Subgenomic Research Articles

Can self-testing be enhanced to hasten safe return of healthcare workers in pandemics? Random order, open label trial using two manufacturers' SARS-CoV-2 lateral flow devices concurrently and nested viral culture study.

Covid-19 healthcare worker testing, isolation and quarantine policies had to balance risks to patients from the virus and from staff absence. The emergence of the Omicron variant led to dangerous levels of key-worker absence globally. We evaluated whether using two manufacturers' lateral flow tests (LFTs) concurrently improved SARS-CoV-2 Omicron detection significantly and was acceptable to hospital staff. In a nested study, to understand risks of return to work after a 5-day isolation/quarantine period, we examined virus culture 5-7 days after positive test or significant exposure. Fully-vaccinated Liverpool (UK) University Hospitals staff participated (February-May 2022) in a random-order, open-label trial testing whether dual LFTs improved SARS-CoV-2 detection, and whether dual swabbing was acceptable to users. Participants used nose-throat swab Innova and nose-only swab Orient Gene LFTs in daily randomised order for 10 days. A user-experience questionnaire was administered on exit. Selected participants gave swabs for viral culture on days 5-7 after symptom onset or first positive test. Cultures were considered positive if cytopathic effect was apparent or SARS-CoV-2 N gene sub-genomic RNA was detected. Two hundred and twenty-six individuals reported 1466 pairs of LFT results. Tests disagreed in 127 cases (8.7%). Orient Gene was more likely (78 cf. 49; OR: 2.1, 1.1-4.1; P = 0.03) to be positive. If Innova was swabbed second, it was less likely to agree with a positive Orient Gene result (OR: 2.7, 1.3-5.2; P = 0.005); swabbing first with Innova made no significant difference (OR: 1.1, 0.5-2.3; P = 0.85). Orient Gene positive Innova negative result-pairs became more frequent over time (OR: 1.2, 1.1-1.3; P < 0.001). Of individuals completing the exit questionnaire, 90.7% reported dual swabbing was easy, 57.1% said it was no barrier to their daily routine and 65.6% preferred dual testing. Respondents had more confidence in dual versus single test results. Viral cultures from days 5-7 were positive for 6/31 (19.4%, 7.5%-37.5%) and indeterminate for 11/31 (35.5%, 19.2%-54.6%) LFT-positive participants, indicating they were likely still infectious. Dual brand testing increased LFT detection of SARS-CoV-2 antigen by a small but meaningful margin and was acceptable to hospital workers. Viral cultures demonstrated that policies recommending safe return to work ~ 5 days after Omicron infection/exposure were flawed. Key-workers should be prepared for dynamic self-testing protocols in future pandemics. https://www.isrctn.com/ISRCTN47058442 (26 January 2022).

Preferential cleavage of the coronavirus defective viral genome by cellular endoribonuclease with characteristics of RNase L

In testing whether coronavirus defective viral genome 12.7 (DVG12.7) with transcription regulating sequence (TRS) can synthesize subgenomic mRNA (sgmRNA) in coronavirus-infected cells, it was unexpectedly found by Northern blot assay that not only sgmRNA (designated sgmDVG 12.7) but also an RNA fragment with a size less than sgmDVG 12.7 was identified. A subsequent study demonstrated that the identified RNA fragment (designated clvDVG) was a cleaved RNA product originating from DVG12.7, and the cleaved sites were located in the loop region of stem‒loop structure and after UU and UA dinucleotides. clvDVG was also identified in mock-infected HRT-18 cells transfected with DVG12.7 transcript, indicating that cellular endoribonuclease is responsible for the cleavage. In addition, the sequence and structure surrounding the cleavage sites can affect the cleavage efficiency of DVG12.7. The cleavage features are therefore consistent with the general criteria for RNA cleavage by cellular RNase L. Furthermore, both the cleavage of rRNA and the synthesis of clvDVG were also identified in A549 cells. Because (i) the cleavage sites occurred predominantly after single-stranded UA and UU dinucleotides, (ii) the sequence and structure surrounding the cleavage sites affected the cleavage efficiency, (iii) the cleavage of rRNA is an index of the activation of RNase L, and (iv) the cleavage of both rRNA and DVG12.7 was identified in A549 cells, the results together indicated that the preferential cleavage of DVG12.7 is correlated with cellular endoribonuclease with the characteristics of RNase L and such cleavage features have not been previously characterized in coronaviruses.

