Negative-sense RNA Research Articles

Ever-increasing viral diversity associated with the red imported fire ant Solenopsis invicta (Formicidae: Hymenoptera)

BackgroundAdvances in sequencing and analysis tools have facilitated discovery of many new viruses from invertebrates, including ants. Solenopsis invicta is an invasive ant that has quickly spread worldwide causing significant ecological and economic impacts. Its virome has begun to be characterized pertaining to potential use of viruses as natural enemies. Although the S. invicta virome is the best characterized among ants, most studies have been performed in its native range, with less information from invaded areas.MethodsUsing a metatranscriptome approach, we further identified and molecularly characterized virus sequences associated with S. invicta, in two introduced areas, U.S and Taiwan. The data set used here was obtained from different stages (larvae, pupa, and adults) of S. invicta life cycle. Publicly available RNA sequences from GenBank’s Sequence Read Archive were downloaded and de novo assembled using CLC Genomics Workbench 20.0.1. Contigs were compared against the non-redundant protein sequences and those showing similarity to viral sequences were further analyzed.ResultsWe characterized five putative new viruses associated with S. invicta transcriptomes. Sequence comparisons revealed extensive divergence across ORFs and genomic regions with most of them sharing less than 40% amino acid identity with those closest homologous sequences previously characterized. The first negative-sense single-stranded RNA virus genomic sequences included in the orders Bunyavirales and Mononegavirales are reported. In addition, two positive single-strand virus genome sequences and one single strand DNA virus genome sequence were also identified. While the presence of a putative tenuivirus associated with S. invicta was previously suggested to be a contamination, here we characterized and present strong evidence that Solenopsis invicta virus 14 (SINV-14) is a tenui-like virus that has a long-term association with the ant. Furthermore, based on virus sequence abundance compared to housekeeping genes, phylogenetic relationships, and completeness of viral coding sequences, our results suggest that four of five virus sequences reported, those being SINV-14, SINV-15, SINV-16 and SINV-17, may be associated to viruses actively replicating in the ant S. invicta.ConclusionsThe present study expands our knowledge about viral diversity associated with S. invicta in introduced areas with potential to be used as biological control agents, which will require further biological characterization.

Revisiting Orthotospovirus phylogeny using full-genome data and testing the contribution of selection, recombination and segment reassortment in the origin of members of new species.

The family Tospoviridae of the order Bunyavirales is constituted of tri-segmented negative-sense single-stranded RNA viruses that infect plants and are also able to replicate in their insect vectors in a persistent manner. The family is composed of a single genus, Orthotospovirus, whose type species is Tomato spotted wilt orthotospovirus. Previous studies assessing the phylogenetic relationships within this genus were based on partial genomic sequences, resulting in unresolved clades and a poor assessment of the roles of recombination and segment reassortment during mixed infections. Full genome sequences of members of recognized Orthotospovirus species are now available at NCBI. In this study, we examined 67 complete genome sequences from members of 22 species. Our study confirms the existence of four phylogroups (A to D), grouped in two major clades (A-B and C-D) within the genus. We found strong evidence that within-segment recombination events and reassortment of segments during mixed infections have been involved in the origin of new orthotospoviruses. Also, selection pressures were analyzed for each gene, and evidence of positive selection was found in all genes.

Human DDX56 protein interacts with influenza A virus NS1 protein and stimulates the virus replication.

Influenza A viruses (IAV) are enveloped viruses carrying a single-stranded negative-sense RNA genome. Detection of host proteins having a relationship with IAV and revealing of the role of these proteins in the viral replication are of great importance in keeping IAV infections under control. Consequently, the importance of human DDX56, which is determined to be associated with a viral NS1 with a yeast two-hybrid assay, was investigated for IAV replication. The viral replication in knocked down cells for the DDX56 gene was evaluated. The NS1 was co-precipitated with the DDX56 protein in lysates of cells transiently expressing DDX56 and NS1 or infected with the viruses, showing that NS1 and DDX56 interact in mammalian cells. Viral NS1 showed a tendency to co-localize with DDX56 in the cells, transiently expressing both of these proteins, which supports the IP and two-hybrid assays results. The data obtained with in silico predictions supported the in vitro protein interaction results. The viral replication was significantly reduced in the DDX56-knockdown cells comparing with that in the control cells. In conclusion, human DDX56 protein interacts with the IAV NS1 protein in both yeast and mammalian cells and has a positive regulatory effect on IAV replication. However, the mechanism of DDX56 on IAV replication requires further elucidation.

