Small RNA Viruses Research Articles

The COVID-19 pandemic and the use of MS disease-modifying therapies

The COVID-19 pandemic and the use of MS disease-modifying therapies

The Tomato spotted wilt virus (TSWV) Genome is Differentially Targeted in TSWV-Infected Tomato (Solanum lycopersicum) with or without Sw-5 Gene.

Tospoviruses cause significant losses to a wide range of agronomic and horticultural crops worldwide. The type member, Tomato spotted wilt tospovirus (TSWV), causes systemic infection in susceptible tomato cultivars, whereas its infection is localized in cultivars carrying the Sw-5 resistance gene. The response to TSWV infection in tomato cultivars with or without Sw-5 was determined at the virus small RNA level in the locally infected leaf. Predicted reads were aligned to TSWV reference sequences. The TSWV genome was found to be differentially processed among each of the three-viral genomic RNAs—Large (L), Medium (M) and Small (S)—in the Sw-5(+) compared to Sw-5(−) genotypes. In the Sw-5(+) cultivar, the L RNA had the highest number of viral small-interfering RNAs (vsiRNAs), whereas in the Sw-5(−) cultivar the number was higher in the S RNA. Among the three-viral genomic RNAs, the distribution of hotspots showed a higher number of reads per million reads of vsiRNAs of 21 and 22 nt class at the 5′ and 3′ ends of the L and the S RNAs, with less coverage in the M RNA. In the Sw-5(−) cultivar, the nature of the 5′ nucleotide-end in the siRNAs varied significantly; reads with 5′-adenine-end were most abundant in the mock control, whereas cytosine and uracil were more abundant in the infected plants. No such differences were seen in case of the resistant genotype. Findings provided insights into the response of tomato cultivars to TSWV infection.

T-Cell Immunity against the Hepatitis C Virus: A Persistent Research Priority in an Era of Highly Effective Therapy.

Approximately 70% of acute hepatitis C virus (HCV) infections become chronic, indicating that the virus is exceptionally well adapted to persist in humans with otherwise normal immune function. Robust, lifelong replication of this small RNA virus does not require a generalized failure of immunity. HCV effectively subverts innate and adaptive host defenses while leaving immunity against other viruses intact. Here, the role of CD4+ and CD8+ T-cell responses in control of HCV infection and their failure to prevent virus persistence in most individuals are reviewed. Two issues of practical importance remain priorities in an era of highly effective antiviral therapy for chronic hepatitis C. First, the characteristics of successful T-cell responses that promote resolution of HCV infection are considered, as they will underpin development of vaccines that prevent HCV persistence. Second, defects in T-cell immunity that facilitate HCV persistence and whether they are reversed after antiviral cure to provide protection from reinfection are also addressed.

Genetic variability and phylogenetic analysis of Lagovirus europaeus strains GI.1 (RHDV) and GI.2 (RHDV2) based on the RNA-dependent RNA polymerase (RdRp) coding gene.

Lagovirus europaeus GI.1 (RHDV-rabbit haemorrhagic disease virus) and GI.2 (RHDV2-rabbit haemorrhagic disease virus 2), family Caliciviridae, genus Lagovirus, are etiological factors of the rabbit haemorrhagic disease (RHD). This small RNA virus is a great model for tracking the variability and evolution of RNA viruses, because it uses an RNA-dependent RNA polymerase (RdRp) to replicate its own genetic material. This polymerase determines the fidelity and the rates of replication and mutation of the virus, conditioning its adaptation to the environment and even to a new host, and thus influencing evolution of the virus. The aim of this study was to determine the genetic variability and phylogenetic relationships of 105 Lagovirus europaeus strains with different genotypes based on the RdRp gene. The strains came from around the world in the years of 1987-2017. The aforementioned group of 105 strains included 14 strains whose RdRp sequences were obtained and analysed in this study, and the rest were retrieved from GenBank: 74 strains classified as genotype GI.1 (RHDV), 14 as GI.2 (RHDV2), 2 strains of Lagovirus europaeus not assigned to any genotype, and a MRCV strain, the sequences of which were collected from GenBank. Among the 14 strains whose RdRp sequences were obtained in this study, the highest variability was presented in the Austrian 237 strain from 2004. The genetic distance between the Austrian 237 strain and the remaining thirteen analysed strains ranged from 0.117 to 0.123 (from 11.7% to 12.3% nucleotide substitutions). The lowest variability, however, was recorded for Hungarian, Czech and Austrian strains. On the phylogenetic tree, the 14 analysed strains were allocated into GI.1c (G2), GI.1d (G3-G5) and GI.1a (RHDVa). Analysis of the genetic variability of the 105 strains of Lagovirus europaeus indicated a growing genetic distance between the strains, both in time and location. Phylogenetic analysis showed a division of the strains into seven groups, dictated by the chronology, geographical location and evolutionary events in the history of the virus, such as mutations and recombinations.

