SARS-CoV-2 Disrupts Splicing, Translation, and Protein Trafficking to Suppress Host Defenses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently identified coronavirus that causes the respiratory disease known as coronavirus disease 2019 (COVID-19). Despite the urgent need, we still do not fully understand the molecular basis of SARS-CoV-2 pathogenesis. Here, we comprehensively define the interactions between SARS-CoV-2 proteins and human RNAs. NSP16 binds to the mRNA recognition domains of the U1 and U2 splicing RNAs and acts to suppress global mRNA splicing upon SARS-CoV-2 infection. NSP1 binds to 18S ribosomal RNA in the mRNA entry channel of the ribosome and leads to global inhibition of mRNA translation upon infection. Finally, NSP8 and NSP9 bind to the 7SL RNA in the signal recognition particle and interfere with protein trafficking to the cell membrane upon infection. Disruption of each of these essential cellular functions acts to suppress the interferon response to viral infection. Our results uncover a multipronged strategy utilized by SARS-CoV-2 to antagonize essential cellular processes to suppress host defenses.

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step.

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.

SARS-CoV-2 triggers DNA damage response in Vero E6 cells.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus responsible for the current COVID-19 pandemic and has now infected more than 200 million people with more than 4 million deaths globally. Recent data suggest that symptoms and general malaise may continue long after the infection has ended in recovered patients, suggesting that SARS-CoV-2 infection has profound consequences in the host cells. Here we report that SARS-CoV-2 infection can trigger a DNA damage response (DDR) in African green monkey kidney cells (Vero E6). We observed a transcriptional upregulation of the Ataxia telangiectasia and Rad3 related protein (ATR) in infected cells. In addition, we observed enhanced phosphorylation of CHK1, a downstream effector of the ATR DNA damage response, as well as H2AX. Strikingly, SARS-CoV-2 infection lowered the expression of TRF2 shelterin-protein complex, and reduced telomere lengths in infected Vero E6 cells. Thus, our observations suggest SARS-CoV-2 may have pathological consequences to host cells beyond evoking an immunopathogenic immune response.

NEDD4 family ubiquitin ligases associate with LCMV Z's PPXY domain and are required for virus budding, but not via direct ubiquitination of Z.

Viral late domains are used by many viruses to recruit the cellular endosomal sorting complex required for transport (ESCRT) to mediate membrane scission during viral budding. Unlike the P(S/T)AP and YPX(1-3)L late domains, which interact directly with the ESCRT proteins Tsg101 and ALIX, the molecular linkage connecting the PPXY late domain to ESCRT proteins is unclear. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) matrix protein, Z, contains only one late domain, PPXY. We previously found that this domain in LCMV Z, as well as the ESCRT pathway, are required for the release of defective interfering (DI) particles but not infectious virus. To better understand the molecular mechanism of ESCRT recruitment by the PPXY late domain, affinity purification-mass spectrometry was used to identify host proteins that interact with the Z proteins of the Old World mammarenaviruses LCMV and Lassa virus. Several Nedd4 family E3 ubiquitin ligases interact with these matrix proteins and in the case of LCMV Z, the interaction was PPXY-dependent. We demonstrated that these ligases directly ubiquitinate LCMV Z and mapped the specific lysine residues modified. A recombinant LCMV containing a Z that cannot be ubiquitinated maintained its ability to produce both infectious virus and DI particles, suggesting that direct ubiquitination of LCMV Z alone is insufficient for recruiting ESCRT proteins to mediate virus release. However, Nedd4 ligases appear to be important for DI particle release suggesting that ubiquitination of targets other than the Z protein itself is required for efficient viral ESCRT recruitment.

Bacterial Lipoproteins Constitute the TLR2-Stimulating Activity of Serum Amyloid A.

Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these proteins. In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in Escherichia coli, hSAA1 produced from eukaryotic cells did not promote proinflammatory cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of E. coli-derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins. Treatment of hSAA1 with lipoprotein lipase or addition of a lipopeptide to eukaryotic cell-derived hSAA1 inhibited or induced the production of TNF-α from macrophages, respectively. Our results suggest that a function of SAA is in the binding of TLR2-stimulating bacterial proteins, including lipoproteins, and demand that future studies of SAA employ a recombinant protein derived from eukaryotic cells.

A Proteomics Survey of Junín Virus Interactions with Human Proteins Reveals Host Factors Required for Arenavirus Replication.

Arenaviruses are negative-strand, enveloped RNA viruses that cause significant human disease. In particular, Junín mammarenavirus (JUNV) is the etiologic agent of Argentine hemorrhagic fever. At present, little is known about the cellular proteins that the arenavirus matrix protein (Z) hijacks to accomplish its various functions, including driving the process of virus release. Furthermore, there is little knowledge regarding host proteins incorporated into arenavirus particles and their importance for virion function. To address these deficiencies, we used mass spectrometry to identify human proteins that (i) interact with the JUNV matrix protein inside cells or within virus-like particles (VLPs) and/or (ii) are incorporated into bona fide JUNV strain Candid#1 particles. Bioinformatics analyses revealed that multiple classes of human proteins were overrepresented in the data sets, including ribosomal proteins, Ras superfamily proteins, and endosomal sorting complex required for transport (ESCRT) proteins. Several of these proteins were required for the propagation of JUNV (ADP ribosylation factor 1 [ARF1], ATPase, H+ transporting, lysosomal 38-kDa, V0 subunit d1 [ATP6V0D1], and peroxiredoxin 3 [PRDX3]), lymphocytic choriomeningitis mammarenavirus (LCMV) (Rab5c), or both viruses (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide [ATP5B] and IMP dehydrogenase 2 [IMPDH2]). Furthermore, we show that the release of infectious JUNV particles, but not LCMV particles, requires a functional ESCRT pathway and that ATP5B and IMPDH2 are required for JUNV budding. In summary, we have provided a large-scale map of host machinery that associates with JUNV and identified key human proteins required for its propagation. This data set provides a resource for the field to guide antiviral target discovery and to better understand the biology of the arenavirus matrix protein and the importance of host proteins for virion function.IMPORTANCE Arenaviruses are deadly human pathogens for which there are no U.S. Food and Drug Administration-approved vaccines and only limited treatment options. Little is known about the host proteins that are incorporated into arenavirus particles or that associate with its multifunctional matrix protein. Using Junín mammarenavirus (JUNV), the causative agent of Argentine hemorrhagic fever, as a model organism, we mapped the human proteins that are incorporated into JUNV particles or that associate with the JUNV matrix protein. Functional analysis revealed host machinery that is required for JUNV propagation, including the cellular ESCRT pathway. This study improves our understanding of critical arenavirus-host interactions and provides a data set that will guide future studies to better understand arenavirus pathogenesis and identify novel host proteins that can be therapeutically targeted.

Visualization of Arenavirus RNA Species in Individual Cells by Single-Molecule Fluorescence In Situ Hybridization Suggests a Model of Cyclical Infection and Clearance during Persistence.

