Flaviviridae family has the 3D structural information resolved for such regions. Hence, future efforts to solve full-length RdRP structures containing these regions and to dissect the functional contribution of them are necessary to improve the overall understanding of the RdRP proteins as an evolutionarily integrated group, and our analyses here may serve as a guideline for selecting representative RdRP systems in these studies.The RNA-dependent RNA polymerases (RdRPs) encoded by the RNA viruses are a unique class of nucleic acid polymerases. Each viral RdRP contains a 500–600 residue catalytic module with palm, fingers, and thumb domains forming an encircled human right hand architecture. Seven polymerase catalytic motifs are located in the RdRP palm and fingers domains, comprising the most conserved parts of the RdRP and are responsible for the RNA-only specificity in catalysis. Functional regions are often found fused to the RdRP catalytic module, resulting in a high level of diversity in RdRP global structure and regulatory mechanism. In this review, we surveyed all 46 RdRP-sequence available virus families of the positive-strand RNA viruses listed in the 2018b collection of the International Committee on Virus Taxonomy (ICTV) and chose a total of 49 RdRPs as representatives. By locating hallmark residues in RdRP catalytic motifs and by referencing structural and functional information in the literature, we were able to estimate the N- and C-terminal boundaries of the catalytic module in these RdRPs, which in turn serve as reference points to predict additional functional regions beyond the catalytic module. Interestingly, a large number of virus families may have additional regions fused to the RdRP N-terminus, while only a few of them have such regions on the C-terminal side of the RdRP. The current knowledge on these additional regions, either in three-dimensional (3D) structure or in function, is quite limited. In the five RdRP-structure available virus families in the positive-strand RNA viruses, only the  The main purpose of this work is to place the current knowledge of these RdRPs in a broader context and to facilitate future studies in RdRPs with representative primary structure and/or with distinct functions beyond the catalytic module, thereby help improve the overall understanding of RdRP structure, function, and evolution. Based on sequence availability in the United States National Center for Biotechnology Information (NCBI) database2, one representative RdRP amino acid sequence was chosen for each virus family by giving the priority to the ICTV-suggested type species  to define conserved sites.First identified in the 1950s in the mengovirus and poliovirus (PV) related studies , 1962, tion mode . Howeverion mode , often d species . As an eA large number of RdRPs from the positive-strand RNA viruses are proteolytic products of viral polyproteins . Since nPermutotetraviridae family has a different motif order of G-F-C-A-B-D-E. While its spatial organization of the motifs is consistent with that of other RdRPs, the folding topology is permutated (Birnaviridae family in the double-stranded (ds) RNA virus category -mediated initiation mechanism for genome replication and a polyprotein coding strategy that are often found in the positive-strand RNA viruses  distributed within the palm (motifs A–E) and fingers (motifs F–G)  see an . RdRPs srmutated . A similcategory . These e viruses . These opol and the hepatitis C virus (HCV) NS5B to help estimate the boundaries of the catalytic module using conserved residues in motifs A/B/C/F as the reference points . The thumb domain usually starts from 50–60 residues after the XGDD sequence and ends at the C-terminal boundary of the catalytic module. The primer-dependent 3Dpol contains four helices in the thumb, while the de novo NS5B contains seven. If compared to 3Dpol, NS5B has one insertion between the third and fourth helices, three extra helices after the fourth helix, and a C-terminal extension   , 3. Amon viruses . Among t viruses , althoug regions . The N-t regions . Based o regions . The N-t regions . Collectde novo RdRPs tend to have important initiation elements located at its C-terminus . Both of these factors could reduce the opportunity for RdRPs to recruit additional regions to their C-termini during their evolution. Among these RdRPs containing additional functional regions, some of them have long drawn attentions in the related field but without much advances in structure and related functional characterization of the additional regions. The Coronaviridae nsp12 and Arteriviridae nsp9 have a ∼200–400 residue N-terminal region with both the structure and function remaining elusive   due to a UAG stop codon within the RdRP ORF  , while much fewer showed up at the C-terminal side (4 out of 37 families with defined C-terminus) . The pre elusive . The Cor copies) , may hav copies) , and wer copies) . Howevere solved . The neafunction . The Kitl region . The Togproteins . The N-tRdRP ORF . Howeverlum (ER) . The ∼90lum (ER) . TherefoViral RdRPs represent a unique nucleic acid polymerase class of and is the only class that does not involve DNA in any stages of the synthesis. To preserve their RNA template and ribonucleotide triphosphate (rNTP) substrate specificity, the seven catalytic motifs are the central segments to preserve during virus evolution. While we have mainly focused on the diversity and variations beyond the catalytic modules, attentions may also be drawn to the variations within the catalytic module but excluding the catalytic motifs. For the representative RdRPs that we surveyed in this study, the spacing between certain catalytic motifs could vary to a great extent. For example, the residue distance between the conserved motif B serine and the motif C aspartic acid in these RdRPs ranges from 30 to 94, corresponding to a motif spacing of 12–76 residues , 2. We hPicornaviridae and 14 in Tombusviridae), suggesting high-level of genome and RdRP diversity within individual families. Moreover, RdRP primary structure diversity and genome-level diversity may not be consistent. The Flaviviridae is such an example with only four genera but three drastically different primary RdRP structures. Therefore, choosing one representative RdRP for each family (with the Flaviviridae as the only exception) may not be sufficient and ideal. Thirdly, the boundary estimation of the catalytic module is only a crude assessment. For example, the distance between the N-terminal boundary of the TaV RdRP catalytic module to the conserved motif F lysine is at least 100 residues longer than estimated distance using the 150-residue criterion used in our analyses (Although we tried to survey the positive-strand RNA virus RdRPs in a comprehensive manner, there are still several limitations in our analyses. Firstly, the virus species that have not been assigned at the virus family level are not included in our analyses. Secondly, some virus families contain a large number of virus genera (e.g., 47 in analyses .In summary, we collected representative RdRPs encoded by positive-strand RNA viruses mainly at the level of virus family. By locating highly conserved residues within catalytic motifs within the RdRP ORF and by referencing structural and functional information of RdRPs in the literature, we tried to estimate the boundaries of the RdRP catalytic module in the full-length RdRP. Numerous regions beyond the RdRP catalytic module exist and many of them have either structure, or function, or both to be determined. Collectively, the global structure and regulatory functions related to regions beyond the catalytic module of the positive-strand RNA virus polymerases are quite diverse, and the current knowledge of these proteins is limited to only a few virus families. One purpose of our analyses is to provide a general guideline for researchers interested in these RdRP proteins and related viral systems to selectively or systematically investigate RdRPs with representative features. The global view of the positive-strand RNA virus RdRPs will continue to evolve with new virus species assigned, new structures determined, new functional regions identified, and new mechanisms dissected.Both authors surveyed the literature, analyzed the data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Viruses are widely used to fabricate nanomaterials in the field of nanotechnology. Plant viruses are of great interest to the nanotechnology field because of their symmetry, polyvalency, homogeneous size distribution, and ability to self-assemble. This homogeneity can be used to obtain the high uniformity of the templated material and its related properties. In this paper, the variety of nanomaterials generated in rod-like and spherical plant viruses is highlighted for the cowpea chlorotic mottle virus (CCMV), cowpea mosaic virus (CPMV), brome mosaic virus (BMV), and tobacco mosaic virus (TMV). Their recent studies on developing nanomaterials in a wide range of applications from biomedicine and catalysts to biosensors are reviewed. Bionanoscience is a combination of biology and nanotechnology that is used to develop biomaterials, devices, and methodologies at the nanoscale. According to its tremendous applications, nanobiotechnology has become a rapidly developing area of science research . NanosysViruses are protein cages that are used for the production of novel nanomaterials in a very precise and controlled fashion. Viruses have a variety of distinct shapes, most commonly icosahedrons and rod-shaped. Genetic and chemical modification enables the insertion or replacement of selected amino acids on virus capsids for uses ranging from bioconjugation to mineralization.In recent years there has been increased interest in the development of plant viruses as biotemplates for beneficial uses. The applications have been diverse, ranging from biomedicine to sensors. Characteristics that make many plant viruses attractive for these studies are their relative simplicity, including ease of purification, the lack of membranes, and simple one or two protein capsid assemblies that are structurally defined. In addition, as plant viruses are phytophages that utilize plants as hosts, they are mostly non-infectious to animals and humans. Furthermore, plant viruses often have wide range of stabilities to temperature, pH, salt, chemicals, and protease degradation. They also differ in terms of structural plasticity assembly and disassembly parameters and electrostatic interactions. The icosahedral (sphere-like) viruses include cowpea chlorotic mottle virus (CCMV), cowpea mosaic virus (CPMV), brome mosaic virus (BMV), and the rod-shaped tobacco mosaic virus (TMV) have been investigated as ideal platform in variety of applications in nanotechnology . Three dThe cowpea chlorotic mottle virus (CCMV) belongs to the Bromovirus group of the Bromoviridae family . CCMV caThe most profound property of the CCMV is that the viral capsid can undergo a pH and metal ion-dependent structural transition called gating, which increases its porosity and allows materials to enter. At pH > 6.5, and in the absence of divalent metal ions, the viral capsid undergoes a reversible swelling (an approximate 10% increase in diameter), allowing the ions to freely diffuse into the interior cavity. The swelling capsid has open pores of 2 nm in diameter with 60 separate permitting ions to dispread through the interior and exterior ,9.The cowpea mosaic virus (CPMV) is a member of the Comovirus group of plant viruses. It can also be used to strengthen the growth of inorganic materials . Its strThe brome mosaic virus (BMV) was first extracted from bromegrass in 1942 . Its genThe BMV has a special genome that is classified into three kinds of 5′-capped RNAs . RNA1 (3The tobacco mosaic virus (TMV) is a helical plant virus consisting of 2130 identical subunits arranged helically around an RNA genome in its native form. The TMV is 300 nm in length and 18 nm in diameter . It is aViruses offer three different surfaces that can be exploited: the exterior, the interior, and the interface between the subunits. These interfaces can be used to conduct chemical and genetic modifications for a variety of purposes. The external capsid surfaces possess a large number of selectively addressable amino acids allowing decoration with a large number of molecules. The functional groups on the capsid surface, amine groups from