The activation of antiviral RNA interference not only exists in neural progenitor cells but also in somatic cells in mammals

ABSTRACT The RNA interference (RNAi) pathway directs an important antiviral immunity mechanism in plants and invertebrates. Recently, we and others have demonstrated that the antiviral RNAi response is also conserved in mammals, at least to five distinct RNA viruses, including Zika virus (ZIKV). ZIKV may preferentially infect neuronal progenitor cells (NPCs) in the developing foetal brain. Ex vivo ZIKV infection induces RNAi-mediated antiviral response in human NPCs, but not in the more differentiated NPCs or somatic cells. However, litter is known about the in vivo property or function of the virus-derived small-interfering RNAs (vsiRNAs) targeting ZIKV. Here we report a surprising observation: different from ex vivo observations, viral small RNAs (vsRNAs) targeting ZIKV were produced in vivo upon infection in both central neuron system (CNS) and muscle tissues. In addition, our findings demonstrate the production of canonical vsiRNAs in murine CNS upon antiviral RNAi activation by Sindbis virus (SINV), suggesting the possibility of antiviral immune strategy applied by mammals in the CNS.

Detection and partial molecular characterization of Picobirnavirus in swine from the state of Minas Gerais, Brazil.

Picobirnavirus (PBV) is a small two-segmented double-stranded RNA (dsRNA) virus that has been identified in diarrheic feces of a large range of animal hosts, including humans. For this reason, PBV has been recognized as an opportunistic agent of gastrointestinal disease. Even under these circumstances, there is a lack of studies regarding this pathogen. Not outstanding, in Brazil, the single description of the PBV occurrence in pigs was provided in the 1980s. Hence, this study aimed to verify the PBV occurrence in Brazilian swine farms and to perform molecular characterization of the identified strains. High genetic variability was found in the analyzed sequences. Further studies comprehending the infection of swine by PBV in Brazilian herds should be performed to provide more accurate information on its epidemiology and to discuss the role of the virus in gastrointestinal diseases.

Carbon dots for effective photodynamic inactivation of virus.

The antiviral function of carbon dots (CDots) with visible light exposure was evaluated, for which the model bacteriophages MS2 as a surrogate of small RNA viruses were used. The results show clearly that the visible light-activated CDots are highly effective in diminishing the infectivity of MS2 in both low and high titer samples to the host E. coli cells, and the antiviral effects are dot concentration- and treatment time-dependent. The action of CDots apparently causes no significant damage to the structural integrity and morphology of the MS2 phage or the breakdown of the capsid proteins, but does result in the protein carbonylation (a commonly used indicator for protein oxidation) and the degradation of viral genomic RNA. Mechanistically the results may be understood in the framework of photodynamic effects that are associated with the unique excited state properties and processes of CDots. Opportunities for potentially broad applications of CDots coupled with visible/natural light in the prevention and control of viral transmission and spread are highlighted and discussed.

Lessons learned from adenovirus (1970-2019).

Animal viruses are well recognized for their ability to uncover fundamental cell and molecular processes, and adenovirus certainly provides a prime example. This review illustrates the lessons learned from studying adenovirus over the past five decades. We take a look back at the key studies of adenovirus structure and biophysical properties, which revealed the mechanisms of adenovirus association with antibody, cell receptor, and immune molecules that regulate infection. In addition, we discuss the critical contribution of studies of adenovirus gene expression to elucidation of fundamental reactions in pre-mRNA processing and its regulation. Other pioneering studies furnished the first examples of protein-primed initiation of DNA synthesis and viral small RNAs. As a nonenveloped virus, adenoviruses have furnished insights into the modes of virus attachment, entry, and penetration of host cells, and we discuss the diversity of cell receptors that support these processes, as well as membrane penetration. As a result of these extensive studies, adenovirus vectors were among the first to be developed for therapeutic applications. We highlight some of the early (unsuccessful) trials and the lessons learned from them.

Host Cell Calpains Can Cleave Structural Proteins from the Enterovirus Polyprotein.

Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C’s cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.

In vitro production of synthetic viral RNAs and their delivery into mammalian cells and the application of viral RNAs in the study of innate interferon responses.

