Genomic epidemiology and evolution of rhinovirus in western Washington State, 2021-22 (preprint)

Background: Human rhinoviruses (RV) primarily cause the common cold, but infection outcomes vary from subclinical to severe cases, including asthma exacerbations and fatal pneumonia in immunocompromised individuals. 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 understanding RV true genomic diversity. Methods: We sequenced 1,003 RV genomes from nasal swabs of symptomatic and asymptomatic individuals to explore viral evolution during two epidemiologically distinct periods in Washington State: when the COVID 19 pandemic affected the circulation of other seasonal respiratory viruses except for RV (from February to July 2021), and when the seasonal viruses reemerged with the severe RSV and influenza outbreak (November and December 2022). We constructed maximum likelihood and BEAST phylodynamic trees to characterize intra-genotype evolution. Results: We detected 100 of 168 known genotypes, identified two new genotypes (A111 and C59), and observed inter-genotypic 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 diversity revealed co-circulation of divergent clades within genotypes with high amino acid constraints throughout polyprotein. Discussion: Our study underscores the dynamic nature of RV genomic epidemiology within a localized geographic region, as more than 20% of existing genotypes within each RV species co-circulated each month. Our findings also emphasize the importance of investigating correlations between rhinovirus genotypes and serotypes to understand long-term immunity and cross-protection.

Quantifying how single dose Ad26.COV2.S vaccine efficacy depends on Spike sequence features (preprint)

It is of interest to pinpoint SARS-CoV-2 sequence features defining vaccine resistance. In the ENSEMBLE randomized, placebo-controlled phase 3 trial, estimated single-dose Ad26.COV2.S vaccine efficacy (VE) was 56% against moderate to severe–critical COVID-19. SARS-CoV-2 Spike sequences were measured from 484 vaccine and 1,067 placebo recipients who acquired COVID-19 during the trial. In Latin America, where Spike diversity was greatest, VE was significantly lower against Lambda than against Reference and against all non-Lambda variants [family-wise error rate (FWER) p < 0.05]. VE also differed by residue match vs. mismatch to the vaccine-strain residue at 16 amino acid positions (4 FWER p < 0.05; 12 q-value ≤ 0.20). VE significantly decreased with physicochemical-weighted Hamming distance to the vaccine-strain sequence for Spike, receptor-binding domain, N-terminal domain, and S1 (FWER p < 0.001); differed (FWER ≤ 0.05) by distance to the vaccine strain measured by 9 different antibody-epitope escape scores and by 4 NTD neutralization-impacting features; and decreased (p = 0.011) with neutralization resistance level to vaccine recipient sera. VE against severe–critical COVID-19 was stable across most sequence features but lower against viruses with greatest distances. These results help map antigenic specificity of in vivo vaccine protection.

SARS-CoV-2 seroprevalence and longitudinal antibody response following natural infection in pregnancy (preprint)

Importance: Antenatal care provides unique opportunities to assess SARS-CoV-2 seroprevalence and antibody response duration after natural infection detected during pregnancy; transplacental antibody transfer may inform peripartum and neonatal protection. Objective: Estimate seroprevalence and durability of antibodies from natural infection (anti-nucleocapsid (anti-N) IgG) among pregnant people, and evaluate transplacental transfer efficiency. Design: Seroprevalence study: cross-sectional SARS-CoV-2 antibody screening among pregnant people December 9, 2020-June 19, 2021. Cohort study: Pregnant people screened anti-N IgG+ by Abbott Architect chemiluminescent immunoassay in seroprevalence study or identified through medical records with RT-PCR+ or antigen positive results enrolled in a prospective cohort December 9, 2020-June 30, 2022 to longitudinally measure anti-N IgG responses. We collected cord blood and assessed transplacental transfer of maternally-derived anti-N antibodies. Setting: Three hospitals and 14 affiliated clinics providing antenatal and delivery care, Seattle, Washington metropolitan area. Participants: Seroprevalence study: pregnant people were screened for SAR-CoV-2 anti-N IgG during routine care. Cohort study: Pregnant people with evidence of prior SARS-CoV-2 infection (screened anti-N IgG+ from seroprevalence study or identified with a RT-PCR+ or antigen positive result from medical records) were enrolled in a cohort study to longitudinally measure anti-N IgG responses. Exposure(s) (for observational studies): COVID-19 diagnosis, symptoms, and disease severity. Main Outcome(s) and Measure(s): Presence and durability of SARS-CoV-2 anti-N IgG, transplacental transfer of maternally-derived anti-N IgG. Results: Of 1289 pregnant people screened in the seroprevalence study, 5% (65) tested SARS-CoV-2 anti-N IgG+, including 39 (60%) without prior RT-PCR+ or antigen positive results and 53 (82%) without symptoms. Among 89 participants enrolled in the cohort study, 73 (82%) had anti-N IgG+ results during pregnancy. Among 49 participants with delivery samples 33 (67%) were anti-N IgG negative by delivery. Of 24 remaining anti-N IgG+ at delivery with paired cord blood samples, 12 (50%) had efficient transplacental anti-N IgG antibody transfer. Median time from first anti-N IgG to below positive antibody threshold was 17 weeks and did not differ by prior RT-PCR+ or antigen positive status. Conclusions and Relevance: Maternally-derived SARS-CoV-2 antibodies to natural infection may wane before delivery. Vaccines are recommended for pregnant persons to reduce severe illness and confer protection to infants.