lysine, and carboxylate groups from aspartic and glutamic acids offer precise sites for chemical modification using various techniques such as covalent coupling ,30,31, cSmall molecules have access to the viral inner cavity with imperceptibility to large molecules. Based on this property, it is possible to allow the internal cavity of virus-like particles (VLPs) to be used as a nanotemplate and nanoreactor. In addition, the virus exterior surface is a powerful platform not only for chemical modification and genetic modification, but also for multivalent ligand display. A key component of assembly is the virus architecture interface, which provides additional ways to manipulate the capsid architecture.A series of amino acid side chains  are located on the protein shell of the viral particles and these amino acid side chains are applicable to fluorescent and medical imaging dyes, proteins, or small molecule therapeutics and sensors or cell-penetrating peptides of the desired reagents for chemical-conjugated reactive sites. Targeting the conjugation site at a particular location on the inner or outer surface can be accomplished by genetic engineering of new residues. For example, through this method, a three dimensional (3D) array of Au nanoparticles (1.4–5 nm) can be produced on the CPMV surface ,40. The The CCMV has a positive inner surface, which hinders iron nanoparticle synthesis. However, protein engineering can change the charge of the inner surface through mutagenesis. After that, iron compound nanoparticle synthesis is possible inside the CCMV . In concAccurately patterning and integrating biotemplates into devices to take advantage of the nanosize and enhanced functionality is a great challenge in nanoscience applications. Here are some methods to enhance the applications of several functionalized PVN surfaces.Using evaporation methods with drop-and-dry or solvent impregnation, the surface alignments of the icosahedral and filamentous plant-based PVNs have been accomplished. The PVN concentration and the intensity of the surface attractive force affect the formation of the PVN film ,44. The Surface displayed metal nanoparticles exhibit unique material properties and an increased surface area of the nanoscale catalysts present enhanced activities. An important way to control the catalytic reactions is reflected by the ability to adjust the position, size, and spatial density of the catalyst. PVN provides a unique backbone for the production, anchoring, and display of key nanocatalytic materials. For instance, Yang et al. studied the production and display of Pd nanoparticles of a defined size with a diameter of 5–15 nm; their distribution can be controlled by TMV PVN . The TMVThe icosahedral viruses are highly consistent 3D building blocks suitable for multidimensional nano- and mesoscale assembly ,52. For N-terminal 1cys, which is recessed in the groove and partially covered by the C-terminal arm of the CP, this site may inhibit direct contact between the cysteine-derived thiol groups and the metal surface, except at the 3′ end of the virion rod where the thiol group is sufficiently exposed to make direct surface contact. ELD mineralization of the subsequent surface-assembled TMV1cys yielded a uniformly coated virus template comprising a fused metal coating with a thickness of 15 nm. In addition, the virus stick vertically assembled to the surface, so the available area significantly increased by an order of magnitude . Th. Th62]. Overcoming the problems associated with the abnormal metallization of nontemplated particles can be represented by surface-assembled virus particles prior to the inorganic coating . AttachmVirus-based nanoparticles (VNPs) have received great attention in recent years; plant viruses have been used in materials science research and have the potential for application in nanomedicine ,64. As v3+) is frequently applied as a MRI contrast agent, however, it has to be used in a chelated form to reduce the toxicity, usually with DOTA tetraazacyclododecane tetraacetic acid (DOTA) or diethylenetriamine pentaacetic acid (DTPA) [T1 relaxivity relative to free Gd (DOTA) complexes after loading with gadolinium. These reagents, determined as nontoxic in vivo, were used for the MRI imaging of live mice [3+ or other lanthanides can increase the relaxation rate, differences in MRI signals can be detected regardless of where they are present, and the contrast level by many factors can be finally obtained. A multifunctional MRI contrast and photodynamic therapy agent  has been incorporated inside capsids of the CCMV protein which represent the first step towards the consecution of fully self-assembled protein cage nanoparticles for multimodal imaging and therapy [Magnetic resonance imaging (MRI) is a technique that visualizes the structure and metabolites within the capsid and therefore has the potential to detect physiological processes in vivo. Among them, the contrast agent and macromolecular platform can further enhance imaging sensitivity. Paramagnetic gadolinium (Gdd (DTPA) . After cd (DTPA) . Finn and (DTPA)  that shoive mice . The inc therapy .The covalent attachment of chelated gadolinium ions to the TMV leads to the enhanced ionic relaxivity of the Gd ions based on reduced tumbling rates. Multivalent display leads to a relaxivity per nanoparticle that is four times higher than other VNP contrast agents . SteinmeThe application of plant viruses to drug delivery is still at an advanced stage of development. However, the plant virus can be devised with new properties based on the unique requirements of the targeted drug delivery. After chemical functionalization of the surface of plant virus particle, they have the property of stealth, thereby extending their circulatory half-life or targeting ability in the body to help deliver the drug to the diseased tissue. The amino acid sequence of the viral coat protein is genetically manipulated to produce a protein cage of a particular chemical structure and the conformation of the protein cage in the external environment can be directed to disassemble and then be reassembled in vitro to exchange the genomic material of the native viral foreign cargo .Without further replication, plant viruses enter mammalian cells, which make them suitable as therapeutic delivery systems. For implementation in nanomedicine, the therapeutic cargo is either loaded into the inner cavity or conjugated to the interior or exterior surfaces of the capsid. Viral nanoparticle surfaces can be chemically or genetically modified to target particular cells including cancer cells and specific cells of the immune system. Currently, virus-based nanoparticles have been widely used in vaccine platforms ,76,77,78VNPs have been used as nanocarriers for a variety of clinically approved chemotherapeutics. Complexation of the therapeutic compound with VNPs possesses several advantages, including an improvement in pharmacokinetics and enhanced drug delivery to disease sites while overcoming off-target effects . DoxorubNoncovalent methods allow for higher loading efficiency as more cargo molecules can be loaded in the entire cavity when compared to the covalent attachment, which uses the internal exposed side chains to conjugate the cargos . For exa2, which enables mixing of the drug and the medium; at lowered pH, the pores close and thereby capture the drug. Using this property, phthalocyanine (ZnPc), a light-absorbing molecule, was encapsulated into CCMV capsids. The ZnPc-containing CCMV capsids were eaten by RAW 264.7 macrophage cells that led to their deaths, indicating the applicability of CCMV-ZnPc for photodynamic therapy (PDT) [Staphylococcus aureus, a pathogenic bacterium, was inactivated through treatment with dual-functionalized CCMV with a photosensitizer and a targeting ligand [Environmentally-triggered structural changes of nanocages, for instance CCMV, allow the entry of the drug into their hollow interiors by changes in pH or ionic strength. The pores are open at neutral pH in the presence of CaClpy (PDT) . PDT is py (PDT) ,105. Stag ligand .Pseudozyma antarctica lipase B and EGFP) in the CCMV capsid to study the activity of this structure. In the case of enzymes, studies have shown that in the encapsulation process, the apparent increase in the overall rate of reaction is almost uninfluenced by the amount of enzyme in the capsid. This result is due to the very high limiting molarity of the enzyme in the capsid, leading to very rapid formation of the enzyme-substrate complex, and highlights the importance of a small volume for efficient multi-enzyme cascade catalysis [Enzymes can also be loaded into the virus capsid to create nanoreactors. In nature, enzymes are found in the limited space of cells. Encapsulation of the enzyme within the viral capsid provides the possibility of studying enzymatic behavior in a cell-mimicking environment. In artificial nanoreactors, the envelopment of enzymes as a biomimetic system are expected to help us understand the interaction of enzymes in active and confined spaces. The capsid of the cowpea chlorotic mottle virus (CCMV), a man-made nanoreactor, can be used to encapsulate a variety of proteins within it. By controlling the encapsulation process, it is able to load the exact amount of protein  ions in the apoferritin cavity . In thisEncapsulation of enzymes or synthetic polymers is different and because of their larger size; they struggle to diffuse through the holes of the protein cage. However, some RNA plant viruses can be reversibly disassembled/assembled by changing the medium pH and salt concentration in order to provide a simple mechanism to bind large species. The most widely studied virus is the cowpea chlorotic mottle virus (CCMV)  with an 3O4 has been synthesized in rod-like tobacco mosaic viruses by Ludwigs and coworkers, which demonstrated superior catalytic performance in the oxygen evolution reaction when compared to a commercial nanopowder [Recently, mineral Conopowder .N-terminus and at an introduced cysteine residue with two different chromophores that undergo FRET to produce a “light harvesting rod” [Viral capsids provide a means to precisely position chromophores with spatial control at the sub-nanometer level. The tobacco mosaic virus coat protein can serve as a convenient template for the synthesis of chromophores into light-harvesting arrays ,113,114.ing rod”   binding peptides which selectively bind TNT in solution and thus the diffusion onto a sensing electrode reduced. The measured TNT electrochemical reduction peaks were shown to correspond to the concentration of TNT . This seVibrio cholera O139, these viral nanoparticles markedly increased assay sensitivity. In a DNA microarray assay, the EF CPMV mutant functionalized with fluorescent dye (Cy5) and NeutrAvidin (NA) was used as a recognition element for hybridized DNA containing biotin. These NA-Cy5-CPMV particles provided a strong signal intensity with low background with a detection sensitivity of 101 genome copies [CPMV particles have also been used as sensors for the detection of DNA and toxins ,136,137.e copies . CPMV haPlant virus-based nanoparticles that are nanocages or nanorods assembled from viral capsid proteins have been widely used as templates to guide the preparation of complex nanostructures. In this review, we described the structural and unique biochemical properties of several plant viruses such as the cowpea chlorotic mottle virus (CCMV), cowpea mosaic virus (CPMV), brome mosaic virus (BMV), and tobacco mosaic virus (TMV). The advantages of plant virus nanoparticles as biological templates in chemical modification and genetic engineering were also analyzed. Nanomaterials developed using viral nanoparticles have superior properties over traditional materials and their potential has been integrated into traditional manufacturing processes that are reflected in microfabrication processes without disrupting their structure or activity. There are many exciting opportunities for potential applications of VNPs or VLPs in biotechnology. Further studies are required to explore the unique self-assembly architectures of more plant viruses to produce new materials for application in the nano biotechnology. In addition, de novo protein engineering and in silico techniques have been rapidly developing in recent years and can play an important role in engineering supermolecular nanomaterials for specific applications.


