2.5. PCR and Sequencing

All conventional PCR amplification reactions were carried out in a GeneAmp^® PCR System 2700 cycler (Applied Biosystems, Foster City, CA, USA) using 0.5 μL dNTP mix (10 mM each), 0.125 μL Taq DNA polymerase, 2.5 μL Taq 10× buffer (all from Invitrogen), 1 μL of forward and reverse primers (20 μM) (Sigma), and 2 μL of cDNA in a final volume of 25 μL.

The specific primers used for each NNV genome fragment were designed targeting highly conserved regions of the RGNNV genotype, as the most predominant worldwide and in the particular geographical area where the outbreak was located [35]. The multiple sequence alignments used to identify such regions were created using the bioinformatic tool Clustal Omega hosted at the server of the European Molecular Biology Laboratory of the European Bioinformatics Institute (EMBL-EBI, Hinxton, UK, http://www.ebi.ac.uk/Tools/msa/clustalo/) (accessed on 16 May 2021) [53]. For this task, the RGNNV complete sequences sharing less than 99% identity that were available at the GenBank database were retrieved (National Center for Biotechnology Information, NCBI, National Institutes of Health, NIH, Bethesda, MD, USA) (Tables S1 and S2 for RNA1 and RNA2 sequences, respectively) (accessed on 16 May 2021). Thus, the respective forward and reverse primer sequences chosen were 5′-CCGATATCACGATGAGTTCAC-3′ and 5′-CTTAGCCCAGCCAATGTC-3′ for RNA1 (rdrp gene) and 5′-AATGGTACGCAAAGGTGA-3′ and 5′-GCACTAGGGAACCGGAT-3′ for RNA2 (cp gene). These alignments and the locations of the primer sequences within them are shown in Figures S1 and S2 for RNA1 and RNA2, respectively. A search using the Basic Local Alignment Search Tool (BLAST, NCBI) with the primer sequences showed no genomic cross-reactivity with other virus families.

The amplification conditions consisted of a denaturing step at 94 °C for 1 min followed by 35 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min, followed by a final extension step of 72 °C for 7 min. PCR products (5 μL) were visualized on a 1.5% agarose gel stained with SYBR Safe^® (Invitrogen). The 1 kb DNA ladder from Promega (Madison, WI, USA) was used as a size marker. Finally, the bands obtained were excised and purified using the ‘‘Isolate PCR and Gel’’ kit (Bioline, London, UK), before being commercially sequenced (Sistemas Genómicos S.L., Valencia, Spain) (Figure S3).