Negative-sense RNA Research Articles

A detailed analysis of codon usages bias and influencing factors in the nucleocapsid gene of Nipah Virus

Several outbreaks of Nipah Virus (NiV) have recently been reported in various parts of the world including India. The 18.2-kb size single-stranded negative-sense RNA genome of NiV has six genes that encode nine proteins. Among nine, the nucleoprotein (N) protein is the most abundant viral protein. The relative availability of N protein is a determining factor for the activation of genome encapsidation, replicase activity, and regulating viral RNA synthesis. In this study, we have conducted a codon usage analysis of N protein-encoding gene (N gene) of NiV. The relative synonymous codon usage (RSCU) values, in combination with an ENC value of 51.048, represented low codon usage bias in the N gene. The effect of mutational pressure on codon usage bias was confirmed by significant correlations of GC3s, G3s, C3s, A3s, U3s, and ENC values with whole nucleotide contents (GC%, G%, C%, A%, and U%). Correlation study of GC3s, G3s, C3s, A3s, and U3s with axis values of correspondence analysis (CA) also supported the role of mutational pressure. The correlation study of Gravy values with GC3s, G3s, C3s, A3s, and U3s revealed the presence of natural selection in addition to mutational pressure on codon usage bias. Moreover, NiV codon adaptation index (CAI) value higher than their corresponding expected CAI (eCAI) values against human (CAI, 0.726; eCAI, 0.715), pig (CAI, 0.837; eCAI, 0.819), and bat (CAI, 0.762; eCAI, 0.750) also indicated natural selection play role on codon usage bias. Additionally, geographical distribution, and evolutionary processes also influenced the codon usage bias to some extent.

ICTV Virus Taxonomy Profile: Xinmoviridae 2023.

Xinmoviridae is a family of viruses with negative-sense RNA genomes of 9-14 kilobases. Xinmovirids typically infect beneficial and pest insects but their host range has not yet been investigated systematically and hence may be broader. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family of Xinmoviridae, which is available at ictv.global/report/xinmoviridae.

ICTV Virus Taxonomy Profile: Tosoviridae 2023.

Tosoviridae is a family of negative-sense RNA viruses with genomes totaling about 12.3 kb that have been found in turtles. The tosovirid genome consists of two segments, each with two open reading frames (ORFs) in ambisense orientation. The small (S) segment encodes a nucleoprotein (NP) and a glycoprotein precursor (GPC); the large (L) segment encodes an L protein containing an RNA-directed RNA polymerase (RdRP) domain and a zinc-binding (Z) protein. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Tosoviridae, which is available at ictv.global/report/tosoviridae.

ICTV Virus Taxonomy Profile: Amnoonviridae 2023.

Amnoonviridae is a family of negative-sense RNA viruses with genomes totalling about 10.3 kb. These viruses have been found in fish. The amnoonvirid genome consists of 10 segments, each with at least 1 open reading frame (ORF). The RNA1-3 ORFs encode the three subunits of the viral polymerase. The RNA4 ORF encodes a nucleoprotein. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Amnoonviridae, which is available at ictv.global/report/amnoonviridae.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.

ICTV Virus Taxonomy Profile: Sunviridae 2023.

Sunviridae is a family of negative-sense RNA viruses with genomes of about 17.2 kb that have been found in snakes. The sunvirid genome comprises nonsegmented RNA with six open reading frames (ORFs) >1 kb that are predicted to encode six proteins. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Sunviridae, which is available at ictv.global/report/sunviridae.

ICTV Virus Taxonomy Profile: Qinviridae 2023.

Qinviridae is a family of negative-sense RNA viruses with genomes of 7.3-8.2 kb that have been associated with crustaceans, insects, gastropods, and nematodes. The qinvirid genome consists of two segments, each with at least one open reading frame (ORF). The large (L) segment ORF encodes a large protein containing an RNA-directed RNA polymerase (RdRP) domain. The small (S) segment ORF encodes a nucleocapsid protein. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Qinviridae, which is available at ictv.global/report/qinviridae.

ICTV Virus Taxonomy Profile: Yueviridae 2023.

Yueviridae is a family of negative-sense RNA viruses with genomes of 7.8-8.2 kb that have been associated with crustaceans, insects, stramenopiles and plants. The yuevirid genome consist of two segments, each with at least one ORF. The large (L) segment ORF encodes a large protein containing an RNA-directed RNA polymerase domain. The small (S) segment ORF encodes a nucleocapsid protein. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Yueviridae, which is available at http://www.ictv.global/report/yueviridae.

Surveillance and Genetic Analysis of Jamestown Canyon Virus in New York State: 2001-2022.

