[show abstract][hide abstract] ABSTRACT: Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of porcine reproductive and respiratory syndrome (PRRS) and porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome (PMWS) in pigs. Coinfection with highly pathogenic PRRSV (HP-PRRSV) and PCV2 in the field has recently become extensive in some Asian countries. A synergistic pathogenicity between PRRSV and PCV2 infections has previously been reported. However, the consequences of the sequential infection of pigs with these two viruses are unknown.
Thirty 35-day-old piglets were randomly divided into six groups (n = 5 each): HP-PRRSV/PCV2 (group 1, inoculated with HP-PRRSV, then inoculated with PCV2 one week later), PCV2/HP-PRRSV (group 2, inoculated with PCV2, then inoculated with HP-PRRSV one week later), HP-PRRSV+PCV2 (group 3, inoculated with HP-PRRSV and PCV2 concurrently), HP-PRRSV (group 4, inoculated with HP-PRRSV), PCV2 (group 5, inoculated with PCV2), and the control (group 6, uninfected). This experiment lasted 28 days. Clinical symptoms and rectal temperatures were recorded each day after inoculation, body weight was recorded weekly, and serum samples were obtained for viral nucleic acid quantification and antibody titration. Variations in CD3+, CD4+ CD8--, CD3+, CD4--, and CD8+ cells, natural killer (NK) cells, and mononuclear cells were determined by flow cytometry. The serum concentrations of interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), and macrophage granulocyte-colony stimulating factor (GM-CSF) were determined. Pathological changes in different tissues from the experimentally infected pigs were recorded.
The piglets in group 1 had the highest viral loads, the lowest antibody titers, the most-severe clinical signs, and the highest mortality (3/5, 60%; the mortality in the other groups was 0%), and interstitial pneumonia was more severe in this group compare to the other HP-PRRSV infected groups. The serum levels of IFN-gamma, TNF-alpha, IL-10, and GM-CSF varied (increased or decreased) most widely in group 1, as did each immunocyte subgroup.
HP-PRRSV infection followed by PCV2 infection enhanced the replication of both viruses in the experimental piglets and led to more-severe clinical signs and lesions, indicating greater synergistic effects during the sequential infection of piglets with HP-PRRSV and then PCV2.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Torque teno sus viruses (TTSuVs) are non-enveloped viruses and have single-stranded, negative sense circular DNA genomes and are widely distributed in pigs. But till now, the prevalence of TTSuVs with porcine circovirus type 2 (PCV2) in pig herds of China is not very clear; and the genetic variation among different TTSuVs isolate is very large and need to divide the subgroups. In this study, the co-infection with TTSuVs and porcine circovrius (PCV) in the pig population of China was investigated and the subgroups of all TTSuVs genomes in Genbank were divided. RESULTS: Results showed that the rate of co-infection with TTSuV1 and TTSuV2 reached 75% in PCV2-positive samples. Also Two TTSuV1 and four TTSuV2 isolates genome sequences were obtained, and the similarity of all TTSuV1 and TTSuV2 genomic sequences in GenBank were compared. Phylogenetic trees indicated that both the TTSuV1 and TTSuV2 sequences could be divided into four genotypes. Interestingly, the sub-genotypes TTSuV1d, TTSuV2c and TTSuV2d exist only in the pig population of China. CONCLUSIONS: This study demonstrates that co-infection with TTSuVs and PCVs is very common in the pig population of China, in which the viruses maybe contribute to clinical diseases cooperatively. In addition, three new subgroups of TTSuVs emerged in China for the first time and a high level of variation among different isolates of TTSuV1 and TTSuV2 was indicated by their genetic diversity.
