Search

The COVID-19 pandemic has created a worldwide public health crisis that has since resulted in 6.8 million reported deaths. The pandemic prompted the immediate response of researchers around the world to engage in rapid vaccine development, surveillance programs, and antiviral testing, which resulted in the delivery of multiple vaccines and repurposed antiviral drug candidates. However, the emergence of new highly transmissible SARS-CoV-2 variants has renewed the desire for discovering new antiviral drug candidates with high efficacy against the emerging variants of concern. Traditional antiviral testing methods employ the plaque-reduction neutralization tests (PRNTs), plaque assays, or RT-PCR analysis, but each assay can be tedious and time-consuming, requiring days to visualize and count plaques, or to prepare cell extractions for PCR analysis. In recent years, plate-based image cytometers have demonstrated high-throughput vaccine screening methods, which can be adopted for screening potential antiviral drug candidates. In this work, we developed a high-throughput antiviral testing method employing the Celigo Image Cytometer to investigate the efficacy of antiviral drug candidates on SARS-CoV-2 infectivity using a fluorescent reporter virus and their safety by measuring the cytotoxicity effects on the healthy host cell line using fluorescent viability stains. The Celigo Image Cytometer can provide an efficient and robust method to rapidly identify potential antiviral drugs to effectively combat the rapidly spreading SARS-CoV-2 virus and its variants during the pandemic.

ImportancePregnant women are at increased risk of severe COVID-19, but the contribution of viral RNA load, the presence of infectious virus, and mucosal antibody responses remain understudied.ObjectiveTo evaluate the association of COVID-19 outcomes following confirmed infection with vaccination status, mucosal antibody responses, infectious virus recovery and viral RNA levels in pregnant compared with non-pregnant women.DesignA retrospective observational cohort study of remnant clinical specimens from SARS-CoV-2 infected patients between October 2020-May 2022.SettingFive acute care hospitals within the Johns Hopkins Health System (JHHS) in the Baltimore, MD-Washington, DC area.ParticipantsParticipants included confirmed SARS-CoV-2 infected pregnant women and matched non-pregnant women (matching criteria included age, race/ethnicity, and vaccination status).ExposureSARS-CoV-2 infection, with documentation of SARS-CoV-2 mRNA vaccination.Main Outcome(s)The primary dependent measures were clinical COVID-19 outcomes, infectious virus recovery, viral RNA levels, and mucosal anti-spike (S) IgG titers from upper respiratory tract samples. Clinical outcomes were compared using odds ratios (OR), and measures of virus and antibody were compared using either Fisher’s exact test, two-way ANOVA, or regression analyses. Results were stratified according to pregnancy, vaccination status, maternal age, trimester of pregnancy, and infecting SARS-CoV-2 variant.Results(s)A total of 452 individuals (117 pregnant and 335 non-pregnant) were included in the study, with both vaccinated and unvaccinated individuals represented. Pregnant women were at increased risk of hospitalization (OR = 4.2; CI = 2.0-8.6), ICU admittance, (OR = 4.5; CI = 1.2-14.2), and of being placed on supplemental oxygen therapy (OR = 3.1; CI =1.3-6.9). An age-associated decrease in anti-S IgG titer and corresponding increase in viral RNA levels (P< 0.001) was observed in vaccinated pregnant, but not non-pregnant, women. Individuals in their 3rdtrimester had higher anti-S IgG titers and lower viral RNA levels (P< 0.05) than those in their 1stor 2ndtrimesters. Pregnant individuals experiencing breakthrough infections due to the omicron variant had reduced anti-S IgG compared to non-pregnant women (P< 0.05).Conclusions and RelevanceIn this cohort study, vaccination status, maternal age, trimester of pregnancy, and infecting SARS-CoV-2 variant were each identified as drivers of differences in mucosal anti-S IgG responses in pregnant compared with non-pregnant women. Observed increased severity of COVID-19 and reduced mucosal antibody responses particularly among pregnant participants infected with the Omicron variant suggest that maintaining high levels of SARS-CoV-2 immunity may be important for protection of this at-risk population.Key PointsQuestionIs greater COVID-19 disease severity during pregnancy associated with either reduced mucosal antibody responses to SARS-CoV-2 or increased viral RNA levels?FindingIn a retrospective cohort of pregnant and non-pregnant women with confirmed SARS-CoV-2 infection, we observed that (1) disease severity, including ICU admission, was greater among pregnant than non-pregnant women; (2) vaccination was associated with reduced recovery of infectious virus in non-pregnant women but not in pregnant women; (3) increased nasopharyngeal viral RNA levels were associated with reduced mucosal IgG antibody responses in pregnant women; and (4) greater maternal age was associated with reduced mucosal IgG responses and increased viral RNA levels, especially among women infected with the Omicron variant.MeaningThe findings of this study provide novel evidence that, during pregnancy, lower mucosal antibody responses are associated with reduced control of SARS-CoV-2, including variants of concern, and greater disease severity, especially with increasing maternal age. Reduced mucosal antibody responses among vaccinated pregnant women highlight the need for bivalent booster doses during pregnancy.

