Discussion
In recent studies, Rotavirus A 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 YPDS mostly affecting young pigeons worldwide (Raue et al.,
2005). PiCV has been reported to be prevalent in at least fourteen
countries. In China, PiCV was first detected in meat pigeons in 2009 (Yu
et al., 2009). In recent years, the epidemiological survey showed that
the positive rates of PiCV infection in Chinese meat pigeons were
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 in the racing pigeons is unknown. The main objective of this
work was to evaluate the genetic diversity and epidemiology of PiCV
strains circulated in the racing pigeons of China. Positive samples were
detected from seven provinces and the prevalence rates among these
provinces were variant. Overall, our data implied that PiCV was also
widely distributed in diseased racing pigeons and healthy racing pigeons
in China for the first time.
Previous studies showed that the length of PiCV genome was 2031-2043 nt
(Loiko et al., 2018; Sarker et al., 2019; Wang et al., 2017). However,
our findings first showed that the 2030 nt (n=1), 2044 nt (n=2), and
2045 nt (n=1) complete genome were present in the PiCV positive samples.
Despite many genetic diversities were found in the genome of PiCV
strains, no mutation was found in the conserved nonanucleotide motif
(TAGTATTAC) (Mankertz et al., 2000; Todd et al., 2001) located at the
apex of a potential stem-loop which was putatively associated with the
initiation of rolling circle replication (RCR) (Mankertz et al., 2000;
Todd et al., 2001). Previous studies have shown many genetic diversities
in the cap gene (Cságola et al., 2012; Stenzel et al., 2014a;
Wang et al., 2017). Our findings showed that the 90 identifiedcap genes exhibited a higher diversity as compared with the
reference strains. Some unique amino acid substitutions at 28 different
positions were observed among the Cap proteins. In addition, thecap nucleotide sequence with 828 nt in length encoding a novel
Cap protein of 275 amino acids was first identified in two Chinese PiCV
strains, TY3/SN/2016/MW181931 (clade E) and WL4/SN/2018/MW181959 (clade
E). Higher diversity of Cap protein versus the Rep protein is due to the
fact that the Cap protein, as the protein shell of the virus, is exposed
to the host’s immune cells, resulting in a stimulation of a cascade of
immune responses. An interaction with the immune system may result in
adaptive mutations in the Cap protein. Some mutations can change the
structure of Cap protein. This may lead to a better binding of virus
with the receptors of target cells, which increases the infection
ability of virus in cells. Moreover, these mutations may also protect
virus from being neutralized by antibodies (Bassami et al., 1998;
Bennett et al., 2006; Stenzel et al., 2017). In this study, we
identified many mutations in both N- and C-terminus of the Cap protein.
According to the investigations on Cap protein of BFDV, the
arginine-rich N-terminus has two functions (nuclear localization and
nucleic acid binding) that enable as penetration of the viral genome
into the host cell nucleus through nuclear pore complex (Heath et al.,
2006). Like BFDV, the N-terminus of PiCV Cap protein is also rich in
arginine and has been predicted to be a nuclear localization signal and
a nucleic acid binding domain by software, which means that this region
is highly possible to be functionally similar to the corresponding
region of the Cap protein of BFDV (Lai et al., 2014). Moreover, the
results of this study showed that most of the amino acid deletion sites
in Cap protein were concentrated in the N-terminus and many novel
mutations also have been identified in this region. However, due to the
lack of relevant experimental evidence, we cannot infer whether these
mutations and deletions could affect the function of Cap protein. The
Cap protein has been used as a coating antigen in ELISA due to its
antigenic activity and can be recognized by PiCV specific antibodies
(Daum et al., 2009; Stenzel et al., 2016). If the N-terminal region of
the PiCV Cap protein, which is located within the capsid, resembles the
corresponding region in BFDV,
the
N-terminal amino acid mutation and deletion may not affect the ability
to bind to the antibody as a coating antigen (Daum et al., 2009). In
addition, there are also many mutations in the C-terminus where most
sites are composed of hydrophilic amino acids. It means that this region
may locate on the outside of the protein and may have ability for
binding with antibody. Therefore, mutations of this region may lead to
changes in antigenicity. Cap protein, which contains neutralizing
antibody epitopes, can be considered as a potential antigen
candidate
in sub-unit vaccine development (Gai et al., 2020; Stenzel et al.,
2018). Moreover, the sub-unit vaccines based on PCV2 recombinant capsid
proteins are successfully used in the prevention of PCV2-SD (Blanchard
et al., 2003; Fort et al., 2009; Li et al., 2015; Zhu et al., 2016). It
means that the development of a sub-unit vaccine may protect pigeons
from infection with PiCV (Stenzel et al., 2018). Due to a high genetic
diversity of Cap proteins may lead to differences in antigenicity
between different strains, sub-unit vaccines may also be diversified in
future. To sum up, since there is
little research on the function of PiCV Cap protein, the specific
meaning of these mutations cannot be accurately explained. Therefore,
further experiments are needed to explain if these mutations and
deletions affect the function of Cap protein. According to the obtained
nucleotide sequences of cap gene, we found that ATT and GTG
existed in the position of the start codon site through sequence
alignment. In addition, several ATG was found inside cap genes.