[show abstract][hide abstract] ABSTRACT: Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs. A monoclonal antibody (mAb) 8E4 against the PCV2 capsid protein has the capacity to neutralize the virus. However, this mAb can only react with some PCV2a strains (LG, CL, and JF2; mAb 8E4-positive strains), but does not cross-react with some PCV2b strains (YJ and JF; mAb 8E4-negative strains). In the present study, site-directed mutagenesis was performed targeting the external amino acids of the capsid proteins, which are different between mAb 8E4-positive and -negative strains. A mutation of arginine to alanine at position 59 in the capsid protein of strain JF allowed the mutant to be recognized and neutralized by mAb 8E4. Likewise, mutations of arginine to alanine at position 59 together with alanine to threonine at position 60 in the capsid protein of the YJ strain resulted in a gain of neutralization and recognition by mAb 8E4. Here, we demonstrated that the amino acids at positions 59 and 60 in the capsid protein of PCV2 participate in the formation of conformational neutralizing epitopes and mutations at positions 59 or 59/60 result in novel neutralizing epitopes of mAb 8E4-negative strains. This study provides valuable information for further in-depth mapping of the conformational neutralizing epitopes, clarification of antigenic differences among PCV2 strains, and development of a useful vaccine candidate for control of PCV2-associated diseases.
[show abstract][hide abstract] ABSTRACT: Porcine circovirus type 1 (PCV1) has been identified as a contaminant of porcine kidney cell line (PK-15). Serological evidence and genetic studies have suggested that PCV1 is widespread in domestic pigs. In this study, monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were generated against a recombinant PCV1 Cap protein (PCV1-Cap), which was expressed using the baculovirus system. PEPSCAN analysis was used to identify epitopes on the PCV1-Cap with mAbs and pAbs. Three linear B-cell epitopes, including residues (85)GGTNPLP(91), (162)FTPKPELDKTIDWFHPNNK(180) and (219)YVQFREFILKDPLNK(233), specific for PCV1-Cap, were finely defined. These results will facilitate future investigations into antigenic differences and differential diagnosis between PCV1 and PCV2.
Archives of Virology 03/2012; 157(7):1339-44. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Porcine circovirus type 2 (PCV2) is considered to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), which has become a serious economic problem for the swine industry worldwide. The major genotypes, PCV2a and PCV2b, are highly prevalent in the pig population and are present worldwide. However, another newly emerging PCV2b genotype mutant, which has a mutation in its ORF2-encoded capsid protein, has been sporadically present in China, as well as in other countries. It is therefore important to determine the relative virulence of the newly emerging PCV2b genotype mutant, compared with the existing PCV2a and PCV2b genotypes, and to investigate whether the newly emerging mutant virus induces more severe illness.
Twenty healthy, 30-day-old, commercial piglets served as controls or were challenged with PCV2a, PCV2b and the newly emerging mutant virus. A series of indexes representing different parameters were adopted to evaluate virulence, including clinical signs, serological detection, viral load and distribution, changes in immune cell subsets in the peripheral blood, and evaluation of pathological lesions. The newly emerging PCV2 mutant demonstrated more severe signs compatible with PMWS, characterized by wasting, coughing, dyspnea, diarrhea, rough hair-coat and depression. Moreover, the pathological lesions and viremia, as well as the viral loads in lymph nodes, tonsils and spleen, were significantly more severe (P<0.05) for piglets challenged with the newly emerging mutant compared with those in the groups challenged with PCV2a and PCV2b. In addition, a significantly lower average daily weight gain (P<0.05) was recorded in the group challenged with the newly emerging PCV2 mutant than in the groups challenged with the prevailing PCV2a and PCV2b.
This is believed to be the first report to confirm the enhanced virulence of the newly emerging PCV2 mutant in vivo.
PLoS ONE 01/2012; 7(7):e41463. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two recombinant mutants of porcine circovirus type 2 (PCV2), which resulted from replacement of a genomic fragment containing the open reading frame 2 (ORF2) of genotype PCV2b with that of genotype PCV2a, were obtained initially from co-infection with PCV2a and 2b genotype viruses in vitro. The two mutant viruses contained the ORF1 sequence from genotype PCV2b and the ORF2 sequence from genotype PCV2a. They were designated according to the nomenclature proposed by Grau et al., indicating the origin of the ORF1 sequence first and that of the ORF2 sequence second, i.e., PCV2b(JF11)/2a(CL1) and PCV2b(YJ)/2a(CL1). The replication efficiencies of the two PCV2 recombinant mutants were enhanced significantly and their antigenicities were altered significantly in vitro when compared with their parental strains.