The human sialidase enzymes (or neuraminidases, NEU1-4) are glycoside hydrolases that catalyze the removal of sialic acid residues from glycoconjugates, including many bioactive glycoproteins and glycolipids. Through their physiochemical effect on glycoconjugates, sialic acid residues are thought to play vital roles in the control of cellular signaling. In previous studies, it was demonstrated that NEU1-4 activity was increased in cells infected with dengue virus serotype 2 (DENV2). Additionally, it was demonstrated that the DENV2 NS1 protein was sufficient for inducing increased NEU1-4 activity in both in vivo and in vitro models, and that this increased activity was linked to endothelial hyperpermeability and vascular leakage, a hallmark of severe dengue disease. However, the role of increased NEU1-4 activity in the viral lifecycle was not understood. Here, we used siRNA-mediated loss of function studies to evaluate the effect of inhibition of sialidase activity on the DENV2 lifecycle. Our analyses uncovered that apart from their importance for viral pathogenesis, NEU1-4 activity was vital for DENV2 viral replication and egress. Moreover, we characterized the inter-relationship between NEU 1-4, and determined that there was a transcriptional dependency of NEU1-3 on NEU4.

Host-targeted therapeutics to control viral infection are gaining prominence given the vulnerability of viral replication at select host-interaction points and the limited possibility of developing drug resistant mutants. Nevertheless, the chemical and biological impact of many host-targeted therapeutics on both the cell and virus has not been elucidated and remains a key complication. Previously, it has been demonstrated that inhibition of fatty acid metabolism has significant antiviral potential. Here, we use a multidisciplinary approach to demonstrate how inhibition of fatty acid biosynthesis creates a metabolically refractory environment that drives viral dependence on alternate metabolic pathways for survival. By profiling the global metabolic landscape following inhibition of fatty acid biosynthesis, we identified additional biochemical pathways that, when inhibited in combination with fatty acid biosynthesis, displayed increased antiviral potential. Our studies also demonstrated that there was a direct link between changes in cellular chemical composition and the ultrastructural membrane architecture induced by viral gene products. Utilizing inhibitors to change these metabolic environments significantly impacted early viral replication and disrupted the membrane architecture critical for the viral life cycle. Here, we have defined at a molecular level how shifting metabolic landscapes can be exploited to identify combinations of therapeutics that have a greater antiviral effect.Author SummaryDengue viruses are transmitted by Aedes aegypti mosquitoes which are prevalent in the tropical and subtropical regions of the world. These viruses cause over 350 million infections annually. There are no antivirals to combat infection and the only vaccine available is suboptimal. Since these viruses are obligate pathogens, they hijack lipid metabolic pathways in host cells to drive new lipid synthesis critically required for their replication. Mechanisms of how lipid synthesis impacts viral replication is unknown. These viruses also rearrange cellular membranes to form platforms for assembly of viral replication complexes. Here, for the first time, we show that virus-hijacking of de novo fatty acid biosynthesis pathways is required for the formation of membranous replication platforms and if inhibited disrupted synthesis of replicative form viral RNA. Importantly, these inhibitors drastically rearranged the metabolic landscape of the cell resulting in an activation of compensatory nucleotide synthesis pathways that allowed the virus to survive at a low level through the inhibition. However, if both pathways were inhibited in combination, infectious virus release was reduced to below detection limits. The study demonstrates how understanding the metabolic landscape altered by specific inhibitors can lead to the discovery of compensatory metabolic pathways and targets that in combination can enhance intervention efficacy.

