Viral RNA Abundance Research Articles

SARS-CoV-2 surveillance in US wastewater: Leading indicators and data variability analysis in 2023–2024

Wastewater-Based Epidemiology (WBE) has become a powerful tool for assessing disease occurrence in communities. This study investigates the coronavirus disease 2019 (COVID-19) epidemic in the United States during 2023–2024 using wastewater data from 189 wastewater treatment plants in 40 states and the District of Columbia. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and pepper-mild mottle virus normalized SARS-CoV-2 RNA concentration data were compared with COVID-19 hospitalization admission data at both national and state levels. We further investigate temporal features in wastewater viral RNA abundance, with peak timing and cross-correlation lag analyses indicating that wastewater SARS-CoV-2 RNA concentrations precede hospitalization admissions by 2 to 12 days. Lastly, we demonstrate that wastewater treatment plant size has a significant effect on the variability of measured SARS-CoV-2 RNA concentrations. This study highlights the effectiveness of WBE as a non-invasive, timely and resource-efficient disease monitoring strategy, especially in the context of declining COVID-19 clinical reporting.

Virus-derived circular RNAs populate hepatitis C virus–infected cells

It is known that pre-mRNAs in eukaryotic cells can be processed to circular RNAs by a backsplicing mechanism. Circular RNAs have great stability and can sequester proteins or small RNAs to exert functions on cellular pathways. Because viruses often exploit host pathways, we explored whether the RNA genome of the cytoplasmic hepatitis C virus is processed to yield virus-derived circRNAs (vcircRNAs). Computational analyses of RNA-seq experiments predicted that the viral RNA genome is fragmented to generate hundreds of vcircRNAs. More than a dozen of them were experimentally verified by rolling-circle amplification. VcircRNAs that contained the viral internal ribosome entry site were found to be translated into proteins that displayed proviral functions. Furthermore, two highly abundant, nontranslated vcircRNAs were shown to enhance viral RNA abundance. These findings argue that novel vcircRNA molecules modulate viral amplification in cells infected by a cytoplasmic RNA virus.

A Genetic Circuit Design for Targeted Viral RNA Degradation.

Advances in synthetic biology have led to the design of biological parts that can be assembled in different ways to perform specific functions. For example, genetic circuits can be designed to execute specific therapeutic functions, including gene therapy or targeted detection and the destruction of invading viruses. Viral infections are difficult to manage through drug treatment. Due to their high mutation rates and their ability to hijack the host's ribosomes to make viral proteins, very few therapeutic options are available. One approach to addressing this problem is to disrupt the process of converting viral RNA into proteins, thereby disrupting the mechanism for assembling new viral particles that could infect other cells. This can be done by ensuring precise control over the abundance of viral RNA (vRNA) inside host cells by designing biological circuits to target vRNA for degradation. RNA-binding proteins (RBPs) have become important biological devices in regulating RNA processing. Incorporating naturally upregulated RBPs into a gene circuit could be advantageous because such a circuit could mimic the natural pathway for RNA degradation. This review highlights the process of viral RNA degradation and different approaches to designing genetic circuits. We also provide a customizable template for designing genetic circuits that utilize RBPs as transcription activators for viral RNA degradation, with the overall goal of taking advantage of the natural functions of RBPs in host cells to activate targeted viral RNA degradation.

Lymphoid tissues contribute to plasma viral clonotypes early after antiretroviral therapy interruption in SIV-infected rhesus macaques.

