Porcine Circovirus (PCV), a non-enveloped virus with single-stranded and circular
DNA (Jiang et al., 2010) was a member of the
Circoviridae family (Lukert et al., 1995) and
it can be classified into two types (PCV1 and 2) according to its antigenicity,
pathogenicity and genomic difference (Allan et al.,
1998; Morozov et al., 1998; Larochelle
et al., 2002). PCV1 is considered non-pathogenic (Allan
et al., 1994; Olvera et al., 2007)
while PCV2 is described as the pivotal and primary causative agent of Postweaning
Multisystemic Wasting Syndrome (PMWS) (Ellis et al.,
1998; Fenaux et al., 2002, 2003;
Ma et al., 2007; Shang et
al., 2007; Li et al., 2010) and it also
is associated with Porcine Dermatitis and Nephro-pathy Syndrome (PDNS), porcine
reproductive disorders and other disease syndromes (Ladekjaer-Mikkelsen
et al., 2001; Meehan et al., 2001;
Segales et al., 2005; Shang
et al., 2007).
PMWS is a newly emerging and economically important disease in pigs originally
reported in Canada (Allan et al., 1998; Ellis
et al., 1998; Allan and Ellis, 2000; Wen
et al., 2005; Stevenson et al., 2007).
It has been acknowledged in many European countries, some Asian countries and
the US (Wen et al., 2005; Liu
et al., 2007; Stevenson et al., 2007).
Typically, clinical PMWS associated with PCV2 affects pigs from 5-12 weeks old
(Wen et al., 2005; Jiang
et al., 2010) with 5-50% morbidity and nearly 100% mortality (Morozov
et al., 1998; Shen et al., 2009; Jiang
et al., 2010). Clinical signs of the disease include pallor, fever,
jaundice and progressive weight loss, together with respiratory and digestive
disorders (Clark, 1997; Liu
et al., 2007; Olvera et al., 2007; Shen
et al., 2009). It can also be pathologically characterized by lymphocyte
depletion and granulomatous inflammation of lymphoid tissues (Rosell
et al., 1999; Olvera et al., 2007).
Now a days, PMWS has caused great concern throughout the swine industry because
of its rapid, Simultaneous and worldwide emergence as well as the uncertain
transmission mode and high mortality rates in pigs (Jiang
et al., 2010).
Based on nucleotide or protein sequences of the complete genome or single (or
combined) gene, phylogenetic and evolutionary studies are of increasing importance
in molecular epidemiological studies on viral and bacterial pathogens (Jiang
et al., 2010). The genome of PCV2 contains 1.767-1.768 Nucleotides
(nt) which comprise 6 Open Reading Frames (ORFs) encoding putative proteins
(Meehan et al., 1998; Stevenson
et al., 2007). However, only 3 proteins encoded by ORF1, ORF2 and
ORF3 have been detected mainly in PCV2 infected cells (Cheung,
2003). Among them, ORF1 encodes 2 replication associated proteins (Rep and
Rep) (Mankertz et al., 1998; Cheung,
2003; Shang et al., 2007), ORF2 encodes a
viral Capsid Protein (Cap) which is involved in the host immune responses (Meehan
et al., 1998; Mahe et al., 2000;
Nawagitgul et al., 2000; Shang
et al., 2007). Previous studies indicated that the PCV2 was a kind
of multi-genotype virus based on the ORF2 and the whole genomic sequences as
well (Larochelle et al., 2002; Olvera
et al., 2007) which would increase viral complexity in the PMWS
pigs in different epidemic regions or eras (Shuai et
al., 2007; Segales et al., 2008; Wang
et al., 2009).
To monitor the latest genotypic variation of PCV2 in PMWS pigs in China, especially in the high-production provinces, 17 PCV2 strains were collected from 11 epidemic provinces in China in 2008 and 2009. The present study described the complete genomic sequence analysis of these 17 PCV2 strains including their genetic variations and genotypes.
MATERIALS AND METHODS
Field samples and DNA preparation: From January 2008 to December 2009,
clinical samples (lungs, lymph nodes, tonsil, livers and spleens) of pigs presenting
clinical signs and lesions associated with PMWS were collected from different
farms of 11 provinces in China. PCR and histopathologic analysis were used to
confirm the samples and the PCV2 positive samples were used for genome amplification.Representative
PCV2 stains with detailed information including the designation, date of isolation,
geographic origin, genome size, GenBankTM accession number are shown
in Table 1.
For tissue samples, homogenates comprising 1:5 tissue/Phosphate Buffer Saline (PBS) were obtained after grinding in a glass homogenizer and were disrupted by freeze-thawing three times. The supernatants containing viruses were recovered by centrifugation and then were treated using penicillin and streptomycin and filter (0.22 μm). The final solutions were added to the cell monolayer of PK-15 cells for 1.5-2 h infection at 37°C and then discarded the infection solution and added maintenance media to culture the PCV2 virus. After 3-5 blind passages, the supernatants of cultural cells were used to extract total viral DNA with DNAZol extraction kit (Invitrogen) according to the manufacturers recommendations. RNA was removed with the RNAase treatment step of the kit. DNA was recovered with centrifugation and then it was dried and dissolved in sterile water.
