar geographical distributions of infected fish, reflecting differences in epidemiological patterns of transmission and infection dynamics for each of the viruses ( Figure 2) .
cord-325954-rhrkr97h	2020	Along with positive SARS-CoV-2 RNA in nasopharyngeal swabs, viral RNA was detectable at high concentration for >3 weeks in fecal samples from 12 mildly symptomatic and asymptomatic children with COVID-19 in Seoul, South Korea. For this study, we included all children <18 years of age who were confirmed to have COVID-19 by positive results for SARS-CoV-2 in combined nasopharyngeal and oropharyngeal swab specimens and who were hospitalized in Seoul Metropolitan Government-Seoul National University Boramae Medical Center during March 8-April 28, 2020. In comparison, the median initial fecal RNA load was 7.68 (range <4.10-10.27) log 10 copies/mL and Along with positive SARS-CoV-2 RNA in nasopharyngeal swabs, viral RNA was detectable at high concentration for >3 weeks in fecal samples from 12 mildly symptomatic and asymptomatic children with COVID-19 in Seoul, South Korea. In addition, the RNA load in feces remained steadily high, whereas that in nasopharyngeal swab specimens and saliva declined with time in both symptomatic and asymptomatic children.
cord-325958-1v1pg2z0	2020	In this study, a total of 38 conjunctival samples from 38 patients, including 12 healthy conjunctiva, 12 melanoma, 7 squamous cell carcinoma and 7 papilloma samples, were analyzed using high‐throughput RNA sequencing to assess mRNA expression of the SARS‐CoV‐2 receptor ACE2 and its cofactors including TMPRSS2, ANPEP, DPP4, and ENPEP. Since the outbreak, many studies described ACE2 expression across human tissues, including lung, stomach, ileum, colon, liver and kidney 8, 9 , supporting the clinical observation that SARS-CoV-2 can infect multiple organs. To obtain information on transcription of ACE2 and associated molecules required for cell entry by SARS-CoV-2, existing datasets of 38 conjunctival samples from 38 patients were included in this study. This study shows that ACE2, which is the main receptor for SARS-CoV-2 6 , is not significantly expressed in healthy and diseased human conjunctival samples.
cord-325966-0g7a9s5z	2020	Some candidate drugs targeting different levels and stages of human responses against COVID-19 such as cell membrane fusion, RNA-dependent RNA polymerase, viral protease inhibitor, interleukin 6 blocker, and convalescent plasma may improve the clinical outcomes of critical COVID-19 patients. However, these clinical, laboratory, and imaging findings are nonspecific and cannot differentiate COVID-19 from other viral respiratory infections; viral diagnostic methods specific for SARS-CoV-2 should be applied for disease confirmation. An open-label study published in 2004 suggested, by comparison with a control group that received only ribavirin, that the addition of lopinavir-ritonavir (400 mg and 100 mg, respectively) to ribavirin reduced the risk of adverse clinical outcomes (acute respiratory distress syndrome or death) and viral load among patients with SARS [29] . Some available candidate drugs targeting different levels of human responses to COVID-19, such as cell membrane fusion, RNA-dependent RNA polymerase, viral protease inhibitor, IL-6 blocker and convalescent plasma, may improve the clinical outcomes of critical COVID-19 patients.
cord-326017-qw4qynqv	2020	Considering this, we have summarized diverse research areas covering the current known biological properties of SARS-CoV-2, diagnostic tools for detection, therapeutic measurements for possible treatment, and prevention techniques to stop further spreading of this pandemic. Considering this, we have summarized diverse research areas covering the current known biological properties of SARS-CoV-2, diagnostic tools for detection, therapeutic measurements for possible treatment, and prevention techniques to stop further spreading of this pandemic. Overall, real-time RT-PCR based method enables developing a high-throughput testing for rapid, on-demand, low-cost, reliable, quantitative detection technique against COVID-19 in clinical settings [39] . Another newly developed method, SARS-CoV-2 DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR), was found to perform simultaneous reverse transcription and isothermal amplification by (i) RT-LAMP for RNA extracted (for nasopharyngeal or oropharyngeal swabs), (ii) Cas12 detection of predefined coronavirus sequences, and (iii) cleavage of a reporter molecule confirms, which detects the virus [56] .
cord-326217-ji0njeha	2018	We found that both inhibition of GSK3 function by synthetic inhibitors as well as silencing of GSK3β gene expression resulted in a decrease of HCV replication and infectious particle production, whereas silencing of the GSK3α isoform had no relevant effect on the HCV life cycle. To assess whether both GSK3 isoforms are required for HCV replication, we silenced GSK3α and / or GSK3β gene expression by transfecting siRNAs and quantified HCV RNA in Huh-7.5 cells harboring HCV subgenomic replicon (Con1) or infectious HCV (Jc1). Given the well-known dependency of HCV on miR-122 (Jopling et al., 2005 (Jopling et al., , 2008 Machlin et al., 2011) and the regulatory circuit between insulin-like growth factor 1 receptor, GSK3β, and miR-122 identified previously (Zeng et al., 2010) , we assessed the effect of GSK3 inhibition on miR-122 expression in Huh-7.5 cells harboring a subgenomic HCV replicon.
cord-326225-crtpzad7	2014	This procedure utilized primers composed of 20 bases of known sequence with 8 random bases at the 3′-end that also served as an identifying barcode that allowed the differentiation each viral library following pooling and sequencing. There is a wealth of information in these isolates, but up till now, it has been time consuming and expensive to sequence these viral genomes, often requiring sets of strain-specific primers for PCR amplification and sequencing. These primers were developed so that the 20 base known sequence was used for PCR amplification of the library as well as served as a barcode for identifying each viral library following pooling and sequencing. This virus, a BVDV 1b strain isolated from alpaca (GenBank accession JX297520.1; Table 2 , library 3, barcode 10), was assembled from Ion Torrent data and was found to have only 1 base difference from the sequence determined earlier (data not shown). One virus, library 1, barcode 9, had only 658 viral sequence reads but 94.4% of the genome was assembled.
cord-326257-rcv8sh22	2020	C->U transitions underpinned almost half of the amino acid differences between SARS-CoV-2 variants, and occurred preferentially in both 5''U/A and 3''U/A flanking sequence contexts comparable to favoured motifs of human APOBEC3 proteins. Importance The evidence that much of sequence change in SARS-CoV-2 and other coronaviruses may be driven by a host APOBEC-like editing process has profound implications for understanding their short and long term evolution. The possibility that the initial diversity within a viral population was largely host-induced would have major implications for 70 evolutionary reconstruction of SARS-CoV-2 variants in the current pandemic, as well as in our understanding both of host antiviral pathways against coronaviruses and the longer term shaping effects on their genome composition. To formally analyse 105 the excess of C->U transitions we calculated an index of asymmetry (frequency[C->U] / f[U->C]) x (fU/fC) and compared this with degrees of sequence divergence and dN/dS ratio in SARS-CoV-2 and other coronavirus datasets (Fig. 2B, 2C ).
cord-326719-p1ma4akz	2003	Coronaviruses have several advantages as vectors over other viral expression systems: (1) coronaviruses are single-stranded RNA viruses that replicate within the cytoplasm without a DNA intermediary, making integration of the virus genome into the host cell chromosome unlikely, (2) these viruses have the largest RNA virus genome and, in principle, have room for the insertion of large foreign genes, (3) a pleiotropic secretory immune response is best induced by the stimulation of gut-associated lymphoid tissues, (4) the tropism of coronaviruses may be modified by manipulation of the spike (S) protein allowing engineering of the tropism of the vector, (5) non-pathogenic coronavirus strains infecting most species of interest (human, porcine, bovine, canine, feline, and avian) are available to develop expression systems, and (6) infectious coronavirus cDNA clones are available to design expression systems.
cord-326911-va3x6au2	2020	We developed an open-source method called Magneticnanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Using 36 COVID-19 patient samples, 2 wastewater samples and 1 human pathogens control sample, we showed that MAVRICS rivals commercial kits in validated diagnostic tests of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. To date, molecular diagnosis of COVID-19 predominantly relies on detection of SARS-CoV-2 RNA using real-time reverse transcription polymerase chain reaction (rRT-PCR) assays, such as those approved by the US Centers for Disease Control and Prevention (US CDC) 1 . MAVRICS performed on par or better than commercial RNA extraction kits in rRT-PCR detection of SARS-CoV-2, influenza viruses and respiratory syncytial virus in various clinical and environmental samples. . https://doi.org/10.1101/2020.06.28.20141945 doi: medRxiv preprint Next, we aimed to develop an efficient SiMNP-based RNA extraction protocol using the contrived SARS-CoV-2 samples and US CDC 2019-nCoV_N1 and N3 rRT-PCR assays.
cord-327000-oyg3oyx1	2020	This review highlights the immune evasion mechanisms employed by PEDV, which provides insights for the better understanding of PEDV-host interactions and developing effective vaccines and antivirals against CoVs. Porcine epidemic diarrhea virus (PEDV) is the etiological agent of porcine epidemic diarrhea (PED) that causes an acute and highly contagious enteric disease of swine characterized by vomiting, diarrhea, dehydration, and anorexia in pigs of all ages, especially resulting in severe diarrhea and high mortality rate in piglets. Nsp3 is the largest nsp protein, containing two papain-like protease (PLP1 and PLP2) domains, of which PEDV PLP2 acts as a viral deubiquitinase (DUB), to negatively regulate type I IFN signaling [80] . The evasive strategies utilized by PEDV are classified into four major types: (1) inhibition of RLRs-mediated IFN production pathways, (2) inhibition of the activation of transcription factors responsible for IFN induction, (3) disruption of the signal cascades induced by IFN, and (4) hiding its viral RNA to avoid the exposure of viral RNA to immune sensors.
cord-327024-1k5jucae	2018	18 reported the detection of avian nephritis virus infection in Croatian goose flocks and provided evidence that this AstV was associated with stunting and prehatching mortality of goose embryos. To determine the potential genetic mutation(s) that might occur during the goose embryo passage, the initial virus genome was sequenced using the total RNA extracted from the clinical case tissue homogenate. When the samples were tested by RT-PCR for virus shedding evaluation, the AAstV specific RNA was sequentially detected from the cloacal swabs of infected goslings from 2 to 12 dpi (Fig. 6 ). To evaluate the potential adaptive mutation (s) of the virus that might occur during the process of goose embryo passage, we sequenced the complete genome of initial virus using the total RNA extracted from the clinical case tissue homogenate of kidney, spleen, and liver using the method described above.
cord-327259-7o7fs4yb	2020	We aim to evaluate pooling tests in experimental procedures, as well as perform in silico statistical modeling analysis validated with specimen samples obtained from a mass testing program of Industry Federation of the State of Rio de Janeiro (Brazil). This data was validated with the results obtained in our mass testing program: statistical modeling predicted a cost saving of 48.0%, which in practice, was 51.5%, already considering the expenditures with pool sampling that were analyzed individually. To assess the advantages of the pooling approach, we used previous qRT-PCR results obtained in the diagnostic analyses performed with industrial workers of Rio de Janeiro state as a base to calculate the prevalence rates (%) of positive cases and to build the statistical modeling methodology. Our study adopted the statistical modeling approach and validated the data with pooling biological samples for COVID-19 diagnostic, confirming that the pool size must be selected according to the prevalence rate of positive cases in the population (Figure 2) .
cord-327272-fspxett8	2020	The new human coronavirus named SARS-CoV-2 is a positive-sense RNA virus for which no specific drugs are currently available. A knowledge-based analysis strongly suggests a possible repositioning of the anti-HCV direct antiviral agent (DAA) Sofosbuvir as treatment for SARS-CoV-2. The only positive-sense RNA virus, for which a very effective drug targeting specifically the RdRp is available and approved world-wide for clinical use, is hepatitis C virus (HCV). All these sequence and structural modelling evidences strongly support the concept that the SARS-CoV-2 RdRp is much more similar to the one from HCV than the one from negative-sense Influenza and Ebola RNA viruses. Therefore, repositioning of Sofosbuvir (Sovaldi®; Epclusa® by Gilead), the inhibitor of the HCV NS5B RdRp protein, as antiviral in the treatment of the SARS-CoV-2 infection has an extremely high potentiality of success, as recently postulated by others [17] , and is suggested as a potential drug for the treatment of COVID-19 in the very recent EASL-ESCMID position paper [18] .
cord-327518-yilv9z2m	2011	In coronaviruses, proteolytic processing results in the production of 15 (in viruses belonging to the species Avian coronavirus) or 16 mature products, commonly referred to as non-structural proteins (nsp''s) and numbered according to their position -from N-to C-terminus -in the viral polyproteins ( Figure 1 ). Apart from their relatively close phylogenetic relationship, the only general characteristics that would set them apart from other coronaviruses are (i) a unique type of nsp1, distinct in size and sequence from betacoronavirus nsp1 and without apparent counterpart in the gammacoronaviruses, and (ii) the presence of a commonly-shared accessory gene (designated ORF3 in most alphacoronavirus species, ORF3b and 3c in TGEV and in FCoV/CCoV, respectively) for a dispensable multi-spanning alphacoronavirus membrane protein (αmp). The structural proteins are expressed from three sg mRNA species that are 3 co-terminal with the genome and believed to be produced via a process of discontinuous minus-strand RNA synthesis similar to that of coronaviruses ( Figure 12 ).
cord-327660-p1b07b4t	2018	The current RdRp tree topology combined with gene gain-loss reconstruction suggests the following evolutionary scenario for branch 1 ( Fig. 2A) : a levivirus-like ancestor that, like the extant members of the Leviviridae, possessed a capsid protein unrelated to SJR-CP (19, 52) gave rise to naked eukaryotic RNA replicons known as "mitoviruses" and "narnaviruses." These replicons consist of a single RdRp gene (Fig. 2B ) and replicate in mitochondria and in the cytosol of the host cells of fungal and invertebrate hosts, respectively (the latter hosts were identified in metaviromic holobiont analyses) (14, 53) . This genome architecture could hint at an ancestral flavivirus genome that was assembled from genes borrowed from preexisting viruses, one of which possessed a divergent "tombus-like virus" RdRp. Although the origins of branch 3 are murky, major trends in its subsequent evolution clearly included lineage-specific gene capture, starting with helicases and CapEs in the ancestors of the major lineages and followed by diverse genes in smaller groups (Fig. 4B) .
cord-327855-txryqil7	2003	Analysis of total cellular RNA from HM175/18f infected FrhK4 cells by denaturing agarose gel electrophoresis and Northern blot hybridization revealed extensive degradation of both the 28S and 18S ribosomal RNA (rRNA) molecules. In this study, we report that infection of FrhK4 cells with the HAV cp strain HM175/18f results in the degradation of ribosomal RNA (rRNA) and the reduction of several cellular mRNAs including β-actin and GAPDH. Degradation of rRNA is a feature of virus infection in interferon (IFN) treated cells and is believed to be due to the availability of double stranded RNA (dsRNA) during replication or transcription of the viral genome, resulting in the activation of the RNase L pathway [58, 59] . While the role of a viral protein in the activation of 2-5A/RNase L pathway in 18f or CBV1 infected FrhK4 cells cannot be ruled out, previous reports with EMC virus and the studies involving dsRNA clearly suggests a similar mechanism of rRNA degradation reported here.
cord-327997-noqbcxua	2020	We predict the SARS-CoV-2 RNA genome and sgRNAs to be enriched towards the host mitochondrial matrix and nucleolus, and that the 5'' and 3'' viral untranslated regions contain the strongest, most distinct localization signals. As previously discussed, since much of the APEX-seq mitochondrial data used to train RNA-GPS actually consists of nuclear-encoded transcripts likely picked up as the APEX-COX4 fusion protein is transported to the mitochondria, we hypothesize that our predicted mitochondrial residency is alluding to similarity in localization pathways, rather than localization destination. To further validate the robustness of these results, we also trained a different predictive algorithm (a recurrent neural network, see STAR Methods for additional details) on the APEX-seq data and performed a similar set of experiments, comparing SARS-CoV-2 dominant subcellular residency predictions to human and coronavirus baselines ( Figure S3A /B).
cord-328042-e1is656g	2020	The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Here, we show that the magnetic bead-based protocol yields RNA extracts comparable to the commercially available QIAcube viral RNA extraction kit, as determined by the commonly applied detection methods RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP) [16] .
cord-328085-7wp18qb6	2020	This indicates that these compounds have a good binding affinity with RdRp. The resulting top 10 ligand conformations were evaluated for its binding mode and molecular interaction with active site residues of RdRP (Table 1 ). The first compound, namely, Chlorohexidine with binding free energy -10.11 Kcal/mol efficiently accommodated in the active site of RdRp and do molecular mimicry of nucleotides in terms of substructure interactions with RdRp residues (Figure 2(A) ). The second lead molecule, Ergotamine bound conformation, showed slight variation in the side-chain orientation with respect to Naldemedine binding in the active site of Nsp15 (Figure 4) . Thus, based on stability and molecular interaction and binding mode, Alectinib acts as lead molecule to design novel RdRp inhibitor to block the viral replication. The differences in NSP15 residues interaction with Naldemedine in simulated structure and predicted docked pose is due to flipped orientation of Naldemedine (Supporting Information Fig. S4B and Figure 4(B) ).
cord-328252-dk54w8z9	2019	Whether PA-X also degrades viral dsRNA species to prevent recognition by cytosolic RNA sensors is not entirely clear, but mutant viruses in which this PA-X protein was expressed in significantly lower amounts elicited higher levels of innate immune response; for example, IFN-beta production was much higher in these infections [71] . This indeed suggests that PA-X, besides having a role in the degradation of cellular mRNAs, may also degrade viral RNA to prevent recognition by innate immune sensors and activation of innate immune responses, similar to what was shown for the CoVs. To my knowledge, an endoribonuclease has not been identified in the RSV genome, so this virus may use alternative innate immune evasion strategies, as discussed elsewhere in this review. [91] suggested that RSV specifically targets mRNA encoding surfactant protein A, an innate immune factor with an important role in the epithelial tissue of the lung, which directly binds to virus particles to cause their destruction by host defense mechanisms.
cord-328259-3g4klpyg	2020	Despite the overrepresentation of dsRNA viruses, our results show that Santiago''s sewage RNA virosphere was composed mostly of unknown sequences (88%), while known viral sequences were dominated by viruses that infect bacteria (60%), invertebrates (37%) and humans (2.4%). Viral sequences identified as Partitiviridae-like viruses included in the "unclassified RNA viruses ShiM-2016" category in the NCBI taxonomy (~25% abundance; Figure 2B ) and Totiviriade family were also highly abundant in treated and untreated sewage samples from the EU [5, 7] . Therefore, the abundance of these viruses in the Trebal metagenome can expand the known sequence space associated with this family (only 10 genomes are currently available in the NCBI database) and contribute to a better understanding of the bacteriophage biology related to RNA genomes. Taken together, our results show that metagenomic surveys of RNA viruses in sewage samples and the use of HMMs could uncover extraordinary viral diversity through the detection of remote homologs in these human-impacted environments.
cord-328300-zehltghv	2014	CoVs encode the nucleocapsid (N) protein, a major structural protein that plays multiple roles in the virus replication cycle and forms a ribonucleoprotein complex with the viral RNA through the N protein''s N-terminal domain (N-NTD). We report the crystal structures of HCoV-OC43 N-NTD complexed with ribonucleoside 5′-monophosphates as a model for understanding the molecular interactions that govern CoV N-NTD binding to RNA. To begin to elucidate how RNA and the N protein interact, we determined the crystal structure of HCoV-OC43 N-NTD complexed with AMP. These amino acids are sequentially and structurally conserved in other HCoV N proteins ( Figure S2 , Supporting Information); therefore, they are likely essential for RNA recognition and interaction in all coronavirus N proteins. Previous studies indicated that the positively charged amino acid, Arg 106, located at the cleft in the HCoV-OC43 N-NTD structure, is conserved in all CoV N proteins and interacts nonspecifically with the RNA phosphate backbone.
cord-328460-thx9zh11	2012	Miniaturized isothermal amplification systems will be then illustrated including nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), multiple displacement amplification (MDA) and recombinase polymerase amplification (RPA). In addition, the use of droplet-based microfluidic systems for PCR enables the amplification and the analysis of individual target sequences to be performed in a significantly lower time as a consequence of a higher thermal cycling speed. These include nucleic acid sequence-based amplification (NASBA), loop-mediated amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA) [7] . Unlike other electrochemical techniques [56], based on the immobilization of an oligonucleotide probe onto an electrode, hybridization of a complementary target sequence, and transduction of the hybridization event [57], the microfluidic detection of LAMP amplicons [55] does not require probe immobilization and bacteria detection can be accomplished in a single chamber without DNA extraction and purification steps, since the used isothermal temperature (66 °C) provides enough thermal shock to lyse the E.
cord-328471-oz99upzz	2020	In this global health emergency, drug repurposing (or repositioning) is one of the fast track option that involves screening of existing FDA approved drugs for the identification of potential molecules that can disrupt the function of key proteins of the SARS-CoV-2 and can be used for treatment against COVID-19. Whereas, Demoxytocin showed ten H-bonds with both active site Asp760 and Asp761 and other key residues e.g. Trp617, Tyr619, Lys621, Ser682, Glu811, Lys621, Tyr619, Trp617, Ser682 and Glu811 with dock score -9.68kcal/mol and ligand efficiency of -0.142 (Supplementary Figure S3) . Colistin (polymyxin E, polypeptide antibiotics) showed most of the H-bonding with Lys551, Trp617, Tyr619, Asp618, Ser682, Asp684, Asn691, and both catalytic residues i.e. Asp760, Asp761, with a docking score of -9.24kcal/mol and ligand efficiency of -0.113 (Supplementary Figure S5) . Examorelin, Lypressin, Ornipressin, and Colistin are also common drugs in both form of RdRp. Only one H-bond with His810 and other non-covalent interactions were observed for Examorelin showed a docking score of -12.139 kcal/mol and ligand efficiency of -0.187.
cord-328633-c31xsyeo	2012	Most RT-PCR protocols rely on two DNA polymerase (Pol) enzymes; a retroviral reverse transcriptase (RT) to copy RNA into cDNA and a thermostable DNA Pol to amplify the target sequence. Despite their wide use and general reliability, existing twoenzyme RT-PCR systems have several documented performance problems attributed to deficiencies inherent in retroviral RTs: 1) poor reagent stability, 2) low fidelity, 3) frequent rearrangements during cDNA synthesis, 4) secondary enzymatic activities (i.e. RNase H and strand switching), 5) bias for specific primers and templates, and 6) inhibition of PCR Pol enzymes [3, 4, 5, 6, 7] . We describe the discovery and biochemical attributes of one of these, 3173 Pol, its inherent RT activity and its incorporation into a single-enzyme PyroScriptH 2X RT-PCR Master Mix. The sensitivity, specificity and overall performance of this mix were compared to available one-and two-enzyme systems using a control MS2 RNA bacteriophage template, the clinically-relevant influenza A RNA and commonly used reference mRNA transcripts.
cord-328659-miujzgtd	2020	title: Mutation landscape of SARS-CoV-2 reveals five mutually exclusive clusters of leading and trailing single nucleotide substitutions Furthermore, clustering analysis revealed unique geographical distribution of SARS-CoV-2 variants defined by their mutation profile. The rapid global spread of SARS-CoV-2 in a short period of time and the availability of a large number of fully sequenced genomes provide us with a unique opportunity of understanding the short-term temporal evolution of this virus in humans in a near real-time scale. By this approach we propose the classification of the SARS-CoV-2 virus genomes into 5 mutually exclusive lineages with unique set of co-occurring mutations and geographic distribution. Our analysis revealed a total of 40 nucleotide substitutions which occurred at > 1% in the SARS-CoV-2 genomes (Table 1 and Figure 1A ). We consider a specific mutation or a set of cooccurring mutations as "lineage-defining" for SARS-CoV-2, only when they are present in at least 2% (n=30) of the sequences analysed.
cord-328686-5ik5em5a	2020	In the present study, SARS-CoV-2 RNA was concentrated from wastewater, sludge, surface water, ground water, and soil samples of municipal and hospital wastewater systems and related environment in Wuhan during the COVID-19 middle and low risk periods, and the viral RNA copies quantified using RT-qPCR. From the findings of this study, during the middle risk period, one influent sample and three secondary treatment effluents collected from Waste Water Treatment Plant 2 (WWTP2), as well as two influent samples from wastewater system of Hospital 2 were SARS-CoV-2 RNA positive. . https://doi.org/10.1101/2020.08.19.20172924 doi: medRxiv preprint From the findings of this study, during the middle risk period, positive samples were detected both in 83 municipal and hospital wastewater systems. . https://doi.org/10.1101/2020.08.19.20172924 doi: medRxiv preprint Although SARS-CoV-2 RNA surveillance in wastewaters is a useful WBE drive, the public health risk associated 109 with water cycle is unclear since viral particles infectivity in sewage and faeces is yet to be determined in 110 addition to its probable fecal-oral transmission.
cord-328737-6mcefqn5	2020	We here describe for the first time a novel nucleic acid amplification system based on nano-gap active resonators that can be used for in various molecular diagnostic applications, which requires a label-free and real-time detection with rapidity and high sensitivity. First, SRSN active disk resonators were used as photoluminescence sensors for the PL peaks J o u r n a l P r e -p r o o f generated by the direct amplification of nucleic acids on the sensor surface in a label-free and real-time manner. These complexes bind to the target nucleic acids and enable the strand exchange which will begin the amplification process both on the surface of disk resonator (solid) and solution simultaneously ( Fig. 1-rectangle) , with the temperature maintained at isothermal conditions (38°C or 43°C for DNA or RNA, respectively). This photoluminescence sensor enables label-free and real-time amplification and direct detection of either bacterial DNA or viral RNA on the resonator surface.
cord-328768-2qk884x2	2020	Here, we aimed to compare the sensitivity and sample and reagent contamination of three extraction methods used for viral mNGS: two automated platforms (eMAG; MagNA Pure 24, MP24) and the manual QIAamp Viral RNA Mini Kit (QIAamp). The development of metagenomics next-generation sequencing (mNGS) has enabled the exploration of whole viral nucleic acids within a clinical sample (human virome) in order to detect pathogens not targeted by conventional PCR and to identify emerging viruses [1] [2] [3] [4] [5] [6] [7] [8] . The aim of this study was to compare two automated extraction platforms commonly used in diagnostic laboratories, the eMAG (bioMérieux, Marcy-l Étoile, France) and the MagNA Pure 24 (MP24) (Roche, Basel, Switzerland), and one manual QIAamp Viral RNA Mini Kit extraction (Qiagen, Hilden, Germany), which is among one of the most popular methods used in research laboratories.
cord-328947-3l9ydspz	2020	CHIKV is no exception, the type-I interferon (IFN) response has been demonstrated to be key in controlling CHIKV infection [21] [22] [23] [24] [25] [26] and primary sensing of the virus is via the pattern recognition receptor (PRR) retinoic acid-inducible gene I (RIG-I)-mitochondrial antiviral signaling protein (MAVS) axis [24, 26] . The lack of cGAS degradation seen in the exogenous expression system when compared to the infected cells shows that although there is a clear interaction of cGAS with nsP4, the degradation of cGAS observed is not mediated by the non-structural proteins of CHIKV, but must require other viral or host factors. Chikungunya virus inhibits DNA sensing by degrading cGAS both mock and infected cells by 4 hpi suggesting that the stress produced during the process of infection (mock or CHIKV) in HFF-1s stimulates increased translation at early timepoints ( Fig 4D, lanes 2 and 5) .
cord-328960-46zui1sl	2020	Particle classification yielded a 3D reconstruction at a nominal resolution of 2.9 Å and led to a refined structure of the RdRp-RNA complex (Extended Data Figures 1 and 2) . The structure resembles that of the free enzyme 16 , but also reveals large additional protein regions in nsp8 that became ordered upon RNA binding and interact with RNA far outside the core enzyme (Extended Data Figure 3a ). The supernatant containing nsp12 was filtered using a 5-μm syringe filter, followed by filtration with a 0.8-µm syringe filter (Millipore) and applied onto a HisTrap HP 5 mL (GE Healthcare), preequilibrated in lysis buffer (300 mM NaCl, 50 mM Na-HEPES pH 7.4, 10 % (v/v) glycerol, 30 mM imidazole pH 8.0, 3 mM MgCl2, 5 mM β-mercaptoethanol, 0.284 µg ml-1 leupeptin, 1.37 µg ml-1 pepstatin, 0.17 mg ml-1 PMSF, and 0.33 mg ml-1 benzamidine).
cord-329041-coryaz2s	2019	These data further extend the known breadth and antiviral activity of RDV to include both contemporary human and highly divergent zoonotic CoV and potentially enhance our ability to fight future emerging CoV. We previously reported the antiviral activity of RDV against a genetically diverse panel of human endemic, emerging and zoonotic CoV including HCoV-NL63 (alpha 1b), mouse hepatitis virus (MHV, beta 2a), SARS-CoV and related Bat CoVs WIV1 and SHC014 (beta 2b), as well as MERS-CoV and related Bat CoV HKU5 (beta 2c) (Agostini et al., 2018; Sheahan et al., 2017) . Inhibition of viral protease has also been evaluated with lopinavir, a protease inhibitor designed for human immunodeficiency virus, which like chloroquine exerts a moderate antiviral effect on CoV replication (EC 50 values: MERS-CoV 8 μM, SARS-CoV 17.1 μM, HCoV-229E 6.6 μM) (de Wilde et al., 2014) .
cord-329102-2y49kcwu	2020	We evaluated the robustness of our in-cell data derived genome-wide model by varying two critical RNA folding parameters used by RNAstructure: 1) the maximum allowed distance for base pairing and 2) the threshold for DMS signal normalization. Previous studies that computationally predicted genome-wide SARS-Cov-2 RNA structures used 1) RNAz, a thermodynamic-based model that additionally takes sequence alignment and considers base pairing conservation (Gruber et al., 2010; Rangan, Zheludev and Das, 2020) , and 2) Contrafold, which predicts RNA secondary structures without physics-based models and instead uses learned parameters based on known structures (Do, Woods and Batzoglou, 2006) . Interestingly, in silico predictions of the RNA structure of the SARS-CoV-2 genome using RNAz (Rangan, Zheludev and Das, 2020) and ScanFold (Andrews et al., 2020) do not find the 3-stem pseudoknot but instead support our in-cell model of Alternative Stem 1.
cord-329107-43e2lkht	2020	Nonsense-mediated messenger RNA (mRNA) decay (NMD) is a surveillance pathway used by cells to control the quality mRNAs and to fine-tune transcript abundance. Disturbance of this control mechanism caused by genetic mutations or dys-regulation of the NMD pathway can lead to pathologies, including neurological disorders, immune diseases and cancers. Nonsense-mediated mRNA decay (NMD) is a critical cellular surveillance mechanism that recognizes and eliminates aberrant RNAs containing premature termination codons (PTC) or abnormally long 3 untranslated regions (UTRs). Nonsense-mediated mRNA decay is a critical RNA quality control and plays a vital role in the recognition of PTCs in transcripts, as well as in the regular homeostasis of the transcriptome. Understanding the mechanism of nonsense-mediated mRNA decay might lead to new ways to use surveillance in cancer therapy or other PTC-associated genetic diseases. Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon Hypoxic Inhibition of Nonsense-Mediated RNA Decay Regulates Gene Expression and the Integrated Stress Response
cord-329311-p68kr4ga	2020	title: SARS-CoV-2 RNA in plasma is associated with ICU admission and mortality in patients hospitalized with COVID-19 Routine biochemistry was taken at admission and study-specific samples of EDTA plasma and serum were taken at three time points; baseline (enrollment), day 3 (1 day) and day 9 ( 2 days) in patients who were still hospitalized (details in Supplementary Figure 1) . SARS-CoV-2 RNAemia was detected in at least one sample in 58/123 (47%) patients, and in a significantly higher proportion of patients who were admitted to the ICU or died (80% vs. RNAemia was significantly more frequent at all time points in patients who reached the primary endpoint, whereas RNA loads were significantly higher at baseline and day 3 ( Table 1 , Supplementary Figure 2A ). In this prospective study of patients hospitalized with COVID-19 we detected SARS-CoV-2 RNAemia in 47% of included patients, and a significantly higher frequency of RNAemia and higher RNA loads in and similarly found that RNAemia was associated with ICU admission and hospital mortality [3] .
cord-329361-0mpbau1b	2012	Inside eukaryotic cells, small RNA duplexes, called small interfering RNAs (siRNAs), activate a conserved RNA interference (RNAi) pathway which leads to specific degradation of complementary target mRNAs through base-pairing recognition. The practical use of RNAi therapy for HIV infection will depend on overcoming several challenges, including the ability to establish long-term expression of siRNA without off-target effects and the capacity to counteract mutant escape viruses. [4] Hence, in plants and Drosophila, when a cell they have sequence specificity for silencing mRNAs, these small is infected by a virus, an RNAi response is triggered by the foreign RNAs potentially represent a future class of antiviral drugs. [42] silencing of viral RNA and transient suppression of HIV replicaobserved that an siRNA targeted to nef rapidly elicited the emertion over a period of 3 to 4 days in single round infection of gence of siRNA-resistant viruses with point mutations in the nef cultured cells have been achieved.