Lipid-specific oligomerization of the Marburg virus matrix protein VP40 is regulated by two distinct interfaces for virion assembly

Marburg virus (MARV) is a lipid-enveloped virus harboring a negative-sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in sub-Saharan Africa. MARV assembles and buds from the host cell plasma membrane where MARV matrix protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain [NTD] and C-terminal domain [CTD]) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include protein trafficking to the plasma membrane, homo-multimerization that induced protein enrichment, plasma membrane fluidity changes, and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wildtype mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD–NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD–CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target protein–protein or lipid–protein interactions to inhibit virus budding.

Mutations designed to modify the NS gene mRNA secondary structure affect influenza A pathogenicity in vivo

The influenza A virus genome consists of eight segments of negative-sense RNA that encode up to 18 proteins. During the process of viral replication, positive-sense (+)RNA (cRNA) or messenger RNA (mRNA) is synthesized. Today, there is only a partial understanding of the function of several secondary structures within vRNA and cRNA promoters, and splice sites in the M and NS genes. The most precise secondary structure of (+)RNA has been determined for the NS segment of influenza A virus. The influenza A virus NS gene features two regions with a conserved mRNA secondary structure located near splice sites. Here, we compared 4 variants of the A/Puerto Rico/8/1934 strain featuring different combinations of secondary structures at the NS segment (+)RNA regions 82-148 and 497-564. We found that RNA structures did not affect viral replication in cell culture. However, one of the viruses demonstrated lower NS1 and NEP expression levels during early stage cell infection as well as reduced pathogenicity in mice compared to other variants. In particular, this virus is characterized by an RNA hairpin in the 82-148 region and a stable hairpin in the 497-564 region.

Significantly Improved Recovery of Recombinant Sonchus Yellow Net Rhabdovirus by Expressing the Negative-Strand Genomic RNA

Generation of recombinant negative-stranded RNA viruses (NSVs) from plasmids involves in vivo reconstitution of biologically active nucleocapsids and faces a unique antisense problem where the negative-sense viral genomic RNAs can hybridize to viral messenger RNAs. To overcome this problem, a positive-sense RNA approach has been devised through expression of viral antigenomic (ag)RNA and core proteins for assembly of antigenomic nucleocapsids. Although this detour strategy works for many NSVs, the process is still inefficient. Using Sonchus yellow net rhabdovirus (SYNV) as a model; here, we develop a negative-sense genomic RNA-based approach that increased rescue efficiency by two orders of magnitude compared to the conventional agRNA approach. The system relied on suppression of double-stranded RNA induced antiviral responses by co-expression of plant viruses-encoded RNA silencing suppressors or animal viruses-encoded double-stranded RNA antagonists. With the improved approach, we were able to recover a highly attenuated SYNV mutant with a deletion in the matrix protein gene which otherwise could not be rescued via the agRNA approach. Reverse genetics analyses of the generated mutant virus provided insights into SYNV virion assembly and morphogenesis. This approach may potentially be applicable to other NSVs of plants or animals.

TMEM16A/ANO1 calcium-activated chloride channel as a novel target for the treatment of human respiratory syncytial virus infection

IntroductionHuman respiratory syncytial virus (HRSV) is a common cause of respiratory tract infections (RTIs) globally and is one of the most fatal infectious diseases for infants in developing countries. Of...

Long-term persistence of infectious Zika virus: Inflammation and behavioral sequela in mice.

