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).