SARS-CoV-2 diversity and transmission on a university campus across two academic years during the pandemic (preprint)

Institutions of higher education (IHEs) have been a focus of SARS-CoV-2 transmission studies but there is limited information on how viral diversity and transmission at IHEs changed as the pandemic progressed. Here we analyze 3606 viral genomes from unique COVID-19 episodes collected at a public university in Seattle, Washington (WA) from September 2020 to September 2022. Across the study period, we found evidence of frequent viral transmission among university affiliates with 60% (n=2153) of viral genomes from campus specimens genetically identical to at least one other campus specimen. Moreover, viruses from students were observed in transmission clusters at a higher frequency than in the overall dataset while viruses from symptomatic infections were observed in transmission clusters at a lower frequency. Though only a small percentage of community viruses were identified as possible descendants of viruses isolated in university study specimens, phylodynamic modelling suggested a high rate of transmission events from campus into the local community, particularly during the 2021-2022 academic year. We conclude that viral transmission was common within the university population throughout the study period but that not all university affiliates were equally likely to be involved. In addition, the transmission rate from campus into the surrounding community may have increased during the second year of the study, possibly due to return to in-person instruction.

Local-Scale phylodynamics reveal differential community impact of SARS-CoV-2 in metropolitan US county (preprint)

SARS-CoV-2 transmission is largely driven by heterogeneous dynamics at a local scale, leaving local health departments to design interventions with limited information. We analyzed SARS-CoV-2 genomes sampled between February 2020 and March 2022 jointly with epidemiological and cell phone mobility data to investigate fine scale spatiotemporal SARS-CoV-2 transmission dynamics in King County, Washington, a diverse, metropolitan US county. We applied an approximate structured coalescent approach to model transmission within and between North King County and South King County alongside the rate of outside introductions into the county. Our phylodynamic analyses reveal that following stay-at-home orders, the epidemic trajectories of North and South King County began to diverge. We find that South King County consistently had more reported and estimated cases, COVID-19 hospitalizations, and longer persistence of local viral transmission when compared to North King County, where viral importations from outside drove a larger proportion of new cases. Using mobility and demographic data, we also find that South King County experienced a more modest and less sustained reduction in mobility following stay-at-home orders than North King County, while also bearing more socioeconomic inequities that might contribute to a disproportionate burden of SARS-CoV-2 transmission. Overall, our findings suggest a role for local-scale phylodynamics in understanding the heterogeneous transmission landscape. One Sentence SummaryAnalysis of SARS-CoV-2 genomes in King County, Washington show that diverse areas in the same metropolitan region can have different epidemic dynamics.

Trends in risk factors and symptoms associated with SARS-CoV-2 and Rhinovirus test positivity in King County, Washington: A Test-Negative Design Study of the Greater Seattle Coronavirus Assessment Network (preprint)

ImportanceFew US studies have reexamined risk factors for SARS-CoV-2 positivity in the context of widespread vaccination and new variants or considered risk factors for co-circulating endemic viruses, such as rhinovirus. ObjectiveTo understand how risk factors and symptoms associated with SARS-CoV-2 test positivity changed over the course of the pandemic and to compare these to the factors associated with rhinovirus test positivity. DesignThis test-negative design study used multivariable logistic regression to assess associations between SARS-CoV-2 and rhinovirus test positivity and self-reported demographic and symptom variables over a 22-month period. SettingKing County, Washington, June 2020-April 2022 Participants23,278 symptomatic individuals of all ages enrolled in a cross-sectional community surveillance study. ExposuresSelf-reported data for 15 demographic and health behavior variables and 16 symptoms. Main Outcome(s) and Measure(s)RT-PCR confirmed SARS-CoV-2 or rhinovirus infection. ResultsClose contact with a SARS-CoV-2 case (adjusted odds ratio, aOR 4.3, 95% CI 3.7-5.0) and loss of smell/taste (aOR 3.7, 95% CI 3.0-4.5) were the variables most associated with SARS-CoV-2 test positivity, but both attenuated during the Omicron period. Contact with a vaccinated case (aOR 2.4, 95% CI 1.7-3.3) was associated with a lower odds of test positivity than contact with an unvaccinated case (aOR 4.4, 95% CI 2.7-7.3). Sore throat was associated with Omicron infection (aOR 2.3, 95% CI 1.6-3.2) but not Delta. Vaccine effectiveness for participants fully vaccinated with a booster dose was 43% (95% CI 11-63%) for Omicron and 92% (95% CI 61-100%) for Delta. Variables associated with rhinovirus test positivity included age <12 years (aOR 4.0, 95% CI 3.5-4.6) and reporting a runny or stuffy nose (aOR 4.6, 95% CI 4.1-5.2). Race, region, and household crowding were significantly associated with both SARS-CoV-2 and rhinovirus test positivity. Conclusions and RelevanceEstimated risk factors and symptoms associated with SARS-CoV-2 infection have changed over time. There was a shift in reported symptoms between the Delta and Omicron variants as well as reductions in the protection provided by vaccines. Racial and socioeconomic disparities persisted in the third year of SARS-CoV-2 circulation and were also present in rhinovirus infection, although the causal pathways remain unclear. Trends in testing behavior and availability may influence these results. Key Points QuestionWhat are the characteristics associated with SARS-CoV-2 and rhinovirus infection? FindingsIn this test-negative design study of 23,278 participants, reporting close contact with a SARS-CoV-2 case was the strongest risk factor associated with test positivity. Loss of smell and taste was associated with the Delta variant, but not the Omicron variant. Vaccination and prior infection provided greater protection against Delta infection than Omicron Infection. Young age was the strongest predictor of rhinovirus positivity. Sociodemographic disparities were present for both SARS-CoV-2 and rhinovirus. MeaningMonitoring factors associated with respiratory pathogen test positivity remains important to identify at-risk populations in the post-SARS-CoV-2 pandemic period.