The neurodevelopmental defects associated with ZIKV infections early in pregnancy are well documented, however the potential defects and long-term consequences associated with milder infections in late pregnancy and perinatal period are less well understood. To model these, we challenged 1 day old (P1) immunocompetent C57BL/6 mice with ZIKV. The animals developed a transient neurological syndrome including unsteady gait, kinetic tremors, severe ataxia and seizures 10–15 days post-infection (dpi) but symptoms subsided after a week, and most animals survived. Despite apparent recovery, MRI of convalescent mice show reduced cerebellar volume that correlates with altered coordination and motor function as well as hyperactivity and impulsivity. Persistent mRNA levels of pro-inflammatory genes including Cd80, Il-1α, and Ifn-γ together with Cd3, Cd8 and perforin (PrfA), suggested persistence of low-grade inflammation. Surprisingly, the brain parenchyma of convalescent mice harbor multiple small discrete foci with viral antigen, active apoptotic processes in neurons, and cellular infiltrates, surrounded by activated astrocytes and microglia as late as 1-year post-infection. Detection of negative-sense strand viral RNA and isolation of infectious virus derived from these convalescent mice by blinded passage in Vero cells confirmed long-term persistence of replicating ZIKV in CNS of convalescent mice. Although the infection appears to persist in defined reservoirs within CNS, the resulting inflammation could increase the risk of neurodegenerative disorders. This raises concern regarding possible long-term effects in asymptomatic children exposed to the virus and suggests that long-term neurological and behavioral monitoring as well as anti-viral treatment to clear virus from the CNS may be useful in patients exposed to ZIKV at an early age.

In Vitro Primer-Based RNA Elongation and Promoter Fine Mapping of the Respiratory Syncytial Virus.

Respiratory syncytial virus (RSV) is a nonsegmented negative-sense (NNS) RNA virus and shares a similar RNA synthesis strategy with other members of NNS RNA viruses, such as measles, rabies virus, and Ebola virus. RSV RNA synthesis is catalyzed by a multifunctional RNA-dependent RNA polymerase (RdRP), which is composed of a large (L) protein that catalyzes three distinct enzymatic functions and an essential coenzyme phosphoprotein (P). Here, we successfully prepared highly pure, full-length, wild-type and mutant RSV polymerase (L-P) complexes. We demonstrated that the RSV polymerase could carry out both de novo and primer-based RNA synthesis. We defined the minimal length of the RNA template for in vitro de novo RNA synthesis using the purified RSV polymerase as 8 nucleotides (nt), shorter than previously reported. We showed that the RSV polymerase catalyzed primer-dependent RNA elongation with different lengths of primers on both short (10-nt) and long (25-nt) RNA templates. We compared the sequence specificity of different viral promoters and identified positions 3, 5, and 8 of the promoter sequence as essential to the in vitro RSV polymerase activity, consistent with the results previously mapped with the in vivo minigenome assay. Overall, these findings agree well with those of previous biochemical studies and extend our understanding of the promoter sequence and the mechanism of RSV RNA synthesis.IMPORTANCE As a major human pathogen, RSV affects 3.4 million children worldwide annually. However, no effective antivirals or vaccines are available. An in-depth mechanistic understanding of the RSV RNA synthesis machinery remains a high priority among the NNS RNA viruses. There is a strong public health need for research on this virus, due to major fundamental gaps in our understanding of NNS RNA virus replication. As the key enzyme executing transcription and replication of the virus, the RSV RdRP is a logical target for novel antiviral drugs. Therefore, exploring the primer-dependent RNA elongation extends our mechanistic understanding of the RSV RNA synthesis. Further fine mapping of the promoter sequence paves the way to better understand the function and structure of the RSV polymerase.

All About the RNA: Interferon-Stimulated Genes That Interfere With Viral RNA Processes.

Interferon (IFN) signaling induces the expression of a wide array of genes, collectively referred to as IFN-stimulated genes (ISGs) that generally function to inhibit viral replication. RNA viruses are frequently targeted by ISGs through recognition of viral replicative intermediates and molecular features associated with viral genomes, or the lack of molecular features associated with host mRNAs. The ISGs reviewed here primarily inhibit viral replication in an RNA-centric manner, working to sense, degrade, or repress expression of viral RNA. This review focuses on dissecting how these ISGs exhibit multiple antiviral mechanisms, often through use of varied co-factors, highlighting the complexity of the type I IFN response. Specifically, these ISGs can mediate antiviral effects through viral RNA degradation, viral translation inhibition, or both. While the OAS/RNase L pathway globally degrades RNA and arrests translation, ISG20 and ZAP employ targeted RNA degradation and translation inhibition to block viral replication. Meanwhile, SHFL targets translation by inhibiting -1 ribosomal frameshifting, which is required by many RNA viruses. Finally, a number of E3 ligases inhibit viral transcription, an attractive antiviral target during the lifecycle of negative-sense RNA viruses which must transcribe their genome prior to translation. Through this review, we aim to provide an updated perspective on how these ISGs work together to form a complex network of antiviral arsenals targeting viral RNA processes.