cord-329366-xuszdrsa	2020	In this study, we show that CoV EndoU activity limits the abundance and length of the polyuridine (polyU) extension on 5′-polyU-containing, negative-sense (PUN) RNAs for both the beta-CoV mouse hepatitis virus strain A59 (MHV-A59) and the alpha-CoV PEDV. Overall, we propose a mechanism for EndoU, which is to cleave polyU sequences from PUN RNAs, thus limiting the formation of a PAMP and impeding the ability of MDA5 to activate the innate immune response to infection. EndoU Activity Limits Abundance and Length of PUN RNAs. Previous studies showed that the 5′ end of the CoV negative-sense RNA contains polyU extensions (35) , and that EndoU cleaves at uridine residues (22, 25, (27) (28) (29) (30) . We found that the products generated from positive-sense RNA were similar between wild-type and EndoUmut viruses, consistent with our previous results indicating that the polyA tail is not cleaved by EndoU activity (Fig. 5C ).
cord-329429-ur8g68vp	2018	Within the gamma-CoVs the main representative is avian coronavirus, a taxonomic name which includes the highly contagious infectious bronchitis viruses (IBVs) in chickens and similar viruses infecting other domestic birds such as turkeys, guinea fowls, or quails. The methods adopted in monitoring studies of CoVs in different avian species are mainly based on detection of conservative regions within the viral replicase, nucleocapsid genes, and 3''UTR or 5''UTR. The purpose of this review is to summarise recent discoveries in the areas of epidemiology and diagnosis of CoVs in avian species and to understand the role of wild birds in the virus distribution. This taxonomic name includes IBV which causes a highly contagious disease of chickens, and genetically similar viruses isolated from other domestic galliformes: turkey coronavirus (TCoV), responsible for turkey enteritis, and the more recently detected guinea fowl coronavirus (GfCoV), the aetiological factor of fulminating disease in this species (2, 6, 27) .
cord-329493-ueqlhgn0	2003	A new infectious disease, known as severe acute respiratory syndrome (SARS), appeared in the Guangdong province of southern China in 2002. When Thiel and colleagues 20 isolated one genomic and eight subgenomic RNAs from the FRA strain and sequenced their 5′ ends, they identified a conserved sequence (5′ACGAAC3′) that was located in coronaviruses: S, spike protein; E, envelope protein; M, membrane glycoprotein; and N, nucleocapsid protein. Alternatively, these antigens could be delivered by DNA immunization by Figure 6 | The S1 domain of SARS-CoV spike is structurally related to group 2 coronaviruses. Schematic representation of cysteine positions in the S1 domains of group 1, 2 and 3 coronaviruses, compared with the SARS-CoV spike protein. The complete genome sequence of a SARS-CoV isolate (FRA) and experimental data on its key RNA elements and protein functions are described. Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection
cord-329494-cdn52epy	2009	The efficacy of this Z2-siRNA against JUNV was also demonstrated in virus-infected cells, by testing infectious virus plaque formation (92.8% JUNV yield reduction), viral RNA level or antigen expression, as well as in cells transfected with Z-specific reporter plasmids (91% reduction in expression of Z-EGFP fusion protein). (E) Expression of viral antigens in Vero cells transfected with Z2-siRNA or X-siRNA and infected with JUNV was detected by immunofluorescence assay using a rabbit anti-JUNV polyclonal serum. The efficacy of this agent against JUNV was also demonstrated in virus-infected cells by testing infectious virus plaque formation, viral RNA or antigen expression, as well as in cells transfected with Z-specific reporter plasmids. The present study represents the first report of virus inhibition mediated by RNA interference for a New World arenavirus, a group in the family including four agents of severe viral hemorrhagic fevers in South America (JUNV, Machupo, Sabiá and Guanarito viruses).
cord-329504-91te3nu8	2020	A general indication of how well the atomic model fits the measurement data can be obtained by comparing the deposited R-factors to results from PDB-REDO (10) (including Whatcheck (11)) to determine the overall density fit as well as many other diagnostics. Our remodelled structure is offering a valuable structural basis for future studies, such as in-silico docking and drug design targeting at SARS-CoV-2 RdRp (34), as well as for computational modelling or simulations to investigate the molecular mechanism of viral replication (31, 35, 36) . This has included a number of posts on our homepage aimed at non-scientists and live streaming the reprocessing of data on Twitch, as well as the design, production, and public release of an accurate 3D printed model of SARS-CoV-2 based on deposited structures for use as a prop for outreach activities.
cord-329527-0rlotyz3	2018	In addition to this, a fatal case attributed to the H1N1 pandemic infection was reported, and the clinical finding showed that the cause of death was an intracerebral thrombosis and hemorrhage with presence of the virus in the brain, but not in lungs or CSF (Simon et al., 2013 ; Figure 2) . In another approximation to understand the etiologic agent causing myelopathy post-influenza-like syndrome, CSF obtained from a patient with this disease was inoculated in several cell lines, previously reported to be permissive for the growth FIGURE 2 | Influenza virus (IV) spreads from the lungs to the CNS through the vagus nerve promoting an inflammatory state. As described so far, CoVs are respiratory viruses that exhibit neurotropic capacities that not only allows them to achieve latency and avoid the immune response of the host, but also have neurological implications that can complicate the disease associated to its infection. Although there are extensive case reports that indicate neurological manifestations associated to hMPV-infection in humans, further studies are required in mice models to characterize this disease.
cord-329618-kywhulpc	2016	To elucidate the effect of type I IFN on the Jak/stat pathway in salmonid alphavirus subtype 3 (SAV3) infected macrophage/dendritic like TO-cells derived from Atlantic salmon (Salmo salar L) headkidney leukocytes, we used a differential transcriptome analysis by RNA-seq and the Kyoto encyclopedia of genes and genomes (KEGGs) pathway analysis to generate a repertoire of de novo assembled genes from type I IFN treated and non-treated TO-cells infected with SAV3. CONCLUSION: Data presented in this study shows that SAV3 infection downregulates several genes of the Jak/stat pathway, which could be an immune evasion strategy, used to block the transcription of antiviral genes that would interfere with SAV3 replication in TO-cells. Hence, in this study we used the KEGG pathway analysis software which is a third generation PT based software to identify networks of genes that form the Jak/stat signaling pathway from a de novo assembled transcriptome with the aim to better understand the modulation of genes expressed by TO cells infected with SAV3.
cord-329687-vhi4tbnc	2020	title: A comparative evaluation of dye-based and probe-based RT-qPCR assay for the screening of SARS-CoV-2 using individual and pooled-sample testing. However, the most efficient system for massive surveillance, the pre-RNA extraction pooling approach, was obtained with the probe-based assay in test up to 10 samples adding 13.5 uL of RNA template. In this study, we compared a dye-based and probe-based real-time RT-qPCR assay for the economic and rapid detection and quantification of SARS-CoV-2 in human samples by individual and pooling testing. Our results showed that using the dye-based assay describe here, the SARS-CoV-2 can be detected up to 50 viral copies (a dilution of 10 copies/µL) of the RNA template, showing a similar analytical sensitivity obtained with the probe-based standard technique ( Table 2) showed no significant differences in CT values between pooled and individual samples, suggesting that sensitivity was not affected by pooling specimens, regardless of the viral load (Wacharapluesadee et al, 2020).
cord-329707-89zyu8bl	2006	We constructed recombinant adenoviral vectors that can express shRNAs, which inhibited the expression of SARS-CoV genes effectively in mammalian cells. METHODS: In this study, we designed several plasmids that express small hairpin RNA molecules (shRNA) specifically targeting to the genes encoding for the SARS-CoV nucleocapsid (N) protein and envelope (E) protein, respectively. The effects of adenovirus-delivered small hairpin RNA on SARS-CoV gene expression were determined by RT-PCR, Western blot, and luciferase activity assays. RESULTS: The levels of viral mRNAs and viral proteins of the targets were significantly decreased or completely inhibited in cell lines after being infected with the recombinant adenoviruses that expressed specific shRNA molecules. CONCLUSIONS: Since many cell types can be efficiently infected by adenovirus, recombinant adenoviruses could serve as an alternative powerful tool for shRNA delivery and for gene suppression, especially when the targeted cells are resistant to transfection by DNA or RNA.
cord-329710-vqorb6j7	2020	We highlight past and recent findings in essential coronavirus proteins, including RNA polymerase machinery, proteases, and fusion proteins, that offer opportunities for the design of novel inhibitors of SARS-CoV-2 infection. Many recent scientific reviews and essays have outlined vaccine efforts, as well as viral and host targets that are the focus of current campaigns aimed at redirecting clinically used compounds for COVID-19. The FDA-approved COVID-19 drug, remdesivir, is a nucleotide analog originally developed to treat Ebola infections (caused by another single-stranded RNA virus) and recently shown to inhibit the SARS-CoV-2 RdRP. HIV protease inhibitors lopinavir and ritonavir, included in the SOLIDARITY trial despite mixed reviews in the clinic, have been predicted to bind SARS-CoV-1 and CoV-2 3CL pro (96% sequence identity) based on computational studies. Using a recently solved crystal structure of the HR1 and HR2 domains of the SARS-CoV-2 S protein, lipidated peptide fusion inhibitors have been designed that inhibit pseudovirus infection of cells with IC 50 values in the single-digit nanomolar range.
cord-329794-msxrdhb3	2004	Here, we provide evidences that RNAi targeting at coronavirus RNA-dependent RNA polymerase (RDRP) using short hairpin RNA (shRNA) expression plasmids can specifically inhibit expression of extraneous coronavirus RDRP in 293 and HeLa cells. Here, we provide the evidence that RNAi targeting at coronavirus RDRP using short hairpin RNA (shRNA) expression plasmids can specifically inhibit expression of extraneous coronavirus RDRP in 293 and HeLa cells. Design of specific shRNA expression plasmids targeting at coronavirus RDRP Coronavirus isolated from SARS patients has a large genomic RNA (approximately 30 kb). After transfecton, about 40-90% of RDRP gene expression was inhibited, depending on the sequence of the shRNA inserts, based on RT-PCR analysis in both HeLa and 293 cells transfected with RDRP (data not shown). shRNA reduced the expression of SARS RDRP mRNA in 293 and HeLa cells As shown in Fig. 1A , based on the RT-PCR analysis, the expression of extraneous RDRP gene was observed peaking at 48 h after the transfection.
cord-329866-io9fvy58	2019	With the aim to contribute to fill this diagnostic gap, a total of 61 effusions from cats with suspected effusive FIP were collected intra-vitam for detection of feline coronavirus (FCoV) antibodies and RNA by means of indirect immunofluorescence (IIF) assay and real-time RT-PCR (qRT-PCR), respectively. Fifty-one (48 ascitic and 3 pleuric fluids) of the 61 tested samples had FCoV antibody (Table 2 and Fig. 1 ), although only 37 positive effusions contained antibody levels ≥ 1:1600, which are considered highly suggestive of FIP diagnosis (Hartmann et al., 2003) . A recent paper (Meli et al., 2013) has investigated the agreement between FCoV antibody titres and RNA detection in the effusions of 13 cats with confirmed FIP, showing a correlation between high amounts of virus and lower signals in IIF assay, likely due to the fact that antibodies bound to viral antigens of the effusions are not able to bind to the antigens of the FCoV-infected cells used in serological tests.
cord-330045-4gj9d181	2020	We recruited hospitalized patients with COVID-19 from 2 designated provincial emergency hospitals for e merging infectious diseases in Guangdong, China, and tested specimens by real-time reverse transcription PCR (rRT-PCR) to estimate the duration of the detection of SARS-CoV-2 RNA in various body fluids, using an accelerated failure time (AFT)-based modeling study. We used parametric Weibull regression models (AFT) to estimate the time until the loss of SARS-CoV-2 RNA detection in each body fluid and reported findings in medians and 95th percentiles using R software version 3.6.1 with flexsurv, survival, and survminer packages (9) . We used Weibull models to estimate the median and the 95th percentile for the time until the loss of SARS-CoV-2 RNA detection in swab, sputum, and fecal samples (Table; Figures 1, 2) . In this study, we estimated the time for COVID-19 case-patients to clear SARS-CoV-2 RNA in the acute phase of infection through an AFT-based modeling study.
cord-330131-yfhrmbvx	2020	In this article, we show, in the specific case of SARS-CoV-2, that the role of cytosine-based metabolites used as cell growth coordinators is central to understanding both innate antiviral immunity and the evolution of the virus. Here we (i) highlight the deviation of SARS-CoV-2 RNA chemical composition compared with that of its human host; (ii) formulate a hypothesis grounded on the canonical organization of cytosine metabolism as a way to coordinate non-homothetic growth of cells-i.e., the simultaneous growth of the cytoplasm (three dimensions), the membrane (two dimensions) and the genome (one dimension)-, and point out the emergence of the endogenous antinucleotide viperin as a cognate adaptive antiviral metabolite and (iii) predict evolutionary trends of CoV-2 for maximizing compositional fitness-which seem to show up in ongoing mutation survey of radiative evolution.
cord-330200-l6bnxi40	2020	title: Long period dynamics of viral load and antibodies for SARS-CoV-2 infection: an observational cohort study ABSTRACT OBJECTIVE To investigate the dynamics of viral RNA, IgM, and IgG and their relationships in patients with SARS-CoV-2 pneumonia over an 8-week period. Only two articles report dynamics of SARS-CoV-2 viral RNA and antibodies with serial samples, but the observation periods are within 30 days. In this study, we investigated the profiles of viral RNA, IgM, and IgG in a group of patients with confirmed SARS-CoV-2 pneumonia over an 8-week period after symptom onset. Demographic data, symptom onset time, clinical features, radiological findings, routine laboratory results, and the results of SARS-CoV-2 viral RNA in throat swabs, sputum, and stool samples were recorded during hospitalization and follow-up. We investigated the serial viral load and dynamics of antibodies from patients infected with SARS-CoV-2 over an eight-week period following the onset of symptoms.
cord-330213-reb9vo7x	2020	From sequencing three isolates, we derive a robust identification of SARS-CoV-2 modification sites within a physiologically relevant host cell type. A comparison of our data with the DRS data from a previous SARS-CoV-2 isolate, both raised in monkey renal cells, reveals consistent RNA modifications across the viral genome. The long RNA sequencing reads generated for this study cover the entire SARS-CoV-2 genomic RNA as well as the different ORFs (Fig 1b,c, Fig. S1b ). Two sets of Galaxy workflows based on Tombo (16) and Nanocompore (17) tools were designed to compute the modification scores from the DRS data (Table S3) . Figure 5 : Direct RNA sequencing raw electrical signals of downsampled reads obtained from unmodified RNA (IVT, black), from samples generated for this study and from isolate from a published korean data set (Fr1-3 and Kr, red).
cord-330800-s91zfzfi	2020	e nucleic acid amplification tests are very popular in the diagnosis of viral infections caused by several viruses, including hepatitis C virus (HCV), human immunodeficiency virus (HIV), dengue virus, Epstein-Barr virus (EBV), influenza viruses, Zika virus (ZIKV), Ebola virus, and coronavirus [26] [27] [28] [29] [30] [31] [32] . Owing to high sensitivity and specificity, short turnaround time for results, and ease of performance [33, 61] , most laboratories across the globe employ real-time PCR for the detection and quantification of medical DNA and RNA viruses in clinical specimens. For example, Co-Diagnostics (Salt Lake City, USA) has developed real-time RT-PCR kit (Logix Smart COVID-19 test) for qualitative detection of nucleic acid from the SARS-CoV-2 in lower respiratory samples (e.g., bronchoalveolar lavage, sputum, and tracheal aspirate) and upper respiratory specimens (e.g., oropharyngeal swabs, nasal swabs, and nasopharyngeal swabs). LAMP is another isothermal nucleic acid amplification method that is extensively utilized for sensitive, specific, rapid, and cost-effective detection of both DNA and RNA viruses in human specimens.
cord-330847-a84pcc9z	1992	Northern and Southern hybridization with oligonucleotide probes specific for either α or γ leader sequences indicated that CV17 γ cDNA clones are representative of native CV17 γ RNAs. Furthermore, bioassays indicated that in vitro transcripts derived from these γ cDNA clones were infectious when coinoculated with in vitro transcripts of full-length α and β cDNA clones. A "bandaid" cloning strategy, which exploits the conservation of the 3'' terminal sequence, as well as the unique nature of the 5'' ends of the BSMV RNAs, has been used previously to obtain full-length cDNA clones from which infectious in vitro transcripts can be produced (3, 7). Analysis of the resulting cDNA clones suggested that CV17 RNA y is a naturally occurring chimeric recombinant, composed of a 70nucleotide (nt) a-specific leader sequence preceding a r-specific coding region. 3. ldentrfrcatron of barley stripe mosaic virus strarn CV17 LY and Type strain y-specific leader sequences In 15 putative CV17 y cDNA clones and native RNAs of strains CV17, CV42, and ND1 8.
cord-330954-ft14aa2n	2020	The discharged patients must meet the following criteria: (1) the body temperature returns to normal for more than 3 days; (2) respiratory symptoms improve significantly; (3) pulmonary imaging shows that the acute exudative lesions were significantly absorbed and improved; (4) the SARS-CoV-2 ribonucleic acid (RNA) detection results of two consecutive respiratory specimens are negative (sampling interval should be at least 24 h). We recorded the clinical manifestations, RNA detection results of oropharyngeal swab specimens, SARS-CoV-2 IgM−IgG antibodies detection results (data were collected on the 28th day after discharge), chest computed tomography (CT) images and medication of these patients. A case report of four mild-to-moderate COVID-19 patients (all were medical staff) showed that all convalescent patients without clinical symptoms presented RP findings of RNA detection in throat swab specimens at 5−13 days after discharge [14] . Another study found that among 62 COVID-19 convalescent medical staff, two patients without clinical symptoms showed RP findings of RNA detection in throat swab specimens at 5−6 days after discharge [15] .
cord-331066-ediowz4s	2020	Due to transitional symmetry of a helix, weakly specific cooperative interaction between ssRNA and nucleocapsid proteins leads to the natural selection of specific quasi-periodic assembly/packaging signals in the related genomic sequence. Therefore, the putative weakly specific assembly/packaging signals in the genomic RNA of coronaviruses should be coordinated with the parameters of the helical nucleocapsid (such as the helix pitch, inner and outer diameters) which are established by cryoelectron microscopy (cryo-EM) and other structural methods. In this article, we provide methods for the detection and comparative analysis of assembly/packaging signals in the genomic RNA of the coronaviruses SARS-CoV and SARS-CoV-2 and describe main results of our study. The abundance of quasi-periodic patterns in the genomic DNA/RNA sequences can be assessed by the spectral entropy (Balakirev et al., 2003 (Balakirev et al., , 2005 (Balakirev et al., , 2014 Chechetkin, 2011; Chechetkin & Lobzin, 1996; Chechetkin & Turygin, 1994) .
cord-331076-ak481qew	2020	In our previous study, we examined the top 10 most frequent mutations in the SARS-CoV-2 nsp12, and identified that four of them are associated with an increase in mutation density in two genes, the membrane glycoprotein (M) and the envelope glycoprotein (E) (the combination of which is hereafter referred to as MoE, as we previously described), which are under less selective pressure, and mutations in these genes are potential markers of reduced replication fidelity (Eskier et al., 2020a) . To identify the trends in SARS-CoV-2 mutation load over time, we calculated the average mutation density per day for all isolates for whole genome, S gene and MoE regions, capping outliers at the 95th and 5th percentile values to minimize the potential effects of sequencing errors (Fig. 1) . Three of the five most common nsp14 mutations, namely 18060C>T, 18736T>C and 18877C>T are associated with increases in both genome-wide mutational load, as well as MoE status, an alternative indicator of mutational rate and virus evolution.
cord-331414-i0oxm5mr	2020	To examine this, GFP was inserted into TC-83, a live-attenuated vaccine for the alphavirus Venezuelan equine encephalitis virus, as well as a low-fidelity variant of TC-83, and passaged until fluorescence was no longer observed. To examine reporter gene stability, three independent replicates of TC-83 GFP and low-fidelity TC-83 GFP were passaged 10 times on Vero cells using an approximate MOI of 0.5 ( Figure 1A ). All three TC-83 GFP replicates contained deletion variants present at a low frequency that were able to selectively remove the GFP reporter gene by passage 10 (Figure 2A) . To better understand the mechanisms underlying the conserved removal of the low-fidelity TC-83 GFP gene, M-fold was used to determine the RNA secondary structure of the virus genome ( Figure 4C-E) . To characterize the role of RNA structure of the virus genome in RNA recombination site selection, we modeled the three most common deletions that occurred at high levels during low-fidelity TC-83 GFP passaging.
cord-331509-p19dg1jw	2020	title: Porcine epidemic diarrhea virus: Viral RNA detection and quantification using a validated one-step real time RT-PCR In this study we described the development and validation of a SYBR™ Green one-step RT-qPCR according to the French norm NF U47-600, for the detection and quantification of PEDV viral RNA. Specificity and sensitivity, limit of detection (LoD), limit of quantification (LQ), linearity, intra and inter assay variability were evaluated using transcribed RNA and fecal and jejunum matrices spiked with virus. For a rapid, accurate and reliable diagnosis of PED in the veterinary laboratory, a method for the detection of PEDV viral RNA has been developed and more importantly validated according to the "Association Francaise de NORmalisation" (AFNOR) French NF U47-600 norm entitled "requirement and recommendation for the implementation, development and validation of PCR in animal health" (AFNOR, 2015a; AFNOR, 2015b) . This method should help harmonize detection and quantification of viral RNA from PEDV belonging to both S-non-INDEL and S-INDEL strains in both field and experimental settings.
cord-331607-2h56vb0n	2018	reported the first 3D architecture of the membrane-bound (+) RNA viral replication compartments in FHV-infected Drosophila cells (Kopek et al., 2007) , several 3D models of cellular remodeling during plant virus infection have been characterized. Confocal microscopy analysis showed that actin patches are closely associated with large p33-containing replication organelle-like structures in yeast cells and that such patches are present throughout the large replication compartments in plant cells, suggesting that actin plays a role in recruiting viral and cellular components (e.g., lipid) for VRC assembly (Nawaz-Ul-Rehman et al., 2016; Xu and Nagy, 2016) . The examples given above provide a good illustration of a common theme in membrane remodeling and the formation of spherules/vesicles: viral proteins, sometimes with the involvement of viral RNAs, recruit host factors that are diverted from their original functions and used to create virus replication factories (Diaz and Wang, 2014; Laliberté and Zheng, 2014; Wang, 2015; Nagy, 2016) . Formation of plant RNA virus replication complexes on membranes: role of an endoplasmic reticulumtargeted viral protein
cord-331680-qlzhtxs0	2020	In accordance with the purpose of the study, the following tasks were set: -to select the nucleotide sequence of the virus that is supposed to be inhibited, -to carry out the synthesis of oligonucleotide, -to determine cytotoxicity and antiviral activity in an in vitro experiment on cell culture. The assessment of antiviral activity of the drug in addition to cytopathic action was also taken into account by reducing the infectious titer of the virus in the culture of Vero cells E6 according to PCR RNA SARS-CoV-2, determined by the threshold of the number of reaction cycles (cycle treshold, Ct) in various dilutions of the study drug. Determination of the antiviral efficacy of the antisense oligonucleotide according to the treatment scheme (administration of the drug 24 hours after infection) was taken into account by the decrease in the infectious titer of the virus in the culture of Vero E6 cells by the cytopathic effect.
cord-331802-wo462anq	2015	Herein, we report that the nonstructural protein 2C(ATPase) of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. Herein, we report that although bacterially expressed EV71 2C ATPase did not exhibit any RNA remodeling activity, eukaryotically expressed EV71 2C ATPase functions not only as an RNA helicase that unwinds RNA helices from 3 0 to 5 0 in an ATP-dependent manner but also as an RNA chaperone that destabilizes helices from either direction and facilitates strand annealing and complex RNA structure formation independently of ATP.
cord-331916-n744pymd	2006	Specific inhibition of endogenous or pathogen mRNA by RNAi can be triggered by the introduction of 21 -23 nucleotide (nt) duplexes of RNA (siRNA) or by transcription of DNA precursor into short hairpin RNAs (shRNA) homologous to target sequences (Brummelkamp et al., 2002; Elbashir et al., 2001a; Paddison et al., 2002) , opening up possibilities for controlling replicative processes of pathogens. To further examine the ability of ORF1 shRNA to suppress viral gene expression in PCV2-infected PK15 cells, single nucleotide mutations were introduced into pSIR3 sequence as shown in Fig. 4A . As shown in Fig. 4B , a mutant with one nucleotide mutation in the 5V-end of the sense strand (pSIR3-m1) still had an effective reduction in the synthesis of ORF1 and ORF2 proteins, whereas with a nucleotide substitution at the center (pSIR3-m2) or 3V-end (pSIR3-m3) of the sense sequence, the resultant shRNAs failed to significantly suppress expression of PCV2 ORF1 and ORF2 in the transfected cells, further verifying the specificity of RNAi as demonstrated (Amarzguioui et al., 2003; Harborth et al., 2003) .
cord-332003-67e9fchy	2015	Although challenged later on as detailed in Section 7, this mechanism and the possibility to use such so called cell-penetrating peptides (CPPs) as non-viral delivery vectors for biomolecules, fostered a very large interest. Since the chemical conjugation and purification of negatively charged ONs with the most popular cationic CPPs has turned out to be difficult, most applications have concerned chargeneutral ON analogues such as Peptide Nucleic Acids (PNAs) and PMO (see Section 3). Unexpectedly, in a well-characterized HeLa 705 cell assay with a positive read-out ( Fig. 1) , splicing redirection using PNA or PMO oligomers conjugated to various standard CPPs (Tat, Penetratin or oligo-arginines) was not achieved in our research groups [57] . This led to the development of several arginine-rich peptides as PMO conjugates for use in muscle cells and in vivo mouse models of DMD as outlined in Section 4.
cord-332006-if46jycd	2009	Three years later, Tuschl and co-workers published their celebrated proof-of-principle experiment demonstrating that synthetic small interfering RNA (siRNA) could achieve sequence-specific gene knockdown in a mammalian cell line 2 . Toxicity of certain cationic lipid particles has been reported both in vitro and in vivo [76] [77] [78] , and certain synthetic agents have been found to induce a gene signature of their own that might increase the off-target effects of siRNA 79, 80 . Tumour growth in a mouse model of metastatic Ewing''s sarcoma was shown to be inhibited by the systemic delivery of nanoparticles formed by cyclodextrin, the targeting ligand transferrin, and siRNA specific for the EWS-FLI1 fusion gene commonly associated with the condition. Toxicogenomics of non-viral drug delivery systems for RNAi: potential impact on siRNA-mediated gene silencing activity and specificity
cord-332024-jk983q4p	2005	We describe a diagnostic system for rapid, sensitive, multiplex discrimination of microbial gene sequences and report its application for detecting 22 respiratory pathogens in clinical samples. We describe a diagnostic system for rapid, sensitive, multiplex discrimination of microbial gene sequences and report its application for detecting 22 respiratory pathogens in clinical samples. To address the need for sensitive multiplex assays in diagnostic molecular microbiology, we created a polymerase chain reaction (PCR) platform in which microbial gene targets are coded by a library of 64 distinct Masscode tags (Qiagen Masscode technology, Qiagen, Hilden, Germany). Multiplex primer sets were designed to identify up to 22 respiratory pathogens in a single Mass Tag PCR reaction; sensitivity was established by using synthetic DNA and RNA standards as well as titered viral stocks; the utility of Mass Tag PCR was determined in blinded analysis of previously diagnosed clinical specimens.
cord-332270-fusfdkjw	2020	The ongoing search for valid biomarkers for AD is being carried out globally in at least a dozen major geriatric, bioinformatic, neurobiological, neuro-genetic and neurological bioscience arenas: (i) those involving the age, gender, and geriatrics of the ''prospectiveAD patient''; (ii) in the genetics and epigenetics of the AD patient including messenger RNA (mRNA) and microRNA (miRNA) signaling patterns, complexity and genomic methylation research; (iii) in multiple biofluids from AD patients including the blood (plasma/serum) of the systemic circulation, the glymphatic system, the cerebrospinal fluid (CSF) and/or urine; (iv); through the detailed analysis of molecular cargos from both biofluids and tissue-compartmentalized exosomes and extracellular microvesicles (EXs and EMVs); (v) throughout the peripheral nervous system (PNS; typically using skin biopsies); (vi) via clinically-based geriatric, psychiatric, and neurological assessment and testing; (vii) via advances in neuro-radiological labeling techniques and neuroimaging technologies including CAT, PET, PET-SN, MRI, fMRI; UHF-MRI, DOT, MEG, SPECT, cranial ultrasound, functional ultrasound (fUS) imaging, and immunohistochemistry involving confocal laser scanning microscopy and other advanced microscopic and neuroimaging techniques; (viii) from the quantitation and characterization of the load of microbial and microbial-derived components in the AD-affected brain; (ix) via the identification, quantitation, and characterization of AD-specific lesions including amyloid peptide-enriched SPs and NFTs; (x) after post-mortem examination and biopsies of AD cases, again matched up against those same biomarkers in age-and gender-matched neurologically normal controls to corroborate the prospective diagnosis of AD; (xi) via the comprehensive analysis of the potential contribution of overlapping progressive, age-related neurological disorders to AD-type change; and lastly (xii), through the assessment of the socioeconomic, environmental, and lifestyle factors of the ''prospectiveAD patient'' ( Table 1 ).
cord-332356-au7s3dmp	2011	The results indicate that the binding ability of Gn-CT towards nucleic acids is rather unspecific since in addition to genomic RNA, also unrelated RNA as well as single-stranded DNA was able to interact with Gn-CTs of PUUV and TULV. Purified, lysed and micrococcal nuclease (MNase)-treated virus preparation and in vitro transcribed and radiolabeled genomic PUUV S segment were allowed to form RNA-protein complexes prior to UV cross-linking. The capability of structural proteins to bind RNA was analyzed by incubating purified, lysed and micrococcal nuclease (MNase)-treated virus preparations with radioactive PUUV S segment RNA in the case of PUUV ( The samples were immunoprecipitated with MAbs or PAbs specific to N, Gn or Gc and retained radioactivity on protein G beads after washing was measured by scintillation counting. The mapping indicated that the same peptides that were previously shown to interact with RNP and N protein (Hepojoki et al., 2010b) were capable of binding also nucleic acids (Fig. 5A ).
cord-332484-qy8vj6uu	2009	This review describes examples where both intracellular bacteria (Salmonella, Chlamydia and Legionella) and viruses (picornaviruses and hepatitis C) recruit membrane vesicles to sites of replication by modulating proteins that control the secretory pathway. Intracellular bacteria remain within membrane-bound vacuoles to avoid delivery to lysosomes and then recruit vesicles from the secretory pathway to provide nutrients necessary for microbial growth and cell division. Many viruses generate densely packed membrane vesicles to shield them from recognition by cellular defence pathways that recognise double-stranded RNA, and at the same time the membranes provide a platform to recruit viral and host proteins required for replication [3] [4] [5] . This review describes how recent work on intracellular bacteria such as Salmonella, Chlamydia and Leigionella draws parallels with studies on (+) strand RNA viruses where microbial proteins recruit membranes by modulating proteins that control the secretory pathway.
cord-332632-u2ud0vmq	2016	In particular, the unique extension of ''self'' to include the viral genome by degrading immunostimulatory viral RNA by E(rns) is reminiscent of various host nucleases that are important to prevent inappropriate IFN activation by the host''s own nucleic acids in autoimmune diseases such as Aicardi-Goutières syndrome or systemic lupus erythematosus. Thus, the survival strategy of BVDV consists of being non-cytopathogenic and producing less dsRNA than its cp counterpart, and expressing the IFN antagonists N pro as the first protein in order to reduce or even avoid IFN production in infected cells and E rns to degrade immunostimulatory viral RNA before they might activate the host''s PRRs. Notably, both pestiviral IFN antagonists are not only required to constantly maintain innate immunotolerance during persistent infections, but they also play an important role in acute infections [25] .
cord-332710-2s14knw6	1996	The capacity of coronaviruses to undergo recombination may be related to its mRNA transcription mechanism, which involves discontinuous RNA synthesis, suggesting the nonprocessive nature of the viral polymerase. The first coronavirus recombinant was isolated by coinfecting temperature-sensitive (ts) mutants of two mouse hepatitis virus (MHV) strains, A59 and JHM, and selecting progeny viruses which grew at the nonpermissive temperature. In contrast, clear-cut evidence of recombination has been obtained for natural isolates of avian infectious bronchitis virus (IBV), many of which have recombination between different strains in the spike protein gene or the 3''-end of viral RNA. 33 In this case, the viral RNA containing the sequence of the transfected RNA fragments was detected by reverse transcription-polymerase chain reaction (RT-PCR), although the actual recombinants could not be isolated because of lack of selection markers.
cord-332747-u46xryoo	2018	To determine which aspects of the HCV replication cycle are limited by lipin1 silencing, single cycle infection experiments were conducted by inoculating control and lipin1-deficient cell cultures at MOI 10 with genotype 2a D183 virus. Once cultures reached >95% of HCV-positive cells, they were transduced with lentiviral vectors expressing control, HCV RNA-targeting or LPIN1-specific shRNAs. At day 7 post-transduction, cells were split and samples of the cells and supernatants were collected 24 hours later to determine infectious virus production rate by infectivity titration HCV (C) and RNA levels by RT-qPCR (D). This reduced abundance is illustrated by a significant reduction in the fraction of cells displaying vesicular structures in lipin1-deficient cell cultures (Fig 7H) despite comparable transfection efficiency and viral protein expression levels, indicating that lipin1 may be required in a critical step leading to formation of the HCV-induced vesicular compartment.
