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

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.

Fig. 1. Novel SARS-CoV-2 lineages from wastewater.

A Schematic of SARS-CoV-2 and the amplification locations. B Distribution of SARS-COV-2 variants based on patient sequences (patient data obtained from GISAID) and wastewater surveillance. Polymorphisms detected from amplicon sequencing that were used to assign sequences to lineages are shown in the legend. The variants detected from the 14 NYC WWTPs were weighted by flowrate to generate a city-wide average distribution. C Novel lineages detected from WWTPs. Schematic highlights shared sequences identified from WWTP 10, 11, and 3 are shown. The percent of the sequences from each date that contained the indicated polymorphisms is shown below each lineage. The viral copies/L corresponding to each date are shown in Supplementary Table 3. Some sequences have additional polymorphisms not listed. WNY lineage designations are shown for sequences used for tropism and antibody neutralization analysis. Source data are provided as a Source Data file.

Fig. 2. ACE2 usage by WNY lineages.

A Schematic of lineages and pseudovirion production. WNY1 = E484A/F486P/S494P/Q498Y/H519N/F572N, WNY2 = Q493K/S494P/Q498Y/H519N/T572N, WNY3 = K417T/K444T/E484A/F590Y/Q498H, WNY4 = K417T/N439K/K444N/Y449R/L452R/N460K/S477N/Δ484/F486V/S494T/G496V/Q498Y/N501T/G504D/505H/H519Q. The indicated mutations were introduced into a codon-optimized SARS-COV-2 expression construct. These constructs were used to produce lentiviral pseudovirions containing a Gaussia luciferase reporter. Pseudoviruses containing SARS-COV-2 Spike with N501Y/A570D were used as a control as this is known to be capable of infecting rodent cells. Pseudoviruses were used to transduce 293FT+TMRPSS2 stably transduced with human, mouse, or rat ACE2. The average and standard deviation from three independent experiments is shown. A two-way ANOVA revealed significant differences in receptor utilization (F = 17.81, DF = 3, 74; P < 0.0001). Source data are provided as a Source Data file.

Fig. 3. Antibody resistance to monoclonal neutralizing antibodies and patient plasma.

Lentiviral reporter pseudoviruses containing Gaussia luciferase were generated with parent (D614G), WNY1, WNY2, WNY3, or WNY4 Spike proteins. These pseudoviruses were treated with 2-fold dilutions of indicated monoclonal neutralizing antibody or patient serum and used to infect 293FT+TMPRSS2+human ACE2. Gaussia luciferase levels were quantitated approximately 2–3 days of post-transduction. Representative examples of three experiments with monoclonal antibodies performed in triplicate are shown. Infection was normalized to the wells infected with pseudovirus alone. Patient plasma Neutralization IC50 titers were calculated using nonlinear regression (Inhibitor vs. normalized response—variable slope) in GraphPad Prism 9.0. The number indicates the mean fold of reduction in IC50 and SD. Wilcoxon matched-pairs signed rank tests, a two-tailed test, were performed for paired comparisons with significance levels as follows: WNY3 patient p = 0.0049, WNY4 patient p = 0.001, and WNY4 vaccinated = 0.0068. Source data are provided as a Source Data file.