Overriding the complement barrier: The ups and downs in the Rhabdovirus's battle for survival

Background: Rhabdoviruses are non-segmented, negative sense RNA viruses and are potent pathogens causing infection in humans and animals, e.g., rabies virus, Chandipura virus. Vesicular stomatitis virus (VSV) is a prototypic rhabdovirus. The complement system is a potent barrier against an array of pathogens including VSV. Complement activation by VSV involves the classical pathway resulting in virus neutralization. However, VSV signature responsible for complement activation is unknown. Being the only surface glycoprotein, we investigated the role of G protein in complement activation. We also investigated the mechanism of modulation of human complement by VSV which included the recruitment of the host membrane associated regulators of complement activation (RCA). Methods and materials: Cell surface activation of complement by VSV was carried out by infecting Vero cells with wt-VSV or transfecting with VSV-G plasmid (pCAGGS-G) and treating with normal human serum followed by immunofluorescence microscopy and flow cytometry. G protein from the virion and infected cells was purified by affinity-chromatography and validated using silver staining. Complement binding and activation by the purified protein was tested using multiple biochemical approaches. The specificity of RCA modulation by VSV was assessed by performing time course experiments in VSV infected HeLa cells followed by western blotting and RT-PCR. RCA incorporation and their relative function in virion budding out at specific time points were validated by cofactor and decay accelerating activity. Results: Immunofluorescence and flow cytometric analysis of VSV infected and G transfected cells showed significant deposition of active complement proteins C3b and C4b suggesting a role for the G protein. Interaction of VSV-G with C3/C4 components of complement was further confirmed by ELISA. Fluid phase activation assays with purified G confirmed the role of G in binding to and activating complement proteins, further supported by SPR studies. Western blotting, FACS experiments with infected HeLa cells showed preference of CD55 over CD46 in complement modulation by VSV. The inactivation of C3b and decay acceleration of C3 convertase by the virion associated CD46 and CD55 conferred VSV with resistance against complement. Conclusion: The VSV-G protein plays a significant role in complement activation pathways. Acknowledgement: Effective modulation of complement neutralization is supported by the RCA CD55.

Comparative characterization of the reassortant Orthobunyavirus Ngari with putative parental viruses, Bunyamwera and Batai: in vitro characterization and ex vivo stability.

Bunyamwera (BUNV), Batai (BATV) and Ngari (NRIV) are mosquito-borne viruses that are members of the genus Orthobunyavirus in the order Bunyavirales. These three viruses are enveloped with single-stranded, negative-sense RNA genomes consiting of three segments, denoted as Small (S), Medium (M) and Large (L). Ngari is thought to be the natural reassortant progeny of Bunyamwera and Batai viruses. The relationship between these ‘parental’ viruses and the ‘progeny’ poses an interesting question, especially given that there is overlap in their respective transmission ecologies, but differences in their infection host ranges and pathogenesis. We compared the in vivo kinetics of these three viruses in a common laboratory system and found no significant difference in growth kinetics. There was, however, a tendency of BATV to have smaller plaques than either BUNV or NRIV. Furthermore, we determined that all three viruses are stable in extracellular conditions and retain infectivity for a week in non-cellular media, which has public health and biosafety implications. The study of this understudied group of viruses addresses a need for basic characterization of viruses that have not yet reached epidemic transmission intensity, but that have the potential due to their infectivity to both human and animal hosts. These results lay the groundwork for future studies of these neglected viruses of potential public and One Health importance.

Steps of the Replication Cycle of the Viral Haemorrhagic Septicaemia Virus (VHSV) Affecting Its Virulence on Fish.

