Metagenomics characterization of respiratory viral RNA pathogens in children under five years with severe acute respiratory infection in the Free State, South Africa.

Prompt detection of viral respiratory pathogens is crucial in managing respiratory infection including severe acute respiratory infection (SARI). Metagenomics next-generation sequencing (mNGS) and bioinformatics analyses remain reliable strategies for diagnostic and surveillance purposes. This study evaluated the diagnostic utility of mNGS using multiple analysis tools compared with multiplex real-time PCR for the detection of viral respiratory pathogens in children under 5 years with SARI. Nasopharyngeal swabs collected in viral transport media from 84 children admitted with SARI as per the World Health Organization definition between December 2020 and August 2021 in the Free State Province, South Africa, were used in this study. The obtained specimens were subjected to mNGS using the Illumina MiSeq system, and bioinformatics analysis was performed using three web-based analysis tools; Genome Detective, One Codex and Twist Respiratory Viral Research Panel. With average reads of 211323, mNGS detected viral pathogens in 82 (97.6%) of the 84 patients. Viral aetiologies were established in nine previously undetected/missed cases with an additional bacterial aetiology (Neisseria meningitidis) detected in one patient. Furthermore, mNGS enabled the much needed viral genotypic and subtype differentiation and provided significant information on bacterial co-infection despite enrichment for RNA viruses. Sequences of nonhuman viruses, bacteriophages, and endogenous retrovirus K113 (constituting the respiratory virome) were also uncovered. Notably, mNGS had lower detectability rate for severe acute respiratory syndrome coronavirus 2 (missing 18/32 cases). This study suggests that mNGS, combined with multiple/improved bioinformatics tools, is practically feasible for increased viral and bacterial pathogen detection in SARI, especially in cases where no aetiological agent could be identified by available traditional methods.

Current trends in RNA virus detection through metatranscriptome sequencing data.

With advances in sequencing technology, metatranscriptome sequencing from a variety of environmental and biological sources has revealed the existence of various previously unknown RNA viruses. This review presents recent major RNA virome studies sampled from invertebrate and vertebrate species as well as aquatic environments. In particular, we focus on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and related RNA virus identification through metatranscriptome sequencing analyses. Recently developed bioinformatics software and databases for RNA virus identification are introduced. A relationship between newly identified RNA viruses and endogenous viral elements in host genomes is also discussed.

Molecular Interaction of Nonsense-Mediated mRNA Decay with Viruses.

The virus-host interaction is dynamic and evolutionary. Viruses have to fight with hosts to establish successful infection. Eukaryotic hosts are equipped with multiple defenses against incoming viruses. One of the host antiviral defenses is the nonsense-mediated mRNA decay (NMD), an evolutionarily conserved mechanism for RNA quality control in eukaryotic cells. NMD ensures the accuracy of mRNA translation by removing the abnormal mRNAs harboring pre-matured stop codons. Many RNA viruses have a genome that contains internal stop codon(s) (iTC). Akin to the premature termination codon in aberrant RNA transcripts, the presence of iTC would activate NMD to degrade iTC-containing viral genomes. A couple of viruses have been reported to be sensitive to the NMD-mediated antiviral defense, while some viruses have evolved with specific cis-acting RNA features or trans-acting viral proteins to overcome or escape from NMD. Recently, increasing light has been shed on the NMD-virus interaction. This review summarizes the current scenario of NMD-mediated viral RNA degradation and classifies various molecular means by which viruses compromise the NMD-mediated antiviral defense for better infection in their hosts.

Generation and Functional Analysis of Defective Viral Genomes during SARS-CoV-2 Infection.

