Tracking cryptic SARS-CoV-2 lineages detected in NYC wastewater

Davida S Smyth,Anna Gao,Marc C Johnson,Kristen Cheung,Caitlyn Guldenpfennig,Michelle Markman,Maddie Graham,Clayton Rushford,Mónica Trujillo ,Reinier Suarez,Irene Hoxie,Yue Guan,Fabrizio Spagnolo,Kaung Myat San,Sherin Kannoly,Terri D Lyddon,Mark A Daniëls ,Nanami Kubota,Devon A Gregory,Geena Sompanya,Emma Teixeiro,John J Dennehy

doi:10.1038/s41467-022-28246-3

Abstract

Tracking SARS-CoV-2 genetic diversity is strongly indicated because diversifying selection may lead to the emergence of novel variants resistant to naturally acquired or vaccine-induced immunity. To monitor New York City (NYC) for the presence of novel variants, we deep sequence most of the receptor binding domain coding sequence of the S protein of SARS-CoV-2 isolated from the New York City wastewater. Here we report detecting increasing frequencies of novel cryptic SARS-CoV-2 lineages not recognized in GISAID’s EpiCoV database. These lineages contain mutations that had been rarely observed in clinical samples, including Q493K, Q498Y, E484A, and T572N and share many mutations with the Omicron variant of concern. Some of these mutations expand the tropism of SARS-CoV-2 pseudoviruses by allowing infection of cells expressing the human, mouse, or rat ACE2 receptor. Finally, pseudoviruses containing the spike amino acid sequence of these lineages were resistant to different classes of receptor binding domain neutralizing monoclonal antibodies. We offer several hypotheses for the anomalous presence of these lineages, including the possibility that these lineages are derived from unsampled human COVID-19 infections or that they indicate the presence of a non-human animal reservoir.

Highlights

The cohort of participants were selected based on equivalent levels of antibodies to SARS-CoV-2 RBD, age, or gender did not contribute to differences between samples (Table 2)
A tertiary PCR (50 μL) was performed to add adapter sequences required for Illumina cluster generation with forward and reverse primers (0.2 μM each), dNTPs (200 μM each) (New England Biolabs, N0447L) and Phusion High-Fidelity DNA Polymerase (1U) (New England Biolabs, M0530L)
Pooled amplicons were purified by addition of Axygen AxyPrep MagPCR Clean-up beads (Axygen, MAGPCR-CL-50) in a 1.0 ratio to purify final amplicons

Summary

Introduction

Primary RT-PCR amplification was performed as follows: 25 °C (2:00) + 50 °C (20:00) + 95 °C (2:00) + [95 °C (0:15) + 55 °C (0:30) + 72 °C (1:00)] × 25 cycles using the MiSeq primary PCR primers (Supplementary Table 1). Secondary PCR (25 μL) was performed on RBD amplifications using 5 μL of the primary PCR as template with MiSeq nested gene specific primers containing 5′ adapter sequences (Supplementary Table 1) (0.5 μM each), dNTPs (100 μM each) (New England Biolabs, N0447L) and Q5 DNA polymerase (New England Biolabs, M0541S). A tertiary PCR (50 μL) was performed to add adapter sequences required for Illumina cluster generation with forward and reverse primers (0.2 μM each), dNTPs (200 μM each) (New England Biolabs, N0447L) and Phusion High-Fidelity DNA Polymerase (1U) (New England Biolabs, M0530L).

Methods

Results

Conclusion