Simple SummaryReplication studies are frequently based on viral production, which provides limited information to understand certain processes. Therefore, to discover which failures in the viral haemorrhagic septicaemia virus (VHSV) replication cycle might be involved in the differences in its virulence on fish, a different approach has been taken. Our results have demonstrated that adsorption and morphogenesis are the steps most involved in the modulation of virulence, although failures in the synthesis step were also observed. As a potential application of our results, we believe that this kind of knowledge relating in vivo virulence to in vitro markers could help reduce the need for experimental infections in animals, representing a step forward in ethical issues.The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the viral replication cycle could affect VHSV virulence in fish, including adsorption, RNA synthesis and morphogenesis (including viral release). Notably, differences among strains in any step of the replication cycle were mostly strain-specific and reflected only in part the in vivo phenotype (high and low virulent). Our data, therefore, support the need for further studies aimed to construct completely avirulent VHSV recombinants targeting a combination of genes rather than a single one in order to study the mechanisms of genes interplay and their effect on viral phenotype in vitro and in vivo.

The Induction of an Effective dsRNA-Mediated Resistance Against Tomato Spotted Wilt Virus by Exogenous Application of Double-Stranded RNA Largely Depends on the Selection of the Viral RNA Target Region.

Tomato spotted wilt virus (TSWV) is a devastating plant pathogen, causing huge crop losses worldwide. Unfortunately, due to its wide host range and emergence of resistance breaking strains, its management is challenging. Up to now, resistance to TSWV infection based on RNA interference (RNAi) has been achieved only in transgenic plants expressing parts of the viral genome or artificial microRNAs targeting it. Exogenous application of double-stranded RNAs (dsRNAs) for inducing virus resistance in plants, namely RNAi-based vaccination, represents an attractive and promising alternative, already shown to be effective against different positive-sense RNA viruses and viroids. In the present study, the protection efficacy of exogenous application of dsRNAs targeting the nucleocapsid (N) or the movement protein (NSm) coding genes of the negative-sense RNA virus TSWV was evaluated in Nicotiana benthamiana as model plant and in tomato as economically important crop. Most of the plants treated with N-targeting dsRNAs, but not with NSm-targeting dsRNAs, remained asymptomatic until 40 (N. benthamiana) and 63 (tomato) dpi, while the remaining ones showed a significant delay in systemic symptoms appearance. The different efficacy of N- and NSm-targeting dsRNAs in protecting plants is discussed in the light of their processing, mobility and biological role. These results indicate that the RNAi-based vaccination is effective also against negative-sense RNA viruses but emphasize that the choice of the target viral sequence in designing RNAi-based vaccines is crucial for its success.

Type I interferon-dependent response of zebrafish larvae during tilapia lake virus (TiLV) infection

Tilapia lake virus (TiLV; genus: Tilapinevirus, family: Amnoonviridae) is a recently characterised enveloped virus with a linear, negative-sense single-stranded RNA genome, which causes high mortality in tilapia species. In the present study, we demonstrated that zebrafish (Danio rerio) larvae are susceptible to TiLV infection upon systemic injection. TiLV replicated in zebrafish larvae and caused their high mortality (of about 70%). Histopathological examination revealed that TiLV infection caused pathological abnormalities in zebrafish larvae that were well visible within the brain. Moreover, gene expression analysis revealed that TiLV infection induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I (ddx58), tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), and pro-inflammatory cytokine (il-1β). We also demonstrated the protective role of the recombinant zebrafish IFNϕ1 on the survival of zebrafish larvae during TiLV infection. Our results show the importance of type I IFN response during TiLV infection in zebrafish larvae and demonstrate that zebrafish is a good model organism to study interactions between TiLV - a newly emerging in aquaculture virus, and fish host.

Host ANP32A mediates the assembly of the influenza virus replicase.

Aquatic birds represent a vast reservoir from which novel pandemic influenza A viruses can emerge1. Influenza viruses contain a negative-sense segmented RNA genome which is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein (vRNP) complexes2,3. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA poorly in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity4. Interestingly, host adaptive mutations, particularly a glutamic acid to lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit (PB2627), allow avian FluPolA to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here, we report cryo-EM structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain (LRR) of ANP32A. The C-terminal low complexity acidic region (LCAR) of ANP32A inserts between the two juxtaposed PB2627 domains of the asymmetric FluPolA dimer, providing insight into the mechanism behind the PB2E627K adaptive mutation in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a vRNP.