Defective viral genomes (DVGs) have been identified in many RNA viruses as a major factor influencing antiviral immune response and viral pathogenesis. However, the generation and function of DVGs in SARS-CoV-2 infection are less known. In this study, we elucidated DVG generation in SARS-CoV-2 and its relationship with host antiviral immune response. We observed DVGs ubiquitously from transcriptome sequencing (RNA-seq) data sets of in vitro infections and autopsy lung tissues of COVID-19 patients. Four genomic hot spots were identified for DVG recombination, and RNA secondary structures were suggested to mediate DVG formation. Functionally, bulk and single-cell RNA-seq analysis indicated the interferon (IFN) stimulation of SARS-CoV-2 DVGs. We further applied our criteria to the next-generation sequencing (NGS) data set from a published cohort study and observed a significantly higher amount and frequency of DVG in symptomatic patients than those in asymptomatic patients. Finally, we observed exceptionally diverse DVG populations in one immunosuppressive patient up to 140 days after the first positive test of COVID-19, suggesting for the first time an association between DVGs and persistent viral infections in SARS-CoV-2. Together, our findings strongly suggest a critical role of DVGs in modulating host IFN responses and symptom development, calling for further inquiry into the mechanisms of DVG generation and into how DVGs modulate host responses and infection outcome during SARS-CoV-2 infection. IMPORTANCE Defective viral genomes (DVGs) are generated ubiquitously in many RNA viruses, including SARS-CoV-2. Their interference activity to full-length viruses and IFN stimulation provide the potential for them to be used in novel antiviral therapies and vaccine development. SARS-CoV-2 DVGs are generated through the recombination of two discontinuous genomic fragments by viral polymerase complex, and this recombination is also one of the major mechanisms for the emergence of new coronaviruses. Focusing on the generation and function of SARS-CoV-2 DVGs, these studies identify new hot spots for nonhomologous recombination and strongly suggest that the secondary structures within viral genomes mediate the recombination. Furthermore, these studies provide the first evidence for IFN stimulation activity of de novo DVGs during natural SARS-CoV-2 infection. These findings set up the foundation for further mechanism studies of SARS-CoV-2 recombination and provide evidence to harness the immunostimulatory potential of DVGs in the development of a vaccine and antivirals for SARS-CoV-2.

Analysis of 3.5 million SARS-CoV-2 sequences reveals unique mutational trends with consistent nucleotide and codon frequencies.

BACKGROUND: Since the onset of the SARS-CoV-2 pandemic, bioinformatic analyses have been performed to understand the nucleotide and synonymous codon usage features and mutational patterns of the virus. However, comparatively few have attempted to perform such analyses on a considerably large cohort of viral genomes while organizing the plethora of available sequence data for a month-by-month analysis to observe changes over time. Here, we aimed to perform sequence composition and mutation analysis of SARS-CoV-2, separating sequences by gene, clade, and timepoints, and contrast the mutational profile of SARS-CoV-2 to other comparable RNA viruses. METHODS: Using a cleaned, filtered, and pre-aligned dataset of over 3.5 million sequences downloaded from the GISAID database, we computed nucleotide and codon usage statistics, including calculation of relative synonymous codon usage values. We then calculated codon adaptation index (CAI) changes and a nonsynonymous/synonymous mutation ratio (dN/dS) over time for our dataset. Finally, we compiled information on the types of mutations occurring for SARS-CoV-2 and other comparable RNA viruses, and generated heatmaps showing codon and nucleotide composition at high entropy positions along the Spike sequence. RESULTS: We show that nucleotide and codon usage metrics remain relatively consistent over the 32-month span, though there are significant differences between clades within each gene at various timepoints. CAI and dN/dS values vary substantially between different timepoints and different genes, with Spike gene on average showing both the highest CAI and dN/dS values. Mutational analysis showed that SARS-CoV-2 Spike has a higher proportion of nonsynonymous mutations than analogous genes in other RNA viruses, with nonsynonymous mutations outnumbering synonymous ones by up to 20:1. However, at several specific positions, synonymous mutations were overwhelmingly predominant. CONCLUSIONS: Our multifaceted analysis covering both the composition and mutation signature of SARS-CoV-2 gives valuable insight into the nucleotide frequency and codon usage heterogeneity of SARS-CoV-2 over time, and its unique mutational profile compared to other RNA viruses.

A multiplex-NGS approach to identifying respiratory RNA viruses during the COVID-19 pandemic.

A methodological approach based on reverse transcription (RT)-multiplex PCR followed by next-generation sequencing (NGS) was implemented to identify multiple respiratory RNA viruses simultaneously. A convenience sampling from respiratory surveillance and SARS-CoV-2 diagnosis in 2020 and 2021 in Montevideo, Uruguay, was analyzed. The results revealed the cocirculation of SARS-CoV-2 with human rhinovirus (hRV) A, B and C, human respiratory syncytial virus (hRSV) B, influenza A virus, and metapneumovirus B1. SARS-CoV-2 coinfections with hRV or hRSV B and influenza A virus coinfections with hRV C were identified in adults and/or children. This methodology combines the benefits of multiplex genomic amplification with the sensitivity and information provided by NGS. An advantage is that additional viral targets can be incorporated, making it a helpful tool to investigate the cocirculation and coinfections of respiratory viruses in pandemic and post-pandemic contexts.