Jamestown Canyon virus (JCV) (Peribunyavirdae; Orthobunyavirus) is a mosquito-borne pathogen endemic to North America. The genome is composed of three segmented negative-sense RNA fragments designated as small, medium, and large. Jamestown Canyon virus is an emerging threat to public health, and infection in humans can cause severe neurological diseases, including encephalitis and meningitis. We report JCV mosquito surveillance data from 2001 to 2022 in New York state. Jamestown Canyon virus was detected in 12 mosquito species, with the greatest prevalence in Aedes canadensis and Anopheles punctipennis. Detection fluctuated annually, with the highest levels recorded in 2020. Overall, JCV infection rates were significantly greater from 2012 to 2022 compared with 2001 to 2011. Full-genome sequencing and phylogenetic analysis were also performed with representative JCV isolates collected from 2003 to 2022. These data demonstrated the circulation of numerous genetic variants, broad geographic separation, and the first identification of lineage B JCV in New York state in 2022.

SND1 binds SARS-CoV-2 negative-sense RNA and promotes viral RNA synthesis through NSP9

Regulation of viral RNA biogenesis is fundamental to productive SARS-CoV-2 infection. To characterize host RNA-binding proteins (RBPs) involved in this process, we biochemically identified proteins bound to genomic and subgenomic SARS-CoV-2 RNAs. We find that the host protein SND1 binds the 5' end of negative-sense viral RNA and is required for SARS-CoV-2 RNA synthesis. SND1-depleted cells form smaller replication organelles and display diminished virus growth kinetics. We discover that NSP9, a viral RBP and direct SND1 interaction partner, is covalently linked to the 5' ends of positive- and negative-sense RNAs produced during infection. These linkages occur at replication-transcription initiation sites, consistent with NSP9 priming viral RNA synthesis. Mechanistically, SND1 remodels NSP9 occupancy and alters the covalent linkage of NSP9 to initiating nucleotides in viral RNA. Our findings implicate NSP9 in the initiation of SARS-CoV-2 RNA synthesis and unravel an unsuspected role of a cellular protein in orchestrating viral RNA production.

Novel Antiviral Molecules against Ebola Virus Infection.

Infection with Ebola virus (EBOV) is responsible for hemorrhagic fever in humans with a high mortality rate. Combined efforts of prevention and therapeutic intervention are required to tackle highly variable RNA viruses, whose infections often lead to outbreaks. Here, we have screened the 2P2I3D chemical library using a nanoluciferase-based protein complementation assay (NPCA) and isolated two compounds that disrupt the interaction of the EBOV protein fragment VP35IID with the N-terminus of the dsRNA-binding proteins PKR and PACT, involved in IFN response and/or intrinsic immunity, respectively. The two compounds inhibited EBOV infection in cell culture as well as infection by measles virus (MV) independently of IFN induction. Consequently, we propose that the compounds are antiviral by restoring intrinsic immunity driven by PACT. Given that PACT is highly conserved across mammals, our data support further testing of the compounds in other species, as well as against other negative-sense RNA viruses.

Plant and animal positive-sense single-stranded RNA viruses encode small proteins important for viral infection in their negative-sense strand

Positive-sense single-stranded RNA (+ssRNA) viruses, the most abundant viruses of eukaryotes in nature, require the synthesis of negative-sense RNA (−RNA) using their genomic (positive-sense) RNA (+RNA) as a template for replication. Based on current evidence, viral proteins are translated via viral +RNAs, whereas −RNA is considered to be a viral replication intermediate without coding capacity. Here, we report that plant and animal +ssRNA viruses contain small open reading frames (ORFs) in their −RNA (reverse ORFs [rORFs]). Using turnip mosaic virus (TuMV) as a model for plant +ssRNA viruses, we demonstrate that small proteins encoded by rORFs display specific subcellular localizations, and confirm the presence of rORF2 in infected cells through mass spectrometry analysis. The protein encoded by TuMV rORF2 forms punctuate granules that are localized in the perinuclear region and co-localized with viral replication complexes. The rORF2 protein can directly interact with the viral RNA-dependent RNA polymerase, and mutation of rORF2 completely abolishes virus infection, whereas ectopic expression of rORF2 rescues the mutant virus. Furthermore, we show that several rORFs in the −RNA of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have the ability to suppress type I interferon production and facilitate the infection of vesicular stomatitis virus. In addition, we provide evidence that TuMV might utilize internal ribosome entry sites to translate these small rORFs. Taken together, these findings indicate that the −RNA of +ssRNA viruses can also have the coding capacity and that small proteins encoded therein play critical roles in viral infection, revealing a viral proteome larger than previously thought.

Segmented, Negative-Sense RNA Viruses of Humans: Genetic Systems and Experimental Uses of Reporter Strains.

