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

Abstract 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.

I salute the intention of the authors to try to fix cut-off values for infectious patients but I would be more careful on the assertion of "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". It is true that in this study, virus was not isolated from any of the samples below a Ct of 31 or negative in the developed sgE assay but all those assays are done on cell culture. We do not know how the transmission could occur for those samples from human to human. Being able to fix a cut-off in Ct value for a define PCR/RT-PCR system would be a great improvement for SARS-CoV-2 infected patient having to stay in quarantine. It is even more important for Ebola positive patients in Africa who has to stay in quarantine in precarious conditions under tents, warm temperatures and without privacy for long period because they still positive by RT-PCR. Unfortunately, fix those values would need a very high number of experiments, including animal experiment.
-Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? No

Significance (Required)
-Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
This new primer/probe system will participate to the accurate diagnostic of SARS-CoV-2. The comparison with the existing methods is relevant to highlight the strengths and weaknesses of each system. Comparison of isolation of SARS-CoV-2 on commonly used Vero E6 with Vero E6-TMPRSS2 will lead to a great improvement of the isolation method for SARS-CoV-2.
-Place the work in the context of the existing literature (provide references, where appropriate). Properly done in the introduction of the paper. Compared with other assays, the WHO total E RNA assay had better parameters when used at a cutoff Ct value of 31 (PPV of 61%). Overall, this manuscript provides a novel primer probe set for RT-PCR diagnostic assay and conducted comparisons with other assays on the same clinical samples. There are some areas that the authors should address prior to publication. **Major comments:** -The authors repeatedly tout VeroE6 TMRSS2 cells as supporting higher viral infection. Therefore, the authors should address why one clinical isolate (E16) was culturable in VeroE6 but not VeroE6 TMRSS2. Was this experiment repeated multiple times? What are the reasons for this discrepancy? -The authors' argument at lines 166-169 is not supported by the data in Fig. 2. The levels of viral RNA between VeroE6 and VeroE6 TMRSS2 appear to show similar trends in the supernatant across the time course but the infectious viral levels are dramatically different. This discordance between FFU levels and RNA levels cannot be explained by instability of viral particles alone. Have the authors looked into differences in viral particles produced from these two cell lines? The authors should collect virus particles from these two cell lines and conduct the stability experiment in Fig 2D to directly test the hypothesis that indeed the drop seen in FFU in VeroE6 TMRSS2 is due to instability.
-The evidence for the packaging of sgE RNA into virions is weak. GAPDH detection by PCR is not a proof that the concentration process did not pellet RNA nonspecifically. First, the authors should provide ample information about viral isolation process at line 379 including rotor, centrifuge and speed utilized. In addition, ribosomes typically stay intact following viral lysis (and can be found in supernatant after release from dead cells). Actively translating ribosomes can contain sgE RNA as well. The authors should consider detecting ribosomal RNAs in their samples to rule out the possibility of contaminating ribosomes. In addition, the authors should strongly consider repeating the experiment with high EDTA concentration to break up ribosomes and only pellet virions. **Minor comments:** -At line 197, the authors refer to "viruses" with lower levels of SARS-CoV2 RNA. This is incorrect and should be changed to "isolates" as the SARS-CoV2 virus particle does not package variable amount of genomic RNA.
-The authors statement on lines 210-212 does not seem to be supported clearly by -The authors should expand on the methodology for creating ROC curves at line 467.

Significance (Required)
This study is significant because it assesses the utility of several clinical assays for the measurement of viral RNA and correlating it with culturable virus. This is important in the field because it helps to identify methods whereby infectivity can be predicted from a simple diagnostic test. This is important to know as a virologist working in the SARS-CoV2 field. It is also important from a public health perspective to better define quarantine requirements for persons testing positive. While the study provided a new primer probe set, it appears that the already available WHO total E RNA assay is superior in predicting infectivity and this study provides further evidence to support this notion.

General Statements [optional]
This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.
We would like to thank the Editor(s) and Reviewers for their time evaluating our submission. We appreciate their insightful suggestions. Both reviewers acknowledged the timely significance of the study and confirmed that we carried out experiments with the appropriate methodologies and rigor. By directly addressing their critiques, we believe the manuscript has been strengthened, with conclusions that are strongly supported by the data.