Lymphocytic choriomeningitis mammarenavirus (LCMV) is an enveloped, negative-strand RNA virus that causes serious disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. Different models have been proposed to describe how arenaviruses regulate the replication and transcription of their bisegmented, single-stranded RNA genomes, particularly during persistent infection. However, these models were based largely on viral RNA profiling data derived from entire populations of cells. To better understand LCMV replication and transcription at the single-cell level, we established a high-throughput, single-molecule fluorescence in situ hybridization (smFISH) image acquisition and analysis pipeline and examined viral RNA species at discrete time points from virus entry through the late stages of persistent infection in vitro We observed the transcription of viral nucleoprotein and polymerase mRNAs from the incoming S and L segment genomic RNAs, respectively, within 1 h of infection, whereas the transcription of glycoprotein mRNA from the S segment antigenome required ∼4 to 6 h. This confirms the temporal separation of viral gene expression expected due to the ambisense coding strategy of arenaviruses and also suggests that antigenomic RNA contained in virions is not transcriptionally active upon entry. Viral replication and transcription peaked at 36 h postinfection, followed by a progressive loss of viral RNAs over the next several days. During persistence, the majority of cells showed repeating cyclical waves of viral transcription and replication followed by the clearance of viral RNA. Thus, our data support a model of LCMV persistence whereby infected cells can spontaneously clear infection and become reinfected by viral reservoir cells that remain in the population.IMPORTANCE Arenaviruses are human pathogens that can establish asymptomatic, lifelong infections in their rodent reservoirs. Several models have been proposed to explain how arenavirus spread is restricted within host rodents, including the periodic accumulation and loss of replication-competent, but transcriptionally incompetent, viral genomes. A limitation of previous studies was the inability to enumerate viral RNA species at the single-cell level. We developed a high-throughput, smFISH assay and used it to quantitate lymphocytic choriomeningitis mammarenavirus (LCMV) replicative and transcriptional RNA species in individual cells at distinct time points following infection. Our findings support a model whereby productively infected cells can clear infection, including viral RNAs and antigen, and later be reinfected. This information improves our understanding of the timing and possible regulation of LCMV genome replication and transcription during infection. Importantly, the smFISH assay and data analysis pipeline developed here is easily adaptable to other RNA viruses.

The use of novel epitope-tagged arenaviruses reveals that Rab5c-positive endosomal membranes are targeted by the LCMV matrix protein.

We report the development of recombinant New World (Junín; JUNV) and Old World (lymphocytic choriomeningitis virus; LCMV) mammarenaviruses that encode an HA-tagged matrix protein (Z). These viruses permit the robust affinity purification of Z from infected cells or virions, as well as the detection of Z by immunofluorescent microscopy. Importantly, the HA-tagged viruses grow with wild-type kinetics in a multi-cycle growth assay. Using these viruses, we report a novel description of JUNV Z localization in infected cells, as well as the first description of colocalization between LCMV Z and the GTPase Rab5c. This latter result, when combined with our previous findings that LCMV genome and glycoprotein also colocalize with Rab5c, suggest that LCMV may target Rab5c-positive membranes for preassembly of virus particles prior to budding. The recombinant viruses reported here will provide the field with new tools to better study Z protein functionality and identify key Z protein interactions with host machinery.

A Map of the Arenavirus Nucleoprotein-Host Protein Interactome Reveals that Junín Virus Selectively Impairs the Antiviral Activity of Double-Stranded RNA-Activated Protein Kinase (PKR).

Arenaviruses are enveloped negative-strand RNA viruses that cause significant human disease. These viruses encode only four proteins to accomplish the viral life cycle, so each arenavirus protein likely plays unappreciated accessory roles during infection. Here we used immunoprecipitation and mass spectrometry to identify human proteins that interact with the nucleoproteins (NPs) of the Old World arenavirus lymphocytic choriomeningitis virus (LCMV) and the New World arenavirus Junín virus (JUNV) strain Candid #1. Bioinformatic analysis of the identified protein partners of NP revealed that host translation appears to be a key biological process engaged during infection. In particular, NP associates with the double-stranded RNA (dsRNA)-activated protein kinase (PKR), a well-characterized antiviral protein that inhibits cap-dependent protein translation initiation via phosphorylation of eIF2α. JUNV infection leads to increased expression of PKR as well as its redistribution to viral replication and transcription factories. Further, phosphorylation of PKR, which is a prerequisite for its ability to phosphorylate eIF2α, is readily induced by JUNV. However, JUNV prevents this pool of activated PKR from phosphorylating eIF2α, even following exposure to the synthetic dsRNA poly(I·C), a potent PKR agonist. This blockade of PKR function is highly specific, as LCMV is unable to similarly inhibit eIF2α phosphorylation. JUNV's ability to antagonize the antiviral activity of PKR appears to be complete, as silencing of PKR expression has no impact on viral propagation. In summary, we provide a detailed map of the host machinery engaged by arenavirus NPs and identify an antiviral pathway that is subverted by JUNV.IMPORTANCE Arenaviruses are important human pathogens for which FDA-approved vaccines do not exist and effective antiviral therapeutics are needed. Design of antiviral treatment options and elucidation of the mechanistic basis of disease pathogenesis will depend on an increased basic understanding of these viruses and, in particular, their interactions with the host cell machinery. Identifying host proteins critical for the viral life cycle and/or pathogenesis represents a useful strategy to uncover new drug targets. This study reveals, for the first time, the extensive human protein interactome of arenavirus nucleoproteins and uncovers a potent antiviral host protein that is neutralized during Junín virus infection. In so doing, it shows further insight into the interplay between the virus and the host innate immune response and provides an important data set for the field.