Genomic characterization of respiratory syncytial virus 2022-2023 outbreak in Washington State, USA (preprint)

Mitigation measures against the COVID-19 pandemic affected the RSV seasonality and led to an unexpectedly high number of RSV cases in Washington State since October 2022. Here we describe the RSV genomic characteristics and evolutionary relationship of 2022 outbreak compared to the previous RSV outbreaks in the region and globally.

Genomic analysis of early spread of monkeypox virus in Washington State (preprint)

Genomic analysis of the monkeypox virus outbreak in Washington State using 109 viral genome sequences collected from July-August 2022 shows low overall genetic diversity, multiple introductions into the state with ongoing community transmission, and potential for coinfection of an individual by multiple strains. Biography Dr. Roychoudhury is Acting Instructor and Director of COVID-19 NGS in the Department of Laboratory Medicine and Pathology at the University of Washington and Associate in the Vaccine and Infectious Disease Division at the Fred Hutchinson Cancer Research Center. Her primary research interests are pathogen genomics and mathematical models of viral evolution and host-pathogen interactions.

Nebulized fusion inhibitory peptide protects cynomolgus macaques from measles virus infection (preprint)

Measles is the most contagious airborne viral infection and the leading cause of child death among vaccine-preventable diseases. We show here that aerosolized lipopeptide fusion inhibitors, derived from heptad-repeat regions of the measles virus (MeV) fusion protein, block respiratory MeV infection in a non-human primate model, the cynomolgus macaque. We used a custom-designed mesh nebulizer to ensure efficient aerosol delivery of peptides to the respiratory tract and demonstrated the absence of adverse effects and lung pathology in macaques. The nebulized peptide efficiently prevented MeV infection, resulting in the complete absence of MeV RNA, MeV-infected cells, and MeV-specific humoral responses in treated animals. This strategy provides an additional shield which complements vaccination to fight against respiratory infection, presenting a proof-of-concept for the aerosol delivery of fusion inhibitory peptides to protect against measles and other airborne viruses, including SARS-CoV-2, in case of high-risk exposure, that can be readily translated to human trials.

Variants in nucleocapsid protein and endoRNase are found to associate with severe COVID-19 in a case-control study in Washington State, USA (preprint)

SARS-CoV-2 is spreading worldwide with continuously evolving variants, some of which occur in the Spike protein and appear to increase the viral transmissibility. However, variants that cause severe COVID-19 or lead to other breakthroughs have not been well characterized. To discover such viral variants, we assembled a cohort of 683 COVID-19 patients; 388 inpatients (“cases”) and 295 outpatients (“controls”) from April to August 2020 using electronically captured COVID test request forms and sequenced their viral genomes. To improve the analytic power, we accessed 7,137 viral sequences in Washington State to filter out viral single nucleotide variants (SNVs) that did not have significant expansions over the collection period. Applying this filter led to the identification of 53 SNVs that were statistically significant, of which 13 SNVs each had 3 or more variant copies in the discovery cohort. Correlating these selected SNVs with case/control status, eight SNVs were found to significantly associate with inpatient status (q-values<0.01). Using temporal synchrony, we identified a four SNV-haplotype (t19839-g28881-g28882-g28883) which was significantly associated with case/control status (Fisher’s exact p=2.84*10 −11 ) that appeared in April 2020, peaked in June, and persisted into January 2021. This association was replicated (OR=5.46, p-value=4.71*10 −12 ) in an independent cohort of 964 COVID-19 patients (June 1, 2020 to March 31, 2021). The haplotype included a synonymous change N73N in endoRNase, and three non-synonymous changes coding residues R203K, R203S and G204R in the nucleocapsid protein. This discovery points to the potential functional role of the nucleocapsid protein in triggering “cytokine storms” and severe COVID-19 that led to hospitalization. The study further emphasizes a need for tracking and analyzing viral sequences in correlations with clinical status.

