Determining the impact of vaccination on SARS-CoV-2 RT-PCR cycle threshold values and infectious viral titers

ABSTRACT Background: As the COVID-19 pandemic continues, efforts to better understand SARS-CoV-2 viral shedding and transmission in both unvaccinated and vaccinated populations remain critical to informing public health policies and vaccine development. The utility of using RT-PCR cycle threshold values (CT values) as a proxy for infectious viral titers from individuals infected with SARS-CoV-2 is yet to be fully understood. This retrospective observational cohort study compares quantitative infectious viral titers derived from a focus-forming viral titer assay with SARS-CoV-2 RT-PCR CT values in both unvaccinated and vaccinated individuals infected with the Delta strain. Methods: Nasopharyngeal swabs positive for SARS-CoV-2 by RT-PCR with a CT value < 27 collected from June 26th to October 17th of 2021 at the University of Vermont Medical Center Clinical Laboratory for which vaccination records were available were included. Partially vaccinated and individuals < 18 years of age were excluded. Infectious viral titers were determined using a micro-focus forming assay under BSL-3 containment. Results: 119 specimens from 22 unvaccinated and 97 vaccinated individuals met all inclusion criteria and had sufficient residual volume to undergo viral titering. A negative correlation between RT-PCR CT values and viral titers was observed in both unvaccinated and vaccinated groups. No difference in mean CT value or viral titer was detected between vaccinated and unvaccinated groups. Viral titers did not change as a function of time since vaccination. Conclusions: Our results add to the growing body of knowledge regarding the correlation of SARS-CoV-2 RNA levels and levels of infectious virus. At similar CT values, vaccination does not appear to impact an individual's potential infectivity.

Viral infectivity in pediatric SARS-CoV-2 clinical samples does not vary by age

At the start of the SARS-CoV-2 pandemic, there was much uncertainty about the role of children in infection and transmission dynamics. Through the course of the pandemic, it became clear that children were susceptible to SARS-CoV-2 infection, however they were experiencing a notable lack of severe disease outcomes as compared to the adult population. This trend held true with the emergence of new SARS-CoV-2 variants, even in pediatric populations that were ineligible to be vaccinated. The difference in disease outcomes has prompted questions about the virologic features of SARS-CoV-2 infection in this population. In order to determine if there was any difference in the infectivity of the virus produced by children infected with COVID-19, we compared viral RNA levels (clinical RT-qPCR CTs) and infectious virus titers from 144 SARS-CoV-2 positive clinical samples collected from children ages 0 to 18 years old. We found that age had no impact on the infectiousness of SARS-CoV-2 within our cohort, with children of all ages able to produce high levels of infectious virus.

Viral infectivity in pediatric SARS-CoV-2 clinical samples does not vary by age

At the start of the SARS-CoV-2 pandemic, there was much uncertainty about the role of children in infection and transmission dynamics. Through the course of the pandemic, it became clear that children were susceptible to SARS-CoV-2 infection, however they were experiencing a notable lack of severe disease outcomes as compared to the adult population. This trend held true with the emergence of new SARS-CoV-2 variants, even in pediatric populations that were ineligible to be vaccinated. The difference in disease outcomes has prompted questions about the virologic features of SARS-CoV-2 infection in this population. In order to determine if there was any difference in the infectivity of the virus produced by children infected with COVID-19, we compared viral RNA levels (clinical RT-qPCR CTs) and infectious virus titers from 144 SARS-CoV-2 positive clinical samples collected from children ages 0 to 18 years old. We found that age had no impact on the infectiousness of SARS-CoV-2 within our cohort, with children of all ages able to produce high levels of infectious virus.

Viral infectivity in pediatric SARS-CoV-2 clinical samples does not vary by age

At the start of the SARS-CoV-2 pandemic, there was much uncertainty about the role of children in infection and transmission dynamics. Through the course of the pandemic, it became clear that children were susceptible to SARS-CoV-2 infection, however they were experiencing a notable lack of severe disease outcomes as compared to the adult population. This trend held true with the emergence of new SARS-CoV-2 variants, even in pediatric populations that were ineligible to be vaccinated. The difference in disease outcomes has prompted questions about the virologic features of SARS-CoV-2 infection in this population. In order to determine if there was any difference in the infectivity of the virus produced by children infected with COVID-19, we compared viral RNA levels (clinical RT-qPCR CTs) and infectious virus titers from 144 SARS-CoV-2 positive clinical samples collected from children ages 0 to 18 years old. We found that age had no impact on the infectiousness of SARS-CoV-2 within our cohort, with children of all ages able to produce high levels of infectious virus.

Measuring infectious SARS-CoV-2 in clinical samples reveals a higher viral titer:RNA ratio for Delta and Epsilon vs. Alpha variants.

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.

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.

Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2.

There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).

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.