Mapping the emergence of SARS-CoV-2 Omicron variants on a university campus (preprint)

Novel variants continue to emerge in the SARS-CoV-2 pandemic. University testing programs may provide timely epidemiologic and genomic surveillance data to inform public health responses. We conducted testing from September 2021 to February 2022 in a university population under vaccination and indoor mask mandates. A total of 3,048 of 24,393 individuals tested positive for SARS-CoV-2 by RT-PCR; whole genome sequencing identified 209 Delta and 1,730 Omicron genomes of the 1,939 total sequenced. Compared to Delta, Omicron had a shorter median serial interval between genetically identical, symptomatic infections within households (2 versus 6 days, P=0.021). Omicron also demonstrated a greater peak reproductive number (2.4 versus 1.8) and a 1.07 (95% confidence interval: 0.58, 1.57; P<0.0001) higher mean cycle threshold value. Despite near universal vaccination and stringent mitigation measures, Omicron rapidly displaced the Delta variant to become the predominant viral strain and led to a surge in cases in a university population.

Interactions among 17 respiratory pathogens: a cross-sectional study using clinical and community surveillance data (preprint)

Background Co-circulating respiratory pathogens can interfere with or promote each other, leading to important effects on disease epidemiology. Estimating the magnitude of pathogen-pathogen interactions from clinical specimens is challenging because sampling from symptomatic individuals can create biased estimates. Methods We conducted an observational, cross-sectional study using samples collected by the Seattle Flu Study between 11 November 2018 and 20 August 2021. Samples that tested positive via RT-qPCR for at least one of 17 potential respiratory pathogens were included in this study. Semi-quantitative cycle threshold (Ct) values were used to measure pathogen load. Differences in pathogen load between monoinfected and coinfected samples were assessed using linear regression adjusting for age, season, and recruitment channel. Results 21,686 samples were positive for at least one potential pathogen. Most prevalent were rhinovirus (33·5%), Streptococcus pneumoniae ( SPn , 29·0%), SARS-CoV-2 (13.8%) and influenza A/H1N1 (9·6%). 140 potential pathogen pairs were included for analysis, and 56 (40%) pairs yielded significant Ct differences (p < 0.01) between monoinfected and co-infected samples. We observed no virus-virus pairs showing evidence of significant facilitating interactions, and found significant viral load decrease among 37 of 108 (34%) assessed pairs. Samples positive with SPn and a virus were consistently associated with increased SPn load. Conclusions Viral load data can be used to overcome sampling bias in studies of pathogen-pathogen interactions. When applied to respiratory pathogens, we found evidence of viral- SPn facilitation and several examples of viral-viral interference. Multipathogen surveillance is a cost-efficient data collection approach, with added clinical and epidemiological informational value over single-pathogen testing, but requires careful analysis to mitigate selection bias.

Comparable specimen collection from both ends of at-home mid-turbinate swabs (preprint)

Unsupervised upper respiratory specimen collection is a key factor in the ability to massively scale SARS-CoV-2 testing. But there is concern that unsupervised specimen collection may produce inferior samples. Across two studies that included unsupervised at-home mid-turbinate specimen collection, ∼1% of participants used the wrong end of the swab. We found that molecular detection of respiratory pathogens and a human biomarker were comparable between specimens collected from the handle of the swab and those collected correctly. Older participants were more likely to use the swab backwards. Our results suggest that errors made during home-collection of nasal specimens do not preclude molecular detection of pathogens and specialized swabs may be an unnecessary luxury during a pandemic.