[show abstract][hide abstract] ABSTRACT: Porcine circovirus type 2 (PCV2) is a major causal agent of post-weaning multisystemic wasting syndrome in piglets. To investigate the feasibility of PCV2 expressing an exogenous epitope, a 14-amino-acid V5 epitope derived from simian parainfluenza virus type 5, was inserted into the C terminus of the capsid protein. Recombinant PCV2 expressing the V5 epitope, recPCV2/CL-V5, was rescued by transfecting an infectious clone into PK-15 cells and was characterised by an immunoperoxidase monolayer assay (IPMA), a serum neutralisation assay (SNA), a capture enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy. The V5 epitope was detected in the recombinant marker virus by IPMA and capture ELISA. Furthermore, there was no detectable difference in the antigenicity of the recombinant marker virus compared with the parental virus by IPMA and SNA using PCV2-positive serum and the neutralising monoclonal antibody 1D2. However, recPCV2/CL-V5 marker virus could be differentiated from the parental virus by PCR, IPMA and capture ELISA. The recombinant marker virus was stable on multiplication through 10 passages in PK-15 cells, with a maximum titre of 10(6.25) 50% tissue culture infective dose (TCID(50))/ml. BALB/c mice were inoculated with the recombinant or parental virus via the intranasal and intraperitoneal routes. The parental and recombinant viruses both could replicate in mice, cause microscopic pathological changes, and induce mice to generate anti-PCV2 antibodies. Furthermore, the recombinant marker virus could also induce anti-V5 epitope tag antibodies. These results indicated that V5 epitope could be displayed on the surface of the capsid protein by inserting its gene just before stop codon of open reading frame 2. More importantly, insertion of the V5 epitope did not seem to interfere with biological characterisation of the recPCV2/CL-V5 marker virus.
Virus Research 05/2011; 161(2):115-23. · 2.75 Impact Factor
[show abstract][hide abstract] ABSTRACT: Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic syndrome in pigs. The capsid (Cap) protein encoded by ORF2 is the main structural protein involved in the host immune protective response to PCV2. It is therefore important to map the antigenic epitopes of the PCV2 Cap protein.
In this study, 5 monoclonal antibodies (mAbs) against the recombinant PCV2 Cap protein, expressed by the baculovirus system in Sf21 insect cells, were generated. The antigenic epitope recognized by these mAbs was located in the Cap A protein by Western blot analysis using 4 overlapping minifragments covering the Cap protein expressed in Escherichia coli. To locate the epitope more accurately, 3 sets of overlapping peptides were synthesized.
The results demonstrated that 4 of the 5 mAbs recognized the same core epitope ((26)RPWLVHPRHRY(36)) located in the nuclear localization signal (NLS) region at the N terminus of the Cap protein. The other mAb (1D2) reacted with the recombinant Cap protein only, indicating that it recognizes a potential conformational epitope. This mAb demonstrated a neutralizing effect on PCV2.
This is the first study to identify an antigenic epitope in the NLS region of the PCV2 Cap protein using mAbs. The results of this study will facilitate future investigations into the mechanism and function of nuclear localization of this protein.
[show abstract][hide abstract] ABSTRACT: A monoclonal antibody (Mab)-based blocking ELISA was developed for the detection of serum neutralizing antibodies to porcine circovirus type 2 (PCV2). The Mab with neutralizing activity, which was produced by immunizing a recombinant capsid protein of PCV2 expressed in insect cells, was used as the detector antibody. The assay was evaluated in comparison with a serum neutralization assay, and its sensitivity and specificity were determined to be 98.8% and 88.5%, respectively. A significant positive correlation was found between results of the blocking ELISA and the serum neutralization assay (r=0.9381). The assay was verified by testing experimental and commercial pig sera. A longitudinal antibody profile showed that serum neutralizing antibodies were detected 2 weeks after vaccination and that the detection rate reached 100% at 4 weeks. The serum neutralizing antibody profile showed a decrease from the age of 4 to 13 weeks, and seroconversion after 13 weeks in pigs from a commercial pig farm. Additionally, the positive detection rate in 703 sera collected from nine commercial pig farms was 73%. This report demonstrates that the assay is a simple, specific, sensitive and convenient method for epidemiological surveys and evaluations of serum neutralizing antibodies against PCV2.
Journal of virological methods 10/2010; 171(1):26-33. · 2.13 Impact Factor