Point-by-point description of the revisions
This section is mandatory. Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. Authors developed a novel primer/probe set for detection of subgenomic (sgE) transcripts for SARS-CoV-2 with the aim to develop a system that may predict the presence of infectious virus in patient samples. After studying the specificity and sensitivity of their system, they compared it with already validated/published systems for diagnostic of SARS-CoV-2 infection. Interestingly, they also studied the effect of the conditions of isolation. They showed Vero E6 expressing TMPRSS2 (Vero E6-TMPRSS2) to be more sensitive to infection than Vero E6, allowing a higher number of isolation from patient samples. They also showed their system to be more 14th Oct 2021 Authors' response to reviewer comments (transferred files) Full Revision sensitive than a previously published sgE system as well as than a negative-strand RNA assay but less sensitive than the WHO/Charité primer/probe set. Anyway, all samples containing infectious particles (successful virus isolation on Vero E6-TMPRSS2) were detected with their primer/probe system contrary to the other tested sgE assay. They showed the negative strand assay to be unlikely to detect virus genetic material in samples which nevertheless contain infectious particles. **Major comments:** -Are the key conclusions convincing?
I salute the intention of the authors to try to fix cut-off values for infectious patients but I would be more careful on the assertion of "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". It is true that in this study, virus was not isolated from any of the samples below a Ct of 31 or negative in the developed sgE assay but all those assays are done on cell culture. We do not know how the transmission could occur for those samples from human to human. Being able to fix a cut-off in Ct value for a define PCR/RT-PCR system would be a great improvement for SARS-CoV-2 infected patient having to stay in quarantine. It is even more important for Ebola positive patients in Africa who has to stay in quarantine in precarious conditions under tents, warm temperatures and without privacy for long period because they still positive by RT-PCR. Unfortunately, fix those values would need a very high number of experiments, including animal experiment.
We appreciate the reviewer's acknowledgment of the significance of this issue. We agree that in vivo animal experiments to more precisely determine the lowest infectious or transmissible dose would be valuable. But such experiments are outside the scope of the current study. To acknowledge the reviewer's important point regarding the unavoidable limitations of cell culture systems, we have modified the abstract (line 51) to say "an effective rule out test for the presence of culturable virus," a conclusion that is fully supported by our data.
-Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? No We apologize for this omission and have included the requested details in the methods. We seed a separate well for each time point and collected the entire supernatant for a given time point, rather than replacing media. We added the following text to the methods section (

with AVE lysis buffer) and viral titration (by focus assay). Dead cells/debris that was pelleted after clarifying supernatants was combined with cells scraped from each well into PBS and spun again to obtain a pellet of all cell material from each timepoint. This pellet was then lysed in AVE viral lysis buffer for RNA extraction."
-Stability of infectious SARS-CoV-2: I am very surprise by your results on stability of cultured virus, knowing we observed a decreased of SARS-CoV-2 titer in our lab after freezing/thawing steps. Do you freeze cell supernatant directly or do you prepare your samples another way? Please state it in the Methods part We measured the stability after freeze/thaw for our normal high concentration viral stocks. Our viral stocks are grown in DMEM with 10% FBS, 1% HEPES, 1% pen/strep, and clarified before use. It is possible that lab-lab variation in the media components or HEPES concentration used to prepare viral stocks explains the differences seen in our work vs the reviewer's lab. We have added the following additional detail to the methods section (lines 415-418) of the manuscript to clarify how these experiments were performed: "High concentration viral stocks (prepared as above in DMEM, 10% FBS, 1% HEPES, 1% pen/strep) were used to measure viral stability over time and after multiple freeze-thaw cycles. Stocks were stored at the indicated temperatures in the dark and aliquots were removed at the indicated days or after each freeze-thaw cycle for measuring infectious virus by focus assay." -Are the experiments adequately replicated and statistical analysis adequate? Yes **Minor comments:** -Specific experimental issues that are easily addressable. Figure 2C and D: Instead of Ct values in cells, it would be more relevant to normalize these results with an endogenous gene and present results as fold change to mock-infected cells. Because you affirm that the level of RNA decline than stay stable over the time but you also Full Revision note there is CPE. If you have less cells but same level of viral RNA, it means you have an increase in the RNA level in alive cells. We have measured the GAPDH level in these cells over time, and that data is included as gray lines in Fig 2 C&D (pasted below). As we are combining the cell pellet from clarified supernatants with the cells that remain adherent to the dish for each harvested timepoint we expect to be harvesting the majority of cells/cell debris for each time point. The levels of GAPDH remain broadly similar over the viral growth curve, with no drop in RNA levels.
It would have been interesting to have the results of isolation at different time-point of treatment for patient samples ( figure 3A and B) to see if the virus is stable in samples We have access to only limited volume (several hundred µl) of residual patient sample which would make it technically challenging to compare multiple days of storage conditions/ temperatures. Unfortunately, we do not have any remaining sample volume for the specimens used in this study, and so we are unable to perform additional isolations at other times/temperatures. While we agree this would be an interesting line of future inquiry, we feel it is outside the scope of the current study.
-Are prior studies referenced appropriately? Yes -Are the text and figures clear and accurate? Yes.
Line 140: "this delay in virus and RNA production". You do not talk about RNA yet... We have removed "and RNA" from this sentence and replaced with "infectious virus production".
Line 156 to 163: sgE RNA detected in cell free supernatant. Can't it come from lysed cells?
We have replaced "cell-free" with "clarified".

