Quantstudio design and analysis software version 1

RNA was isolated from the cells using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. UltraPlex 1-Step ToughMix (QuantaBio, Beverly, MA, USA) was used along with 2019-nCoV CDC Probe and Primer Kit for SARS-CoV-2 (Catalog: KIT-nCoV-PP1-1000) for the CoV-2 qPCR reactions. Human ACE-2 primer/probe and 18S Ribosomal RNA control (Applied Biosystems, Waltham, MA, USA) were used for the hACE-2 qPCR reactions. QuantStudio 3 Real-Time PCR machine (Applied Biosystems, Waltham, MA, USA) was used with QuantStudio Design and Analysis software version 1.5.1 (Applied Biosystems, Waltham, MA, USA) for analysis. Results are expressed as CoV-2 or ACE-2 expression determined using 2^−(ΔCt) method with 18S ribosome as the endogenous control.

Cells were scraped and collected in Buffer AVL with carrier RNA (Qiagen, Hilden, Germany). RNA was then isolated from the samples using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer’s instructions. RNA was also isolated from a heat-inactivated cell lysate and supernate containing SARS-CoV-2 isolate USA-WAI/2020 (BEI Resources, Manassas, VA, USA) with known genome equivalents of virus, which was used as a standard during qPCR.
UltraPlex 1-Step ToughMix (QuantaBio, Beverly, MA, USA) was used along with 2019-nCoV CDC Probe and Primer Kit for SARS-CoV-2 (Catalog: KIT-nCoV-PP1-1000) for the qRT-PCR reactions. QuantStudio 3 Real-Time PCR machine (Applied Biosystems, Waltham, MA, USA) was used with QuantStudio Design and analysis software version 1.5.1 (Applied Biosystems, Waltham, MA, USA) for analysis. Results are expressed as log of genome equivalents/mL.

DNA extracts from filters from the core sampling (see above) were diluted 100 times, and 1 µL was then used for qPCR analysis. EhV abundance was determined by qPCR for the major capsid protein (mcp) gene: 5′-acgcaccctcaatgtatggaagg-3′ (mcp1F) and 5′-rtscrgccaactcagcagtcgt -3′ (mcp94Rv). All reactions were carried out in technical triplicates using water as a negative control. For all reactions, Platinum SYBER Green qPCR SuperMix-UDG with ROX (Invitrogen, Carlsbad, CA, USA) was used as described by the manufacturer. Reactions were performed on a QuantStudio 5 Real-Time PCR System equipped with the QuantStudio Design and Analysis Software version 1.5.1 (Applied Biosystems, Foster City, CA, USA) as follows: 50 °C for 2 min, 95 °C for 5 min, 40 cycles of 95 °C for 15 s, and 60 °C for 30 s. Results were calibrated against serial dilutions of EhV201 DNA at known concentrations, enabling exact enumeration of viruses. Samples showing multiple peaks in melting curve analysis or peaks that were not corresponding to the standard curves were omitted. Data are available in ref. ⁷⁴ . A comparison of viral counts based on flow-cytometry and qPCR is shown in Supplementary Fig. 2.