Associations between SARS-CoV-2 variants and risk of COVID-19 hospitalization among confirmed cases in Washington State: a retrospective cohort study (preprint)

BackgroundThe COVID-19 pandemic is now dominated by variant lineages; the resulting impact on disease severity remains unclear. Using a retrospective cohort study, we assessed the risk of hospitalization following infection with nine variants of concern or interest (VOC/VOI). MethodsOur study includes individuals with positive SARS-CoV-2 RT-PCR in the Washington Disease Reporting System and with available viral genome data, from December 1, 2020 to July 30, 2021. The main analysis was restricted to cases with specimens collected through sentinel surveillance. Using a Cox proportional hazards model with mixed effects, we estimated hazard ratios (HR) for the risk of hospitalization following infection with a VOC/VOI, adjusting for age, sex, and vaccination status. FindingsOf the 27,814 cases, 23,170 (83.3%) were sequenced through sentinel surveillance, of which 726 (3.1%) were hospitalized due to COVID-19. Higher hospitalization risk was found for infections with Gamma (HR 3.17, 95% CI 2.15-4.67), Beta (HR: 2.97, 95% CI 1.65-5.35), Delta (HR: 2.30, 95% CI 1.69-3.15), and Alpha (HR 1.59, 95% CI 1.26-1.99) compared to infections with an ancestral lineage. Following VOC infection, unvaccinated patients show a similar higher hospitalization risk, while vaccinated patients show no significant difference in risk, both when compared to unvaccinated, ancestral lineage cases. InterpretationInfection with a VOC results in a higher hospitalization risk, with an active vaccination attenuating that risk. Our findings support promoting hospital preparedness, vaccination, and robust genomic surveillance.

Oral prodrug of remdesivir parent GS-441524 is efficacious against SARS-CoV-2 and a variant of concern in ferrets (preprint)

Remdesivir is the only small-molecule antiviral approved to date for COVID-19 treatment, but its wider use is limited by intravenous delivery. An orally bioavailable remdesivir analog may boost therapeutic benefit by facilitating early administration to non-hospitalized patients. This study characterized the anti-SARS-CoV-2 efficacy of GS-621763, an oral prodrug of remdesivir parent nucleoside GS-441524. Both GS-621763 and GS-441524 inhibited SARS-CoV-2, including variants of concern (VoC) in cell culture. Oral GS-621763 was efficiently converted to plasma metabolite GS-441524, and in lungs to the triphosphate metabolite identical to that generated by remdesivir, demonstrating a consistent mechanism of activity. Twice-daily oral administration of 10 mg/kg GS-621763 reduced SARS-CoV-2 burden to near-undetectable levels. When dosed therapeutically against VoC P.1 gamma (γ), oral GS-621763 blocked virus replication and prevented transmission to untreated contact animals. These results demonstrate therapeutic efficacy of a much-needed orally bioavailable analog of remdesivir in a relevant animal model of SARS-CoV-2 infection.  

Predicting Infectivity: Comparing Four PCR-based Assays to Detect Culturable SARS-CoV-2 in Clinical Samples (preprint)

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 C T 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 C T 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 C T 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.

Hydroxychloroquine with or Without Azithromycin for Treatment of Early SARS-CoV-2 Infection Among High-Risk Outpatient Adults: A Randomized Clinical Trial (preprint)

