Wild poliovirus type 1 (WPV1) and wild poliovirus type 3 (WPV3) cell culture isolates used in this study were WEAF-B (West African group B) polioviruses isolated from two stool specimens from Nigeria. Sabin 1 (SL1), Sabin 2 (SL2), and Sabin 3 (SL3) viruses were obtained front the National Institute for Biological Standards and Control (NIBSC, Hertfordshire, United Kingdom). To prepare WPV1, WPV3, SL1, SL2, and SL3 virus stocks, human rhabdomyosarcoma cells (RD) cells were seeded in 75 cm² culture flasks at a density of 3.0 × 10⁵ cells/mL in growth media (minimum essential medium with Earle’s salts [MEM; Gibco, Rockville, MD], supplemented with 1 M HEPES, 1.5 g/L sodium bicarbonate, 100 U/mL penicillin/streptomycin, and 10 % fetal bovine serum) and incubated at 37 °C in a CO₂ incubator. Cells were grown to about 90 % confluency, and culture media was then replaced with maintenance media (MEM with Earle’s salts supplemented with 1 M HEPES, 1.5 g/L sodium bicarbonate, 100 U/mL penicillin/streptomycin, and 2 % fetal bovine serum). Cells were inoculated at multiplicity of infection of 0.2 with WPV1, WPV3, SL1, SL2, and SL3 isolates and incubated for 3 days at 37 °C in a CO₂ incubator. Flasks were frozen and thawed twice to lyse cells and release virus. Supernatants were collected and centrifuged at 10,000 x g for 15 min to pellet debris. Clarified virus stocks were titered, aliquoted and stored at −20 °C until further use.

To prepare 10 % stool suspensions, approximately 0.5 g of stool was added to 5 mL of serum-free MEM and 0.5 g of glass beads. Chloroform (250 μL) was added and the suspension was shaken for 30 min on a mechanical shaker (Heidolph North America, Wood Dale, IL). Following shaking, the suspension was centrifuged at 1800 x g for 30 min at 4 °C to pellet insoluble material. Clarified suspensions were titered, aliquoted, and stored at −20 °C until use. RNA was extracted front each stool suspension using the Quick Viral RNA kit (Zymo Research, Irvine, CA) and the poliovirus serotype and genotype were confirmed by rRT-PCR (Gerloff et al., 2017).

Extraction buffers and kits used in this study include: Trizol LS (Cat No. 10296098; Thermo Fisher, Carlsbad, CA); QIAamp Viral RNA Mini Kit (Cat No. 52906; QIAGEN, Germantown, MD); Quick-RNA Viral Kit (Cat No. R1035; Zymo Research); High Pure Viral Nucleic Acid Kit (Cat. No. 11858874001; Roche, Wilmington, MA); MagMAX Lysis/Binding Buffer (Cat. No. 8500; part of the MagMAX Pathogen RNA/DNA Kit; Applied Biosystems, Foster City, CA); UNEX (Cat No. 726288-260; VWR, Radnor, PA). The QIAamp Viral RNA Mini Kit is the most commonly used kit for poliovirus RNA extraction from cell culture isolates in the Global Poliovirus Laboratory Network (GPLN). Trizol LS and the Qiagen Viral RNA Mini Kit are popular among many non-poliovirus laboratories for RNA extraction and have been used by several inactivation studies (Esona et al., 2013; Kochel et al., 2017; Ngo et al., 2017). Universal extraction buffer (UNEX) was developed at the Centers for Disease Control and Prevention for nucleic acid extraction from environmental samples and has previously been shown to inactivate SL1 (Cromeans et al., 2019; Hill et al., 2015). Guanidine thiocyanate (Cat. No. G9277), 2-(N-morpholino)ethanesulfonic acid (MES) hydrate (Cat No. M8250), ethanol (Cat. No. E7023), and Amicon Ultra-15 100,000 NMWL centrifugal filters (Cat. No. UFC9100) were all purchased from Millipore Sigma (St. Louis, MO).