Patterns of Cellular Immunity Associated with Experimental Infection with rDEN2Δ30 (Tonga/74) Support Its Suitability as a Human Dengue Virus Challenge Strain.

A deletion variant of the dengue virus (DENV) serotype 2 (DENV2) Tonga/74 strain lacking 30 nucleotides from its 3' untranslated region (rDEN2Δ30) has previously been established for use in a controlled human DENV challenge model. To evaluate if this model is appropriate for the derivation of correlates of protection for DENV vaccines on the basis of cellular immunity, we wanted to compare the cellular immune response to this challenge strain to the response induced by natural infection. To achieve this, we predicted HLA class I- and class II-restricted peptides from rDEN2Δ30 and used them in a gamma interferon enzyme-linked immunosorbent spot assay to interrogate CD8+ and CD4+ T cell responses in healthy volunteers infected with rDEN2Δ30. At the level of CD8 responses, vigorous ex vivo responses were detected in approximately 80% of donors. These responses were similar in terms of the magnitude and the numbers of epitopes recognized to the responses previously observed in peripheral blood mononuclear cells from donors from regions where DENV is hyperendemic. The similarity extended to the immunodominance hierarchy of the DENV nonstructural proteins, with NS3, NS5, and NS1 being dominant in both donor cohorts. At the CD4 level, the responses to rDEN2Δ30 vaccination were less vigorous than those to natural DENV infection and were more focused on nonstructural proteins. The epitopes recognized following rDEN2Δ30 infection and natural infection were largely overlapping for both the CD8 (100%) and CD4 (85%) responses. Finally, rDEN2Δ30 induced stronger CD8 responses than other, more attenuated DENV isolates.IMPORTANCE The lack of a known correlate of protection and the failure of a neutralizing antibody to correlate with protection against dengue virus have highlighted the need for a human DENV challenge model to better evaluate the candidate live attenuated dengue vaccines. In this study, we sought to characterize the immune profiles of rDEN2Δ30-infected subjects and to compare the profiles with those for subjects from areas where DENV is hyperendemic. Our data demonstrate that T cell responses to rDENV2Δ30 are largely similar to those to natural infection in terms of specificity, highlighting that the response to this virus in humans is appropriate as a model for the T cell response to primary DENV2 infection.

The Lymphocytic Choriomeningitis Virus Matrix Protein PPXY Late Domain Drives the Production of Defective Interfering Particles.

