Detection and amplification of APPV genome from positive samples
From October 2017 to May 2019, clinical tissue samples (including brain, liver, lymph node and spleen from each piglet) from fifty-three less than one-week old piglets from eighteen pig farms were detected by polymerase chain reaction (PCR) or reverse transcription-PCR (RT-PCR). All the samples were negative for CSFV, porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), PCV3, porcine pseudorabies virus (PRV), Japanese encephalitis virus (JEV) and porcine parvovirus (PPV), while 41 samples were positive for APPV (41/53, 77.36%) (Figure 1).
APPV complete genome was amplified by 8 pairs of specific primers (Table 1) to amplify 8 overlapping fragments encompassing the open reading frame (ORF) and by rapid amplification of cDNA ends (RACE) to amplify 5′ untranslated region (UTR) and 3′ UTR from APPV positive cDNA samples (Figure 2). After sequencing and assembling, the complete genomic sequences of five APPV strains were obtained and were 11 534-11 565 nucleotides (nt) in full-length, with a 5′ UTR of 358-378 nt, followed by a single large ORF and a 3′ UTR of 268-279 nt. The ORF was 10 908 nt in length, which encoded a polyprotein of 3 635 amino acids (aa). The polyprotein was composed of twelve proteins, including four structural proteins (C, Erns, E1 and E2) and eight nonstructural proteins (Npro, P7, N2, NS3, NS4A, NS4B, NS5A and NS5B) (Table 2). Five APPV strains obtained from this study were named as GX04/2017, GX01-2018, GX02-2018, GX01-2019 and GX02-2019, respectively. The genomic sequences of these identified strains have been submitted to the GenBank database of National Center for Biotechnology Information (NCBI) under accession number MH102210, MH715893, MK453045, MN564752 and MN729215, respectively.
Sequence analyses of APPV genome
Sequence analysis revealed that the nucleotide identities of the complete genome, ORF, Npro, Erns and E2 genes among six APPV strains from Guangxi province, including five strains from this study and one from other researchers, were 83.3-97.5%, 83.0-98.2%, 79.6-97.2%, 80.8-98.4% and 83.4-96.8%, respectively; the amino acid identities of ORF, Npro, Erns and E2 genes were 91.7-99.1%, 81.1-97.8%, 89.5-99.0% and 90.5-96.3%, respectively (Table 3). The nucleotide identities of the complete genome, ORF, Npro, Erns and E2 genes among six APPV strains from Guangxi province and the reference strains were 77.7-97.7%, 80.8-98.6%, 77.4-99.3%, 80.3-98.9% and 79.8-98.9%, respectively, while the amino acid identities of ORF, Npro, Erns and E2 genes were 90.6-99.3%, 79.4-99.4%, 88.1-100% and 88.0-98.8%, respectively (Table 3).
Phylogenetic analyses of APPV genome
To investigate the evolutionary relationship and genetic diversity of the APPV strains circulating in Guangxi province, the phylogenetic trees were constructed using the complete genome, Npro, Erns and E2 gene sequences of the six strains from Guangxi and other published reference strains available in GenBank (Table 4). All APPV strains could be grouped into four clusters based on the complete genomic sequences (subgroup O1-O4 in Figure 3A), Npro gene (subgroup N1-N4 in Figure 3B), Erns gene (subgroup S1-S4 in Figure 3C) and E2 gene (subgroup E1-E4 in Figure 3D). It was noteworthy that phylogenetic tree based on the complete genomic sequences showed that GX01-2019, APPV GX-Ch2016, GX01-2018 and GX02-2018 strains, together with KU16-2 strain from Korea, Bavaria S5/9 strain from Germany and AUT-2016_C strain from Austria, belonged to one branch (subgroup O1); GX04/2017 strain, together with 000515 strain from USA, KU16-6 strain from Korea, belonged to another branch (subgroup O2); GX02-2019 strain, together with most strains from Guangdong and other provinces in China, belonged to another branch (subgroup O3). The phylogenetic trees based on Npro, Erns and E2 gene sequences showed quite similar topology to that of complete genome with a little disagreement. The above results showed that all six strains from Guangxi province existed a high degree of genetic diversity and obviously different evolutionary relationship.
Evolution analyses of APPV epidemic strains
To evaluate the recombination events of APPV strains during the process of evolution, the complete genomic sequences of 58 APPV strains form different countries available in GenBank (Table 4) were analyzed using Recombination Detection Program 4 (RDP4) and Simplot 3.5.1. The results showed that there existed recombination detection signs in six strains, including GD-BZ01-2018 (MH493896), GD-DH01-2018 (MH493895), YN01/2017 (MH378079), JX-JM01-2018A01 (MG792803), GD-LN-2017.04 (MK216753) and GD2 (KX950763) (Table 5), and all of them came from China. No sign of recombination was detected in all six strains from Guangxi province (Figure 4).
Evolutionary estimation based on the complete genome, Npro, Erns and E2 gene sequences of 58 APPV strains form different countries was conducted by Bayesian analysis. The results indicated that APPV genomic sequences evolved at a mean rate of 1.37×10-4 (95% HPD 5.12×10-6-3.02×10-4) substitutions/site/year (s/s/y), and the mean rates of molecular evolution of Npro, Erns and E2 gene sequences were 1.00×10-4 (8.47×10-5-1.17×10-4), 1.56×10-4 (9.81×10-5-2.20×10-4) and 1.01×10-4 (8.17×10-5-1.22×10-4) s/s/y, respectively. Bayesian inferences (BI) estimated that the tMRCA of complete genomes of APPV epidemic strains existed 1700.5 (95% HPD 228.4-4654.5) years ago, and the tMRCAs of Npro, Erns and E2 gene sequences were 1495.5 (1404.3-1597.4), 1390.8 (821.9-2032.2) and 1823.0 (1723.2-1918.1) years ago, respectively.