One volume (200 μL) of sample was mixed and incubated with selected commercial nucleic acid extraction buffers according to the sample-to-buffer ratios outlined in the kit protocol (Table 1) or with four volumes of 4 M guanidine thiocyanate (in 0.1 M MES, pH 6.0) for 5–30 min at room temperature (24–26 °C). Ethanol was added after mixing the sample with Buffer AVL, UNEX Buffer, or 4 M guanidine thiocyanate buffer. To remove the extraction buffer and reduce cytotoxicity, samples were diluted in 10–12 mL PBS and centrifuged in an Amicon Ultra-15 100,000 NMWL filter at 1800 x g for 10 min. This process was repeated 2–4 additional times until the residual extraction buffer was removed. The resulting retentates, ranging front 120–200 μL, were used in CCID₅₀ assays or tested in blind passages to assess the extent of inactivation.

For CCID₅₀ assays, RD cells were diluted in maintenance medium and seeded at 1.5 × 10⁴ cells per well in 96-well plates. Treated samples were serially diluted 1:10 in serum-free MEM and added (100 μL per well) to cells freshly plated in maintenance medium. For each experiment, a positive control in which virus was incubated with PBS alone in addition to a negative control consisting of extraction buffer alone was included to assess cytotoxicity; controls were treated identically to the virus samples, including buffer exchange by centrifugal filtration. Plates were incubated at 37 °C and monitored for cytopathic effect (CPE) after 5 days. Viral titer was calculated according to the Spearman-Kärber method (World Health Organization, 2004). For experiments with treated stool suspensions, RD cells were seeded at 1.25 × 10⁵ cells per well in 24-well plates and incubated at 37 °C. When cells reached at least 90 % confluence, the medium was replaced with maintenance medium. Half of each treated stool suspension or control was added undiluted to duplicate wells. Wells were visually scored for the presence or absence of CPE using an inverted microscope after plates were incubated for 5 days at 37 °C. For blind passage experiments, RD cells were seeded in 25 cm² flasks at 1.25 × 10⁶ cells per flask and grown until 90 % confluence. The medium was replaced by 5 mL of maintenance medium prior to addition of treated samples. Inoculated flasks were checked for CPE using an inverted microscope after incubation at 37 °C for 5 days. Approximately 1 mL of supernatant from a negative flask was transferred to a fresh confluent flask containing 4 mL of maintenance medium. These flasks were monitored for CPE using an inverted microscope after another incubation for 5 days at 37 °C.

Several commercial nucleic acid extraction buffers were selected (Table 1) to initially test whether they could inactivate a high titer (10^8.6 CCID₅₀/0.1 mL) WPV1 isolate. All buffers used in this study contain at least 4 M guanidine thiocyanate, with the exception of Trizol LS, which contains 1.3–3.4 M guanidine thiocyanate, and High Pure Binding Buffer, which has none (Table 1). Each extraction buffer was mixed with 200 μL of WPV1 isolate according to the sample-to-buffer ratios recommended by the manufacturers (Table 1) and incubated for 30 min to ensure maximal inactivation. Samples were then diluted with PBS, the lysis buffer was removed using an Amicon filter, and virus was titered in a CCID₅₀ assay. Treatment with Viral RNA Buffer, Trizol LS, MagMAX Lysis Buffer alone inactivated WPV1 below the limit of detection of the assay (10^0.5 CCID₅₀/0.1 mL; Table 2). Incubation with Buffer AVL or UNEX alone reduced the WPV1 titer by 5.4 and 7.2 log₁₀ respectively, but the addition of ethanol (to a final concentration of 44 % (v/v) or 50 % (v/v), respectively) was necessary to reduce WPV1 titer below the limit of detection. Moreover, WPV3, SL1, and SL3 isolates were also inactivated by Buffer AVL with the addition of ethanol to 44 % v/v (Fig. 1). Contrary to the other buffers tested, High Pure Binding Buffer had no effect on WPV1 titer. Proteinase K treatment after incubation in High Pure Binding Buffer, however, was sufficient to fully inactivate the virus.