Cytokines are small proteins, mostly secreted into the extracellular environment, that bind to specific cell surface receptors, which mediate cell differentiation, migration, growth, and death. Gene expression and cellular release of cytokines are strictly regulated to assure proper function of cells, tissues, and organs. Upon virus infection, a cell starts producing type I interferons (IFN) and inflammatory cytokines (ICs) to restrict spread and replication of the respective virus. Ideally, the virus is completely eliminated by the immune system and the antiviral mechanisms are turned off within a reasonable time frame. However, there are different scenarios where this process does not work efficiently or does not happen at all, leading to cytokine-mediated organ dysfunction and tissue damage.Bohmwald et al. describe in detail where and which cytokines are induced during human RSV infection and their potential contribution to damage of not only the lung but also the brain. The pathophysiological production of ICs is most probably also due to the ability of RSV to induce the expression of Toll-like receptor (TLR) 4 in human airway epithelial cells, which normally do not respond to endotoxin  or influenza virus. In a comprehensive review article, ndotoxin .1 (AFB1), a mycotoxin produced by Aspergillus flavus, increases TLR4 expression as well as TLR2, IL-1β, and IL-6 expression in human monocyte-derived dendritic cells  and mice infected with swine influenza virus (SIV) subtype H1N1. Importantly, they show that AFB1 exacerbates lung damage in mice during SIV infection, caused by a TLR4-dependent increase in viral replication and TNF levels. Consequently, uptake of AFB1, for example by contaminated food  strains, but not with human adapted IAV strains, leads to constitutive phosphorylation of tripartite motif (TRIM) 28 at S473 and increased production of IFN-β, IL-6, and IL-8. TRIM28 negatively regulates transcription in a number of ways  strain , which hLehmann et al. provide a model to understand the complexity of these pathologies based on recent cell biology findings about systemic distribution of the viral Nef protein and improved understanding of C-C motif chemokine ligand 2 (CCL2) dependent transendothelial cell migration. The focus of this Mini Review is on the brain, where non-physiological induction of CCL2 expression not only drives encephalitis but also affects signaling and survival of neuronal cells. Indeed, antiviral and anti-inflammatory cytokine expression have to be well-coordinated during a viral infection in order to enable elimination of the pathogen on the one hand, and to avoid tissue and organ damage on the other hand. This problem is discussed by Savarin and Bergmann on the basis of a murine model of encephalomyelitis induced by a neurotropic strain of mouse hepatitis virus where the interplay of IFN and IL-10 dictates the extent of viral control and tissue pathology.The cellular tropism of human and simian immunodeficiency virus is limited, but dysfunctions and damages also occur in the hearts, lungs, kidneys, and brains of infected individuals. Liu et al. found that functional TRIM21 is important for high level type I IFN expression in vitro and in vivo. Additionally, they show that TRIM21 deficiency leads to higher viral titers, stronger cardiac and pancreatic damages, and higher levels of ICs including CCL2 in mice infected with CVB3. Meyer et al. detected increased levels of colony stimulating factor 1 (CSF-1) in heart biopsies of patients with myocarditis, and by using nanoparticle-encapsulated siRNA directed against CSF-1 they could decrease CVB3-induced monocyte infiltration and heart damage in mice. Beling and Kespohl suggest that therapeutic targeting the proteasome could help to prevent immunopathology of the heart, which can be triggered by many different viruses. Theiler's murine encephalitis virus (TMEV) induces myocarditis in mice, but this virus can also induce demyelination of neurons depending on the mouse strain. A Hypothesis and Theory article by Omura et al. summarizes findings about the different nature of these diseases and provides evidence that TMEV induces cell-type specific innate immune responses and distinct organ-specific pathology. Thus, the choice of the right animal model to study virus induced immunopathology can be challenging. For that, Manickam et al. provide a thorough review of non-human primate models for understanding the extent of cytokine-mediated tissue damage during many different types of virus infection, including dengue virus, HCMV, hepatitis B and C virus, HIV, influenza virus, and Zika virus.Type I IFN is required to restrict coxsackievirus B3 (CVB3) replication , and LiuCollectively, this Research Topic introduces some of the complex virus-host interactions that can tip the scales toward immunopathology. The common themes that emerge from this collection include the potential for use of cytokines as markers of disease and the manipulation of certain cellular molecules as therapeutic options.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ixodes ricinus ticks in south-eastern Finland. Screening of sera from patients suspected for tick-borne encephalitis for Jingmen tick virus-like virus RNA and antibodies revealed no human cases. The presence of ALSV in common European ticks warrants further investigations on its role as a human pathogen.The newly identified tick-borne Alongshan virus (ALSV), a segmented Jingmen virus group flavivirus, was recently associated with human disease in China. We report the detection of ALSV RNA in  Ixodes ricinus ticks in Kotka archipelago, south-eastern Finland.Recent reports have associated two members of Jingmen virus group, Alongshan virus (ALSV) and Jingmen tick virus (JMTV), to febrile disease in humans ,2. Here Rhipicephalus microplus ticks in Hubei Province, China [R. microplus and cattle in Brazil, i.e. the Mogiana tick virus (MGTV) [Amblyomma javanense, Dermacentor silvarium and I. persulcatus ticks as well as humans in China [I. persulcatus and isolated from febrile patient sera in Heilongjian Province [Flavivirus. The other two segments appear to originate from an unknown ancestor. Together, the viruses form a separate and diverse group tentatively called the Jingmen virus group in the family Flaviviridae [In 2010, a novel segmented tick-borne RNA virus, JMTV, was detected in e, China . Subsequs (MGTV) -6; humans (MGTV) ; Amblyomin China . Recent iviridae .I. ricinus ticks collected from the Kotka archipelago in 2017 and 2018 for the presence of JMTV-like RNA. We found another positive tick from a neighbouring Kuutsalo island in the Kotka archipelago, and obtained the full genome using next-generation sequencing. The viruses (GenBank accession numbers MN107153 to MN107160) cluster together with ALSV (MH158415 to MH158418) from Heilongjian Province, China, and form a cluster distinct from the other members JMTV group, including the strains found in Kosovo (MH133313 to MH133324) [In 2019, while performing a metatranscriptomic analysis of ticks collected in 2011 from Haapasaari island, Kotka archipelago, south-eastern Finland, we detected a full genome of JMTV-like virus together with tick-borne encephalitis virus (TBEV) genome. Thereafter, we used RT-PCR to screen 198 The emerging reports on the association of JMTV-like viruses with human disease in China ,2 led usTicks were homogenised and RNA was extracted as described previously . Total nSynthetic gene constructs encoding JTMV glycoproteins VP1a, VP1b, membrane and capsid proteins were cloned into pCAGGS/MCS . The recTick homogenates were treated with a mixture of micrococcal nuclease  and benzonase  for 1 hour at 37 °C, followed by RNA extraction using TriPure Isolation reagent . RRNA was removed using a NEBNext rRNA Depletion Kit (New England BioLabs) according to the manufacturer’s protocol. The sequencing library was prepared using a NEBNext Ultra II RNA Library Prep Kit (New England BioLabs). The library fragment sizes were measured using agarose gel electrophoresis and the concentrations using Qubit dsDNA BR Assay Kit  and NEBNext Library Quant Kit for Illumina (New England BioLabs). Sequencing was conducted using MiSeq Reagent Kit V2 with 150 bp reads.Raw sequence reads were trimmed and low-quality, quality score < 15, and short, < 36 nt, sequences were removed using Trimmomatic . ThereafComplete genome sequences of all available tick-borne JMTV-like viruses were downloaded from GenBank (accessed June 2019). The amino acid sequences were aligned using the ClustalW algorithm followed by manual refinement. In addition, NS5 sequences of the representatives of all flavivirus species were retrieved from NCBI Reference Sequence Database (RefSeq) and aligned with MAFFT programme version 7  using E-The phylogenetic trees were constructed using the Bayesian Markov chain Monte Carlo (MCMC) method, implemented in MrBayes version 3.2  with twoI. ricinus ticks, a tick species that is common across the European continent. Despite apparent ALSV circulation in the south-eastern archipelago of Finland, no ALSV RNA or antibodies to selected recombinant ALSV proteins were found in ca 900 Finnish patients suspected for TBEV infection in recent years. While our results suggest low human infection pressure, further research using other methods, including properly evaluated ALSV antibody tests, and focusing on other geographic areas and patient cohorts beyond meningitis or encephalitis cases is needed.Our findings show that ALSV, a newly described tick-borne human pathogen, is also present in south-eastern Finland. Notably, ALSV was detected in


Zika virus infection has recently attracted the attention of medical community. While clinical manifestations of the infection in adult cases are not severe and disease is not associated with high mortality rates, Zika virus infection can have an impact on fetal development and lead to severe neurodevelopmental abnormalities.To gain insight into different aspects of Zika virus infection, a comprehensive literature review was performed. With regard to epidemiology and geographical distribution of Zika virus infection, relevant information was extracted from CDC and WHO websites.In this review, we discuss different basic and clinical aspects of Zika virus infection including virology, epidemiology and pathogenesis of disease. Laboratory methods required for the diagnosis of disease together with ethical issues associated with Zika virus infection will also be discussed in detail.Herein, we have tried to provide a multi-faceted view of Zika virus infection, with greater emphasis on disease status in Eastern Mediterranean Region. Flaviviruses are responsible for several important human diseases including Dengue, West Nile and Yellow fevers, Japanese encephalitis, Tick-borne encephalitis, and Zika fever. The major route of transmission for most Flaviviruses is through arthropod vectors and central nervous system injury and hemorrhagic fevers represent major clinical outcomes. In recent years, Zika virus infections have attracted the attention of international medical community, chiefly because of their role in causing microcephaly and other neurodevelopmental abnormalities which occur as a consequence of maternal infections .Zika virus was first isolated in 1947 in the Zika forest in Uganda from a rhesus macaque . SerologIn this review, we have discussed the current knowledge about various aspects of Zika virus infection and recent developments including antiviral treatments for this infection. The first four sections of this review cover virology, epidemiology, pathogenesis, and laboratory diagnosis of Zika virus infection. While Zika virus infection is a global concern, in the epidemiology section we have put some more emphasis on findings from Eastern Mediterranean Region (EMRO) countries. Considering the ethical considerations related to feto-maternal and neurodevelopmental dimensions of the Zika virus infection, the final section of this review provides a detailed account of ethical aspects of Zika virus infection .Flaviviridae family of viruses , the capsid (C) and the precursor of the membrane (prM)) and seven nonstructural proteins  . The ssRFlaviviridae family despite significant differences in tissue tropism, transmission, and pathogenesis , ZINC33683341 and ZINC49605556 (both in silico designed) has been proposed. The other approaches include the inhibition of endosomal fusion using compounds that reduce the acidity of endolysosomal vesicles like chloroquine  and disrupting