Enzymatic amplification and sequencing: The complete genome of PCV2
was amplified with primers P1 (- CCGggatccCCGCGGGCTGGCTGAACTTTTAAAAGT-3) and
P2 (5-CAT ctcg ag ACCCGCGG AAA TTTCT GA CAAACGTTACA-3).
||PCV2 strains sequenced in the present study
||Genome sequences of PCV2 strains deposited in the GenBank
The primer pairs were designed using software Oligo (Version 6.0) according
to the published sequences of PCV2 in the GenBankTM (Table
2). PCR mix (25 μL) included 2.5 μM of each primer, 2.5 μL
of 10xrTaq buffer, 0.2 mM of each dNTP, 1.25U of rTaq DNA polymerase (TAKARA)
and 1 μL of DNA sample in a thermocycler (Biometra). The PCR was performed
with the following conditions: an initial denaturation at 94°C for 35 min,
followed by 94°C for 1 min (denaturation); 54°C for 1 min (annealing);
72°C for 1.5 min (extension) for 35 cycles then with a final extension at
72°C for 10 min. These optimized cycling conditions for the specific and
efficient amplification of individual DNA fragments were obtained after varying
annealing temperatures). Samples without genomic DNA (no-DNA controls) or with
host genomic DNA (host-DNA controls) were included in each amplification run
and in no case were amplicons detected in the no-DNA and host-DNA controls.
Each amplicon (5 μL) was examined by agarose gel electrophoresis to validate
amplification efficiency (Zhao et al., 2009a,
The PCR products were purified using spin columns (Wizard PCR-Preps DNA Purification System, Promega) and ligated with pGEM-T Easy plasmid vector (Promega) according to the manufacturers recommendations. The recombinant plasmid was then transformed into Escherichia coli DH5α competent cells (Promega) and positive transformants were selected and checked by PCR amplification. Cell cultures with confirmed recombinant plasmid were cultured and sent to Songon Co. Ltd. (Shanghai) for sequencing. For each sample, 3 colonies were sequenced from both ends.
Molecular genetic analysis: Sequences of the complete genome were aligned
using the Clustal X 1.81 (Thompson et al., 1997)
and mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml).
Alignments were checked by eye and modifications were made as required. Megalign
procedure within the DNAStar 5.0 (Burland, 2000) was
used to analyze sequence identity and to calculate base composition and the
divergence. The Neighbor-Joining (NJ) method in Mega version 4.0 (Tamura
et al., 2007) was used to examine the genetic relationship under
default settings. The consensus tree was obtained after bootstrap analysis,
with 1,000 replications and with values above 50% reported. We divided PCV2
genotypes with the following criteria as described by previous studies (Segales
et al., 2008; Wang et al., 2009):
when the ORF2 genetic distance between them is >0.035 and agrees with the
distance between viral sequence groups in the phylogenetic trees revealed by
complete genomic sequences, the two PCV2 genotypes are regarded as different.
Phylograms were drawn using the Tree View program version 1.65 (Page,
RESULTS AND DISCUSSION
Sequence analysis of nucleotide and amino acid: Genomic DNA was prepared
from 17 stains in 11 epidemic provinces of China. The complete genomes were
amplified successfully from all of the samples. The amplicons were subjected
to agarose gel electrophoresis, sequenced and then deposited into the GenBank
with accession numbers HM776437-HM776453 (Table 1). No size
variation was detected in all of the genomic sequences which were 1.767 bp in
length. The A+T contents of complete genomic sequences were 51.10-51.78%, slight
higher than G+C contents. All the complete genomic sequences of PCV2 in the
present study contained at least six Open Reading Frames (ORFs), encoding 2
major proteins (Rep and Cap) as previous studies reported (Shang
et al., 2007; Wang et al., 2009).
The comparison of complete genomic sequences revealed 95.6-99.9% identity in
the 17 PCV strains and the most variable regions were within 1.000-1.700 nt
(located in the coding region of ORF2) (Fig. 1) which was
consistent with previous study in China (Wang et al.,
2009). Among these viruses, ZJ-38 and HLJ-10 showed the highest identity
with a percentage nearly to 100%. Comparative analysis of amino acids of the
two ORFs revealed that the variation extend of ORF2 (93.1-100%) was greater
than ORF1 (98.4-100%) and the third codon position showed more variable than
the first and second sites.