The rebound-competent viral reservoir, composed of a virus that is able to persist during antiretroviral therapy (ART) and mediate reactivation of systemic viral replication and rebound viremia after ART interruption (ATI), remains the biggest obstacle to treating HIV infection. A better understanding of the cellular and tissue origins and the dynamics of viral populations that initiate rebound upon ATI could help develop therapeutic strategies for reducing the rebound-competent viral reservoir. In this study, barcoded simian immunodeficiency virus (SIV), SIVmac239M, was used to infect rhesus macaques to enable monitoring of viral barcode clonotypes contributing to virus detectable in plasma after ATI. Blood and tissues from secondary lymphoid organs (spleen, mesenteric lymph nodes, and inguinal lymph nodes) and from the colon, ileum, lung, liver, and brain were analyzed using viral barcode sequencing, intact proviral DNA assay, single-cell RNA sequencing, and combined CODEX and RNAscope in situ hybridization. Four of seven animals had viral barcodes detectable by deep sequencing of plasma at necropsy, although plasma viral RNA remained below 22 copies per milliliter. Among the tissues studied, mesenteric lymph nodes, inguinal lymph nodes, and spleen contained viral barcodes detected in plasma. CD4+ T cells were the main cell type harboring viral RNA after ATI. Furthermore, T cell zones in lymphoid tissues showed higher viral RNA abundance than B cell zones for most animals. These findings are consistent with lymphoid tissues contributing to the virus present in plasma early after ATI.

Single-cell transcriptomics identifies prothymosin α restriction of HIV-1 in vivo.

Host restriction factors play key roles in innate antiviral defense, but it remains poorly understood which of them restricts HIV-1 in vivo. Here, we used single-cell transcriptomic analysis to identify host factors associated with HIV-1 control during acute infection by correlating host gene expression with viral RNA abundance within individual cells. Wide sequencing of cells from one participant with the highest plasma viral load revealed that intracellular viral RNA transcription correlates inversely with expression of the gene PTMA, which encodes prothymosin α. This association was genome-wide significant (Padjusted < 0.05) and was validated in 28 additional participants from Thailand and the Americas with HIV-1 CRF01_AE and subtype B infections, respectively. Overexpression of prothymosin α in vitro confirmed that this cellular factor inhibits HIV-1 transcription and infectious virus production. Our results identify prothymosin α as a host factor that restricts HIV-1 infection in vivo, which has implications for viral transmission and cure strategies.

The P323L substitution in the SARS-CoV-2 polymerase (NSP12) confers a selective advantage during infection

BackgroundThe mutational landscape of SARS-CoV-2 varies at the dominant viral genome sequence and minor genomic variant population. During the COVID-19 pandemic, an early substitution in the genome was the D614G change in the spike protein, associated with an increase in transmissibility. Genomes with D614G are accompanied by a P323L substitution in the viral polymerase (NSP12). However, P323L is not thought to be under strong selective pressure.ResultsInvestigation of P323L/D614G substitutions in the population shows rapid emergence during the containment phase and early surge phase during the first wave. These substitutions emerge from minor genomic variants which become dominant viral genome sequence. This is investigated in vivo and in vitro using SARS-CoV-2 with P323 and D614 in the dominant genome sequence and L323 and G614 in the minor variant population. During infection, there is rapid selection of L323 into the dominant viral genome sequence but not G614. Reverse genetics is used to create two viruses (either P323 or L323) with the same genetic background. L323 shows greater abundance of viral RNA and proteins and a smaller plaque morphology than P323.ConclusionsThese data suggest that P323L is an important contribution in the emergence of variants with transmission advantages. Sequence analysis of viral populations suggests it may be possible to predict the emergence of a new variant based on tracking the frequency of minor variant genomes. The ability to predict an emerging variant of SARS-CoV-2 in the global landscape may aid in the evaluation of medical countermeasures and non-pharmaceutical interventions.

Multi-omic analysis characterizes molecular susceptibility of receptors to SARS-CoV-2 spike protein