the electrostatic interaction between cell and virus membrane using squalamine  .Flaviviridae, antiviral agents targeted their helicase could also be used for the Zika virus  is a RdRP inhibitor approved by FDA for chronic HCV treatment. It also inhibits Zika virus replication –17. Zikaka virus . Likewiska virus  as well.Despite the many studies done on Zika antiviral treatments, so far no FDA approved category A drug has been found safe to use in mothers and fetuses .Before the first large outbreak of Zika virus infection on Yap Island, Federated States of Micronesia , only spAn increased incidence of cases of the Guillain– Barré syndrome (GBS) was reported after a larger epidemic of Zika virus infection in French Polynesia in 2013 . In 2014In early 2015, several cases presenting a “dengue-like syndrome” investigated in Brazil (a non-Dengue virus and non-Chikungunya virus infection) and Zika virus was detected by reverse transcription polymerase chain reaction (RT-PCR) assay and confirmed by DNA sequencing , 26. TheOn Jul 2015, in the State of Bahía, Brazil, an increase in the number of Guillain-Barré cases in which half of them had reported symptoms consistent with Zika virus infection .In Jan 2016, an unusually significant increase of GBS was reported in El Salvador. An Emergency Committee was convened by the Director-General of WHO, under the International Health Regulations (2005) on 1 Feb 2016, and finally announced “the recent cluster of microcephaly and other neurologic disorders reported in Brazil to be a PHEIC .Aedes mosquitoes: Djibouti, Egypt, Oman, Pakistan, Saudi Arabia, Somalia, Sudan and Yemen. Some of these countries are particularly vulnerable to emerging of Zika virus, because of the fragility of health systems, weakness of disease surveillance systems, the inadequacy of response capacities, and a suboptimal level of public health preparedness Content source: Centers for Disease Control and Prevention, Sep 2018 updated.  . In vitrFollowing cell entry, Zika virus induces strong interferon responses in infected cells. Studies on Zika virus-infected human primary fibroblasts have shown strong upregulation of interferon-beta transcripts 24 to 48 h after infection . A mildeProductive infection of dermal fibroblasts and dendritic cells together with insufficient control of infection by innate and adaptive immune mechanisms usually leads to Zika virus viremia, which underlies the non-specific clinical symptoms which might last for a few days. In a pregnant woman, maternal viremia might lead to fetal viremia. It is not well known how the virus is transmitted to developing nervous system following the establishment of fetal viremia. While in circulation, Zika virus can infect fetal monocytes and these monocytes can carry the infection to the developing nervous system. Studies on fetal brain tissue derived from Zika virus-infected mothers who have undergone abortion have shown viral particles in neural cells, indicating that the virus can proliferate inside neural cells in a developing brain . While iDepending on the purpose of the investigation, laboratory diagnosis of Zika virus can be conducted by virus isolation, antigen detection, viral RNA detection with molecular assays and anti-Zika virus antibodies detection with serological assays .The first isolation of Zika virus was performed by intracerebral mouse inoculation considered as the reference assay for arboviruses isolation , 36. AmoAntigen detection is a valuable assay to approved Zika virus infection in autopsy tissues. Using immunohistochemistry (IHC) technique, Zika virus antigen has been detected in brain and placental tissues from congenitally infected newborns with microcephaly and miscarriages , 42. RecZika virus RNA is detectable in different types of body fluids such as blood (serum or plasma) –50, urinReverse transcriptase PCR (RT-PCR) is highly sensitive and specific and known as the “gold standard” for ZIKV diagnosis . RegardiMolecular diagnosis of Zika virus infection in human usually performs on plasma or serum specimens within the first week after onset of clinical symptoms . AlthougDespite the fact that the molecular diagnosis possesses high sensitivity and specificity, short period of viremia can negatively affect the Zika virus RNA detection . TherefoDetection of anti-Zika virus antibodies can be performed by different serological tests including complement fixation, haemagglutination inhibition, immunofluorescence (IF) assay, ELISA and neutralization tests , 35. TypSo far there is no FDA approved a serological test for Zika virus. However, FDA has authorized the use of five serological assays with emergency use authorization for detection of anti-Zika virus IgM antibody including IgM antibody capture ELISA (Zika MAC-ELISA) (CDC) , ZIKV DeBecause of cross-reactivity with other Flaviviruses such as dengue and yellow fever viruses, and the possibility of nonspecific reactivity, results of IgM detection assays should be interpreted with caution and positive or equivocal results must be confirmed by plaque-reduction neutralization testing (PRNT) .Zika virus has been classified as a risk group 2 human pathogen. Therefore, diagnostic laboratory tests should be performed in a Biosafety Level 2 (BSL-2) facility . The virAddressing ethical questions and moral conflicts of Zika virus infection may be essential to develop a comprehensive approach to control this public health crisis . TherefoNational and international health authorities should observe the principle of veracity, by clear and transparent informing system for raising the public awareness regarding the real situation . At the The fear raised from high probability of congenital problems, mainly microcephaly, of neonates delivered by mothers with Zika virus infection in their first trimester of pregnancy , puts abTogether with Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), Zika virus infection qualifies as a “newly emerging” infectious disease, with the potential to cause serious public health issues. Unlike the other “newly emerging” infections which can lead to severe morbidity and mortality in infected adults or pediatric hosts, Zika infection does not pose a significant threat to infected adults and its risk is more due to the potential to cause fetal abnormalities, provided that the infection occurs during pregnancy. Indeed, among the four recent PHEIC declarations by WHO , Zika is unique in the sense that it is a member of the TORCH group of infections; i.e. the group of pathogens with the ability to lead to congenital infections/anomalies. This might influence both disease prevention and management strategies as well as raising ethical and sociological issues. Thus, increased awareness of the medical community together with improvements in vector control and disease surveillance systems are of utmost importance for controlling any potential Zika virus-related threats in different countries.Ethical issues  have been completely observed by the authors.


The availability of these data will greatly expedite research on G-quadruplex in viruses, with the possibility to accelerate finding therapeutic opportunities to numerous and some fearsome human diseases.G-quadruplexes are non-canonical nucleic-acid structures that control transcription, replication, and recombination in organisms. G-quadruplexes are present in eukaryotes, prokaryotes, and viruses. In the latter, mounting evidence indicates their key biological activity. Since data on viruses are scattered, we here present a comprehensive analysis of potential quadruplex-forming sequences (PQS) in the genome of all known viruses that can infect humans. We show that occurrence and location of PQSs are features characteristic of each virus class and family. Our statistical analysis proves that their presence within the viral genome is orderly arranged, as indicated by the possibility to correctly assign up to two-thirds of viruses to their exact class based on the PQS classification. For each virus we provide: i) the list of all PQS present in the genome (positive and negative strands), ii) their position in the viral genome, iii) the degree of conservation among strains of each PQS in its genome context, iv) the statistical significance of PQS abundance. This information is accessible from a database to allow the easy navigation of the results:  in silico directly from the nucleotide sequence. Several computational methods exist for this purpose, but they are all limited to the analysis of independent sequences. Since viral genomes can be highly variable, G-quadruplexes with important functional roles are expected to be conserved among strains and isolates belonging to the same viral species. Here we aimed at characterizing the potential quadruplex-forming sequences (PQS) content in the genome of viral human pathogens and assess their degree of conservation in each viral species. We demonstrate that many viruses possess more PQSs than expected from their nucleotide composition and some of them are highly conserved within single viral species, claiming some biological roles. We provide a website where the results of our analyses are displayed for each virus with interactive graphics. This work is intended as a resource that can guide scientists in the choice of the most promising candidates for functional characterization.G-quadruplexes are nucleic acid non-canonical structures that have been implicated in the regulation of different biological processes of many organisms. Their presence has been demonstrated also in several viral pathogens, providing new insights into viruses’ biology and potentially serving as drug targets. Although experimental validation is needed to confirm the actual folding of G-quadruplexes, they can be inferred  We decided to expand the search to PQSs with very short islands and quite extended loops for the following reasons: first, the folding of PQS with GG-islands has been previously demonstrated in viruses ; second,PQSs were searched in the positive and negative strand of each virus genome sequence, since both filaments are present and important in different stages of the viral replicative cycle of all virus classes. As the length of virus genomes greatly varies, i.e. from 235,646 nucleotides (nts) of the human cytomegalovirus (HCMV) to 1,682 nts of hepatitis delta virus (HDV), we reported the number of PQS independently of the genome length by normalizing their number per 1,000 nts . The PQSloop_conservation value) was calculated as the difference between G4_SCI and LSC. Positive and negative loop_conservation scores indicate, respectively, lower and higher conservation of connecting regions compared to the conservation of G-islands. Values close to zero mean that both G-islands and connecting loops show the same level of sequence conservation. In http://www.medcomp.medicina.unipd.it/main_site/doku.php?id=g4virus .Then, we evaluated the conservation of PQSs among different strains of each viral species, hypothesizing that the presence of a conserved PQS within a less conserved genome environment could be an indication of a G4 with a biological function . To alloTo assess the results, we retrieved from the literature all the available experimentally validated G4s detected in human viruses. All patterns were confirmed also by our analysis and the complete list is reported in G4 formation may be largely affected by G/C content, which greatly varies in viral genomes (from 76% of Cercopithecine 2 herpes virus to 27% of Yaba like disease virus). Moreover, it has been shown that some di- and trinucleotides are over- or under-represented in certain viruses , 42 and,To check whether the presence of PQSs was statistically relevant or whether it occurred by pure chance, we compared the results obtained from real viral genomes with those obtained by two different simulation strategies. The first one (single nucleotide assembling) assumes that the occurrence of each DNA base in the genome is independent ; the secFor each virus and simulation strategy, we produced 10,000 random sequences, which were screened with our PQSs detection pipeline. Real and simulated data were compared by computing a P-value, defined as twice the smaller proportion of simulated sequences that exhibit, respectively, a higher and lower count of PQSs as compared to the median value of all the available complete genome sequences for a certain virus. Hence, a P-value close to 1 means that the median PQS content in real viral sequences is not significant if compared to a random distribution; conversely, a