To date, at least 6 linear immunodominant regions and 5 overlapping conformational
B lymphocyte epitopes within 47-85, 156-202 and 230-233 residues in the PCV2
Cap were revealed by PEPSCAN analysis and mAbs scanning (Mahe
et al., 2000; Shang et al., 2007;
Lekcharoensuk et al., 2004). Previous study also
showed that 2 immunodominant regions encoded by PCV2 ORF1 (amino acid residues
81-100 and 201-220) and 1 region encoded by PCV2 ORF3 (amino acid residues 31-50)
appeared to be able to induce T lymphocyte proliferation in porcine Peripheral
Blood Mononuclear Cells (PBMCs) from infected pigs (Stevenson
et al., 2007). It suggested that the T lymphocytes responses to PCV2
are primarily directed toward epitopes of the nonstructural proteins of ORF1
and ORF3. Comparative analysis of these regions detected 13 mutations (Table
3) in ORF2, 2 in ORF1 and 1 in ORF3 and T lymphocyte epitopes were more
conserved than those of B lymphocytes (Table 3).
Phylogenetic relationships among PCV2 sequences: The NJ phylogenetic
tree of the 17 Chinese PCV2 strains sequenced in the present study, together
with 66 PCV2 strains available in the GenBankTM (Table
2) with complete genomic nucleotide sequences, are shown in Fig.
||Comparative analysis of complete genomic sequences of PCV2
in China using mVISTA
||Mutations in the sequences of T and B lymphocyte epitopes
All of these Chinese PCV2 strains were isolated from 25 epidemic provinces,
respectively. Phylogenetic analysis showed 6 novel genotypes of PCV2 in addition
to the 5 known geneotypes (PCV-2a, PCV-2b, PCV-2c, PCV-2d, PCV-2e) reported
(Segales et al., 2008; Wang
et al., 2009). The sequences were segregated into two large groups
A and B. The upper part of the tree contained 2 genotypes (PCV-2d and PCV-2b)
in previous studies and 2 novel genotypes (unidentified type 2 and 3). Three
genotypes (PCV-2a, PCV-2c, PCV-2e) with 3 novel genotypes were in the other
clade. Of these genotypes, PCV-2b, PCV-2d and the 3 unidentified genotypes were
the most prevailing, within 13, 17 and 16 epidemic provinces, respectively (Table
4). For the Chinese PCV2 strains in this study, 5 strains were in PCV-2b,
6 strains were in PCV-2d, 1 strains in PCV-2e and other 5 strains were in novel
genotypes but no strains were in genotypes of PCV-2a and PCV-2c (Fig.
The study of the evolution of the genetic diversity of virus strains and the
existence of variants are very important. It provides a better understanding
of the pathogenesis of these potentially emerging diseases and has implications
for the development of prophylactic measures (Larochelle
et al., 2002). On the other hand, point mutation and recombination
are major mechanism of viral evolution (Ma et al.,
In the present study, complete genomes of 17 PCV2 strains in 11 epidemic provinces
were sequenced and analyzed. The sequences contained at least 6 ORFs and it
was found that the Cap protein encoded by ORF2 was the most variable regions
and it exhibited a higher rate of variation (the lowest homology reported being
90%) compared with ORF1 which was consistent with previous studies (Brunborg
et al. 2004; Wen et al., 2005; Wang
et al., 2009). The result of the present study further confirmed
that capsid protein was the major structural protein responsible for viral pathogenecity
(Hamel et al., 2000; Mankertz
et al., 2000; Larochelle et al., 2002).
||Phylogenetic analysis of 17 PCV2 strains in this study and
66 strains isolates from other regions of China or other countries based
on the complete genomic nucleotide sequences. The tree was constructed using
Neighbor-Joining (NJ) algorithm by MEGA 4.0. (▲)Reference sequences
for genotype PCV-2a; (▼) reference sequences for genotype PCV-2b;
(●) reference sequences for genotype PCV-2c; (w) reference sequences
for genotype PCV-2d; (♦) reference sequences for genotype PCV-2e
||Genotypes of PCV2 in different provinces in China
Simultaneously, deduced amino acid sequences alignment obtained mutations in
epitopes of T and B lymphocytes as previous reports (Larochelle
et al., 2002; Wen et al., 2005). Previous
studies indicated that the genetic variations of PCV2 might be associated with
geographic origin rather than with differences in tropism (Fenaux
et al., 2000; Meehan et al., 2001).
However, the study showed that the genotypes of PCV2 were not relation to their
locations. PCV2 strains from different provinces contained some of the same
mutation sites, while strains from the same province have different variations.
Phylogenetic tree based on complete genomic sequences also showed that strains
from the same provinces were categorized into different genotypes, while same
genotypes were in different orgins. The genotypes in the same geographical orgins
would be changed with years. These results could be one of the reasons for immunity
complexity of PCV2 infection.
The present study examined genetic variations and genotypes of PCV2 strains from 11 epidemic provinces in China in 2008 and 2009 based on complete genomic sequence. Analyses of these sequences revealed low level genetic differences of PCV2 strains in China. Phylogenetic relationships analyses showed that there are novel genotypes of PCV2 existed in China. These results would have important implications for the effective prevention and control of PCV2 infection.
Project support was provided by the Ministry of Agriculture Program for prevention and monitoring of animal disease (NY200609-03) to DK Chen and the Special Funds for Talents in Northwest A and F University to GH Zhao.