In the post COVID-19 era, new SARS-CoV-2 variant strains may continue emerging and long COVID is poised to be another public health challenge. Deciphering the molecular susceptibility of receptors to SARS-CoV-2 spike protein is critical for understanding the immune responses in COVID-19 and the rationale of multi-organ injuries. Currently, such systematic exploration remains limited. Here, we conduct multi-omic analysis of protein binding affinities, transcriptomic expressions, and single-cell atlases to characterize the molecular susceptibility of receptors to SARS-CoV-2 spike protein. Initial affinity analysis explains the domination of delta and omicron variants and demonstrates the strongest affinities between BSG (CD147) receptor and most variants. Further transcriptomic data analysis on 4100 experimental samples and single-cell atlases of 1.4 million cells suggest the potential involvement of BSG in multi-organ injuries and long COVID, and explain the high prevalence of COVID-19 in elders as well as the different risks for patients with underlying diseases. Correlation analysis validated moderate associations between BSG and viral RNA abundance in multiple cell types. Moreover, similar patterns were observed in primates and validated in proteomic expressions. Overall, our findings implicate important therapeutic targets for the development of receptor-specific vaccines and drugs for COVID-19.

Macrophage Depletion Reactivates Fecal Virus Shedding following Resolution of Acute Hepatitis A in Ifnar1-/- Mice.

Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1-/- mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm3 in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4+ or CD8+ T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifnγ, Tnfα, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1-/- mice and that macrophages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1-/- mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1-/- mice and may have relevance to relapsing hepatitis A in humans.

Spatial mapping of the total transcriptome by in situ polyadenylation

Spatial transcriptomics reveals the spatial context of gene expression, but current methods are limited to assaying polyadenylated (A-tailed) RNA transcripts. Here we demonstrate that enzymatic in situ polyadenylation of RNA enables detection of the full spectrum of RNAs, expanding the scope of sequencing-based spatial transcriptomics to the total transcriptome. We demonstrate that our spatial total RNA-sequencing (STRS) approach captures coding RNAs, noncoding RNAs and viral RNAs. We apply STRS to study skeletal muscle regeneration and viral-induced myocarditis. Our analyses reveal the spatial patterns of noncoding RNA expression with near-cellular resolution, identify spatially defined expression of noncoding transcripts in skeletal muscle regeneration and highlight host transcriptional responses associated with local viral RNA abundance. STRS requires adding only one step to the widely used Visium spatial total RNA-sequencing protocol from 10x Genomics, and thus could be easily adopted to enable new insights into spatial gene regulation and biology.

RPLP1, an NS4B-interacting protein, enhances production of CSFV through promoting translation of viral genome

ABSTRACT Classical swine fever virus (CSFV), the etiological agent of classical swine fever (CSF), causes serious financial losses to the pig industry. Using yeast two-hybrid screening, we have previously identified ribosomal protein RPLP1 as a potential binding partner of CSFV NS4B. In this study, the interaction between host RPLP1 and CSFV NS4B was further characterized by co-immunoprecipitation (co-IP), glutathione S-transferase (GST) pulldown, and confocal microscopy. In addition, lentivirus-mediated shRNA knockdown of RPLP1 drastically attenuated CSFV growth, while stable overexpression of RPLP1 markedly enhanced CSFV production. Moreover, cellular RPLP1 expression was found to be significantly up-regulated along with CSFV infection. Dual-luciferase reporter assay showed that depletion of RPLP1 had no effects on the activity of CSFV internal ribosome entry site (IRES). In the first life cycle of CSFV, further studies revealed that RPLP1 depletion did not influence the intracellular viral RNA abundance but diminished the intracellular and extracellular progeny virus titers as well as the viral E2 protein expression, which indicates that RPLP1 is crucial for CSFV genome translation. In summary, this study demonstrated that RPLP1 interacts with CSFV NS4B and enhances virus production via promoting translation of viral genome.

Targeted proteomics as a tool to detect SARS-CoV-2 proteins in clinical specimens.

The rapid, sensitive and specific detection of SARS-CoV-2 is critical in responding to the current COVID-19 outbreak. In this proof-of-concept study, we explored the potential of targeted mass spectrometry (MS) based proteomics for the detection of SARS-CoV-2 proteins in both research samples and clinical specimens. First, we assessed the limit of detection for several SARS-CoV-2 proteins by parallel reaction monitoring (PRM) MS in infected Vero E6 cells. For tryptic peptides of Nucleocapsid protein, the limit of detection was estimated to be in the mid-attomole range (9E-13 g). Next, this PRM methodology was applied to the detection of viral proteins in various COVID-19 patient clinical specimens, such as sputum and nasopharyngeal swabs. SARS-CoV-2 proteins were detected in these samples with high sensitivity in all specimens with PCR Ct values <24 and in several samples with higher CT values. A clear relationship was observed between summed MS peak intensities for SARS-CoV-2 proteins and Ct values reflecting the abundance of viral RNA. Taken together, these results suggest that targeted MS based proteomics may have the potential to be used as an additional tool in COVID-19 diagnostics.

