Discussion
In recent studies, RVA G18P[17] had been confirmed as a primary cause of YPDS-like diseases in domestic pigeons (Rubbenstroth et al., 2020). Furthermore, PiCV is also supposed to be an etiological agent of a multifactorial disease named young pigeon disease syndrome (YPDS) affecting mostly young pigeons worldwide (Raue et al., 2005). PiCV had been reported to be prevalent in at least fourteen countries. In China, PiCV has been first detected in meat pigeons since 2009 (Yu et al., 2009). In recent years, the epidemiology survey showed that the positive rates of PiCV infection in Chinese meat pigeons are 19.67% and 75.3% in the poultry farms of eastern China in 2009 and 2015 (Wang et al., 2017; Zhang et al., 2015), implying that PiCV is widely distributed among meat pigeon populations in eastern China. However, the epidemiology and distribution of PiCV infection in the racing pigeons is unknown. The main objective of this work was to evaluate the genetic diversity and epidemiology of PiCV strains circulating in the racing pigeons of China. Positive samples were detected from seven provinces and the prevalence rates among provinces are variant. The results showed that the positive rates of PiCV were 59.0% (23/39) at the club level and 19.3% (120/622) at the sample level. The data also indicated the positive rate in sick pigeons (positive rate, 27.4%) is higher than that of the apparently healthy pigeons (positive rate, 18.6%). Overall, our data for the first time implied that PiCV is also widely distributed in diseased racing pigeons and apparently healthy racing pigeons in China.
The previous studies had shown that the PiCV genome is 2031-2043 nucleotide in length and contains two major identified ORFs (ORF-V1 and ORF-C1) (Loiko et al., 2018). The most common genome sizes obtained in our study were encompassing 2037 (n=21), and 2042 (n=13) nucleotides, respectively. Furthermore, our findings first showed that the 2030 nt (n=1), 2044 nt (n=2), and 2045 nt (n=1) complete genome fragments were also existed in the PiCV positive samples. In addition, four unique nucleotide substitution (CG-GCGA) was first found in the intergenic region between the rep andcap stop codons of two strains (SX1/SN/2017/MW181932 and YB4/SN/2018/MW181981) compared with the 67 PiCV strains obtained in this study and the other reference PiCV strains. In this study, we also investigated whether PiCV strains isolated from a single club or from the same province were identical or similar to each other and evaluated the degree of their relatedness. Sequence analysis demonstrated that the sequence homologies between them were relatively low. The nucleotide sequence data revealed genetic diversity in the genome of the Chinese field PiCV strains, even though the viruses were obtained from the same place. Interesting, two different isolates (QYQX1/HE/2018/MW181909, QYQX2/HE/2018/MW181910) with lower identity (75.5% nucleotide identity and 76.8% amino acid identity) in the cap gene were detected from the same pigeon. These results indicated that different strains were present in the same club. Though many genetic diversities were found in the genome of PiCV strains, no mutation was found in the conserved nonanucleotide motif (TAGTATTAC), located between the start codons of genes encoding rep and cap proteins. These data increase the understanding of the genetic diversities and the prevalence of PiCV in China. However, the pathogenicity and molecular characterization of the different PiCV still need to be validated in the future.