cord-332844-2se4d1yp	2015	Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase. Generation of an infectious cDNA clone is a key step in developing a reverse genetics system for RNA viruses, especially for positive-strand RNA viruses, because its genome acts as viral mRNA that is translated into proteins by host cell ribosomes.
cord-332992-8rmqg4rf	2020	Although SARS-CoV-2 particles/components have been detected in, for example, endothelial cells, the digestive tract and the liver, not all extrarespiratory manifestations of COVID-19 are necessarily caused by direct viral injury but may also be the consequence of the hypoxaemia, (hyper)inflammatory response, neuroendocrine imbalance and other pathophysiological changes induced by the airway infection [43] . Factors that may contribute to the thrombophilia observed in severely ill COVID-19 patients include the following: (1) a disturbed balance between pro-and anticoagulant activities due to excessive production of proinflammatory cytokines, activation of complement, formation of neutrophil extracellular traps and activation of platelets; (2) inflammation-related endothelial activation; (3) death of SARS-CoV-2-infected endothelial cells; (4) endothelial dysfunction caused by unbalanced angiotensin IIangiotensin II type-1 receptor signalling; (5) formation of prothrombotic antiphospholipid antibodies; (6) immobility-associated reduction of blood flow; (7) hypoxia due to respiratory impairment resulting from SARS-CoV-2-induced lung injury [79] [80] [81] .
cord-333080-qytwbsne	2020	Altogether, TGF-beta signaling pathway as well as hub miRNAs, and LncRNAs involve during SARS-CoV pathogenesis can be considered as potential therapeutic targets. Developing functional computational models and networks to predict potential SARS-CoV -miRNA/lncRNA association may benefit not only the understanding of COVID-19 mechanism at the noncoding RNA level, but also the detection of disease biomarkers for disease diagnosis, treatment, prognosis, and prevention. Since multiple ways of interaction between miRNAs, lncRNAs, and mRNA have been reported to play key roles in determining the cellular functions during viral infection, it is essential to discover these interactions in an integrated fashion to comprehensively decipher the networks and key regulatory noncoding-RNA hubs underpinning the pathology of SARS-CoV. Our in-silico analysis has built a network of protein-protein interaction between the Human SARS coronavirus (SARS-CoV) and host proteome (Figure 1) , as well as strong miRNA-mRNA-lncRNA crosstalk ( Figure 4 and Table 2 ) possibly modulating the human response to the viral infection.
cord-333261-knj2rrut	2011	To facilitate the collections of HRV sequences over a number of years, a virology experiment was designed in which students test nasal lavage samples to look for HRV infection. The extensive data available on HRV genomes enables many bioinformatics opportunities for students, including alignment of genome sequences to look for mutations at the RNA level and differences among protein sequences. Students can examine differences in the HRV serotypes over several years of data regarding a university population to identify HRV mutations that have occurred and their severity in causing symptoms in the host. In this inquiry-based laboratory project, students use a variety of techniques, including RNA isolation, cDNA synthesis, qPCR, and agarose gel electrophoresis to look for the presence of HRV in nasal lavage samples from human subjects. By analyzing surveys in which subjects indicate severity of their symptoms, stress factors, and average hours of rest per night, students can identify possible contributors to HRV infection.
cord-333429-bq7kfpby	2020	title: Clinical characteristics and factors associated with long-term viral excretion in patients with SARS-CoV-2 infection: a single center 28-day study Male sex (HR, 0.58 [95% CI, 0.35-0.98]), immunoglobulin use (HR, 0.42 [95% CI, 0.24-0.76]), APACHE II score (HR, 0.89 [95% CI, 0.84-0.96]), and lymphocyte count (HR, 1.81 [95% CI, 1.05-3.1]) were independent factors associated with a prolonged duration of SARS-CoV-2 shedding. We identified that male sex, immunoglobulin use, APACHE II score, and lymphopenia were independent risk factors associated with the duration of SARS-CoV-2 RNA shedding, whereas ARV A c c e p t e d M a n u s c r i p t combination therapy and corticosteroid treatment were not independent factors. In conclusion, we found that male sex, immunoglobulin use, APACHE II score, and lymphopenia were independent risk factors associated with the duration of SARS-CoV-2 RNA shedding, whereas ARV combination therapy and corticosteroid treatment were not.
cord-333473-c1lykari	2016	Also, it has been employed in the study of the replication of large DNA viruses; namely, human cytomegalovirus [17] [18] , Kaposi''s sarcoma-associated herpesvirus [19] , herpes simplex virus 1 [20] , vaccinia virus [21] , and bacteriophage lambda [22] , providing insights into the temporal regulation of gene expression in these viruses and identifying numerous previously unrecognized translated ORFs, including novel protein-coding ORFs and short regulatory uORFs. In this paper, we describe the first analysis of RNA virus replication and gene expression by ribosome profiling (and parallel RNASeq), using MHV as a model system. The latter are calculated on a per gene (rather than per transcript) basis, using RNASeq and RiboSeq reads contained entirely within annotated CDS regions (i.e. excluding 5 0 and 3 0 UTRs and also RPFs accumulating at or near to initiation or termination sites), and, like the virus values, are expressed relative to the mean levels for the cell (due to normalization by library size).
cord-333515-llqpfhwg	2020	Their serial plasma samples (n = 535) collected during the hospitalization period were tested for total antibodies (Ab), IgM and IgG against SARS-CoV-2 using immunoassays. To mitigate this knowledge gap, and to provide scientific analysis on the benefit of antibody testing when used in combination with the current RNA testing, this study investigates the dynamics of total antibody (Ab), IgM and IgG antibody against SARS-CoV-2 in serial blood samples collected from 173 confirmed COVID-19 patients and provides discussion on the clinical value of antibody testing. A total of 535 plasma samples collected during the hospitalization period of the 173 patients were tested for antibodies against SARS-CoV-2. In addition to the diagnosis value of Ab test, our study revealed a strong positive correlation between clinical severity and antibody titer since 2-week after illness onset, for the first time in COVID-19 patients.
cord-333524-a6p6ma8r	2020	19 The current most common diagnostic method used to identify SARS-CoV-2 infection is a molecular technique for detecting viral RNA through nucleic acid amplification, RT-PCR. Nucleic acid amplification tests (NAATs) are the most common diagnostic tests used to detect pathogens, and many of the current SARS-CoV-2 detection techniques are primarily based on NAATs. 21 NAATs involve nucleic acid amplification, a process that initiates with a small quantity of starting nucleic acids and uses primers that target specific, short nucleic acid sequences in conjunction with enzymes to amplify or increase the quantity of starting nucleic acids. 34 This test incorporates a nested nucleic acid amplification technique showing higher sensitivity of detection than LAMP alone and conventional RT-PCR for minimally processed SARS-CoV-2 samples. 55 The technique first uses RT-LAMP for reverse transcription and isothermal amplification of viral RNA, and then employs the Cas12a enzyme to identify sequences of SARS-CoV-2, allowing cleavage of a reporter molecule ( Figure 5 ).
cord-333547-88dkh6xd	2020	Testing SARS-CoV-2 viral loads in wastewater has recently emerged as a method of tracking the prevalence of the virus and an early-warning tool for predicting outbreaks in the future. A limited number of studies have shown that the shedding period of SARS-CoV-2 in stool samples varies considerably, and can still be detected up to 27.9 ± 10.7 days after infection in some cases [9, 11] . Consequently, the main objectives of this study were: (i) to detect the presence of SARS-CoV-2 virus in municipal (untreated) wastewater and treated effluents of wastewater treatment plants (WWTPs) in the UAE; (ii) to quantify the viral concentration in viral gene copies per liter; and (iii) to explore whether these measurements mirror infections in the population in order to comment on the utility of this method to track the epidemiology of the disease.
cord-333636-h2sg6shp	2003	The histidine-tagged VP3 was affinity-purified and used to study its ability to bind RNA molecules using three complementary methods: (i) Northwestern blotting; (ii) binding of VP3 protein-RNA complexes to nitrocellulose membranes; and (iii) electrophoretic mobility shift assays. Briefly, increasing amounts (from 5 × 10 −2 mM to 5 × 10 −6 mM) of purified VP3 or BSA, used as a control for non-specific interaction, were mixed with 10 6 cpm of 32 P-labeled ssRNA probe B (5 × 10 −6 mM) in binding reaction buffer (50 mM Tris, 150 mM KCl, 150 mM NaCl 10% Glycerol, 0,01% Triton X 100), in 16 µl final volume, so the VP3/RNA molar ratio in these experiments varied from 10 4 to 1. Competition assays based on nitrocellulose binding were carried out using virus-derived radiolabeled ssRNA probes together with increasing concentrations of non-radioactive competitor RNA and DNA probes.
cord-333979-bx2xspbe	2020	title: High-throughput extraction of SARS-CoV-2 RNA from nasopharyngeal swabs using solid-phase reverse immobilization beads We developed a method using a widely available lysis buffer coupled with solid-phase reverse immobilization (SPRI) beads to extract viral RNA from swabs collected in viral transport medium (VTM) which can be performed manually or on a Hamilton STAR liquid-handling robot. Retrospective, residual nasopharyngeal specimens that previously tested positive or negative for respiratory syncytial virus (RSV) using the Cepheid GeneXpert Flu/RSV kit or the Fast Track Diagnostics Respiratory Pathogens 21 PCR (FTD-RESP21) were extracted with our new approach on a Hamilton STAR liquid handling robot with 8 CO-RE channels. When applying these conditions to 204 clinical samples, we only observed a 2.5% inhibition of the RT-qPCR, indicating that our method is suitable for large-scale use in testing laboratories. In this manuscript, we demonstrate the feasibility of using reagents commonly available in molecular biology and next-generation sequencing laboratories for the extraction of SARS-CoV-2 viral RNA for the diagnosis of COVID-19.
cord-334082-fyxn0g3v	2015	This scheme in turn places two requirements on the nucleic acid: it must be replicated in the virus-producing cell, to provide the genetic material encased in the progeny virus particles; and it must encode the proteins needed for the production of the progeny particles, including at a minimum the structural proteins from which the particles will be assembled. The DNAs of ssDNA viruses are replicated by a mechanism similar to ''rolling circle'' replication, involving synthesis of dsDNA intermediates containing multiple tandem copies of the viral genome. These viruses are unique in that their genomic RNA is translated immediately upon infection; that is, the virus particle is simply a package that introduces an mRNA molecule into the cell. When the particle infects a new host cell, the RNA-dependent DNA polymerase or ''reverse transcriptase'' in the virus copies this RNA into dsDNA.
cord-334123-wb45ww7f	1989	A third and earlier study proposed that an RNA pseudoknot is recognized by a DNA binding protein that autogenously regulates translation of its mRNA (McPheeters et al., 1988) . The tRNA-like structures at the 3'' ends of plant viral RNAs provide one example where the pseudoknot format may be necessary to form a substrate that is specifically recognized by an enzyme. Previous structural mapping experiments by Draper and co-workers suggested a model for the 5'' region of the mRNA which constitutes the S4 binding site; it envisions a hairpin helix and loop that encompasses nucleotides 19 to 72. The second study provides evidence that pseudoknot formation in a viral mRNA is required for frameshift suppression of a termination codon that, in turn, allows a fusion protein to be synthesized from two overlapping reading frames. This element encodes a stem-loop structure that starts six nucleotides downstream from the proposed site of frameshifting, near the end of the first reading frame.
cord-334299-0zn1z7rc	2020	title: Surveillance of SARS-CoV-2 RNA in wastewater: Methods optimisation and quality control are crucial for generating reliable public health information However, in order to reliably interpret data produced from these efforts for informing public health interventions, additional quality control information and standardization in sampling design, sample processing, and data interpretation and reporting is needed. The review highlights areas for potential standardization including considerations related to sampling timing and frequency relative to peak fecal loading times; inclusion of appropriate information on sample volume collected; sample collection points; transport and storage conditions; sample concentration and processing; RNA extraction process and performance; effective volumes; PCR inhibition; process controls throughout sample collection and processing; PCR standard curve performance; and recovery efficiency testing. In view of this need, we recommend methodological and quality assurance approaches for SARS-CoV-2 RNA detection in 158 wastewater using molecular methods.
cord-334315-ymkrgj0h	2013	This review describes the myriad ways that viruses deal with the general host RNA decay machinery that is active in the cell immediately upon viral infection-turning what, at first, appears to be very hostile territory for a foreign transcript into a sort of ''''promised land'''' for viral gene expression. Disparate RNA viruses have, therefore, evolved unique mechanisms by which they disarm host RNA decay pathways by inactivating or proteolytically degrading important nucleases to promote productive viral infections. In addition, to make a cell more amenable to virus production, these virally encoded nucleases may also create a new ''''sandbox'''' in the cytoplasm for viral RNAs by initiating the large-scale decay of cellular mRNAs and dramatically altering the landscape of host gene expression. Considering the importance of RNA stability in regulating transcript abundance, the inactivation or commandeering of cellular RNA decay factors by viruses is likely to significantly alter host gene expression.
cord-334394-qgyzk7th	2020	To address the ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 and expand the known sequence diversity of viruses, we aligned pangenomes for coronaviruses (CoV) and other viral families to 5.6 petabases of public sequencing data from 3.8 million biologically diverse samples. To expand the known repertoire of viruses and catalyse global virus discovery, in particular for Coronaviridae (CoV) family, we developed the Serratus cloud computing architecture for ultra-high throughput sequence alignment. We aligned 3,837,755 public RNA-seq, meta-genome, meta-virome and meta-transcriptome datasets (termed a sequencing run [5] ) against a collection of viral family pangenomes comprising all GenBank CoV records clustered at 99% identity plus all non-retroviral RefSeq records for vertebrate viruses (see Methods and Extended Table 1 ). We performed de novo assembly on 52,772 runs potentially containing CoV sequencing reads by combining 37,131 SRA accessions identified by the Serratus search with 18,584 identified by an ongoing cataloguing initiative of the SRA called STAT [5] .
cord-334463-nvu5tqxb	2012	We found that drugs, small interfering RNAs (siRNAs), and dominant negative mutants that target factors required for clathrin-mediated endocytosis, including clathrin and dynamin, inhibited both EV7 infection and internalization of virions from the cell surface. We previously (19) observed that, to infect polarized epithelium, CVB3 binds DAF on the cell surface, moves to the tight junction, and then enters the cell by an unusual mechanism that is independent of both dynamin and clathrin but which requires caveolin; contact with CAR in the tight junction leads to an essential conformational change in the CVB3 virion, but disruption of the capsid does not appear to occur until the virus has moved to an unidentified intracellular compartment. We had previously observed that CVB3 infection of Caco-2 cells occurs by a complex process that depends on caveolin but not dynamin-2, appears to be independent of clathrin-mediated endocytosis, and is inhibited by depletion of membrane cholesterol (19) .
cord-334771-uy3s6443	2004	Samples obtained were: 54 blood samples, 22 throat swabs, ten CSF samples, and one brain aspirate from 55 patients with encephalitis; five blood samples and nine throat swabs from 13 fever cases; and ten blood samples and one throat swab from ten family contacts (including specimens from the brother and mother of a patient who Methods Cell lines and peripheral blood lymphocyte co-cultures were used to isolate the causative agent from clinical samples. The confirmed Chandipura virus encephalitis group consisted of individuals from whose samples we isolated the virus, viral RNA, or reactive IgM antibodies. The viruses isolated in different cell lines from clinical samples from patients with encephalitis were confirmed as Chandipura virus with various techniques including complement fixation, neutralisation test, and immunofluorescence assay. Moreover, the presence of Chandipura virus RNA in nine patients with encephalitis, all from samples obtained before day 4 after onset of illness, suggests an early viraemic phase of the infection process.
cord-334891-4jgtxg07	2020	This study is hoped to rationalize the comparative binding and sensing of SARS-CoV-2 mRNA towards the intracellular TLRs, considering the solvent-based force-fields operational in the cytosolic aqueous microenvironment that predominantly drive these reactions. Our in-silico study on the binding of all mRNAs with the intracellular TLRs shown that the mRNA of NSP10, S2, and E proteins of SARS-CoV-2 are potent enough to bind with TLR3, TLR9, and TLR7 and trigger downstream cascade reactions, and may be used as an option for validation of therapeutic option and immunomodulation against COVID-19. The binding of Spike protein with the human ACE2 receptor triggers the pathogenesis 3 of the SARS-CoV-2, leading to the activation of TLRs to activate the proliferation and 4 production of pro-inflammatory cytokines causing cytokine storm, those results in 5 inflammations.
cord-334947-pa0p5dif	2014	Since residue 483 is conserved among alphaviruses, we examined the analogous mutations in Sindbis virus (SINV), which also reduced polymerase fidelity and generated replication defects in mosquito cells. To estimate the population diversity of variants by deep sequencing, cDNA libraries were prepared by Superscript III from RNA extracted from virus generated in BHK-21 or C6/36 cells, and the viral genome was amplified using a high fidelity polymerase (Phusion) to generate 5 overlapping amplicons 2-3 kb in length. To address the possibility that the fitness cost of defective replication, observed in mosquito cell culture, would favor the reversion of these mutant polymerases to wildtype, we deep sequenced virus from the body of an individual mosquito that presented the median titer from each group.
cord-335040-1qa6pe4v	2020	The SARS-CoV-2 non-structural protein 10 (nsp10) displays high sequence similarity with its SARS homologue, which binds to and stimulates the 3′-to-5′ exoribonuclease and the 2′-O-methlytransferase activities of nsps 14 and 16, respectively. The crystal structure and solution behaviour of nsp10 will not only form the basis for understanding the role of SARS-CoV-2 nsp10 as a central player of the viral RNA capping apparatus, but will also serve as a basis for the development of inhibitors of nsp10, interfering with crucial functions of the replication–transcription complex and virus replication. observed SARS nsp10 in the same space group as reported here, I213, reporting a monomer in the asymmetric unit but a dimer in solution, as was determined by size exclusion Residues shaded in red are fully conserved, while residues with text in red indicate a change to a similar residue. We determined the crystal structure and behaviour in solution of SARS-CoV-2 nsp10 in its unbound form.
cord-335067-tg66h99q	2013	The realisation that many newly discovered and/or emerging pathogens have zoonotic origins has prompted much interest in the nature of the non-human reservoirs and biological characteristics that enable pathogens to jump between host species. 1 Here, we focus on an important group of pathogens -RNA viruses -and consider how taxonomic diversity and various aspects of ecological diversity -host range, route of infection and transmissibility -are related to one another. 2 We catalogue ICTV-recognised RNA virus species for which we can find convincing evidence of natural human infection in the peer-reviewed scientific literature (see Ref. 3 for more methodological details). Our procedure reveals a total of 158 human infective RNA virus species from 47 genera and 17 families (with one genus unassigned to a family). The overlap is also illustrated in that 47 out of 60 recognised mammal virus genera contain human infective species, as do 17 out of 19 families. There are 68 species of vector-borne RNA virus that can infect humans.
cord-335231-617e5dcy	2008	During an outbreak in northern Sweden of nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome, we collected saliva and plasma from 14 hospitalized NE patients with verifi ed Puumala virus (PUUV) infection. During an outbreak in northern Sweden of nephropathia epidemica (NE), a milder form of hemorrhagic fever with renal syndrome, we collected saliva and plasma from 14 hospitalized NE patients with verifi ed Puumala virus (PUUV) infection. For this reason, we collected saliva from patients during an NE outbreak in northern Sweden and analyzed the samples for the presence and levels of PUUV RNA by using a real-time reverse transcription-PCR (RT-PCR) assay. Furthermore, we detected no inhibition of real-time RT-PCR in saliva or plasma when we analyzed our patient samples by using an internal positive control (data not shown).
cord-335377-zrbn637z	2012	Furthermore, the inability to dimerize caused by the silent codon change in Stem 3 of SARS-CoV changed the viral growth kinetics and affected the levels of genomic and subgenomic RNA in infected cells. We further show that kissing dimer formation plays a role in frameshift-stimulation and modulates the relative abundance of full-length and subgenomic viral RNAs. Plasmids containing wild-type pseudoknot as well as the ÁS3 pk mutant were described in Plant et al (1) . Our previous NMR analysis of exchangeable imino protons of the SARS-CoV pseudoknot ( Figure 1A , wild-type pk) provided unequivocal evidence for the existence of Stem 3 (1). Surprisingly, in the context of the SARS-CoV Stem 3 sequence, 5 0 -cuug-3 0 tetraloop-capped mutants readily formed extended duplex structures as revealed by native gel and NMR analysis.
cord-335441-bj3me7p8	2010	Five of the six ducks excreted viral RNA in their feces on the first day post-inoculation (PI) and all samples (feces, cloacal and oral swabs) from all birds were positive on the second day PI (Figures 4 and S1 ). Intermittent and moderate (high ct-values) viral RNA shedding was detected for all birds in water, fecal or cloacal samples between day 1 and 7 after H7N7 re-inoculation ( Figure 4 ). Active H5 infection was confirmed only in one duck, by expression of H5-specific antibodies and detection of viral RNA in the various sample types (feces, water, oral and cloacal swabs) with a pattern similar to the H5-inoculated control bird. Eight 3-month-old male wild-type mallards (Anas platyrhynchos) of approximately the same body mass and size (measurement of the left wing, the right tarsus length and the distance from bill tip to back of the skull) were selected from a Swedish duck farm known from previous successive sampling to be free from IAV infection.
cord-335443-iv2gs3kg	2020	Here, we combine crystallography, biochemical and whole cell assays, and show that this compound inhibits SARS-CoV-2 Nsp15 and interacts with the uridine binding pocket of the enzyme''s active site, providing basis for the uracil scaffold-based drug development. For SARS-CoV it was reported that Nsp15 cleaves highly conserved non-translated RNA on (+) sense strand showing that both RNA sequence and structure are important for cleavage 6, 7 . The enzyme cleaves efficiently eicosamer 5''GAACU¯CAU¯GGACCU¯U¯GGCAG3'' at all four uridine sites (Fig. 1) , as well as synthetic EndoU substrate ( 5′-6-FAM-dArU¯dAdA -6-TAMRA-3′ ) 8 in the presence of Mn 2+ and the reaction rate increases with metal ion concentration. SARS-CoV-2 Nsp15 protein was crystallized with 5''UMP, 3''UMP, 5''GpU and Tipiracil using methods described previously 8 and the structures were determined at 1.82 Å, 1.85 Å, 1.97 Å and 1.85 Å, respectively. In the crystal structure of Nsp15/5''GpU, the dinucleoside monophosphate binds to the active site with uracil interacting with Tyr343 and Ser294 (Fig. 4B ), as seen in the Nsp15/5''UMP complex.
cord-335482-nx7odchj	1984	We have observed marked reduction of infectivity in the course od serial undiluted passages of JHM virus in DBT cell culture. To avoid contamination of the standard virus stock with DI particles, plaque purified MHV (JHM strain) (Hirano et aL, 1981; Makino et al, 1983) was propagated on DBT cells at a multiplicity of infection (m.o.i.) of 0.0002 and at 15 hr postinfection (p.i.) culture fluid was harvested and clarified by low speed centrifugation. Preparation of the Standard JHM Virus for Interference Assay JHM virus was propagated on DBT cells after infection at an m.o.i. of 1.0 and was harvested at 14 hr p.i. The culture fluid was clarified by centrifugation at 8000 rpm for 30 min. To determine if the reduction in the yield of infectious virus was due to the presence of DI particles, a''n interference analysis was performed with several culture fluid samples at different passage levels.
cord-335614-qh98622y	2019	These genes and metabolites were linked to NIBV-infection related processes, including immune response, signal transduction, peroxisome, purine, and amino acid metabolism. Taken together, our research comprehensively describes the host responses during NIBV infection and provides new clues for further dissection of specific gene functions, metabolite affections, and the role of gut microbiota during chicken gout. The results of PCA and OPLA-DA analysis showed that there was an obvious separation between the content of the Con and Dis groups, revealing significant changes in the concentrations of metabolites in the kidney induced by NIBV infection. In addition, the transcriptomic analysis showed that NIBV infection also activated the RIG-I-like receptor signalling pathway (Figure 3f , signal 2), which included the transcriptional upregulation of genes such as MDA5, IPS-1, TRAF3, and IκB. In the present study, the ABCG2 mRNA was downregulated in the model group chicken kidneys, partially explaining the significantly increased uric acid levels caused by NIBV infection.
cord-335864-392xmrq0	2020	With recent developments in sequencing methods and information analysis, an increasing number of novel ncRNAs have been identified, including long noncoding RNAs (lncRNAs) [5, 6] , circular RNAs (circRNAs) [7, 8] , and novel small ncRNAs [9] [10] [11] . Most circRNAs derived from mRNA back-splicing lose translational capacity because of the lack of effective ORFs or ribosome entry approaches, while a few circRNAs from coding or Sno-lncRNAs maintain their stability by their classical stem-loop structures of snoRNAs. c Alternative splicing within circRNAs. d A number of novel small ncRNAs derived from rRNAs (rRFs), tRNAs (tsRNAs), and snoRNAs (sdRNAs) have also been found to be enriched in RNA-induced silencing complexes (RISCs) and function in a miRNA-like pathway This approach can provide in-depth analysis of transcripts from target regions of genome.
cord-336012-8klkojpo	2020	Unlike RT-qPCR, SARS-CoV-2 Whole Genome Sequencing (cWGS) has the added advantage of identifying cryptic origins of the virus, and the extent of community-based transmissions versus new viral introductions, which can in turn influence public health policy decisions. Methods We performed shotgun transcriptome sequencing using RNA extracted from nasopharyngeal swabs of patients with COVID-19, and compared it to targeted SARS-CoV-2 full genome amplification and sequencing with respect to virus detection, scalability, and cost-effectiveness. Conclusions SARS-CoV-2 whole genome sequencing is a practical, cost-effective, and powerful approach for population-based surveillance and control of viral transmission in the next phase of the COVID-19 pandemic. Here we show that cWGS is cost-effective and is highly scalable when using a target enrichment sequencing method, and we also demonstrate its utility in tracking the origin of SARS-CoV-2 transmission.
cord-336093-ic6q6ke8	2014	Abbreviations: SARS, severe acute respiratory syndrome; SARS-CoV, SARS coronavirus; nsp, nonstructural protein; N7-MTase, guanine-N7-methyltransferase; 2 0 -O-MTase, 2 0 -O-methyltransferase; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-L-homocysteine; ATA, aurintricarboxylic acid; IC 50 , inhibitory concentration at 50% activity. A single transformed colony of the YBS40 strain containing plasmids expressing human N7-MTase (MT-Human), SARS-CoV N7-MTase (MT-SARS), N7-MTases of other coronaviruses (MT-MHV, MT-TGEV, and MT-IBV), and the pMceK294A vector as control (representing the yeast N7-MTase [MT-Yeast]), were inoculated separately into 5 ml of a basic medium (Min SD Base) lacking tryptophan and incubated at 30°C for 21-24 h until reaching a similar final cell density in the stationary phase (0.5-1.0 Â 10 8 cells/ml) (Chrebet et al., 2005) . Although AdoHcy, ATA and sinefungin, were previously reported to be inhibitors of coronavirus RNA MTases in vitro , only sinefungin significantly suppressed the growth of the MT-yeast, MT-human, and MT-SARS yeast cells (Fig. 3 ).
cord-336319-8068s9a3	2005	Indirect mechanisms include reduction in cellular guanosine triphosphate (GTP) pools via inosine monophosphate dehydrogenase (IMPDH) inhibition, and an immunomodulatory effect in which a T-helper type 1 (antiviral) immune response is maintained. Direct mechanisms include inhibition of RNA capping activity, direct inhibition of viral polymerases, and increased mutation frequency via incorporation of ribavirin into newly synthesised genomes leading to error catastrophe. For all three compounds, a linear correlation was noted between GTP pool inhibition and antiviral activity as measured by viral RNA synthesis, as well as antiviral effect measured by reduction in CPE, suggesting that IMPDH inhibition is the primary mechanism of antiviral activity for all three compounds. Although direct biochemical evidence was not obtained, this finding suggested that ribavirin inhibits the capping of RNA genomes, either by interfering with the guanylyltransferase or methyltransferase activities (both of which are thought to be encoded by nsP1) or potentially by being incorporated as a cap analogue, which may impact translation of the RNA.
cord-336447-hpnkou41	2020	Despite multiple publications and increasing knowledge regarding the biological secrets of SARS-CoV-2, as of the writing of this paper, there is neither an approved vaccine nor medication to prevent infection or cure for this highly infectious disease. 7, 8 This paper reviews the microbiological, clinical, and epidemiological characteristics of the coronavirus disease 2019 (COVID-19) pandemic, as well as its socio-economic impact. In the early days of the pandemic great effort was invested into understanding the life cycle of SARS-CoV-2, 9 so as to provide a basis for discovery of an effective vaccine to prevent COVID-19 and/or a safe and efficacious drug to cure it, or at the least, to ameliorate its symptoms, shorten its duration, and/ or block its mechanism of transmission. 59 Unfortunately, to date, no human genetic markers predisposing to SARS-CoV-2 infection, nor the severity of COVID-19, have been found-although recent isolated exceptions to this statement can be found.
cord-336554-n8n5ii5k	2020	Number of drugs such as remdesivir, favipiravir, ribavirin, lopinavir, ritonavir, darunavir, arbidol, chloroquine, hydroxychloroquine, tocilizumab and interferons have shown inhibitory effects against the SARS-CoV2 in-vitro as well as in clinical conditions. Outbreaks of novel emerging infections such as coronavirus disease 2019 (COVID19) have unique challenges in front of the health professionals to select appropriate therapeutics/pharmacological treatments in the clinical setup with very little time available for the new drug discovery [3] . Currently, with the lack of effective agents against SARS-CoV2 as well as public-health emergency, WHO has identified some therapies which doctors and researchers believe are the most promising, such as a combination of two HIV drugs (lopinavir and ritonavir), anti-malarial drugs (chloroquine and hydroxychloroquine), and an experimental antiviral compound remdesivir. Ribavirin at a dose rate of 500 mg 2-3 times/day in combination with other drugs such as lopinavir/ritonavir or interferon (IFN)-α through intravenous route for not more than 10 days made the SARS-CoV2 infected patients more resistant to respiratory distress syndrome as well as death [41] .
cord-336560-m5u6ryy9	2020	Our results reveal the importance of STAT2-dependent interferon responses in the pathogenesis and virus control during SARS-CoV-2 infection and may help rationalizing new strategies for the treatment of COVID-19 patients. The lack of readily accessible serum markers or the absence of overt disease symptoms in hamsters prompted us to establish a non-invasive means to score for lung infection and SARS-CoV-2 induced lung disease by computed tomography (CT) as used in standard patient care to aid COVID-19 diagnosis with high sensitivity and monitor progression/recovery 7, 33, 35, 36 . Similar as in humans 37 , semiquantitative lung pathology scores were obtained from high-resolution chest micro-CT scans of freebreathing animals 38 The increase in replication of SARS-CoV-2 seen in IL28R-a -/hamsters, on one hand, combined with a tempered inflammatory response and lung injury as compared to WT hamsters, on the other hand, is in line with the role of type III IFN plays during respiratory virus infections, including SARS-CoV-1 53 .
cord-336628-0evl3wnd	2020	Consistently, secreted cytokine profiles from both severe COVID-19 patients and SARS-CoV-2 infected lung epithelial cells, were enriched for pro-inflammatory cytokines and lacked type I/III IFNs. We also demonstrate that SARS-CoV-2 infection leads specifically to NF-κB but not IRF3 nuclear localization and that poly(I:C)-induced pathway activation is attenuated in infected cells. To confirm that the lack of IFN response in Calu-3 or A549-ACE2 cells infected with SARS-CoV-2 was not due to defects in the activation of innate immune pathways, we To test if IFNs could limit virus replication even after establishment of infection, A549-ACE2 cells were treated with high levels of various IFNs at the time point of infection or 6 h thereafter. these results indicate that SARS-CoV2-infection triggers the cGAS-STING pathway, leading to NF-κB-mediated induction of pro-inflammatory cytokines, and that this response can be controlled with STING inhibitors.
cord-336775-d4hi9myk	2020	Abstract Human Coronaviruses (HCoV), periodically emerging across the world, are potential threat to humans such as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) – diseases termed as COVID-19. Hence, acute respiratory distress syndrome (ARDS) is caused by cytokine storm that triggers a destruction in host cells via immune system and subsequently results into multiple organs failure or death as stated in case of SARS-CoV-2 outbreak; similar observations were noted in case of SARS-CoV infection (Kumar et al., 2020) . When developing novel therapeutic strategies to check the immunoregulatory cytokines such as TNFβ and IL6, investigation should be considered on the viral strain and targeted organ specificity; for example, SARS-CoV-2 has more affinity to ACE2 which are scattering on different organs like lung and kidney while MERS-like CoV can even infect T-cells. Tumor necrosis factor-alpha convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensin-converting enzyme-2 (ACE2)
cord-336986-rmxin1da	2019	Eight different compounds, all nucleoside analogues, could presently be considered as potential drug candidates for the treatment of Ebola virus (EBOV) and/or other hemorrhagic fever virus (HFV) infections. Abstract: Eight differentc ompounds, all nucleoside analogues, could presently be considered as potential drug candidates for the treatment of Ebola virus (EBOV) and/oro ther hemorrhagic fever virus (HFV) infections.T hey can be considered as either (i)adenine analogues (3-deazaneplanocin A, galidesivir,G S-6620 andr emdesivir) or (ii)guanine analogues containing the carboxamide entity (ribavirin, EICAR, pyrazofurin and favipiravir). [26] It wasa lso found active against other emerging viruses such as respiratory syncytial virus (RSV) and hepatitis Cv irus (HCV),a nd the presence of the 1''-cyano group in remdesivir wasf ound to be critical in providing selectivity toward the viral (RNA) polymerases.
