Respiratory viral infections (RVIs) cause substantial morbidity in pediatric transplant recipients. Previous efforts to establish the epidemiology of RVIs are mainly retrospective, with limited evaluation of association between pre-transplant screening of respiratory viruses and future RVIs. The VIPER study is a prospective, multicenter cohort study investigating the epidemiology and impact of RVIs in these patients.Figure 1Clinical Presentation of Respiratory Viral Infections Post Transplant Patients undergoing Hematopoietic Cell Transplantation (HCT) and Solid Organ Transplant (SOT) at 26 US institutions were enrolled at transplant and monitored for RVIs for a year. Nasal swabs (NS) were collected at baseline and first clinical RVI, and clinical data extracted from medical records. Baseline NS were tested for viruses using standard real time PCR methods (OpenArray, ThermoFischer Scientific) and compared with clinical PCR results at first RVI. We summarize the preliminary clinical data and analyze the association between baseline testing and the first RVI with univariate logistic regression. The study is conducted through the Pediatric Infectious Diseases Transplant Network (PIDTRAN).Figure 2Respiratory Viruses Detected at Baseline and at First Respiratory Viral Infection Episode 157 HCT and 174 SOT recipients were included. Demographic and transplant characteristics are described in Table 1. HCT patients had more RVIs vs SOT (77% vs 69%). Median time from transplant to first RVI was 99 days for HCT and 112 days for SOT. Upper respiratory tract infections (URTI) were common in both groups (Figure 1) but more SOT than HCT recipients progressed to lower respiratory tract infection (LRTI). Rhinovirus/enterovirus (RhV/EV) were the most frequent viruses in HCT and SOT, both at baseline and first RVI (Figure 2). Among LRTI, the most frequent viruses were RhV/EV (48%), Adenovirus (16%) and RSV (13%). Only baseline RhV/EV predicted RhV/EV at first RVI (OR:2.9, CI:1.2-6.9, p=0.02). Many transplant recipients carry respiratory viruses at the time of transplant. Most post- transplant RVIs were URTI. RhV/EV were the most frequent virus detected overall, including in LRTI. RhV/EV at baseline predicted subsequent RhV/EV RVI. No other virus demonstrated this association. Analysis is limited by the current sample size, but the continuing accrual will allow for a more robust analysis. Lara A. Danziger-Isakov, MD, MPH, AiCuris.: Grant/Research Support|Ansun BioPharma: Grant/Research Support|Astellas Pharma Global Development, Inc: Advisor/Consultant|Astellas Pharma Global Development, Inc: Grant/Research Support|kamada: Advisor/Consultant|Merck Sharp & Dohme Corporation: Grant/Research Support|Pfizer (Any division): Grant/Research Support|Takeda Pharmaceuticals: Grant/Research Support

BACKGROUND:Human rhinoviruses (RVs) primarily cause the common cold, but infection outcomes vary from subclinical to severe cases, including asthma exacerbations and fatal pneumonia in individuals who are immunocompromised. To date, therapeutic strategies have been hindered by the high diversity of serotypes. Global surveillance efforts have traditionally focused on sequencing VP1 or VP2/VP4 genetic regions, leaving gaps in our understanding of RV genomic diversity. METHODS:We sequenced 1078 RV genomes from nasal swabs of symptomatic and asymptomatic individuals to explore viral evolution during 2 epidemiologically distinct periods in Washington State: when the COVID-19 pandemic affected the circulation of other seasonal respiratory viruses except for RV (February-July 2021) and when the seasonal viruses reemerged with the severe outbreak of respiratory syncytial virus and influenza (November-December 2022). We constructed maximum likelihood and BEAST phylodynamic trees to characterize intragenotype evolution. RESULTS:We detected 99 of 168 known genotypes and observed intergenotypic recombination and genotype cluster swapping from 2021 to 2022. We found a significant association between the presence of symptoms and viral load but not with RV species or genotype. Phylodynamic trees, polyprotein selection pressure, and Shannon entropy revealed cocirculation of divergent clades within genotypes with high amino acid constraints throughout the polyprotein. CONCLUSIONS:Our study underscores the dynamic nature of RV genomic epidemiology within a localized geographic region, as >20% of existing genotypes within each RV species cocirculated each studied month. Our findings also emphasize the importance of investigating correlations between RV genotypes and serotypes to understand long-term immunity and cross-protection.