Full Revision
We have modified this text to read according to the Reviewer's suggestion.
Ligne 258: Fig 6A and B We have added the missing reference to Fig 6B as requested. -Do you have suggestions that would help the authors improve the presentation of their data and conclusions? No Reviewer #1 (Significance (Required)): -Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.
This new primer/probe system will participate to the accurate diagnostic of SARS-CoV-2. The comparison with the existing methods is relevant to highlight the strengths and weaknesses of each system. Comparison of isolation of SARS-CoV-2 on commonly used Vero E6 with Vero E6-TMPRSS2 will lead to a great improvement of the isolation method for SARS-CoV-2.
We appreciate the Reviewer's assessment of the significance of our study and the improvement in our isolation method compared to the existing standard of using Vero E6 cells.

Full Revision
While this new assay identified 100% of culturable isolates, only 56% of isolates testing positive actually had culturable virus. Compared with other assays, the WHO total E RNA assay had better parameters when used at a cutoff Ct value of 31 (PPV of 61%). Overall, this manuscript provides a novel primer probe set for RT-PCR diagnostic assay and conducted comparisons with other assays on the same clinical samples. There are some areas that the authors should address prior to publication. **Major comments:** -The authors repeatedly tout VeroE6 TMRSS2 cells as supporting higher viral infection. Therefore, the authors should address why one clinical isolate (E16) was culturable in VeroE6 but not VeroE6 TMRSS2. Was this experiment repeated multiple times? What are the reasons for this discrepancy?
We did not have sufficient residual sample volume to repeat isolation attempts of any clinical specimen, so we are limited to a single data point for each cell line. It is possible that this sample had levels of infectious virus at the limit of detection, and stochastic probability meant infectious virus was only present in the aliquot used to infect the Vero E6 (rather than Vero E6-TMPRSS2) cells. It is also possible that viral adaptation/evolution occurred in the VeroE6 well that allowed this virus to successfully grow, but we do not have sequencing data or remaining nucleic acids to test this theory.
-The authors' argument at lines 166-169 is not supported by the data in Fig. 2. The levels of viral RNA between VeroE6 and VeroE6 TMRSS2 appear to show similar trends in the supernatant across the time course but the infectious viral levels are dramatically different. This discordance between FFU levels and RNA levels cannot be explained by instability of viral particles alone. Have the authors looked into differences in viral particles produced from these two cell lines? The authors should collect virus particles from these two cell lines and conduct the stability experiment in Fig 2D to directly test the hypothesis that indeed the drop seen in FFU in VeroE6 TMRSS2 is due to instability.
We apologize for the confusion. We did not intend to make claims about differences in particle stability as a result of the cell line used for viral production, but rather to highlight a general observation that RNA was more stable than infectious virus. This is more obvious in the TMRPSS2 cell line, as replication is faster and more synchronized than in Vero E6 cells (the TMRPSS2 cells are largely dead by day 4, whereas infection progresses more slowly in Vero E6 cells so that new virions continue to be produced during the measured time period). We have added clarifying text at line 167-169, "We observed that SARS-CoV-2 RNA species persist for much longer than infectious virus in cell culture time course experiments, a feature that was most obvious in Vero E6 TMRPSS-2 cells due to their viral kinetics but is likely not cell specific (Fig 2)." -The evidence for the packaging of sgE RNA into virions is weak. GAPDH detection by PCR is Full Revision not a proof that the concentration process did not pellet RNA nonspecifically. First, the authors should provide ample information about viral isolation process at line 379 including rotor, centrifuge and speed utilized. In addition, ribosomes typically stay intact following viral lysis (and can be found in supernatant after release from dead cells). Actively translating ribosomes can contain sgE RNA as well. The authors should consider detecting ribosomal RNAs in their samples to rule out the possibility of contaminating ribosomes. In addition, the authors should strongly consider repeating the experiment with high EDTA concentration to break up ribosomes and only pellet virions.
