Quantitative measurement of infectious virus in SARS-CoV-2 Alpha, Delta and Epsilon variants reveals higher infectivity (viral titer:RNA ratio) in clinical samples containing the Delta and Epsilon variants
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.
SIGNIFICANCE STATEMENT
Current and future SARS-CoV-2 variants threaten our ability to control the COVID-19 pandemic. Variants with increased transmission, higher viral loads, or greater immune evasion are of particular concern. Viral loads are currently measured by the amount of viral RNA in a clinical sample rather than the amount of infectious virus. We measured both RNA and infectious virus levels directly in a set of 165 clinical specimens from Alpha, Epsilon or Delta variants. Our data shows that Delta is more infectious compared to Alpha, with ∼ six times as much infectious virus for the same amount of RNA. This increase in infectivity suggests increased measures (vaccination, masking, distancing, ventilation) are needed to control Delta compared to Alpha.
Article activity feed
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Our take
In this preprint study which has not yet been peer-reviewed, researchers found that clinical specimens from persons infected with the Delta and Episilon variant had higher infectious viral titers (i.e., amount of replication competent virus) and infectivity (amount of replication competent virus per viral RNA copy) than clinical specimens from persons infected with the Alpha variant. This study provides biological rationale for increased transmission and spread of the Delta variant as compared to other SARS-CoV-2 variants. While this study used rigorous laboratory methods, it was limited by a lack of epidemiological data on specimens, which limits our understanding of how important factors like age or vaccination status may also impact viral RNA levels and infectious viral titer.
Study design
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Our take
In this preprint study which has not yet been peer-reviewed, researchers found that clinical specimens from persons infected with the Delta and Episilon variant had higher infectious viral titers (i.e., amount of replication competent virus) and infectivity (amount of replication competent virus per viral RNA copy) than clinical specimens from persons infected with the Alpha variant. This study provides biological rationale for increased transmission and spread of the Delta variant as compared to other SARS-CoV-2 variants. While this study used rigorous laboratory methods, it was limited by a lack of epidemiological data on specimens, which limits our understanding of how important factors like age or vaccination status may also impact viral RNA levels and infectious viral titer.
Study design
cross-sectional
Study population and setting
This cross-sectional study used 165 clinical specimens (e.g., nasal swabs) identified to be positive for SARS-CoV-2 at the University of Washington Virology Laboratory on March 25, 2021, August 3, 2021, or August 25, 2021. Aliquots of all included specimens were frozen within two days of collection. RT-PCR was used to measure total and subgenomic E RNA levels of SARS-CoV-2. A micro-focus forming assay, which involves growing live virus, was used to measure infectious viral titers of SARS-CoV-2.
Summary of main findings
For each variant, the amount of total and E subgenomic viral RNA levels (i.e., cycle threshold values) were positively correlated with the amount of replication competent virus. Compared to the Alpha variant, the Epsilon and Delta variants had a greater number of infectious units per quantity of total RNA (i.e., infectivity) and subgenomic E RNA.
Study strengths
The study measured infectious viral titers and total and subgenomic viral RNA levels, and compared these values across variants.
Limitations
The major limitation of the study is the lack of clinical and epidemiological data that may impact viral titers (e.g., age, time since symptom onset and vaccination status). Additionally, since not all variants were widely circulating at the same time in the U.S., the included samples were not collected from the same population, nor would sample characteristics that may impact viral titers and culturable virus likely be the same across time when the samples were collected (ex: vaccination status).
Value added
This novel investigation using a micro-focus forming assay demonstrated that the Delta and Epsilon variants of SARS-CoV-2 may be more biologically infectious than the Alpha variant. This information can be useful in understanding how interventions such as masking, ventilation, etc. may need to be increased in response to increased infectivity of specific variants.
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SciScore for 10.1101/2021.09.07.21263229: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The use of deidentified positive specimens for the above studies was approved by the University of Washington Institutional Review Board (STUDY00010205) Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cells were permeabilized with 0.1% 100X Triton in 1× PBS for 15 min and then incubated with a primary, cross-reactive rabbit anti-SARS-CoV N monoclonal antibody (Sinobiological, distributed by Thermo Fisher, Cat. #40143-R001 at a dilution of 1:20,000) followed by a peroxidase-labelled goat anti-rabbit antibody (SeraCare, Milford, MA, USA, … SciScore for 10.1101/2021.09.07.21263229: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: The use of deidentified positive specimens for the above studies was approved by the University of Washington Institutional Review Board (STUDY00010205) Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cells were permeabilized with 0.1% 100X Triton in 1× PBS for 15 min and then incubated with a primary, cross-reactive rabbit anti-SARS-CoV N monoclonal antibody (Sinobiological, distributed by Thermo Fisher, Cat. #40143-R001 at a dilution of 1:20,000) followed by a peroxidase-labelled goat anti-rabbit antibody (SeraCare, Milford, MA, USA, Cat. #5220-0336 diluted to 1:4,000) and then the peroxidase substrate (SeraCare, Cat. #5510-0030). anti-SARS-CoV Nsuggested: Noneanti-rabbitsuggested: (SeraCare KPL Cat# 5220-0336, RRID:AB_2857917)Experimental Models: Cell Lines Sentences Resources Serial ten-fold dilutions of clinical sample were used to inoculate Vero E6-TMPRSS2 cell monolayers (60,000 cells/well seeded one day prior) in 96□well white polystyrene microplates (Falcon, Cat. Vero E6-TMPRSS2suggested: NoneRecombinant DNA Sentences Resources RT-PCR reactions used one of two sets of primers/probes: 1) WHO-E, using the E_Sarbeco-F/R/P set34; 2) Mills-sgE, using sgLeader-F2, sgE-R, and sgE-P23. sgE-P23suggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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