The antiviral endoribonuclease, RNase L, is a vital component of the mammalian innate immune response that destroys host and viral RNA to reduce viral gene expression. Herein, we show that a consequence of RNase L-mediated decay of cytoplasmic host RNAs is the widespread re-localization of RNA-binding proteins (RBPs) from the cytoplasm to the nucleus, due to the presence of nuclear RNA. Concurrently, we observe global alterations to host RNA processing in the nucleus, including alterations of splicing and 3’ end formation, with the latter leading to downstream of gene (DoG) transcripts. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and coincide with the retention of their mRNAs in the nucleus. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is fundamentally dictated by the availability of RNA in each compartment and thus viral infections that trigger cytoplasmic RNA degradation alter RNA processing due to the nuclear influx of RNA binding proteins.

Nestled within the Rocky Mountain National Forest, 114 scientists and students gathered at Colorado State University’s Mountain Campus for this year’s 21st annual Rocky Mountain National Virology Association meeting. This 3-day retreat consisted of 31 talks and 30 poster presentations discussing advances in research pertaining to viral and prion diseases. The keynote address provided a timely discussion on zoonotic coronaviruses, lessons learned, and the path forward towards predicting, preparing, and preventing future viral disease outbreaks. Other invited speakers discussed advances in SARS-CoV-2 surveillance, molecular interactions involved in flavivirus genome assembly, evaluation of ethnomedicines for their efficacy against infectious diseases, multi-omic analyses to define risk factors associated with long COVID, the role that interferon lambda plays in control of viral pathogenesis, cell-fusion-dependent pathogenesis of varicella zoster virus, and advances in the development of a vaccine platform against prion diseases. On behalf of the Rocky Mountain Virology Association, this report summarizes select presentations.

The recent global Zika epidemics have revealed the significant threat that mosquito-borne viruses pose. There are currently no effective vaccines or prophylactics to prevent Zika virus (ZIKV) infection. Limiting exposure to infected mosquitoes is the best way to reduce disease incidence. Recent studies have focused on targeting mosquito reproduction and immune responses to reduce transmission. Previous work has evaluated the effect of insulin signaling on antiviral JAK/STAT and RNAi in vector mosquitoes. Specifically, insulin-fed mosquitoes resulted in reduced virus replication in an RNAi-independent, ERK-mediated JAK/STAT-dependent mechanism. In this work, we demonstrate that targeting insulin signaling through the repurposing of small molecule drugs results in the activation of both RNAi and JAK/STAT antiviral pathways. ZIKV-infected Aedes aegypti were fed blood containing demethylasterriquinone B1 (DMAQ-B1), a potent insulin mimetic, in combination with AKT inhibitor VIII. Activation of this coordinated response additively reduced ZIKV levels in Aedes aegypti. This effect included a quantitatively greater reduction in salivary gland ZIKV levels up to 11 d post-bloodmeal ingestion, relative to single pathway activation. Together, our study indicates the potential for field delivery of these small molecules to substantially reduce virus transmission from mosquito to human. As infections like Zika virus are becoming more burdensome and prevalent, understanding how to control this family of viruses in the insect vector is an important issue in public health.

The antiviral endoribonuclease, RNase L, is activated by the mammalian innate immune response to destroy host and viral RNA to ultimately reduce viral gene expression. Herein, we show that RNase L and RNase L-mediated mRNA decay are primarily localized to the cytoplasm. Consequently, RNA-binding proteins (RBPs) translocate from the cytoplasm to the nucleus upon RNase L activation due to the presence of intact nuclear RNA. The re-localization of RBPs to the nucleus coincides with global alterations to RNA processing in the nucleus. While affecting many host mRNAs, these alterations are pronounced in mRNAs encoding type I and type III interferons and correlate with their retention in the nucleus and reduction in interferon protein production. Similar RNA processing defects also occur during infection with either dengue virus or SARS-CoV-2 when RNase L is activated. These findings reveal that the distribution of RBPs between the nucleus and cytosol is dictated by the availability of RNA in each compartment. Thus, viral infections that trigger RNase L-mediated cytoplasmic RNA in the cytoplasm also alter RNA processing in the nucleus, resulting in an ingenious multi-step immune block to protein biogenesis.