A Novel Coronavirus and a Broad Range of Viruses in Kenyan Cave Bats.

BACKGROUND AND METHODS: To investigate virus diversity in hot zones of probable pathogen spillover, 54 oral-fecal swabs were processed from five bat species collected from three cave systems in Kenya, using metagenome sequencing. RESULTS: Viruses belonging to the Astroviridae, Circoviridae, Coronaviridae, Dicistroviridae, Herpesviridae and Retroviridae were detected, with unclassified viruses. Retroviral sequences were prevalent; 74.1% of all samples were positive, with distinct correlations between virus, site and host bat species. Detected retroviruses comprised Myotis myotis, Myotis ricketti, Myotis daubentonii and Galidia endogenous retroviruses, murine leukemia virus-related virus and Rhinolophus ferrumequinum retrovirus (RFRV). A near-complete genome of a local RFRV strain with identical genome organization and 2.8% nucleotide divergence from the prototype isolate was characterized. Bat coronavirus sequences were detected with a prevalence of 24.1%, where analyses on the ORF1ab region revealed a novel alphacoronavirus lineage. Astrovirus sequences were detected in 25.9%of all samples, with considerable diversity. In 9.2% of the samples, other viruses including Actinidia yellowing virus 2, bat betaherpesvirus, Bole tick virus 4, Cyclovirus and Rhopalosiphum padi virus were identified. CONCLUSIONS: Further monitoring of bats across Kenya is essential to facilitate early recognition of possibly emergent zoonotic viruses.

Advances in Molecular Genetics Enabling Studies of Highly Pathogenic RNA Viruses.

Experimental work with viruses that are highly pathogenic for humans and animals requires specialized Biosafety Level 3 or 4 facilities. Such pathogens include some spectacular but also rather seldomly studied examples such as Ebola virus (requiring BSL-4), more wide-spread and commonly studied viruses such as HIV, and the most recent example, SARS-CoV-2, which causes COVID-19. A common characteristic of these virus examples is that their genomes consist of single-stranded RNA, which requires the conversion of their genomes into a DNA copy for easy manipulation; this can be performed to study the viral life cycle in detail, develop novel therapies and vaccines, and monitor the disease course over time for chronic virus infections. We summarize the recent advances in such new genetic applications for RNA viruses in Switzerland over the last 25 years, from the early days of the HIV/AIDS epidemic to the most recent developments in research on the SARS-CoV-2 coronavirus. We highlight game-changing collaborative efforts between clinical and molecular disciplines in HIV research on the path to optimal clinical disease management. Moreover, we summarize how the modern technical evolution enabled the molecular studies of emerging RNA viruses, confirming that Switzerland is at the forefront of SARS-CoV-2 research and potentially other newly emerging viruses.

Revisiting Viral RNA-Dependent RNA Polymerases: Insights from Recent Structural Studies.

RNA-dependent RNA polymerases (RdRPs) represent a distinctive yet versatile class of nucleic acid polymerases encoded by RNA viruses for the replication and transcription of their genome. The structure of the RdRP is comparable to that of a cupped right hand consisting of fingers, palm, and thumb subdomains. Despite the presence of a common structural core, the RdRPs differ significantly in the mechanistic details of RNA binding and polymerization. The present review aims at exploring these incongruities in light of recent structural studies of RdRP complexes with diverse cofactors, RNA moieties, analogs, and inhibitors.

Viral cross-class transmission results in disease of a phytopathogenic fungus.

