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Porcine circovirus type 2 (PCV2) has been confirmed as the primary cause of postweaning multisystemic wasting syndrome (PMWS). However, in the field, PMWS is seen only in a small percentage of pigs infected with PCV2. The overall objective of the study reported here was to determine whether host genetic differences in the susceptibility to PCV2-associated disease exist among selected breeds of pigs. This study included Duroc (n = 23), Landrace (n = 19), and Large White (n = 21) pigs. The pigs were infected intranasally and intramuscularly at 5-7 weeks of age with PCV2. A portion of the pigs (31/63; 30.2%) had low passively acquired PCV2 antibodies at the time of infection. There were no differences in mean weight gain, rectal temperature, or respiratory score. Clinical disease compatible with PMWS was observed only in the Landrace pigs. Most of the PCV2-infected pigs had enlarged lymph nodes, and individual Duroc and Landrace pigs had mottled tan lungs. PCV2-associated lymphoid depletion and granulomatous inflammation were observed in pigs of all breeds. Three of 19 Landrace pigs and none of the Duroc or Large White pigs developed severe lymphoid lesions associated with large amounts of intralesional PCV2 antigen typical of PMWS. Compared with seronegative Landrace pigs, Landrace pigs that had low maternal antibodies at the time of PCV2 inoculation had significantly (P < 0.05) less-severe PCV2-associated lesions. The results suggest a predisposition of the Landrace pigs of this study to PCV2-induced disease and lesions, and that low levels of passively acquired antibodies are protective.

To determine whether there is an effect of the timing of vaccination on porcine circovirus type 2 (PCV-2) replication and PCV-2-associated lesions, 78 pigs were randomly assigned to eight groups: group 1 (10 pigs) was vaccinated with a commercial Mycoplasma hyopneumoniae vaccine at two and four weeks of age, group 2 (nine pigs) was vaccinated at four and six weeks of age, group 3 (10 pigs) at six and eight weeks of age and group 4 (10 pigs) at eight and 10 weeks of age; group 5 (nine pigs) was vaccinated once with a double dose at four weeks of age, and group 6 (10 pigs) was vaccinated once with a double dose at eight weeks of age. Groups 7 and 8, both of 10 pigs, were not vaccinated. At eight weeks of age, the pigs in groups 1 to 7 were inoculated with PCV-2. Fourteen days after they had been inoculated, the pigs in groups 1, 4 and 5 had significantly (P<0.05) more copies of the PCV-2 genome in their serum than the unvaccinated pigs. Microscopically, 14 of the 68 inoculated pigs had normal lymphoid tissues, 40 had mild PCV-2-associated lymphoid lesions and 14 had moderate lesions. The mean overall lymphoid lesions (lymphoid depletion, granulomatous inflammation, and quantity of PCV-2 antigen in spleen, tonsil, and five lymph nodes) were significantly (P<0.05) more severe in groups 4 and 5 than in groups 2, 3, 7 and 8.

