Evolutionary Trajectories of SARS-CoV-2 Alpha and Delta Variants in White-Tailed Deer in Pennsylvania

Introductory paragraphThe SARS-CoV-2 pandemic likely began by viral spillover from animals to humans1-3; today multiple animal species are known to be susceptible to infection4-8. White-tailed deer, Odocoileus virginianus are infected in North America at substantial levels9-11, and genomic data suggests that a variant in deer may have spilled back to humans12,13. Here we characterize SARS-CoV-2 in deer from Pennsylvania (PA) sampled during fall and winter 2021. Of 123 nasal swab samples analyzed by RT-qPCR, 20 (16.3%) were positive for SARS-CoV-2. Seven whole-genome sequences were obtained, together with six more partial spike sequences. These annotated as alpha and delta variants, the first reported observations of these lineages in deer, documenting multiple new jumps from humans to deer. The alpha lineage persisted in deer after its displacement by delta in humans, and deer-derived alpha variants diverged significantly from those in humans, consistent with a distinctive evolutionary trajectory in deer.

Detection and Interspecies Comparison of SARS-CoV-2 Delta Variant (AY.3) in Feces from a Domestic Cat and Human Samples

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have spilled over from humans to companion and wild animals since the inception of the global COVID-19 pandemic. However, whole genome sequencing data of the viral genomes that infect non-human animal species has been scant. Here, we detected and sequenced a SARS-CoV-2 delta variant (AY.3) in fecal samples from an 11-year-old domestic house cat previously exposed to an owner who tested positive for SARS-CoV-2. Molecular testing of two fecal samples collected 7 days apart yielded relatively high levels of viral RNA. Sequencing of the feline-derived viral genomes showed the two to be identical, and differing by between 4 and 14 single nucleotide polymorphisms in pairwise comparisons to human-derived lineage AY.3 sequences collected in the same geographic area and time period. However, several mutations unique to the feline samples reveal their divergence from this cohort on phylogenetic analysis. These results demonstrate continued spillover infections of emerging SARS-CoV-2 variants that threaten human and animal health, as well as highlight the importance of collecting fecal samples when testing for SARS-CoV-2 in animals. To the authors knowledge, this is the first published case of a SARS-CoV-2 delta variant in a domestic cat in the United States.

SARS-CoV-2 variants associated with vaccine breakthrough in the Delaware Valley through summer 2021

The severe acute respiratory coronavirus-2 (SARS-CoV-2) is the cause of the global outbreak of COVID-19. Evidence suggests that the virus is evolving to allow efficient spread through the human population, including vaccinated individuals. Here we report a study of viral variants from surveillance of the Delaware Valley, including the city of Philadelphia, and variants infecting vaccinated subjects. We sequenced and analyzed complete viral genomes from 2621 surveillance samples from March 2020 to September 2021 and compared them to genome sequences from 159 vaccine breakthroughs. In the early spring of 2020, all detected variants were of the B.1 and closely related lineages. A mixture of lineages followed, notably including B.1.243 followed by B.1.1.7 (alpha), with other lineages present at lower levels. Later isolations were dominated by B.1.617.2 (delta) and other delta lineages; delta was the exclusive variant present by the last time sampled. To investigate whether any variants appeared preferentially in vaccine breakthroughs, we devised a model based on Bayesian autoregressive moving average logistic multinomial regression to allow rigorous comparison. This revealed that B.1.617.2 (delta) showed three-fold enrichment in vaccine breakthrough cases (odds ratio of 3; 95% credible interval 0.89-11). Viral point substitutions could also be associated with vaccine breakthroughs, notably the N501Y substitution found in the alpha, beta and gamma variants (odds ratio 2.04; 95% credible interval of 1.25-3.18). This study thus provides a detailed picture of viral evolution in the Delaware Valley and a geographically matched analysis of vaccine breakthroughs; it also introduces a rigorous statistical approach to interrogating enrichment of viral variants. ImportanceSARS-CoV-2 vaccination is highly effective at reducing viral infection, hospitalization and death. However, vaccine breakthrough infections have been widely observed, raising the question of whether particular viral variants or viral mutations are associated with breakthrough. Here we report analysis of 2621 surveillance isolates from people diagnosed with COVID-19 in the Delaware Valley in South Eastern Pennsylvania, allowing rigorous comparison to 159 vaccine breakthrough case specimens. Our best estimate is a three-fold enrichment for some lineages of delta among breakthroughs, and enrichment of a notable spike substitution, N501Y. We introduce statistical methods that should be widely useful for evaluating vaccine breakthroughs and other viral phenotypes.

Molecular beacons allow specific RT-LAMP detection of B.1.1.7 variant SARS-CoV-2

Over the course of the COVID-19 pandemic, several SARS-CoV-2 genetic variants of concern have appeared and spread throughout the world. Detection and identification of these variants is important to understanding and controlling their rapid spread. Current detection methods for a particularly concerning variant, B.1.1.7, require expensive qPCR machines and depend on the absence of a signal rather than a positive indicator of variant presence. Here we report an assay using a pair of molecular beacons paired with reverse transcription loop mediated amplification to allow isothermal amplification from saliva to specifically detect B.1.1.7 and other variants which contain a characteristic deletion in the gene encoding the viral spike protein. This assay is specific, affordable and allows multiplexing with other SARS-CoV-2 LAMP primer sets.

LAMP-BEAC: Detection of SARS-CoV-2 RNA Using RT-LAMP and Molecular Beacons

BackgroundRapid spread of SARS-CoV-2 has led to a global pandemic, resulting in the need for rapid assays to allow diagnosis and prevention of transmission. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) provides a gold standard assay for SARS-CoV-2 RNA, but tests are expensive and supply chains are potentially fragile, motivating interest in additional assay methods. Reverse Transcription and Loop-Mediated Isothermal Amplification (RT-LAMP) provides an alternative that uses orthogonal and often less expensive reagents without the need for thermocyclers. The presence of SARS-CoV-2 RNA is typically detected using dyes to report bulk amplification of DNA; however, a common artifact is nonspecific DNA amplification, which complicates detection. ResultsHere we describe the design and testing of molecular beacons, which allow sequence-specific detection of SARS-CoV-2 genomes with improved discrimination in simple reaction mixtures. To optimize beacons for RT-LAMP, multiple locked nucleic acid monomers were incorporated to elevate melting temperatures. We also show how beacons with different fluorescent labels can allow convenient multiplex detection of several amplicons in "single pot" reactions, including incorporation of a human RNA LAMP-BEAC assay to confirm sample integrity. Comparison of LAMP-BEAC and RT-qPCR on clinical saliva samples showed good concordance between assays. To facilitate implementation, we developed custom polymerases for LAMP-BEAC and inexpensive purification procedures, which also facilitates increasing sensitivity by increasing reaction volumes. ConclusionsLAMP-BEAC thus provides an affordable and simple SARS-CoV-2 RNA assay suitable for population screening; implementation of the assay has allowed robust screening of thousands of saliva samples per week.