Since no available cell lines have been found to culture PiCV in
vitro (Huang et al., 2021; Santos et al., 2020; Todd, 2004) and lack of
commercial antibodies against PiCV Cap protein, it is hard to verify
that indeed a full-length natural Cap protein is translated from the
described alternative start codons, rather than a truncated version from
a downstream in frame of ATG by experiments now. Therefore, the ATT and
GTG were highly suspected to be the start codons of the Cap protein
through sequence analysis in this study. A similar approach has been
employed for identifying the alternative start codon of Cap protein in
other circoviruses, such as GuCV (Todd et al., 2007) and BFDV (Bassami
et al., 2001). Moreover, previous studies had confirmed that codons
differing from ATG in a single position can support translation
initiation in eukaryotes (Peabody, 1989; Wei, J. et al., 2013). Thus, it
still needs more experiments to identify if the two non-ATG start codons
can be actually used for translation initiation by Cap protein of PiCV.
In all 68 identified Rep proteins, three amino acid motifs named FTLNNP
(position 41-46), HLQGF (position 78-82), and YCSK (position 116-119)
that putatively associated with RCR (Mankertz et al., 2000; Mankertz et
al., 1998) were completely conserved. In addition, a fourth motif, which
was putatively associated with dNTPase activity, namely GKS (position
197-199) (Mankertz et al., 2000; Mankertz et al., 1998), was also
completely conserved. These sequence characteristics help understand the
genetic diversity of PiCV. However, the pathogenicity of different PiCV
strains still need to be validated in future.
In the present study, the 90 PiCV strains were divided into seven clades
(A, B, C, E, G, H, and I) based on a phylogenetic analysis of capgene. Additionally, we found that PiCV strains isolated from the same
club belonged to different clades and shared a low sequence identity.
These data suggested that the infection and evolution of PiCV in Chinese
racing pigeons might have different evolutionary origins. This may
result from the fact that the import of racing pigeons from all around
the world to China is very significant and a lack of oversight in the
international pigeon trade (racing pigeons mainly) (Ashton, 1984;
Stenzel et al., 2012). Interestingly, two
isolates,QYQX1/HE/2018/MW181909 (clade A) and QYQX2/HE/2018/MW181910
(clade C), with lower identity (75.5% nucleotide identity and 76.8%
amino acid identity) in the cap gene were detected from the same
pigeon, suggesting a horizontal transmission occurred among the racing
pigeons in the same racing clubs (Gerdes, 1993; Woods et al., 1993).
The complexity of epidemic PiCV
strains in China may cause difficulties to protect pigeon from infection
by vaccines in future.
Viral
recombination
had been proved to play a significant role in the evolution of many
ssDNA viruses (Lefeuvre et al., 2009). The extensive recombination
events had been reported in PiCV genomes and other circoviruses, such as
PCV (Kleymann et al., 2020; Wei, C. et al., 2019) and BFDV (Julian et
al., 2013; Varsani et al., 2011). Similar to the previous reports, 31
recombination events were detected in the 67 identified PiCV strains in
this study. Thus, the recombination seemed to be a key mechanism for
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 infection in racing
pigeons was widespread in northern China and revealed the
characteristics of the PiCV genome. Furthermore, the identified PiCV
strains displayed a high genetic diversity. Our data also demonstrated
that PiCV in Chinese racing pigeons had an extensive recombination for
the first time. Thus, these data could increase our understanding of the
epidemiology and
genetic
variation of PiCV circulated in northern China and evolutionary
relationships
among
different strains.