cord-337026-osgi06o4	2020	Given the uprising number of publications and case reports of COVID-19 patients showing conjunctivitis [61, 62] and the history of other coronaviruses that are found in tears, we have to consider the possibility of a separate, alternative viral mechanism through which the virus can enter the patient''s organism through epithelial cells of the eye [63] . The growing evidence on COVID-19 and its ocular implications and manifestations, in both animals and humans, is covered by many interesting reviews, all published 5 to 6 months after the novel coronavirus'' outbreak [64] [65] [66] [67] [68] , something that reveals the need to understand the virus from different perspectiveswhich at first may have seemed secondary in priority-in order to be able to reach a treatment.
cord-337199-mbv8kd1k	2012	This review outlines the essential steps required for the clinical translation of RNAi-based respiratory therapies including disease and RNA target selection, siRNA design, respiratory barriers, and delivery solutions. 1 In vitro screening and selection of siRNA candidates against potential targets such as pathogenic or host factors; 2 optimize siRNA designs with high silencing activity and avoid immune recognition, increase stability, and reduce off-target effects; 3 evaluation of cytokine profile and gene silencing efficiency in preclinical disease models using either naked or a suitable delivery system; and 4 clinical trials to evaluate toxicity and biological activity of lead formulations Considerable efforts have attempted to harness the "exogenous" or "endogenous" branches of the RNAi pathway for therapeutic purposes. The authors suggested that the observed therapeutic effect reported in early studies was most likely due to activation of the innate immune response by the recognition of viral-specific nonmodified siRNA sequences by toll-like receptors (TLRs) instead of an RNAi-mediated effect.
cord-337285-t6qr41wc	2007	Using cell culture systems, several cellular proteins have been identified as effective molecules for HCV RNA replication (Table 1) . [66] reported that lovastatin (LOV), one of the HMG-CoA reductase inhibitors, inhibited HCV RNA replication in HCV replicon-harboring cells. Depletion of the GGPP by statins may inhibit the geranylgeranylation of cellular proteins such as FBL2 and cause the anti-HCV effect in the cells. During the development of IFN therapy for patients with CH-C, the lack of a robust method of HCV RNA replication in cell culture has hampered research into the HCV life cycle and the discovery of potent new anti-HCV reagents. VX-950, a novel hepatitis C virus (HCV) NS3-4A protease inhibitor, exhibits potent antiviral activities in HCV replicon cells Selectable subgenomic and genome-length dicistronic RNAs derived from an infectious molecular clone of the HCV-N strain of hepatitis C virus replicate efficiently in cultured Huh7 cells
cord-337339-0vkigjv2	2020	We propose that comparative assessment in young versus aged hamsters of SARS-CoV-2 vaccines and treatments may yield valuable information, as this small-animal model appears to mirror age-dependent differences in human patients. Moreover, transgenic mice expressing human ACE2 represent a lethal SARS-CoV-2 infection model resulting in significant weight loss and permitting robust virus replication in the respiratory tract including the lungs [20] . In contrast to SARS-CoV-2 titers, histopathological changes differed markedly between young and aged Syrian hamsters over time: younger animals launched more severe reactions at early time points after infection, while lesions and inflammation in the lungs became more pronounced and widespread at later time points in the elderly. Based on the data presented here, we propose that comparative preclinical assessments of SARS-CoV-2 vaccines and other treatment options in young versus aged hamsters may yield valuable and relevant results, as this small animal model appears to mimic age-dependent differences in humans.
cord-337361-salby0fu	2013	In some viruses, the frequency of homologous crossing-over is very high and practically every replicated viral RNA molecule can be considered as chimerical in nature, as we have demonstrated for brome mosaic virus (BMV) RNAs (Urbanowicz et al., 2005) . The generally accepted mechanism of RNA recombination is currently explained by a copy-choice model where the viral RNA polymerase (RdRp) complex in mRNA viruses [reverse transcriptase (RT) in retroviruses] changes templates during synthesis of the nascent strand (Galetto et al., 2006) . Among the factors known to promote replicase to switch are sequence homologies between recombination substrates along with secondary structures at the crossover sites, as demonstrated with the BMV and other systems (Figlerowicz and Bujarski, 1998; Nagy et al., 1999b) . Comparison among three plant RNA virus replication systems (TBSV, BMV, and dianthoviruses) reveals general patterns within the stepwise process of viral replicase complex assembly which requires concerted involvement of protein-protein, RNA-protein, and protein-lipid interactions (Mine and Okuno, 2012) .
cord-337508-nfzaw8gg	2020	authors: Kirkland, P.D.; Frost, M.J. title: The impact of viral transport media on PCR assay results for the detection of nucleic acid from SARS-CoV-2 With the widespread introduction of molecular based diagnostic assays, especially real time PCR (qPCR), studies have been undertaken to evaluate the stability of viruses in VTMs, particularly in commercially prepared products, while being held at a range of temperatures. After becoming aware of concerns of variable results for the same samples in different assays, we initiated a study to compare the stability of SARS-CoV-2 RNA in several commercially manufactured VTMs and an in-house product. The results of these studies clearly indicate that the commercially prepared VTM solutions have had an adverse impact on the ability to detect both SARS-CoV-2 and influenza RNA. The impact of viral transport media on PCR assay results for the detection of nucleic acid from SARS-CoV-2 and other viruses
cord-337673-1nau263l	2006	Recently, small interfering RNA (siRNA) has shown promise in the protection from viral invasion, as it can inhibit the expression of viral antigens and accessory genes as well as control the transcription and replication of the viral genome. Genes encoding vital proteins in reproducing SARS-CoV virions can be chosen for chemotherapeutic intervention (e.g., those coding for S, 3C-like protease [3CLpro], RNA-dependent RNA polymerase and possibly other gene products involved in viral-protein-mediated processes) [81] first demonstrated that siRNA was able to silence the replicase of SARS-CoV (1a region of the genome) and that this approach was effective in vitro against SARS-CoV. [82] subsequently observed that vector-based siRNAs could inhibit the replication of SARS-CoV, and showed that expression in the plasmid, pSUPER, of siRNAs specifically targeting viral RNA polymerases could block the cytopathic effects of SARS-CoV on Vero cells. [86] showed that three chemically synthesised siRNA duplexes targeting viral RNA polymerases, and one targeting the S gene potently inhibited SARS-CoV infection and replication in fetal rhesus kidney cells (FRhK-4) .
cord-337879-liqhbqxl	2012	Sequences from GB virus C (GBV-C), a flavivirus not previously isolated from brain, were enriched in one of the MS samples. This study shows the feasibility of deep sequencing for the detection of occult viral infections in the brains of deceased persons with MS. Multiple sclerosis (MS) is a chronic demyelinating disease of unknown cause, which affects the brain and spinal cord of about 400,000 individuals in the U.S. A number of viral infections of the CNS can lead to demyelination, including distemper (dogs), measles (SSPE, humans), and influenza (humans). To enhance the detection of non-human sequences, RNA samples that passed the quality control step above were subjected to rRNA removal using the RiboMinus kit (Invitrogen Inc., Carlsbad, CA). One subject who died with primary-progressive MS had .1000 36 bp sequences detected that mapped to GBV-C virus (hepatitis G), a human flavivirus not known to cause any persistent disease and never before detected in human brain.
cord-337899-w5zh40gv	2017	Recently, several protease inhibitors have been developed using computeraided drug design methodologies (Gupta, 2013; Gupta et al., 1983; Wlodawer and Vondrasek, 1998) , synthetic approaches, high-throughput screening method (Mayr and Bojanic, 2009) , and drug reposition-based approaches (Sundberg, 2000) , which could possibly target the NSPs responsible for virus replication. The former ligand is therefore supposed to be a promising inhibitor of NSP2 protease of CHIKV virus, and has been emerged as one of the promising antiviral drug candidates with potential symptomatic and disease-modifying effects. Exploring the polymerase activity of Chikungunya viral non structural protein 4 (nsP4) using molecular modeling, epharmacophore and docking studies. Molecular modeling and docking study to elucidate novel Chikungunya virus nsP2 protease inhibitors Exploring the polymerase activity of Chikungunya viral nonstructural protein 4 (nsP4) using molecular modeling, e-pharmacophore and docking studies
cord-337976-c2auspti	1983	A cDNA probe representing the genome of mouse hepatitis virus (MHV) strain A59 (MHV-A59) was used to measure nucleotide sequence homologies among murine and human coronaviruses and the SD and SK coronaviruses isolated by Burks et al. Since SD and SK were isolated by inoculation of multiple sclerosis (MS) central nervous system (CNS) tissue into mice or cultured mouse cells, it is important to determine their relationships to other murine and human coronavirus isolates. Some strains of HCV such as 0C43, are antigenically related to murine coronaviruses such as MHV strain JHM (McIntosh, 1974; Gerdes et al., 1981a, b) and may be grown in the brains of suckling mice (McIntosh et al., 1967) . We have further compared murine and human coronaviruses and SD and SK by using molecular hybridization of virus-specific RNA with cDNA probes. RNA extracted either from brain homogenates of OC43-infected suckling mice or from HRT cells infected with OC43 shows homology with A59 cDNA when assayed by blot hybridization.
cord-337998-08tknscm	2016	Selective 2′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3′ stem-loop (nucleotides 1868–1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. 3E-5E-GFP minigenome RNA secondary structure and host protein interactions were examined using selective 2 -hydroxyl acylation analyzed by primer extension (SHAPE) (38, 39) , antisense-interfered SHAPE (aiSHAPE) (40) , electrophoretic mobility shift assays (EMSA), siRNA, and mutational analysis, using both the 3E-5E-GFP minigenome system and EBOV reverse genetics. The secondary structure of the EBOV 3E-5E-GFP minigenome RNA predicted by RNAstructure software version 5.7 (48) and chemical probing data from SHAPE were used to generate 10 three-dimensional (3D) models for the trailer-to-leader panhandle interaction in the wt-EBOV genome and variants, A30U and A26U/A30U, using open-source RNAComposer, version 1.0 (http://rnacomposer.cs.put.poznan.pl/).
cord-338083-77re4l0w	2005	The genetic diversity that occurs among isolates of BVDV is characteristic of RNA viruses that exist in nature as quasispecies (a swarm of viral mutants). However, altered base sequence in this region of the viral genome has been identified after passage of the virus in cell culture, and has been detected in viral RNA that was extracted from tissues of an infected animal [20, 21] . The selection of the antigenic variants likely occurred during the acute infection of the dams of those PI cattle and resulted in transplacental transmission of slightly different BVDV to a group of fetuses. Genetic and antigenic variability in bovine viral diarrhea virus (BVDV) isolates from Belgium Pathogenesis of primary respiratory disease induced by isolates from a new genetic cluster of bovine viral diarrhea virus type I Clinical and immunologic responses of vaccinated and unvaccinated calves to infection with a virulent type-II isolate of bovine viral diarrhea virus
cord-338307-vfutmwxq	1983	The pattern of three major structural proteins and their organization in the virion as shown for MHV-A59 in Fig. 3 is generally applicable to most other species of coronaviruses (reviewed in detail by Siddell et al., 1982) . (1981b) , using a cDNA probe prepared against the mRNA of MHV-A59 which coded for the nucleocapsid protein, obtained evidence of 7 0 4 0 % homology by analysis of hybridization kinetics of viral RNA from cells infected with MHV-1, -3, -S, and -JHM. t 1980) demonstrated that different size classes of poly(A)containing intracellular MHV-JHM RNAs fractionated on sucroseformamide gradients directed the synthesis of different structural proteins in cell-free translation systems. The studies showed that, like avian coronaviruses, for the murine coronavirus MHV-A59, the oligonucleotides of each of the subgenomic RNAs were included within the next larger species, starting from the 3'' end of the genome.
cord-338345-mr1orklo	2016	Unless stated otherwise, for this specific experiment, we used the 5=-RNA-20 partially duplex substrate (see Fig. S1 in the supplemental material), since previous coronavirus helicase, i.e., SARS-CoV nsp13, was shown to have a 5=-to-3= directionality (29) . To determine whether nucleic acid unwinding by M-nsp13 depends on the loading strand length and to determine the minimum length of the 5= singlestranded overhang required for efficient helicase activity, we designed five partially duplex RNA substrates with single-stranded 5= ends of various lengths (between 0 and 20 bases) (see Fig. S1 in the supplemental material; Fig. 6 ) but with dsRNA duplex regions of the same length. Overall, these results suggest that M-nsp13''s ability to unwind the 5=-RNA-20 partially duplex substrate is likely due to residual cellular ATP purified with the helicase as well as to a long overhang length, which enhances M-nsp13''s unwinding activity.
cord-338358-ppjxo2di	2016	title: Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs Pairs of CRISPR guide RNAs that flanked the start codon and polyadenylated Cas9 were co-injected into the cytoplasm of zygotes and cultured in vitro to the blastocyst stage. In a previous study, CRISPR/Cas9 RNA injection of pig zygotes resulted in 100 % of the piglets having biallelic DNA edits of the targeted CD163 or CD1D gene (Whitworth et al. Zygote injection with CRISPR/Cas9 guide RNA was used to efficiently (100 %) create pigs with a biallelic edit of the TMPRSS2 gene for use as a biomedical model (Fig. 2e, f) . The sex ratio measured in the resulting blastocysts and piglets was not significantly affected by in vitro culture or CRISPR/Cas9 RNA injection.
cord-338582-o976nab9	2010	Genome sequencing has allowed efficient, sensitive, and specific diagnostic assays to be developed based on the detection of nucleic acids. PCR uses the highly specific molecular recognition ability of Watson-Crick base pairing to provide the selectivity needed for a nucleic acid probe to bind to a targeted DNA sequence and allow for its exponential amplification. It has been used to develop rapid diagnostic tests for several pathogenic viruses with singlestranded RNA genomes, including influenza A, 13 footand-mouth disease virus, 14 and severe acute respiratory syndrome (SARS)-associated coronavirus. DNA microarrays also permit relatively rapid interrogation of a clinical sample against thousands of genetic targets, allowing for simultaneous detection and discrimination among hundreds of pathogenic agents of veterinary interest. Unlike PCR technology where the target agent must be known to use specific test primers, microarrays can allow for the rapid diagnosis of multiple pathogenic agents in disease outbreaks and epidemics of unknown etiology.
cord-338680-wwlttymp	2020	Due to the high prevalence of infection during the peak of the outbreak, one of the suggested strategies to prevent healthcare transmission was to screen all patients on admission by a single combined nose and throat swab assessed for SARS-CoV-2 RNA to allow segregation into COVID-19 positive and non COVID-19 cohort wards. The latter included assessment of the utility of a single combined throat and nose swab (CTNS) for patient placement, delayed RNA positivity, COVID-19 patients as sources of infection, self-reported COVID-19 sickness absence among hospital staff hospital bed occupancy, community incidence, and the incidence of other significant hospital-acquired infections. NHS England released its reporting criteria in May 2020 (written communication described in supplementary data) following which cases were also classified as per date of the SARS-CoV-2 RNA detection. Correlation between weekly incidence of HA-COVID-19 (including late indeterminate cases) and staff self-reported sickness absence, delayed RNA positive cases, community incidence and Trust COVID-19 bed occupancy is displayed in figure 3.
cord-338727-1kodz527	2014	Many ribonucleases (RNases) are able to inhibit the reproduction of viruses in infected cell cultures and laboratory animals, but the molecular mechanisms of their antiviral activity remain unclear. Therefore, the formation of RNA fragments enhanced by RNase L, followed by their interaction with RIG I and MDA5, activates transcription factor NF κB and triggers transcription of interferon β gene, which prevents virus replication and stimulates the growth of immune system cells [9] . Previously, onconase, an RNases from oocytes of the leopard frog Rana pipiens, efficiently suppresses the replication of HIV 1 due to the selective degrada tion of viral RNA, which exhibits no pronounced cytotoxic effect on infected human cells [22] . At the first stage, when binase meets the virus outside cell, its catalytic activity is not inhibited by the natural RNase and it may destroy viral RNA (Fig. 3, C) . Ribonucleases in HIV type 1 inhibition: Effect of recombinant RNases on infection of primary T cells and immune activation induced RNase gene and protein expression
cord-338812-q24jycgk	2011	At present, we know that the nucleus contains the chromatin territories that mainly consist of DNA-protein complexes, as well as several distinct interchromosomal compartments (bodies) that support molecular activities, including replication, transcription and processing of nucleic acids (Fig. 1) . For many years the classical view of these components focused on their crucial role for the biogenesis of ribosomal subunits, but proteomic analysis of human nucleoli showed the presence of about 4500 nucleolar proteins with different functions, thus revealing the multifunctional nature of the nucleolus (Ahmad et al., 2009) . The previous decade of extensive studies implicated PML NBs in stress response (Maul et al., 1995) , gene regulation (Zhong et al., 2000) , oncogenesis (Salomoni and Pandolfi, 2002) , cell senescence (Bischof et al., 2002) , DNA damage repair (Dellaire and Bazett-Jones, 2004) , apoptosis (Takahashi et al., 2004) as well as in antiviral defence mechanisms (Everett and Chelbi-Alix, 2007) .
cord-338901-1kzy7rts	2020	According to the information that we have collected so far, this article provides an overview of potential therapeutic drugs and compounds with much attention, including favipiravir and hydroxychloroquine, as well as traditional Chinese medicine, which have been reported with good clinical treatment effects. In these 155 pooled clinical trials, a number of approved chemical and biomacromolecule drugs have been used in COVID-19 treatment clinical trials for drug repurposing, most of which are nucleotide analogs and protease inhibitors against other viral pathogens, including influenza virus, HIV and HCV. In vitro studies have shown that lopinavir/ritonavir can inhibit the replication of MERS-CoV and SARS-CoV and exert antiviral effects [22] [23] [24] [25] . In the latest "Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia", it is recommended to use ribavirin at a dose of 500 mg each time for adults and in combination with interferon or lopinavir/ritonavir, with 2-3 intravenous infusions daily. In vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment acute respiratory syndrome Coronavirus 2 (SARS-CoV-2)
cord-339172-210dwhgj	2008	Specific þRNA virus replicase subunits are targeted to the membranes of particular cell organelles that are subsequently modified into characteristic structures with which viral RNA synthesis is associated. We used electron microscopy and tomography for the three-dimensional imaging of the membrane alterations induced by severe acute respiratory syndrome (SARS)-coronavirus, a member of the virus group with the largest RNA genome known to date. The lumen of this unique membrane network contains numerous large (diameter 250-300 nm) ''''inner vesicles,'''' which were formerly thought to reside in isolated DMVs. Intriguingly, although the interior of these vesicles does not appear to be connected to the cytosol, it labels abundantly for double-stranded RNA, which presumably is present at the site of viral RNA synthesis. In some of our images, the SARS-CoV-induced CM appeared to be continuous with both DMV outer membranes ( Figure 2D ; inset) and ER cisternae, suggesting a link to the viral RTC also in coronaviruses.
cord-339209-oe8onyr9	2014	The organization of each genome was similar to that described previously for the mesoniviruses (NDiV, CavV, HanaV, NseV and MenoV), featuring a long 5''-untranslated region (5''-UTR) of 359 to 370 nt, six major long open reading frames (ORFs), and a long terminal region of 1780 to 1804 nt preceding the poly[A] tail ( Figure 2 ). To determine the phylogenetic relationships of the newly identified insect viruses, maximum likelihood (ML) phylogenetic trees were constructed based on the amino acid alignments of ORF2a (unprocessed S protein) and a concatenated region of the highly conserved domains within ORF1ab (3CL pro , RdRp and ZnHel1). A Clustal X alignment of the mesonivirus ORF3a proteins and individual structural analyses using SignalP and TMHMM and NetNGlyc (www.expasy.org) indicated that each is a class I transmembrane glycoprotein with a predicted N-termimal signal peptide, an ectodomain containing a conserved set of 6 cysteine residues and a single conserved N-glycosylation site, a transmembrane domain and a C-terminal cytoplasmic domain ( Figure 4A, 4D) .
cord-339288-y8woqsii	2016	Self-replicating RNA derived from the genomes of positive strand RNA viruses represents a powerful tool for both molecular studies on virus biology and approaches to novel safe and effective vaccines. Three years later, the performance of poliovirus cDNA clones could be significantly ameliorated through the introduction of SV40 transcription and replication signals and transfection of the resulting construct into cells expressing the SV40 large T antigen [14] , thus ensuring replication of the DNA-plasmid in eukaryotic cells leading to a higher yield of viral RNA and recovered virus (Fig. 2, left part) . The resulting virus CP7_E2alf was only able to infect pigs and thus displayed the Fig. 3 Generation of a chimeric viral genome from two parental RNAs. On the level of a cDNA construct, one protein-coding sequence is replaced by the corresponding gene of the other virus (principle used for the pestivirus vaccine CP7_E2alf [58] ).
cord-339431-kyr5lv15	2020	In the case of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are several mechanisms that would make miRNAs impact the virus, like interfering with replication, translation and even modulating the host expression. In this study, we performed a machine learning based miRNA prediction analysis for the SARS-CoV-2 genome to identify miRNA-like hairpins and searched for potential miRNA – based interactions between the viral miRNAs and human genes and human miRNAs and viral genes. Although there are studies regarding to the viral replication and their interaction with host innate immune system, the role of miRNA-mediated RNA-silencing in SARS-CoV-2 infection has not been enlightened yet. In this study, SARS-CoV-2 genome was searched for miRNA-like sequences and potential host-virus interactions based on miRNA actions were analyzed. In our study, we have also identified possible miRNA like small RNAs from SARS-CoV-2 genome which target important human genes.
cord-339456-82iks0xf	2015	title: Methods for Preparation of MS2 Phage-Like Particles and Their Utilization as Process Control Viruses in RT-PCR and qRT-PCR Detection of RNA Viruses From Food Matrices and Clinical Specimens The technology for production of MS2 phage-like particles is theoretically well established, uses the knowledge gained from the study of the familiar bacteriophage MS2 and utilizes many different approaches for the construction of the various process control viruses. Also MS2 phage-like particles have been used as the process control virus for the detection of pathogenic RNA viruses in clinical samples. prepared MS2 phage-like particles, also called armored RNA (aRNA), that carried the consensus RNA sequence from human immunodeficiency virus type 1 (HIV-1) packaged in the capsid which can serve as quantitative standard in detection of HIV-1 (Pasloske et al. MS2 phage-like particles carrying the plant-specific ribulose-1,5-bisphosphate carboxyl small subunit (rbcs) gene fragment were used as the process control virus in qRT-PCR detection of severe acute respiratory syndrome coronavirus (SARS-CoV) .
cord-339782-rybjc58j	2020	The study evidenced that most patients tested positive for more than two weeks and that persistence of viral RNA is not necessarily associated with severe disease but may result from a weaker immune response instead. In men, the first negative test took 24 ± 9 days (range: 7 -46) and in women it took 25 ± 9 days (range: 9 -44), P>0.05, In an attempt to understand why some patients maintained positive tests for longer, we correlated the detection of SARS-CoV-2 RNA with the host immune response to virus infection. The lack of information regarding persistence of virus RNA and infectivity, disease severity and immune response, supports the current guidance of viral clearance confirmation prior to patient transference out of dedicated COVID-19 wards and of ending isolation in mild illness patients.
cord-339976-tg2jkss7	2004	title: Detection and Monitoring of SARS Coronavirus in the Plasma and Peripheral Blood Lymphocytes of Patients with Severe Acute Respiratory Syndrome Reliable and sensitive determination of the SARS CoV load would aid in the early identification of infected individuals, provide guidance for treatment (especially the use of steroid hormones and antiviral agents), and aid in monitoring of a patient''s clinical course and outcome. The method could detect the CoV load during the SARS course, as demonstrated in Fig. 1B , representative data from the 44 patients tested. High frequency of point mutations clustered within the adenosine triphosphatebinding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance Serial analysis of the plasma concentration of SARS coronavirus RNA in pediatric patients with severe acute respiratory syndrome Quantitative analysis and prognostic implication of SARS coronavirus RNA in the plasma and serum of patients with severe acute respiratory syndrome
cord-340046-kgbvld0y	2011	BACKGROUND: Exhaled breath condensate (EBC) sampling has been considered an inventive and novel method for the isolation of respiratory viruses. RESULTS: Viral screening resulted in the detection of 4 different viruses in EBC and/or nasal swabs: Rhinovirus, Human Respiratory Syncytial Virus B, Influenza A and Influenza B. This observation has created a growing interest in the use of EBC as a new sampling method for the screening of respiratory viruses infecting the upper airways. The aim of this study was to investigate whether the EBC collection method was suited for the efficient condensation of aerosolised virus particles during normal breathing and to explore the isolation of respiratory viruses in the condensate. In this study, 102 EBCs were collected from otherwise healthy volunteers showing respiratory or flu-like symptoms (defined in Table 1 ), using a commercially available condenser (RTube™, Respiratory Research Inc., Charlottesville, Virginia, USA). Collection of exhaled breath condensates is a novel and non-invasive method for obtaining samples of the upper respiratory tract.
cord-340189-jo38hjqa	2020	If you are infectious for 4 days, then you will infect four others on average, which is on the high end of the R 0 values for SARS-CoV-2 in the absence of physical distancing. Assuming entry of the virus to the cells is rapid (we estimate 10 min for SARS-CoV-2), the time it takes to produce progeny can be estimated by quantifying the lag between inoculation and the appearance of new intracellular virions, also known as the ''eclipse period''. While both the time to complete a replication cycle and the burst size may vary significantly in an animal host due to factors including the type of cell infected or the action of the immune system, these numbers provide us with an approximate quantitative view of the viral life-cycle at the cellular level. (Hirano et al., 1976) : "The average per-cell yield of active virus was estimated to be about 6-7 Â 10 2 plaque-forming units." This data is for MHV, so more research is needed to verify these values for SARS-CoV-2.
cord-340325-0oh40b6r	2020	
cord-340422-8f5xe4zc	2001	Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to a group of RNA viruses that establish persistent infections. IFN-γ mRNA expression was observed in the lymph nodes and lungs of pigs infected with wild-type PRRSV strain SDSU-23983. The purpose of this study was to evaluate the production of IFN-␥ in pigs following infection with PRRSV and to characterize the mechanism of IFN-␥ action following infection of MARC-145 cells with wild-type and culture adapted PRRSV isolates. The results show that IFN-␥ efficiently inhibits PRRSV replication in MARC-145 cells, and at least part of the antiviral activity may be through PKR. IFN-␥ mRNA expression was detected in the lymph nodes from two of the 3 pigs infected with the wild-type P6 virus (Fig. 1) . Consistent with other reports describing the presence of cell-mediated immunity during PRRSV infection [4, 31] , we observed IFN-␥ mRNA expression in lymph nodes and lungs of PRRSV-infected pigs (Fig. 1) . IFN gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages
cord-340423-f8ab7413	2016	
cord-340475-h0q1m3ed	2014	Further validation showed that ISR2, 8, and 12 expression mimics that of their neighboring genes GBP1, IRF1, and IL6, respectively, all related to the IFN response.These genes are induced in response to different doses of IFNα2 in different cell lines at early (ISR2 or 8) or later (ISR12) time points. HuH7 cells were treated for 6, 24, 48, or 72 h with increasing doses of IFNα2 up to 10,000 units/ml, and the expression levels of GBP1, IRF1, BST2, OAS, IL6, and ISG15 were evaluated by qRT-PCR (Figure 1) .
cord-340489-yo3cp5vs	2008	Die Wirksamkeit von BVDU bei VZV-Infektionen (Varizellen und Zoster) immunkompromittierter Patienten ist durchaus sehr gut und vergleichbar der von i.v. verabreichtem Aciclovir, jedoch fällt die Nutzen-Risiko-Betrachtung insgesamt auch bei VZV-Therapie zu Gunsten von Aciclovir aus, da BVDU eher mutagen zu sein scheint und nicht zusammen mit 5-Fluorouracil (Zytostatikum) gegeben werden darf. In klinischen Studien konnte durch Anwendung von ACV bei EBV-Infektionen auch die Virusausscheidung deutlich vermindert werden, ein wesentlicher Einfluss auf den Krankheitsverlauf ließ sich nicht erreichen. Typisch für viele opportunistische Erreger ist, dass sie weit verbreitet sind und nach einer Primärinfektion, die bereits vor der HIV-Infektion stattfindet, zu latenten Infektionen führen. Die Prophylaxe von Infektionen bereits vor deren erstem Auftreten (Primärprophylaxe) oder nach der ersten Episode (Sekundärprophylaxe) ist weiterhin eine wichtige Aufgabe bei der Betreuung HIV-positiver Patienten, auch wenn opportunistische Infektionen durch die antiretrovirale Therapie insgesamt seltener geworden sind.
cord-340554-7cwp2xbw	2019	
cord-341029-49360l2a	2015	
cord-341034-2oigu75k	2012	We found that the energy regulator AMPK, which potently inhibits fatty acid synthesis, restricts infection of the Bunyavirus, Rift Valley Fever Virus (RVFV), an important re-emerging arthropod-borne human pathogen for which there are no effective vaccines or therapeutics. Furthermore, we found that AMPK is activated during RVFV infection, leading to the phosphorylation and inhibition of acetyl-CoA carboxylase, the first rate-limiting enzyme in fatty acid synthesis. Consistent with a role for AMPK both in early events during viral replication and in spread as measured by plaque assay Figure 1A ), we observed an increase in viral infection at early time points before virus spread, as well as increased spread in cells lacking AMPK by monitoring the production of RVFV N protein over time by microscopy ( Figure S1A-B) . We have found that energy-mediated activation of AMPK restricts infection of the Bunyavirus Rift Valley fever virus by decreasing levels of fatty acid synthesis.
cord-341050-hnuogpqn	2016	
cord-341071-nwrl1qws	2020	
cord-341171-s8vkgdhf	2020	
cord-341176-83khavoh	2020	
cord-341321-paucodwz	2008	
cord-341324-f9g9gitn	2020	This includes for instance cooperating in PRR recognition of viral PAMPs, stabilizing signaling complexes to improve their resistance to degradation, stopping virus entry, blocking viral capsid formation, impairing trafficking and budding of virions from the infected cells, but also modulating the IFN response to avoid the toxicity of these potent immune mediators. The phosphorylated STAT1/STAT2 heterodimer associates with interferon regulatory factor 9 (IRF9) to form the transcriptional factor complex ISGF3, which translocate to the nucleus and binds the IFN-response elements (ISRE) in ISG promoters leading to the expression of ISG products [36] (Fig. 2 The oligoadenylate synthetase (OAS)-latent RNase (RNase L) pathway is another IFN-inducible pathway that provides the cell with an effector mechanism upon recognition of viral dsRNA (reviewed in [44] ).
cord-341330-31ngknq4	2020	
cord-341342-kyavg4vu	1992	The RNase sensitivity of the ability of N protein to migrate into nondenaturing gels was not due to possible contaminating protease activity in the RNase preparations, since the same RNase-treated samples of translated N 148 P.S. Masters protein showed no degradation when analyzed by SDS-PAGE (Fig. 3, lanes an, lower) . Since the N mRNAs synthesized from the transcription vectors shown in Fig. 1 contained this portion of the leader sequence, it seemed possible that the N-RNA complex observed in nondenaturing PAGE was that of translated N protein binding to its own mRNA. That the observed complex was not due to translated N protein binding to the leader portion of its own mRNA was also supported by the observation that full-length N protein translated from a construct in which the MHV leader sequence was entirely deleted was also able to migrate into nondenaturing gels (data not shown).
cord-341502-jlzufa28	2020	The second pool of 275 oligos ("Probe II") covers the remaining region (21563:29872, NC_045512.2) which is shared by both the gRNA and sgRNAs. To first check whether our method specifically captures the viral RNP complexes, we compared the resulting purification from Vero cells infected with SARS-CoV-2 (BetaCoV/Korea/KCDC03/2020) at MOI 0.1 for 24 hours (Kim et al., 2020b ) by either Probe I or Probe II. In combination, we define these 109 proteins as the "SARS-CoV-2 RNA interactome." 37 host proteins such as CSDE1 (Unr), EIF4H, FUBP3, G3BP2, PABPC1, ZC3HAV1 were enriched in both the Probe I and Probe II RNP capture experiments on infected cells ( Figure 1F ), thus identifying a robust set of the "core SARS-CoV-2 RNA interactome." Gene ontology (GO) term enrichment analysis revealed that these host factors are involved in RNA stability control, mRNA function, and viral process ( Figure S1F ). To measure the impact of these host proteins on coronavirus RNAs, we conducted knockdown experiments and infected Calu-3 cells with SARS-CoV-2 ( Figure 5A and 5B).
cord-341513-e6p3lrlf	2017	title: Microarray Analysis Identifies the Potential Role of Long Non-Coding RNA in Regulating Neuroinflammation during Japanese Encephalitis Virus Infection To determine the role of lncRNAs in inflammatory cytokine production, the cells were transfected with siE52329, siN54010 or non-specific control siRNA, and then infected with JEV. To examine the role of lncRNA E52329 and N54010 in regulating the kinase activity of MKK4/JNK pathway, BV2 cells were transfected with siE52329, siN54010 or non-specific control siRNA, and then infected with JEV. The results of our study reveal the first experimental evidence demonstrating the complex regulation of lncRNAs by JEV infection in mice brain and microglial cells. Third, the integration of microarray platform, quantitative real-time PCR, GO analysis, pathways analysis, and lncRNA-mRNA coexpression network analysis has allowed us to conduct an active comparative genomics and bioinformatics study to reveal host lncRNAs expression patterns associated with JEV infection.
