2 MATERIALS AND METHODS
2.1 Routine diagnosis
Tissues of the CNS (cerebellum), mesenteric lymph node, and tonsil from two 3-day-old piglets that showed severe signs of CT and moribund were collected and examined by reverse transcription (RT)-polymerase chain reaction (PCR) for pathogens of neurological disorders, such as porcine teschovirus (Krumbholz et al., 2003), porcine sapelovirus (Zell et al., 2000), porcine astrovirus (Chu et al., 2008), and APPV (Postel et al., 2016), and by real-time RT-PCR (qRT-PCR) for porcine circovirus 2 (Sunaga et al., 2019), pseudorabies virus (Ma et al., 2008), porcine hemagglutinating encephalomyelitis virus (Sunaga et al., 2019),Haemophilus parasuis (Turni et al., 2010), andStreptococcus suis (Bonifait et al., 2014). Tissues were minced by scissors, diluted 1:10 in phosphate-buffered saline, and homogenized for 20 sec at 3,200 rpm in the presence of three stainless steel beads (φ4 mm) by using a bead crusher μT-12 (TAITEC, Inc.). To obtain the supernatant, organ emulsions were centrifuged at 12,000 g for 5 min. Viral RNA and DNA and bacterial DNA were extracted from the supernatant using an IndiSpin Pathogen Kit (Indical Bioscience). For conventional RT-PCR, cDNA synthesis was carried out using PrimeScript™ RT Master Mix (Takara Bio). PCR was performed using GoTaq® G2 Hot Start Taq Polymerase (Promega). qRT-PCR and PCR were performed using a One Step PrimeScript RT-PCR Kit (Takara Bio) for RNA viruses and Premix Ex Taq (Takara Bio) for DNA viruses and bacteria, and performed as described previously (Sunaga et al., 2019).
2.2 Pathological examination
The cerebrum, medulla oblongata, lymph nodes, and spleen of the two CT-affected piglets that were examined by RT-PCR were fixed with 10% buffered formalin, and embedded in paraffin by a conventional method. Sections were prepared from the blocks and stained with hematoxylin and eosin.
2.3 RT-PCR, 5’ rapid amplification of cDNA end (5’RACE), and sequencing for whole genome sequence determination
Total RNA was extracted from CNS tissue using a QIAamp viral RNA mini kit (Qiagen). RT-PCR was carried out as described above using the primers listed in Supplementary Figure 1. The amplification of the 5’ end of the virus genome was performed by the RACE method using a commercial kit (Invitrogen) and three reverse primers, APPV5’R1, APPV5’R2, and APPV5’R3 (Supplementary Figure 1). The A-Plus™ Poly(A) Polymerase Tailing Kit (CELLSCRIPT) and a reverse primer, TX30SXN (Oka et al., 2017), were employed for the amplification of the 3’ end of the APPV genome.
2.4 Retrospective investigation
A retrospective investigation for the detection of APPV RNA was performed using a total of 399 samples; they consisted of the supernatants of homogenized organ tissues or sera that were collected from 1-day-old to 3-year-old pigs and had been submitted to the Pig Clinical Center in Azabu University for the diagnosis of diseases from 2005 to 2020. Viral RNA was extracted using an IndiSpin Pathogen Kit or QIAamp viral RNA mini kit. cDNA was synthesized using PrimeScript™ RT Master Mix. SYBR Green qRT-PCR was carried out using FastStart Essential DNA Green Master (Roche Diagnostics) with the primer pair reported by Postel et al. (2016). qRT-PCR-positive samples were subjected to amplification of the whole genome through conventional RT-PCR using the primers listed in Supplementary Figure 1, and by Sanger sequencing.
2.5 Genome analysis
Phylogenetic analysis using the complete nucleotide (nt) sequences of APPV was performed by the maximum likelihood method with the best fit model (GTR+G+I) in MEGA7 (Kumar et al., 2016). For the maximum likelihood tree analyses of the partial genome of 5’UTR-Npro (using 329 nt), Erns-E1 (197 nt), NS3 (159 nt), NS5b (330 nt), and NS5b (258 nt), K2+G, T92+G+I, K2+G, TN93+I, and K2+G+I were used for the best fit models. Pairwise nt sequence identities were calculated using CLC Genomics Workbench 7.5.5 (CLC Bio). Similarity plot analysis was performed using SimPlot software V.3.5.1 (Lole et al., 1999).