SARS-CoV-2 infection perturbs the gastrointestinal tract and induces modest microbial translocation across the intestinal barrier.

SARS-CoV-2 infects via the respiratory tract, but COVID-19 includes an array of non-respiratory symptoms, among them gastrointestinal (GI) manifestations such as vomiting and diarrhea. Here we investigated the GI pathology of SARS-CoV-2 infections in rhesus macaques and humans. Macaques experienced mild infection with USA-WA1/2020 and shed viral RNA in the respiratory tract and stool, including subgenomic RNA indicative of replication in the GI tract. Intestinal immune cell populations were disturbed, with significantly fewer proliferating (Ki67+) jejunal B cells in SARS-CoV-2-infected macaques than uninfected ones. Modest translocation of bacteria/bacterial antigen was observed across the colonic epithelium, with a corresponding significant increase in plasma soluble CD14 (sCD14) that may be induced by LPS. Human plasma demonstrated significant decreases in interleukin (IL)-6 and sCD14 upon recovery from COVID-19, suggesting resolution of inflammation and response to translocated bacteria. sCD14 significantly positively correlated with zonulin, an indicator of gut barrier integrity, and IL-6. These results demonstrate that GI perturbations such as microbial translocation can occur in even mild SARS-CoV-2 infections and may contribute to the COVID-19 inflammatory state.IMPORTANCEThis study investigates gastrointestinal (GI) barrier disruption in SARS-CoV-2 infections and how it may contribute to disease. We observed bacteria or bacterial products crossing from the colon interior (the lumen) to the lamina propria during SARS-CoV-2 infection in macaques. Bacteria/bacterial products are tolerated in the lumen but may induce immune responses if they translocate to the lamina propria. We also observed a significant increase in soluble CD14, which is associated with an immune response to bacterial products. In addition, we observed that humans recovering from COVID-19 experienced a significant decrease in soluble CD14, as well as the inflammatory marker interleukin (IL)-6. IL-6 and sCD14 correlated significantly across macaque and human samples. These findings suggest that SARS-CoV-2 infection results in GI barrier disruption that permits microbial translocation and a corresponding immune response. These findings could aid in developing interventions to improve COVID-19 patient outcomes.

Pan-flavivirus analysis reveals sfRNA-independent, 3' UTR-biased siRNA production from an insect-specific flavivirus.

RNA interference (RNAi) plays an essential role in mosquito antiviral immunity, but it is not known whether viral small interfering RNA (siRNA) profiles differ between mosquito-borne and mosquito-specific viruses. A pan-Orthoflavivirus analysis in Aedes albopictus cells revealed that viral siRNAs were evenly distributed across the viral genome of most representatives of the Flavivirus genus. In contrast, siRNA production was biased toward the 3' untranslated region (UTR) of the genomes of classical insect-specific flaviviruses (cISF), which was most pronounced for Kamiti River virus (KRV), a virus with a unique, 1.2 kb long 3' UTR. KRV-derived siRNAs were produced in high quantities and almost exclusively mapped to the 3' UTR. We mapped the 5' end of KRV subgenomic flavivirus RNAs (sfRNAs), products of the 5'-3' exoribonuclease XRN1/Pacman stalling on secondary RNA structures in the 3' UTR of the viral genome. We found that KRV produces high copy numbers of a long, 1,017 nt sfRNA1 and a short, 421 nt sfRNA2, corresponding to two predicted XRN1-resistant elements. Expression of both sfRNA1 and sfRNA2 was reduced in Pacman-deficient Aedes albopictus cells; however, this did not correlate with a shift in viral siRNA profiles. We suggest that cISFs, particularly KRV, developed a unique mechanism to produce high amounts of siRNAs as a decoy for the antiviral RNAi response in an sfRNA-independent manner.IMPORTANCEThe Flavivirus genus contains diverse mosquito viruses ranging from insect-specific viruses circulating exclusively in mosquito populations to mosquito-borne viruses that cause disease in humans and animals. Studying the mechanisms of virus replication and antiviral immunity in mosquitoes is important to understand arbovirus transmission and may inform the development of disease control strategies. In insects, RNA interference (RNAi) provides broad antiviral activity and constitutes a major immune response against viruses. Comparing diverse members of the Flavivirus genus, we found that all flaviviruses are targeted by RNAi. However, the insect-specific Kamiti River virus was unique in that small interfering RNAs are highly skewed toward its uniquely long 3' untranslated region. These results suggest that mosquito-specific viruses have evolved unique mechanisms for genome replication and immune evasion.