P-value close to 0 means that PQS content is highly significant. This interpretation holds independently of the length of the genome and/or of the prevalence of either G/C bases or G-islands, as we compare the number of PQSs in a viral genome with the one we would expect in a simulated genome of the same length and of either the same base or G-island composition. To account for possible high discreteness of the data, a less conservative version of the P-value, called the mid-P value , was useSagiyama virus and Human coronavirus HKU1 are depleted of PQSs belonging to GG-islands category in the positive genome strand when compared with both simulation strategies based on single nucleotide assembling and GG-island reshuffling. In addition, Human parainfluenza virus 2 is poor of PQSs made of GG-island in the positive genome strand but is enriched in both GG- and GGG-type PQSs in the negative strand.Our data show that most members of the dsDNA, ssDNA, and ssRNA (RT) present a highly significant content of PQSs formed by GG-, GGG- and/or GGGG-islands in one or both strands. ssRNA (-) and ssRNA (+) classes are heterogeneous since some viruses are highly significant in any PQS category (from GG- to GGGG-islands), while others are not (see below). The presence of PQSs in members of the dsRNA group is notably less significant. Interestingly, few viruses display a smaller amount of PQSs than expected: both Overall, if we consider the viruses that contain at least one PQS in either the real or the simulated genomes, we observe that the increase in G-islands’ length corresponds to a decrease in the absolute number of viruses containing PQSs, but it also corresponds to a dramatic increase in the fraction of them that is statistically significant.Herpesviridae is not only the one with the highest PQS content, but most of its members display significantly more PQSs than expected in both genome strands and in all considered G-island lengths. Notably, some of the viruses belonging to Herpesviridae and showing the highest G/C content are statistically enriched in PQSs. This suggests that simply having a high G/C content is not a sufficient condition to justify the presence of such a high number of PQSs. Other viral families that are consistently enriched in PQSs are Adenoviridae and Papillomaviridae, especially in GG- (both strands) and GGG-island (positive strand) types. Poxviridae and Parvoviridae show an enrichment of GG-type PQSs in both genome strands, whereas the same pattern is enriched in the positive strand of all Anelloviridae members and in the negative strand of most Paramyxoviridae and Retroviridae viruses. All other families are generally not enriched in PQSs in any of the evaluated categories, with only a few exceptions that are listed in the following: L segments of Lassa virus and Lymphocytic choriomeningitis virus (Arenaviridae), Wu and Merkel cell polyomaviruses (Polyomaviridae), Salivirus (Picornaviridae), M and S segments of respectively Crimean-Congo hemorrhagic fever virus and Rift Valley fever virus (Bunyaviridae).By looking at the family level of viral classification, which is far more homogeneous than the Baltimore groups, some virus families emerge as prominently enriched in PQSs. Among them, By comparing the results obtained independently from the two simulation strategies it is possible to draw additional conclusions. First, in most cases the results are concordant, meaning that both simulations show similar trends in the statistical significance. Nonetheless, the overall number of viruses whose PQS content is significantly different with respect to simulated data is higher when real viral genomes are compared to those generated by single nucleotide assembling. This difference indicates that viral genome k-mer composition is indeed affecting the probability of randomly finding PQSs, at least in a proportion of viruses as shown in Poxviridae family, namely Molluscum contagiosum virus and Orf virus, which are enriched in GG- and GGG-type PQSs in both strands of their genomes if compared with the islands reshuffling simulation but show the opposite behavior when compared with the single nucleotide assembling , repeat regions (RR), 5’- and 3’-untranslated (UTR), and promoter regions. While CDS and RR are explicitly defined in RefSeq and GenBank databases, the annotation of UTRs and promoters is more inconsistent, being defined only for some viral species. For this reason, the annotations of genes and CDSs were exploited to indirectly extract the coordinates of 5’–and 3’–regulatory regions see –S5 Figs.In this line of thinking, we asked whether the observed number of PQSs, and more precisely its statistical significance with respect to the two random assembling scenarios, is representative for a specific Baltimore class. To answer this question, we checked whether it is possible to classify each virus to one of the six classes considered, that is, dsDNA, ssDNA, dsRNA, ssRNA (+), ssRNA (-) and ssRNA (RT), based on the information of how significant its median PQS counts are. We used a classifier built on multinomial logistic regression, as this method is both interpretable and robust to unbalanced group sizes as long as the group sizes are large enough. To avoid the latter drawback, we excluded from the model fit the hepatitis B virus, the only virus classified as dsDNA (RT), and the two unclassified hepatitis delta and hepatitis E viruses. Six features were used to classify the viruses, i.e. the six mid-P values  which qualify the PQS content of the real viral sequences. The values were multiplied by 1 or -1 depending on whether the median PQS count was over- or under-represented. Since real and corresponding simulated sequences contain the same base or G-islands composition, the classification model based on PQS content does not depend on the highly variable genome length and G/C content in the different virus classes but is specifically designed on the peculiar presence or absence of PQSs in each viral class. Furthermore, 34 viruses with no PQS count in all three G-island types in both the positive and negative strand and non-significant mid-P values at the 10% level were excluded from the analysis. We re-classified every viral genome used in our assessment using the discriminant function obtained from a leave-one-out analysis. This latter technique allowed us to accurately estimate how our classifier performs without the need to split our data into a training and a test set. The corresponding confusion matrix is given in In this work we provide: i) the list of PQSs present in all human virus genomes (both positive and negative strands), ii) their position in the viral genome, iii) the degree of conservation of both G-islands and loops vs. the genome, iv) the statistical significance of PQS abundance in each virus. Our data show that viruses belonging to dsDNA, ssDNA, ssRNA (RT) and, to a less extent, ssRNA (+) and ssRNA (-) display the largest presence of GG-, GGG- and GGGG-type PQSs  The statistical significance of PQSs found in real sequences tends to decrease when G-islands reshuffling (ISL) is compared with the corresponding single nucleotide assembling (SN), as is appreciable from the heatmap in a priori but up to two-thirds of unequivocal assignments suggest that for some viruses the PQS content works as a distinctive feature. iv) PQS localization shows differences in some virus classes, but this outcome is still incomplete due to lack of information in the databases about virus genomic features and partitioning into regulatory and coding regions.We evidenced some general trends and exceptions that are worth noting if seen in comparative terms among all viruses considered in this study. Starting from the general features we noted: i) high G/C content is not sufficient per se to generate a high number of PQSs, as observed in G/C rich members of Molluscum contagiosum virus and Orf virus, are the only ones belonging to genera other than the Orthopoxvirus within the Poxviridae family that cause skin lesions. Finally, dsRNA and dsDNA (RT) viruses are notably poor in PQSs and with mostly null statistical significance; however, single PQSs are highly conserved (e.g. rotavirus a segment 6), therefore still conveying potential biological interest.Some other interesting observations are worth reporting as either special cases or exceptions. To start with, the ssRNA (-) group is the most heterogeneous one, since some viral species are significantly enriched in PQSs up to the most extended G-island type (GGGG), while others lack this feature. Surprisingly, two viruses of the dsDNA group, which was generally highly enriched in PQSs, show a significantly lower presence of PQSs than expected in a random sequence with the same G/C content SN, , even thHerpesviridae family of dsDNA viruses. In this case, most PQSs reported here and also previously described . The complete list of selected reference sequences is reported in The complete list of viral species able to infect humans was retrieved from usearch8 , using al’ is the minimum number of G in every G-island of a PQS and ‘d’ is the maximum distance allowed between two consecutive G-islands, the following combinations of ‘l’ and ‘d’ were searched: l = 2 and d = 7; l = 3 and d = 12; l = 4 and d = 12. Patterns in the negative strands of viral genomes were searched by looking for cytosines (Cs) instead of Gs. The conservation of each PQS in the multiple aligned genomes of the viruses was determined by looking at the conservation not only of the G-islands but also their connecting loops. We computed different indexes to measure the nucleotide sequence conservation of viral genomes and PQSs:PQSs were searched in all multiple-aligned nucleotide sequences with an in-house developed tool, as previously described , 52. BriG4_scaffold_conservation_index (G4_SCI): it is referred to the G-islands. For each virus and for every detected PQS, it is calculated as the percentage of independent genomes maintaining the corresponding G-islands:G_islands is the number of sequences possessing the G-islands in a certain genome position and Ntot is the total number of sequences available for the virus.Loop_conservation: it is the difference between G4_SCI and the local conservation of the viral sequence spanning the PQS (LSCG4).G4 is calculated as the average of LSC windows overlapping the PQS. LSC measure is computed within a sliding window of fixed length , averaging the conservation values of each position extracted from the multiple sequence alignments with Jalview , exploiting Pandas [LSC Jalview . They arregions) . All chag Pandas  and Numpg Pandas . Multiplg Pandas  and genog Pandas . Unless To determine whether the presence of PQSs in a virus is a conserved feature or it is only a consequence of its nucleotide composition, simulated viral genomes were generated and compared with real data. Two different strategies were adopted to generate simulated data:Single nucleotide assembling (SN). A computational approach was adopted where, in analogy to Huppert and Balasubramanian [p(G), the probability of any base being G. In our approach, we also accounted for the probability of cytosines (p(C)) and additionally assumed that adenine (A) and thymine (T) bases were equally likely to occur. As all four probabilities need to sum up to one, the statistical reference model is a multinomial distribution with probability vector (p(G), p(C), p(A), p(T)). We hence took as many independent draws from this multinomial distribution as the number of nucleotides in the reference viral genome (p(G) and p(C) vary for each virus and reflect the prevalence of G and C bases present in that virus, while the remaining proportion is equally split to give p(A) and p(T). For each virus, 10,000 independent sequences were produced in silico with this method; the ‘sample’ R command with replacement was used and the provided parameters were the genome length and the relative abundance of the four nucleotides in the real genomes.ramanian , the virl genome . The proG-islands reshuffling (ISL). For each virus, we generated a scaffold of length n made of only As, with n corresponding to the length of the virus reference genome; then, we replaced di-, tri-, or tetra-nucleotides, at random positions and without overlaps, with as many GG-, GGG-, GGGG-, CC-, CCC-, CCCC-islands as in the reference sequence, respectively. Overall, we generated 10,000 independent sequences for three different simulated datasets, one for each island length.The simulated sequences were scanned for