Arenaviruses cause severe diseases in humans but establish asymptomatic, lifelong infections in rodent reservoirs. Persistently-infected rodents harbor high levels of defective interfering (DI) particles, which are thought to be important for establishing persistence and mitigating virus-induced cytopathic effect. Little is known about what drives the production of DI particles. We show that neither the PPXY late domain encoded within the lymphocytic choriomeningitis virus (LCMV) matrix protein nor a functional endosomal sorting complex transport (ESCRT) pathway is absolutely required for the generation of standard infectious virus particles. In contrast, DI particle release critically requires the PPXY late domain and is ESCRT-dependent. Additionally, the terminal tyrosine in the PPXY motif is reversibly phosphorylated and our findings indicate that this posttranslational modification may regulate DI particle formation. Thus we have uncovered a new role for the PPXY late domain and a possible mechanism for its regulation.

Visualization of the lymphocytic choriomeningitis mammarenavirus (LCMV) genome reveals the early endosome as a possible site for genome replication and viral particle pre-assembly.

We report a fluorescence in situ hybridization (FISH) assay that allows the visualization of lymphocytic choriomeningitis mammarenavirus (LCMV) genomic RNAs in individual cells. We show that viral S segment genomic and antigenomic RNA, along with viral nucleoprotein, colocalize in subcellular structures we presume to be viral replication factories. These viral RNA structures are highly dynamic during acute infection, with the many small foci seen early coalescing into larger perinuclear foci later in infection. These late-forming perinuclear viral RNA aggregates are located near the cellular microtubule organizing centre and colocalize with the early endosomal marker Rab5c and the viral glycoprotein in a proportion of infected cells. We propose that the virus is using the surface of a cellular membrane-bound organelle as a site for the pre-assembly of viral components, including genomic RNA and viral glycoprotein, prior to their transport to the plasma membrane, where new particles will bud.

A novel phosphoserine motif in the LCMV matrix protein Z regulates the release of infectious virus and defective interfering particles.

We report that the lymphocytic choriomeningitis virus (LCMV) matrix protein, which drives viral budding, is phosphorylated at serine 41 (S41). A recombinant (r)LCMV bearing a phosphomimetic mutation (S41D) was impaired in infectious and defective interfering (DI) particle release, while a non-phosphorylatable mutant (S41A) was not. The S41D mutant was disproportionately impaired in its ability to release DI particles relative to infectious particles. Thus, DI particle production by LCMV may be dynamically regulated via phosphorylation of S41.

CD8+ memory T cells appear exhausted within hours of acute virus infection.

CD8(+) memory T cells are abundant and are activated in a near-synchronous manner by infection, thereby providing a unique opportunity to evaluate the coordinate functional and phenotypic changes that occur in vivo within hours of viral challenge. Using two disparate virus challenges of mice, we show that splenic CD8(+) memory T cells rapidly produced IFN-γ in vivo; however, within 18-24 h, IFN-γ synthesis was terminated and remained undetectable for ≥ 48 h. A similar on/off response was observed in CD8(+) memory T cells in the peritoneal cavity. Cessation of IFN-γ production in vivo occurred despite the continued presence of immunostimulatory viral Ag, indicating that the initial IFN-γ response had been actively downregulated and that the cells had been rendered refractory to subsequent in vivo Ag contact. Downregulation of IFN-γ synthesis was accompanied by the upregulation of inhibitory receptor expression on the T cells, and ex vivo analyses using synthetic peptides revealed a concurrent hierarchical loss of cytokine responsiveness (IL-2, then TNF, then IFN-γ) taking place during the first 24 h following Ag contact. Thus, within hours of virus challenge, CD8(+) memory T cells display the standard hallmarks of T cell exhaustion, a phenotype that previously was associated only with chronic diseases and that is generally viewed as a gradually developing and pathological change in T cell function. Our data suggest that, instead, the "exhaustion" phenotype is a rapid and normal physiological T cell response.

SARS-CoV-2 Omicron: Viral Evolution, Immune Evasion, and Alternative Durable Therapeutic Strategies.