The Feline calicivirus capsid protein VP1 is a client of the molecular chaperone Hsp90.

Feline calicivirus (FCV) icosahedral viral capsids are composed of dozens of structural subunits that rely on cellular chaperones to self-assemble in an orderly fashion. Here, we report that the heat shock protein 90 (Hsp90) inhibition significantly reduced FCV particle production, suggesting a role in the replicative cycle. We found that Hsp90 inhibition was not related to the synthesis or stability of the early proteins that translate from the gRNA nor to the minor capsid protein VP2 but with a reduction in the major capsid protein VP1 levels, both translated late in infection from the subgenomic RNAs. Reduction in VP1 levels was observed despite an augment of the leader of the capsid (LC)-VP1 precursor levels, from which the LC and VP1 proteins are produced after proteolytic processing by NS6/7. The direct interaction of VP1 with Hsp90 was observed in infected cells. These results suggest that upon release from the polyprotein precursor, VP1 becomes a client of Hsp90 and that this interaction is required for an efficient FCV replicative cycle.

Subgenomic flaviviral RNAs and human proteins: in silico exploration of anti-host defense mechanisms

Flaviviruses pose significant global health threats, infecting over 300 million people annually. Among their evasion strategies, the production of subgenomic flaviviral RNAs (sfRNAs) from the 3′ UTR of viral genomes is particularly notable. Utilizing a comprehensive in silico approach with the catRAPID algorithm, we analyzed over 300,000 interactions between sfRNAs and human proteins derived from more than 8000 flavivirus genomes, including Dengue, Zika, Yellow Fever, West Nile, and Japanese Encephalitis viruses. By providing the first extensive atlas of sfRNA interactions, we offer new insights into how flaviviruses can manipulate host cellular machinery to facilitate viral survival and persistence. Our study not only validated known interactions but also revealed novel human proteins that could be involved in sfRNA-mediated host defense evasion, including helicases, splicing factors, and chemokines. These findings significantly expand the known interactome of sfRNAs with human proteins, underscoring their role in modulating host cellular pathways. Intriguingly, we predict interaction with stress granules, a critical component of the cellular response to viral infection, suggesting a mechanism by which flaviviruses inhibit their formation to evade host defenses. Moreover, a set of highly-interacting proteins in common among the sfRNAs showed predictive power to identify sfRNA-forming regions, highlighting how protein signatures could be used to annotate viruses. This atlas not only serves as a resource for exploring therapeutic targets but also aids in the identification of sfRNA biomarkers for improved flavivirus diagnostics.

Magnesium Regulates RNA Ring Dynamics and Folding in Subgenomic Flaviviral RNA.

Mosquito-borne flaviviruses including dengue, Zika, yellow fever, and regional encephalitis produce a large amount of short subgenomic flaviviral RNAs during infection. A segment of these RNAs named as xrRNA1 features a multi-pseudoknot (PK)-associated structure, which resists the host cell enzyme (XRN1) from degrading the viral RNA. We investigate how this long-range RNA PK folds in the presence of counterions, specifically in a mix of monovalent (K+) and divalent (Mg2+) salts at physiological concentrations. In this study, we use extensive explicit solvent molecular dynamics (MD) simulations to characterize the RNA ion environment of the folded RNA conformation, as determined by the crystal structure. This allowed us to identify the precise locations of various coordinated RNA-Mg2+ interactions, including inner-sphere/chelated and outer-sphere coordinated Mg2+. Given that RNA folding involves large-scale conformational changes, making it challenging to explore through classical MD simulations, we investigate the folding mechanism of xrRNA1 using an all-atom structure-based RNA model with a hybrid implicit-explicit treatment of the ion environment via the dynamic counterion condensation model, both with and without physiological Mg2+ concentration. The study reveals potential folding pathways for this xrRNA1, which is consistent with the results obtained from optical tweezer experiments. The equilibrium and free energy simulations both capture a dynamic equilibrium between the ring-open and ring-close states of the RNA, driven by a long-range PK interaction. Free energy calculations reveal that with the addition of Mg2+ ions, the equilibrium shifts more toward the ring-close state. A detailed analysis of the free energy pathways and ion-mediated contact probability map highlights the critical role of Mg2+ in bridging G50 and A33. This Mg2+-mediated connection helps form the long-range PK which in turn controls the transition between the ring-open and ring-close states. The study underscores the critical role of Mg2+ in the RNA folding transition, highlighting specific locations of Mg2+ contributing to the stabilization of long-range PK connections likely to enhance the robustness of Xrn1 resistance of flaviviral xrRNAs.