The transcriptional induction of interferon (IFN) genes is a key feature of the mammalian antiviral response that limits viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFN proteins in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Here, we performed single-molecule RNA visualization to examine the expression and localization of host mRNAs during SARS-CoV-2 infection. Our data show that the biogenesis of type I and type III IFN mRNAs is inhibited at multiple steps during SARS-CoV-2 infection. First, translocation of the interferon regulatory factor 3 (IRF3) transcription factor to the nucleus is limited in response to SARS-CoV-2, indicating that SARS-CoV-2 inhibits RLR-MAVS signaling and thus weakens transcriptional induction of IFN genes. Second, we observed that IFN mRNAs primarily localize to the site of transcription in most SARS-CoV-2 infected cells, suggesting that SARS-CoV-2 either inhibits the release of IFN mRNAs from their sites of transcription and/or triggers decay of IFN mRNAs in the nucleus upon exiting the site of transcription. Lastly, nuclear-cytoplasmic transport of IFN mRNAs is inhibited during SARS-CoV-2 infection, which we propose is a consequence of widespread degradation of host cytoplasmic basal mRNAs in the early stages of SARS-CoV-2 replication by the SARS-CoV-2 Nsp1 protein, as well as the host antiviral endoribonuclease, RNase L. Importantly, IFN mRNAs can escape SARS-CoV-2-mediated degradation if they reach the cytoplasm, making rescue of mRNA export a viable means for promoting the immune response to SARS-CoV-2.

A key feature of the mammalian antiviral response is the transcriptional induction of interferon (IFN) genes, which encode for secreted proteins that prime the antiviral response to limit viral replication and dissemination. A hallmark of severe COVID-19 disease caused by SARS-CoV-2 is the low presence of IFNs in patient serum despite elevated levels of IFN-encoding mRNAs, indicative of post-transcriptional inhibition of IFN protein production. Herein, we show SARS-CoV-2 infection limits type I and type III IFN biogenesis by preventing the release of mRNA from their sites of transcription and/or triggering their nuclear degradation. Additionally, SARS-CoV-2 infection inhibits nuclear-cytoplasmic transport of IFN mRNAs as a consequence of widespread cytosolic mRNA degradation mediated by both the host antiviral endoribonuclease, RNase L, and by the SARS-CoV-2 protein, Nsp1. Thus, inhibition of host and/or viral Nsp1-mediated mRNA decay, as well as IFN treatments, may reduce viral-associated pathogenesis by promoting the innate immune response. Funding Information: Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number F32AI145112 (J.M.B), funds from HHMI (Roy Parker), and support provided by the Office of the Vice President for Research and the Dept. of Microbiology, Immunology and Pathology at Colorado State University (Rushika Perera). Declaration of Interests: Roy Parker is a founder and consultant of Faze Medicines.

Due to the COVID-19 pandemic and multiple devastating forest fires, the 2020 meeting of the Rocky Mountain Virology Association was held virtually. The three-day gathering featured talks describing recent advances in virology and prion research. The keynote presentation described the measles virus paradox of immune suppression and life-long immunity. Special invited speakers presented information concerning visualizing antiviral effector cell biology in mucosal tissues, uncovering the T-cell tropism of Epstein-Barr virus type 2, a history and current survey of coronavirus spike proteins, a summary of Zika virus vaccination and immunity, the innate immune response to flavivirus infections, a discussion concerning prion disease as it relates to multiple system atrophy, and clues for discussing virology with the non-virologist. On behalf of the Rocky Mountain Virology Association, this report summarizes selected presentations.

This autumn, approximately 100 scientists and students from the Rocky Mountain area along with invited speakers attended the 18th annual meeting of the Rocky Mountain Virology Association that was held at the Colorado State University Mountain Campus. The two-day gathering featured 31 talks and 33 posters all of which focused on specific areas of current virology and prion protein research. Since the keynote presentation focused on the oligoadenylate synthetase-ribonuclease L pathway the main area of focus was on host–virus interactions, however other areas of interest included virus vectors, current models of virus infections, prevention and treatment of virus infections, separate sessions on RNA viruses and prion proteins, and a special talk highlighting various attributes of targeted next-generation sequencing. The meeting was held at the peak of the fall Aspen colors surrounded by five mountains >11000 ft (3.3 km) where the secluded campus provided the ideal setting for extended discussions and outdoor exercise. On behalf of the Rocky Mountain Virology Association, this report summarizes 42 selected presentations.