Viral genome sequencing places White House COVID-19 outbreak into phylogenetic context (preprint)

In October 2020, an outbreak of at least 50 COVID-19 cases was reported surrounding individuals employed at or visiting the White House. Here, we applied genomic epidemiology to investigate the origins of this outbreak. We enrolled two individuals with exposures linked to the White House COVID-19 outbreak into an IRB-approved research study and sequenced their SARS-CoV-2 infections. We find these viral sequences are highly genetically similar to each other, but are distinct from over 160,000 publicly available SARS-CoV-2 genomes, possessing 5 nucleotide mutations that differentiate this lineage from all other circulating lineages sequenced to date. We estimate this lineage has a common ancestor in the USA in April or May 2020, but its whereabouts for the past 5 to 6 months are not clear. Looking forwards, sequencing of additional community SARS-CoV-2 infections collected in the USA prior to October 2020 may reveal linked infections and shed light on its geographic ancestry. In sequencing of SARS-CoV-2 infections collected after October 2020, the relative rarity of this constellation of mutations may make it possible to identify infections that likely descend from the White House COVID-19 outbreak.

Discovery of five HIV nucleoside analog reverse-transcriptase inhibitors (NRTIs) as potent inhibitors against the RNA-dependent RNA polymerase (RdRp) of SARS-CoV and 2019-nCoV (preprint)

The outbreak of SARS in 2002-2003 caused by SARS-CoV, and the pandemic of COVID-19 in 2020 caused by 2019-nCoV (SARS-CoV-2), have threatened human health globally and raised the urgency to develop effective antivirals against the viruses. In this study, we expressed and purified the RNA-dependent RNA polymerase (RdRp) nsp12 of SARS-CoV and developed a primer extension assay for the evaluation of nsp12 activity. We found that nsp12 could efficiently extend single-stranded RNA, while having low activity towards double-stranded RNA. Nsp12 required a catalytic metal (Mg2+ or Mn2+) for polymerase activity and the activity was also K+-dependent, while Na+ promoted pyrophosphorylation, the reverse process of polymerization. To identify antivirals against nsp12, a competitive assay was developed containing 4 natural rNTPs and a nucleotide analog, and the inhibitory effects of 24 FDA-approved nucleotide analogs were evaluated in their corresponding active triphosphate forms. Ten of the analogs, including 2 HIV NRTIs, could inhibit the RNA extension of nsp12 by more than 40%. The 10 hits were verified which showed dose-dependent inhibition. In addition, the 24 nucleotide analogs were screened on SARS-CoV primase nsp8 which revealed stavudine and remdesivir were specific inhibitors to nsp12. Furthermore, the 2 HIV NRTIs were evaluated on 2019-nCoV nsp12 which showed inhibition as well. Then we expanded the evaluation to all 8 FDA-approved HIV NRTIs and discovered 5 of them, tenofovir, stavudine, abacavir, zidovudine and zalcitabine, could inhibit the RNA extension by nsp12 of SARS-CoV and 2019-nCoV. In conclusion, 5 FDA-approved HIV NRTIs inhibited the RNA extension by nsp12 and were promising candidates for the treatment of SARS and COVID-19.

Viral genomes reveal patterns of the SARS-CoV-2 outbreak in Washington State (preprint)

The rapid spread of SARS-CoV-2 has gravely impacted societies around the world. Outbreaks in different parts of the globe are shaped by repeated introductions of new lineages and subsequent local transmission of those lineages. Here, we sequenced 3940 SARS-CoV-2 viral genomes from Washington State to characterize how the spread of SARS-CoV-2 in Washington State (USA) was shaped by differences in timing of mitigation strategies across counties, as well as by repeated introductions of viral lineages into the state. Additionally, we show that the increase in frequency of a potentially more transmissible viral variant (614G) over time can potentially be explained by regional mobility differences and multiple introductions of 614G, but not the other variant (614D) into the state. At an individual level, we see evidence of higher viral loads in patients infected with the 614G variant. However, using clinical records data, we do not find any evidence that the 614G variant impacts clinical severity or patient outcomes. Overall, this suggests that at least to date, the behavior of individuals has been more important in shaping the course of the pandemic than changes in the virus.