T cells protect against hepatitis A virus infection and limit infection-induced liver injury

Hepatitis A virus (HAV) is a common cause of enterically transmitted viral hepatitis. In non-immune individuals, infection results in typically transient but occasionally fulminant and fatal inflammatory liver injury. Virus-specific T cell frequencies peak when liver damage is at its zenith, leading to the prevalent notion that T cells exacerbate liver disease, as suspected for other hepatotropic virus infections. However, the overall contribution of T cells to the control of HAV and the pathogenesis of hepatitis A is unclear and has been impeded by a historic lack of small animal models. Ifnar1-/- mice are highly permissive for HAV and develop pathogenesis that recapitulates many features of hepatitis A. Using this model, we identified HAV-specific CD8+ and CD4+ T cells by epitope mapping, and then used tetramers andfunctional assays to quantify T cells in the liver at multiple times after infection. We assessed the relationships between HAV-specific T cell frequency, viral RNA amounts, and liver pathogenesis. A large population of virus-specific T cells accumulated within the livers of Ifnar1-/- mice during the first 1-2 weeks of infection and persisted over time. HAV replication was enhanced and liver disease exacerbated when mice were depleted of T cells. Conversely, immunization with a peptide vaccine increased virus-specific CD8+ T cell frequencies in the liver, reduced viral RNA abundance, and lessened liver injury. These data show that T cells protect against HAV-mediated liver injury and can be targeted to improve liver health. Hepatitis A virus is a leading cause of acute viral hepatitis worldwide. T cells were thought to contribute to liver injury during acute infection. We now show that virus-specific T cells protect against infection and limit liver injury.

Traces of SARS-CoV-2 RNA in Peripheral Blood Cells of Patients with COVID-19.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third virus that caused coronavirus-related outbreaks over the past 20 years. The outbreak was first reported in December 2019 in Wuhan, China, but rapidly progressed into a pandemic of an unprecedented scale since the 1918 flu pandemic. Besides respiratory complications in patients with COVID-19, clinical characterization of severe infection cases showed several other comorbidities, including multiple organ failure, and septic shock. To better understand the systemic pathogenesis of COVID-19, we interrogated the virus's presence in the peripheral blood cells, which might provide a form of trafficking or hiding to the virus. By analyzing >2 billion sequence reads of high-throughput transcriptome sequence data from 180 samples of patients with active SARS-CoV-2 infection or healthy controls collected from 6 studies, we found evidence of traces of SARS-CoV-2 RNA in peripheral blood mononuclear cells in two samples from two independent studies. In contrast, the viral RNA was abundant in bronchoalveolar lavage specimens from the same patients. We also devised a “viral spike-to-actin” RNA normalization as a metric to compare across various samples and minimize errors caused by intersample variability in total human RNA abundance. Our observation suggests immune presentation and discounts the possibility of extensive viral infection of lymphocytes or monocytes.