Negative-stranded RNA viruses are a large group of viruses that encode their genomes in RNA across multiple segments in an orientation antisense to messenger RNA. Their members infect broad ranges of hosts, and there are a number of notable human pathogens. Here, we examine the development of reverse genetic systems as applied to these virus families, emphasizing conserved approaches illustrated by some of the prominent members that cause significant human disease. We also describe the utility of their genetic systems in the development of reporter strains of the viruses and some biological insights made possible by their use. To conclude the review, we highlight some possible future uses of reporter viruses that not only will increase our basic understanding of how these viruses replicate and cause disease but also could inform the development of new approaches to therapeutically intervene.

Complete Genome Characterization of Penicillimonavirus gammaplasmoparae, a Bipartite Member of the Family Mymonaviridae.

In this study, we identified Plasmopara-viticola-lesion-associated mononegaambi virus 3 (recently classified as Penicillimonavirus gammaplasmoparae), a fungi-associated mymonavirus, in grapevine plants showing an unusual upward curling symptomatology on the leaves and premature decline. Mymonaviridae is a family comprising nine genera of negative-sense single-stranded RNA viruses infecting filamentous fungi, although few of them have been associated with oomycetes, plants, and insects. Although the first mymonavirus genome description was reported a decade ago, the genome organization of several genera in the family, including the genus Penicillimonavirus, has remained unclear to date. We have determined the complete genome of P. gammaplasmoparae, which represents the first complete genomic sequence for this genus. Moreover, we provide strong evidence that P. gammaplasmoparae genome is bipartite and comprises two RNA molecules of around 6150 and 4560 nt. Our results indicate that the grapevine powdery mildew pathogen, Erysiphe necator, was also present in the analyzed plants and suggest P. gammaplasmoparae could be infecting this fungus. However, whether the fungus and/or the mycovirus are associated with the symptomatology that initially prompted these efforts remains to be determined.

The First Virome of a Russian Vineyard.

Among other pathogens, more than 80 viruses infect grapevine. The aim of this work was to study the virome diversity of grapevine viruses and mycoviruses of a vineyard using high-throughput sequencing technologies. The grapevine virome was studied in symptomatic vines of the Rkatsiteli cultivar (V. vinifera) collected at the vineyards of the Krasnodar Krai in Russia. Ribosomal-depleted total RNA and isolated small RNAs were used for library preparation and high-throughput sequencing. Six grapevine-infecting viruses and two viroids were validated by RT-PCR and analyzed phylogenetically. We identified the presence of grapevine leafroll-associated virus 3, grapevine Pinot gris virus, grapevine virus T, grapevine rupestris stem-pitting-associated virus, grapevine fleck virus, and grapevine rupestris vein feathering virus, as well as two viroids, grapevine yellow speckle viroid 1 and hop stunt viroid. We also studied the mycovirome of the vineyard and identified nine viruses with single-stranded positive-sense RNA genomes: alternaria arborescens mitovirus 1, botrytis cinerea mitovirus 1, botrytis cinerea mitovirus 2, botrytis cinerea mitovirus 3, botrytis cinerea mitovirus 4, sclerotinia sclerotiorum mitovirus 3, botrytis cinerea hypovirus 1, grapevine-associated narnavirus 1, and botrytis virus F. In addition, we identified botrytis cinerea hypovirus 1 satellite-like RNA and two single-stranded negative-sense RNA viruses. This is the first study of grapevine mycoviruses in Russia. The obtained result will contribute to the development of biocontrol strategies in the future.

Structural dynamics of the RNA dependent RNA polymerase of H1N1 strain affecting humans: a bioinformatics approach

The Influenza flu is a pandemic disease that renders the highest risk factor to the society due to its efficient ability of airborne transmission. Studies on the H1N1 strain gained significant focus, since its pandemic outbreak in 2009 and particularly the computational studies on its structural elements significantly aided in revealing their functional uniqueness. Among the 10 structural proteins of H1N1, the RNA-dependent RNA polymerase (RdRp) heterotrimeric protein complex, which is responsible for the synthesis of viral RNA (vRNA) from the negative-sense RNA genome of the virus, is the focus of the present study. This study aimed to investigate the structural dynamics of the RdRp complex with particular emphasis on the reported 17 mutations. The mutant strain is more stabilized by strong concerted residue-residue interactions at both intra- and inter- monomeric levels. In comparison, the mutant strain is structurally flexible with enhanced stabilizing interactions. The structural dynamics of RdRp are significantly governed by the dynamics of the (i) endonuclease domain of PA, (ii) RNA-entry region of PB1 and (iii) cap-binding region of PB2. Explicitly, the cap binding region of PB2 expresses (i) a concerted motion with the RNA-entry region, along with (ii) an anti-correlated motion with the endonuclease domain of the PA subunit, which further supports the stable dynamics of cap-binding towards RNA binding. These findings contribute to the understanding of the structural dynamics associated with the pandemic and mutant structures of RdRp and render a basic knowledge for further development of novel inhibitors towards influenza flu affected humans. Communicated by Ramaswamy H. Sarma