We have added additional experimental details (rotor, centrifuge and speed) describing how the viral concentration step was performed (line 389-394), "Viral RNA (courtesy of David Bauer, The Francis Crick Institute, UK) from concentrated SARS-CoV-2 (England02 strain, B lineage 'Wuhan-like') was obtained by clarifying viral supernatants (2 x 4000 rpm for 30 mins at 4°C in a Beckman Allegra X-30R centrifuge with a SX4400 rotor), overlaying clarified media onto a 30% sucrose/PBS cushion (1/4 th tube volume) and concentrating by ultracentrifugation in a Beckman ultra XPN-90 centrifuge with SW32TI rotor for 90 min at 25,500 rpm at 4°C. Pellets were then resuspended in buffer and extracted with TRIzol LS." We thank the reviewer for their suggestion of including an additional control, and we have added an 18S primer-probe set (see new Figure  8). This data, while not as pronounced as the GAPDH control, suggests that the ultracentrifugation step has removed significant amounts of 18S RNA (though the clarified supernatants retain similar amounts of 18S RNA as the cells, suggesting that clarification alone is not sufficient to remove contaminating ribosomes). While we agree that repeating the ultracentrifuge concentration with high concentrations of EDTA is an interesting line of inquiry we feel it is outside the scope of this manuscript (and we face additional technical restrictions to pursue this as we currently lack access to an ultracentrifuge at BSL-3). We have updated the discussion to include the possibility of residual ribosome-protected fragments of sgE as a potential alternative interpretation (line 350-352). **Minor comments:** -At line 197, the authors refer to "viruses" with lower levels of SARS-CoV2 RNA. This is incorrect and should be changed to "isolates" as the SARS-CoV2 virus particle does not package variable amount of genomic RNA.
We have changed this to "clinical specimens" for clarity.
-The authors statement on lines 210-212 does not seem to be supported clearly by Fig. 5. The authors should consider including trendlines as well as other analyses that help show the correlation between viral RNA vs FFU. In addition, the authors should label the Y-axis clearly for Fig. 5.
We have added clarifying labels to both the X and Y axes. Due to the limited sample volume we were unable to directly measure the infectious titers from the clinical samples used in this study, and thus the FFU/mL represents the titer post-isolation while the C T represents the amount of RNA pre-isolation. Nonetheless, we do see broad trends (ie, the colored dots are generally arranged in rainbow order from left to right, though we agree there is variation within this trend). We have also modified the text at lines 212-217 to reflect the reviewer's concern-"Greater initial viral RNA levels was broadly associated with faster viral growth in both cell lines (seen in the progression of colors from left to right), however we saw significant variation within these trends. Our data suggests that when standard SARS-CoV-2 RNA RT-PCR values are the only available data for patient or population-level viral loads, they are useful in gauging the presence of infectious virus in patient NP samples (Fig 5)." -The authors should expand on the methodology for creating ROC curves at line 467.
We have included the following text in the methods section for ROC curve analysis: "ROC curves were generated using R [43]. For each potential scoring marker (CT_e, CT_sge1, CT_sge2, neg_e,) samples were ordered by that marker, followed by culturable status. The false-positive rate was calculated as the cumulative count of culturable samples (after ordering by marker intensity) divided by the total count of culturable samples; the true positive rate was calculated as the cumulative count of non-culturable samples (after ordering) divided by the total count of non-culturable samples. The false positive rate was plotted on the X axis of the ROC curves and the true positive rate on the Y axis."