Abstract S03-03: Cancer patients display diminished viral RNA clearance and altered T cell responses during SARS-CoV-2 infection

Abstract Cancer patients display immunomodulation related to malignancy and anti-cancer therapies, but how these factors impact COVID-19 remains unknown. To investigate immune responses in cancer patients with COVID-19, we undertook a prospective case-control study, enrolling hospitalized solid tumor patients with acute COVID-19, as well as age-, gender-, and comorbidity-matched COVID-19 patients without cancer as controls. Using biospecimens collected during hospitalization, we performed virologic measurements as well as in-depth immunophenotyping of cellular, antibody and cytokine responses. We enrolled 17 cancer patients (cases) admitted to Yale-New Haven Hospital between March 15 and June 30, 2020 with COVID-19, as well as 17 matched non-cancer patients (controls) admitted with COVID-19. No significant differences were observed between cases and controls based on patient characteristics (age, gender, race, co-morbidities, smoking history, days from symptom onset to COVID-19 diagnosis) or outcomes (COVID-19 severity, length of hospital stay, rate of intubation or mortality). The most common primary tumor sites were lung (4/17) and gastrointestinal (4/17); all cases had received cancer-directed therapy within 6 months of COVID-19 diagnosis, with 13/17 receiving treatment less than 1 month prior to hospitalization. Three of 17 cases had received immune checkpoint inhibitor therapies. Despite having similar SARS-CoV-2 viral RNA loads at the time of COVID-19 diagnosis when compared with controls, cancer cases had increased viral RNA abundance during hospitalization, suggesting slower clearance. Antibody responses against SARS-CoV-2 were preserved in cancer cases, with cases displaying similar levels of IgM and IgG antibodies directed against SARS-CoV-2 epitopes compared to controls. Cytokine profiling revealed higher plasma levels of CCL3, IL1A and CXCL12 in cancer cases compared to controls. Using flow cytometric immunophenotyping, we found that innate immune and non-T cell adaptive immune parameters were similar between cases and controls hospitalized with COVID-19. However, among cancer cases on conventional therapies, T cell lymphopenia was more profound, and these cases demonstrated higher levels of CD8+ exhausted (CD8+CD45RA−PD1+TIM3+), CD8+GranzymeB+ and CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ activated T cells when compared with controls; interestingly, these differences were not observed in patients who had received immune checkpoint inhibition. Thus, we found reduced viral RNA clearance and specific alterations in T cell and cytokine responses in cancer patients hospitalized with COVID-19 compared with matched controls with COVID-19. This dysregulated T cell response in cancer patients, which may reflect immune modulation due to chronic antigen stimulation as well as cancer therapies, may lead to altered virologic and clinical outcomes in this population. Citation Format: Michael Chiorazzi, Erin Silva, Kristina Brower, Patrick Wong, Carolina Lucas, Jon Klein, Feimei Liu, Maura Nakahata, Jun Zhao, Nur-Taz Rahman, Camila Odio, Santos Bermejo, Shelli F. Farhadian, Charles Dela Cruz, Arnau Casanovas-Massana, John Fournier, Catherine Muenker, Anne L. Wyllie, Chantal B.F. Vogels, Chaney C. Kalinich, Mary E. Petrone, Isabel M. Ott, Anne E. Watkins, Adam J. Moore, Tara Alpert, Yuval Kluger, Aaron Ring, Nathan D. Grubaugh, Akiko Iwasaki, Albert I. Ko, Roy S. Herbst. Cancer patients display diminished viral RNA clearance and altered T cell responses during SARS-CoV-2 infection [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr S03-03.

Single-cell analysis of arthritogenic alphavirus-infected human synovial fibroblasts links low abundance of viral RNA to induction of innate immunity and arthralgia-associated gene expression

ABSTRACT Infection by (re-)emerging RNA arboviruses including Chikungunya virus (CHIKV) and Mayaro virus primarily cause acute febrile disease and transient polyarthralgia. However, in a significant subset of infected individuals, debilitating arthralgia persists for weeks over months up to years. The underlying immunopathogenesis of chronification of arthralgia upon primary RNA-viral infection remains unclear. Here, we analysed cell-intrinsic responses to ex vivo arthritogenic alphaviral infection of primary human synovial fibroblasts isolated from knee joints, one the most affected joint types during acute and chronic CHIKV disease. Synovial fibroblasts were susceptible and permissive to alphaviral infection. Base-line and exogenously added type I interferon (IFN) partially and potently restricted infection, respectively. RNA-seq revealed a CHIKV infection-induced transcriptional profile that comprised upregulation of expression of several hundred IFN-stimulated and arthralgia-mediating genes. Single-cell virus-inclusive RNA-seq uncovered a fine-tuned switch from induction to repression of cell-intrinsic immune responses depending on the abundance of viral RNA in an individual cell. Specifically, responses were most pronounced in cells displaying low-to-intermediate amounts of viral RNA and absence of virus-encoded, fluorescent reporter protein expression, arguing for efficient counteraction of innate immunity in cells expressing viral antagonists at sufficient quantities. In summary, cell-intrinsic sensing of viral RNA that potentially persists or replicates at low levels in synovial fibroblasts and other target cell types in vivo may contribute to the chronic arthralgia induced by alphaviral infections. Our findings might advance our understanding of the immunopathophysiology of long-term pathogenesis of RNA-viral infections.