Interspecies transmission of viruses is a well-known phenomenon in animals and plants whether via contacts or vectors. In fungi, interspecies transmission between distantly related fungi is often suspected but rarely experimentally documented and may have practical implications. A newly described double-strand RNA (dsRNA) virus found asymptomatic in the phytopathogenic fungus Leptosphaeria biglobosa of cruciferous crops was successfully transmitted to an evolutionarily distant, broad-host range pathogen Botrytis cinerea. Leptosphaeria biglobosa botybirnavirus 1 (LbBV1) was characterized in L. biglobosa strain GZJS-19. Its infection in L. biglobosa was asymptomatic, as no significant differences in radial mycelial growth and pathogenicity were observed between LbBV1-infected and LbBV1-free strains. However, cross-species transmission of LbBV1 from L. biglobosa to infection in B. cinerea resulted in the hypovirulence of the recipient B. cinerea strain t-459-V. The cross-species transmission was succeeded only by inoculation of mixed spores of L. biglobosa and B. cinerea on PDA or on stems of oilseed rape with the efficiency of 4.6% and 18.8%, respectively. To investigate viral cross-species transmission between L. biglobosa and B. cinerea in nature, RNA sequencing was carried out on L. biglobosa and B. cinerea isolates obtained from Brassica samples co-infected by these two pathogens and showed that at least two mycoviruses were detected in both fungal groups. These results indicate that cross-species transmission of mycoviruses may occur frequently in nature and result in the phenotypical changes of newly invaded phytopathogenic fungi. This study also provides new insights for using asymptomatic mycoviruses as biocontrol agent.

Patent intelligence of RNA viruses: Implications for combating emerging and re-emerging RNA virus based infectious diseases.

The recent outbreak of one of the RNA viruses (2019-nCoV) has affected most of the population and the fatalities reported may label it as a modern-day scourge. Active research on RNA virus infections and vaccine development had more commercial impact which leads to an increase in patent filings. Patents are a goldmine of information whose mining yields crucial technological inputs for further research. In this research article, we have investigated both the patent applications and granted patents, to identify the technological trends and their impact on 2019-nCoV infection using biotechnology-related keywords such as genes, proteins, nucleic acid etc. related to the RNA virus infection. In our research, patent analysis was majorly focused on prospecting for patent data related to the RNA virus infections. Our patent analysis specifically identified spike protein (S protein) and nucleocapsid proteins (N proteins) as the most actively researched macromolecules for vaccine and/or drug development for diagnosis and treatment of RNA virus based infectious diseases. The outcomes of this patent intelligence study will be useful for the researchers who are actively working in the area of vaccine or drug development for RNA virus-based infections including 2019-nCoV and other emerging and re-emerging viral infections in the near future.

Decoding the Role of Temperature in RNA Virus Infections.

RNA viruses include respiratory viruses, such as coronaviruses and influenza viruses, as well as vector-borne viruses, like dengue and West Nile virus. RNA viruses like these encounter various environments when they copy themselves and spread from cell to cell or host to host. Ex vivo differences, such as geographical location and humidity, affect their stability and transmission, while in vivo differences, such as pH and host gene expression, impact viral receptor binding, viral replication, and the host immune response against the viral infection. A critical factor affecting RNA viruses both ex vivo and in vivo, and defining the outcome of viral infections and the direction of viral evolution, is temperature. In this minireview, we discuss the impact of temperature on viral replication, stability, transmission, and adaptation, as well as the host innate immune response. Improving our understanding of how RNA viruses function, survive, and spread at different temperatures will improve our models of viral replication and transmission risk analyses.

Computational and Experimental Approaches to Study the RNA Secondary Structures of RNA Viruses.

Most pandemics of recent decades can be traced to RNA viruses, including HIV, SARS, influenza, dengue, Zika, and SARS-CoV-2. These RNA viruses impose considerable social and economic burdens on our society, resulting in a high number of deaths and high treatment costs. As these RNA viruses utilize an RNA genome, which is important for different stages of the viral life cycle, including replication, translation, and packaging, studying how the genome folds is important to understand virus function. In this review, we summarize recent advances in computational and high-throughput RNA structure-mapping approaches and their use in understanding structures within RNA virus genomes. In particular, we focus on the genome structures of the dengue, Zika, and SARS-CoV-2 viruses due to recent significant outbreaks of these viruses around the world.

Characterization of Pipistrellus pygmaeus Bat Virome from Sweden.