To determine the effects of porcine circovirus type 2 (PCV2) maternal antibodies on and response to experimental PCV2 infection, 24 piglets were divided into four groups on the basis of the enzyme-linked immunosorbent assay titers of PCV2 maternal antibodies: group A (n = 6; sample/positive [S/P] ratio, <0.2), group B (n = 5; S/P ratio, >0.2 to <0.5), and groups C (n = 8) and D (n = 5) (S/P ratio, >0.5). Piglets in groups A, B, and C were inoculated with PCV2 at day 0 and challenged with PCV2 at day 42. Group D piglets were not exposed to PCV2 at day 0 but were challenged at day 42. Before challenge, seroconversion to PCV2 antibodies occurred in five of six group A piglets, and the antibody level rose above the cutoff level in one of five group B piglets. Viremia was detected in five of six, four of five, and two of eight pigs in groups A, B, and C, respectively. After challenge, PCV2 DNA was detectable from 7 to 21 days postchallenge in the sera from six of six, four of five, three of eight, and five of five pigs in groups A, B, C, and D, respectively. The results indicated that protection against PCV2 infection conferred by maternal antibodies is titer dependent: higher titers are generally protective, but low titers are not.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. To identify potential genetic determinants for virulence and replication, we serially passaged a PCV2 isolate 120 times in PK-15 cells. The viruses harvested at virus passages 1 (VP1) and 120 (VP120) were biologically, genetically, and experimentally characterized. The PCV2 VP120 virus replicated in PK-15 cells to a titer similar to that of the PK-15 cell line-derived nonpathogenic PCV1 but replicated more efficiently than PCV2 VP1 with a difference of about 1 log unit in the titers. The complete genomic sequences of viruses at passages 0, 30, 60, 90, and 120 were determined. After 120 passages, only two nucleotide mutations were identified in the entire genome, and both were located in the capsid gene: the mutations were located at nucleotide positions 328 (C328G) and 573 (A573C). The C328G mutation, in which a proline at position 110 of the capsid protein changed to an alanine (P110A), occurred at passage 30 and remained in the subsequent passages. The second mutation, A573C, resulting in a change from an arginine to a serine at position 191 (R191S), appeared at passage 120. To experimentally characterize the VP120 virus, 31 specific-pathogen-free pigs were randomly divided into three groups. Ten pigs in group 1 received phosphate-buffered saline as negative controls. Each pig in group 2 (11 pigs) was inoculated intramuscularly and intranasally with 10(4.9) 50% tissue culture infective doses (TCID(50)) of PCV2 VP120. Each pig in group 3 (10 pigs) was similarly inoculated with 10(4.9) TCID(50) of PCV2 VP1. Viremia was detected in 9 of 10 pigs in the PCV2 VP1 group with a mean duration of 3 weeks, but in only 4 of 11 pigs in the PCV2 VP120 group with a mean duration of 1.6 weeks. The PCV2 genomic copy numbers in serum in the PCV2 VP1 group were significantly higher than those in the PCV2 VP120 group (P < 0.0001). Gross and histopathologic lesions in pigs inoculated with PCV2 VP1 were more severe than those inoculated with PCV2 VP120 at both day 21 and 42 necropsies (P = 0.0032 and P = 0.0274, respectively). Taken together, the results from this study indicated that the P110A and R191S mutations in the capsid of PCV2 enhanced the growth ability of PCV2 in vitro and attenuated the virus in vivo. This finding has important implications for PCV2 vaccine development.

Non-pathogenic porcine circovirus type 1 (PCV1) and pathogenic PCV2 are widespread in swine herds. In this study, the detection and characterization of PCV1 and PCV2 DNA from porcine-derived commercial pepsin are reported. The complete genomic sequences of the pepsin-derived PCV1 and PCV2 share 76 % nucleotide sequence identity with each other and 95-99 % identity with respective North American PCV1 and PCV2 isolates. However, the PCV-contaminated pepsin lacks infectivity in PK-15 cells. To further assess the infectivity of the contaminating pepsin in vivo, 16 5-week-old, specific-pathogen-free pigs were divided randomly into three groups: pigs in group 1 (n=5) were each inoculated intramuscularly and intranasally with 4 ml PBS buffer as negative controls, those in group 2 (n=6) were each inoculated with 400 mg contaminated pepsin dissolved in 4 ml PBS and those in group 3 (n=5) were each inoculated with 4 x 10(4.3) TCID(50) PCV2 as positive controls. PCV2 viraemia, seroconversion and pathological lesions were detected in group 3 pigs, but not in group 1 or 2 pigs, confirming that the contaminating PCVs were non-infectious. Nevertheless, the detection of PCV DNA in a porcine-derived commercial product raises concern for potential human infection through xenotransplantation.