Sensitive detection and quantification of SARS-CoV-2 by multiplex droplet digital RT-PCR

The purpose of this study is to develop a one-step droplet digital RT-PCR (RT-ddPCR) multiplex assay that allows for sensitive quantification of SARS-CoV-2 RNA with respect to human-derived RNA and could be used for screening and monitoring of Covid-19 patients. A one-step RT-ddPCR multiplex assay was developed for simultaneous detection of SARS-CoV-2 E, RdRp and N viral RNA, and human Rpp30 DNA and GUSB mRNA, for internal nucleic acid (NA) extraction and RT-PCR control. Dilution series of viral RNA transcripts were prepared in water and total NA extract of Covid-19-negative patients. As reference assay, an E-GUSB duplex RT-PCR was used. GUSB mRNA detection was used to set validity criteria to assure viral RNA and RT-PCR assay quality and to enable quantification of SARS-CoV-2 RNA. In a background of at least 100 GUSB mRNA copies, 5 copies of viral RNA are reliably detectable and 10 copies viral RNA copies are reliably quantifiable. It was found that assay sensitivity of the RT-ddPCR was not affected by the total NA background while assay sensitivity of the gold standard RT-PCR assay is drastically decreased when SARS-CoV-2 copies were detected in a background of total NA extract compared with water. The present study describes a robust and sensitive one-step ddRT-PCR multiplex assay for reliable quantification of SARS-CoV-2 RNA. By determining the fractional abundance of viral RNA with respect to a human housekeeping gene, viral loads from different samples can be compared, what could be used to investigate the infectiveness and to monitor Covid-19 patients.

Challenges of Studying the Human Virome - Relevant Emerging Technologies.

In this review we provide an overview of current challenges and advances in bacteriophage research within the growing field of viromics. In particular, we discuss, from a human virome study perspective, the current and emerging technologies available, their limitations in terms of de novo discoveries, and possible solutions to overcome present experimental and computational biases associated with low abundance of viral DNA or RNA. We summarize recent breakthroughs in metagenomics assembling tools and single-cell analysis, which have the potential to increase our understanding of phage biology, diversity, and interactions with both the microbial community and the human body. We expect that these recent and future advances in the field of viromics will have a strong impact on how we develop phage-based therapeutic approaches.

Zika Virus Infects Primary Human Glial Cells and Results in Neuroinflammation and Cell Death