cord-341541-3l6tjf3t	2020	title: Molecular characterization of infectious bronchitis virus based on RNA‐dependent RNA polymerase gene Extensive rate of variations in in spike glycoprotein subunit gene of infectious bronchitis virus (IBV) caused challenges for counting variants for differentiation of infected from vaccinated birds and addressing the variants of unknown significance. The study aimed at investigating the possibility use of RNA‐dependent RNA polymerase gene (RdRp) as a target for molecular characterization of IBV strains in Iran. Phylogenetic analysis of RdRp gene sequences resulted in clustering the IBV strains related to each area. Using RdRp, as a genetic marker eliminates the challenges arise from the enormous variations that making difficult the discrimination between field and vaccine strains as well as affiliation of certain variants to various geographical areas. Based on the RT-PCR detection and sequence analysis of IBV RdRp gene, an overall prevalence of field strains estimated as 44.2%.
cord-341804-rnj3wtg4	2020	This article reviewed the clinical use, mechanism and efficacy of the clinically approved drugs recommended in the Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (DTPNCP) released by National Health Commission of P.R.China, and the novel therapeutic agents now undergoing clinical trials approved by China National Medical Products Administration (NMPA) to evaluate experimental treatment for COVID-19. However, more evidence is needed either for 4 supporting or opposing the systemic therapeutic administration of glucocorticoids in 5 patients with SARS-CoV-2 infection (Qin et al., 2020 a variety of immune cells 20 and improves the immunity, while IFN-β takes effect by inhibiting the adsorption of certain 1 viruses, enhancing phagocytosis of natural killer cells and mononuclear macrophages Tocilizumab is a recombinant humanized anti-IL-6 receptor (IL-6R) monoclonal antibody, 21 13 which can specifically bind to soluble and membrane-bound IL-6 receptors and inhibit 1 signal transduction mediated by IL-6, thereby reducing inflammation and blocking cytokine 2 storm caused by COVID-19 (Scheinecker et al., 2009) .
cord-342117-r2chpw7y	2010	The antiviral effect of siRNA was evaluated by measurement of cell survival rate using the MTT method and viral RNA was quantitated with real-time RT-PCR. CONCLUSIONS: Our data showed that synthetic siRNA against the DEN-1 membrane glycoprotein precursor gene effectively inhibited DEN-1 viral RNA replication and increased C6/36 cell survival rate. Therefore, in this study, we designed and synthesized 21 nt siRNAs against the DEN-I viral PrM gene, and investigated their inhibition effects of dengue virus replication in transfected C6/36 cells. These data indicate that siRNA against DEN viral genome can effectively inhibit viral RNA replication in the C6/36 cells, protect host cell from viral attack, suggesting its potential role in prevention and treatment of dengue fever. Our data showed that synthetic siRNA against the DEN membrane glycoprotein precursor gene effectively inhibited DEN viral RNA replication and increased C6/36 cell survival rate. Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference
cord-342145-cq6xe5r7	2020	The SARS-CoV-2 diagnostic pipeline that has proven to be successful and that is currently used in many test centers consists of three steps: collecting nasopharyngeal or oropharyngeal swab specimens, isolation of total RNA, and specific detection of the viral genome by RT-qPCR. During the early phase of the COVID-19 pandemic (early March 2020) in Germany, we tested the sensitivity and specificity of a colorimetric RT-LAMP assay for detecting SARS-CoV-2 RNA in clinical RNA samples isolated from pharyngeal swab specimens collected from individuals being tested for COVID-19 (and provided by the Heidelberg University Hospital''s diagnostic laboratory after removal of an aliquot for SARS-CoV-2 RNA testing by RT-qPCR) (fig. For samples with a CT ≤ 30 as measured by RT-qPCR with E-Sarbeco primers, we found overall satisfactory sensitivity and specificity values for SARS-CoV-2 RNA detection by the RT-LAMP assay using RNA samples isolated from pharyngeal swab specimens ( Fig. 3 and Table 1 ).
cord-342189-ya05m58o	2020	Here, we comprehensively define the interactions between each SARS-CoV-2 protein and human RNAs. We show that 10 viral proteins form highly specific interactions with mRNAs or ncRNAs, including those involved in progressive steps of host cell protein production. We show J o u r n a l P r e -p r o o f 5 that NSP16 binds to the mRNA recognition domains of the U1 and U2 RNA components of the spliceosome and acts to suppress global mRNA splicing in SARS-CoV-2-infected human cells. We identified several pathogenic functions of SARS-CoV-2 in human cells -including global inhibition of host mRNA splicing, protein translation, and membrane protein trafficking -and described the molecular mechanisms by which the virus acts to disrupt these essential cell processes.
cord-342344-jjnf4yje	2020	Diagnostic assays for the presence of SARS-CoV-2 currently use real-time reverse transcriptase PCR (RT-qPCR) to yield a binary (positive or negative) result based on an amplification cycle threshold (Ct) value 9-12 . Current PCR-based assays can detect the presence of very short SARS-CoV-2 RNA sequences but do not distinguish whether these sequences are derived from longer molecules present in the sample at the time of collection. To address these issues, we explored using droplet digital PCR (ddPCR) 19, 20 to more precisely quantify SARS-CoV-2 RNA in biological samples and evaluate the extent to which positive results reflect larger, intact viral nucleic acids. The results yielded definitive evidence of linkage: although only 822 of the 12,220 droplets (6.7%) were positive for either N1 or N2, 75% of the droplets that were positive for N2 (HEX) were also positive for N1 (FAM) (Fig. 2a, Table 1 ); we estimate (using a formula we previously described 21 , which accounts for chance co-encapsulation) that 71% of the detected RNA sequences were physically linked.
cord-342412-azkamnpa	2005	This paper focuses on the information in the database for pathogens that impact global public health, emerging infectious organisms, and bioterrorist threat agents. For example, the Centers for Disease Control and Prevention (CDC) maintains an ever-changing list of notifiable diseases, the National Institute of Allergy and Infectious Disease (NIAID) lists agents with potential for use in bioterrorist attacks, and the Department of Health and Human Services (HHS) maintains a list of critical human pathogens. This article focuses on the information in the database for pathogens that impact global public health, emerging infectious organisms, and bioterrorist threat agents. It provides a compilation of lists, taken from the database, of important and/or regulated biological agents from a number of agencies including HHS, the United States Department of Agriculture (USDA), the CDC, the World Health Organization (WHO), the NIAID, and other sources.
cord-342456-5gp3cry0	2020	Utilizing expression patterns of SARS-CoV-2-infected cells, we identified a region in gene expression space that was unique to virus infection and inversely proportional to the transcriptional footprint of known compounds characterized in the Library of Integrated Network-based Cellular Signatures. These signatures were then used as queries against the LINCS L1000 dataset, a collection of gene expression profiles generated following the administration of >20,000 bioactive compounds including >1,000 FDA-approved drugs to human cell lines at a variety of different times and concentrations (Subramanian et al., 2017) With L1000FWD , we could identify reciprocal transcriptional signatures generated between SARS-CoV-2 infection and a given compound. Overall, based on the L1000 data, these seven compounds influence the same pharmacological high-dimensional gene expression signature space and are predicted to disrupt key cellular processes that are modulated in response to SARS-CoV-2 infection.
cord-342634-4ouhdjsr	2010	In brief, the group of proteins with RNA chaperone activity includes proteins that, first, open up misfolded structures without requirement of ATP and that, second, are dispensable once the RNA has been folded. coli showed that 1/3 of the tested proteins possesses strong RNA chaperone activity in vitro in the trans-splicing assay [21] . E. coli contains nine members of the csp family and CspA, the major cold-shock protein and CspE were identified to interact non-specifically with RNA molecules and to possess nucleic acid melting activities [44] [45] [46] . Later, a detailed study on possible functions of Ro RNPs, which are Ro ribonucleoprotein complexes, composed of a small noncoding cytoplasmic RNA, termed Y RNA and its protein partners was conducted: besides the permanently associated proteins Ro60 and La, subpopulations of Ro-RNPs also contain hnRNP I and hnRNP K, both of which exhibited strong RNA chaperone activity in vitro in the transsplicing and the cis-splicing assay [22] .
cord-342649-ysossker	2012	The relationship between viral load, disease severity and antiviral immune activation in infants suffering from respiratory syncytial virus (RSV)-associated bronchiolitis has not been well identified. The main objective of this study was to determine the existence of a correlation between RSV load and disease severity and also between different clinical markers and mRNA levels of the interferon stimulated gene (ISG)56 in infants hospitalized for bronchiolitis. Results indicated that viral load was positively related to the clinical severity of bronchiolitis, the length of hospital stay, the levels of glycemia and the relative gene expression of ISG56, whereas an inverse correlation was observed with the levels of hemoglobin. In the framework of a study aimed at understanding the pathogenesis of RSV infection and at further characterizing viral and host factors involved in determining the severity of bronchiolitis, we addressed whether any diVerences in RSV-RNA levels in the airway tracts of infants with bronchiolitis might explain the broad clinical spectrum of RSVassociated bronchiolitis.
cord-342653-bpyc2gbl	2020	While E3 ligases are often thought to negatively regulate the stability of the target molecule by Ub-mediated proteasomal targeting, many TRIMs have been shown to enhance innate immune signaling pathways [15] , through both proteasome-dependent and -independent mechanisms. The study of RIG-I and RIPLET interaction provides a detailed example of how TRIM-like proteins utilize bivalency and CC for regulating substrate selectivity, higher-order oligomerization and innate immune function. Given that an increasing number of receptors and signaling molecules in the innate immune system are shown to multimerize upon activation [77] , it is tempting to speculate that TRIM/TRIM-like proteins may utilize multimer-specific substrate recognition as a common mechanism for regulating their immune functions. The avidity-driven substrate recognition mechanism of TRIM/TRIM-like proteins would thus ensure more precise control of innate immune signaling and restriction functions.
cord-342676-ykog278j	2016	To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We wished to investigate the possibility that similar specific secondary structures are present within HCV RNAs destined for packaging, and whether their interactions with Core cooperatively drive the RNA encapsidation and nucleocapsid assembly processes. A mutant genome unable to form such structures displays impaired viral infectivity whilst RNA replication is unaffected, indicating these motifs may interact specifically with Core. Role of RNA structures in genome terminal sequences of the hepatitis C virus for replication and assembly
cord-342681-pqzcy9wu	2020	Among 11 patients in Thailand infected with severe acute respiratory syndrome coronavirus 2, we detected viral RNA in upper respiratory specimens a median of 14 days after illness onset and 9 days after fever resolution. During the study period, Thailand''s discharge criteria for hospitalized COV-ID-19 patients required resolution of clinical signs and symptoms and 2 respiratory specimens without detectable SARS-CoV-2 RNA collected >24 hours apart. Clinical resolution occurred a median of 12 (9-13.5 ) days after illness onset, and these patients had detectable SARS-CoV-2 RNA in upper respiratory tract specimens for a median of 14 (9-26) days after illness onset (Table 2) . However, patients became afebrile 6 days after illness onset, with a median of 9 (3-19.75 ) additional days of detectable SARS-CoV-2 RNA in respiratory specimens after resolution of fever ( Table 2 ). Other studies have described asymptomatic patients with upper respiratory specimens positive for SARS-CoV-2 (9), and evidence suggests such cases pose a risk for transmission (10) (11) (12) .
cord-342756-rgm9ffpk	2020	Here, we aimed at presenting a critical view of ongoing drug repurposing efforts for COVID-19 as well as discussing opportunities for development of new treatments based on current knowledge of the mechanism of infection and potential targets within. In the following topic, we will review SARS-CoV-2 structure and mechanism of infection in order to discuss molecular targets from the virus or its human host that are being considered for drug repurposing and perhaps future development of new drugs. (128) Its role as a functional receptor of SARS-CoV-2 S protein in host cells makes this protein a potential drug target to treat COVID-19. (138) TMPRSS2 has a major role in SARS-CoV-2 cell entry and replication, and thus represents an interesting therapeutic target since its inhibitors could potentially block virus infection in its initial stages. (199) A robust preclinical drug discovery pipeline comprising in vitro, and in vivo models of SARS-CoV-2 infection is particularly important to identify new antivirals for human COVID-19 treatment.
cord-342800-62jklwiy	2020	The RNActive ® vaccine platform designed by CureVac used co-delivered RNA and protamine complex as the adjuvant to induce Th1 T cell responses, and naked, unmodified, and sequence-optimized mRNA as the antigen to develop mRNA vaccines [54] . used LNP to deliver self-amplified RNA vaccines, which caused the mRNA expression level in mice to be significantly higher than that of naked mRNA, CD4 + , and CD8 + T cell immune responses were also effectively induced. Overall development steps of those vaccines are (1) constructing the core antigen-encoding mRNA sequence optimized or combined based on selected antigen(s) from the target pathogen; (2) trying and choosing a proper combination of mRNA construction type, adjuvants, carrier materials and the route of administration; (3) detecting in vivo expression of the encoded antigen and the level of elicited immune responses; (4) providing research and demonstrations of immune induction mechanisms.
cord-342901-ca2xxkb2	2015	Though PTB was the first, a wide spectrum of factors, largely nuclear RBPs, have also been proposed as poliovirus ITAFs, including Lupus autoantigen (La) (Meerovitch et al., 1993 (Meerovitch et al., , 1989 , poly(rC)binding proteins (PCBPs) (Blyn et al., 1996; Gamarnik and Andino, 1997) , upstream of N-ras (UNR) (Anderson et al., 2007; Boussadia et al., 2003; Hunt et al., 1999) , SRp20 (Bedard et al., 2007) and glycyl-tRNA synthetase (GARS) (Andreev et al., 2012) (Table 1 ). In addition, certain ITAFs play crucial roles in regulating the conversion of translation-competent genomic RNAs into replicationcompetent RNAs. PTB was known as a nuclear splicing factor when its role as an ITAF of poliovirus and EMCV was discovered; a classic hijacked nuclear protein pressed into a new role required for the virus.
cord-342902-y1v8wzxq	2020	Here, we show that clofazimine, an anti-leprosy drug with a favorable safety and pharmacokinetics profile, possesses pan-coronaviral inhibitory activity, and can antagonize SARS-CoV-2 replication in multiple in vitro systems, including the human embryonic stem cell-derived cardiomyocytes and ex vivo lung cultures. In a hamster model of SARS-CoV-2 pathogenesis, prophylactic or therapeutic administration of clofazimine significantly reduced viral load in the lung and fecal viral shedding, and also prevented cytokine storm associated with viral infection. Since clofazimine is orally bioavailable and has a comparatively low manufacturing cost, it is an attractive clinical candidate for outpatient treatment and remdesivir-based combinatorial therapy for hospitalized COVID-19 patients, particularly in developing countries. We found that co-application of clofazimine and remdesivir impacts SARS-CoV-2 replication in a manner that extends beyond the additive combinatorial activity predicted by the Bliss independence model (maximal Bliss Synergy Score of 44.28; Figure 5a , Extended Data Figure 2) , and indicates these two drugs harbor a synergistic antiviral relationship.
cord-343221-e29of29o	2017	Specifically, we show that genetically engineered mutants of mouse hepatitis virus (MHV) and human coronavirus 229E (HCoV-229E), respectively, that encode an EndoU active-site substitution known to abolish nucleolytic activity, were severely attenuated and elicited an immediate and simultaneous activation of host cell dsRNA sensors. The severe attenuation of MHV H277A and HCoV-229E H250A replication in wild-type murine and human macrophages, respectively, precluded the use of primary macrophages to assess the sensitivity of EndoU-deficient coronaviruses to IFN-I pre-treatment. Our data suggest that direct restriction of replication of EndoU-deficient coronaviruses is mediated by RNase L-mediated RNA degradation and inhibition of host cell translation through activation of PKR. Notably, also in this case Xrn1 is required to further process the de-capped mRNAs. Our data show that early during coronavirus infection the EndoU activity conceptually fulfils the same task, namely the removal of dsRNA that would otherwise trigger host cell dsRNA responses, such as IFN-β expression and activation of PKR and OAS/RNase L.
cord-343350-04e6wvov	2009	White spot syndrome virus (WSSV) infects specific hemocytes of the shrimp Penaeus merguiensis Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon Time-course and levels of apoptosis in various tissues of black tiger shrimp Penaeus monodon infected with white-spot syndrome virus Protein expression profiling of the shrimp cellular response to white spot syndrome virus infection Cloning and characterization of a caspase gene from black tiger shrimp (Penaeus monodon)-infected with white spot syndrome virus (WSSV) Immunological responses of Penaeus monodon to DNA vaccine and its efficacy to protect shrimp against white spot syndrome virus (WSSV) Multiple envelope proteins are involved in white spot syndrome virus (WSSV) infection in crayfish Identification of white spot syndrome virus (WSSV) envelope proteins involved in shrimp infection DNA fragmentation, an indicator of apoptosis, in cultured black tiger shrimp Penaeus monodon infected with white spot syndrome virus (WSSV)
cord-343448-xhm97wy2	2020	In brief, RNAi works through gene silencing, degrading the mRNA for a specific protein through the coordinated action of double-stranded RNA and a complex biochemical machinery it activates (Fig 1) . The spearhead is mRNA-1273, which encodes a prefusion-stabilized form of the spike (S) protein of SARS-CoV-2, developed at an unusually fast rate (first volunteer injected on Mach 16, only 65 days after the publication of the virus sequence) by Moderna, a biotech based in Cambridge, Massachusetts (https://www.modernatx.c om/). Multi-component proteins that would be impossible to target with other systems are accessible with RNA technology, as recently demonstrated by the generation of a multi-antigenic mRNA vaccine encoding human cytomegalovirus glycoproteins gB and pentameric complex (John et al, 2018) . CV9202, for example, is a mRNA encoding six antigens usually expressed in non-small-cell lung cancer (NSCLC), developed by CureVac, a biotech based in Tübingen, Germany (https://www.curevac.com/), in collaboration with Boehringer Ingelheim.
cord-343470-w215pzdc	2020	Eukaryotic gene expression is regulated not only by genomic enhancers and promoters, but also by covalent modifications added to both chromatin and RNAs. Whereas cellular gene expression may be either enhanced or inhibited by specific epigenetic modifications deposited on histones (in particular, histone H3), these epigenetic modifications can also repress viral gene expression, potentially functioning as a potent antiviral innate immune response in DNA virus-infected cells. First, the viral protein VP16, which is packaged into the tegument layer of incoming virions, recruits host proteins, including host-cell factor 1 (HCF-1) and octamer-binding factor (Oct-1), in order to form a complex that recruits the histone demethylases lysine-specific demethylase 1 (LSD1) and Jumonji domain 2 (JMJD2) family members as a means to remove repressive H3K9 marks from viral immediate early promoters 42 Upon entry into the cell nucleus, the DNA of many viruses initiates the replication process adjacent to subnuclear structures called pro-myelocytic leukaemia nuclear bodies (PML-NBs).
cord-343604-v986m9jd	2020	title: In silico pharmacokinetic and molecular docking studies of natural flavonoids and synthetic indole chalcones against essential proteins of SARS-CoV-2 Hence, these flavonoids and structurally similar indole chalcones derivatives were studied in silico for their pharmacokinetic properties including absorption, distribution, metabolism, excretion, toxicity (ADMET) and anti-SARS-CoV-2 properties against their proteins, namely, RNA dependent RNA polymerase (rdrp), main protease (M(pro)) and Spike (S) protein via homology modelling and docking. Functional/structural roles of amino acid residues of SARS-CoV-2 proteins and, the effect of flavonoid and indole chalcone interactions which may cause disease suppression are discussed. The in vitro anti-SARS-CoV-2 activity of these 30 compounds needs to be studied further for complete understanding and confirmation of their inhibitory potential. Coronavirus main 403 proteinase (3CLpro) structure: basis for design of anti-SARS drugs Structural basis for inhibition 675 of the RNA-dependent RNA polymerase from SARS-CoV-2 by remdesivir
cord-343632-cv3qgno3	2020	Here we report a method to identify SARS-CoV-2 (COVID-19) virus RNA from purified RNA or cell lysis using loop-mediated isothermal amplification (LAMP) using a visual, colorimetric detection. Here we describe a molecular diagnostic approach for SARS-CoV-2 RNA detection using loop-mediated isothermal amplification (LAMP) and simple visual detection of amplification for potential use in rapid, field applications. This study describes testing and validation of 5 sets LAMP primers targeting two fragments of the SARS-CoV-2 genome using short (~300bp) RNA fragments made with in vitro transcription and RNA samples from patients. https://doi.org/10.1101/2020.02.26.20028373 doi: medRxiv preprint With current diagnostic methods, e.g. RT-qPCR, purified RNA is used in the input. Although a small number of samples were tested here, the colorimetric LAMP assay enables reliable SARS-CoV-2 detection without sophisticated instrumentation, matching the RT-qPCR performance in field and point-of-care settings. /2020 In conclusion, colorimetric LAMP provides a simple, rapid method for SARS-CoV-2 RNA detection.
cord-343662-scn7b4c6	2018	CROSSalign is based on the combination of the Computational Recognition Of Secondary Structure (CROSS) algorithm to predict the RNA secondary structure profile at single-nucleotide resolution and the Dynamic Time Warping (DTW) method to align profiles of different lengths. CROSSalign, available at our webpages http://service.tartaglialab.com//new_submission/ crossalign, is based on the combination of two methods: (1) Computational Recognition Of Secondary Structure (CROSS), which is an algorithm trained on experimental data to predict RNA secondary structure profiles without sequence length restrictions and at single-nucleotide resolution (Delli Ponti et al., 2017) ; (2) the Dynamic Time Warping (DTW) algorithm to assess the similarity of two profiles of different lengths (Giorgino, 2009) . We developed the CROSSalign method based on the combination of the CROSS algorithm to predict the RNA secondary structure at single-nucleotide resolution (Delli Ponti et al., 2017) and the DTW algorithm to align profiles of different lengths (Giorgino, 2009 ).
cord-343918-5yk1j4ms	2008	For inferring phylogeny, the differences between aligned sequences of genomes and proteins are quantified and depicted in the form of a tree, in which contemporary species and their intermediate and common ancestors occupy, respectively, the terminal nodes, internal nodes, and the root. Phylogenetic analysis is used in a wide range of studies to address both applied and fundamental issues of virus research, including epidemiology, diagnostics, forensic studies, phylogeography, origin, evolution, and taxonomy of viruses. With the latter virus, poor sampling of the coronavirus diversity in the SARS-CoV lineage at the time, some uncertainty over the relationship between phylogeny and taxonomy of coronaviruses, and the complexity of phylogenetic analysis of a virus data set including isolated distant lineages led to considerable controversy over the exact evolutionary position of SARS-CoV among coronaviruses.
cord-343963-99rd3o79	2014	13, 14 Upon infection by viruses such as HCV, viral RNA is first sensed by cellular pattern recognition receptors (PRRs), and the PRR-mediated recruitment of adaptor proteins and the activation of downstream signaling lead to IFN production. First, we briefly discuss the signaling triggered by the retinoic acid-inducible gene 1-like receptor (RLR) and the Toll-like receptor (TLR), which leads to type I IFN synthesis and IFN-mediated signaling pathway activation, resulting in the expression of a variety of effector ISGs. We also summarize the strategies that HCV uses to escape IFN antiviral surveillance. 156 demonstrated that HCVinduced SG formation is IFN-and PKR-dependent and is inversely correlated with the induction of ISG proteins, such as myxovirus resistance gene A (MxA) and Ub-like (UBL)specific protease 18 (USP18), in HCV-infected cells without affecting the mRNA levels of these ISGs. Furthermore, the SG proteins TIA-1, TIAR and G3BP1 have been shown to play a critical role in HCV replication and infectious virus production.
cord-344006-0iq9s94n	2020	All rights reserved Like other coronaviruses, SARS-CoV-2 is a single-stranded, positive-sense RNA virus that uses spike proteins to bind to human lung epithelial cells (Fig. 2) [67] . Upon membrane fusion, the RNA of the coronavirus genome is released into the host cell cytoplasm via an early endosome -unlike SARS-CoV, which employs a late endosome and therefore must cross higher barriers of antiviral host immunity -where it is translated into a replication-translation complex that in turn translates sub-genomic RNA into accessory and structural proteins (Fig. 3) [82-84]. The Vivalytic VRI (viral respiratory tract infections) COVID-19 Test System pioneered by Bosch and Randox Laboratories is similar to the Abbott RealTime SARS-CoV-2 assay in that it reduces hands-on time and can confirm a positive test within 2.5 hours with a reported 95% accuracy [100]. More specific assays have now emerged that are proving very useful in providing a fuller picture of the rates of asymptomatic or mild SARS-Cov2 infection, through detection of anti-viral antibodies that persist for months and even years after the virus has been cleared [107] .
cord-344321-fjer281d	2020	Aptamers, first raised by two research groups independently in 1990 [1, 2] , are single-stranded DNA (ssDNA) or RNA sequences obtained through systematic evolution of ligands by exponential enrichment (SELEX) that can fold into secondary and three-dimensional shapes, enabling them to recognize various target molecules with high specificity and affinity, including proteins [3, 4] , small molecules [5, 6] , metal ions [7, 8] , bacterial cells [9, 10] , viruses [11, 12] , cancer cells [13, 14] , and even tissues [15] . developed a novel QD-Apt conjugate to be loaded with Dox for the targeted delivery of drugs to PC cells based on the mechanism of binary (Bi)-FRET (Fig. 15A) [215] . The Apt-Dox-Lip complex showed selective internalization and enhanced cytotoxicity to MCF-7 breast cancer cells and xenograft MCF-7 breast tumors in nude mice, suggesting that AS1411 aptamer-functionalized liposomes can specifically bind nucleolin overexpressed on the MCF-7 cell surface, and implement drug delivery with high selectivity.
cord-344410-yo9libo0	2011	A core palm structure containing the four motifs (A-D) found in all classes of polymerases [1] is present in the C-terminal region of nsp9; therefore, it is possible to identify RdRp catalytic activity by testing whether the conserved SDD motif (at residues 3050 to 3052 of nsp9) itself is essential for virus replication (Figure 1) . To study the impact of mutations in the SDD motif on PRRSV transcription, RT-PCR analysis of the sg mRNA7s was performed on the total RNAs obtained from mock-transfected BHK-21 cells and cells transfected with pMDD, pSAD, pSED, pSND, pSGD, pSDA, pSDE, pSDN, pSGA, pGND, pGDD, and pAPRRS, using a forward primer located in the leader region and a reverse primer in ORF7. The mutant PRRSV full-length cDNA clones pMDD, pSAD, pSED, pSND, pSGD, pSDA, pSDE, pSDN, pGDN, and pSGA carrying mutations specifying the SDD motif in the viral replicase (Table 1) , were transfected into BHK-21 cells and failed to produce sg mRNA7s (Figure 2) .
cord-344421-rmnck42f	2018	Suckling piglets shed kobuvirus from one week of age, but an association between peak of viral shedding (10(6.42)–10(7.01) copies/swab) and diarrheic signs was not observed during a follow-up study. Third-generation sequencing using MinION (Oxford Nanopore Technologies, ONT) might be a useful and affordable diagnostic tool for swine veterinary medicine as it allows rapid sample preparation and real-time sequence analysis. The shedding of porcine kobuvirus, RVA and rotavirus C (RVC) was quantitatively investigated in 5 suckling pigs of the same farm from which the diarrheic feces originated. The third-generation sequencing device MinION (ONT), holds promise as a diagnostic platform, as it allows real-time sequencing and analyses of all DNA/RNA in a sample, theoretically without needing pre-amplification of viral nucleic acids. In the present study, a novel RT-qPCR assay, targeting the conserved 3D gene encoding the RNA-dependent-RNA-polymerase, was developed and used to assess, for the first time, longitudinal quantitative shedding kinetics of porcine kobuvirus in pigs under field conditions.
cord-344464-if6js43s	2002	title: The complete genome sequence of gill-associated virus of Penaeus monodon prawns indicates a gene organisation unique among nidoviruses(*): Brief Report We report here a 5596 nt sequence comprising the 3′-end of the (+) ssRNA genome of gill-associated virus (GAV), an invertebrate nidovirus of Penaeus monodon prawns. The sequence extends from a subgenomic RNA start site 35 nt upstream of the 4923 nt ORF3 gene to a 3′-poly(A) tail of the 26235 nt genome of GAV. Completion of the genome sequence of GAV has revealed a gene organisation unique among nidoviruses in there is no discrete membrane protein gene and that the putative ORF3 spike glycoprotein gene resides downstream of a gene (ORF2) encoding a structural protein associated with nucleocapsids. A contig of a 5596 nt sequence from the GAV genomic 3 -poly(A) tail to an intergenic 5 -sgRNA start site 35 nt upstream of the ORF3 start codon [10] was deduced from overlapping cDNA clones (Figs.
cord-344636-go5cw92q	2020	In this work, we developed a COVID-19 diagnosis kit for the rapid detection of SARS-CoV-2, using one-step reverse transcription and loop-mediated isothermal amplification (RT-LAMP). Positive amplification products were obtained even for 2 copies of the synthetic viral DNA fragment template in 30 min when using the S17 primers (lane 4 in Fig. 1D ), demonstrating that the LAMP reaction was rapid and sensitive. To assess the potential of RT-LAMP in detecting RNA virus of SARS-CoV-2, we then tested the performance of these primers with synthesized RNA fragments of the N gene, S gene and Orf1ab gene obtained from in vitro transcription (Appendix S1). The ultimate aim is to develop an enclosed device that integrates RNA extraction, purification, reverse transcription (RT) and loop-mediated isothermal amplification (LAMP) to detect the SARS-CoV-2 virus directly from a throat swab sample.
cord-344714-0cam9ipf	2020	Here, we reviewed the capacity of well-known (e.g. quercetin, baicalin, luteolin, hesperetin, gallocatechin gallate, epigallocatechin gallate) and uncommon (e.g. scutellarein, amentoflavone, papyriflavonol A) flavonoids, secondary metabolites widely present in plant tissues with antioxidant and anti-microbial functions, to inhibit key proteins involved in coronavirus infective cycle, such as PL(pro), 3CL(pro), NTPase/helicase. Inhibition of TMPRSS2 and Furin protease activities can be considered an interesting therapeutic option against coronavirus infection, especially COVID-19, allowing the block and/or prevention of SARS-CoV-2 infection, as recently reported [28] . Based on these observations, it is not surprising that molecular docking approach, summarized in Fig. 3 , supports the role of flavonoids in the inhibition of SARS-CoV 3CL pro by binding His41 and Cys145 of the catalytic site and other active site residues (e.g., Met49, Gly143, His163, His164, Glu166, Pro168, and Gln89), stimulating their validation by in vitro and in vivo studies.
cord-344749-omzhhr0k	2020	Cell culture-based virus isolation has been accepted as a "gold standard" in the detection and identification of viruses and is the technique by which all other test methods have been compared [35] . A novel method for dengue virus detection and antibody screening using a graphene-polymer based electrochemical biosensor Chitosan-carbon nanofiber modified single-use graphite electrodes developed for electrochemical detection of DNA hybridization related to hepatitis B virus A sensitive electrochemical biosensor for specific DNA sequence detection based on flower-like VS2, graphene and Au nanoparticles signal amplification Electrochemical DNA biosensor based on a tetrahedral nanostructure probe for the detection of avian influenza A (H7N9) virus Electrochemical DNA biosensor based on gold nanorods for detecting hepatitis B virus Electrochemical-DNA biosensor development based on a modified carbon electrode with gold nanoparticles for influenza a (H1N1) detection: effect of spacer Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen
cord-344782-ond1ziu5	2018	Nucleic acid sequencing of the virus isolate has identified the entire genome and indicates that this is a novel nidovirus that has a low level of nucleotide similarity to recognised nidoviruses. Following the detection of the novel virus, in November 2015 (about 6 months after the cessation of the outbreak) an intensive survey of the parts of the river where affected turtles had been detected [2] was undertaken by groups of biologists and ecologists and samples collected from a wide range of aquatic species and some terrestrial animals (n = 360) to establish the size of the remaining population and whether any other animals were carrying this virus. BRV, as a novel nidovirus, was isolated from tissues of diseased animals, very high levels of viral RNA were detected in tissues with marked pathological changes and in situ hybridisation assays demonstrated the presence of specific viral RNA in lesions in kidneys and eye tissue-two of the main affected organs.
cord-345157-fhmhpobi	2018	Herein, we focus on several possible mechanisms of infection-induced host RNA turnover, which seems to be a common strategy for both prokaryotic and eukaryotic viruses during the very early stage of infection and a potential application of live cell imaging on its visualization. Many viruses also impair the translation of cellular mRNA [1e3], one of the mechanisms during the shift of gene expression from host to virus, a process termed "host shutoff", in order to prevent the production of anti-viral, host protecting proteins [4] . Moreover, Gaglia et al.''s work showed that viral encoded proteins trigger host mRNA degradation by a primary endonucleolytic cleavage causing shutoff of host gene expression and a host exonuclease such as Xrn1, an important 5 0 to 3 0 exonuclease in human cells, were required in subsequent completion of host mRNA turnover [5] .