BACKGROUND:Although antivirals remain important for the treatment COVID-19, methods to assess treatment efficacy are lacking. Here, we investigated the impact of remdesivir on viral dynamics and their contribution to understanding antiviral efficacy in the multicenter Adaptive COVID-19 Treatment Trial 1, which randomized patients to remdesivir or placebo. METHODS:Longitudinal specimens collected during hospitalization from a substudy of 642 patients with COVID-19 were measured for viral RNA (upper respiratory tract and plasma), viral nucleocapsid antigen (serum), and host immunologic markers. Associations with clinical outcomes and response to therapy were assessed. RESULTS:Higher baseline plasma viral loads were associated with poorer clinical outcomes, and decreases in viral RNA and antigen in blood but not the upper respiratory tract correlated with enhanced benefit from remdesivir. The treatment effect of remdesivir was most pronounced in patients with elevated baseline nucleocapsid antigen levels: the recovery rate ratio was 1.95 (95% CI, 1.40-2.71) for levels >245 pg/mL vs 1.04 (95% CI, .76-1.42) for levels <245 pg/mL. Remdesivir also accelerated the rate of viral RNA and antigen clearance in blood, and patients whose blood levels decreased were more likely to recover and survive. CONCLUSIONS:Reductions in SARS-CoV-2 RNA and antigen levels in blood correlated with clinical benefit from antiviral therapy. CLINICAL TRIAL REGISTRATION:NCT04280705 (ClinicalTrials.gov).

ABSTRACT Filoviruses are some of the most lethal viruses in the modern world, and increasing numbers of filovirus species and genera have been discovered in recent years. Despite the potential severity of filovirus outbreaks in the human population, comparably few sensitive pan-filovirus RT-PCR assays have been described that might facilitate early detection and prevention. Here, we present a new pan-filovirus RT-PCR assay targeting the L polymerase gene for detection of all known mammalian filoviruses. We demonstrate the detection of 10 synthetic filovirus RNA templates with analytical sensitivity ranging from 178 to 3,354 copies/mL, without cross-reactivity on 10 non-filoviral human viral species. We verified assay performance on 10 inactivated filovirus isolates, yielding initial sensitivities of 0.012–44.17 TCID50/mL. We coupled this broadly reactive RT-PCR with a deep sequencing workflow that is amenable to high-throughput pooling to maximize detection and discovery potential. In summary, this pan-filovirus RT-PCR assay targets the most conserved filovirus gene, offers the widest breadth of coverage to date, and may help in the detection and discovery of novel filoviruses.IMPORTANCEFiloviruses remain some of the most mysterious viruses known to the world, with extremely high lethality rates and significant pandemic potential. Yet comparably few filovirus species and genera have been discovered to date and questions surround the definitive host species for zoonotic infections. Here, we describe a novel broadly reactive RT-PCR assay targeting the conserved L polymerase gene for high-throughput screening for filoviruses in a variety of clinical and environmental specimens. We demonstrate the assay can detect all known mammalian filoviruses and determine the sensitivity and specificity of the assay on synthetic RNA sequences, inactivated filovirus isolates, and non-filoviral species.

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

BACKGROUND:In spring of 2022, an outbreak of monkeypox (mpox) spread worldwide. Here, we describe performance characteristics of monkeypox virus (MPXV)-specific and pan-orthopoxvirus qPCR assays for clinical use.METHODS:We validated probe-based qPCR assays targeting MPXV-specific loci F3L and G2R (genes MPXVgp052/OPG065 and MPXVgp002 and gp190/OPG002, respectively) and a pan-orthopoxvirus assay targeting the E9L locus (MPXVgp057/OPG071). Clinical samples and synthetic controls were extracted using the Roche MP96 or Promega Maxwell 48 instrument. qPCR was performed on the AB7500 thermocycler. Synthetic control DNA and high concentration clinical samples were quantified by droplet PCR. Cross-reactivity was evaluated for camelpox and cowpox genomic DNA, vaccinia culture supernatant, and HSV- and VZV-positive clinical specimens. We also tested the performance of the F3L assay using dry swabs, Aptima vaginal and rectal swabs, nasopharyngeal, rectal, and oral swabs, cerebrospinal fluid, plasma, serum, whole blood, breastmilk, urine, saliva, and semen.RESULTS:The MPXV-F3L assay is reproducible at a limit of detection (LoD) of 65.6 copies/mL of viral DNA in viral transport medium/universal transport medium (VTM/UTM), or 3.3 copies/PCR reaction. No cross-reactivity with herpesviruses or other poxviruses was observed. MPXV-F3L detects MPXV DNA in alternative specimen types, with an LoD ranging between 260-1000 copies/mL, or 5.7-10 copies/PCR reaction. In clinical swab VTM specimens, MPXV-F3L and MPXV-G2R assays outperformed OPXV-E9L by an average of 2.4 and 2.8 Cts, respectively. MPXV-G2R outperformed MPXV-F3L by 0.4 Cts, consistent with presence of two copies of G2R present in labile inverted terminal repeats (ITRs) of MPXV genome.CONCLUSIONS:MPXV is readily detected by qPCR using three clinically validated assays.

Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Surprisingly, we initially detected a number of N1 and/or N2 positive samples with high cycle threshold values, though after conducting environmental swabbing of the laboratory and verifying with a second independent primer set (WHO-E) and PCR without reverse transcriptase, we showed that these were false positives due to plasmid contamination from a construct expressing the N gene in the general laboratory environment. Our final results indicate that no sampled wildlife were positive for SARS-CoV-2 RNA, and highlight the importance of physically separate locations for the processing of samples for surveillance and experiments that require the use of plasmid DNA containing the target RNA sequence. These negative findings are surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American’s wildlife populations.

aDepartment of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, USA bVaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA cHelix, San Mateo, California, USA dThermo Fisher Scientific, South San Francisco, California, USA eRosalind Bio, San Diego, California, USA fNaviDx, LLC, Newport Beach, California, USA gPersonalized Science, LLC, South Burlington, Vermont, USA

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.

ABSTRACTAmplicon-based sequencing methods have been central in characterizing the diversity, transmission and evolution of SARS-CoV-2, but need to be rigorously assessed for clinical utility. Here, 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 a 95% limit of detection of ≥ 40.08 SARS-CoV-2 copies/PCR reaction. Breadth of genome recovery was evaluated across a range of Ct values (11.3 – 36.7, median 21.6). Out of 428 positive samples, 406 (94.9%) generated genomes with < 10% Ns, with a mean genome coverage of 13,545X ± SD 8,382X. No genomes were recovered from PCR-negative specimens (n = 30), or from specimens positive for non-SARS-CoV-2 respiratory viruses (n = 20). Compared to 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.

Background The circulation of respiratory viruses poses a significant health risk among those residing in congregate settings. Data are limited on seasonal human coronavirus (HCoV) infections in homeless shelter settings. Methods We analysed data from a clinical trial and SARS-CoV-2 surveillance study at 23 homeless shelter sites in King County, Washington between October 2019-May 2021. Eligible participants were shelter residents aged >= 3 months with acute respiratory illness. We collected enrolment data and nasal samples for respiratory virus testing using multiplex RT-PCR platform including HCoV. Beginning April 1, 2020, eligibility expanded to shelter residents and staff regardless of symptoms. HCoV species was determined by RT-PCR with speciesspecific primers, OpenArray assay or genomic sequencing for samples with an OpenArray relative cycle threshold <22. Findings Of the 14,464 samples from 3281 participants between October 2019-May 2021, 107 were positive for HCoV from 90 participants (median age 40 years, range: 0 cent 9-81 years, 38% female). HCoV-HKU1 was the most common species identified before and after community-wide mitigation. No HCoV-positive samples were identified between May 2020-December 2020. Adults aged =50 years had the highest detection of HCoV (11%) among viruspositive samples among all age-groups. Species and sequence data showed diversity between and within HCoV species over the study period. Interpretation HCoV infections occurred in all congregate homeless shelter site age-groups with the greatest proportion among those aged >= 50 years. Species and sequencing data highlight the complexity of HCoV epidemiology within and between shelters sites. Copyright (c) 2022 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

AbstractWhile detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by diagnostic reverse-transcription polymerase chain reaction (RT-PCR) is highly sensitive for viral RNA, the nucleic acid amplification of subgenomic RNAs (sgRNAs) that are the product of viral replication may more accurately identify replication. We characterized the diagnostic RNA and sgRNA detection by RT-PCR from nasal swab samples collected daily by participants in postexposure prophylaxis or treatment studies for SARS-CoV-2. Among 1932 RT-PCR–positive swab samples with sgRNA tests, 40% (767) had detectable sgRNA. Above a diagnostic RNA viral load (VL) threshold of 5.1 log10 copies/mL, 96% of samples had detectable sgRNA with VLs that followed a linear trend. The trajectories of diagnostic RNA and sgRNA VLs differed, with 80% peaking on the same day but duration of sgRNA detection being shorter (8 vs 14 days). With a large sample of daily swab samples we provide comparative sgRNA kinetics and a diagnostic RNA threshold that correlates with replicating virus independent of symptoms or duration of illness.

