Negative-sense Single-stranded RNA Research Articles

Non-Ebola Filoviruses: Potential Threats to Global Health Security

Filoviruses are negative-sense single-stranded RNA viruses often associated with severe and highly lethal hemorrhagic fever in humans and nonhuman primates, with case fatality rates as high as 90%. Of the known filoviruses, Ebola virus (EBOV), the prototype of the genus Orthoebolavirus, has been a major public health concern as it frequently causes outbreaks and was associated with an unprecedented outbreak in several Western African countries in 2013–2016, affecting 28,610 people, 11,308 of whom died. Thereafter, filovirus research mostly focused on EBOV, paying less attention to other equally deadly orthoebolaviruses (Sudan, Bundibugyo, and Taï Forest viruses) and orthomarburgviruses (Marburg and Ravn viruses). Some of these filoviruses have emerged in nonendemic areas, as exemplified by four Marburg disease outbreaks recorded in Guinea, Ghana, Tanzania, and Equatorial Guinea between 2021 and 2023. Similarly, the Sudan virus has reemerged in Uganda 10 years after the last recorded outbreak. Moreover, several novel bat-derived filoviruses have been discovered in the last 15 years (Lloviu virus, Bombali virus, Měnglà virus, and Dehong virus), most of which are poorly characterized but may display a wide host range. These novel viruses have the potential to cause outbreaks in humans. Several gaps are yet to be addressed regarding known and emerging filoviruses. These gaps include the virus ecology and pathogenicity, mechanisms of zoonotic transmission, host range and susceptibility, and the development of specific medical countermeasures. In this review, we summarize the current knowledge on non-Ebola filoviruses (Bombali virus, Bundibugyo virus, Reston virus, Sudan virus, Tai Forest virus, Marburg virus, Ravn virus, Lloviu virus, Měnglà virus, and Dehong virus) and suggest some strategies to accelerate specific countermeasure development.

Comparative analysis of the taxonomic classification criteria for a number of groups of pathogenic DNA and RNA viruses based on genomic data

The basis for criteria of the taxonomic classification of DNA and RNA viruses based on data of the genomic sequencing are viewed in this review. The genomic sequences of viruses, which have genome represented by double-stranded DNA (orthopoxviruses as example), positive-sense single-stranded RNA (alphaviruses and flaviviruses as example), non-segmented negative-sense single-stranded RNA (filoviruses as example), segmented negative-sense single-stranded RNA (arenaviruses and phleboviruses as example) are analyzed. The levels of genetic variability that determine the assignment of compared viruses to taxa of various orders are established for each group of viruses.

Molecular Characterization of a Novel Rubodvirus Infecting Raspberries.

A novel negative-sense single-stranded RNA virus showing genetic similarity to viruses of the genus Rubodvirus has been found in raspberry plants in the Czech Republic and has tentatively been named raspberry rubodvirus 1 (RaRV1). Phylogenetic analysis confirmed its clustering within the group, albeit distantly related to other members. A screening of 679 plant and 168 arthropod samples from the Czech Republic and Norway revealed RaRV1 in 10 raspberry shrubs, one batch of Aphis idaei, and one individual of Orius minutus. Furthermore, a distinct isolate of this virus was found, sharing 95% amino acid identity in both the full nucleoprotein and partial sequence of the RNA-dependent RNA polymerase gene sequences, meeting the species demarcation criteria. This discovery marks the first reported instance of a rubodvirus infecting raspberry plants. Although transmission experiments under experimental conditions were unsuccessful, positive detection of the virus in some insects suggests their potential role as vectors for the virus.

RNA N6-methyladenosine methylation in influenza A virus infection.

Influenza A virus (IAV) is a negative-sense single-stranded RNA virus that causes acute lung injury and acute respiratory distress syndrome, posing a serious threat to both animal and human health. N6-methyladenosine (m6A), a prevalent and abundant post-transcriptional methylation of RNA in eukaryotes, plays a crucial regulatory role in IAV infection by altering viral RNA and cellular transcripts to affect viral infection and the host immune response. This review focuses on the molecular mechanisms underlying m6A modification and its regulatory function in the context of IAV infection and the host immune response. This will provide a better understanding of virus-host interactions and offer insights into potential anti-IAV strategies.