cord-345204-ch0e6lzl	2020	The method uses fluorescent sensors (i.e. molecular beacons) designed to detect COVID-19 RNA or any RNA of interest, concurrent with an internal control without the need for amplification. The molecular beacons are stem-loop structures in which a ~10 nucleotide loop region has the complementary sequence of a region of the target RNA, and a fluorophore and quencher are placed on the 5'' and 3'' ends of the stem. Here, we designed a COVID-19 beacon that is completely quenched in its native form and undergoes a 50-fold increase in fluorescence when exposed to nanomolar amounts of synthetic viral oligonucleotide. Fluorescence increases from beacon responses signals are rapid and can be reversed by the addition of inexpensive ssDNA with a sequence identical to the loop region, or high salt if attached to a matrix.
cord-345302-wbkfjz8r	2016	This paper attempts to characterize the viral pathogens associated with 2–3-week-old RSS-affected and unaffected broiler chickens using next-generation sequencing and comparative metagenomics. Of specific interest is the enteric viral population associated with RSS of which many RNA and DNA viruses have been implicated and co-infections of multiple viruses such as rotavirus, chicken astrovirus (CAstV), avian nephritis virus (ANV), and reoviruses have been detected in birds affected with RSS or with poor performance (Reynolds et al., 1986; Guy, 1998; Kang et al., 2012) . Other enteric pathogens associated with avian malabsorption diseases include reoviruses, parvoviruses, and members of the family Caliciviridae; many of which have also been observed in broiler chickens Smyth et al., 2007; Pantin-Jackwood et al., 2008 , Wolf et al., 2011 .
cord-345371-pjbviagq	2020	The rationale for drug selection was mainly, though not exclusively, based either i) on the activity against other coronaviruses or RNA viruses in order to potentially hamper viral entry and replication in the epithelial cells of the airways, and/or ii) on the ability to modulate the excessive inflammatory reaction deriving from dysregulated host immune responses against the SARS-CoV-2. Here, we review the recently published literature on the pharmacological treatments used so far and/or undergoing evaluation in clinical trials, with focus on the biochemical mechanisms of action of repurposed or investigational drugs, classified as agents directly targeting the virus ( Figure 1 and Table 1 ) and those used to treat the respiratory distress and inflammation associated with the cytokine release syndrome ( Figure 2 and Table 2 ).
cord-345413-bsd32j8r	2019	To determine viral RNA replication in A72 cells, A72, MDCK, DH82, and Fcwf-4 cells were infected with rC3663-Nluc virus at an MOI of 0.01, followed by real-time RT-PCR analysis of RNA extracted at 24, 48, and 72 hpi. Next, we determined the levels of production of infectious virus particles from rC3663-Nluc-infected A72 cells by collecting the culture supernatants at 24, 48, and 72 hpi and measuring viral titers by plaque assays with Fcwf-4 cells (Fig. 3E ). Thus, to determine expression levels of sg N mRNA (sg mRNA6), total RNAs extracted from rC3663 virus-infected or mock-infected Fcwf-4 and A72 cells at 48 and 72 hpi were subjected to Northern blot analysis with a specific type I FCoV 3= untranslated region (3=UTR) probe (Fig. 5A) . We further examined the expression levels of viral sg mRNAs in cells infected with the parental C3663 strain or type I FCoV strain Yayoi using Northern blot analysis with specific RNA probes against the 3=-UTR.
cord-345630-bam3pa70	1991	authors: Lee, Han-Jung; Shieh, Chien-Kou; Gorbalenya, Alexander E.; Koonin, Eugene V.; La Monica, Nicola; Tuler, Jeremy; Bagdzhadzhyan, Anush; Lai, Michael M.C. title: The complete sequence (22 kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase Third, the 3''-half of the gene 1 sequences of IBV and MHV-A59 contains the sequence motifs for RNA polymerase and helicase, which are the activities expected to be involved in RNA synthesis Gorbalenya et a/., 198913; Bredenbeek et a/., 1990) . The alignment of amino acids in ORF la of MHV-JHM and IBV showed that there are four possible stretches of moderate homology which are separated by highly diverged sequences (Fig. 8) . Although ORF la is highly diverged between MHV-JHM and IBV, common functional domains could be identified in this ORF of both viruses by detailed amino acid sequence analysis (see Materials and Methods) (Fig. 9 ).
cord-345647-h3imwhss	2020	To identify the possible etiologic agents of disease in the four pangolins, eight meta-transcriptomic libraries from blood, liver, spleen, lung, kidney, and fecal samples were constructed, generating a total of 306,908,179 paired-end sequence reads. De novo assembly revealed the high abundance of a pestivirusand coltivirus-like virus in all the meta-transcriptomic libraries of the pangolin 1-Dongyang and 2-Lishui, representing 6-80 and 1-29 per cent of total viral contigs, respectively (Supplementary Table S2 ). Considering that they are related, yet clearly genetically distinct, from known members of Pestivirus and Coltivirus (see below), we designated these two newly identified viruses as Dongyang pangolin virus (DYPV) and Lishui pangolin virus (LSPV), respectively, reflecting their hosts species and the geographic location of sampling. Consequently, DYPV was identified in the heart, liver, spleen, lung, kidney, brain, blood, throat swab, and fecal sampled from pangolin 1-Dongyang, as well as the nymph ticks (Amblyomma javanense) collected from this animal.
cord-345654-vyz6f3he	2016	Virus emergence requires overlap between host populations, alterations in virus genetics to permit infection of new hosts, and adaptation to novel hosts such that between‐host transmission is sustainable, all of which are the purview of the fields of ecology and evolution. I argue that, while virus acquisition of the ability to infect new hosts is not difficult, limited evolutionary trajectories to sustained virus between‐host transmission and the combined effects of mutational meltdown, bottlenecking, demographic stochasticity, density dependence, and genetic erosion in ecological sinks limit most emergence events to dead‐end spillover infections. Virus quasispecies may facilitate host range expansion Viruses are among the smallest nucleic acid-based replicating entities and possess characteristics associated with exceptionally fast evolutionary change: small genomes, short generation times, high mutation rates, large population sizes, high levels of genetic diversity, and strong selection pressures.
cord-345817-rrf3dbnb	2011	
cord-345863-j01l71dh	2020	Considering the importance of genetics and host immune responses in viral infections, the goal of this study was to elucidate host gene expression in macrophages using RNA sequencing. Our results highlight individual host responses playing an important role, consistent with the fact that few cats develop feline infectious peritonitis despite a common presence of enteric FCoV. Cultured macrophages were infected with the FIPV 79-1146 for 2 and 17 h before RNA was collected for the expression analysis of host genes and viral reads. Viral RNA load per sample in both raw read count summation and normalized fragments per kilobase million (FPKM) average from all virus'' genes combined, obtained in cellular extracts of either macrophages or CRFK cells infected with FIPV, at 2 and 17 h post-infection. In contrast to the macrophages exposed to the virus, RNA sequencing showed several log-fold increases of viral isolates in the CRFK cells used as replication controls from 2 to 17 h (Table 1 ).
cord-345898-a6vt8kso	2016	There are three types of cell-based reporter systems that express certain reporter protein for positive-sense single strand RNA virus infections. An RVP is a type of virus-like particle (VLP) composed of viral structural proteins and a self-replicating replicon RNA containing a reporter gene [17, 63] . However, when the cells were infected with the specific virus, the viral RdRp was provided by the virus and the defective replicon was activated, resulting in high-level expression of the reporter gene, which could be easily examined [44] . However, when the cells were infected with the specific virus, the viral RdRp was provided by the virus and the defective replicon was activated, resulting in high-level expression of the reporter gene, which could be easily examined [44] . Development of Dengue type-2 virus replicons expressing GFP reporter gene in study of viral RNA replication
cord-345957-wuk2arf9	2018	When compared to the only recorded BKoV genome sequence (BKoV/U-1/AB084788), the studied strain showed 94 amino acid (aa) substitutions through its entire polyprotein (2463 aa), one nucleotide (nt) insertion and one nt deletion in the 2B gene and 4-nt deletions in the UTRs (2 each). Initially, kobuviruses included Aichivirus (AiV), bovine kobuvirus (BKoV), and porcine kobuvirus (PKoV) based on the hosts infected e.g. humans, cattle, 1 3 and swine, respectively. In this study, we report BKoV infections among cattle populations of Egypt, the full-length genome of one Egyptian strain (BKoV/ Egy-1/ KY407744) as well as the phylogenetic analysis of BKoV strains from Cairo and Sharkia provinces. The RNA samples (n = 36) were subjected to RT-PCR for the detection of BKoV using degenerate primers (Univ-Kobu-F and Univ-Kobu-R), which amplify a conserved region in the 3D (RdRp) gene of all kobuviruses [12] ( Table 1 ).
cord-346138-ip42zcld	2020	The results highlight how the pharyngeal swab is highly sensitive in the first phase of the disease, while in the advanced stages, other specimens should be considered, such as sputum, or even stool to detect SARS-CoV-2. Several authors therefore suppose an infection of the gastrointestinal tract by the virus (11, 24) , with its continuous elimination with the feces which has been reported to last from 1 to 12 days (24) and in some cases, viral RNA were detected in feces or anal swabs even after the respiratory tests became negative (11, 22, 24) . The reference method for testing positivity to SARS-CoV-2 infection is represented by the pharyngeal swab that is taken from the patient''s nasopharynx or oropharynx and, through an RT-PCR analyzed for the presence of viral RNA (8) .
cord-346267-l08ld2cy	2011	Over longer periods of evolutionary time (i.e., along long internal branches on a phylogenetic tree), evidence of evolution at the nucleotide level could be lost, which could bias tMRCA estimates towards younger dates (i.e., underestimate the length of these branches). The results presented here demonstrate that not accounting for purifying selection may bias tMRCA estimation in RNA viruses towards more recent dates, and a degree of correction can be realized by employing more realistic codon-based substitution models, capable of partially accounting for the biasing effect of purifying selection. Using BMCMC analysis, we inferred the substitution rate and root age under a standard nucleotide substitution model (GTR + Γ 4 ) for each of our three viral data sets: MeV/RPV/PPRV nucleoprotein, EBOV glycoprotein, and AIV neuraminidase (table 1) . The nucleotide substitution model (GTR + Γ 4 ) underestimated the length of branches simulated under empirical (IFEL) selection regimes inferred from the three viral data sets ( fig.
cord-346314-o9fjpqaj	2012	
cord-346514-vyo8l14p	2013	The polyadenylation signal in the 3′ UTR of BaMV was shown to be involved in the synthesis of minus-strand viral RNA and regulating the length of the poly(A) tail (Chen et al., 2005) . To test whether the replicase preparation could polyadenylate exogenous plus-strand RNA templates, 5′ end-labeled radioactive BaMV plus-strand RNA substrate containing the entire 3′-UTR and 7, 15, or 40 adenylates (r138/7A, r138/15A, or r138/40A, respectively), were subjected to the polyadenylation activity assay. BaMV minigenomes were demonstrated to be suitable templates for an in vitro replication assays (Huang et al., 2009) ; therefore, the exogenous plus-strand or minus-strand BaMV minigenome was added to the reaction to help determine whether the poly(A) tail could be added to the 3′ end of preformed or newly transcribed plus-strand RNA, respectively.
cord-346544-kk7qyn4w	2020	Background: Laboratory diagnosis of SARS-CoV-2 infection (the cause of COVID-19) uses PCR to detect viral RNA (vRNA) in respiratory samples. . https://doi.org/10.1101/2020.05.21.20105486 doi: medRxiv preprint prevalence of vRNA detection in blood, serum or plasma, noting whether this attribute was correlated with clinical or laboratory phenotypes of disease, and recording Ct values when these were reported. We collected 212 serum samples through the microbiology department at Oxford University Hospitals NHS Foundation Trust, OUH NHSFT, comprising adults with a diagnosis of COVID-19, confirmed by SARS-CoV-2 detection by a clinical diagnostic microbiology laboratory using RT-PCR on a respiratory swab, classified in three groups as follows: Based on a systematic review of the literature, together with our own data, we estimate that SARS-CoV-2 RNA may be present at low copy numbers in ~10% of blood samples obtained from individuals with COVID-19 prior to day 28, most of which arise at earlier timepoints and in the setting of more severe disease.
cord-346697-ixho9t5g	2019	
cord-346853-0c1qdjb5	2007	
cord-346916-jj4l9ydl	2020	Moreover, despite the molecular mimicry set by RNA viruses to resemble cellular mRNAs and escape host recognition, the viral nucleic acid still needs to embark on a long journey through a hostile cell environment and must overcome the obstacles put in place by the host antiviral system in order to be translated and replicated. Another example, is the zinc-finger antiviral protein (ZAP), which binds vRNAs containing a ZAP response element (ZRE) and induces RNA degradation via interaction of its N-terminal domain with host decay machinery mediated [75] (Fig. 1 ). In fact, IRES elements present in the genome of different families of RNA viruses lack overall conserved features [146, 147] .The classification of viral IRESs in four types stems from their structural organization, their respective dependence on sets of translation initiation factors, and whether they use scanning or instead directly recruit ribosomes to the start codon [148] (Fig. 2) .
cord-346930-gl573ip9	2020	Although multiple drugs show promise in the treatment of COVID-19 via either inhibiting viral replication or preventing fusion of the virus to the ACE2 receptors, further investigation is still warranted and necessary before the admission of any type of pharmaceutical agent. This review explores various drugs and their mechanism of action which are either currently being used in clinical trials or may be used in the future for the treatment of COVID-19. Since the emergence of the virus in China in December of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread across the globe resulting in the current global pandemic. Arbidol (also known as Umifenovir) is a promising repurposed antiviral agent with a unique mechanism of action targeting the S protein/ACE2 interaction and inhibiting membrane fusion of the viral envelope to the host cell [7] .
cord-346978-ubkqny8j	2020	
cord-347128-6lyoz8nn	2020	O-acetylated SAs interact with the lectin-like spike glycoprotein of SARS CoV-2 for the initial attachment of viruses to enter into the host cells. In RNA viruses, the S glycoprotein (PDB: 6VSB) is the biggest protein, heavily glycosylated and its N-terminal domain (NTD) sequence binds to the host receptor to enter the ER of host cells. However, MERS-CoV does not have a similar enzyme and thus MER-CoV binding to SA receptors is mediated by energetically reversible interactions of the lipid rafts with increased SA receptors [75] , thus enhancing dipeptidyl peptidase 4 (DPP4) or carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) recognition power and viral entry [76] and membrane-associated 78-kDa glucose-regulated protein (GRP78) [77] . Entry of host cells needs binding of S glycoproteins to the CEACAM receptor, forming S-protein-mediated membrane fusion. For example, impairment of ACE2 receptor glycosylation does not influence S-glycoprotein-ACE2 interaction, however, SARS-CoV-2 virus entry into respiratory epithelial host cells was downregulated [133] .
cord-347221-g98q9cga	2020	This review mainly focuses on various nucleic acid-based biologically active molecules and their therapeutic potentials in developing vaccines for SARS-CoV-2. This review mainly focuses on various nucleic acid-based biologically active molecules and their therapeutic potentials in developing vaccines for SARS-CoV-2. This phenomenon of producing an effective immunity is particularly important in their use against the development of nucleic acid based therapeutic drugs for the treatment of SARS-CoV-2. Nucleic acid-based therapies, especially, RNA therapies including RNAi (RNA interference), siRNAs (small interfering RNA) and RNA aptamers, Ribozymes and ASOs (antisense oligonucleotides) target and neutralize the crucial components of the virus-like specific mRNA molecules, viral proteins like E (envelope), M (membrane), or N (nucleocapsid), or SARS helicase, etc. The nucleic acid-based vaccination technologies involve the use of RNA (mRNA) [34] or plasmid DNA, which encodes for antigen.
cord-347302-ylnb6qfl	2016	Replication organelles (ROs) are membranous structures that not only harbor viral proteins but also contain a specific array of hijacked host factors that create a unique lipid microenvironment optimal for genome replication. Whatever function GBF1 plays in enterovirus replication, it might be related to the functioning rather than the formation of ROs, because poliovirus proteins expressed in the presence of BFA induced membrane rearrangements indistinguishable from those found in infected cells [43] . The Importance of MCSs for Controlling the Metabolism of Other Lipids in Infected Cells Besides mediating cholesterol/PI4P exchange, OSBP regulates the homeostasis of other lipids via MCSs. Notably, OSBP regulates the ceramide transfer protein (CERT)-dependent shuttling of ceramide (a precursor of sphingomyelin, a lipid that associates with cholesterol in membrane microdomains) from ER to trans-Golgi [70] . Since RO membranes are enriched in PI4P, ORP5 and/or ORP8 may also be recruited to ER-RO MCSs and transport phosphatidylserine to ROs, from where it may transition to the autophagosome-like DMVs. Enterovirus replication has been associated with alterations of other lipid metabolic pathways as well.
cord-347351-emdj66vj	2020	Originating from a single travel-associated primary case from China, the first documented chain of multiple human-to-human transmissions of SARS-CoV-2 outside of Asia allowed a detailed study of transmission events and identified several factors (e.g. cumulative face-toface contact, direct contact with secretions or body fluids of a patient, personal protective equipment) to classify contacts as low or high risk [32] . In the close surrounding of COVID-19 patients in hospitals SARS-CoV-2 RNA is detected more frequently compared to surfaces outside the patient rooms but samples were so far consistently negative for infectious virus. General disinfection of frequently touched surfaces in the public such as shopping carts or door handles is, however, unlikely to add any protective value because even in COVID-19 wards inanimate surfaces were mainly contaminated in the permanent and immediate surrounding of symptomatic patients (detection of viral RNA, not of infectious virus) and only rarely one room away [138] suggesting that the risk to find SARS-CoV-2 on frequently touched surfaces in the public is low.
cord-347472-n6811ens	2020	The US Centers for Disease Control and Prevention (CDC) have specified and given emergency use authorization (EUA) for a SARS-CoV-2 molecular diagnostic used to detect viral RNA in clinical samples (2) . Genomic DNA is co-purified in quantities sufficient to generate strong positive signals for the CDC-specified extraction control during work-up of clinical RNA specimens. To test for the presence of control-affecting DNA, qPCR reactions lacking reverse transcriptase were performed on SARS-CoV-2-positive clinical samples using the CDC-specified RP primer and probe. All clinical samples tested generated unambiguous extraction control positive signals in the absence of reverse transcription, a reaction context that could not have detected virus an RNA virus ( Fig. 2A) Single-digit copies of genomic DNA are sufficient to generate a positive control signal using the CDC-designed assay. Due to the presence of co-purifying genomic DNA in clinical samples, loss of RNA integrity leads to false-negative results using the CDC-specified control.
cord-347532-n51qv9pp	2013	To the Editor: Bats play a critical role in the transmission and origin of zoonotic diseases, primarily viral zoonoses associated with high casefatality rates, including those caused by Nipah virus (NiV) and severe acute respiratory syndrome (SARS)-like coronavirus (CoV) infections (1) . To assess pathogens in bat guano, we examined bat guano from a cave in the Khao Chong Phran Non-hunting Area (KCP-NHA) in Ratchaburi Province, Thailand, where bat guano was sold as agricultural fertilizer, for the presence of NiV, CoV, and H. The detection of CoVs in bat guano from the KCP-NHA cave in Ratchaburi was consistent with the previous finding of alphacoronavirus from Hipposideros armiger bats from the same province in 2007, but those researchers tested fresh bat feces (9) . Duplex nested RT-PCR for detection of Nipah virus RNA from urine specimens of bats
cord-347710-ff64y6ef	2020	hnRNPs involve in a large number of cellular processes, including chromatin remodelling, transcription regulation, RNP assembly and stabilization, RNA export, virus replication, histone-like nucleoid structuring, and even intracellular immunity. 6, 13 It is well known that Hsp90 not only interacts and contributes to RNA polymerase assembly and nuclear import of some (−) ssRNA viruses (e.g., PB2 of influenza virus), but plays crucial roles in the folding process of viral capsid proteins and virion assemblies as well. 17, 18 As a critical component of cellular protein surveillance, the ATP-dependent molecular chaperone protects cells from damage caused by stress and takes part in a number of folding processes, including folding of newly synthesized polypeptides, recognition and refolding of misfolded or aggregated proteins, solubilization or degradation of proteins, transporting proteins, assembly or disassembly of oligomeric protein complexes, and the regulation of certain natively folded proteins.
cord-347917-fmb5nyxu	2020	
cord-347992-coby2m6e	2010	
cord-348147-leni23pa	2007	Sequence analysis of the full-length and partial genomes obtained from naturally infected mice yielded valuable data on genetic diversity of murine noroviruses. These specimens were obtained from 28 different mouse lines, including immunocompetent mice, lines with muta(4), b-TCR À=À (2), RAG2=gChain À=À (5), B6 Casp À=À (2), CD36=SRA À=À (2), CCL3 À=À (2), LFA À=À (2), LyZ À=À (2), PSEN tg (2), Bl6 wt (4) a GE genome equivalents; for more than one positive sample the mean was calculated b Includes partial sequences of the ORFs (underlined isolates; ORF1, RNA polymerase gene; ORF2, capsid gene) Ã Contains both regions tions in a number of immune effector molecules, and transgenic mouse lines (Table 1) . Like human noroviruses, the present sequence data indicate that murine norovirus strains are also genetically diverse and can, therefore, be subdivided in genetic clusters.
cord-348204-365z3qxz	2013	Real time RT-qPCR developed focusing on 2 up-regulated genes (PD-L1 and A3H) together with an apoptosis associated gene PD-1 expressions in FIPV infected CRFK cells and in PBMCs from healthy and FIP diagnosed cats produced concordant results with transcriptome data. FIPV infected cells also showed high up-regulation of PD-1 expression at 3 h.p.i. and moderately up-regulated at 12 h.p.i but were being down-regulated at 6, 9, 24 and 48 h.p.i. Meanwhile, PD-L1 gene was consistently down-regulated from 3 hours to 48 h.p.i. Peripheral Blood Mononuclear Cells (PBMCs), obtained from cats with clinical signs associated with FIP (Table 4) , were purified and analysed with real-time PCR. The reference feline genome sequence assembly of transcriptome analysis of early infection (3 h.p.i.) of CRFK cells with FIPV 79-1146 showed that the expressions of 215 transcripts (0.8% of the trimmed annotated) were statistically significant, based on Kal''s Z test.
cord-348243-e5tdb08v	2020	METHODS: To avoid these obstacles, we tested PCR-independent methods for the detection of SARS-CoV-2 RNA from primary material (nasopharyngeal swabs) including reverse transcription loop-mediated isothermal amplification (RT-LAMP) and specific high-sensitivity enzymatic reporter unlocking (SHERLOCK). To allow for the comparison of different nucleic acid detection methods for SARS-CoV-2 we collected redundant material from nasopharyngeal swabs obtained for qPCR testing in clinical routine due to suspected COVID-19. We first tested two recently described assays for SARS-CoV-2 detection on isolated RNA from patient samples. In summary, our multiplex RT-LAMP protocol is a simple and sensitive way to detect SARS-CoV-2 RNA from clinical samples. Currently, a test based on our multiplexed RT-LAMP assay would-in contrast to a good specificity-most likely miss to identify those infected patients with very low amounts of viral RNA in the nose or throat and would not yet reach the sensitivity of the gold-standard qPCR assays.
cord-348669-mizygp4j	2016	title: Characterization of a Pathogenic Full-Length cDNA Clone and Transmission Model for Porcine Epidemic Diarrhea Virus Strain PC22A The infectious-clone-derived PEDV (icPEDV) replicated as efficiently as the parental virus in cell culture and in pigs, resulting in lethal disease in vivo. Importantly, recombinant PEDV was rapidly transmitted to uninoculated pigs via indirect contact, demonstrating virulence and efficient transmission while replicating phenotypes seen in the wild-type virus. While the recombinant icPEDV replicated the clinical phenotypes of parental PC22A in vivo, icPEDV-⌬ORF3-RFP infection resulted in a partial attenuation in pigs based on lower diarrhea scores. Both the parental PEDV PC22A strain and its derivative recombinant cloned virus were genetically stable and fully pathogenic in neonatal gnotobiotic pigs, demonstrating that icPEDV provides not only a strategy that allows for the systematic evaluation of the role of viral genes in pathogenesis, tropism, and virulence but also a translational platform for the development of rationally attenuated live virus vaccines.
cord-348777-pk9y6vfp	2020	title: Effect of Corticosteroid Therapy on the Duration of SARS-CoV-2 Clearance in Patients with Mild COVID-19: A Retrospective Cohort Study This study aims to investigate the association between corticosteroid therapy and the duration of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clearance among patients with mild COVID-19. Our observational results revealed that corticosteroid therapy had no positive effect on the durations of SARS-CoV-2 RNA clearance among patients with mild COVID-19. Results from this study suggested that patients with mild COVID-19 may not benefit from corticosteroid therapy in terms of the duration of SARS-CoV-2 clearance.
cord-348799-qu4zin3o	2019	This lack of specificity was not present in cells however, given that the ectopic expression of ISG20 did not lead to the indiscriminate degradation of cellular RNAs (ribosomal as well as small nucleolar RNAs previously shown to be associated to ISG20 [2] ), nor of an Hepatitis C virus (HCV)-derived Luciferase reporter mRNA produced in vitro and then transfected into cells (S4C and S4D Fig) , suggesting that in cells the RNase activity of ISG20 is tightly controlled. Self mimicry allows the escape of target genes'' mRNAs from the effects of ISG20 VSV infection and ectopic DNA or RNA transfection do not bear much in common apart from the fact that both represent artificial injections into the cell of non-self genetic material from without. To further determine whether ISG20 acted on translation initiation and more specifically through specific initiation factors (eIFs), capped and polyadenylated mRNAs coding luciferase under the control of several 5'' untranslated regions (5'' UTRs) were generated in vitro and directly transfected in ISG20-expressing cells (Fig 3D) .
cord-348815-lthz75oc	2009	Successful applications in animal models have already led to the initiation of RNAi‐based clinical trials as a new therapeutic option.[Image: see text] Only ten years ago Andrew Fire and Craig Mello were able to show that double‐stranded RNA molecules could inhibit the expression of homologous genes in eukaryotes. This Review provides an overview of the molecular processes involved, with a particular focus on the posttranscriptional inhibition of gene expression in mammalian cells, the possible applications in research, and the results of the first clinical studies. Antisense and RNAi strategies have many things in common, such as the necessity to identify suitable binding sequences on the target RNA, the stabilization of the oligonucleotide by chemical modification, or the transport of the negatively charged polymer across the cell membrane. [19] There are, however, important differences between the two technologies: Antisense oligonucleotides are single-stranded (modified) DNA molecules, which primarily induce the cleavage of the target RNA in the cell nucleus by activation of RNase H.
cord-348860-zaimorg0	2008	The genomics era has revolutionized the biological sciences and has heralded the emergence of new ''omics'' methodologies such as transcriptomics (study of the gene expression and expression levels of mRNAs at a given time and condition), proteomics (study of the entire protein content of a cell/tissue under various conditions, their structure and functions), metabolomics (study of the metabolite profi le of different cellular processes), phosphoproteomics (a branch of proteomics that characterizes proteins that are phosphorylated), interactomics/system biology (a science that unifi es transcriptomics, proteomics and metabolomics to look at the organism as a whole) and so on. DNA microarrays, proteomics and bioinformatic analysis are routinely used to analyze changes in host and viral gene and protein expression that occur in a virus infected cell [25] .
cord-349011-kxhpdvri	2019	
cord-349042-u9svz7pf	2018	The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. Examination of expression profiles of single live cells has shown that linear aRNA amplification neither results in occurs after synthesis of double-stranded cDNA, when T7 RNA polymerase is added and aRNA is transcribed from the cDNA template. 45 developed a method to facilitate aRNA detection of antibody-antigen interactions by covalently attaching a double-stranded cDNA that contains a T7 RNA polymerase promoter in front of a reporter sequence to a specific antibody.
cord-349249-jwvz1ux2	2019	
cord-349341-ap5n6ijl	2007	The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5''-triphosphate incorporation localized between inner and outer mitochondrial membranes inside ∼50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. To localize more precisely FHV RNA synthesis in relation to spherules, we incubated mitochondria isolated from uninfected and FHV-infected Drosophila cells with a nucleotide mix including 5-bromouridine 5''-triphosphate (BrUTP) and performed immunogold labeling EM with an antibody recognizing BrU incorporated into RNA, but not unincorporated BrUTP. To generate 3-D surface maps of the virus-induced membrane rearrangements associated with FHV RNA replication, we manually traced the inner and outer mitochondrial membranes (including spherules) over ;100 adjacent, 2.2nm-spaced virtual sections of selected tomographic reconstructions, and we used a computer-generated mesh overlay to join these tracings into continuous surfaces ( Figure 4 ).
cord-349358-leicos9j	2006	During the past few years, it has been demonstrated that RNAi, induced by specifically designed double‐stranded RNA (dsRNA) molecules, can silence gene expression of human viral pathogens both in acute and chronic viral infections. Likewise, expression vectors of siRNAs specific for two different regions of the WNV genome protected 293T cells from WNV infection, and significantly reduced viral RNA replication and virus production [35] . From the reports on the use of siRNA against human viral pathogens causing acute disease, we could learn that for each specific pathogen infecting a specific cell lineage or tissue, we would probably need to perform an indepth assessment, with proper in vitro and in vivo models, and develop specific delivery systems. The most challenging part of RNAi approaches for chronic viral infections is to design the best delivery method that would facilitate the targeting of the specific organ/cells with the appropriate expression system, for durable intracellular levels of gene-silencing effect.
cord-349417-vn7q8wc4	2006	Activation of the coronavirus replication complex involves extensive proteolytic processing of the replicase polyproteins to produce 16 (in IBV: 15) mature products called nonstructural proteins (nsp) 1 to 16 (reviewed in Refs. The N-terminal regions of the coronavirus polyproteins, which are poorly conserved among the coronavirus groups I, II, and III, are cleaved at two (in IBV) or three sites (in all other coronaviruses) by one (IBV and SARS-CoV) or two zinc-finger-containing papain-like cysteine proteases called PL1 pro and PL2 pro . The observed pattern of conservation in different nidovirus families suggests a functional hierarchy for the newly identified RNA-processing activities, with the manganese ion-dependent uridylate-specific endoribonuclease, NendoU, playing a central role. Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication
cord-349623-dw5o9i59	2020	Methods: Optimal direct protocol was selected by comparing RT-qPCR performance under a set of thermal (65, 70, and 95° for 5, 10, and 30 min) and amplification conditions (3 or 3.5 uL loading volume; 2 commercial RT-qPCR kits with a limit of detection below 10 copies/reaction) in nasopharyngeal swabs stored at 4°C in sterile Weise''s buffer pH 7.2. For the standard protocol, routinely used in the laboratory for the detection of SARS-CoV-2, an aliquot of 180 ul of the sample from the nasopharyngeal swab, including 10 ul of extraction control, was used to extract RNA with the MagNA Pure 96 DNA and Viral NA LV Kit (Roche Diagnostics, Cat. No. For the standardization of the direct SARS-CoV-2 detection protocol without RNA extraction steps, 50 ul aliquots from the primary sample (nasopharyngeal swabs) of 5 anonymized patients were subjected to heat shock (65, 70, or 95 • C) during different incubation times (5, 10, or 30 min), and then were quickly placed at 4 • C until the moment of amplification.
cord-349672-2kt7xw8i	2020	Here, based on available structural and biochemical data, we discuss how a type IA topoisomerase may recognize and bind single-stranded DNA or RNA to initiate its required catalytic function. In type IA topoisomerases, the hydroxyl group in the side chain of the active site tyrosine residue is responsible for the first nucleophilic attack on the scissile phosphate of a single-stranded DNA resulting in the cleavage of the G-strand and the formation of the transient 5 -phospho-tyrosyl covalent linkage [66] . Though the type IA topoisomerase core domain that forms the characteristic torus structure contains all the highly conserved motifs responsible for G-strand binding and cleavage religation, the C-terminal domains of bacterial topoisomerase I have been shown to be required for removing negative supercoils from DNA rapidly in a processive mechanism [61, 72, [84] [85] [86] [87] .
cord-349684-2tioh80m	2020	The present study compared the effects of exposing SARS-CoV-2 to aqueous suspensions of Si3N4 and aluminum nitride (AlN) particles and two controls, (i.e., a suspension of copper (Cu) particles (positive control) and a sham treatment (negative control)). In (c) and (d), results of RT-PCR tests for supernatants after 10 min exposure of virus suspension to Cu, AlN, and Si3N4 powders for viral N gene "set 1" and "set 2" primers are shown, respectively. The present work is the first to show that compounds capable of endogenous nitrogen-release, such as Si3N4 and AlN, can inactivate the SARS-CoV-2 virus at least as effectively as Cu. These results suggest that multiple antiviral mechanisms may be operative, such as RNA fragmentation, and in the case of Cu, direct metal ion toxicity; but while Cu and AlN supernatants demonstrated strong and partial cellular lysis, respectively, Si3N4 provoked no metabolic alterations.