RNA viruses of Crithidia bombi, a parasite of bumblebees

Leishbuviridae (Bunyavirales) are a diverse monophyletic group of negative-sense single-stranded RNA virus infecting parasitic flagellates of the family Trypanosomatidae. The presence of RNA viruses in trypanosomatids can influence the virulence of the latter. Here, we performed a screening for viruses in Crithidia bombi – a common parasite of important pollinators Bombus spp. (bumblebees) that negatively affects its host in stressful conditions. The majority (8/10) of C. bombi isolates collected in Europe and North America were positive for a virus that we named Crithidia bombi leishbuvirus 1 with high conservation of amino acid sequences between isolates. The results of our comparative phylogenetic analyses of the trypanosomatids and their viruses suggest that the high mobility of bumblebees and frequent coinfections by different strains of C. bombi determine an extensive viral exchange between the latter.

ICTV Virus Taxonomy Profile: Phenuiviridae 2023.

The family Phenuiviridae comprises viruses with 2-8 segments of negative-sense or ambisense RNA, comprising 8.1-25.1 kb in total. Virions are typically enveloped with spherical or pleomorphic morphology but can also be non-enveloped filaments. Phenuivirids infect animals including livestock and humans, birds, plants or fungi, as well as arthropods that serve as single hosts or act as biological vectors for transmission to animals or plants. Phenuivirids include important pathogens of humans, livestock, seafood and agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Phenuiviridae, which is available at ictv.global/report/phenuiviridae.

Molecular and genetic characterization of bovine parainfluenza type 3 European field and vaccine strains.

Bovine Parainfluenza Type 3 virus (BPIV-3) is an enveloped, non-segmented single-stranded, negative-sense RNA virus belonging to the Paramyxoviridae family (genus Respirovirus) with a well-known role in Bovine Respiratory Disease (BRD) onset. Being isolated for the first time in 1959, BPIV-3 currently circulates worldwide in cattle herds and is routinely tested in suspected BRD cases. Different commercial vaccines are available to prevent infection and/or to reduce the clinical signs associated with BPIV-3 infection, which are essential to prevent secondary infections. Despite years of molecular surveillance, a very limited number of complete genome sequences were made publicly available, preventing thus the understanding of the genetic diversity of the circulating strains in the field. In addition, no data about the genetic identity between field and vaccine strains is currently available. In this study, we sequenced the full-genome and genetically characterized BPIV-3 strains isolated from animals displaying respiratory illness in France and Sweden, as well as the vaccine strains contained in three different commercialized vaccines. Our results show that the sequences from France and Sweden belong to genotype C. However, a third sequence from Sweden from 2017 clustered within genotype A. The sequencing of vaccine strains revealed that two of the vaccine strains clustered within genotype C, whereas the third vaccine strain belonged to genotype A. Altogether, our findings suggest that both genotypes A and C circulate in Europe and that BPIV-3 field and vaccine strains are genetically divergent. Our sequencing results could be useful to better understand the genetic differences between the circulating field and vaccine BPIV-3 strains. This is crucial for a correct interpretation of diagnostic findings and for the assessment of BPIV-3 prevalence in cattle population.

Virome analysis of an ectomycorrhizal fungus Suillus luteus revealing potential evolutionary implications.

Suillus luteus is a widespread edible ectomycorrhizal fungus that holds significant importance in both ecological and economic value. Mycoviruses are ubiquitous infectious agents hosted in different fungi, with some known to exert beneficial or detrimental effects on their hosts. However, mycoviruses hosted in ectomycorrhizal fungi remain poorly studied. To address this gap in knowledge, we employed next-generation sequencing (NGS) to investigate the virome of S. luteus. Using BLASTp analysis and phylogenetic tree construction, we identified 33 mycovirus species, with over half of them belonging to the phylum Lenarviricota, and 29 of these viruses were novel. These mycoviruses were further grouped into 11 lineages, with the discovery of a new negative-sense single-stranded RNA viral family in the order Bunyavirales. In addition, our findings suggest the occurrence of cross-species transmission (CST) between the fungus and ticks, shedding light on potential evolutionary events that have shaped the viral community in different hosts. This study is not only the first study to characterize mycoviruses in S. luteus but highlights the enormous diversity of mycoviruses and their implications for virus evolution.