Reviewer #2 (Significance (Required)):
This study is significant because it assesses the utility of several clinical assays for the measurement of viral RNA and correlating it with culturable virus. This is important in the field because it helps to identify methods whereby infectivity can be predicted from a simple diagnostic test. This is important to know as a virologist working in the SARS-CoV2 field. It is also important from a public health perspective to better define quarantine requirements for persons testing positive. While the study provided a new primer probe set, it appears that the already available WHO total E RNA assay is superior in predicting infectivity and this study provides further evidence to support this notion. We appreciate the Reviewer's assessment that this study is significant and provides information of high interest to SARS-CoV-2 virologists that also has important public health implications. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1) In the main manuscript file, please do the following: -Correct/answer the track changes suggested by our data editors by working from the attached document.
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B-Statistics and general methods
the assay(s) and method(s) used to carry out the reported observations and measurements an explicit mention of the biological and chemical entity(ies) that are being measured. an explicit mention of the biological and chemical entity(ies) that are altered/varied/perturbed in a controlled manner.
a statement of how many times the experiment shown was independently replicated in the laboratory.
Any descriptions too long for the figure legend should be included in the methods section and/or with the source data.
In the pink boxes below, please ensure that the answers to the following questions are reported in the manuscript itself. Every question should be answered. If the question is not relevant to your research, please write NA (non applicable). We encourage you to include a specific subsection in the methods section for statistics, reagents, animal models and human subjects.
definitions of statistical methods and measures: a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.).
The data shown in figures should satisfy the following conditions: Source Data should be included to report the data underlying graphs. Please follow the guidelines set out in the author ship guidelines on Data Presentation.
Please fill out these boxes ê (Do not worry if you cannot see all your text once you press return) a specification of the experimental system investigated (eg cell line, species name).
Sample size was based on previous literature and what was feasible to obtain during a period of high positivity rates and limited testing capability in the Seattle area.
graphs include clearly labeled error bars for independent experiments and sample sizes. Unless justified, error bars should not be shown for technical replicates. if n< 5, the individual data points from each experiment should be plotted and any statistical test employed should be justified the exact sample size (n) for each experimental group/condition, given as a number, not a range; Each figure caption should contain the following information, for each panel where they are relevant:

Captions
There are no animal studies in this manuscript.
The only samples excluded from analysis are listed in the supplemental dataset, and were excluded because CPE was likely the result of contamination from neighboring wells during isolation (based on the kinetics, plate placement and initial RNA levels of samples during the course of isolation) No samples were allocated to specific groups.

Manuscript Number: EMM-2021-15290
The Fisher Exact test used is appropriate for a 2x2 contingency table and provides exact p values.
The Fisher Exact test assumes that the binary data are independent, and does not make any assumptions about normality. The data were drawn from independent biological samples.
Graphs show SEM; estimation of within-group variance does not apply to 2x2 contingency tables for the Fisher exact test.
NA RNA level (cycle threshold) of clinical specimens was blinded from investigator performing BSL3 titer work.

Data
the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner. figure panels include only data points, measurements or observations that can be compared to each other in a scientifically meaningful way.

E-Human Subjects
Vero E6 and Vero E6-TMPRSS2 cells were routinely tested for mycoplasma contamination by DAPI staining, no cytoplasmic staining was observed. Vero E6 cells were obtained directly from Dr. J.L. Whitton, Vero E6 TMPRSS-2 cells were obtained from the JCRB Cell Bank (JCRB No. JCRB1819). Neither cell line was recently authenticated.
Graphs show SEM, they are broadly similar.
The rabbit anti-SARS-CoV-2 nucleocapsid antibody used in this study for viral focus assays was obtained from Sinobiological, distributed by Thermo Fisher, Cat #40143-R001). The use of this antibody for this assay is first described in the following publication: Graham et al, 202 (PMID: 33072323).
NA NA NA G-Dual use research of concern F-Data Accessibility The use of deindentified positive specimens for the above study was approved by the University of Washington Institutional Review Board under a consent waiver (STUDY00010205) The use of deindentified samples in this study was approved by the University of Washington Institutional Review Board under a consent waiver, and were determined to be exempt.
There are no photos in this study.

No
The data in this paper is available in the supplemental dataset. There is no remaining volume of the clinical specimans or extracted RNA used in this work.
NA NA NA All the data is included in the manuscript and supplemental materials.
Data is available in the supplemental dataset.
Not applicable for this data type.
The source code used to generate the ROC curves is available at github.com/emilybrucelab.