the presence of PQSs as previously described. The 10,000 counts obtained for each simulation formed the empirical distribution for PQS prevalence under the hypothesis of random assembling of the genome in the SN and ISL models respectively. The mid-P value was calculated using a homemade function. The semiparametric classifier used to assign the virus to its exact class relying on its PQS content was based on the ‘multinom’ function of the R package ‘nnet’.gene − Scds; 3’–regulatory = Ecds—Egene. For the genes in the negative strand of viral genomes we defined: 5’–regulatory = Scds—Sgene; 3’–regulatory = Egene—Ecds. Sgene, Scds, Egene and Ecds are the Start (S) and End (E) of genes and CDSs as extracted from the feature tables. These newly defined features contain both UTRs and promoters. Since the positive genomic strands are deposited in RefSeq for most of the viruses belonging to the ssRNA (-) class, the following sequences available as negative strands were converted into their inverse complement, together with the coordinates of their genomic features: Junin arenavirus segment S (NC_005081) and segment L (NC_005080), Lassa virus segment L (NC_004297), lymphocytic choriomeningitis virus segment S (GQ862982), Machupo virus segment S (AY924208) and L (AY624354), Pichinde virus segment S (NC_006447), Rift Valley fever virus segment S (NC_014395), and Toscana virus segment S (NC_006318). The overlap extent between PQSs and genomic features was computed by intersecting the genomic coordinates of each PQS with the genomic features extracted from the corresponding virus, and a positive count was recorded every time an overlap of at least one nucleotide was detected. Finally, to compare the enrichment in different feature classes, characterized by different sizes, a normalization step was performed. The total extension of each feature class  was calculated by summing the lengths of individual features. The total count of PQS overlapping a feature class was then divided by the total length of the class and multiplied by a factor 1,000 to obtain the number of PQS present every 1,000 nucleotides. This procedure was performed considering only the PQSs conserved in at least 80% of sequences for each viral species. All feature tables files were manually revised to fix inconsistencies in the use of keywords and coordinates.The feature tables containing viral genome annotations were downloaded from RefSeq or GenBank for all the reference sequences reported in S1 FigSegment diagrams of mid-P values obtained by comparing the PQS content detected in real and simulated viral genomes. Simulated viruses have the same nucleotide composition of the real ones, but different order. The three G-island types considered in the positive (+) and negative (-) strands of all human virus genomes are grouped in the 7 Baltimore classes. From left to right, each segment represents one of the three G-islands  in the positive  and negative  strands; the radius of a segment corresponds to 1 minus the mid-P value. Thus, full segments indicate highly significant PQSs, whereas null segments indicate non-significant PQSs, with respect to the random sequences.(TIF)Click here for additional data file.S2 FigSegment diagrams of mid-P values obtained by comparing the PQS content detected in real and simulated viral genomes. Simulated viruses are obtained by reshuffling the positions of their GG-, GGG- or GGGG-islands. The three G-island types considered in the positive (+) and negative (-) strands of all human virus genomes are grouped in the 7 Baltimore classes. From left to right, each segment represents one of the three G-islands  in the positive  and negative  strands; the radius of a segment corresponds to 1 minus the mid-P value. Thus, full segments indicate highly significant PQSs, whereas null segments indicate non-significant PQSs, with respect to the random sequences.(TIF)Click here for additional data file.S3 FigRepresentative figure of GG-island PQSs that overlap with genomic features. The bar charts report the distribution of PQSs in genomic features where available and annotated in the database. The number of PQSs per 1kb is reported on the x-axis both for the positive (orange) and negative (blue) strands. The four features considered are coding sequences (CDS), repeat regions (RR), and regulatory regions at the 5’ and 3’ ends.(TIF)Click here for additional data file.S4 FigRepresentative figure of GGG-island PQSs that overlap with genomic features. The bar charts report the distribution of PQSs in genomic features where available and annotated in the database. The number of PQSs per 1kb is reported on the x-axis both for the positive (orange) and negative (blue) strands. The four features considered are coding sequences (CDS), repeat regions (RR), and regulatory regions at the 5’ and 3’ ends.(TIF)Click here for additional data file.S5 FigRepresentative figure of GGGG-island PQSs that overlap with genomic features. The bar charts report the distribution of PQSs in genomic features where available and annotated in the database. The number of PQSs per 1kb is reported on the x-axis both for the positive (orange) and negative (blue) strands. The four features considered are coding sequences (CDS), repeat regions (RR), and regulatory regions at the 5’ and 3’ ends.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


We detected Zika virus RNA in rectal swab samples from 10 patients by using real-time reverse transcription PCR, and we isolated the virus from 1 patient. The longest interval from symptom onset to detection was 14 days. These findings are applicable to diagnosis and infection prevention recommendations. In early 2015, Zika virus was identified in Brazil and spread across nearly the whole continent, affecting thousands of persons . Laboratory confirmation of infection was based on real-time reverse transcription PCR (rRT-PCR) performed on samples  from persons in whom a rash developed https://www.thermofisher.com) and stored at −80°C until processing. After collection, specimens were kept refrigerated and transported within 2 hours to the laboratory, where they were maintained at −80°C. Zika virus detection was performed by rRT-PCR by processing 200 μL of each specimen for RNA extraction through an automated nucleic acid purification platform by using the Maxwell 16 Viral Total Nucleic Acid Purification Kit  in a final volume of 70 μL. For rRT-PCR, we used the commercially available ZDC Kit from Instituto de Tecnologia em Imunobiológicos Biomanguinhos, approved by Agência Nacional de Vigilância Sanitária/ANVISA . We considered positive those samples that displayed positive amplification in the ZDC Kit internal control reaction  and those samples in which the target amplification was detected within 38 amplification cycles, as previously described  at specific intervals from patients identified as Zika virus positive after the screening visit . VaginalWe report 10 Zika virus–infected patients from 2 locations in Brazil. Of these, 3 patients were identified in Recife, northeastern Brazil, and 7 were from Manaus, northern Brazil. The targeted Zika virus amplicons were found in the plasma of 9 patients and in the urine of 7 patients, all tested at screening visits. We also tested all plasma and urine samples for dengue and chikungunya virus by rRT-PCR; results were negative. Of the 10 Zika virus–infected patients, 9 were symptomatic and 1 (a Zika virus–positive household contact) was asymptomatic at enrollment but subsequently reported muscular weakness and irritability on day 5 and arthralgia on day 7 after Zika virus identification. Median patient age was 31.5 years; 7 patients were nonpregnant women, and 3 were men . All patt) was 34.92 in plasma (interquartile range [IQR] 33.57–36.78) and 31.57 in urine (IQR 30.34–34.92). The longest duration of persistence in saliva was 21 days. Zika virus RNA was detected in rectal swab samples from all patients (median Ct 34.36 [IQR 30.08–36.07]); positive results were obtained at >1 time for 4 patients. Vaginal swab sample results were positive for 2 patients (t of 28.77 after 6 days of incubation at 37°C.For all patients, Zika virus RNA was detected >1 time in different body fluids. The longest interval for Zika virus–positive results by rRT-PCR in plasma and urine was 14 days after symptom onset; median cycle threshold value (Cpatients . We atteOur detection of Zika virus RNA in human rectal swab samples demonstrates the presence of virus RNA in the anorectal mucosa of naturally infected patients. Because the anorectal mucosa is a major entry site for HIV-1 and other sexually transmitted disease organisms (Recently, 2 studies explored the mucosa as an experimental entry site for Zika virus. Macaques exposed to high doses of the virus by direct inoculation on palatine tonsils, nasal mucosa, and conjunctival mucosa became infected, whereas virus-naive animals exposed to saliva from Zika virus–infected macaques, containing a 20-fold lower virus concentration, remained uninfected (In patients with yellow fever, mucocutaneous bleeding with virus presence has been reported (


Bunyavirales: Arenaviridae), obtained from a wild rodent Oligoryzomys mattogrossae captured in Mato Grosso do Sul state, Brazil. The genome of this virus showed strong similarity to highly pathogenic mammarenavirus from South America.Here, we report the complete genome sequence of the Aporé virus ( Arenaviridae family currently comprises 41 viral species, classified into three genera: mammarenavirus (35 species), reptarenavirus (five species), and hartmanivirus (one specie).,Mammarenaviruses are negative sense bi-segmented RNA viruses each segment encodes genes for two non-overlapping reading frames in ambisense polarity.Neacomys guianae), Cupixi virus , Flexal virus (unidentified oryzomyini rodent), Sabiá virus , Oliveros virus (Necromys lasiurus), Latino virus , Pinhal virus  and Xapuri virus (Neacomys musseri).,,,Oligoryzomys mattogrossae (= Oligoryzomys fornesi), captured in Cassilândia municipality, Mato Grosso do Sul state, Midwest, Brazil.To date, eight mammarenavirus were detected in Brazil: Amaparí virus . Isolated RNA from one rodent liver sample was treated with DNAse I (Life Technologies) following the manufacturer’s instructions and depleted of ribosomal RNA using NEBNext rRNA Depletion Kit (New England BioLabs inc). A library was constructed with the Nextera XT Library Preparation Kit (Illumina) using 2 x 250 bp paired-end protocol on the MiSeq platform (Illumina). Sequencing reads were assembled A classical arenavirus bi-segmented genome was identified, each segment encoding two open reading frames (ORFs) in an ambisense organisation with an intergenic region containing a predicted stem-loop region between the ORFs. Full S segment (3.4 kb) encoded genes for two inferred proteins: nucleoprotein (562 aa) and a glycoprotein precursor (GPC) (489 aa), which is normally post-translationally processed into the envelope glycoproteins G1 (197 aa) and G2 (234 aa) and the stable signal peptide (SSP - 58 aa). The L segment (7.2 kb) encoded genes for zinc-binding matrix protein (99 aa) and the RNA-dependent RNA polymerase (2155 aa). Additional features commonly observed in mammarenavirus genomes include the conservation of the 3′-5′ termini and the presence of an L-domain motif within the Z protein.,Nucleotide sequences and deduced amino acid of the new virus were compared to those of other mammarenaviruses species. A nucleotide sequence divergence of 28% for S and 25% and 26% for L segment was found between the new virus and Chapare (from Bolivia) and Sabiá (from Brazil) viruses, respectively.O. mattogrossae, SABV and CHAPV. Thus, the results of the phylogenetic analysis also indicates that it represents a novel virus within the Clade B New World mammarenavirus  (Pairwise sequence comparison (PASC) was performed on both segments reinforcing the close relation between the new discovered virus from viridae) . Therefo,O. mattogrossae rodents and APOV.In recent years, novel arenaviruses have been identified expanding our knowledge about their genetic diversity, geographic range and host association. Herein we show that APOV is closely related to two highly pathogenic arenaviruses from South America that were recovered from fatal cases of hemorrhagic fever, whose reservoirs remain unknown.Nucleotide sequence accession numbers - The complete genome sequence of Aporé virus has been deposited in GenBank under the following accession numbers: MF317490 for the S segment, and MF317491 for the L segment.