Mammalian cells express different types of RNA molecules that can be classified as protein coding RNAs (mRNA) and non-coding RNAs (ncRNAs) the latter of which have housekeeping and regulatory functions in cells. Cellular RNAs are not recognized by cellular pattern recognition receptors (PRRs) and innate immunity is not activated. RNA viruses encode and express RNA molecules that usually differ from cell-specific RNAs and they include for instance 5'capped and 5'mono- and triphosphorylated RNAs, small viral RNAs and viral RNA-protein complexes called vRNPs. These molecules are recognized by certain members of Toll-like receptor (TLR) and RIG-I-like receptor (RLR) families leading to activation of innate immune responses and the production of antiviral cytokines, such as type I and type III interferons (IFNs). Virus-specific ssRNA and dsRNA molecules that mimic the viral genomic RNAs or their replication intermediates can efficiently be produced by bacteriophage T7 DNA-dependent RNA polymerase and bacteriophage phi6 RNA-dependent RNA polymerase, respectively. These molecules can then be delivered into mammalian cells and the mechanisms of activation of innate immune responses can be studied. In addition, synthetic viral dsRNAs can be processed to small interfering RNAs (siRNAs) by a Dicer enzyme to produce a swarm of antiviral siRNAs. Here we describe the biology of RNAs, their in vitro production and delivery into mammalian cells as well as how these molecules can be used to inhibit virus replication and to study the mechanisms of activation of the innate immune system.

Bioinformatically-predicted varicella zoster virus small non-coding RNAs are expressed in lytically-infected epithelial cells and neurons

Most herpesviruses use both host and viral small non-coding RNAs (sncRNA), especially microRNA, to modulate infection. Bioinformatic analyses of NGS data obtained from Varicella Zoster virus (VZV)-infected cells predicted 24 VZVsncRNA, seven of which were confirmed to be expressed in infected fibroblasts and neurons using stem-loop quantitative reverse transcription PCR (SL-PCR). We here assayed for the expression of all 24 of the bioinformatically predicted VZVsncRNA in cells productively infected by VZV using SL-PCR. 23 of the 24 predicted sequences were detected in VZV-infected ARPE19 cells and 19 of the 24 sequences in infected human neurons generated by two methods from embryonic stem cells. We also show that blocking one of two newly-tested VZV-encoded sncRNA using locked nucleotide antagonists significantly increased viral replication. These findings suggest that further study of VZV encoded sncRNA could elucidate an additional level of regulation of the life cycle of this pathogenic human herpesvirus.

Immune Response Modulation by Caliciviruses.

Noroviruses and Sapoviruses, classified in the Caliciviridae family, are small positive-stranded RNA viruses, considered nowadays the leading cause of acute gastroenteritis globally in both children and adults. Although most noroviruses have been associated with gastrointestinal disease in humans, almost 50 years after its discovery, there is still a lack of comprehensive evidence regarding its biology and pathogenesis mainly because they can be neither conveniently grown in cultured cells nor propagated in animal models. However, other members of this family such as Feline calicivirus (FCV), Murine norovirus (MNV), Rabbit hemorrhagic disease virus (RHDV), and Porcine sapovirus (PS), from which there are accessible propagation systems, have been useful to study the calicivirus replication strategies. Using cell cultures and animal models, many of the functions of the viral proteins in the viral replication cycles have been well-characterized. Moreover, evidence of the role of viral proteins from different members of the family in the establishment of infection has been generated and the mechanism of their immunopathogenesis begins to be understood. In this review, we discuss different aspects of how caliciviruses are implicated in membrane rearrangements, apoptosis, and evasion of the immune responses, highlighting some of the pathogenic mechanisms triggered by different members of the Caliciviridae family.

No evidence for viral small RNA production and antiviral function of Argonaute 2 in human cells

RNA interference (RNAi) has strong antiviral activity in a range of animal phyla, but the extent to which RNAi controls virus infection in chordates, and specifically mammals remains incompletely understood. Here we analyze the antiviral activity of RNAi against a number of positive-sense RNA viruses using Argonaute-2 deficient human cells. In line with absence of virus-derived siRNAs, Sindbis virus, yellow fever virus, and encephalomyocarditis virus replicated with similar kinetics in wildtype cells and Argonaute-2 deficient cells. Coxsackievirus B3 (CVB3) carrying mutations in the viral 3A protein, previously proposed to be a virus-encoded suppressor of RNAi in another picornavirus, human enterovirus 71, had a strong replication defect in wildtype cells. However, this defect was not rescued in Argonaute-2 deficient cells, arguing against a role of CVB3 3A as an RNAi suppressor. In agreement, neither infection with wildtype nor 3A mutant CVB3 resulted in small RNA production with the hallmarks of canonical vsiRNAs. Together, our results argue against strong antiviral activity of RNAi under these experimental conditions, but do not exclude that antiviral RNAi may be functional under other cellular, experimental, or physiological conditions in mammals.