The objectives of this study were to investigate the interactions between Mycoplasma hyopneumoniae and porcine circovirus type 2 (PCV2) and to establish a model for studying the pathogenesis of and testing intervention strategies for the control of PCV2-associated porcine respiratory disease complex (PRDC). Sixty-seven pigs were randomly assigned to four groups. Group 1 (n=17) pigs served as controls, group 2 (n=17) pigs were inoculated with M. hyopneumoniae, group 3 (n=17) pigs were dual infected with M. hyopneumoniae and PCV2, and group 4 (n=16) pigs were inoculated with PCV2. Pigs were inoculated intratracheally with M. hyopneumoniae at 4 weeks of age followed by intranasal inoculation with PCV2 at 6 weeks of age. Dual-infected pigs had moderate dyspnea, lethargy, and reduced weight gain. The overall severity of macroscopic lung lesions, PCV2-associated microscopic lesions in lung and lymphoid tissues, and the amount of PCV2-antigen associated with these lesions were significantly (P <0.05) higher in dual-infected pigs compared with all other groups. Four of 17 (23.5%) dual-infected pigs had decreased growth rate and severe lymphoid depletion and granulomatous lymphadenitis associated with high amounts of PCV2-antigen consistent with postweaning multisystemic wasting syndrome (PMWS). PCV2-antigen in lung tissue was most often associated with M. hyopneumoniae-induced peribronchial lymphoid hyperplasia, suggesting that this is an important site for PCV2 replication in the lung. This study indicates that M. hyopneumoniae potentiates the severity of PCV2-associated lung and lymphoid lesions, increases the amount and prolongs the presence of PCV2-antigen, and increases the incidence of PMWS in pigs.

Porcine circovirus type 2 (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas PCV1 is nonpathogenic. We previously demonstrated that a chimeric PCV1-2 virus (with the immunogenic capsid gene of PCV2 cloned into the backbone of PCV1) induces an antibody response to the PCV2 capsid protein and is attenuated in pigs. Here, we report that the attenuated chimeric PCV1-2 induces protective immunity to wild-type PCV2 challenge in pigs. A total of 48 specific-pathogen-free piglets were randomly and equally assigned to four groups of 12 pigs each. Pigs in group 1 were vaccinated by intramuscular injection with 200 microg of the chimeric PCV1-2 infectious DNA clone. Pigs in group 2 were vaccinated by intralymphoid injection with 200 microg of a chimeric PCV1-2 infectious DNA clone. Pigs in group 3 were vaccinated by intramuscular injection with 10(3.5) 50% tissue culture infective doses (TCID(50)) of the chimeric PCV1-2 live virus. Pigs in group 4 were not vaccinated and served as controls. By 42 days postvaccination (DPV), the majority of pigs had seroconverted to PCV2 capsid antibody. At 42 DPV, all pigs were challenged intranasally and intramuscularly with 2 x 10(4.5) TCID(50) of a wild-type pathogenic PCV2 virus. By 21 days postchallenge (DPC), 9 out of the 12 group 4 pigs were viremic for PCV2. Vaccinated animals in groups 1 to 3 had no detectable PCV2 viremia after challenge. At 21 DPC the lymph nodes in the nonvaccinated pigs were larger (P < 0.05) than those of vaccinated pigs. The PCV2 genomic copy loads in lymph nodes were reduced (P < 0.0001) in vaccinated pigs. Moderate amounts of PCV2 antigen were detected in most lymphoid tissues of nonvaccinated pigs but in only 1 of 36 vaccinated pigs. Mild-to-severe lymphoid depletion and histiocytic replacement were detected in lymphoid tissues in the majority of nonvaccinated group 4 pigs but in only a few vaccinated group 1 to 3 pigs. The data from this study indicated that when given intramuscularly in pigs, the attenuated chimeric PCV1-2 live virus, as well as the chimeric PCV1-2 infectious DNA clone, induces protective immunity against PCV2 infection and could potentially serve as an effective vaccine.

The objectives of this study were to determine if coinfection of segregated early weaned (SEW) pigs with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) induces an increase in the incidence of post-weaning multisystemic wasting syndrome (PMWS) compared to singular PCV2 infection, and to determine if vaccination against PPV protects pigs against PMWS associated with PCV2/PPV coinfection in SEW pigs. Seventy, 3-week-old, SEW pigs were randomly assigned to one of the five groups. Pigs in group 1 (n = 14) served as the negative controls, group 2 pigs (n = 14) were inoculated with PCV2, group 3 pigs (n = 12) were inoculated with PPV, groups 4 (n = 16) and 5 (n = 14) pigs were inoculated with both PCV2 and PPV. Pigs in groups 1-3 and 5 were vaccinated with two doses of a killed parvovirus-leptospira-erysipelothrix (PLE) vaccine prior to inoculation. The PCV2/PPV-coinfected pigs (groups 4 and 5) had significantly (P < 0.05) higher and more persistent fevers than the singular PCV2-infected pigs. One pig in each of the coinfected groups developed clinical disease (fever, respiratory disease, jaundice, weight loss) consistent with PMWS. Lymphoid depletion was significantly (P < 0.05) more severe in the dually-infected pigs at 42 days post-inoculation (DPI). Vaccinated, coinfected pigs (group 5) remained viremic significantly (P < 0.05) longer and had higher copy numbers of genomic PCV2 DNA in sera at 28, 35, and 42 DPI compared to the unvaccinated coinfected pigs (group 4). PPV-viremia was detected only in the unvaccinated group 4 pigs. PLE-vaccination prevented PPV-viremia but did not prevent clinical PMWS or reduce the severity of lymphoid depletion in PCV2/PPV-coinfected pigs. Evidence of increased incidence of clinical PMWS due to vaccination was not observed in this model.

