Accelerated SARS-CoV-2 intrahost evolution leading to distinct genotypes during chronic infection

The chronic infection hypothesis for novel SARS-CoV-2 variant emergence is increasingly gaining credence following the appearance of Omicron. Here we investigate intrahost evolution and genetic diversity of lineage B.1.517 during a SARS-CoV-2 chronic infection lasting for 471 days (and still ongoing) with consistently recovered infectious virus and high viral loads. During the infection, we found an accelerated virus evolutionary rate translating to 35 nucleotide substitutions per year, approximately two-fold higher than the global SARS-CoV-2 evolutionary rate. This intrahost evolution led to the emergence and persistence of at least three genetically distinct genotypes suggesting the establishment of spatially structured viral populations continually reseeding different genotypes into the nasopharynx. Finally, using unique molecular indexes for accurate intrahost viral sequencing, we tracked the temporal dynamics of genetic diversity to identify advantageous mutations and highlight hallmark changes for chronic infection. Our findings demonstrate that untreated chronic infections accelerate SARS-CoV-2 evolution, ultimately providing opportunity for the emergence of genetically divergent and potentially highly transmissible variants as seen with Delta and Omicron.

Insights into the limited global spread of the immune evasive SARS-CoV-2 variant Mu

SARS-CoV-2 Variants of Concern (VOCs) continue to reshape the trajectory of the COVID-19 pandemic. However, why some VOCs, like Omicron, become globally dominant while the spread of others is limited is not fully understood. To address this question, we investigated the VOC Mu, which was first identified in Colombia in late 2020. Our study demonstrates that, although Mu is less sensitive to neutralization compared to variants that preceded it, it did not spread significantly outside of South and Central America. Additionally, we find evidence that the response to Mu was impeded by reporting delays and gaps in the global genomic surveillance system. Our findings suggest that immune evasion alone was not sufficient to outcompete highly transmissible variants that were circulating concurrently with Mu. Insights into the complex relationship between genomic and epidemiological characteristics of previous variants should inform our response to variants that are likely to emerge in the future.

Rapid emergence of SARS-CoV-2 Omicron variant is associated with an infection advantage over Delta in vaccinated persons

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continues to shape the coronavirus disease 2019 (Covid-19) pandemic. The detection and rapid spread of the SARS-CoV-2 Omicron variant (lineage B.1.1.529) in Botswana and South Africa became a global concern because it contained 15 mutations in the spike protein immunogenic receptor binding domain and was less neutralized by sera derived from vaccinees compared to the previously dominant Delta variant. To investigate if Omicron is more likely than Delta to cause infections in vaccinated persons, we analyzed 37,877 nasal swab PCR tests conducted from 12-26 December 2021 and calculated the test positivity rates for each variant by vaccination status. We found that the positivity rate among unvaccinated persons was higher for Delta (5.2%) than Omicron (4.5%). We found similar results in persons who received a single vaccine dose. Conversely, our results show that Omicron had higher positivity rates than Delta among those who received two doses within five months (Omicron = 4.7% vs. Delta = 2.6%), two doses more than five months ago (4.2% vs. 2.9%), and three vaccine doses (2.2% vs. 0.9%). Our estimates of Omicron positivity rates in persons receiving one or two vaccine doses were not significantly lower than unvaccinated persons but were 49.7% lower after three doses. In comparison, the reduction in Delta positivity rates from unvaccinated to 2 vaccine doses was 45.6-49.6% and to 3 vaccine doses was 83.2%. Despite the higher positivity rates for Omicron in vaccinated persons, we still found that 91.2% of the Omicron infections in our study occurred in persons who were eligible for 1 or more vaccine doses at the time of PCR testing. In conclusion, escape from vaccine-induced immunity likely contributed to the rapid rise in Omicron infections.

Immunogenicity of heterologous BNT162b2 booster in fully vaccinated individuals with CoronaVac against SARS-CoV-2 variants Delta and Omicron: the Dominican Republic Experience

The recent emergence of the SARS-CoV-2 Omicron variant is raising concerns because of its increased transmissibility and by its numerous spike mutations with potential to evade neutralizing antibodies elicited by COVID-19 vaccines. The Dominican Republic was among the first countries in recommending the administration of a third dose COVID-19 vaccine to address potential waning immunity and reduced effectiveness against variants. Here, we evaluated the effects of a heterologous BNT162b2 mRNA vaccine booster on the humoral immunity of participants that had received a two-dose regimen of CoronaVac, an inactivated vaccine used globally. We found that heterologous CoronaVac prime followed by BNT162b2 booster regimen induces elevated virus-specific antibody levels and potent neutralization activity against the ancestral virus and Delta variant, resembling the titers obtained after two doses of mRNA vaccines. While neutralization of Omicron was undetectable in participants that had received a two-dose regimen of CoronaVac vaccine, BNT162b2 booster resulted in a 1.4-fold increase in neutralization activity against Omicron, compared to two-dose mRNA vaccine. Despite this increase, neutralizing antibody titers were reduced by 6.3-fold and 2.7-fold for Omicron compared to ancestral and Delta variant, respectively. Surprisingly, previous SARS-CoV-2 infection did not affect the neutralizing titers for Omicron in participants that received the heterologous regimen. Our findings have immediate implications for multiples countries that previously used a two-dose regimen of CoronaVac and reinforce the notion that the Omicron variant is associated with immune escape from vaccines or infection-induced immunity, highlighting the global need for vaccine boosters to combat the impact of emerging variants.

