Introduction
Porcine circovirus 3 (PCV3), which belongs to the familyCircoviridae and genus Circovirus , was first identified in
2015 in pigs in the United States displaying myocarditis, multisystemic
inflammation, porcine dermatitis and nephropathy syndrome (PDNS) and
reproductive failure (Palinski et al., 2017; Phan et al., 2016).
Subsequently, PCV3 has been detected in many countries around the world
raising concerns to the swine industry, although the virus has also been
detected in asymptomatic pigs (Gu et al., 2020; Jiang et al., 2019;
Klaumann et al., 2018). PCV3 is a small DNA virus with a 2000 nucleotide
single-stranded circular genome containing three major open-reading
frames (ORFs): ORF1 encoding replicase (rep) protein, ORF2 encoding the
capsid (cap) protein and ORF3 encoding a protein with unknown functions
(Klaumann et al., 2018; Palinski et al., 2017; Qi et al., 2019; Assao et
al., 2021a). The cap protein is considered to be the major structural
protein, which determines the antigenic properties of the virus, showing
only approximately 37% amino acid sequence identity to that of PCV2
(Palinski et al., 2017; Nawagitgul et al., 2000). PCV3 strains have
shown genetic stability and low mutation rates over time and across
countries, with nucleotide similarity ranging from 97 to 100% on
partial or complete sequences (Assao et al., 2021a; Assao et al., 2021b;
Franzo et al., 2020; Plut et al., 2020; Qi et al., 2019). This work
aimed to investigate the genetic diversity of PCV3 strains circulating
in Canada and whether a main PCV3 strain circulating across different
Canadian provinces.{Palinski, 2017 #400;Phan, 2016 #401}{Palinski,
2017 #400;Phan, 2016 #401}{Palinski, 2017 #400;Phan, 2016
#401}{Palinski, 2017 #400;Phan, 2016 #401}
The investigated sample set consisted of 44 samples including oral,
processing and thoracic fluids (25/44), feces (1/44) and tissues
(18/44), which were submitted to diagnostic laboratories including the
Centre de diagnostic vétérinaire de l’Université de Montréal (CDVUM) in
Quebec, Animal Health Laboratory (AHL) in Ontario, and Prairie
Diagnostic Services (PDS) in Saskatchewan (Supplementary Table 1).
Available samples enrolled in this study were from different provinces
including Saskatchewan (2/44), Manitoba (2/44), Quebec (4/44), Alberta
(11/44) and Ontario (25/44) between 2019 and 2021. Nucleic acid
extraction was performed using MagMAXTM CORE Nucleic
Acid Extraction Procedure (ThermoFisher, Texas, USA) as per the
manufacturer’s instructions. PCV3 detection was performed using a
previously described real-time PCR assay (Palinski et al., 2017) and
samples with quantification cycle (Cq) values at or below 30 were
selected for PCR and sequencing of the full PCV3 ORF2 sequences. Two
sets of primers with the first set (Forward 5′-GTG TAC AAT TAT TGC GTT
GGG-3′ and Reverse 5′- AAA ACA CAG CCG TTA CTT CAC C-3′) and the second
set of primers (Forward 5′-GCT TTG TCC TGG GTG AGC G-3′ and Reverse
5′-CCT GCG GCA TCA AAA CAC G-3′) were utilized separately to generate
two overlapping fragments containing the full ORF2 sequence (Arruda et
al., 2019). These two PCRs were carried out using Taq DNA polymerase
master mix (Qiagen, Vilnius, Lithuania). The following cycling
conditions were used: initial activation phase at 94 °C for 10 min; 45
two-step cycles of denaturation at 94 °C for 30 s; annealing at 55 °C
for 30 s and extension at 68 °C for 45 s (Arruda et al., 2019). A 444
and 643 bp PCR products could be visualized using Qiagen QIAxcel
instrument, respectively. Then PCR products were sent to Macrogen
(Seoul, South Korea) for Sanger sequencing.