Camellia sinensis (L.) O. Kuntze) is an economically important woody species. In this study, we collected 26 tea plant samples with typical discoloration symptoms from different tea gardens and performed metagenomic analysis based on next-generation sequencing. Homology annotation and PCR sequencing validation finally identified seven kinds of plant viruses from tea plant. Based on abundance distribution analysis, the two most abundant plant viruses were highlighted. Genetic characterization suggested that they are two novel virus species with relatively high homology to Blueberry necrotic ring blotch virus and American plum line pattern virus. We named the newly discovered viruses tea plant necrotic ring blotch virus (TPNRBV) and tea plant line pattern virus (TPLPV). Evolutionary relationship analysis indicated that TPNRBV and TPLPV should be grouped into the Blunervirus and the Ilarvirus genera, respectively. TPLPV might have same genome activation process with known ilarviruses based on sequence analysis. Moreover, specific primers for both viruses detection were designed and validated. The symptoms and ultrastructure of TPNRBV infected leaves were first recorded. Virus detections in the symptomatic and asymptomatic tissues from field plants showing tea plant necrotic ring blotch disease suggest that TPNRBV has a systemic movement feature. In summary, we first identified seven kinds of putative plant viruses by metagenomic analysis and report two novel viruses being latent pathogens to tea plant. The results will advance our understanding of tea plant virology and have significance for the genetic breeding of tea plants in the future.The tea plant ( Camellia sinensis (L.) O. Kuntze) is a perennial woody species first discovered as a drink and medicine in China approximately 3000 years ago. In addition to China, the tea plant has been widely cultivated in India, Sri Lanka, Kenya, Japan, and other countries. Tea production has great economic importance around the world because tea is second to water in terms of worldwide beverage consumption . The 26 RNAs were combined in equal amounts for RNA-Seq library construction. For PCR validation, 15 plant materials with leaf discoloration were randomly collected in the summer and fall of 2017 in Hangzhou. For systemic movement analysis, different tissues including apical buds, young stems, upper leaves , lower leaves (with reddish-brown rings or blotches), and roots from different diseased tea plants showing necrotic ring blotch symptoms were sampled in the summer of 2018 in Hangzhou, five biological replicates were carried out. Moreover, 15 individual shoots and leaves showing mixed symptoms of necrotic ring blotch disease were collected from different tea plants. RNA isolation and genomic DNA removal were performed as above.Twenty-six tea plant leaves or shoots with typical leaf discoloration  symptoms were collected from different tea gardens in the fall of 2016 in Hangzhou, Zhejiang province, China . Total RNA from individual plant materials was isolated independently using the CTAB method described by ® Stranded Total RNA with Ribo-ZeroTM Gold Kit  was used to remove ribosomal RNA and construct the RNA-Seq library according to the manufacturer’s instructions. The final sequencing library was subjected to quality control using an Agilent 2100 Bioanalyzer . High-throughput RNA sequencing was performed on a HiSeq X Ten (Illumina) sequencer, and paired-end reads of 150 bp were obtained.TruSeq1. The read quality was evaluated using the FastQC program. Non-virus sequences were filtered from clean data by the BBMap (v35.59) program2 with a mismatch rate of 3 bases/read on the basis of the available transcriptome  and genome data of the tea plant  analysis with the non-redundant protein sequence (nr) database, nucleotide collection (nt) database, tea genome database and virus genome database5, the best hits  in each database were assigned to the assembled transcripts of the tea plant. Viruses were classified according to the last virus taxonomy released by the International Committee on Taxonomy of Viruses (ICTV) in 2016. The abundance of each transcript was quantified using the RSEM (v1.2.11) program (Adapter sequences were first removed from harvested raw data using the Cutadapt (v1.15) program, and then low-quality and unpaired reads were trimmed using the Sickle quality-based trimming programea plant . Trinity program .® III First-Strand Synthesis System  according to the manufacturer’s instruction [oligo(dT) was replaced by the degenerate primer N25: 5′-NNNNNNNNNNNNNNNNNNNNNNNNN-3′]. The final products were diluted with 400 μL of ultrapure water for PCR validation. The top-abundant transcript of each annotated virus was used for sequencing validation. Primer pairs were designed by the online program Primer-BLAST6. For PCR, a 10 μL mixture containing 1 μL of cDNA template, 5 μL of 2× master mix  and 0.5 μL of each primer (20 pmol) was set up. PCRs were performed with a program of 95°C for 10 min and 35 cycles of amplification with 95°C for 20 s, appropriate annealing temperature for 10 s, and 70°C for 35 s. Primer and annealing temperature information were provided in Supplementary Material 1. PCR products were monitored by 1.5% gel electrophoresis, and then the main bands were recovered by gel extraction and sequenced on Sanger platform.RNAs were reverse-transcribed into cDNAs using the SuperScript® RACE 5′/3′ kit . A Marathon® cDNA Amplification Kit  was used to amplify the middle, 5′ end, and 3′ end following the kit’s instructions. The primer information for full-length genome sequence cloning is presented in Supplementary Material 2. To confirm the presence of poly(A) tail at 3′ terminus, reverse transcription was redone using the SuperScript® III First-Strand Synthesis System [oligo(dT) in kit was used], and then a forward specific primer together with oligo(dT) was used for the amplification of 3′ terminus fragment. Amplicon was purified and sequenced on Sanger platform. Sequence identity and similarity were detected by the EMBOSS Needle program, an online global alignment tool provided by EMBL-EBI7. Phylogenetic trees were inferred using the neighbor-joining method conducted in MEGA7.0 (8 (Full-length genome sequences were cloned by rapid amplification of cDNA ends (RACE) using the SMARTer MEGA7.0  with 1,0GA7.0 (8 .® III First-Strand Synthesis System [oligo(dT) in kit was used]. PCR reactions were set up using KOD-Plus-Neo system  following the kit’s instructions, and 35 cycles of amplification were carried out. Finally PCR products were monitored by 2% gel electrophoresis. The PCR positive tissues with typical disease spot together with healthy control were separately cut into small pieces and fixed with 2.5% glutaraldehyde overnight at 4°C. The methods for sample preparation, ultrathin section, and electron microscopic examination were carried out following the descriptions by On the basis of full-length genome sequence information of TPNRBV and TPLPV, multiple primer pairs were designed by Primer-BLAST for specific primer selection. cDNAs from all the collected samples were mixed equally and then used as template in the selection. PCR and gel electrophoresis were performed following above descriptions. To detect the systemic movement characteristic of TPNRBV, different tissues, shoots, and leaves collected from diseased plants were monitored using the four specific primer pairs by reverse-transcription (RT)-PCR. RNAs from these tissues were independently reverse-transcribed into cDNAs using the SuperScriptFigure 1, including the albino or chlorina phenomenon in one or two shoots or leaves from healthy, growing tea plants. A total of 108 Gbp of raw data in 643,055,788 paired-end reads were yielded after sequencing (SRA accession number in NCBI: SRP128758), and 102 Gbp of clean data were harvested after quality control and trimming. Finally, after removal of non-virus sequences, only 3 Gbp of sequences, 2.82% of the original sequencing data, were reserved (Table 1). The de novo assembly produced 142,773 contigs with N50 of 365 bp, and the longest contig was 13,897 bp.In this study, 26 samples showing leaf discoloration were collected from tea gardens located in different regions of Hangzhou, China. All the samples were chosen for metagenomic sequencing. Partial samples with typical symptoms are shown in Supplementary Material 3). To filter out false positives, we did sequence homology analysis between virus-annotated contigs and available tea genome sequence, and 27 virus species showed relative high sequence identity with tea genome, which were considered as putative insertion of virus related sequences in the plant genome . Moreover, using the cDNA mixture from 26 collected samples as PCR template, the highest abundant contig of each annotated virus species were further validated by Sanger sequencing. More than one primer pairs were designed for the PCR if no productions or unique bands were obtained. Finally 29 out of 36 contigs were validated . Taking all into account, the seven plant viruses without homology with tea genome and validated by PCR amplification and sequencing were considered as real viruses existing in tea plants. Furthermore, an abundance distribution of the seven plant viruses was presented in Figure 2B. Among the viruses, blueberry necrotic ring blotch virus (BNRBV) and American plum line pattern virus (APLPV) took up the highest transcript abundance, followed by Panax notoginseng virus A (PnVA) and maize-associated totivirus 2 (MATV2), piper DNA virus 2 (PDV2), maize-associated totivirus (MATV), Humulus japonicus latent virus (HJLV).After functional annotation, 80.17% of the contigs were assigned to at least one of the nt, nr, and virus genome databases. Only 1.54% of the contigs were annotated as virus homologous sequences or virus protein-coding genes. Based on abundance quantification and functional annotation, the virus-annotated contigs were removed when their log2 (FPKM) values were lower than 1. Finally, 206 contigs from 36 plant virus species were identified as virus-associated sequences (Table 2). PCR validation was performed to check the positive ones