Background: Treatment options for outpatients with COVID-19 could reduce morbidity and prevent SARS-CoV-2 transmission.Methods: In this randomized, double-blind, three-arm (1:1:1) placebo-equivalent control trial conducted remotely throughout the United States, adult outpatients with laboratory-confirmed SARS-CoV-2 infection were recruited. Participants were randomly assigned to receive HCQ (400mg BID x1day, followed by 200mg BID x9days) or placebo-equivalent (ascorbic acid) and AZ (500mg, then 250mg daily x4days) or placebo-equivalent (folic acid), stratified by risk for progression to severe COVID-19 (high-risk vs. low-risk). Self-collected mid-turbinate nasal swabs for SARS-CoV-2 PCR, FLUPro symptom surveys, EKGs and vital signs collected daily. Primary endpoints were: (a) 14-day progression to lower respiratory tract infection (LRTI), 28-day COVID-19 related hospitalization, or death; (b) 14-day time to viral clearance; secondary endpoints included time to symptom resolution (ClinicalTrials.gov: NCT04354428). Due to the low rate of clinical outcomes, the study was terminated for operational futility.Findings: Between 15th April and 27th July 2020, 231 participants were enrolled and 219 initiated medication a median of 5.9 days after symptom onset. Incident LRTI occurred in six participants (two control, four HCQ/AZ) and COVID-19 related hospitalization in nine (four control, two HCQ, three HCQ/AZ). There were no deaths. Median time to clearance was 5 days (95% CI=4-6) in HCQ, 6 days (95% CI=4-8) in HCQ/AZ, and 8 days (95% CI=6-10) in control. HCQ but not HCQ/AZ had faster time to viral clearance (HR=1.62, 95% CI=1.01-2.60, p=0.047 & HR=1.25, 95% CI=0.75-2.07, p=0.39) compared to control. Among 197 participants who met the COVID-19 definition at enrollment, time to symptom resolution did not differ by group.Interpretation: Neither HCQ nor HCQ/AZ shortened the clinical course of outpatients with COVID-19, and HCQ, but not HCQ/AZ, had only a modest effect on SARS-CoV-2 viral shedding. HCQ and HCQ/AZ are not effective therapies for outpatient treatment of SARV-CoV-2 infection.Trial Registration Number: ClinicalTrials.gov: NCT04354428Funding: The COVID-19 Early Treatment Study was funded by the Bill & Melinda Gates Foundation (INV-017062) through the COVID-19 Therapeutics Accelerator. University of Washington Institute of Translational Health Science (ITHS) grant support (UL1 TR002319), KL2 TR002317, and TL1 TR002318 from NCATS/NIH funded REDCap. The content is solely the responsibility of the authors and does not necessarily represent the views, decisions, or policies of the institutions with which they are affiliated. PAN and MJA were supported by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program. Conflict of Interest: Declaration of interests: CJ reports grants from BMGF for conduct of the study, and grants from CDC and NIH outside of the submitted work. HSK reports funding from NIH. PAN and MJA have a potential financial relationship with AliveCor related to QT assessment using the device, however the investigators would receive no financial benefit for use of the technology for patients at Mayo Clinic or for its use in the current study. AB reports consulting for Gates Ventures and grants from BMGF and NIH outside of the submitted work. HYC reports consulting for BMGF, Pfizer, Ellume, and Merck, and grants from Gates Ventures, NIH, CDC, BMGF, DARPA, Apple Inc., Sanofi-Pasteur, and Roche-Genentech, outside of the submitted work. Ethical Approval: Study visits were conducted via Health Insurance Portability and Accountability Act (HIPAA)-compliant telemedicine. The Western Institutional Review Board (WIRB) approved this study with reliance agreements with collaborating institutions.

Neutralizing antibodies correlate with protection from SARS-CoV-2 in humans during a fishery vessel outbreak with high attack rate (preprint)

The development of vaccines against SARS-CoV-2 would be greatly facilitated by the identification of immunological correlates of protection in humans. However, to date, studies on protective immunity have only been performed in animal models and correlates of protection have not been established in humans. Here, we describe an outbreak of SARS-CoV-2 on a fishing vessel associated with a high attack rate. Predeparture serological and viral RT-PCR testing along with repeat testing after return to shore was available for 120 of the 122 persons on board over a median follow-up of 32.5 days (range 18.8 to 50.5 days). A total of 104 individuals had an RT-PCR positive viral test with Ct <35 or seroconverted during the follow-up period, yielding an attack rate on board of 85.2% (104/122 individuals). Metagenomic sequencing of 39 viral genomes suggested the outbreak originated largely from a single viral clade. Only three crewmembers tested seropositive prior to the boats departure in initial serological screening and also had neutralizing and spike-reactive antibodies in follow-up assays. None of these crewmembers with neutralizing antibody titers showed evidence of bona fide viral infection or experienced any symptoms during the viral outbreak. Therefore, the presence of neutralizing antibodies from prior infection was significantly associated with protection against re-infection (Fishers exact test, p=0.002).

Development and validation of direct RT-LAMP for SARS-CoV-2 (preprint)

We have developed a reverse-transcriptase loop mediated amplification (RT-LAMP) method targeting genes encoding the Spike (S) protein and RNA-dependent RNA polymerase (RdRP) of SARS-CoV-2. The LAMP assay achieves comparable limit of detection as commonly used RT-PCR protocols based on artificial targets, recombinant Sindbis virus, and clinical samples. Clinical validation of single-target (S gene) LAMP (N=120) showed a positive percent agreement (PPA) of 41/42 (97.62%) and negative percent agreement (NPA) of 77/78 (98.72%) compared to reference RT-PCR. Dual-target RT-LAMP (S and RdRP gene) achieved a PPA of 44/48 (91.97%) and NPA 72/72 (100%) when including discrepant samples. The assay can be performed without a formal extraction procedure, with lyophilized reagents which do need cold chain, and is amenable to point-of-care application with visual detection.

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.