Monkeypox virus, the causative agent of the 2022 monkeypox outbreak, is a double-stranded DNA virus in the Orthopoxvirus genus of the Poxviridae family. Genes in terminal regions of Orthopoxvirus genomes mostly code for host-pathogen interaction proteins and are prone to selective pressure and modification events. Using viral whole genome sequencing, we identified twenty-five total clinical samples with ORF-disrupting mutations, including twenty samples encoding nonsense mutations in MPXVgp001/191 (OPG001), MPXVgp004/188 (OPG015), MPXVgp010 (OPG023), MPXVgp030 (OPG042), MPXVgp159 (OPG0178), or MPXVgp161 (OPG181). Additional mutations include a frameshift leading to an alternative C-terminus in MPXVgp010 (OPG023) and an insertion in an adenine homopolymer at the beginning of the annotated ORF for MPXVgp153 (OPG151), encoding a subunit of the RNA polymerase, suggesting the virus may instead use the start codon that encodes Met9 as annotated. Finally, we detected three samples with large (>900 bp) deletions. These included a 913 bp deletion that truncates the C-terminus of MPXVgp010 (OPG023); a 4205 bp deletion that eliminates MPXVgp012 (OPG025), MPXVgp013 (OPG027), and MPXVgp014 (OPG029) and truncates MPXVgp011 (OPG024; D8L) and MPXVgp015 (OPG030); and a 6881 bp deletion that truncates MPXVgp182 (OPG210) and eliminates putative ORFs MPXVgp184, MPXVgp185 (OPG005), and MPXVgp186, as well as MPXVgp187 (OPG016), and MPXVgp188 (OPG015) from the 3' ITR only. MPXVgp182 encodes the monkeypox-specific, highly immunogenic surface glycoprotein B21R which has been proposed as a serological target. Overall, we find greater than one-tenth of our sequenced MPXV isolates have at least one gene inactivating mutation and these genes together comprised greater than one-tenth of annotated MPXV genes. Our findings highlight non-essential genes in monkeypox virus that may be evolving as a result of selective pressure in humans, as well as the limitations of targeting them for therapeutics and diagnostic testing.

Novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between levels of viral RNA and infectious virus for individual variants is unknown. We measured infectious viral titer (using a microfocus-forming assay) and total and subgenomic viral RNA levels (using RT-PCR) in a set of 162 clinical samples containing SARS-CoV-2 Alpha, Delta, and Epsilon variants that were collected in identical swab kits from outpatient test sites and processed soon after collection. We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite this, the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (5.9- and 3.0-fold increase; P < 0.0001, P = 0.014, respectively) or subgenomic E RNA (14.3- and 6.9-fold increase; P < 0.0001, P = 0.004, respectively). In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity for Delta may further explain increased spread, suggesting a need for increased measures to prevent viral transmission.

To determine the epidemiology of human parainfluenza virus in homeless shelters during the COVID-19 pandemic, we analyzed data and sequences from respiratory specimens collected in 23 shelters in Washington, USA, during 2019-2021. Two clusters in children were genetically similar by shelter of origin. Shelter-specific interventions are needed to reduce these infections.

ABSTRACT Background Novel SARS-CoV-2 Variants of Concern (VoC) pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between viral RNA and infectious virus for individual variants is unknown. Methods We measured infectious viral titer (using a micro-focus forming assay) as well as total and subgenomic viral RNA levels (using RT-PCR) in a set of 165 clinical samples containing SARS-CoV-2 Alpha, Delta and Epsilon variants that were processed within two days of collection from the patient. Results We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite the variability we observed for individual samples the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (6 and 4 times as much, p=0.0002 and 0.009 respectively) or subgenomic E RNA (11 and 7 times as much, p<0.0001 and 0.006 respectively). Conclusion In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may also be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity of the Delta variant may further explain increased spread and suggests a need for increased measures to prevent viral transmission.

More than one year into a global pandemic, SARS-CoV-2 is now defined by a variety of rapidly evolving variant lineages. Several FDA authorized molecular diagnostic tests have been impacted by viral variation, while no reports of viral variation affecting antigen test performance have occurred to date. While determining the analytical sensitivity of the Quidel Sofia SARS Antigen FIA test (Sofia 2), we uncovered a high viral load specimen that repeatedly tested negative by this antigen test. Whole genome sequencing of the specimen uncovered two mutations, T205I and D399N, present in the nucleocapsid protein of the isolate. All six SARS-CoV-2 positive clinical specimens available in our laboratory with a D399N nucleocapsid mutation and CT < 31 were not detected by the Sofia 2 but detected by the Abbott BinaxNOW COVID-19 Ag Card, while clinical specimens with the T205I mutation were detected by both assays. Testing of recombinant SARS-CoV-2 nucleocapsid with these variants demonstrated an approximate 1000-fold loss in sensitivity for the Quidel Sofia SARS Antigen FIA test associated with the D399N mutation, while the BinaxNOW and Quidel Quickvue SARS Antigen tests were unaffected by the mutation. The D399N nucleocapsid mutation has been relatively uncommon to date, appearing in only 0.02% of genomes worldwide at time of writing. Our results demonstrate how routine pathogen genomics can be integrated into the clinical microbiology laboratory to investigate diagnostic edge cases, as well as the importance of profiling antigenic diversity outside of the spike protein for SARS-CoV-2 diagnostics.

Multiple rapid antigen (Ag) tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have recently received emergency-use authorization (EUA) from the U.S. Food and Drug Administration (FDA).

ABSTRACTWith 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 12.3-37.5). 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 cut-off of >31 or specifically testing for sgRNA can serve as an effective rule-out test for viral infectivity.