Previous studies have shown that there are many genetic diversities including insertions, deletions and substitutions in the genomes of PiCV, especially in cap gene (Cságola et al., 2012; Stenzel et al., 2014a; Wang et al., 2017). Our findings showed that the capgene of the PiCV strains exhibits the highest diversity among themselves as well as in comparison with the reference strains. The cap gene of PiCV strains obtained in this study were also demonstrated to be highly genetically diverse with the length varied from 813 to 828 nt. In addition, the cap nucleotide sequences with 828 nt in length encoding for a novel cap protein of 275 amino acids was firstly been identified in two Chinese PiCV strains. Codons differing from ATG in a single position, collectively referred to as near-cognate initiation codons, are known to support translation initiation in eukaryotes (Wei, J. et al., 2013). As reported, two groups identified by ATG and ATA initiation codons of the cap gene had been found in the meat pigeons (Wang et al., 2017). In our study, four groups of initiation codons including ATG, ATA, ATT, and GTG were identified in the 90 PiCV strains among the racing pigeons in China. The previous results and our findings presented here showed that most of the PiCV strains were in the ATG group (Loiko et al., 2018; Todd et al., 2008; Wang et al., 2017). However, the ATT, and GTG group was firstly found in initiation codons of the cap gene in comparison with the reference strains in GenBank. Codons other than ATG had been identified to be less efficient initiation codons in Neurospora (N.) crassaand human cells in vivo and in vitro (Wei, J. et al., 2013). Whether the different types of initiation codons of thecap gene have a hierarchy in initiation efficiencies need to be investigated in the future. Moreover, the cap gene of the current PiCV strains exhibits the highest diversity at the nucleotide and amino acid level and the rep gene is relatively conservative. Furthermore, 68 rep and 90 cap protein sequences were subjected to analysis of amino acid mutations, which show that there are numerous unique amino acid substitutions. These above-mentioned data combined with a comparison of entropy (Hx) in cap protein suggest that PiCV strains isolated from China had a higher level of diversity than the PiCV reference strains. These data also suggest that the Chinese PiCV strains may be undergoing more rapid mutational change in the racing pigeons. These data increase the understanding of the genetic diversity of PiCV. However, the pathogenicity and molecular characterization of the different PiCVs still need to be validated.
In the present study, acap- gene-based phylogenetic analysis performed on the 90 cap genes obtained here and the other 126 cap genes available in GenBank revealed that the 90 PiCV strains were divided into seven clades (A, B, C, E, G, H, I). None of the 90 strains belonged to clades D and F, which contained the two Chinese meat pigeons and two Australia feral pigeons, respectively. These data suggested that the infection and evolution of PiCV in Chinese racing pigeons may have different evolutionary origins. This may result from the fact that the import of racing pigeons from all around the globe to China is very significant and the lack of oversight in the international pigeon trade (racing pigeons mainly) (Ashton, 1984; Stenzel et al., 2012). In addition, two novel PiCV strains (TY3/SN/2016/MW181931 and WL4/SN/2018/MW181959) with the ATT start codon and 828 nt in cap length formed a novel clade E with six Chinese PiCV strains from meat pigeons. The above indicates that some Chinese PiCV strains might evolved from the exclusively ancestors. On the other hand, the other two new identified in this study PiCV strains (TF5/SN/2016/MW181901 and WQ3/SN/2018/MW181945) with the GTG start codon form a small subclade with four European isolates in the clade A. Moreover, two different isolates (QYQX1/HE/2018/MW181909 and QYQX2/HE/2018/MW181910) obtained from one sick pigeon were clustered into different clades, which suggested horizontal transmission was occurred among the racing pigeons in the same racing clubs (Gerdes, 1993; Woods et al., 1993).
Viral recombination had been proved to play a significant role in the evolution of many ssDNA viruses (Lefeuvre et al., 2009). The extensive recombination had been reported in PiCV genomes and other circoviruses including PCV (Kleymann et al., 2020; Wei, C. et al., 2019) and BFDV (Julian et al., 2013; Varsani et al., 2011). Similar to the previously reports, a total of 31 recombination events were detected in the genomes of Chinese PiCV strains from the racing pigeons. Moreover, the recombination breakpoint hot plots were found to be located within both the intergenic region between the rep and cap stop codons, and near the virion strand origin of replication. The previous results and our findings presented here demonstrated that the recombination seems to be the key mechanism for the PiCV evolution (Cságola et al., 2012; Loiko et al., 2018; Sarker et al., 2019; Stenzel et al., 2014c).
In conclusion, our study demonstrated that PiCV infections in racing pigeons are widespread in the north of China and reveals characteristics of the PiCV genome. Furthermore, the identified PiCV strains displayed very high genetic diversity. This work has also demonstrated for the first time that PiCV from Chinese racing pigeons had extensive recombination. All the data increase our understanding of epidemiology and genetic variation of the PiCV circulating in China and evolutionary relationships amongst our strains with other worldwide strains. More molecular epidemiological information from other provinces in China and biological meaning of near-cognate codon initiation of cap gene is needed from future research.