The coronavirus nsp14 exoribonuclease interface with the cofactor nsp10 is essential for efficient virus replication and enzymatic activity.

Coronavirus replication requires assembly of a replication transcription complex composed of nonstructural proteins (nsp), including polymerase, helicase, exonuclease, capping enzymes, and non-enzymatic cofactors. The coronavirus nsp14 exoribonuclease mediates several functions in the viral life cycle including genomic and subgenomic RNA synthesis, RNA recombination, RNA proofreading and high-fidelity replication, and native resistance to many nucleoside analogs. The nsp-14 exonuclease activity in vitro requires the non-enzymatic co-factor nsp10, but the determinants and importance the nsp14-10 interactions during viral replication have not been defined. Here we show that for the coronavirus murine hepatitis virus, nsp14 residues at the nsp14-10 interface are essential for efficient viral replication and in vitro exonuclease activity. These results shed new light on the requirements for protein interactions within the coronavirus replication transcription complex, and they may reveal novel non active-site targets for virus inhibition and attenuation.

Oral SARS-CoV-2 host responses predict the early COVID-19 disease course

Oral fluids provide ready detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host responses. This study sought to evaluate relationships between oral virus, oral and systemic anti-SARS-CoV-2-specific antibodies, and symptoms. Oral fluids (saliva/throat wash (saliva/TW)) and serum were collected from asymptomatic and symptomatic, nasopharyngeal (NP) SARS-CoV-2 RT-qPCR+ human participants (n = 45). SARS-CoV-2 RT-qPCR and N-antigen detection by immunoblot and lateral flow assay (LFA) were performed. RT-qPCR for subgenomic RNA (sgRNA) was sequence confirmed. SARS-CoV-2-anti-S protein RBD LFA and ELISA assessed IgM and IgG responses. Structural analysis identified host salivary molecules analogous to SARS-CoV-2-N-antigen. At time of enrollment (baseline, BL), LFA-detected N-antigen in 86% of TW and was immunoblot-confirmed. However, only 3/17 were saliva/TW qPCR+ . Sixty percent of saliva and 83% of TW demonstrated persistent N-antigen at 4 weeks. N-antigen LFA signal in three anti-spike sero-negative participants suggested potential cross-detection of 4 structurally analogous salivary RNA binding proteins (alignment 19–29aa, RMSD 1–1.5 Angstroms). At enrollment, symptomatic participants demonstrated replication-associated sgRNA junctions, were IgG+ (94%/100% in saliva/TW), and IgM+ (63%/54%). At 4 weeks, SARS-CoV-2 IgG (100%/83%) and IgM (80%/67%) persisted. Oral and serum IgG correlated 100% with NP+ PCR status. Cough and fatigue severity (p = 0.010 and 0.018 respectively), and presence of weakness, nausea, and composite upper respiratory symptoms (p = 0.037, 0.005, and 0.017, respectively) were negatively associated with saliva IgM but not TW or serum IgM. Throat wash IgM levels were higher in women compared to men, although the association did not reach statistical significance (median: 290 (female) versus 0.697, p = 0.056). Important to transmission and disease course, oral viral replication and persistence showed clear relationships with select symptoms and early oral IgM responses during early infection. N-antigen cross-reactivity may reflect mimicry of structurally analogous host proteins.