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. We report here the construction and characterization of two chimeric infectious DNA clones of PCV1 and PCV2. The chimeric PCV1-2 clone contains the PCV2 capsid gene cloned in the backbone of the nonpathogenic PCV1 genome. A reciprocal chimeric PCV2-1 DNA clone was also constructed by replacing the PCV2 capsid gene with that of PCV1 in the backbone of the PCV2 genome. The PCV1, PCV2, and chimeric PCV1-2 and PCV2-1 DNA clones were all shown to be infectious in PK-15 cells, and their growth characteristics in vitro were determined and compared. To evaluate the immunogenicity and pathogenicity of the chimeric infectious DNA clones, 40 specific-pathogen-free (SPF) pigs were randomly assigned into five groups of eight pigs each. Group 1 pigs received phosphate-buffered saline as the negative control. Group 2 pigs were each injected in the superficial inguinal lymph nodes with 200 micro g of the PCV1 infectious DNA clone. Group 3 pigs were each similarly injected with 200 micro g of the PCV2 infectious DNA clone, group 4 pigs were each injected with 200 micro g of the chimeric PCV1-2 infectious DNA clone, and group 5 pigs were each injected with 200 micro g of the reciprocal chimeric PCV2-1 infectious DNA clone. As expected, seroconversion to antibodies to the PCV2 capsid antigen was detected in group 3 and group 4 pigs. Group 2 and 5 pigs all seroconverted to PCV1 antibody. Gross and microscopic lesions in various tissues of animals inoculated with the PCV2 infectious DNA clone were significantly more severe than those found in pigs inoculated with PCV1, chimeric PCV1-2, and reciprocal chimeric PCV2-1 infectious DNA clones. These data indicated that the chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genomic backbone induces a specific antibody response to the pathogenic PCV2 capsid antigen but is attenuated in pigs. Future studies are warranted to evaluate the usefulness of the chimeric PCV1-2 infectious DNA clone as a genetically engineered live-attenuated vaccine against PCV2 infection and PMWS.

The objective of this study was to determine whether vaccination with bacterins commonly used in the USA, when administered at a time typical of US protocol, enhances porcine circovirus type 2 (PCV2) replication and the incidence and severity of clinical signs and lesions characteristic of postweaning multisystemic wasting syndrome (PMWS) in conventional pigs. Sixty-one pigs free of PCV2 were randomly assigned to four groups. Groups 1 (n = 15) and 2 (n = 15) pigs served as sham-inoculated negative controls. Groups 3 (n = 14) and 4 (n = 17) pigs were inoculated intralymphoid with PCV2 field isolate ISU-40895. Pigs in groups 2 and 4 were vaccinated with Actinobacillus pleuropneumoniae (APP) and Mycoplasma hyopneumoniae (M. hyopneumoniae) bacterins 21 days before and again 1 day before inoculation with PCV2. Mild transient respiratory disease and diarrhea were observed from 13 to 34 days postinoculation (DPI) in pigs in groups 3 and 4. Half the pigs from each group were necropsied at 22 and 34 DPI, respectively. Moderately enlarged, tan-colored lymph nodes were observed in the majority of pigs in groups 3 and 4. There was a significantly (P < 0.05) longer length of viremia (2.14 +/- 0.26 versus 4.44 +/- 0.23 weeks), a higher copy number of the PCV2 genome in serum, a wider range of tissue distribution of PCV2 antigen, and an increased severity of lymphoid depletion in pigs vaccinated with commercial APP and M. hyopneumoniae vaccines and inoculated with PCV2 compared with PCV2-inoculated unvaccinated pigs. Swine producers and veterinarians may need to consider changes in vaccination protocols in herds with recurrent PCV2-associated PMWS.

