RNA extraction and RT-(semi-)nested-PCR

Viral RNA in the virus concentrates obtained in U.S. and Nepal was extracted using a ZR Viral DNA/RNA Kit (Zymo Research, Irvine, CA, USA) and QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), respectively. Extracted RNA was subjected to RT using a High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. Subsequently, (semi-)nested PCR assays targeting the ORF1b-ORF2 junction regions of CAstV, MLB-AstV, and VA-AstV were performed separately as described previously¹⁷ (Table ^S2 in the Supplementary Information). Amplification of each target gene was confirmed by visualization under a UV lamp after electrophoresis in a 1.5% agarose gel stained by GelRed^TM (Wako, Osaka, Japan). In the first-round PCR, primers AHAstVF1 and AHAstVR1, SF0073 and AHMLBR1, and AHVAF1 and AHVAR1 were used to amplify CAstV, MLB-, and VA-AstV genes, respectively. In the second-round PCR, primers AHAstVF2 and AHAstVR2, F0073 and AHMLBR2, and AHVAF2 and AHVAR2 were used to amplify the first PCR amplicons of CAstV, MLB-, and VA-AstV genes, respectively. Resultant second-round PCR amplicon sizes of CAstV, MLB-AstV, and VA-AstV genes were expected to be 407, 689, and 663 bp, respectively.