cord-349762-f5no10eq	2020	The clinical performances of six molecular diagnostic tests and a rapid antigen test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were clinically evaluated for the diagnosis of coronavirus disease 2019 (COVID-19) in self-collected saliva. SARS-CoV-2 RNA in saliva was detected using a quantitative reverse transcription-PCR (RT-qPCR) laboratory-developed test (LDT), a cobas SARS-CoV-2 high-throughput system, three direct RT-qPCR kits, and reverse transcription–loop-mediated isothermal amplification (RT-LAMP). Here, we describe the clinical performance of various molecular diagnostic methods, including the RT-qPCR LDT, the cobas SARS-CoV-2 high-throughput system, 3 direct RT-qPCR kits, and RT-LAMP, and a commercial SARS-CoV-2 RAT used on self-collected saliva specimens in diagnosing COVID-19. The RT-qPCR LDT, the cobas SARS-CoV-2 high-throughput system, direct RT-qPCR kits (except for one commercial kit), and RT-LAMP showed different sensitivities for detecting viral RNA in saliva specimens, but each can be selectively used according to the clinical setting and facilities if close attention is paid to any false-negative results.
cord-349839-s32d3di2	1992	In the folding of a single-stranded RNA molecule, there are only three ways in which two base-paired segments can be related to each other: two consecutive hairpins; two helices separated by an internal bulge; and, pseudoknots [1]. The first two motifs can be represented as two-dimensional graphs without self-intersections whereas pseudoknots cannot, as they are fundamentally three-dimensional structures in which the four base-paired strands alternate along the sequence of the RNA molecule. The three types of ''classic'' pseudoknots with co-axial stacking of the two base-paired helices and with single-stranded segments crossing the RNA grooves. The third pseudoknot involves the 530 stem-loop structure [19] , which is known to be important for the binding of tRNA to the ribosomal A site [20] , and was recently shown to be essential for ribosomal function [21.o] .
cord-350019-4nlbu54e	2019	Using this extensively studied biological system, we identified the first example of a TLR-stimulated lncRNA, lincRNA-Cox2, which was capable of positively and negatively regulating distinct types of innate immune genes [42] [43] [44] [45] [46] . The majority of lncRNAs studied in immunity were initially identified following RNA-sequencing to examine their expression profiles in specific cell lines or tissues during inflammatory activation. Hence future studies may opt to target TF binding sites, secondary structure and/or polyadenylation sites as a way to more finely dissect the functional portions of lncRNAs. The ease in which Cas9 can be targeted to specific genomic regions sparked the development of a modified (catalytically inactivated) version of the protein fused to the KRAB (Krüppel associated box) chromatin-silencing domain termed CRISPRi [174, 175] (Fig. 3C) . Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites
cord-350040-e8q7wq0h	2006	Here is limned a roadmap to explain RISC assemblyhow there are two types of RNAi, one of which is applicable to humans; how thermodynamic properties of siRNA direct strand selection to confer full RNAi activity; how RISC proteins direct siRNA presentation to its target mRNA; and how these principles can be used to design selective and functional siRNAs. Innate mRNA silencing in mammals is currently the province of microRNAs, single stranded, small RNAs that block translation with short-term survival of target mRNA. Not all siRNAs are active, however, even when their guide strands have perfect complementarity to target mRNAs. Designing siRNAs with single nucleotide specificity requires the guide strand to be incorporated into RISC, in preference to the passenger strand. The complexity of siRNA design is that small changes in siRNA:mRNA complementarity can have profound effects on RNAi effectiveness; mismatches might have few consequences or, as shown above, improve silencing by strand selection.
cord-350083-kldu8q8x	2015	title: Highly conserved regions in Ebola virus RNA dependent RNA polymerase may be act as a universal novel peptide vaccine target: a computational approach METHODS: In the present study, we used the immunoinformatics approach to design a potential epitope-based vaccine against the RNA-dependent RNA polymerase-L of EBOV. To date, information regarding the processing, structure and functions of Ebola virus (EBOV) protein L (EBOL) demonstrates that it is an RNA-dependent RNA polymerase, with the assistance of VP35. In the present study, we have followed immunoinformatics approaches for designing potential conserved epitope candidate for the utility of vaccine development against the deadly Ebola virus, with an expectation of further wet lab validation. Protein variability server predicted the variability of the conserved region of the RNA-dependent RNA polymerase-L ( Fig. 10) to ensure that the proposed epitope is within the invariable region. Design of an epitope-based peptide vaccine against spike protein of human corona virus: an in silico approach
cord-350189-2su7oqbz	2005	A priori, this suggests that LNA may be used to increase the functional half-life of siRNA in vivo by two different mechanisms, e.g. by enhancing the resistance of the constituent RNA strands against degradation by single-stranded RNases and by stabilizing the siRNA duplex structure that is critical for activity. Next, we examined the effect of making single RNA to LNA exchanges at base-paired positions in the antisense strand of the firefly luciferase siLNA1. Although we cannot exclude that these modifications somehow prevent loading of the antisense strand into RISC, we believe this to be unlikely given the functionality of many significantly more modified siLNAs. Rather, as these positions are all close to the site where RNA target cleavage occurs [between pos. The SARS siRNA (Table 1) has identical closing base-pairs at both ends (A:U) making it likely that enough of both the antisense and sense strand would be incorporated into RISC to observe activity on the respective targets.
cord-350286-n7ylgqfu	2020	The results of this analysis are summarized in Table 2 , which clearly shows that most of the SARS-CoV-2 proteins contain at least one MoRF, indicating that disorder does play an important role in the functionality of these viral proteins. As it follows from Figure 3 , these cleavage sites are located within the IDPRs. In Human SARS CoV S protein, fusion peptide (residues 770-788) is located within a flexible region, is characterized by the mean disorder score of 0.232±0.053. Global analysis of intrinsic disorder in the replicase polyprotein 1ab Table 3 represents the PPID mean scores of 15 non-structural proteins (Nsps) derived from the Replicase polyprotein 1ab in SARS-CoV-2, Human SARS CoV, and Bat CoV. Similar to many other non-structural proteins of coronaviruses, Nsp15s from SARS-CoV-2, Human SARS, and Bat CoV are predicted to possess multiple flexible regions but contain virtually no IDPRs (see Figures 32A, 32B, and 32C) .
cord-350342-j4p8235a	2020	All of the SARS-CoV-2-challenged hamsters had detectable viral RNA in pharyngeal swabs at the first time point assayed, 3 dpi, and remained consistent (10 3 to 10 5 molecules of nucleocapsid using a second primer set [N2] per 100 ng RNA) throughout the duration of CyP treatment (Fig. 1C) . Viral RNA and infectious virus were detected in lung tissue from a subset of hamsters collected 13 dpi, on the day of euthanasia of moribund animals (14 to 34 dpi), or after euthanasia at 35 dpi (end of study) ( Fig. 1E and F). Electron microscopy studies were performed on lung sections of SARS-CoV-2infected, CyP-treated hamsters with various lung viral loads (Fig. 1) . Similarly, lung tissue collected at 13 dpi (end of study) indicate comparable levels of viral RNA detected (Fig. 6D ) but significantly reduced infectious virus in Centi-F1 MAb-treated animals (P Ï­ 0.0002; unpaired t test) (Fig. 6E) .
cord-350533-fp1ctpax	2020	Moreover, we identified 2''-deoxy-2''-amino-CTP as a novel inhibitor of CCHFV RdRp. We further show that CC.4 inhibits the CCHFV-associated DUB activity of the full-length L protein and the isolated DUB domain to a similar extent. Inhibition of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein ribavirin-TP or favipiravir-TP are also used as substrates opposite template C (Fig 3, middle) . Inhibition of the Crimean-Congo Hemorrhagic Fever Virus full-length L-protein CCHFV L full-length protein and the isolated OTU domain cleave ubiquitin-AMC in a time dependent manner with similar velocities illustrated by the slopes of the initial linear portion of product formation (Fig 8B) . The identification of small molecule compounds that inhibit viral RNA synthesis relies on the availability of recombinant L protein, which is a multifunctional protein with a high molecular weight of~450 kDa. Here we report the expression of full-length CCHFV L protein that possess active RdRp and DUB domains.
cord-350600-73q8mve4	2005	A method is described for the detection of human coronavirus 229E (HCV 229E) in nasal washings using RNA:RNA filter hybridization. In this paper we describe a specific and sensitive test to detect one of these major groups, HCV 2293, in nasal washings. Briefly, using a method based on that of Gubler and Hoffmann [19831, cDNAs were generated from HCV 2293 RNA isolated from infected C16 cells [Phillpotts, 19831. The results of virus titration and probing of nasal washings from seven volunteers are presented in Table I . Three of the seven volunteers suffered a cold, and all three volunteers had detectable coronavirus RNA in their nasal washings. The results we have obtained show that the detection of HCV 2293 infection by nucleic acid hybridisation is a reliable and specific method. Hybridisation to known quantities of HCV 2293 RNA showed that less than 1 ng of virus-specific RNA from C16 cells infected with HCV 2293 could be detected.
cord-350640-sz6xj5o3	2012	When transfecting our infectious firefly luciferase reporter virus genome Luc-Jc1 [14] into highly permissive Huh-7.5 human hepatoma cells [15] cultured in the presence or absence of serum, we measured comparable levels of luciferase activity 4 to 48 h after transfection ( Figure S1A ). Among the inhibitors tested, U0126 a selective inhibitor of the MAPK/ERK pathway, substantially decreased the production of infectious virus particles as is evident from the dose-dependent reduction of luciferase activity in the inoculated cells ( Figure 1B) . To investigate if PLA2G4A activity is relevant for the production of infectious HCV particles we treated Luc-Jc1 transfected cells with pyrrolidine-2 (Py-2), a specific inhibitor of this type of phospholipase [22, 23] . Irrespective of culturing these cells in the presence or absence of serum, we observed a strong and dose-dependent inhibition of production of infectious particles resulting in a more than 100-fold reduction of luciferase transduction at 5 and 20 mM of Py-2, respectively ( Figure 2B ).
cord-350697-u032yk0z	2020	Lack of effective prophylaxis against COVID-19 has prompted regulatory authorities to propose boosting of immunity of individuals via nutritional supplements. While modern medicine directly confronts an antigen (via vaccination or antibiotic), in comparison nutraceuticals, food supplements, and traditional medicines activate the overall immunity of the human body. Both molecules have a proven history of antiviral activity in both in vitro and in vivo trials thus could be leadings in developing new prophylactic candidates against COVID-19. Thus, these supplements as a part of the food, nutraceutical, or traditional medicines may pave the way towards developing a therapeutic strategy against the COVID-19 pandemic. The success story of TCM is continuously inspiring us to test food supplements and raise individual immunity. Potential mechanisms by which Curcumin and Zinc can exert therapeutic effects against COVID-19. Curcumin inhibits SARS-CoV-2 entry by binding directly to the receptorbinding domain (RBD) of Spike (S) protein of the virus.
cord-350747-5t5xthk6	2005	It was believed until recently that the only possible mechanism of RNA recombination is replicative template switching, with synthesis of a complementary strand starting on one viral RNA molecule and being completed on another. An illustrative example of deletions is provided by defective interfering (DI) genomes, which accumulate in a virus population upon high-multiplicity infections and lack a fragment of the sequence coding for viral proteins [5] [6] [7] . A special role in the variation of RNA viruses is played by recombination, the generation of new genomes from two or more parental RNAs. Recombination between viral RNA molecules was observed for the first time as early as in the 1960s in the poliovirus [14, 15] . In other words, it is possible to assume that some of the mechanisms of nonreplicative RNA recombination play an important role in the evolution of not only viral, but also cell genomes [51, 90] .
cord-350762-rh4zbehk	2015	In this study, we utilize microRNA (miRNA)-expressing constructs (a type of RNA interference) in an attempt to target and knockdown five NDV structural RNAs for nucleoprotein (NP), phosphoprotein (P), matrix (M), fusion (F), and large (L) protein genes. Using pre-miRNA to activate the cellular RNAi pathway, a miRNA can be used to target the messenger RNA of NDV structural proteins, leading to the degradation of the transcripts and inhibiting viral replication [11] . In this study, we attempted to determine if constitutive expression of miRNA sequences targeting the mRNA of five of the structural NDV proteins in chicken embryo fibroblast cells (DF-1) would lead to decreased viral yield after infection, and/or resistance against NDV cytopathic effects. Inhibition of Newcastle disease virus replication by RNA interference targeting the matrix protein gene in chicken embryo fibroblasts
cord-350836-1enteev7	2019	RIG-I (Retinoic acid-inducible gene I) and MDA5 (Melanoma Differentiation-Associated protein 5), collectively known as the RIG-I-like receptors (RLRs), are key protein sensors of the pathogen-associated molecular patterns (PAMPs) in the form of viral double-stranded RNA (dsRNA) motifs to induce expression of type 1 interferons (IFN1) (IFNα and IFNβ) and other pro-inflammatory cytokines during the early stage of viral infection. For the former, siRNA-mediated knock-down (110, 111) , cellular knockout (112) and inhibition by viral protein (109, (113) (114) (115) (116) conditions for TRIM25 in multiple cell types have been shown to change RIG-I cellular localization (110) and to negatively affect RIG-I K63 ubiquitination, association with MAVS and IFN signaling [when the constitutively active RIG-I CARD domain was overexpressed (109, (112) (113) (114) (115) (116) or during viral infection (109, 111, 114) ].
cord-350906-ew04zzh6	2018	10 East-Seletsky et al 10 have taken the advantage and demonstrated the use of Cas13 for the detection of 0.01 nM of λRNA with high specificity, using the signals generated from fluorophore-quencher based reporter RNA molecule ( Figure 1 ). The resultant amplified DNA sequence is then subjected to in vitro T7 transcription, and produced RNA molecule is detected by Cas13-guided reporter assay. 11 For improved multiplexed detection, Cas12a has been used along with Cas13 RNAse. 11 F I G U R E 1 Schematic representation of CRISPR-based diagnostic tool, in the presence of the appropriate gRNA, Cas13 recognizes the target RNA sequence that is complementary. The produced RNA molecule can be recognized via fluorescence signals that are generated by the reporter molecule upon target recognition by the Cas13 RNAse. 4 The cleaved reporter molecule can also be detected in the form of bands through a lateral flow assay.
cord-351115-dy81dtnk	2020	This study addresses both by utilizing evolutionary information from SARS-CoV-2 sequence and structural data to search for actionable functional sites for each protein in the SARS-CoV-2 genome. Here we systematically suggest potential drug target sites for most SARS-CoV-2 proteins based on evolutionary information. This relative ranking re ects the variation entropy of each sequence position within and across the branches of an associated phylogenetic tree, revealing evolutionary pressure points that correspond to functional and structural determinants, and the protein sites at which they often cluster (30) . As in our approach to discover ET drug sites, we combined ET residue ranking information with sequencing data from SARS-CoV-2 isolates to arrive at linear peptides along the proteome that are evolutionarily important and also show little variation in the current outbreak ( Figure S6 , Dataset S5). The data include, for example, multiple sequence alignments, precalculated ET ranks, and predicted epitopes (both linear and structural) for all SARS-CoV-2 proteins.
cord-351365-dc9t3vh3	2016	Consequently, the onset of RBV treatment in chronically HEV-infected individuals can result in two divergent outcomes: viral extinction versus selection of fitness-enhanced viruses. Following an overview of RNA viruses treated with RBV in clinics and a summary of the different antiviral modes of action of this drug, we focus on the mutagenic effect of RBV on HEV intrahost populations, and how HEV is able to overcome lethal mutagenesis. Figure 1 provides an overview of a selection of RNA viruses against which RBV was shown to be active: hepatitis C virus (HCV, Flaviviridae), dengue virus (DENV, Flaviviridae), respiratory syncytial virus (RSV, Paramyxoviridae), influenza A and B virus (Orthomyxoviridae), chikungunya virus (CHIKV, Togaviridae), poliovirus (Picornaviridae), Hantaan virus (Bunyaviridae), and Lassa virus (Arenaviridae) [28, 29] ( Figure 1 ). Furthermore, mechanisms on the virus itself were described by inhibition of the capping efficiency, the viral polymerase, and a mutagenic effect on newly synthesized RNA genomes. A Mutation in the hepatitis E virus RNA polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients
cord-351377-xorj8tnz	2018	Moreover, inoculation with iPEDVPT-P96 elicited comparable levels of anti-PEDV specific plasma IgG and fecal/salivary IgA, neutralizing antibody titers, and similar but less effective immunoprotection against the virulent PEDVPT-P5 challenge compared to the parental PEDVPT-P96. In the present study, an infectious cDNA clone of an attenuated G2b PEDV strain was successfully generated for the first time, and the in vitro and in vivo data indicate that iPEDVPT-P96 is further attenuated but remains immunogenic compared to its parental PEDVPT-P96 viral stock. While one piglet in the PEDVPT-P96 group showed intermittent loose diarrhea (score = 1) and viral shedding at 6 to 11 days post-inoculation (DPI) with the peak viral titer of 1.45 ± 3.24 log 10 RNA copies/mL at 8 DPI (Figure 4) , no evidence of PEDV-associated clinical signs and fecal viral shedding were demonstrated in both iPEDVPT-P96 and mock groups.
cord-351482-hzh5tyoo	2011	The small RNAs identified also included many non-miRNA small RNAs, such as small nucleolar RNAs (snoRNAs), in addition to nonannotated small RNAs. An integrative sequencing analysis of both small RNAs and long transcripts from the same samples showed that the results revealing differential expression of miRNAs during infection were largely due to transcriptional regulation and that the predicted miRNA-mRNA network could modulate global host responses to virus infection in a combinatorial fashion. In total, of 4,473,273 start positions in the genome with at least one uniquely mapped read, we found that about 5% (233,236) gave at least 4 reads of the same length in a sample, resulting in 16,054 nonredundant candidate loci for putative small RNAs. About 1.7% (276/16,054) of the candidate loci (median length, 39 nt) were differentially expressed during SARS-CoV and/or influenza virus infection (see Table S2 and Fig. S4a in the supplemental material); 46 of those candidate loci overlapped with annotated miRNA precursors (miRBase version 16).
cord-351489-tzmev77c	2020	They were first evaluated in our primary screening in VeroE6 cells and then the most potent anti-SARS-CoV-2 antiviral agents were further evaluated using viral antigen expression, viral load reduction, and plaque reduction assays. In addition to remdesivir, lopinavir, and chloroquine, our primary screening additionally identified types I and II recombinant interferons, 25-hydroxycholesterol, and AM580 as the most potent anti-SARS-CoV-2 agents among the 22 antiviral agents. In this primary screening, chloroquine, lopinavir, and remdesivir which were recently reported to have anti-SARS-CoV-2 activity, exhibited about 1.3-2.0 log10 copies/mL reduction in viral RNA load ( Figure 1 ). In comparison, recombinant IFN-β demonstrated the most potent anti-SARS-CoV-2 activity, with Avonex (IFN-β1a), Rebif (IFN-β1a), and Betaferon (IFN-β1b) each achieving about 3 log10 copies/mL reduction in viral load. In our primary screening using a fixed antiviral agent concentration and virus inoculum, we identified recombinant IFNs and lipogenesis modulators to be the most potent anti-SARS-CoV-2 agents among 22 broad-spectrum antivirals.
cord-351520-c5fi2uoh	2010	Recently, we and others identified a new adapter protein called mediator of IRF3 activation (MITA, also known as STING), which plays a critical role in virus-induced type I IFN expression (Ishikawa and Barber, 2008; Zhong et al., 2008) .
cord-351548-jvl63652	2013	Finally, a recent transcriptomic analysis of newly synthesized RNA in MCMV infected fibroblasts [15] applied RNA-Seq technology to study regulation of viral gene expression and identified a very early peak of viral gene transcriptional activity at 1-2 hours post infection followed by rapid cellular countermeasures but did not attempt to re-annotate MCMV genome. The MCMV transcripts identified through our classical cDNA cloning and sequencing (green arrows) and the RNA-Seq expression profiles (gray histograms), showed complementary results to each other but diverged dramatically from current annotations. Because cDNA cloning and RNA-Seq identified significant differences between the MCMV transcriptome and current annotations, we performed an in depth analysis of several genomic regions by northern analyses ( Figure 5 , Figures S2, S3 , S4, S5) using our cDNA clones to generate strand specific riboprobes ( Table 1) .
cord-351559-az4pgi9k	2020	Regulatory roles of long non-coding RNAs (lncRNAs) during viral infection and associated antagonism of host antiviral immune responses has become more evident in last decade. To elucidate possible functions of lncRNAs in the COVID-19 pathobiology, we have utilized RNA-seq dataset of SARS-CoV-2 infected lung epithelial cells. By network enrichment analysis we find that these lncRNAs can directly interact with differentially expressed protein-coding genes ADAR, EDN1, KYNU, MALL, TLR2 and YWHAG; and also AKAP8L, EXOSC5, GDF15, HECTD1, LARP4B, LARP7, MIPOL1, UPF1, MOV10 and PRKAR2A, host genes that interact with SARS-CoV-2 proteins. Conclusions Our investigation determines that deregulated lncRNAs in SARS-CoV-2 infection are involved in viral proliferation, cellular survival, and immune response, ultimately determining disease outcome and this information could drive the search for novel RNA therapeutics as a treatment option.
cord-351837-vasuu70k	2020	title: Rapid incorporation of Favipiravir by the fast and permissive viral RNA polymerase complex results in SARS-CoV-2 lethal mutagenesis It possesses both unusually high nucleotide incorporation rates and high-error rates allowing facile insertion of Favipiravir into viral RNA, provoking C-to-U and G-to-A transitions in the already low cytosine content SARS-CoV-2 genome. This enzyme readily incorporates T-705-ribose-5′-phosphate into viral RNA in vitro, and cell culture based infectious virus studies show an increase in mutations in the presence of Favipiravir. To determine the efficacy and MoA of T-705 against SARS-CoV we first characterised nsp12 primerdependent activity using traditional annealed primer-template (PT) and self-priming hairpin (HP) RNAs that may confer additional stability on the elongation complex ( Supplementary Fig. 1c) . These data reveal that the SARS-CoV nsp12 is the fastest viral RdRp known, with rates significantly faster than the 5-20 s −1 observed for picornaviral polymerases at room temperature [33] [34] [35] and 4-18 s −1 for hepatitis C and dengue virus polymerases at 30 and 37°C 36, 37 .
cord-351854-5s03f0pp	2020	title: Pooled RNA extraction and PCR assay for efficient SARS-CoV-2 detection We have implemented the method in a routine clinical diagnosis setting, and already tested 2,168 individuals for SARS-CoV-2 using 311 RNA extraction and RT-PCR kits. Three such limitations might be: (1) a limit on the number of stages due to the importance of delivering a test result quickly, exemplified by the urgent clinical context of COVID-19 diagnosis; (2) a limit on the ability to dilute samples and still safely identify a single positive sample in a pool; (3) favorability of simple algorithms which may minimize human error in a laboratory setting. . https://doi.org/10.1101/2020.04.17.20069062 doi: medRxiv preprint probability of a sample to be positive by p (prevalence of detectable COVID-19 patients in the relevant population) and the pool size by n. This allows for reliable and efficient screening of large asymptomatic populations for the presence of SARS-CoV-2 infection, even when RNA extraction and RT-PCR reagents are in short supply.
cord-351864-zozrj7w5	2020	Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays that involve a separate RNA extraction and qPCR reaction for each sample with a fixed cost and reaction time. Our workflow, ApharSeq, includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Briefly, we show ( Figure 1 ) that we can introduce barcoded and target-specific reverse transcription primers to the samples, allowing them to hybridize to target RNA molecules already in the lysis buffer, or after a brief RNA cleanup step. Observing the target sequence directly allowed us to identify viral sequence variations in some cases ( Figure 2D ).Cross-Sample Contamination is minimal When pooling samples early on in the protocol, the main concern is that RNA molecules will be erroneously tagged due to residual free primers, or due to other artifacts during RT, PCR, or sequencing.
cord-351920-igmb2yfe	2016	The aims of the study were to investigate the duration and quantity of BCoV shedding in feces and nasal secretions related to clinical signs, the presence of virus in blood and tissues and to test the hypothesis that seropositive calves are not infectious to naïve in-contact calves three weeks after BCoV infection. In two experimental studies, infected calves were not protected against reinfection with a different BCoV strain three weeks after the first challenge, but did not develop clinical signs [19, 20] . The majority of experimental studies have used BCoV inoculation as challenge procedure, which may influence clinical signs and viral shedding, and thereby the transmission potential compared to natural infection. The present study showed that calves infected with BCoV shed viral RNA for five weeks, and harbored viral RNA in intestinal tissues and lymph nodes even longer.
cord-352088-9k01ej6l	2020	The authors show that vaccination with the GP5-Mosaic-based vaccines resulted in cellular reactivity and higher levels of neutralizing antibodies to both VR2332 and MN184C PRRSV strains, whilst vaccination with the GP5-WT vaccines only induced response against the heterologous challenging virus (VR2332). Another important disease in pigs is that caused by the porcine epidemic diarrhea virus (PEDV), a coronavirus responsible of highly contagious intestinal infections that may result in the death of newborn piglets and weight loss in pigs of all ages, and that seriously damages the swine industry. López-Gil and coworkers [7] , by using an approach based on the modified vaccinia Ankara (MVA) encoding the RVFV glycoproteins (rMVAGnGc), extend their previous observations that a single inoculation was sufficient to induce a protective immune response in mice after a lethal viral challenge, which was related to the presence of glycoprotein specific CD8+ cells and a low-level detection of in vitro neutralizing antibodies.
cord-352178-irjhmxsg	2013	Fifteen distinct chimeric viruses were constructed to evaluate both structural and non-structural regions of the genome and infection patterns were determined through artificial infectious feeds in An. gambiae with each of these chimeras. When ONNV non-structural protein 3 (nsP3) replaced nsP3 from CHIKV virus in one of the chimeric viruses, infection rates in An. gambiae went from 0% to 63.5%. Our study analyzed both structural and non-structural regions of the ONNV genome using chimeric viruses and artificially infected Anopheles gambiae mosquitoes. When ONNV non-structural protein 3 (nsP3) replaced nsP3 from chikungunya virus in one of the chimeric viruses, infection rates in An. gambiae went from 0% to 63.5%. Six additional non-structural chimeric viruses were also constructed using a novel type II restriction enzyme cloning strategy to examine the broader genome with respect to ONNV''s unique vector specificity for An. gambiae mosquitoes (Figure 2) .
cord-352200-i05h8csb	2012	title: Transcriptome and Comparative Gene Expression Analysis of Sogatella furcifera (Horváth) in Response to Southern Rice Black-Streaked Dwarf Virus METHODOLOGY/PRINCIPAL FINDINGS: By de novo transcriptome assembling and massive parallel pyrosequencing, we constructed two transcriptomes of WBPH and profiled the alternation of gene expression in response to SRBSDV infection in transcriptional level. As a whole, 81388 distinct unigenes have been identified and the results indicated that SRBSDV infection can potentially perturb primary metabolism and the ubiquitin-proteasome pathway of WBPH and activate immune regulatory systems, such as RNA interfering, autophagy and antimicrobial peptide production. However, some unigenes were obtained only from viruliferous or non-viruliferous samples (data not shown) and we believe these differences may be caused by distinctions that arise from long-term ecological adaptation to virus infection. In addition, GO analysis also showed a similar distribution of gene functions for non-viruliferous and viruliferous WBPH (Figure 4 ), indicating that the number of genes expressed in each GO category was not significantly affected by SRBSDV infection.
cord-352361-jh31omg2	2020	Although several rodents and other small mammals are known as important reservoirs for many viruses, bats (order: Chiroptera) represent the vast majority of identified natural reservoirs of several virus families/species to date [1, 14] . In conclusion, due to the continuous detection of new viruses in bats, the unclear situation regarding additional potential BoDV-1-reservoirs and molecular evidence for co-evolution of bats and bornaviruses, this study was conducted to investigate the potential presence of the most common orthobornaviruses in bats from endemic and non-endemic areas in Germany. Although the bicolored white-toothed shrew has been identified as indigenous reservoir of BoDV-1, other potential reservoirs or animal carriers are still unknown so that further investigations of small mammals including bat species are urgently needed. Distribution of Borna disease virus antigen and RNA in tissues of naturally infected bicolored white-toothed shrews, Crocidura leucodon, supporting their role as reservoir host species
cord-352379-q5inrxcm	2003	Nevertheless, the lack of a firm association of coronaviruses with any serious human illnesses had dampened the public''s interest in this virus family until the sudden emergence of the SARS coronavirus [24, 41, 62] , which caused the first new infectious disease of this millennium. In the SARS virus genome, the organization of gene la-lb, which accounts for more than two-thirds of the viral RNA, is very similar to that of the murine coronavirus MHV, except that it contains only one papain-like protease (PLpro-2) ( fig. Based on the predicted cleavage site specificity, the SARS virus gene la-lb is likely processed into thirteen final protein products. However, the published sequence analysis indicated that the entire SARS virus RNA resembled that of group II viruses; no evidence of recombination was noted [55, 66] . Comparative full-length genome sequence analysis of 14 SARS coronavirus isolates and common mutations associated with putative origins of infection
cord-352465-n746e8qt	2020	Chronic stress might even induce formation of cytotoxic pathological SGs. SGs participate in various biological functions including response to apoptosis, inflammation, immune modulation, and signalling pathways; moreover, SGs are involved in pathogenesis of neurodegenerative diseases, viral infection, aging, cancers and many other diseases. One of the most studied mRNP granules is SGs. SGs are a type of dynamic granular substance formed of mRNA of stagnant translation and RBPs in the cytoplasm of eukaryotic cells, the formation of which is stimulated by various stresses including oxidative stress, heat shock, hypoxia, or viral infection (Fig. 1) . For example, eIF2α phosphorylation-dependent SGs (Type I) induced by sodium arsenite (SA) and bortezomib [40] may protect cells in the stress response, inhibit apoptosis and promote cell survival by the sequestration of signalling molecules, such as RACK1 [41] , ROCK1 [42] and Raptor [43] .
cord-352664-heoj8ji8	2015	In this study, we developed a robust and rapid ''field pathogenomics'' strategy, using transcriptome sequencing of PST-infected wheat leaves to gain insight into the population structure of an emerging pathogen. To characterize the genotypic diversity of PST at the field level, we collected 219 samples of wheat and triticale infected with PST from 17 different counties across the UK in the spring and summer of 2013 ( Figure 1a ; Table S1 in Additional file 1). To determine the relationship between the 2013 PST field isolates and previously prevalent PST populations, the genomes of 14 UK and 7 French purified PST isolates collected between 1978 and 2011 were sequenced using an Illumina whole-genome shotgun approach Figure 2 Identification of wheat varieties using transcriptome data generated directly from PST-infected field samples. We used multivariate discriminant analysis of principal components (DAPC) with the 34,764 biallelic SNP sites to define the population structure and identify groups of genetically related PST isolates.
cord-352768-16vgnq14	2008	Containment of the SARS coronavirus (SCV) outbreak was accompanied by the rapid characterization of this new pathogen''s genome sequence in 2003, encouraging the development of anti-SCV therapeutics using short interfering RNA (siRNA) inhibitors. A pair of siRNA duplexes identified as potent SCV inhibitors in vitro was evaluated for in vivo efficacy and safety in a rhesus macaque SARS model using intranasal administration with clinical viable delivery carrier in three dosing regimens. Observations of SCV-induced SARS-like symptoms, measurements of SCV RNA presence in the respiratory tract, microscopic inspections of lung histopathology, and immunohistochemistry sections from 21 tested macaques consistently demonstrated siRNA-mediated anti-SCV activity. A pair of siRNAs showing prominent prophylactic and therapeutic activities in the cell culture study (29), referred to as siSC2 and siSC5, were further evaluated in vivo, first in mice using a reporter gene assay and subsequently using a clinically acceptable intranasal administration in the recently established rhesus macaque SARS model (23-26).
cord-352814-fcl2g5wr	2011	In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The aim of this work was to develop a real-time PCR assay for diagnosing infection with SARS-related coronaviruses from bat guano in order to use it as a screening tool in epidemiological surveys for the detection of the viruses. The developed SYBR Green real-time PCR techniques were applied to an SARS-like coronavirus survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) which were sampled in Italy in 2009, resulting in a prevalence of coronavirus infection of 42%. After optimisation of the SYBR Green real-time PCR assay, for each run, duplicates of six 10-fold dilutions of the standard plasmid, triplicates of the viral reverse-transcribed RNA of the bat samples, and a no template control were simultaneously subjected to analysis.
cord-352891-ljmkqdzx	2020	title: Comparative Antiviral Activity of Remdesivir and Anti-HIV Nucleoside Analogs against Human Coronavirus 229E (HCoV-229E) Herein, we report the antiviral activity of remdesivir against human coronavirus 229E (HCoV-229E) compared to known anti-HIV agents. These agents included tenofovir (TFV), 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA), alovudine (FLT), lamivudine (3TC), and emtricitabine (FTC), known as nucleoside reverse-transcriptase inhibitors (NRTIs), and a number of 5′-O-fatty acylated anti-HIV nucleoside conjugates. Therefore, HCoV-229E may be a good initial model for the evaluation of antiviral compounds that could have potential applications against other coronaviruses, such as SARS-COV-2, the coronavirus that causes COVID-19. We have previously shown that the conjugation of certain fatty acids to the anti-HIV NRTIs, such as FLT, 3TC and FTC, enhanced activity against X4, R5, cell-associated, and/or multi-drug resistant virus when compared with their parent nucleosides [24] [25] [26] [27] . A series of anti-HIV nucleosides and their fatty acyl derivatives were compared with remdesivir for antiviral activity against HCoV-229E in MRC-5 cells.
