3 RESULTS
3.1 Clinical history and routine
detection of potential swine pathogens
In March 2020, 15 out of approximately 150 litters in a commercial pig
farm, which kept approximately 800 breeding crossbred sows, on the main
island of Japan were affected by CT. Approximately 80% to 90% of the
piglets in each litter showed varying degrees of repetitive constant
tremors of the body. Severely affected piglets with CT displayed
systemic tremors, had difficulty standing and sucking milk, and died,
whereas piglets with mild clinical symptoms recovered and grew normally.
The pre-weaning mortality rates in February, March, and April of 2020
were 6.0%, 5.1%, and 5.4%, respectively. On this farm, pigs with CT
have been sporadically observed from about 15 years prior.
All samples from the two piglets with severe CT were negative for
porcine teschovirus, porcine sapelovirus, porcine astrovirus, porcine
circovirus 2, pseudorabies virus, hemagglutinating encephalomyelitis
virus, and H. parasuis , but were positive for APPV and S.
suis (data not shown).
3.2 Pathological examination
Histopathological investigations revealed many vacuoles in the medulla
oblongata (Figure 1a) and the white matter around the ventriculus
lateralis of the cerebrum (Figure 1b) in one of the two piglets
examined. There was no inflammatory reaction observed as perivascular
infiltration. No lesion was observed in the cerebrum or medulla
oblongata of the other piglet. In both piglets, a decrease in
lymphocytes as well as follicular atrophy in the lymph nodes and spleen
were seen.
3.3 Analysis of the whole genome sequence of the Japanese APPV
Due to the lack of available Japanese APPV genome data in
the NCBI database, we determined
the complete genome sequence of this APPV, which was named Anna/2020,
through RT-PCR and Sanger sequencing. We successfully determined the
complete genome consisting of 11,567 nt sequences (Figure 2a). Anna/2020
possessed an ORF, which encoded a protein of 3,635 aa, flanked by a
5’UTR of 378 nt and a 3’UTR of 281 nt. Phylogenetic tree analysis using
the complete genome nt sequences of Anna/2020 and APPVs from the NCBI
database showed that APPVs were clearly divided into three genotypes
(Figure 2c). Genotype 1 consisted of APPVs from the Americas, Europe,
and Asia, while genotypes 2 and 3 contained only Chinese APPVs.
Anna/2020 falls into genotype 3 with the Chinese APPVs; however, it
branched independently and distantly from the Chinese APPVs (Figure 2c).
Pairwise sequence identity analysis of the complete coding regions
revealed that Anna/2020 exhibited 80.2% to 81.8% and 80.9% to 81.2%
sequence identity to the genotype 1 and genotype 2 APPVs. While there is
94.0% to 99.7% sequence identity among the Chinese APPVs in genotype
3, Anna/2020 showed 87.0% to 89.3% identity to these Chinese APPVs in
genotype 3 (Table 1). Recombination events are an important mechanism by
which viruses acquire genetic diversity, and they have been identified
in APPVs (Guo et al., 2020; Xiao et al., 2016). Thus, to find crossover
sites between Anna/2020 and other
APPVs, similarity plot analysis
was performed using SimPlot software. Anna/2020 exhibited higher
sequence homology to genotype 3 APPVs than to genotype 1 and 2 APPVs
throughout the whole genome, and no recombination event was observed
(Figure 2b).
3.4 Retrospective identification of APPV RNAs
A total of 399 samples obtained from pigs during 2005 to 2020 were
subjected to SYBR Green qRT-PCR to detect APPV RNA. The results showed
that three samples were positive; these samples were collected from
20-day-old, 68-day-old, and 12-week-old pigs without CT that were kept
in Kyusyu in 2007, the main island in 2018, and Kyusyu in 2018,
respectively (Figure 3). Although we tried to determine the whole genome
sequences of these samples, we could obtain only partial genome
sequences, probably due to the low quality and insufficient amount of
samples. The obtained partial sequences were aligned to the complete
genome sequence of Anna/2020 (Figure 4a). Similarity and phylogenetic
analysis using the APPV sequences from the NCBI database revealed that
two APPVs from the samples collected in Kyusyu in 2007 and 2018, named
Miya/2007 and Kago/2018, respectively, were assigned to genotype 3, and
they formed a lineage with Anna/2020 that was distant from the Chinese
APPVs in the phylogenetic trees; in contrast, all analyzed regions of
the APPV detected in the sample collected on the main island in 2018,
named Mae/2018, belonged to genotype 1 (Figure 4b and c).