Infection of animals with a molecular viral clone is critical to study the genetic determinants of viral replication and virulence in the host. Type 2 porcine circovirus (PCV2) has been incriminated as the cause of postweaning multisystemic wasting syndrome (PMWS), an emerging disease in pigs. We report here for the first time the construction and use of an infectious molecular DNA clone of PCV2 to characterize the disease and pathologic lesions associated with PCV2 infection by direct in vivo transfection of pigs with the molecular clone. The PCV2 molecular clone was generated by ligating two copies of the complete PCV2 genome in tandem into the pBluescript SK (pSK) vector and was shown to be infectious in vitro when transfected into PK-15 cells. Forty specific-pathogen-free pigs at 4 weeks of age were randomly assigned to four groups of 10 each. Group 1 pigs served as uninoculated controls. Pigs in group 2 were each inoculated intranasally with about 1.9 x 10(5) 50% tissue culture infective doses of a homogeneous PCV2 live virus stock derived from the molecular clone. Pigs in group 3 were each injected intrahepatically with 200 microg of the cloned PCV2 plasmid DNA, and pigs in group 4 were each injected into the superficial iliac lymph nodes with 200 microg of the cloned PCV2 plasmid DNA. Animals injected with the cloned PCV2 plasmid DNA developed infection resembling that induced by intranasal inoculation with PCV2 live virus stock. Seroconversion to PCV2-specific antibody was detected in the majority of pigs from the three inoculated groups at 35 days postinoculation (DPI). Viremia, beginning at 14 DPI and lasting 2 to 4 weeks, was detected in the majority of the pigs from all three inoculated groups. There were no remarkable clinical signs of PMWS in control or any of the inoculated pigs. Gross lesions in pigs of the three inoculated groups were similar and were characterized by systemically enlarged, tan lymph nodes and lungs that failed to collapse. Histopathological lesions and PCV2-specific antigen were detected in numerous tissues and organs, including brain, lung, heart, kidney, tonsil, lymph nodes, spleen, ileum, and liver of infected pigs. This study more definitively characterizes the clinical course and pathologic lesions exclusively attributable to PCV2 infection. The data from this study indicate that the cloned PCV2 genomic DNA may replace infectious virus for future PCV2 pathogenesis and immunization studies. The data also suggest that PCV2, although essential for development of PMWS, may require other factors or agents to induce the full spectrum of clinical signs and lesions associated with advanced cases of PMWS.

Postweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Increasing evidence indicates that a variant strain of porcine circovirus (PCV), designated type 2 PCV (PCV-2), is responsible for PMWS. To determine the extent of genetic heterogeneity of PCV-2 isolates, the complete genomes of six PCV-2 isolates from different regions of North America were amplified by PCR and sequenced. Sequence and phylogenetic analyses confirmed that two distinct genotypes of PCV exist: nonpathogenic genotype PCV-1 and PMWS-associated genotype PCV-2. However, within the PCV-2 genotype, several minor branches that have been identified appear to be associated with geographic origins. The genomic sequences of two French PCV-2 isolates diverge the most from those of other PCV-2 isolates and form a distinct branch. Other minor but distinguishable branches have also been identified for a Taiwan PCV-2 isolate and two of the Canadian PCV-2 isolates. All the U.S. PCV-2 isolates are closely related, but the Canadian isolates vary, to some extent, in their genomic sequences. The data from this study indicate that although the genome of PCV-2 is generally stable among different isolates, PCV-2 isolates from different geographic regions vary in their genomic sequences. This variation may have important implications for PCV-2 diagnosis and research. On the basis of genetic analyses of available PCV strains, a universal PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed to detect and differentiate between infections with PCV-1 and PCV-2. This PCR-RFLP assay should be useful for studying the pathogenesis of PCV-2, for detecting PCV-2 infection in pigs from different geographic regions, and for screening donor pigs for use in xenotransplantation.