Assessing the inhibitory effects of some secondary amines, thioureas and 1,3-dimethyluracil conjugates of (-)-cytisine and thermopsine on the RNA-dependent RNA polymerase of SARS-CoV-1 and SARS-CoV-2

SARS-CoV-2 causes COVID-19, with symptoms ranging from mild to severe, including pneumonia and death. This beta coronavirus has a 30-kilobase RNA genome and shares about 80 % of its nucleotide sequence with SARS-CoV-1. The replication/transcription complex, essential for viral RNA synthesis, includes RNA-dependent RNA polymerase (RdRp, nsp12) enhanced by nsp7 and nsp8. Antivirals like molnupiravir and remdesivir, which are RdRp inhibitors, treat severe COVID-19 but have limitations, highlighting the need for new therapies. This study assessed (-)-cytisine, methylcytisine, and thermopsine derivatives against SARS-CoV-1 and SARS-CoV-2 in vitro, focusing on their RdRp inhibition. Selected compounds from a previous study were evaluated using a SARS-CoV-2 RNA polymerase assay kit to investigate their structure–activity relationships. Compound 17 (1,3-dimethyluracil conjugate with (-)-cytisine and thermopsine) emerged as a potent inhibitor of SARS-CoV-1 and SARS-CoV-2 RdRp, with an IC50 value of 7.8 μM against SARS-CoV-2 RdRp. It showed a dose-dependent reduction in cytopathic effects in cells infected with SARS-CoV-1 and SARS-CoV-2 replicon-based single-round infectious particles (SRIPs) and significantly inhibited SARS-CoV N protein expression, with EC50 values of 0.12 µM for SARS-CoV-1 and 1.47 µM for SARS-CoV-2 SRIPs. Additionally, compound 17 reduced viral subgenomic RNA levels in a concentration-dependent manner in SRIP-infected cells. The structure–activity relationships of compound 17 with SARS-CoV-1 and SARS-CoV-2 RdRp were also investigated, highlighting it as a promising lead for developing antiviral agents against SARS and COVID-19.

RNase L-induced bodies sequester subgenomic flavivirus RNAs to promote viral RNA decay

Subgenomic flavivirus RNAs (sfRNAs) are structured RNAs encoded by flaviviruses that promote viral infection by inhibiting cellular RNA decay machinery. Herein, we analyze sfRNA production and localization using single-molecule RNA fluorescence in situ hybridization (smRNA-FISH) throughout West Nile virus, Zika virus, or dengue virus serotype 2 infection. We observe that sfRNAs are generated during the RNA replication phase of viral infection in the cytosol and accumulate in processing bodies (P-bodies), which contain RNA decay machinery such as XRN1 and Dcp1b. However, upon activation of the host antiviral endoribonuclease, ribonuclease L (RNase L), sfRNAs re-localize to ribonucleoprotein complexes known as RNase L-induced bodies (RLBs). RLB-mediated sequestration of sfRNAs reduces sfRNA association with RNA decay machinery inP-bodies, which coincides with increased viral RNA decay. These findings establish a functional role forRLBs in enhancing the cell-mediated decay of viral RNA by sequestering functional viral RNA decay products.

Transcriptional kinetics of NADC34-like porcine reproductive and respiratory syndrome virus during cellular infection.

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) employs complex strategies to synthesize subgenomic RNAs (sgRNAs); however, their plasticity and temporal dynamics remain largely unexplored. Using next-generation sequencing (NGS), we examined the high-resolution landscape of the PRRSV subgenome, highlighting considerable heterogeneity in temporal kinetics and transcriptional control and revealing extensive coordination between TRSL-dependent and TRSL-independent sgRNAs. In addition, a comprehensive re-annotation of transcription regulatory sequence (TRS) locations was conducted, clarifying that their usage involved canonical, alternative, and non-canonical splicing events for annotated genes. These insights emphasize that the coding of genetic material in PRRSV is far more intricate than previously anticipated. Collectively, the altered sgRNA phenotype offers distinctive insights into PRRSV transcription and gives additional impetus for mining the functional short- and long-range RNA-RNA interactome at active viral replication sites.

A conserved opal termination codon optimizes a temperature-dependent tradeoff between protein production and processing in alphaviruses.