Transcriptomic alterations in host parasitoid wasps resulting in extended lifespan due to PpNSRV-1 infection

The parasitoid wasp Pteromalus puparum, a pivotal agent in agricultural ecosystem balance, experiences significant alterations in lifespan due to infection by the negative-sense single-stranded RNA virus PpNSRV-1. This study utilized RNA sequencing (RNA-seq) to investigate the genetic mechanism(s) driving lifespan extension in response to this virus. Lifespan analysis, combined with RNA-seq of nearly isogenic lines of infected and uninfected wasps, yields a comprehensive temporal expression profile aligned with the P. puparum genome. Despite a modest overall impact of PpNSRV-1 on gene expression, notable effects were observed on specific gene families, such as the SPOP genes and the cytochrome P450 family. Through Weighted Gene Co-expression Network Analysis (WGCNA), we identified connections between the green and greenyellow modules to viral infection traits, with further functional analyses highlighting the pivotal roles of the Hedgehog, autophagy, AMPK, mTOR, p53, and PI3K-Akt signaling pathways. RNA interference experiment targeting the SPOP gene PPU06594-RA confirmed its role in lifespan modulation, underscoring the importance of Hedgehog pathway in the host’s adaptive response. These insights not only elucidate the complex genetic landscape of host-virus interactions but also open avenues for innovative biological pest control strategies.

Molecular characterization of a single-negative-stranded RNA virus from the rice blast fungus Magnaporthe oryzae isolate NJ39.

A novel negative-sense single-stranded RNA mycovirus, designated as "Magnaporthe oryzae mymonavirus 1" (MoMNV1), was identified in the rice blast fungus Magnaporthe oryzae isolate NJ39. MoMNV1 has a single genomic RNA segment consisting of 10,515 nucleotides, which contains six open reading frames. The largest open reading frame contains 5837 bases and encodes an RNA replicase. The six open reading frames have no overlap and are arranged linearly on the genome, but the spacing of the genes is small, with a maximum of 315 bases and a minimum of 80 bases. Genome comparison and phylogenetic analysis indicated that MoMNV1 is a new member of the genus Penicillimonavirus of the family Mymonaviridae.

Host obesity impacts genetic variation in influenza A viral populations.

Obesity is well established as a risk factor for many noncommunicable diseases; however, its consequences for infectious disease are poorly understood. Here, we investigated the impact of host obesity on influenza A virus (IAV) genetic variation using a diet-induced obesity ferret model and the A/Hong Kong/1073/1999 (H9N2) strain. Using a co-caging study design, we investigated the maintenance, generation, and transmission of intrahost IAV genetic variation by sequencing viral genomic RNA obtained from nasal wash samples over multiple days of infection. We found evidence for an enhanced role of positive selection acting on de novo mutations in obese hosts that led to nonsynonymous changes that rose to high frequency. In addition, we identified numerous cases of mutations throughout the genome that were specific to obese hosts and that were preserved during transmission between hosts. Despite detection of obese-specific variants, the overall viral genetic diversity did not differ significantly between obese and lean hosts. This is likely due to the high supply rate of de novo variation and common evolutionary adaptations to the ferret host regardless of obesity status, which we show are mediated by variation in the hemagglutinin and polymerase genes (PB2 and PB1). We also identified defective viral genomes (DVGs) that were found uniquely in either obese or lean hosts, but the overall DVG diversity and dynamics did not differ between the two groups. Our study suggests that obesity may result in a unique selective environment impacting intrahost IAV evolution, highlighting the need for additional genetic and functional studies to confirm these effects.IMPORTANCEObesity is a chronic health condition characterized by excess adiposity leading to a systemic increase in inflammation and dysregulation of metabolic hormones and immune cell populations. Influenza A virus (IAV) is a highly infectious pathogen responsible for seasonal and pandemic influenza. Host risk factors, including compromised immunity and pre-existing health conditions, can contribute to increased infection susceptibility and disease severity. During viral replication in a host, the negative-sense single-stranded RNA genome of IAV accumulates genetic diversity that may have important consequences for viral evolution and transmission. Our study provides the first insight into the consequences of host obesity on viral genetic diversity and adaptation, suggesting that host factors associated with obesity alter the selective environment experienced by a viral population, thereby impacting the spectrum of genetic variation.