Rapid cost-effective viral genome sequencing by V-seq (preprint)

ABSTRACT Conventional methods for viral genome sequencing largely use metatranscriptomic approaches or, alternatively, enrich for viral genomes by amplicon sequencing with virus-specific PCR or hybridization-based capture. These existing methods are costly, require extensive sample handling time, and have limited throughput. Here, we describe V-seq, an inexpensive, fast, and scalable method that performs targeted viral genome sequencing by multiplexing virus-specific primers at the cDNA synthesis step. We designed densely tiled reverse transcription (RT) primers across the SARS-CoV-2 genome, with a subset of hexamers at the 3’ end to minimize mis-priming from the abundant human rRNA repeats and human RNA PolII transcriptome. We found that overlapping RT primers do not interfere, but rather act in concert to improve viral genome coverage in samples with low viral load. We provide a path to optimize V-seq with SARS-CoV-2 as an example. We anticipate that V-seq can be applied to investigate genome evolution and track outbreaks of RNA viruses in a cost-effective manner. More broadly, the multiplexed RT approach by V-seq can be generalized to other applications of targeted RNA sequencing.

Preliminary support for a 'dry swab, extraction free' protocol for SARS-CoV-2 testing via RT-qPCR (preprint)

The urgent need for massively scaled clinical or surveillance testing for SARS-CoV-2 has necessitated a reconsideration of the methods by which respiratory samples are collected, transported, processed and tested. Conventional testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab, storage of the swab during transport in universal transport medium (UTM), extraction of RNA, and quantitative reverse transcription PCR (RT-qPCR). As testing has scaled across the world, supply chain challenges have emerged across this entire workflow. Here we sought to evaluate how eliminating the UTM storage and RNA extraction steps would impact the results of molecular testing. Using paired mid-turbinate swabs self-collected by 11 individuals with previously established SARS-CoV-2 positivity, we performed a comparison of conventional (swab [->] UTM [->] RNA extraction [->] RT-qPCR) vs. simplified (direct elution from dry swab [->] RT-qPCR) protocols. Our results suggest that dry swabs eluted directly into a simple buffered solution (TE) can support molecular detection of SARS-CoV-2 via endpoint RT-qPCR without substantially compromising sensitivity. Although further confirmation with a larger sample size and variation of other parameters is necessary, these results are encouraging for the possibility of a simplified workflow that could support massively scaled testing for COVID-19 control.

Cryptic transmission of SARS-CoV-2 in Washington State (preprint)

Following its emergence in Wuhan, China, in late November or early December 2019, the SARS-CoV-2 virus has rapidly spread throughout the world. On March 11, 2020, the World Health Organization declared Coronavirus Disease 2019 (COVID-19) a pandemic. Genome sequencing of SARS-CoV-2 strains allows for the reconstruction of transmission history connecting these infections. Here, we analyze 346 SARS-CoV-2 genomes from samples collected between 20 February and 15 March 2020 from infected patients in Washington State, USA. We found that the large majority of SARS-CoV-2 infections sampled during this time frame appeared to have derived from a single introduction event into the state in late January or early February 2020 and subsequent local spread, strongly suggesting cryptic spread of COVID-19 during the months of January and February 2020, before active community surveillance was implemented. We estimate a common ancestor of this outbreak clade as occurring between 18 January and 9 February 2020. From genomic data, we estimate an exponential doubling between 2.4 and 5.1 days. These results highlight the need for large-scale community surveillance for SARS-CoV-2 introductions and spread and the power of pathogen genomics to inform epidemiological understanding.

A Genomic Survey of SARS-CoV-2 Reveals Multiple Introductions into Northern California without a Predominant Lineage (preprint)

The COVID-19 pandemic caused by the novel coronavirus SARS-CoV-2 has spread globally, resulting in >300,000 reported cases worldwide as of March 21st, 2020. Here we investigate the genetic diversity and genomic epidemiology of SARS-CoV-2 in Northern California using samples from returning travelers, cruise ship passengers, and cases of community transmission with unclear infection sources. Virus genomes were sampled from 29 patients diagnosed with COVID-19 infection from Feb 3rd through Mar 15th. Phylogenetic analyses revealed at least 8 different SARS-CoV-2 lineages, suggesting multiple independent introductions of the virus into the state. Virus genomes from passengers on two consecutive excursions of the Grand Princess cruise ship clustered with those from an established epidemic in Washington State, including the WA1 genome representing the first reported case in the United States on January 19th. We also detected evidence for presumptive transmission of SARS-CoV-2 lineages from one community to another. These findings suggest that cryptic transmission of SARS-CoV-2 in Northern California to date is characterized by multiple transmission chains that originate via distinct introductions from international and interstate travel, rather than widespread community transmission of a single predominant lineage. Rapid testing and contact tracing, social distancing, and travel restrictions are measures that will help to slow SARS-CoV-2 spread in California and other regions of the USA.