Alphaviruses are enveloped, single-stranded, positive-sense RNA viruses that often require transmission between arthropod and vertebrate hosts for their sustained propagation. Most alphaviruses encode an opal (UGA) termination codon in nonstructural protein 3 (nsP3) upstream of the viral polymerase, nsP4. The selective constraints underlying the conservation of the opal codon are poorly understood. Using primate and mosquito cells, we explored the role and selective pressure on the nsP3 opal codon through extensive mutational analysis in the prototype alphavirus, Sindbis virus (SINV). We found that the opal codon is highly favored over all other codons in primate cells under native 37°C growth conditions. However, this preference is diminished in mosquito and primate cells grown at a lower temperature. Thus, the primary determinant driving the selection of the opal stop codon is not host genetics but the passaging temperature. We show that the opal codon is preferred over amber and ochre termination codons because it results in the highest translational readthrough and polymerase production. However, substituting the opal codon with sense codons leads to excessive full-length polyprotein (P1234) production, which disrupts optimal nsP polyprotein processing, delays the switch from minus-strand to positive-strand RNA production, and significantly reduces SINV fitness at 37°C; this fitness defect is relieved at lower temperatures. A naturally occurring suppressor mutation unexpectedly compensates for a delayed transition from minus to genomic RNA production by also delaying the subsequent transition between genomic and sub-genomic RNA production. Our study reveals that the opal stop codon is the best solution for alphavirus replication at 37°C, producing enough nsP4 protein to maximize replication without disrupting nsP processing and RNA replication transitions needed for optimal fitness. Our study uncovers the intricate strategy dual-host alphaviruses use at a single codon to optimize fitness.

Improved sub-genomic RNA prediction with the ARTIC protocol.

Viral subgenomic RNA (sgRNA) plays a major role in SARS-COV2's replication, pathogenicity, and evolution. Recent sequencing protocols, such as the ARTIC protocol, have been established. However, due to the viral-specific biological processes, analyzing sgRNA through viral-specific read sequencing data is a computational challenge. Current methods rely on computational tools designed for eukaryote genomes, resulting in a gap in the tools designed specifically for sgRNA detection. To address this, we make two contributions. Firstly, we present sgENERATE, an evaluation pipeline to study the accuracy and efficacy of sgRNA detection tools using the popular ARTIC sequencing protocol. Using sgENERATE, we evaluate periscope, a recently introduced tool that detects sgRNA from ARTIC sequencing data. We find that periscope has biased predictions and high computational costs. Secondly, using the information produced from sgENERATE, we redesign the algorithm in periscope to use multiple references from canonical sgRNAs to mitigate alignment issues and improve sgRNA and non-canonical sgRNA detection. We evaluate periscope and our algorithm, periscope_multi, on simulated and biological sequencing datasets and demonstrate periscope_multi's enhanced sgRNA detection accuracy. Our contribution advances tools for studying viral sgRNA, paving the way for more accurate and efficient analyses in the context of viral RNA discovery.

Sub-genomic flaviviral RNA elements increase the stability and abundance of recombinant AAV vector transcripts.

Viral RNA elements can hijack host cell machinery to control stability of transcripts and consequently, infection. Studies that help better understand such viral elements can provide insights into antiviral strategies and also potentially leverage these features for therapeutic applications. In this study, by incorporating structured flaviviral RNA elements into recombinant adeno-associated viral (AAV) vector genomes, we show improved AAV transcript stability and transgene expression can be achieved, with implications for gene transfer.

Adaptive truncation of the S gene in IBV during chicken embryo passaging plays a crucial role in its attenuation.