Comprehensive surveillance and molecular profiling of viral infections in cultured olive flounder (Paralichthys olivaceus) across Jeju Island, South Korea

To effectively control viral diseases, ongoing research into virus detection and their genetic characteristics is imperative. We conducted an analysis of the prevalence of five species of viruses in olive flounder (Paralichthys olivaceus) farms in Jeju, specifically viral haemorrhagic septicaemia virus (VHSV), lymphocystis disease virus (LCDV), marine birnavirus (MABV), nervous necrosis virus (NNV), and flounder iridovirus (FLIV). Between July and December 2022, a total of 3282 olive flounders were monitored. VHSV and LCDV were detected in 116 and 173 olive flounders, respectively, confirming detection rates of 3.5% and 5.4%. The highest detection rate for VHSV occurred in December (12.5%), while for LCDV, it was in June (13.8%). MABV, NNV, and FLIV were not detected. Phylogenetic analysis of 19 VHSV isolates collected from 2010 to 2022 revealed that all VHSVs belonged to the IVa genotype based on the nucleoprotein gene. Mutations were identified, indicating genetic variability extending to protein mutations. Additionally, continuous and specific nucleotide mutations of VHSV were confirmed on Jeju Island, a central hub for the aquaculture of olive flounder. These observed mutations in VHSV are considered valuable markers for tracking future mutations in subsequent studies. LCDV isolates were classified under genotype II. The results of the phylogenetic analyses of VHSV, a negative-sense single-stranded RNA virus, and LCDV, a double-stranded DNA virus, provide insights into the mutation patterns of marine viruses.

Metabolomics captures the differential metabolites in the replication pathway of snakehead vesiculovirus regulated by glutamine.

Snakehead vesiculovirus (SHVV) is a negative-sense single-stranded RNA virus that infects snakehead fish. This virus leads to illness and mortality, causing significant economic losses in the snakehead aquaculture industry. The replication and spread of SHVV in cells, which requires glutamine as a nitrogen source, is accompanied by alterations in intracellular metabolites. However, the metabolic mechanisms underlying the inhibition of viral replication by glutamine deficiency are poorly understood. This study utilized liquid chromatography-mass spectrometry to measure the differential metabolites between the channel catfish Parasilurus asotus ovary cell line infected with SHVV under glutamine-containing and glutamine-deprived conditions. Results showed that the absence of glutamine regulated 4 distinct metabolic pathways and influenced 9 differential metabolites. The differential metabolites PS(16:0/16:0), 5,10-methylene-THF, and PS(18:0/18:1(9Z)) were involved in amino acid metabolism. In the nuclear metabolism functional pathway, differential metabolites of guanosine were observed. In the carbohydrate metabolism pathway, differential metabolites of UDP-d-galacturonate were detected. In the signal transduction pathway, differential metabolites of SM(d18:1/20:0), SM(d18:1/22:1(13Z)), SM(d18:1/24:1(15 Z)), and sphinganine were found. Among them, PS(18:0/18:1(9Z)), PS(16:0/16:0), and UDP-d-galacturonate were involved in the synthesis of phosphatidylserine and glycoprotein. The compound 5,10-methylene-THF provided raw materials for virus replication, and guanosine and sphingosine are related to virus virulence. The differential metabolites may collectively participate in the replication, packaging, and proliferation of SHVV under glutamine deficiency. This study provides new insights and potential metabolic targets for combating SHVV infection in aquaculture through metabolomics approaches.

The recruitment of TRiC chaperonin in rotavirus viroplasms correlates with virus replication.

