3. RESULT AND D ISCUSSION
Farm workers and all species of farm animals can be infected by circovirus (Li et al., 2011). Multiple studies have indicated that three circovirus species can infect pigs. PCV2 and PCV3 are recognized as the main pathogens in PCV-associated disease (PCVAD) and cause severe economic loss to the swine industry worldwide (Liu et al., 2019; Wang, Cao, et al., 2019; Wen et al., 2018). Recently, a novel and genetically divergent circovirus, PCV4, was considered to be a new pathogen of respiratory disease, diarrhea and PDNS (Zhang et al., 2019).
In the present study, with reference to the method of Zhang et al. (Zhang et al., 2019), a TaqMan® real-time PCR assay was used to detect PCV4 in clinical samples collected between 2015 and 2019 from Guangxi Province, China. The results showed that 5.1% (13/257) of the porcine samples collected from six cities were PCV4 positive. Eight of 93 tissue samples were PCV4 positive, and 5 of 164 serum samples were PCV4 positive. Six porcine samples with PDNS and two porcine samples with respiratory disease were PCV4 positive. Coinfection of pigs with PCV2 and PCV3 is known to exacerbate disease severity. Further detection showed that 9 of 13 (69.2%) samples were coinfected with PCV2 (n=7) or PCV3 (n=3). Sample NN88 from Nanning was coinfected with PCV2 and PCV3 at the same time.
To analyze the genetic relationship between PCV4 strains and other representative circoviruses, three complete genome sequences (GX2020/NN88, GX2020/GL69, GX2020/FCG49) of PCV4 were obtained. All strains in this study had genomes that were 1770 bp long. Similar to PCVs, the three Guangxi strains of PCV4 also contain two major ORFs. ORF1 encodes the Rep protein (296 amino acids), and ORF2 encodes the Cap protein (228 amino acids). The amino (N) terminus of the Rep protein contains 3 conserved amino acid motifs: RCR motif I (FTLNN), RCR motif II (PHLQG) and RCR motif III (YCSK). The C terminus contains the dNTP binding site GVGKS. The PCV4 genome has two noncoding intergenic regions (IRs) between the 5’- and 3’- ends of the two major ORFs. A stem loop was detected in the genome of PCV4, and it contains 11 bases (TTCAGTATTAC) instead of the 9 bases previously reported (Figure 2) (Zhang et al., 2019).
Multiple sequence alignment of the PCV4 strains in this study showed that the sequences in their genomes shared 5.1%–73.8% nucleotide identity with all available reference sequences in the complete genome. Furthermore, the PCV4 strains in this study shared 98.5%–99.1% and 99.7%–99.8% nucleotide identity with other PCV4 strains and each other, respectively. An amino acid sequence comparison showed that the Rep protein and Cap protein the PCV4 strains in this study shared 5.1%–80.5% and 17.8%–70.6% identity with those of all available reference strains. Interestingly, multiple sequence alignment results based on three different methods have confirmed that PCV4 shares the highest homology with MiCV (73.8% for the complete genome, 80.5% for the Rep protein and 70.6% for the Cap protein). However, multiple sequence alignment results showed that PCV4 shares relatively low nucleic acid and amino acid identity (≤ 50%) with PCV1, PCV2, and PCV3 (Table 1).
In circoviruses, Cap is the sole structural protein and contains immunologically important epitopes associated with virus neutralization (Lekcharoensuk et al., 2004; Meng, 2013). Therefore, it has been the main target for vaccines. At present, commercial vaccines against PCV2 have been introduced worldwide, and they have been considered a successful story in veterinary vaccinology (Park et al., 2019). The Cap protein of PCV4 shares a low amino acid sequence similarity (<30%) with that of PCV3. Thus, cross protection seems unlikely. Interestingly, the Cap protein of PCV4 has relatively high homology (approximately 50%) with those of PCV1 and PCV2. Several previous studies have reported the antigenic site (Lekcharoensuk et al., 2004; Mahe et al., 2000; Shang et al., 2009) and receptor binding site (98IRKVKVl03) of the Cap protein of PCV2 (Misinzo et al., 2006). In this study, multiple sequence alignment analysis of the amino acid sequence of the Cap protein revealed that PCV2 and PCV4 have multiple highly homologous antigen sites and identical receptor binding sites (Figure 3). Therefore, PCV2 and PCV4 may have cross-protective immunogenicity.
To promote understanding of the genetic relationship between the different strains identified in the present study, a phylogenetic tree was constructed using the maximum likelihood method based on the complete genome and the amino acid sequences of the Rep and Cap proteins. Interestingly, the results were similar using the different algorithms. The results confirm that PCV4 had a close relationship to MiCV and bat-associated circovirus, followed by PCV1 and PCV2. PCV4 and PCV3 are not in the same branch and are distant from each other (Figure 4). Limited numbers of sequences, however, resisted independent evolution analysis on PCV4. Furthermore, multiple studies have indicated that PCV2 and PCV3 are transmitted to nonporcine hosts, possibly through cross-species transmission routes (Song et al., 2019; X. Wang et al., 2018; J. Zhang et al., 2018). Can PCV4 also infect nonporcine hosts? Further studies are needed to answer this question.
Overall, this research is the first report to detect PCV4 in Guangxi Province, China, and to obtain three complete genomic sequences. Our results combined with those of other reports suggest that PCV4 may commonly circulate within swine herds in South China. This research detected PCV4 in swine with PDNS, and whether PCV4 infection is related to PDNS still needs further research to confirm. Further studies are warranted to elucidate the prevalence and pathogenesis of this novel circovirus.