4 DISCUSSION
The mortality rates of CT clinical disease caused by APPV are generally low, and the economic losses remain unknown; however, severely affected piglets with CT may die due to colostrum intake difficulties and starvation, resulting in a higher pre-weaning mortality rate (Pan et al., 2019a; Stenberg et al., 2020). Clinical signs of CT have been observed since 15 years ago on the farm where Anna/2020 was detected, so the presence of APPV in the piglets might have been causing economic losses for the farm. A routine diagnosis revealed that S. suiswas simultaneously identified with APPV in the CNS of CT-affected piglets. The co-infection of APPV with other viruses, such as porcine circovirus 3 or teschovirus, has been reported (Possatti et al., 2018; Yan et al., 2019). APPV RNA was frequently found in lymphoid organs, suggesting that APPV may play a role in suppressing the host immune system (Pan et al., 2019a). Immune suppression might facilitate the co-infection of APPV with S. suis in CT-affected piglets. The histopathological findings of atrophy and fewer lymphocytes in the spleen and lymph nodes of affected piglets suggested that they were under immunosuppressive conditions.
According to phylogenetic dendrograms and pairwise sequence comparisons, APPVs are subdivided into three genotypes (Choe et al., 2020; Folgueiras-González et al., 2020; Guo et al., 2020; Xie et al., 2019; Yan et al., 2019). Our analyses showed that Anna/2020 belonged to genotype 3; however, Anna/2020 formed an independent branch, and was distantly related to the Chinese genotype 3 strains. Furthermore, Anna/2020 showed 87.0% to 89.3% identity in the complete coding sequences to the other genotype 3 APPVs, while there is 94.0% to 99.7% identity among the Chinese APPV genotype 3 strains. Thus, it is suggested that Anna/2020 constitutes a novel APPV lineage within genotype 3. APPV genotype 3 strains have been reported only from China (Guo et al., 2020; Xie et al., 2019; Yan et al., 2019); thus, Anna/2020 represents the first strain belonging to genotype 3 from a country other than China. Furthermore, no recombination event was found between Anna/2020 and other APPVs from the NCBI database. These findings suggest that Anna/2020 might have independently evolved only in the Japanese pig population. Interestingly, two Japanese APPVs identified in samples from 2007 and 2018 in the retrospective study branched with Anna/2020 and formed a cluster, indicating that the novel genotype 3 lineage of APPVs has been present in Japan since at least 2007. Genotype 3 APPVs were identified in pigs on the main island of Japan and on Kyusyu island, suggesting that the APPVs of the novel genotype 3 lineage may already be prevalent throughout Japan. Retrospective examination also showed the presence of a genotype 1 APPV in Japan. The genotype 1 APPV was identified on a farm in close proximity to the farm where genotype 3 Anna/2020 was detected (Figure 3). Further studies are warranted to clarify the prevalent APPV genotype(s) in Japan.
Three (0.75%) APPV RNAs were found using qRT-PCR in 399 previously obtained samples from pigs without a history of CT. In recent APPV prevalence studies of apparently healthy pigs by qRT-PCR, the APPV detection rates were 2.3% to 22% in Europe, the United States, and Asia (Beer et al., 2017; Hause et al., 2015; Postel et al., 2016, 2017). Our detection rate was lower, and may be attributable to the low quality of the preserved samples or biased sample collection. A recent report showed that 19% of wild boars possessed APPV RNA, suggesting that the boars may be a source of APPV transmission (Cagatay et al., 2018). Further investigations on the prevalence of APPV in pigs and wild boars are needed for estimating the precise prevalence of APPV, and for establishing proper control and prevention measures for APPV infection in Japan.
In conclusion, in the present study, we firstly determined the complete genome sequence of an APPV detected from CT-affected piglets in Japan. Genomic analyses revealed that this strain is a novel APPV that forms a new lineage within genotype 3 APPVs. The retrospective study demonstrated that one genotype 1 and two novel genotype 3 APPVs were identified from pigs without CT, and the novel genotype 3 strains were closely related to Anna/2020. The novel genotype 3 lineage of APPV might have been prevalent in Japan since at least 13 years ago, and evolved only in the Japanese pig population.