Rice Dwarf Virus Small RNA Profiles in Rice and Leafhopper Reveal Distinct Patterns in Cross-Kingdom Hosts.

RNA silencing has evolved as a widespread antiviral strategy in many eukaryotic organisms. Antiviral RNA silencing is mediated by virus-derived small RNAs (vsiRNAs), created by the cleavage of double-stranded viral RNA substrates by Dicer (Dcr) in animals or Dicer-like (DCL) proteins in plants. However, little is known about how the RNA silencing mechanisms of different hosts respond to the same virus infection. We performed high-throughput small RNA sequencing in Nephotettix cincticeps and Oryza sativa infected with Rice dwarf phytoreovirus and analyzed the distinct accumulation of vsiRNAs in these two hosts. The results suggested a potential branch in the evolution of antiviral RNA silencing of insect and plant hosts. The rice vsiRNAs were predominantly 21 and 22 nucleotides (nt) long, suggesting that OsDCL4 and OsDCL2 are involved in their production, whereas 21-nt vsiRNAs dominated in leafhopper, suggesting the involvement of a Dcr-2 homolog. Furthermore, we identified ~50-fold more vsiRNAs in rice than in leafhoppers, which might be partially attributable to the activity of RNA-dependent RNA polymerase 6 (RDR6) in rice and the lack of RDR genes in leafhoppers. Our data established a basis for further comparative studies on the evolution of RNA silencing-based interactions between a virus and its hosts, across kingdoms.

Antiviral defense against Cypovirus 1 (Reoviridae) infection in the silkworm, Bombyx mori.

Recent years have shown a large increase in studies of infection of the silkworm (Bombyx mori) with Cypovirus 1 (previously designated as B. mori cytoplasmic polyhedrosis virus), that causes serious damage in sericulture. Cypovirus 1 has a single-layered capsid that encapsulates a segmented double-strand RNA (dsRNA) genome which are attractive features for the establishment of a biotechnological platform for the production of specialized gene silencing agents, either as recombinant viruses or as viral-like particles with nonreplicative dsRNA cargo. For both combatting viral disease and application of Cypovirus-based pest control, however, a better understanding is needed of the innate immune response caused by Cypovirus infection of the midgut of lepidopteran larvae. Studies of deep sequencing of viral small RNAs have indicated the importance of the RNA interference pathway in the control of Cypovirus infection although many functional aspects still need to be elucidated and conclusive evidence is lacking. A considerable number of transcriptome studies were carried out that revealed a complex response that hitherto remains uncharacterized because of a dearth in functional studies. Also, the uptake mechanism of Cypovirus by the midgut cells remains unclarified because of contrasting mechanisms revealed by electron microscopy and functional studies. The field will benefit from an increase in functional studies that will depend on transgenic silkworm technology and reverse genetics systems for Cypovirus 1.

A 212-nt long RNA structure in the Tobacco necrosis virus-D RNA genome is resistant to Xrn degradation.

Plus-strand RNA viruses can accumulate viral RNA degradation products during infections. Some of these decay intermediates are generated by the cytosolic 5′-to-3′ exoribonuclease Xrn1 (mammals and yeast) or Xrn4 (plants) and are formed when the enzyme stalls on substrate RNAs upon encountering inhibitory RNA structures. Many Xrn-generated RNAs correspond to 3′-terminal segments within the 3′-UTR of viral genomes and perform important functions during infections. Here we have investigated a 3′-terminal small viral RNA (svRNA) generated by Xrn during infections with Tobacco necrosis virus-D (family Tombusviridae). Our results indicate that (i) unlike known stalling RNA structures that are compact and modular, the TNV-D structure encompasses the entire 212 nt of the svRNA and is not functionally transposable, (ii) at least two tertiary interactions within the RNA structure are required for effective Xrn blocking and (iii) most of the svRNA generated in infections is derived from viral polymerase-generated subgenomic mRNA1. In vitro and in vivo analyses allowed for inferences on roles for the svRNA. Our findings provide a new and distinct addition to the growing list of Xrn-resistant viral RNAs and stalling structures found associated with different plant and animal RNA viruses.