Zebrafish (Danio rerio) larvae as a model for real-time studies of propagating VHS virus infection, tissue tropism and neutrophil activity.

Viral haemorrhagic septicaemia virus (VHSV) is a negative-sense single-stranded RNA virus that infects more than 140 different fish species. In this study, zebrafish larvae were employed as in vivo model organisms to investigate progression of disease, the correlation between propagation of the infection and irreversibility of disease, cell tropism and in situ neutrophil activity towards the VHSV-infected cells. A recombinant VHSV strain, encoding "tomato" fluorescence (rVHSV-Tomato), was used in zebrafish to be able to follow the progress of the infection in the live host in real-time. Two-day-old zebrafish larvae were injected into the yolk sac with the recombinant virus. The virus titre peaked 96hr post-infection in zebrafish larvae kept at 18°C, and correlated with 33% mortality and high morbidity among the larvae. By utilizing the transgenic zebrafish line Tg(fli1:GFP)y1 with fluorescently tagged endothelial cells, we were able to demonstrate that the virus initially infected endothelial cells lining the blood vessels. By observing the rVHSV-Tomato infection in the neutrophil reporter zebrafish line Tg(MPX:eGFP)i114 , we inferred that only a subpopulation of the neutrophils responded to the virus infection. We conclude that the zebrafish larvae are suitable for real-time studies of VHS virus infections, allowing in vivo dissection of host-virus interactions at the whole organism level.

RNA Secondary Structure as a First Step for Rational Design of the Oligonucleotides towards Inhibition of Influenza A Virus Replication.

Influenza is an important research subject around the world because of its threat to humanity. Influenza A virus (IAV) causes seasonal epidemics and sporadic, but dangerous pandemics. A rapid antigen changes and recombination of the viral RNA genome contribute to the reduced effectiveness of vaccination and anti-influenza drugs. Hence, there is a necessity to develop new antiviral drugs and strategies to limit the influenza spread. IAV is a single-stranded negative sense RNA virus with a genome (viral RNA—vRNA) consisting of eight segments. Segments within influenza virion are assembled into viral ribonucleoprotein (vRNP) complexes that are independent transcription-replication units. Each step in the influenza life cycle is regulated by the RNA and is dependent on its interplay and dynamics. Therefore, viral RNA can be a proper target to design novel therapeutics. Here, we briefly described examples of anti-influenza strategies based on the antisense oligonucleotide (ASO), small interfering RNA (siRNA), microRNA (miRNA) and catalytic nucleic acids. In particular we focused on the vRNA structure-function relationship as well as presented the advantages of using secondary structure information in predicting therapeutic targets and the potential future of this field.

Meta-Transcriptomic Identification of Divergent Amnoonviridae in Fish.

Tilapia lake virus (TiLV) has caused mass mortalities in farmed and wild tilapia with serious economic and ecological consequences. Until recently, this virus was the sole member of the Amnoonviridae, a family within the order Articulavirales comprising segmented negative-sense RNA viruses. We sought to identify additional viruses within the Amnoonviridae through total RNA sequencing (meta-transcriptomics) and data mining of published transcriptomes. Accordingly, we sampled marine fish species from both Australia and China and discovered several segments of two new viruses within the Amnoonviridae, tentatively called Flavolineata virus and Piscibus virus, respectively. In addition, by mining vertebrate transcriptome data, we identified nine additional virus transcripts matching to multiple genomic segments of TiLV in both marine and freshwater fish. These new viruses retained sequence conservation with the distantly related Orthomyxoviridae in the RdRp subunit PB1, but formed a distinct and diverse phylogenetic group. These data suggest that the Amnoonviridae have a broad host range within fish and that greater animal sampling will identify additional divergent members of the Articulavirales.

The two-stage interaction of Ebola virus VP40 with nucleoprotein results in a switch from viral RNA synthesis to virion assembly/budding

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40–NP interaction within IBs, sequestering VP40 within IBs, reducing VP40–VLP egress, abolishing the incorporation of NC-like structures into VP40–VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.