The replication of rotavirus takes place in cytosolic inclusions termed viroplasms. In these inclusions, the distinct 11 double-stranded RNA genome segments are co-packaged to complete a genome in newly generated virus particles. In this study, we show for the first time that the tailless complex polypeptide I ring complex (TRiC), a cellular chaperonin responsible for the folding of at least 10% of the cytosolic proteins, is a component of viroplasms and is required for the synthesis of the viral negative-sense single-stranded RNA. Specifically, TRiC associates with NSP5 and VP2, the cofactor involved in RNA replication. Our study adds a new component to the current model of rotavirus replication, where TRiC is recruited to viroplasms to assist replication.

Autophagy and Apoptosis in Rabies Virus Replication.

Rabies virus (RABV) is a single-stranded negative-sense RNA virus belonging to the Rhabdoviridae family and Lyssavirus genus, which is highly neurotropic and can infect almost all warm-blooded animals, including humans. Autophagy and apoptosis are two evolutionarily conserved and genetically regulated processes that maintain cellular and organismal homeostasis, respectively. Autophagy recycles unnecessary or dysfunctional intracellular organelles and molecules in a cell, whereas apoptosis eliminates damaged or unwanted cells in an organism. Studies have shown that RABV can induce both autophagy and apoptosis in target cells. To advance our understanding of pathogenesis of rabies, this paper reviews the molecular mechanisms of autophagy and apoptosis induced by RABV and the effects of the two cellular events on RABV replication.

The interaction of influenza A virus RNA polymerase PA subunit with the human β-actin protein

Influenza A viruses are enveloped viruses with a genome of eight single-stranded negative-sense RNA molecules. In virions, RNA segments are found as vRNPs associated with NP proteins. The RdRp enzyme, which catalyzes the replication/transcription of the viral genome, is carried as attached to vRNPs. In this study, it was demonstrated that the PA subunit of the viral RdRp interacts with β-actin proteins by the yeast two-hybrid assay. It was shown that the amino-terminal domains of the β-actin protein bind to the carboxy-terminal moiety of the viral PA protein in the mammalian cells. The results were supported by in silico analysis. Over-expression of the β-actin protein was found to have a negative effect on the viral RdRp activity in mini-replicon, but its mechanism of action has remained unknown. The results suggest that the interaction of β-actin and PA protein, a component of vRNPs, may have a role in the intracellular trafficking of the influenza vRNPs and/or viral transcription.

Identification and characterization of a novel Cytorhabdovirus associated with goji berry (Lycium barbarum L.) crinkle disease.

Goji berry (Lycium barbarum L.) is a traditional Chinese herbal medicinal plant that is extensively cultivated in the arid and semiarid regions of northwest China. In this study, a novel cytorhabdovirus, tentatively named "goji cytorhabdovirus A (GCVA)," was identified from the goji berry plant exhibiting leaf crinkle symptoms through high-throughput sequencing (HTS). GCVA contains a linear, negative sense single-stranded RNA genome of 14,812 nucleotides and encodes six open reading frames in the order of 3' leader-N-P-P4-M-G-L-5' trailer. The genome of GCVA shares the highest nucleotide (nt) identity of 65.80% (16% query coverage) with yerba mate virus A (YmVA) (NC_076472). The N and L proteins also share low amino acid (aa) identities (<35.42 and < 41.23%, respectively) with known cytorhabdoviruses. Typical features of the viruses in the genus Cytorhabdovirus include a highly conserved consensus sequence in the intergenic regions and extensive complementation of the 5' non-coding trailer and the 3' leader. These features were also found in GCVA. These data in combination with a phylogenetic analysis that was based on the aa sequences of the N and L proteins support the proposal that GCVA is a new species in the genus Cytorhabdovirus.

Investigating Liquid-Liquid Phase Separation in Virus-Generated Inclusion Bodies Using Fluorescence Recovery After Photobleaching of Fluorescently Labeled Host Proteins.

Many negative-sense single-stranded RNA viruses within the order Mononegavirales harm humans. A common feature shared among cells infected by these viruses is the formation of subcellular membraneless structures called biomolecular condensates, also known as inclusion bodies (IBs), that form through a process called liquid-liquid phase separation (LLPS). Like many other membraneless organelles, viral IBs enrich a specific subset of viral and host proteins involved in the formation of viral particles. Elucidation of the properties and regulation of these IBs as they mature throughout the viral replication process are important for our understanding of viral replication, which may also lead to the development of alternative antiviral treatments. The protocol outlined in this chapter aims to characterize the intrinsic properties of LLPS within themeasles virus (MeV, a member of Mononegavirales) IBs by using an imaging approach that fluorescently tags an IB-associated host protein. This method uses common laboratory techniques and is generalizable to any host factors as well as other viral systems.