The Activation of Antiviral RNA Interference Not Only Exist in Neural Progenitor Cells But Also in Somatic Cells in Mammals

The RNA interference (RNAi) pathway directs an important antiviral immunity mechanism in plants and invertebrates. Recently, we and others have demonstrated that the induction and suppression of antiviral RNAi response are also conserved in mammals, at least to five distinct RNA viruses, including Zika virus (ZIKV). ZIKV preferentially infects neuronal progenitor cells (NPCs) in the developing fetal brain. Xu et al. have revealed that ex vivo ZIKV infection induces RNAi-mediated antiviral response in human NPCs, but not in the more differentiated NPCs or somatic cells. However, less is known about the in vivo property or function of the virus-derived small-interfering RNAs (vsiRNAs) targeting Zika virus. Here we report a surprising observation: viral small RNAs (vsRNAs) targeting ZIKV were produced in vivo upon infection in both central neuron system (CNS) and muscle tissues. In addition, our findings demonstrate the production of canonical vsiRNAs in murine CNS upon antiviral RNAi activation by Sindbis virus, suggesting the possibility of antiviral immune strategy applied by mammals in the CNS.

Evasion Strategies of Zika Virus Antagonizing Host Innate Immunity

Zika virus is a small enveloped positive-strand RNA virus, which belongs to the Flaviviridae family. The RNA genome of all flaviviruses encodes three structural and seven nonstructural genes, together with 5′- and 3′-untranslated region genes flanking. Accompanying the explosive nature of the Zika outbreak in 2014, there was a devastating congenital neurodevelopmental disease in fetuses. Apart from viral RNA replication, polyprotein cleavage, Zika virus nonstructural proteins also play vital roles in the host's innate immune evasion. In this brief report, we summarize the evasion mechanisms of the viral nonstructural proteins antagonizing host antiviral innate immunity.

Detection of Sacbrood Virus Infecting Apis cerana (AcSBV) Using Polyclonal Antibody Raised against Recombinant VP3 Capsid Protein Expressed in Escherichia coli

In Korea, sacbrood virus infecting Apis cerana (AcSBV) is a small RNA virus belonging to the family Iflaviridae. AcSBV causes larval death, and even the collapse of entire bee colonies and its viral capsid is composed of four structural proteins (VP1, VP2, VP3 and VP4). In this study, we synthesized VP3 gene based on genomic sequences to insert into a His-tag expression plasmid of pET-28a. The resultant recombinant vector was then transformed into Escherichia coli BL21 (DE3) to express the recombinant VP3 protein (rVP3). The rVP3 protein was strongly expressed in an insoluble fraction, with a molecular size of around 26 kDa. Purification of the rVP3 protein using an affinity Ni-NTA resin, followed by SDS-PAGE separation, resulted in a high purity of the protein and used as an antigen to raise the polyclonal antibodies. Mice were then immunized with the purified rVP3 protein and the antiserum collected at 4SUPth/SUP week and 6SUPth/SUP week was able to detect the rVP3 at a concentration of 500 ng and 250 ng. Furthermore, IgG antibody levels reacted with wild-type larva crude protein extract, AcSBV-infected larva crude protein extracts and rVP3 protein were quantitatively analyzed by ELISA and qualitatively analyzed by western blotting. This is the first report on production of polyclonal antibodies directed against the rVP3 protein of AcSBV and its use for detection and diagnosis of virus from field samples.

Why all blood donations should be tested for hepatitis E virus (HEV)

BackgroundHepatitis E is a liver disease caused by a small RNA virus known as hepatitis E virus (HEV). Four major genotypes infect humans, of which genotype 1 and 2 (HEV-1, HEV-2) are endemic mainly in Asia and responsible for waterborne epidemics. HEV-3 and HEV-4 are widely distributed in pigs and can be transmitted to humans mainly by undercooked meat, and contact with pigs. HEV-3 is the main genotype in industrialised countries with moderate climate conditions and object of this debate.Main textWhereas an HEV-3 infection in healthy humans is mostly asymptomatic, HEV-3 can induce chronic infection in immunocompromised individuals and acute-on-chronic liver failure (ACLF) in patients with underlying liver diseases. The number of reported cases of HEV-infections in industrialised nations increased significantly in the last years. Since HEV-3 has been transmitted by blood transfusion to other humans, testing of blood donors has been introduced or introduction is being discussed in some industrialised countries. In this article we summarise the arguments in favour of testing all blood donations for HEV-3.ConclusionThe number of HEV infection in the population and the possibility of HEV transmission by blood transfusion are increasing. Transmission by blood transfusion can be dangerous for the recipients considering their immunosuppressive status, underlying disease or other circumstances requiring blood transfusion. This argues in favour of testing all blood donations for HEV-3 to prevent transmission.