Increasing amounts of data indicate that bats harbor a higher viral diversity relative to other mammalian orders, and they have been recognized as potential reservoirs for pathogenic viruses, such as the Hendra, Nipah, Marburg, and SARS-CoV viruses. Here, we present the first viral metagenomic analysis of Pipistrellus pygmaeus from Uppsala, Sweden. Total RNA was extracted from the saliva and feces of individual bats and analyzed using Illumina sequencing. The results identified sequences related to 51 different viral families, including vertebrate, invertebrate, and plant viruses. These viral families include Coronaviridae, Picornaviridae, Dicistroviridae, Astroviridae, Hepeviridae, Reoviridae, Botourmiaviridae, Lispviridae, Totiviridae, Botoumiaviridae, Parvoviridae, Retroviridae, Adenoviridae, and Partitiviridae, as well as different unclassified viruses. We further characterized three near full-length genome sequences of bat coronaviruses. A phylogenetic analysis showed that these belonged to alphacoronaviruses with the closest similarity (78-99% at the protein level) to Danish and Finnish bat coronaviruses detected in Pipistrellus and Myotis bats. In addition, the full-length and the near full-length genomes of picornavirus were characterized. These showed the closest similarity (88-94% at the protein level) to bat picornaviruses identified in Chinese bats. Altogether, the results of this study show that Swedish Pipistrellus bats harbor a great diversity of viruses, some of which are closely related to mammalian viruses. This study expands our knowledge on the bat population virome and improves our understanding of the evolution and transmission of viruses among bats and to other species.

RespiCoV: Simultaneous identification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and 46 respiratory tract viruses and bacteria by amplicon-based Oxford-Nanopore MinION sequencing.

Since December 2019 the world has been facing the outbreak of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Identification of infected patients and discrimination from other respiratory infections have so far been accomplished by using highly specific real-time PCRs. Here we present a rapid multiplex approach (RespiCoV), combining highly multiplexed PCRs and MinION sequencing suitable for the simultaneous screening for 41 viral and five bacterial agents related to respiratory tract infections, including the human coronaviruses NL63, HKU1, OC43, 229E, Middle East respiratory syndrome coronavirus, SARS-CoV, and SARS-CoV-2. RespiCoV was applied to 150 patient samples with suspected SARS-CoV-2 infection and compared with specific real-time PCR. Additionally, several respiratory tract pathogens were identified in samples tested positive or negative for SARS-CoV-2. Finally, RespiCoV was experimentally compared to the commercial RespiFinder 2SMART multiplex screening assay (PathoFinder, The Netherlands).

Lethal Mutagenesis of RNA Viruses and Approved Drugs with Antiviral Mutagenic Activity.

In RNA viruses, a small increase in their mutation rates can be sufficient to exceed their threshold of viability. Lethal mutagenesis is a therapeutic strategy based on the use of mutagens, driving viral populations to extinction. Extinction catastrophe can be experimentally induced by promutagenic nucleosides in cell culture models. The loss of HIV infectivity has been observed after passage in 5-hydroxydeoxycytidine or 5,6-dihydro-5-aza-2'-deoxycytidine while producing a two-fold increase in the viral mutation frequency. Among approved nucleoside analogs, experiments with polioviruses and other RNA viruses suggested that ribavirin can be mutagenic, although its mechanism of action is not clear. Favipiravir and molnupiravir exert an antiviral effect through lethal mutagenesis. Both drugs are broad-spectrum antiviral agents active against RNA viruses. Favipiravir incorporates into viral RNA, affecting the G→A and C→U transition rates. Molnupiravir (a prodrug of ß-d-N4-hydroxycytidine) has been recently approved for the treatment of SARS-CoV-2 infection. Its triphosphate derivative can be incorporated into viral RNA and extended by the coronavirus RNA polymerase. Incorrect base pairing and inefficient extension by the polymerase promote mutagenesis by increasing the G→A and C→U transition frequencies. Despite having remarkable antiviral action and resilience to drug resistance, carcinogenic risks and genotoxicity are important concerns limiting their extended use in antiviral therapy.

Virome in the cloaca of wild and breeding birds revealed a diversity of significant viruses.