Abstract Zika virus (ZIKV) infection in humans has been linked to serious birth defects, including abnormalities of the brain and eyes. Here, we used two lines of primary human glial cells from the brain and found both are susceptible to ZIKV infection. Viral RNA abundance increased from 6 to 36 h post-infection (hpi). Both cells showed cytopathic effects with cell shedding, suspension and death at 48 hpi. We found increased mRNA levels of IL-6, IL-1b, CXCL10 (IP-10), IL-8 and CCL2 in ZIKV-infected microglial cells, as well as those of TNF-a in astrocytes. Elevated levels of CXCL10 protein were also observed in microglial cells at 48 hpi. These results suggested that glial cells may directly contribute to neuroinflammation by releasing pro-inflammatory cytokines. ZIKV infection can also activate ISGs, as evidenced by increased mRNA levels of IFN-b, OAS1, Mx1 and IFITM1 in infected microglia at 48 hpi. In addition, ZIKV can induce levels of caspase-1 and caspase-3 expression in microglia and astrocytes, respectively. These findings suggest that activation of glial cells contributes to neuroinflammation and induces distinct cell death pathways in different glial cells. Finally, we examined the antiviral effect of azithromycin in infected glial cells and found a dose-dependent effect (0, 10 and 20 uM) on viral reduction. Azithromycin treatment significantly decreased gene expression of TNF-a. Moreover, the antiviral effect of azithromycin was confirmed in mouse models of ZIKV infection. Collectively, our study demonstrated that ZIKV can infect primary human glial cells, leading to inflammatory responses and cell death. Azithromycin treatment inhibits viral load in glial cells and alleviate neuroinflammation during ZIKV infection.

West Nile Virus-Inclusive Single-Cell RNA Sequencing Reveals Heterogeneity in the Type I Interferon Response within Single Cells.

West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus of global importance. Neuroinvasive WNV infection results in encephalitis and can lead to prolonged neurological impairment or death. Type I interferon (IFN-I) is crucial for promoting antiviral defenses through the induction of antiviral effectors, which function to restrict viral replication and spread. However, our understanding of the antiviral response to WNV infection is mostly derived from analysis of bulk cell populations. It is becoming increasingly apparent that substantial heterogeneity in cellular processes exists among individual cells, even within a seemingly homogenous cell population. Here, we present WNV-inclusive single-cell RNA sequencing (scRNA-seq), an approach to examine the transcriptional variation and viral RNA burden across single cells. We observed that only a few cells within the bulk population displayed robust transcription of IFN-β mRNA, and this did not appear to depend on viral RNA abundance within the same cell. Furthermore, we observed considerable transcriptional heterogeneity in the IFN-I response, with genes displaying high unimodal and bimodal expression patterns. Broadly, IFN-stimulated genes negatively correlated with viral RNA abundance, corresponding with a precipitous decline in expression in cells with high viral RNA levels. Altogether, we demonstrated the feasibility and utility of WNV-inclusive scRNA-seq as a high-throughput technique for single-cell transcriptomics and WNV RNA detection. This approach can be implemented in other models to provide insights into the cellular features of protective immunity and identify novel therapeutic targets.IMPORTANCE West Nile virus (WNV) is a clinically relevant pathogen responsible for recurrent epidemics of neuroinvasive disease. Type I interferon is essential for promoting an antiviral response against WNV infection; however, it is unclear how heterogeneity in the antiviral response at the single-cell level impacts viral control. Specifically, conventional approaches lack the ability to distinguish differences across cells with varying viral abundance. The significance of our research is to demonstrate a new technique for studying WNV infection at the single-cell level. We discovered extensive variation in antiviral gene expression and viral abundance across cells. This protocol can be applied to primary cells or in vivo models to better understand the underlying cellular heterogeneity following WNV infection for the development of targeted therapeutic strategies.

Differential and convergent utilization of autophagy components by positive-strand RNA viruses.

Many viruses interface with the autophagy pathway, a highly conserved process for recycling cellular components. For three viral infections in which autophagy constituents are proviral (poliovirus, dengue, and Zika), we developed a panel of knockouts (KOs) of autophagy-related genes to test which components of the canonical pathway are utilized. We discovered that each virus uses a distinct set of initiation components; however, all three viruses utilize autophagy-related gene 9 (ATG9), a lipid scavenging protein, and LC3 (light-chain 3), which is involved in membrane curvature. These results show that viruses use noncanonical routes for membrane sculpting and LC3 recruitment. By measuring viral RNA abundance, we also found that poliovirus utilizes these autophagy components for intracellular growth, while dengue and Zika virus only use autophagy components for post-RNA replication processes. Comparing how RNA viruses manipulate the autophagy pathway reveals new noncanonical autophagy routes, explains the exacerbation of disease by starvation, and uncovers common targets for antiviral drugs.