Because the chemical composition of commercial nucleic extraction buffers varies widely, we next determined whether guanidine thiocyanate alone could inactivate WPV1 isolate. Guanidine thiocyanate concentrations typically used for nucleic acid extractions and found in commercial buffer formulations (see Table 1) range between 4 M (~50 %) and 6 M (~70 %). For this experiment, we chose to test the concentration of 4 M guanidine thiocyanate, which is the lowest concentration in nucleic acid extraction buffers. Four volumes (800 μL) of 4 M guanidine thiocyanate (in 0.1 MES, pH 6.0) were added to 1 vol of WPV1 isolate (200 μL), similar to the QIAamp Viral RNA Mini Kit protocol (Fig. 2). An increasing amount of ethanol (10–50 % v/v final concentration) was also added to assess potential enhancement of inactivation. Ethanol is often added as a part of RNA extraction procedures to facilitate nucleic acid binding to silica columns. It is also a partial protein denaturant and has been shown to inactivate poliovirus at high concentrations (Roberts and Lloyd, 2007). After a 30-minute incubation at room temperature (24–26 °C), residual buffer in the samples was removed by ultrafiltration and the viral titers determined by CCID₅₀ assay. Incubation with 4 M guanidine thiocyanate alone reduced the viral titer by more than 4 log₁₀ (Fig. 2). Adding ethanol to a final concentration of 10 % further reduced the viral titer by approximately 1 log₁₀. Viral titer was below the limit of detection after the addition of at least a 20 % final concentration of ethanol.

Next, nucleic acid extraction buffers were evaluated for their effectiveness in inactivating poliovirus in stool suspensions. Although stool suspensions typically have viral titers lower than isolates, increased organic load due to the presence of bile salts and other compounds may impair viral inactivation. The Quick-RNA Viral Kit and QIAamp Viral RNA Mini Kit were selected for further analysis, as both are widely used by the GPLN and non-poliovirus laboratories for viral RNA extractions from stool suspensions and isolates. Viral RNA Buffer and Buffer AVL with ethanol were evaluated for their ability to inactivate poliovirus in 10 % stool suspensions consisting of either WPV1, SL1, SL3, SL2 or SL2 isolate. The 4 M guanidine thiocyanate buffer with 20 % ethanol was also tested for comparison. The viral titers of the suspensions ranged from to 1.4–4.5 log CCID₅₀ /0.1 mL. One volume (200 μL) of each stool suspension was combined with three volumes of Viral RNA Buffer, four volumes of Buffer AVL and ethanol (44 % final concentration), four volumes of 4 M guanidine thiocyanate in 0.1 M MES, pH 6.0 and ethanol (20 % final concentration), or four volumes of PBS. Samples were incubated for 5 min (Viral RNA Buffer and Buffer AVL with 44 % ethanol) or 30 min (4 M guanidine thiocyanate with 20 % ethanol). To increase sensitivity, treated stool suspensions were concentrated by ultrafiltration and added undiluted to RD cells. There was no CPE in any of the cultures following treatment of the samples with any of the buffers at Day 5, in contrast to those treated with PBS (Table 3).

Finally, as further confirmation of the effectiveness of these methods to inactivate poliovirus in stool suspensions and to increase the possibility of detecting any remaining viable virus, blind passage experiments were performed. A high-titer WPV1 virus stock was spiked into enterovirus-negative stool suspension. Extraction buffers were added to the resulting spiked suspension (10⁷ CCID₅₀ /0.1 mL) and incubated for the minimum amount of time to achieve inactivation previously determined in CCID₅₀ assays (Table 5). Treated suspensions were processed by ultrafiltration, added to RD cells, and CPE was recorded after 5 days. Supernatants from negative flasks were then passaged onto a fresh cell monolayer and the cells were monitored for CPE for an additional 5 days. There was no CPE detected in the first or second passages of WPV1-spiked stool suspension treated with any of the commercial extraction buffers with the incubation times tested (Table 4). However, viable virus was present in the first passage after treatment with a buffer containing 4 M guanidine thiocyanate with 20 % ethanol. Increasing the final ethanol concentration to 30 % with 4 M guanidine thiocyanate resulted in no detectable CPE in the first or second passages.