BACKGROUND: Wild birds may harbor and transmit viruses that are potentially pathogenic to humans, domestic animals, and other wildlife. RESULTS: Using the viral metagenomic approach, we investigated the virome of cloacal swab specimens collected from 3182 birds (the majority of them wild species) consisting of > 87 different species in 10 different orders within the Aves classes. The virus diversity in wild birds was higher than that in breeding birds. We acquired 707 viral genomes from 18 defined families and 4 unclassified virus groups, with 265 virus genomes sharing < 60% protein sequence identities with their best matches in GenBank comprising new virus families, genera, or species. RNA viruses containing the conserved RdRp domain with no phylogenetic affinity to currently defined virus families existed in different bird species. Genomes of the astrovirus, picornavirus, coronavirus, calicivirus, parvovirus, circovirus, retrovirus, and adenovirus families which include known avian pathogens were fully characterized. Putative cross-species transmissions were observed with viruses in wild birds showing > 95% amino acid sequence identity to previously reported viruses in domestic poultry. Genomic recombination was observed for some genomes showing discordant phylogenies based on structural and non-structural regions. Mapping the next-generation sequencing (NGS) data respectively against the 707 genomes revealed that these viruses showed distribution pattern differences among birds with different habitats (breeding or wild), orders, and sampling sites but no significant differences between birds with different behavioral features (migratory and resident). CONCLUSIONS: The existence of a highly diverse virome highlights the challenges in elucidating the evolution, etiology, and ecology of viruses in wild birds. Video Abstract.

Reinventing positive-strand RNA virus reverse genetics.

Reverse genetics is the prospective analysis of how genotype determines phenotype. In a typical experiment, a researcher alters a viral genome, then observes the phenotypic outcome. Among RNA viruses, this approach was first applied to positive-strand RNA viruses in the mid-1970s and over nearly 50 years has become a powerful and widely used approach for dissecting the mechanisms of viral replication and pathogenesis. During this time the global health importance of two virus groups, flaviviruses (genus Flavivirus, family Flaviviridae) and betacoronaviruses (genus Betacoronavirus, subfamily Orthocoronavirinae, family Coronaviridae), have dramatically increased, yet these viruses have genomes that are technically challenging to manipulate. As a result, several new techniques have been developed to overcome these challenges. Here I briefly review key historical aspects of positive-strand RNA virus reverse genetics, describe some recent reverse genetic innovations, particularly as applied to flaviviruses and coronaviruses, and discuss their benefits and limitations within the larger context of rigorous genetic analysis.

Aminoacyl-tRNA Synthetase: A Non-Negligible Molecule in RNA Viral Infection.

Infectious diseases such as the ongoing coronavirus disease 2019 (COVID-19) continue to have a huge impact on global health, and the host-virus interaction remains incompletely understood. To address the global threat, in-depth investigations in pathogenesis are essential for interventions in infectious diseases and vaccine development. Interestingly, aminoacyl-transfer RNA (tRNA) synthetases (aaRSs), an ancient enzyme family that was once considered to play housekeeping roles in protein synthesis, are involved in multiple viral infectious diseases. Many aaRSs in eukaryotes present as the components of a cytoplasmic depot system named the multi-synthetase complex (MSC). Upon viral infections, several components of the MSC are released and exert nonenzymatic activities. Host aaRSs can also be utilized to facilitate viral entry and replication. In addition to their intracellular roles, some aaRSs and aaRS-interacting multi-functional proteins (AIMPs) are secreted as active cytokines or function as "molecule communicators" on the cell surface. The interactions between aaRSs and viruses ultimately affect host innate immune responses or facilitate virus invasion. In this review, we summarized the latest advances of the interactions between aaRSs and RNA viruses, with a particular emphasis on the therapeutic potentials of aaRSs in viral infectious diseases.

Crosstalk between RNA viruses and DNA sensors: Role of the cGAS-STING signalling pathway.

Despite only comprising half of all known viral species, RNA viruses are disproportionately responsible for many of the worst epidemics in human history, including outbreaks of influenza, poliomyelitis, Ebola, and most recently, the coronavirus disease-2019 (COVID-19) pandemic. The propensity for RNA viruses to replicate in cytosolic compartments has led to an evolutionary arms race and the emergence of cytosolic sensors to recognise and initiate the host innate immune response. Although significant progress has been made in identifying and characterising cytosolic RNA sensors as anti-viral innate immune factors, the potential role for cytosolic DNA sensors in RNA viral infection is only recently being appreciated. Among these, the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has attracted increasing attention. The cGAS-STING signalling pathway has emerged as a key innate immune signalling axis that is implicated in diverse human diseases from infectious diseases to neurodegeneration and cancer. Here we review the existing literature on RNA viruses and their reciprocal interactions with the cGAS-STING pathway and share insights into RNA virus diversity by touching on the similarities and differences of RNA viral strategies.