Like all coronaviruses, infectious bronchitis virus, the causative agent of infectious bronchitis in chickens, exhibits a high mutation rate. Adaptive mutations that arise during the production of live attenuated vaccines against IBV often decrease virulence. The specific impact of these mutations on viral pathogenicity, however, has not been fully elucidated. In this study, we identified a mutation at the 3' end of the S gene in an IBV strain that was serially passaged in chicken embryos, and showed that this mutation resulted in a 9-aa truncation of the cytoplasmic tail (CT) of the S protein. This phenomenon of CT truncation has previously been observed in the production of attenuated vaccines against other coronaviruses such as the porcine epidemic diarrhea virus. We next discovered that the 9-aa truncation in the S protein CT resulted in the loss of the endoplasmic-reticulum-retention signal (KKSV). Rescue experiments with recombinant viruses confirmed that the deletion of the KKSV motif impaired the localization of the S protein to the endoplasmic-reticulum-Golgi intermediate compartment (ERGIC) and increased its expression on the cell surface. This significantly reduced the incorporation of the S protein into viral particles, impaired early subgenomic RNA and protein synthesis, and ultimately reduced viral invasion efficiency in CEK cells. In vivo experiments in chickens confirmed the reduced pathogenicity of the mutant IBV strains. Additionally, we showed that the adaptive mutation altered the TRS-B of ORF3 and impacted the transcriptional regulation of this gene. Our findings underscore the significance of this adaptive mutation in the attenuation of IBV infection and provide a novel strategy for the development of live attenuated IBV vaccines.

Ronapreve (REGN-CoV; casirivimab and imdevimab) reduces the viral burden and alters the pulmonary response to the SARS-CoV-2 Delta variant (B.1.617.2) in K18-hACE2 mice using an experimental design reflective of a treatment use case.

With some exceptions, global policymakers have recommended against the use of existing monoclonal antibodies in COVID-19 due to loss of neutralization of newer variants. The purpose of this study was to investigate the impact of Ronapreve on compartmental viral replication using paradigms for susceptible and insusceptible variants. Virological efficacy and impact on pathogenicity was assessed in K18-hACE2 mice inoculated with either the Delta or BA.1 Omicron variants. Ronapreve reduced sub-genomic viral RNA levels in lung and nasal turbinate, 4 and 6 days post-infection, for the Delta variant but not the Omicron variant. It also blocked brain infection, which is seen with high frequency in K18-hACE2 mice after Delta variant infection. At day 6, the inflammatory response to lung infection with the Delta variant was altered to a multifocal granulomatous inflammation in which the virus appeared to be confined. The current study provides evidence of an altered tissue response to SARS-CoV-2 after treatment with a monoclonal antibody combination that retains neutralization activity. These data demonstrate that experimental designs that reflect treatment use cases are achievable in animal models for monoclonal antibodies. Extreme caution should be taken when interpreting prophylactic experimental designs that may not be representative of treatment.IMPORTANCEFollowing the emergence of the SARS-CoV-2 Omicron variant, the WHO recommended against the use of Ronapreve in its COVID-19 treatment guidelines due to a lack of efficacy based on current pharmacokinetic-pharmacodynamic understanding. However, the continued use of Ronapreve, specifically in vulnerable patients, was advocated by some based on in vitro neutralization data. Here, the virological efficacy of Ronapreve was demonstrated in both the lung and brain compartments using Delta as a paradigm for a susceptible variant. Conversely, a lack of virological efficacy was demonstrated for the Omicron variant. Comparable concentrations of both monoclonal antibodies were observed in the plasma of Delta- and Omicron-infected mice. This study made use of a reliable murine model for SARS-CoV-2 infection, an experimental design reflective of treatment, and demonstrated the utility of this approach when assessing the effectiveness of monoclonal antibodies.

Extended remdesivir administration in haematological patients with malignancies and COVID-19 during the Omicron era: safety and outcomes.

To describe the management of haematological patients experiencing prolonged SARS-CoV-2 viral shedding, as the optimal management strategy for this condition remains undetermined. We conducted a retrospective evaluation of our prospectively followed cohort of haematological patients treated with remdesivir for more than 10 days. Starting January 2023, upon COVID-19 diagnosis, the treatment strategy was based on symptoms and PCR cycle threshold (Ct) as follows: (i) when Ct was 25 or less or if the patient had symptoms, a course of remdesivir for at least 10 days, nirmatrelvir/ritonavir for 5 days (whenever possible) and convalescent plasma was administered; and (ii) when the patient was asymptomatic and had a PCR Ct of more than 25, when possible, a course of 5 days of nirmatrelvir/ritonavir was administered. The patient was considered to have achieved viral clearance and, thus, remdesivir was stopped, in either of these cases: (i) PCR negativity, or (ii) subgenomic RNA negativity. From January to November 2023, 18 patients benefited from a safe extended remdesivir administration, resulting in detection of SARS-CoV-2 viral clearance in a median time of 3.5 weeks (IQR 2.6-3.9) (min-max 1.6-8.0). No clinical or biological side effects were detected. No patient died or needed further treatment for their COVID-19 episode. The extended course of remdesivir, combined with other active therapies for COVID-19 infection, was well tolerated. Cure and virus negativity were obtained in all these high-risk patients.

