Magmax 96 viral isolation kit

For titration of infectious virus particles in bloodmeals and saliva, plaque assays were performed with duplicate serial dilutions of 125 μL inoculated on Vero cell monolayers as previously described [55 (link)]. For titration of CHIKV RNA genome copies (gc), CHIKV RNA was extracted from 200 μL of each mosquito tissue homogenate and bloodmeal. Extractions were performed with a MagMax-96 viral isolation kit (Thermo Fisher Scientific) on a MagMax Express-96 particle processer (Thermo Fisher Scientific). All extracted RNA samples were stored at -80°C. Extracted RNA was titrated in triplicate by qRT-PCR with previously described primers (CHIKV 6856, 6981, and 6919-FAM) [51 (link)] on QuantStudio 5 instruments (Thermo Fisher Scientific). Serially diluted CHIKV IVT RNA was used for standard curves. For a sample to be treated as a positive detection, all three qRT-PCR replicates were required to be positive (C_t<40). Biologically discordant qRT-PCR positive results (e.g., CHIKV RNA detected in L/W, but not body) were infrequent (44/735 samples tested) and always low CHIKV RNA titer (S1 Fig), and thus were treated as false positives and excluded from the analyses. The limit of detection was 2 plaque-forming units (PFU) per sample for plaque assays and 25 gc per sample for qRT-PCR.

Total RNA was prepared from viral isolates using a MagMAX™ 96 Viral Isolation Kit (Thermo Fisher Scientific), using 50 μL of collected medium, with KingFisher Flex (Thermo Fisher Scientific) according to the manufacturer’s instructions. Total RNA was also prepared from 100 μL of RNase-treated samples using a MagMAX™ CORE Nucleic Acid Purification Kit (Thermo Fisher Scientific), using 60 μL of elution buffer, with KingFisher Flex (Thermo Fisher Scientific), according to the manufacturer’s instructions.

RS samples were suspended in Minimum Essential Media (Invitrogen, Carlsbad, CA, USA) as a 10% fecal suspension. The RNA was extracted using the MagMax™-96 Viral Isolation kit (Ambion, Austin, TX, USA) then subjected to RT-qPCR with the primers and probe targeting the PEDV N gene [33 (link)]. The detection limit was 10 N gene copies per 20 μL of reaction, corresponding to 4.8 log₁₀ copies per mL of original fecal sample.

Two rectal swabs were suspended in 4 mL Minimum Essential Media (Invitrogen, Carlsbad, CA, USA) as a 10% fecal suspension. The RNA was extracted from 50 μL of clarified (centrifugation at 2095 × g for 30 min at 4 °C) fecal suspensions using MagMax™-96 Viral Isolation kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. The PEDV fecal shedding titers were determined by TaqMan real-time reverse transcription-PCR (RT-qPCR) with the primers and probe targeting the conserved N protein region of PEDV as described previously [23 (link)]. The detection limit was 10 GE per 20 μL of reaction, corresponding to 4.8 log₁₀ GE per mL of the original fecal samples.

Environmental swabs were analyzed for quantitative real-time polymerase chain reaction (qRT-PCR) for PEDV and PRRSV at the Kansas State University Veterinary Diagnostic Laboratory using procedures similar to those described by Elijah et al. (2022) (link). First, 50 µL of supernatant was placed in a deep well plate and RNA was extracted using a Kingfisher Flex magnetic particle processor (Fisher Scientific, Pittsburgh, PA) and a MagMAX-96 Viral Isolation Kit (Life Technologies, Grand Island, NY). The final elution volume was reduced to 60 µL, and extracted RNA was stored at −80 °C until analyzed for PEDV or PRRSV using a qRT-PCR duplex assay with a maximum cycle threshold of 45. Results were reported as the number of samples considered PCR positive and the cycle threshold (Ct) at which either PEDV or PRRSV RNA was detected.

The MagMAX™ 96 Viral Isolation Kit (Life Technologies, Waltham MA, USA) was used to obtain viral RNA from the samples, as described in the instructions provided (1836 M Revision F). A 175-μl volume of sample was used for the extraction. The magnetic bead extractions were completed on a Kingfisher96 instrument (Thermo Scientific, Waltham MA, USA).