among 26 collected samples for the two most enriched plant viruses . The results indicated that 6 of 26 samples might have been infected by BNRBV or a related virus and that 4 of 26 samples might have been infected by APLPV or a related virus. Moreover, one sample might have been infected by both viruses. Based on this information, RACE was performed to obtain full-length genome sequences. After RT-PCR and Sanger sequencing confirmation, a complete genome sequence of 14,979 nt, comprising four segments , and a complete genome sequence of 7,869 nt, comprising three segments , were obtained. Sequence analyses indicated that less than 60% identity and similarity were detected between the two cloned genome sequences compared with the corresponding BNRBV and APLPV sequences at both the nucleotide and amino acid levels . No sequence identity to other known viruses was detected by BLASTN search. These results suggest that two new virus species have been identified in the tea plant. Therefore, we tentatively named the virus that was similar to BNRBV as tea plant necrotic ring blotch virus (TPNRBV) and the virus similar to APLPV as tea plant line pattern virus (TPLPV).According to the homology annotation, five contigs were annotated as BNRBV protein-coding genes, and three as APLPV protein-coding genes . RNA1 was composed of 5922 nt and contained a long ORF spanning nucleotides 135 to 5720 and encoding a putative 212 kDa protein (1861 amino acids) having 22.8% identity with the methyltransferase-helicase encoded by BNRBV RNA1. Correspondingly, the RNA1 of TPNRBV encoding protein contained methyltransferase , cysteine-protease  and helicase  domains like the RNA1 of BNRBV . Moreover, the presence of a poly(A) tail was confirmed at each segment. A phylogenetic tree was constructed based on a putative RNA polymerase encoded by TPNRBV together with BNRBV, BNRBV-RL, and other viruses . The results indicated that TPNRBV had the closest evolutionary relationship with BNRBV and BNTBR-RL (a strain of BNRBV). The phylogenetic analysis suggested that TPNRBV is a tentative new species in the genus Blunervirus.By gene structure analysis, the four segments  of TPNRBV had poly(A) tails of varied lengths on the 3′ ends . Like the TPNRBV, all the segments had a poly(A) tail of different lengths. RNA1 contained a long ORF spanning nucleotides 30 to 3158 and encoding a 117 kDa putative methyltransferase-helicase  with 42.0% identity to the protein encoded by RNA1 of APLPV. RNA2 had a long ORF spanning nucleotides 30–2120 and encoding an 80 kDa putative RNA polymerase  with 41.8% identity to the protein encoded by RNA2 of APLPV. RNA3 contained two large ORFs spanning nucleotides 157–1026 and 1204–1815. The putative protein located in the N-terminus had 289 amino acids , and the putative protein in the C-terminus had 203 amino acids . These proteins had 24.8 and 31.7% identity, respectively, with the movement protein and coat protein encoded by RNA3 of APLPV. The evolutionary relationships of TPLPV with other viruses in the Ilarvirus genus were inferred using the neighbor-joining method based on the RNA polymerases encoded by RNA2 . TPLPV had weak phylogenetic relationships with other members of the Ilarvirus genus. These results suggest that TPLPV is a new member of the Ilarvirus genus and could be classified into the same subgroup as American plum line pattern virus. Among TPLPV, APLPV, Prunus necrotic ringspot virus (PNRSV), apple mosaic virus (ApMV), and prune dwarf virus (PDV), alignment of the coat proteins indicated a high proportion of arginine (R) in N-terminus and a highly conserved region in C-terminus, which located at the same position with the RNA binding domain (RBD) and dimerization region (DR) of PNRSV coat protein, respectively . Particularly, the residue 23 (R) of TPLPV coat protein highly conserved in the RBD. Secondary structure analysis further showed that eight stem-loop structures would form at the 3′UTR of TPLPV RNA3 . Compared with the stem-loops in 3′UTR of PNRSV and APLPV, fewer stem-loops were observed in APLPV, and TPLPV had more similar stem-loop structures with PNRSV . Furthermore, alignment of the TPLPV 3′UTR with APLPV, PNRSV ApMV, and PDV indicated a high conservation among them .The complete genome of TPLPV had three segments of 3373 nt (RNA1), 2354 nt (RNA2), and 2142 nt (RNA3) . Finally, one specific primer pair was selected for each segment of the two viruses, and the sequence information is listed in Table 3. Using the specific primers, we detected the 15 samples randomly collected in Hangzhou in 2017. Three samples were TPNRBV infected, and no sample was infected by TPLPV. The symptoms of TPNRBV-infected leaves varied, including chlorina on the edge of the leaf , mottled chlorina on a branch , and multiple necrotic ring blotches on mature leaves  distributed at the bottom of the tea plant. The necrotic ring blotch symptom can be easily observed in multiple tea cultivars in many tea gardens. Also, the infected plants show same symptom in the second year. Virus distribution studies showed that all the detected tissues including apical buds and roots were TPNRBV positive . Furthermore, 15 individual shoots and leaves showing mixed symptoms of necrotic ring blotch disease were also TPNRBV positive either in symptomatic tissues or in asymptomatic tissues . Particularly, the diseased leaves with necrotic ring blotch symptom were sampled and observed using TEM. Compared with healthy control , multiple virus particles of TPNRBV were detected in cytoplasm of infected-leaf tissue . The particles were in spherical shape with a diameter approximately 85 nm.Both TPNRBV and TPLPV had multiple segments. To accurately determine the presence of TPNRBV or TPLPV in tea plant, multiple primer pairs were designed and validated by PCR analysis using positive samples confirmed in The tea plant has a large geographic distribution in China. The differences in climatic conditions provide diverse ecological environments for tea plant growth and interactions with microorganisms. The 26 samples with discoloration symptoms were collected in Hangzhou. These samples are representative and common in tea-producing areas. In order to determine whether or not putative plant viruses are involved in discolored tea leaves, metagenomic sequencing was performed. NGS-based metagenomic analysis has been recently used to discover plant viruses , 2014. FIlarvirus and BNRBV from genus Blunervirus, there are more tentative viruses identified in Supplementary Material 3, and many of them have not been assigned to genera. Most of the identified viruses have only been detected in few plant species or limited regions. Therefore, it is very difficult to identified plant viruses from tea plant without the application of NGS analysis. Based on genomic identification and molecular characterization, PnVA, MATV, and MATV2 were reported as tentative members of the genus Totivirus, family Totiviridae  have worldwide distribution and can infect herbaceous and woody hosts. In this genus, APLPV, ApMV, PDV, and PNRSV are invasive pathogens of many cultivated fruit trees, such as peach, cherry, prune, apple, and plum (Table 2) had highest abundance and was annotated to putative movement protein 3a based on the best BLASTX hit . Actually, complete RNA3 of TPLPV encodes both movement and coat proteins which have only 24.8 and 31.7% identity, respectively, compared with those of APLPV. Our phylogenetic tree further showed that TPLPV was grouped in the same branch as APLPV, but distinct from known APLPV isolates. Particularly, the 3′ termini of members of family Bromoviridae, including viruses in genera Alfamovirus, Anulavirus, Bromovirus, Cucumovirus, Ilarvirus, and Oleavirus, are not polyadenylated, though generally are highly conserved within a species or isolates, and form strong secondary structures (reported by ICTV)9. However, a poly(A) tail was detected in the 3′ termini of the three segments of TPLPV. This is a unique property within ilarviruses or even family Bromoviridae. Further studies, including the validation of poly(A) tail both in TPNRBV and TPLPV using different strategies to eradicate the possible source of an experimental artifact and the dissection of poly(A) tail-based regulatory mechanism in virus replication, are still required in the future. Generally, the binding of coat protein to the 3′UTR of the genomic RNAs is required for ilarviruses in a phenomenon called genome activation to initiate infection , which supports the observation that a central R residue in consensus sequence (Q/K/R-P/N-T-X-R-S-R/Q-Q/N/S-W/F/Y-A) of RBD is essential for the binding to the RNA  vector systems for the tea plant because stable genetic transformation to downregulate gene expression is still mostly not achievable . It can Taking advantage of NGS, metagenomic analysis was performed to investigate plant viruses in tea plants. Ultimately, seven kinds of plant viruses were identified on the basis of homology annotation and Sanger sequencing. TPNRBV and TPLPV were the two most abundant plant viruses in our tea plant samples. This first report of these two tentative novel viruses includes full-length genome sequence cloning and genetic characterization. Moreover, specific primer pairs for PCR analysis were designed and validated for TPNRBV and TPLPV identification. Sequence conservation analysis and RNA secondary structure prediction assumed that the genome expression of TPLPV may be activated by the binding of coat protein to 3′UTR of the genomic RNAs. RT-PCR detections of TPNRBV in the symptomatic and asymptomatic tissues from field plants showing tea plant necrotic ring blotch disease indicate that TPNRBV can infect tea plants systemically. Particularly, the suspected virus particles of TPNRBV were observed using TEM. Our results advance our understanding of tea plant virology and will be important for the genetic breeding of tea plants in the future.XH, YY, and XW conceived and designed the experiments. XH, WZ, YL, WQ, and LW performed the experiments. XH, FZ, and JZ analyzed the data. XH, WQ, and YW contributed with plant materials. XH wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.