Direct nanopore RNA sequencing of umbra-like virus-infected plants reveals long non-coding RNAs, specific cleavage sites, D-RNAs, foldback RNAs, and temporal- and tissue-specific profiles.

The traditional view of plus (+)-strand RNA virus transcriptomes is that infected cells contain a limited variety of viral RNAs, such as full-length (+)-strand genomic RNA(s), (-)-strand replication intermediate(s), 3' co-terminal subgenomic RNA(s), and viral recombinant defective (D)-RNAs. To ascertain the full complement of viral RNAs associated with the simplest plant viruses, long-read direct RNA nanopore sequencing was used to perform transcriptomic analyses of two related umbra-like viruses: citrus yellow vein-associated virus (CY1) from citrus and CY2 from hemp. Analysis of different timepoints/tissues in CY1- and CY2-infected Nicotiana benthamiana plants and CY2-infected hemp revealed: (i) three 5' co-terminal RNAs of 281 nt, 442 nt and 671 nt, each generated by a different mechanism; (ii) D-RNA populations containing the 671 fragment at their 5'ends; (iii) many full-length genomic RNAs and D-RNAs with identical 3'end 61 nt truncations; (iv) virtually all (-)-strand reads missing 3 nt at their 3' termini; (v) (±) foldback RNAs comprising about one-third of all (-)-strand readsand (vi) a higher proportion of full-length gRNAs in roots than in leaves, suggesting that roots may be functioning as a gRNA reservoir. These findings suggest that viral transcriptomes are much more complex than previously thought.

Evaluation of Viral Subgenomic RNAs and Antigen Presence in SARS-CoV-2 PCR Positive Cases

Polymerase chain reaction (PCR) and antigen test (AgT) are frequently used in the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Routine PCR tests that detect the virus genome cannot determine whether the virus is infectious or not. However, detection of subgenomic RNA (sgRNA) produced during the replication period may indicate active viral infection. Active virus detection can offer various health and economic benefits from isolation time to treatment. Antigen tests are also considered as indicators of infectiousness since they can detect viruses above a certain load amount. The aim of this study was to use two different subgenomic RNAs and antigen test instead of genomic RNA to examine the relationship with each other and the clinic in terms of infectiousness. Evaluating the antigen test together with subgenomic RNA as an indicator of infectiousness may show the importance of this test. SARS-CoV-2 PCR positive 109 naso/oropharyngeal swab samples stored at -80 °C were included in the study. In order to confirm the PCR positivity of these samples, E gene PCR was performed and AgT, and E and N sgRNA quantitative real-time reverse transcription-PCR (RT-qPCR) detection was performed. Of the 109 SARSCoV-2 PCR positive samples, 83 (76.14%) had antigen test positivity, 88 (80.73%) had E gene sgRNA, 96 (88.07%) had N gene sgRNA and 97 (89%) had at least one sgRNA positivity.The antigen test was found positive in 77.3% of the samples in which at least one sgRNA was detected and in 66.7% of the negative samples and this difference was not statistically significant (p= 0.475). The difference between E sgRNA and AgT positivity was significant (p= 0.023). N sgRNA was positive in 98.9% of E sgRNA positive samples and 42.9% of the negative samples and this difference was statistically significant (p= 0.0001). The AgT positivity rate was found to be 98.15% (53/54) for cycle threshold (Ct) value ≤ 25, 57.14% (12/21) for Ct 25-30, and 52.94% (18/34) for Ct ≥ 30. The difference in antigen test positivity between E gRNA Ct value ≤ 25 and > 25, ≤ 29 and > 29, < 30 and ≥ 30 was statistically significant (p= 0.0001). Antigen test positivity appears to be associated with viral load and infectivity, as expected. In our study, it has been shown that sgRNAs and AgT which are indicators of infectiousness can be detected at least 10 days after the symptom period. Using these two tests together could detect infective individuals with higher accuracy and shorten the duration of hospital stay and isolation.