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).