cord-352991-duqkpkll	2013	Although RSV RNA has been detected in serum from patients with RSV lower respiratory disease (LRD) after HCT, the association with clinical outcomes has not been well established in multivariable models. Univariate analysis at day 90 following RSV LRD revealed several factors that were statistically significantly associated with overall mortality and pulmonary death including transplant year, cell source, conditioning regimen, WBC count, monocyte count, platelet count, oxygen requirement at diagnosis, steroid use at diagnosis, palivizumab treatment, and ribavirin treatment following LRD (Supplementary Table 1 ). Hazard ratios and 95% confidence intervals from multivariable models evaluating respiratory syncytial virus (RSV) RNA detection in blood as a risk factor for overall mortality and pulmonary death by day 90 following RSV lower respiratory disease (LRD; n = 92), including P values.
cord-353274-wozwpvpq	2020	In this study we quantified IgG and IgM antibody kinetics and RNA shedding probability during SARS-CoV-2 infection (up to 60 days post symptom onset) by drawing on published data. This formal integration approach enabled us to leverage 3,214 data points from 516 individuals with symptoms ranging from asymptomatic to critical, published in 22 studies, resulting in a quantitative synthesis of diverse data on anti-SARS-CoV-2 antibody patterns and RNA shedding during the early phase of infection. One of the goals of this study is to estimate the means and variation of IgG and IgM seroconversion times (time between symptom onset and first antibody detection) for different assays, antigens, and disease severity. . https://doi.org/10.1101/2020.05.15.20103275 doi: medRxiv preprint weighted bootstrapping procedure integrates all types of data that contain useful information about the timing of seroconversion of different antibodies in day(s) post symptom onset (dpo). The probability of detecting SARS-CoV-2 specific IgG or IgM in plasma or serum samples was estimated by integrating data on whether an individual tested positive or negative on a given dpo.
cord-353290-1wi1dhv6	2020	We investigated possible reasons for the advantage of A-rich sequences including weakened RNA secondary structures, codon usage bias, and selection for a particular amino-acid composition, and conclude that host immune pressures may have led to similar biases in coding sequence composition across very divergent RNA viruses. Nevertheless, RNA viruses do share several common features that drive their evolution: (a) their ultimate dependence on the cell, (b) their high mutation rates, (c) strong purifying selection derived from constraints operating on a small and densely coding genome, and (d) sporadic but powerful positive selection driven by an evolutionary arms race with the host they infect. Two non-mutually exclusive hypotheses may be put forth to explain the consistent pattern of A-richness that we observe: there is selection for more A in viral sequences, and/or there is a mutational bias that leads to more A in genomes of viruses.
cord-353342-2n6kqyeo	2016	title: Viral Shedding and Antibody Response in 37 Patients With Middle East Respiratory Syndrome Coronavirus Infection The Middle East respiratory syndrome (MERS) coronavirus causes isolated cases and outbreaks of severe respiratory disease. We studied 37 adult patients infected with MERS coronavirus for viral load in the lower and upper respiratory tracts (LRT and URT, respectively), blood, stool, and urine. Quantitative data, such as viral loads and antibody titers, could enable comparisons with related diseases, in particular, severe acute respiratory syndrome (SARS), for which studies of natural history were conducted in the aftermath of the 2002-2003 epidemic [7] . DISCUSSION We studied quantitative viral excretion and serum antibody kinetics of a substantial group of hospitalized patients infected with MERS-CoV. Detection of SARS coronavirus in patients with severe acute respiratory syndrome by conventional and real-time quantitative reverse transcription-PCR assays
cord-353475-dtn7h1gj	2020	In this work, via the miRDB database, we determined the target scores of predicted human miRNA to bind with the ss-RNA of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) in general and its spike gene in specific. The exciting findings here that the nucleotide substitution 1841A > G at the viral genomic RNA level, which is an amino acid substation D614G at the spike protein level showed a change in the predicted miRNA sequence from hsa-miR-4793-5p to hsa-miR-3620-3p with an increase in the target score from 91 to 92. To understand the early steps of COVID-19 infection, we predicted miRNAs sequences targeting the submitted 29903bp of viral ss-RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 complete genomic RNA sequence) from the isolate of Wuhan-Hu-J o u r n a l P r e -p r o o f 1.
cord-353484-q7d0ysbo	2020	Given the urgency of the outbreak, we focus here on recent advances in the diagnostics, treatment, and vaccine development for SARS-CoV-2 infection, helping to guide strategies to address the current COVID-19 pandemic. Another type of rapid diagnostic test (RDT) that detects the presence of viral antigens expressed by SARS-CoV-2 virus in a respiratory tract sample is of low complexity and may provide results typically within 30 minutes [68, 69] . Studies in Vero E6 cells have suggested that favipiravir can cripple the SARS-CoV-2 virus (EC50 = 61.88 μM) [88] , and patients with COVID-19 are being recruited in randomized trials to evaluate the efficacy of favipiravir plus other antivirals (e.g., ClinicalTrials.gov: ChiCTR2000029600, ChiCTR2000029544). As no specific therapeutic agents or vaccines are available for COVID-19, this therapy is the only strategy that is immediately available for use to prevent and treat a novel, emerging infectious disease such as SARS-CoV-2 infection [121, 122] .
cord-353524-3w970ycx	2020	Given that SARS-CoV-2 and SARS-CoV share very high identical sequence in their 3CLpro, these HIV protease inhibitors are currently again repurposed for the treatment of COVID-19 (Chinese Clinical Trial Registry: ChiCTR2000029539). 30, 31 The interplay of the ACE receptor in cardiovascular diseases (with the well-known drug class of ACE inhibitors) and as the docking point for SARS-CoV-2 cellular infection is a current point of intense debate and research. For example, the crystal structure of SARS-CoV-2 N protein RNA-binding domain was just published and will give structural insight as a potential drug target. Potential broad spectrum inhibitors of the coronavirus 3CLpro: A virtual screening and structure-based drug design study Severe acute respiratory syndrome coronavirus papain-like novel protease inhibitors: design, synthesis, protein-ligand X-ray structure and biological evaluation Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites
cord-353576-f29kmtot	2020	We then tested 1 l of mouthwash from each of the 20 individuals using two RT-qPCR kits advertised to allow direct detection of SARS-CoV-2 from nasopharyngeal swabs: Luna Universal Probe (NEB, Ipswich, USA) and PrimeDirect (Takara, Kyoto, Japan) as well as another kit, SuperScript III with Platinum Taq (Invitrogen, Waltham, USA). To systematically investigate how the NEB Luna assay performs compared to RNA extraction followed by the Roche assay for mouthwash samples, we investigated 62 gargle lavages from patients that were either negative or presented with various viral loads based on previous investigations. In the first scheme, the samples were tested individually using the direct RT-qPCR protocol and the results were evaluated and reported back to the facility by 7 p.m. To detect any inhibition that the mouthwash samples may introduce into the RT-qPCR reactions, we added a synthesized control RNA that was quantified in parallel with SARS-CoV-2 by a probe .
cord-353640-giznbcpd	2020	RT-PCR results using the ChromaCode HDPCR™ SARS-CoV-2 were compared using the heat-RNA release method and an automated RNA extraction system (EMAG). We compared the diagnostic sensitivity of the heat-RNA release method for detection of SARS-CoV-2 using NP swabs in viral transport media (VTM) to results obtained using an automated RNA extraction system (EMAG ® , bioMerieux, Durham, NC). A total of 174 COVID-19 positive samples were used for the study comprising 87 unique COVID-19 NP including Ct values using the heat-RNA release method were compared to the results obtained using the automated RNA-extraction method with both the LDT and ChromaCode SARS-CoV-2 assays. Comparison of the heat-RNA release method to EMAG ® using the ChromaCode SARS-CoV-2 RUO alone yielded a 94% agreement (81/86 positive samples). We found that the direct detection of SARS-CoV-2 using a 65°C heat inactivation and release step for 20minutes without RNA extraction and purification performed very well compared to the use of an automated RNA extraction instrument.
cord-353703-u86ggw11	2019	We provide evidence that the virus targets the UPR central regulator GRP78 for proteasomal degradation via a mechanism that requires viral glycoprotein GP2a, while both IRE1-XBP1s and PERK-eIF2α-ATF4 signaling branches of the UPR are turned on at early stage of infection. In support of our hypothesis, antibodies to ATF4, but not the isotype control (IgG), could immunoprecipitate viral RNA from infected MARC-145 cells, as detected by RT-qPCR of the PRRSV 5''UTR region ( Fig 7D) . Because GRP78 is the master regulator, its reduced accumulation likely prevents any modulation of the UPR, keeping the ER stress pathways induced for The abundance of viral RNA was assessed by RT-PCR of ORF7, normalized against the house-keeping gene GAPDH, and then compared the benefit of the virus.
cord-353810-mf753ae9	2019	This report also describes a successful application of capture-based purification as a direct RNA sequencing strategy that addresses certain limitations of current strategies in sequencing RNA viral genomes. Based on the mapping rates to human and DENV1 reference genomes for pre and post-capture groups, shown in Table 2 , purification factor was calculated to be 272-fold. The minimum coverage required for variant calling was, as described above, benchmarked to that of Illumina reads so that the effectiveness of our capture-based purification method could be more accurately evaluated based on the higher error read rates of direct RNA sequencing technology. Indeed, after comparison of the postcapture and concentrated post-capture sequencing runs (Table 2) , the 2.5-fold increase in the percentage of reads mapping to DENV1 suggests that scaling our method greatly improved the signal-to-noise ratio of this particular downstream RNA assay.
cord-354003-ko45l1qv	2020	Pioneering efforts to identify the mechanisms regulating translation of 5 0 TOP mRNAs, however, did show that wheat germ extracts contain a repressor that specifically limits translation of 5 0 TOP mRNAs in cell-free translation assays (Biberman and Meyuhas, 1999; Shama and Meyuhas, 1996) , suggesting that plants also discriminately regulate translation of 5 0 TOP mRNAs. Here, we show that the TOR-LARP1-5 0 TOP signaling axis regulates translation in Arabidopsis, impacting expression of a set of deeply conserved 5 0 TOP genes, including translation elongation factors, polyA-binding proteins, karyopherins/importins, and the translationally-controlled tumor protein. TOR-LARP1-5 0 TOP signaling in Arabidopsis seedlings regulates translation of mRNAs that encode deeply conserved eukaryotic proteins, plant lineage-specific proteins, and diverse proteins involved in ribosome biogenesis.
cord-354051-ro3o27pv	2020	title: SARS-CoV-2 RNA concentrations in primary municipal sewage sludge as a leading indicator of COVID-19 outbreak dynamics We report a time course of SARS-CoV-2 RNA concentrations in primary sewage sludge during the Spring COVID-19 outbreak in a northeastern U.S. metropolitan area. As viral shedding can occur before cases are detected, we hypothesize that the time course of SARS-CoV-2 RNA concentrations in primary sewage sludge is a leading indicator of outbreak dynamics within a community served by the treatment plant. SARS-CoV-2 viral RNA concentrations were quantitatively compared with local hospital admission data and community COVID-19 compiled testing data. SARS-CoV-2 RNA sludge concentrations were quantitatively compared with data that are commonly used to track the community progression of COVID-19 including hospital admissions (Figure 2A This study uniquely utilized primary sewage sludge instead of raw wastewater for virus RNA measurements.
cord-354096-x2skguz8	2020	We hypothesized that SARS-CoV-2 infection drives changes in immune cell-derived factors that then interact with receptors expressed by the sensory neuronal innervation of the lung to further promote important aspects of disease severity, including ARDS. We sought to quantify how immune cells might interact with sensory innervation of the lung in COVID-19 using published data from patients, existing RNA sequencing datasets from human dorsal root ganglion neurons and other sources, and a genome-wide ligand-receptor pair database curated for pharmacological interactions relevant for neuro-immune interactions. Additionally, we found that upregulation of transcription factor genes in COVID-19 samples identifies transcription factors associated with alveolar cell types (EHF, PAX9, ELF3, GHRL2) and immune cells (RFX3, SOX5, TP63, HOPX) with functions including regulation of antiviral pathways (NR3C2), based on ARCHS4 database (Lachmann et al., 2018) and the Enrichr gene set enrichment analysis tool (Kuleshov et al., 2016) (Supplementary Table 2 ).
cord-354114-frdsct44	2010	FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The cats were monitored for clinical signs, body weight, faecal virus shedding and serum antibody titres for 70 days post inoculation. In all animals, the amount of virus in the faeces subsequently increased within two days to peak levels of 10 6 -10 8 genomes/lL faeces, after which shedding remained high for an extended period until day 16 and day 23 post inoculation in FECV UCD and FECV RM infected cats, respectively. In order to determine in which part(s) of the gut virus was present during viral persistence, we screened the contents of the entire intestines of two FECV UCD infected cats.
cord-354394-zojhdnlu	2004	We examined oral specimens, including throat wash and saliva, and found large amounts of SARS-CoV RNA in both throat wash (9.58 x 10(2) to 5.93 x 10(6) copies/mL) and saliva (7.08 x 10(3) to 6.38 x 10(8) copies/mL) from all specimens of 17 consecutive probable SARS case-patients, supporting the possibility of transmission through oral droplets. This finding, with the high detection rate a median of 4 days after disease onset and before the development of lung lesions in four patients, suggests that throat wash and saliva should be included in sample collection guidelines for SARS diagnosis. Using a quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) assay and fractionation experiment, we investigated the load of SARS-CoV in these samples and different components of the throat wash. As shown in Table 1 , SARS-CoV RNA was detected in the cell-associated component of the throat wash from all 16 specimens examined.
cord-354398-f3cg8gi1	2020	The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. We validated the modified Invitrogen Forensic DNA Purification kit in extracting in-laboratory propagated SARS-COV-2 RNA by conducting manual and automated extractions on titrations from 15 000 copies to 60 copies of SARS-COV-2 followed by RT-qPCR methods: the commercially available TaqPath One-Step qRT-QP-CR kit (using the N, S, and ORF1b genes) and primers and probes from Metabion, Germany to establish an inhouse RT-qPCR assay based on E, RdRp2 and RdRp4 gene detection as per recommended SARS-COV-2 testing from CDC and WHO.
cord-354407-zzxjv666	2004	title: Structural genomics of the SARS coronavirus: cloning, expression, crystallization and preliminary crystallographic study of the Nsp9 protein The aetiologic agent of the recent epidemics of Severe Acute Respiratory Syndrome (SARS) is a positive‐stranded RNA virus (SARS‐CoV) belonging to the Coronaviridae family and its genome differs substantially from those of other known coronaviruses. The crystal structure of the main (or 3CL) protease of transmissible gastroenteritis virus, a related coronavirus, has been determined and was used to construct a model of the SARS-CoV 3CL protease, facilitating future drug design against this important target (Anand et al., 2003) . In the related mouse hepatitis virus, which is a group 2 coronavirus, the SARS-CoV Nsp9 corresponds to a 12 kDa cleavage product (P1a-12) that is found preferentially in the perinuclear region of infected cells, where it co-localizes with other components of the viral replication complex (Bost et al., 2000) .
cord-354465-5nqrrnqr	1999	Numerical studies based on kinetic folding and a simple extension of the standard energy model show that the global features of the sequence-structure map of RNA do not change when pseudo-knots are introduced into the secondary structure picture. Numerical studies based on kinetic folding and a simple extension of the standard energy model show that the global features of the sequence-structure map of RNA do not change when pseudo-knots are introduced into the secondary structure picture. In case of one particular class of biopolymers, the ribonucleic acid (RNA) molecules, decoding of information stored in the sequence can be properly decomposed into two steps: (i) formation of the secondary structure, that is, of the pattern of Watson-Crick (and GU) base pairs, and (ii) the embedding of the contact structure in three-dimensional space. On the other hand, an increasing number of experimental findings, as well as results from comparative sequence analysis, suggest that pseudo-knots are important structural elements in many RNA molecules (Westhof and Jaeger, 1992) .
cord-354510-jlg5je0s	2020	Methods Nasopharyngeal and oropharyngeal swab samples were collected from SARS-CoV-2-infected patients and from laboratory personnel using a commercially available viral transport solution (VTM) and the denaturing solution (DS) described here. Nasopharyngeal and oropharyngeal swab samples were collected from SARS-CoV-2-infected patients and from laboratory personnel using a commercially available viral transport solution (VTM) and the denaturing solution (DS) described here. 13 Here we describe the use of a simple, virus-inactivating and denaturing solution as part of a swab collection kit, aiming to decrease the infectious potential of the clinical sample and, at the same time, to preserve highly frail RNA molecules during transportation and short-term storage before testing. In order to increase personnel safety, to avoid losing collaborators due to infections by SARS-CoV-2, and at the same time to increase preservation of the RNA contained in clinical samples, we introduced the use of the guanidinecontaining solution as collection and transport media instead of commonly used viral transport media (VTM).
cord-354529-k8p2u7iq	2020	Clinical information of patients was collected from the electronic medical information system of Jinyintan Hospital, including the following factors: demographic data; date of symptom onset, admission, first CP infusion and discharge; laboratory data before and after infusion of CP, including white blood cell count, neutrophil count, lymphocyte count, liver and kidney function test, and inflammatory factors such as high sensitive C-reaction protein (HsCRP); results of SARS-CoV-2 test and cycle threshold value (Ct value) of quantitative reverse transcription-polymerase chain reaction; patients'' status and treatments before or after the CP therapy, including the vital signs, anti-virus therapy, oxygen therapy, and other treatments; total volume dose of CP; pulmonary imaging examination data; information on complications such as transfusion-related adverse reactions. Clinical Benefit and Outcome of Patients with Prolonged Positivity of SARS-CoV-2 RNA after CP Therapy As shown in Table 3 , the median and interquartile ranged total volume of CP transfusion was 400 (200-400) mL in EN group and 400 (400-800) mL in LN group.
cord-354536-c9v9kbw8	2020	This article introduced the origin, virological characteristics and epidemiological overview of SARS-CoV-2, reviewed the currently known drugs that may prevent and treat coronavirus, explained the characteristics of the new coronavirus and provided novel information for the prevention and treatment of COVID-19. 18 In view of the curative effect of ribavirin in the treatment of diseases caused by SARS-CoV and MERS-CoV, 21 it is expected to become one of the effective drugs to treat coronavirus. 16 The "Pneumonitis Diagnosis and Treatment Scheme for New Coronavirus Infection (Trial Version 7)" states that aerosolized interferon alpha can be used as a trial treatment against SARS-CoV-2 virus to improve the virus clearance effect of respiratory mucosa in patients. 64 It has been revealed that chlorpromazine is a broad-spectrum virus inhibitor that can inhibit HCV, alpha virus, and various coronaviruses including human coronavirus 229E, SARS-CoV and MERS-CoV in vitro.
cord-354582-fniymnmf	2020	In this review, we systematically describe the role of reverse genetics technology in studying the effects of coronavirus proteins on viral virulence and innate immunity, cell and tissue tropism and antiviral drug screening. Recently, reverse genetics techniques, including targeted RNA recombination, in vitro ligation and bacterial artificial chromosome systems, vaccinia virus vectors and transformation associated recombination (TAR) cloning, have been successfully used to manipulate the genome of coronaviruses (Fig. 2 ). Using a recombinant SARS-CoV strain with reduced nsp3 de-ADP-ribosylation activity showed that this mutant strain led to virus attenuation in mice but protected them from an otherwise lethal SARS-CoV infection and significantly enhanced the innate immune response, indicating that it is an important virulence factor for SARS-CoV . The N protein plays an important role in viral pathogenesis since BALB/c mice immunized with recombinant virus MVA-MERS-N exhibit stronger T cell responses and anti-N monoclonal antibodies protect mice from lethal infection by MHV (Nakanaga et al., 1986; Veit et al., 2018) .
cord-354733-qxivrhj8	2020	Whole genome sequencing confirmed the virus genotype in patients with prolonged 28 viral RNA shedding and droplet digital PCR (ddPCR) was used to assess the rate of false 29 negative standard of care PCR results. Whole genome sequencing confirmed the virus genotype in patients with prolonged 28 viral RNA shedding and droplet digital PCR (ddPCR) was used to assess the rate of false 29 negative standard of care PCR results. Prolonged viral RNA shedding was associated with recovery of infectious 31 virus in specimens collected up to 20 days after the first positive result in patients who were 32 symptomatic at the time of specimen collection. Infection control personnel and physicians managing COVID-19 patients and patients under 43 investigation (PUI) continue to face several diagnostic dilemmas related to a lack of 44 understanding of the clinical sensitivities of SARS-CoV-2 molecular diagnostics and the 45 correlation between viral RNA detection and shedding of infectious virus.
cord-354824-7fdcu2f0	2020	Evolving research and clinical data regarding the virologic SARS-CoV-2 suggest a potential list of repurposed drugs with appropriate pharmacological effects and therapeutic efficacies in treating COVID-19 patients. This estimated 20% of patients developing more severe disease with SARS-CoV-2 infection are most likely due to genetics, epigenetics, and or other factors, with dampened innate immune response to fight the virus coupled with enhanced viral load leading to cytokine storm, severe inflammatory/oxidative stress response, and severe lung injury secondary to ARDS. Chloroquine can inhibit the entry of SARS-CoV-2 and prevent virus-cell fusion by interfering with glycosylation of ACE2 receptor and its binding with spike protein, suggesting that chloroquine treatment might be more effective in the early stage of infection, before COVID-19 reduces ACE2 expression and activity [30, 38, 39] . Chloroquine diphosphate in two different dosages as adjunctive therapy of hospitalized patients with severe respiratory syndrome in the context of coronavirus (SARS-CoV-2) infection: Preliminary safety results of a randomized, doubleblinded, phase IIb clinical trial (CloroCovid-19 Study)
cord-354829-god79qzw	2020	title: Identification of robust genetic signatures associated with lipopolysaccharide-induced acute lung injury onset and astaxanthin therapeutic effects by integrative analysis of RNA sequencing data and GEO datasets Here we performed a statistical meta-analysis of five publicly available gene expression datasets from LPS-induced ALI mouse models, conducted RNA-sequencing (RNA-seq) to screen differentially expressed genes (DEGs) in response to LPS administration and AST treatment, and integrative analysis to determine robust genetic signatures associated with LPS-induced ALI onset and AST administration. We subsequently integrated the RNA-seq and microarray meta-analysis data, and 11 core DEGs (Timp1, Ly6i, Cxcl13, Irf7, Cxcl5, Ccl7, Isg15, Saa3, Saa1, Tgtp1, and Gbp11) that were upregulated in ALI models and downregulated significantly after AST treatment were identified ( Table 2) . To further identify the robust expression signature related to LPS-induced ALI and investigate the transcriptional changes in response to the treatment of ALI by AST, we performed RNA-seq on three groups of mice and integrated the data with the results of the above mentioned meta-analysis.
cord-355075-ieb35upi	2012	alecto transcriptome provides information on a variety of immune genes not previously identified in any bat species and represents an important starting point for examining the antiviral activity of these molecules. To enrich for sequences corresponding to cytokines and innate immune genes, the second dataset was derived from pooled total RNA obtained from mitogen-stimulated spleen, white blood cells and lymph node and unstimulated thymus and bone marrow obtained from one pregnant female and one adult male flying fox. A full length transcript, encoding a 667 amino acid protein was identified in our bat transcriptome datasets and found to be orthologous to Mx1 based on comparison with known mammalian Mx1 and Mx2 family members (Figure 4a and data not shown). Genes involved in the adaptive immune system, including MHC class I and II genes and T and B cell receptors and co-receptors were highly represented in both the thymus and pooled datasets providing evidence that bats have all of the components necessary to mount an adaptive immune response.
cord-355179-wmfwl2bh	2019	Overall, the data indicate that the antiviral activity of niclosamide during the early stage of the DENV life cycle correlates with the neutralization profile of the low-pH compartments, suggesting that blocking endosomal acidification results in the inhibition of viral genome replication and polyprotein processing, which further impedes viral protein expression and virus production. In this study, we found that neutralization of low-pH intracellular compartments by niclosamide not only inhibited the early stage of the DENV viral life cycle, such as viral RNA replication, independent of the entry step but also the late stage, specifically, the maturation of virus particles into infectious virions. Indeed, niclosamide treatment www.nature.com/scientificreports www.nature.com/scientificreports/ during the first 6 h of infection reduced DENV replication in BHK-21 cells harbouring dengue replicons to a level comparable to that of ribavirin, suggesting that the drug affects viral RNA replication and/or translation independent of its effect on entry, membrane fusion and genome release.
cord-355357-b6aklh44	2015	IMPORTANCE Previous studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase in regulating replication fidelity. Similar to other studies, we previously utilized the RNA mutagens ribavirin and 5-fluorouracil to study drug-resistant mutations in alphaviruses (CHIKV and SINV) and identified residue 483 of the RdRp nsP4 as a key determinant of polymerase fidelity (10, 11) . Furthermore, given that both the CHIKV high-fidelity polymerase variant nsP4 (C483Y) and the novel nsP2 (G641D) variants were isolated in the same antiviral treatment, we addressed their additive roles in resistance to the RNA mutagens ribavirin and 5-fluorouracil (Fig. 1B) . By treating cells during viral infection with each compound, we found that the high-fidelity nsP2 variant and double mutant were more resistant to both nucleotide-depleting compounds ( Fig. 7B and C) , similar to what we observed with ribavirin treatment.
cord-355397-y69bk5jc	2020	Here, we used molecular dynamics (MD) simulations to study the binding of SARS-CoV-2 N-NTD to non-specific (NS) and TRS dsRNAs. We probed dsRNAs'' Watson and Crick (WC) base-pairing over 25 replicas of 100 ns MD simulations, showing that only one N-NTD of dimeric N is enough to destabilize dsRNAs, initiating melting. We calculated the structural model of the N-NTD:dsTRS complex based on the experimental data for the N-NTD interaction with a non-specific dsRNA (5''-CACUGAC-3'') (dsNS) (16) using the HADDOCK 2.2 server (20) . In contrast to the decrease in the number of intramolecular hydrogen bonds between the sense and anti-sense strands of dsRNAs (WC base-pairing) due to N-NTD binding, we observed an increase in the average number of intermolecular hydrogen bonds formed between the nitrogenous bases of dsTRS and N-NTD (protein-RNA interaction) along the 100 ns MD simulation, whereas for dsNS, this average value was constant ( Figure 3B top).
cord-355477-7xd93aqv	2007	abstract: Severe acute respiratory syndrome (SARS) is the first emerging infectious disease of the 21st century that has been highly transmissible and fatal and was caused by a previously unknown coronavirus (SARS‐CoV). Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in SARS patients: implications for pathogenesis and virus transmission pathways Assembly of severe acute respiratory syndrome coronavirus RNA packaging signal into virus-like particles is nucleocapsid dependent Recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein forms a dimer through its C-terminal domain Intracellular localization of the severe acute respiratory syndrome coronavirus nucleocapsid protein: absence of nucleolar accumulation during infection and after expression as a recombinant protein in vero cells The 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in Vero E6 cells The 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in Vero E6 cells
cord-355499-5vj3oasa	2015	title: Transmissible Gastroenteritis Virus (TGEV) Infection Alters the Expression of Cellular MicroRNA Species That Affect Transcription of TGEV Gene 7 In this study, we performed microRNA microarray assay and predicted targets of altered miRNAs. The results showed TGEV infection caused the change of miRNAs profile. In conclusion, differentially expressed miR-4331 that is caused by TGEV infection can suppress transcription of TGEV gene 7 via targeting cellular CDCA7. Overall, we observed that TGEV infection caused the change of miRNA profile and miR-4331 suppressed transcription of TGEV gene 7 via directly targeting CDCA7. The major findings in this study are that TGEV infection leads to the change of cellular miRNAs expression profile, and altered miRNAs regulate transcription of TGEV gene 7 through targeting cellular CDCA7. In conclusion, the results of the present study provide evidence that TGEV infection resulted in altered profiles of miRNAs in PK-15 cells and the differentially expressed miR-4331 was involved in regulation of TGEV transcription by targeting cellular CDCA7.
cord-355676-2y8vowbi	2006	The RNA secondary structure prediction algorithm Vienna RNA 1.5 4 was used to predict the secondary structures of group 1 and group 2 coronavirus 5'' UTRs. A reverse genetic system based on in vitro assembly of cloned cDNAs (A-G) was used to recover wild-type MHV-A59 1000 and mutant viruses. The assembled fragment containing either the wild-type sequence, or with mutations in SL2, was ligated into MHV-A59-1000 RNA was electroporated into BHK-R cells to recover infectious virus as described. Analysis of the entire 30 kb MHV and SARS-CoV genomes reveals that SL2-like stem loops are extremely rare (appearing just 3 and 5 other times, respectively); this suggests an important role in coronavirus replication. Consistent with the predicted structure of the UNR loop, the U49A mutant was viable and produced a virus with a near normal plaque size (Figs 4A-B) .
cord-355743-vjiecd4k	2020	The decoding algorithm takes as input cycle threshold values from qPCR tests on the pools, and returns a result for each sample, along with an estimate of viral load if positive. Once pooled tests are run, the cycle threshold values can be entered into the app which solves for and displays the list of positive and negative samples along with an estimate of viral loads. 16 × 40 pooling matrix: Table 2 shows the emulated cycle threshold (Ct) values for the 16 RT-qPCR tests corresponding to five different trials (0, 1, 2, 3 or 4 positive samples out of a total of 40 samples). 24 × 60 pooling matrix: Table 3 shows the cycle threshold (Ct) values obtained for the 24 RT-qPCR tests corresponding to a trial with 2 positive samples out of a total of 60 samples.
cord-355758-tk7eturq	2020	Background The emergence of a novel coronavirus (SARS-CoV-2) associated with severe acute respiratory disease (COVID-19) has prompted efforts to understand the genetic basis for its unique characteristics and its jump from non-primate hosts to humans. Tests for positive selection can identify apparently nonrandom patterns of mutation accumulation within genomes, highlighting regions where molecular function may have changed during the origin of a species. Several recent studies of the SARS-CoV-2 genome have identified signals of conservation and positive selection within the gene encoding Spike protein based on the ratio of synonymous to nonsynonymous substitution. In addition, we find other likely targets of positive selection within the genome of SARS-CoV-2, specifically within the genes encoding Nsp4 and Nsp16. In Importantly, we also detected signals of positive selection in two additional regions of the 414 SARS-CoV-2 genome, specifically within the genes encoding Nsp4 and Nsp16 (Fig 1A) . Comparative analysis of coronavirus genomic RNA structure reveals 718 conservation in SARS-like coronaviruses.
cord-355913-fhvt1ht1	2016	Little is known about what determines whether a given picornavirus positive-sense RNA molecule will be directed (1) to a replication complex (a structure bound to smooth endoplasmic reticulum), where it serves as template for transcription by RNA-dependent RNA polymerase into negative-sense RNA, or (2) to a ribosome, where it serves as mRNA for translation into protein, or (3) to a procapsid, with which it associates to form a virion. In the case of positive-sense single-stranded RNA viruses, the incoming RNA genome can bind directly to ribosomes and be translated in full or in part without the need for any prior transcription; all other forms of incoming viral RNA must first be transcribed to produce mRNA, in order to begin the process of expression of the infecting viral genome.
cord-356009-emn2w8if	2020	Conclusions: Our review concludes that not only the SARS-CoV-2 can be excreted in the urine in eight ?percent of patients but also its incidence may have associations with the severity of the ?systemic disease, ICU admission, and fatality rates. The searches included medical subject headings (MeSH) and keywords for SARS-CoV-2, COVID, Corona, together with shedding, persistence, urine, urinary, specimen, viral load, or RNA body fluids. We completed the data abstraction process using created forms to record study characteristics, clinical data, and laboratory data including study year and design, country of study origin, total initial population size, test type for disease diagnosis, test type for samples (urine/stool/rectal swab/blood), patients age (including mean and range), number of positive and total patients and/or (wherever applicable) number of positive and total specimens collected for each test category, disease severity, ICU admission, and fatality rate.
cord-356013-pl3tmky8	2005	In addition to the SARS coronavirus (treated separately elsewhere in this volume), the complete genome sequences of six species in the coronavirus genus of the coronavirus family [avian infectious bronchitis virus-Beaudette strain (IBV-Beaudette), bovine coronavirus-ENT strain (BCoV-ENT), human coronavirus-229E strain (HCoV-229E), murine hepatitis virus-A59 strain (MHV-A59), porcine transmissible gastroenteritis-Purdue 115 strain (TGEV-Purdue 115), and porcine epidemic diarrhea virus-CV777 strain (PEDV-CV777)] have now been reported. With regard to the 5 0 UTR it is known that the 5 0 -terminal sequence is required for DI RNA replication ) and at least two stem-loops (stem-loops III and IV in Fig. 4 ) function as higher-order cis-acting signaling elements (Raman et al. cis-Acting sequences required for coronavirus infectious bronchitis virus defective-RNA replication and packaging
cord-356115-vblgotjn	2005	The coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mRNAs in the virus-infected cell. Here, we report a genetic and functional analysis of 19 temperature-sensitive (ts) mutants of Murine hepatitis virus MHV-A59 that are unable to synthesize viral RNA when the infection is initiated and maintained at the non-permissive temperature. We focused our phenotypic analysis on the eight MHV-A59 ts mutants that had been genotyped and began by measuring ''''total'''' viral RNA synthesis in infected cells prior to and following shift from the permissive to the non-permissive temperature. This phenotype resembled that seen with MHV-A59-infected cells treated with CH, and we conclude that the complementation group I mutants are defective in their ability to form active replicase-transcriptase complexes at 40 8C but retain the positive-strand synthesis activity of the complexes formed at the permissive temperature.