Comparative transmissibility of SARS-CoV-2 variants Delta and Alpha in New England, USA

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant quickly rose to dominance in mid-2021, displacing other variants, including Alpha. Studies using data from the United Kingdom and India estimated that Delta was 40-80% more transmissible than Alpha, allowing Delta to become the globally dominant variant. However, it was unclear if the ostensible difference in relative transmissibility was due mostly to innate properties of Deltas infectiousness or differences in the study populations. To investigate, we formed a partnership with SARS-CoV-2 genomic surveillance programs from all six New England US states. By comparing logistic growth rates, we found that Delta emerged 37-163% faster than Alpha in early 2021 (37% Massachusetts, 75% New Hampshire, 95% Maine, 98% Rhode Island, 151% Connecticut, and 163% Vermont). We next computed variant-specific effective reproductive numbers and estimated that Delta was 58-120% more transmissible than Alpha across New England (58% New Hampshire, 68% Massachusetts, 76% Connecticut, 85% Rhode Island, 98% Maine, and 120% Vermont). Finally, using RT-PCR data, we estimated that Delta infections generate on average [~]6 times more viral RNA copies per mL than Alpha infections. Overall, our evidence indicates that Deltas enhanced transmissibility could be attributed to its innate ability to increase infectiousness, but its epidemiological dynamics may vary depending on the underlying immunity and behavior of distinct populations.

Variant abundance estimation for SARS-CoV-2 in wastewater using RNA-Seq quantification

Effectively monitoring the spread of SARS-CoV-2 variants is essential to efforts to counter the ongoing pandemic. Wastewater monitoring of SARS-CoV-2 RNA has proven an effective and efficient technique to approximate COVID-19 case rates in the population. Predicting variant abundances from wastewater, however, is technically challenging. Here we show that by sequencing SARS-CoV-2 RNA in wastewater and applying computational techniques initially used for RNA-Seq quantification, we can estimate the abundance of variants in wastewater samples. We show by sequencing samples from wastewater and clinical isolates in Connecticut U.S.A. between January and April 2021 that the temporal dynamics of variant strains broadly correspond. We further show that this technique can be used with other wastewater sequencing techniques by expanding to samples taken across the United States in a similar timeframe. We find high variability in signal among individual samples, and limited ability to detect the presence of variants with clinical frequencies <10%; nevertheless, the overall trends match what we observed from sequencing clinical samples. Thus, while clinical sequencing remains a more sensitive technique for population surveillance, wastewater sequencing can be used to monitor trends in variant prevalence in situations where clinical sequencing is unavailable or impractical.

Impact of circulating SARS-CoV-2 variants on mRNA vaccine-induced immunity in uninfected and previously infected individuals

The emergence of SARS-CoV-2 variants with mutations in major neutralizing antibody-binding sites can affect humoral immunity induced by infection or vaccination1-6. We analysed the development of anti-SARS-CoV-2 antibody and T cell responses in previously infected (recovered) or uninfected (naive) individuals that received mRNA vaccines to SARS-CoV-2. While previously infected individuals sustained higher antibody titers than uninfected individuals post-vaccination, the latter reached comparable levels of neutralization responses to the ancestral strain than previously infected individuals 7 days after the second vaccine dose. T cell activation markers measured upon spike or nucleocapsid peptide in vitro stimulation showed a progressive increase after vaccination in the time-points analysed. Comprehensive analysis of plasma neutralization using 16 authentic isolates of distinct locally circulating SARS-CoV-2 variants revealed a range of reduction in the neutralization capacity associated with specific mutations in the spike gene: lineages with E484K and N501Y/T (e.g., B.1.351 and P.1) had the greatest reduction, followed by lineages with L452R (e.g., B.1.617.2) or with E484K (without N501Y/T). While both groups retained neutralization capacity against all variants, plasma from previously infected vaccinated individuals displayed overall better neutralization capacity when compared to plasma from uninfected individuals that also received two vaccine doses, pointing to vaccine boosters as a relevant future strategy to alleviate the impact of emerging variants on antibody neutralizing activity.