RNA binding motif 4 inhibits the replication of ebolavirus by directly targeting 3′-leader region of genomic RNA

Ebola virus (EBOV) belongs to Filoviridae family possessing single-stranded negative-sense RNA genome, which is a serious threat to human health. Nowadays, no therapeutics was proved to be successful to efficiently decrease the mortality rate. RNA binding proteins (RBPs) are reported to participate in maintaining cell integrity and regulation of viral replication. However, little is known about whether and how RBPs participate in regulating the life cycle of EBOV. In our study, we found that RNA binding motif protein 4 (RBM4) inhibited the replication of EBOV in HEK293T and Huh-7 cells through suppressing viral mRNA production. And such inhibition was resulted from the directly interaction between the RRM1 domain of RBM4 and the “CU” enrichment elements located in the PE1 and TSS of the 3′-leader region within the viral genome. Simultaneously, RBM4 could upregulate the expression of some cytokines involved in the host innate immune responses to synergistically exert its antiviral function. The findings therefore suggest that RBM4 might serve as a novel target of anti-EBOV strategy.

Novel Tri-Segmented Rhabdoviruses: A Data Mining Expedition Unveils the Cryptic Diversity of Cytorhabdoviruses.

Cytorhabdoviruses (genus Cytorhabdovirus, family Rhabdoviridae) are plant-infecting viruses with enveloped, bacilliform virions. Established members of the genus Cytorhabdovirus have unsegmented single-stranded negative-sense RNA genomes (ca. 10-16 kb) which encode four to ten proteins. Here, by exploring large publicly available metatranscriptomics datasets, we report the identification and genomic characterization of 93 novel viruses with genetic and evolutionary cues of cytorhabdoviruses. Strikingly, five unprecedented viruses with tri-segmented genomes were also identified. This finding represents the first tri-segmented viruses in the family Rhabdoviridae, and they should be classified in a novel genus within this family for which we suggest the name "Trirhavirus". Interestingly, the nucleocapsid and polymerase were the only typical rhabdoviral proteins encoded by those tri-segmented viruses, whereas in three of them, a protein similar to the emaravirus (family Fimoviridae) silencing suppressor was found, while the other predicted proteins had no matches in any sequence databases. Genetic distance and evolutionary insights suggest that all these novel viruses may represent members of novel species. Phylogenetic analyses, of both novel and previously classified plant rhabdoviruses, provide compelling support for the division of the genus Cytorhabdovirus into three distinct genera. This proposed reclassification not only enhances our understanding of the evolutionary dynamics within this group of plant rhabdoviruses but also illuminates the remarkable genomic diversity they encompass. This study not only represents a significant expansion of the genomics of cytorhabdoviruses that will enable future research on the evolutionary peculiarity of this genus but also shows the plasticity in the rhabdovirus genome organization with the discovery of tri-segmented members with a unique evolutionary trajectory.

Molecular characterization of a novel cytorhabdovirus infecting Plumbago indica L.

In 2021, Plumbago indica plants with necrotic spots on their leaves were observed in Beijing, China. Through high-throughput sequencing, we discovered a putative novel member of the genus Cytorhabdovirus, which was provisionally named "plumbago necrotic spot-associated virus" (PNSaV). The full-length negative-sense single-stranded RNA genome of this virus is 13,180 nucleotides in length and contains eight putative open reading frames (ORFs), in the order 3' leader-N-(P')-P-P3-M-G-P6-L-5' trailer. Phylogenetic analysis and pairwise comparisons suggested that PNSaV is most closely related to pastinaca cytorhabdovirus 1, with 59.2% nucleotide sequence identity in the complete genome and 56.4% amino acid sequence identity in the L protein. These findings suggest that PNSaV should be considered a new member of the genus Cytorhabdovirus.

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.

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.

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.