Since the SARS-CoV-2 Omicron virus has gained dominance worldwide, its continual evolution with unpredictable mutations and patterns has revoked all authorized immunotherapeutics. Rapid viral evolution has also necessitated several rounds of vaccine updates in order to provide adequate immune protection. It remains imperative to understand how Omicron evolves into different subvariants and causes immune escape as this could help reevaluate the current intervention strategies mostly implemented in the clinics as emergency measures to counter the pandemic and, importantly, develop new solutions. Here, we provide a review focusing on the major events of Omicron viral evolution, including the features of spike mutation that lead to immune evasion against monoclonal antibody (mAb) therapy and vaccination, and suggest alternative durable options such as the ACE2-based experimental therapies superior to mAbs to address this unprecedented evolution of Omicron virus. In addition, this type of unique ACE2-based virus-trapping molecules can counter all zoonotic SARS coronaviruses, either from unknown animal hosts or from established wild-life reservoirs of SARS-CoV-2, and even seasonal alpha coronavirus NL63 that depends on human ACE2 for infection.

An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV.

The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.

Predicting infectivity: comparing four PCR-based assays to detect culturable SARS-CoV-2 in clinical samples.

With the COVID-19 pandemic caused by SARS-CoV-2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT-PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g., subgenomic RNA [sgRNA]) and replicative intermediates (e.g., negative-strand RNA species). Using a novel primer/probe set for detection of subgenomic (sg)E transcripts, we successfully identified 100% of specimens containing culturable SARS-CoV-2 from a set of 126 clinical samples (total sgE CT values ranging from 12.3 to 37.5). This assay showed superior performance compared to a previously published sgRNA assay and to a negative-strand RNA assay, both of which failed to detect target RNA in a subset of samples from which we isolated live virus. In addition, total levels of viral RNA (genome, negative-strand, and sgE) detected with the WHO/Charité primer-probe set correlated closely with levels of infectious virus. Specifically, infectious virus was not detected in samples with a CT above 31.0. Clinical samples with higher levels of viral RNA also displayed cytopathic effect (CPE) more quickly than those with lower levels of viral RNA. Finally, we found that the infectivity of SARS-CoV-2 samples is significantly dependent on the cell type used for viral isolation, as Vero E6 cells expressing TMRPSS2 extended the analytical sensitivity of isolation by more than 3 CT compared to parental Vero E6 cells and resulted in faster isolation. Our work shows that using a total viral RNA Ct cutoff of > 31 or specifically testing for sgRNA can serve as an effective rule-out test for the presence of culturable virus.

Clinical Performance Characteristics of the Swift Normalase Amplicon Panel for Sensitive Recovery of Severe Acute Respiratory Syndrome Coronavirus 2 Genomes.

Amplicon-based sequencing methods are central in characterizing the diversity, transmission, and evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but need to be rigorously assessed for clinical utility. Herein, we validated the Swift Biosciences' SARS-CoV-2 Swift Normalase Amplicon Panels using remnant clinical specimens. High-quality genomes meeting our established library and sequence quality criteria were recovered from positive specimens, with 95% limit of detection of 40.08 SARS-CoV-2 copies/PCR. Breadth of genome recovery was evaluated across a range of CT values (11.3 to 36.7; median, 21.6). Of 428 positive samples, 413 (96.5%) generated genomes with <10% unknown bases, with a mean genome coverage of 13,545× ± SD 8382×. No genomes were recovered from PCR-negative specimens (n = 30) or from specimens positive for non-SARS-CoV-2 respiratory viruses (n = 20). Compared with whole-genome shotgun metagenomic sequencing (n = 14) or Sanger sequencing for the spike gene (n = 11), pairwise identity between consensus sequences was 100% in all cases, with highly concordant allele frequencies (R2 = 0.99) between Swift and shotgun libraries. When samples from different clades were mixed at varying ratios, expected variants were detected even in 1:99 mixtures. When deployed as a clinical test, 268 tests were performed in the first 23 weeks, with a median turnaround time of 11 days, ordered primarily for outbreak investigations and infection control.

An international, interlaboratory ring trial confirms the feasibility of an extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.