Combining genomic and epidemiological data to compare the transmissibility of SARS-CoV-2 lineages

Emerging SARS-CoV-2 variants have shaped the second year of the COVID-19 pandemic and the public health discourse around effective control measures. Evaluating the public health threat posed by a new variant is essential for appropriately adapting response efforts when community transmission is detected. However, this assessment requires that a true comparison can be made between the new variant and its predecessors because factors other than the virus genotype may influence spread and transmission. In this study, we develop a framework that integrates genomic surveillance data to estimate the relative effective reproduction number (Rt) of co-circulating lineages. We use Connecticut, a state in the northeastern United States in which the SARS-CoV-2 variants B.1.1.7 and B.1.526 co-circulated in early 2021, as a case study for implementing this framework. We find that the Rt of B.1.1.7 was 6-10% larger than that of B.1.526 in Connecticut in the midst of a COVID-19 vaccination campaign. To assess the generalizability of this framework, we apply it to genomic surveillance data from New York City and observe the same trend. Finally, we use discrete phylogeography to demonstrate that while both variants were introduced into Connecticut at comparable frequencies, clades that resulted from introductions of B.1.1.7 were larger than those resulting from B.1.526 introductions. Our framework, which uses open-source methods requiring minimal computational resources, may be used to monitor near real-time variant dynamics in a myriad of settings.

Sequencing SARS-CoV-2 Genomes from Saliva

Genomic sequencing is crucial to understanding the epidemiology and evolution of SARS-CoV-2. Often, genomic studies rely on remnant diagnostic material, typically nasopharyngeal swabs, as input into whole genome SARS-CoV-2 next-generation sequencing pipelines. Saliva has proven to be a safe and stable specimen for the detection of SARS-CoV-2 RNA via traditional diagnostic assays, however saliva is not commonly used for SARS-CoV-2 sequencing. Using the ARTIC Network amplicon-generation approach with sequencing on the Oxford Nanopore MinION, we demonstrate that sequencing SARS-CoV-2 from saliva produces genomes comparable to those from nasopharyngeal swabs, and that RNA extraction is necessary to generate complete genomes from saliva. In this study, we show that saliva is a useful specimen type for genomic studies of SARS-CoV-2.

A stem-loop RNA RIG-I agonist confers prophylactic and therapeutic protection against acute and chronic SARS-CoV-2 infection in mice

As SARS-CoV-2 continues to cause morbidity and mortality around the world, there is an urgent need for the development of effective medical countermeasures. Here, we assessed the antiviral capacity of a minimal RIG-I agonist, stem-loop RNA 14 (SLR14), in viral control, disease prevention, post-infection therapy, and cross-variant protection in mouse models of SARS-CoV-2 infection. A single dose of SLR14 prevented viral replication in the lower respiratory tract and development of severe disease in a type I interferon (IFN-I) dependent manner. SLR14 demonstrated remarkable protective capacity against lethal SARS-CoV-2 infection when used prophylactically and retained considerable efficacy as a therapeutic agent. In immunodeficient mice carrying chronic SARS-CoV-2 infection, SLR14 elicited near-sterilizing innate immunity by inducing IFN-I responses in the absence of the adaptive immune system. In the context of infection with variants of concern (VOC), SLR14 conferred broad protection and uncovered an IFN-I resistance gradient across emerging VOC. These findings demonstrate the therapeutic potential of SLR14 as a host-directed, broad-spectrum antiviral for early post-exposure treatment and for treatment of chronically infected immunosuppressed patients.

Early introductions and community transmission of SARS-CoV-2 variant B.1.1.7 in the United States

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2500 COVID-19 cases associated with this variant have been detected in the US since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight the primary ports of entry for B.1.1.7 in the US and locations of possible underreporting of B.1.1.7 cases. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern

With the emergence of SARS-CoV-2 variants that may increase transmissibility and/or cause escape from immune responses1-3, there is an urgent need for the targeted surveillance of circulating lineages. It was found that the B.1.1.7 (also 501Y.V1) variant first detected in the UK4,5 could be serendipitously detected by the ThermoFisher TaqPath COVID-19 PCR assay because a key deletion in these viruses, spike {Delta}69-70, would cause a "spike gene target failure" (SGTF) result. However, a SGTF result is not definitive for B.1.1.7, and this assay cannot detect other variants of concern that lack spike {Delta}69-70, such as B.1.351 (also 501Y.V2) detected in South Africa6 and P.1 (also 501Y.V3) recently detected in Brazil7. We identified a deletion in the ORF1a gene (ORF1a {Delta}3675-3677) in all three variants, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a {Delta}3675-3677 as the primary target and spike {Delta}69-70 to differentiate, we designed and validated an open source PCR assay to detect SARS-CoV-2 variants of concern8. Our assay can be rapidly deployed in laboratories around the world to enhance surveillance for the local emergence spread of B.1.1.7, B.1.351, and P.1.