The Sanger sequencing chromatograms of PCR products were basecalled and
assembled using Tracy v0.5.9 (Rausch et al., 2020) to obtain full ORF2
sequences. Multiple alignment of ORF2 sequences was performed using
Mafft v7.475 (Katoh and Standley, 2013). To determine the best-fit
nucleotide substitution model, jModelTest2 v2.1.10 (Darriba et al.,
2012) was used to assess model fit to the sequence alignment across 88
nucleotide substitution models based on Bayesian Information Criterion
estimates. The maximum likelihood tree was generated using RAxML-NG
v1.0.3 (Kozlov et al., 2019). Maximum likelihood inference was performed
using the best-fit HKY nucleotide model with invariant sites (+I) and
Gamma distributed rates (+G). The robustness of the phylogenetic tree
nodes was determined by bootstrap analysis of 1000 replicates. The
phylogenetic tree was visualized using the ggtree package v2.4.1 in R
(Yu, 2020). Pairwise sequence alignment and calculation of sequence
identity were conducted using Jalview v2.11.1.4 (Waterhouse et al.,
2009). All 44 sequences were deposited to the National Center for
Biotechnology Information (NCBI) GenBank database (accession numbers
provided in Supplementary Table 1).
Sequence analysis revealed that cap gene sequences of 44 Canadian
strains had 96.43% to 100% nucleotide identity with each other and
88.28% to 100% nucleotide identity with those of 33 reference strains
included in this study. According to the criteria of genetic distance of
6% at the ORF2 level (3% at the complete genome level) and over 90%
bootstrap support, PCV3 is classified into two clades: clade 1 (PCV3a)
and clade 2 (PCV3b) (Franzo et al., 2020). Clade 2 (PCV3b) contains only
two sequences identified from Chinese farms (Franzo et al., 2020). After
removing the reference sequence from clade 2 (PCV3b) (GenBank number
MG372488) included in this study, the resulting pairwise nucleotide
identity across all sequences ranged from 96.74% to 100%. This
supports the statement of genetic stability and low mutation rates of
PCV3 (Arruda et al., 2019; Assao et al., 2021a; Qi et al., 2019). All 44
Canadian sequences in this study were classified into clade 1 (PCV3a)
along with included reference strains (Figure 1), which strengthens the
hypothesis that clade 2 (PCV3b) including only two sequences is
considered to be either recently emerged variants or the last descendant
of previously circulating genotypes (Franzo et al., 2020). The
phylogenetic tree of the cap gene (Figure 1) showed that Canadian
strains were subdivided into seven lineages based on nucleotide
patterns. A total of 12 Canadian strains (Lineage 1) clustered with
strains detected in different countries. A cluster of four Canadian
strains (Lineage 2) clustered with a strain from Malaysia and a cluster
of 11 Canadian strains (Lineage 3) clustered with a strain in USA were
found. Lineage 4 containing only one Canadian strain (12769/2021/MB-CAN)
clustered with strains from China. Lineage 5 and 6 containing six
Canadian strains respectively clustered separately. Lineage 7 containing
four Canadian strains clustered with a strain from Germany. Assao et
al., demostrated that at least six different PCV3 lineages circulating
in North and South America based on amino acid patterns with only one
available Canadian strains in that study (Assao et al., 2021a). Further
study is necessary to determine whether the nucleotide variations in cap
gene, as revealed in this study, could be associated with differences in
the pathogenicity of PCV3 strains.
Considering cap as the major structural protein and the main antigen of
PCV3 (Klaumann et al., 2018), the amino acid sequences of the 44
Canadian PCV3 strains included in this study were compared to the
reference strain NC_031753. Sequence analysis showed that the presence
of 17 amino acid substitutions in the cap protein (Table 1). Common
amino acid changes at A24V, R27K, N56D, T77S, Q98R, L150I (F) and R168K
positions were observed (Table 1). It has been speculated that A24V and
R27K might be related to immune escape and were used for lineage or
subclade classification of PCV3 (Assao et al., 2021a; Gu et al., 2020;
Fu et al., 2018). In this study, mutation of24ARRR27, 24ARRK27and 24VRRK27 was found without
mutation of 24VRRR27, which was
reported previously (Sun et al., 2018). Amino acid change at T77N
position was observed on both sequences from AB and ON, along with other
amino acids changes at R4K, A5P (T, S, G), F7Y, A75T, V134Y, K140T,
S156G (T), L173V and G196E positions across different provinces (Table
1), suggesting the importance of these differences for understanding the
evolutions of these strains.