A porcine circovirus type 2 (PCV2) mutant with 234 amino acids in capsid protein showed more virulence in vivo, compared with classical PCV2a/b strain

Abstract

Background: 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.

Methodology/principal findings: 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.

Conclusions: This is believed to be the first report to confirm the enhanced virulence of the newly emerging PCV2 mutant in vivo.

Figure 1. Clinical pictures of inguinal (A) and submandibular (B) lymph nodes in the different challenge groups.

Moderate to severe atrophy, enlargement and hemorrhage were observed in the inguinal and submandibular lymph nodes, respectively, compared with the control group. More severe clinical signs were observed in the PCV2b/rBDH-challenged group, such as simultaneous presence of atrophy, enlargement and hemorrhage of inguinal and submandibular lymph nodes. a: PCV2a/rCL challenge group; b: PCV2b/rJF challenge group; c: PCV2b/rBDH challenge group; d: control group.

Figure 2. Weight of individual animals from each group (A) and ADWG (B).

A: Weight of individual animals from each group at days 0 and 35, respectively. B: ADWG±SD was calculated for each treatment group throughout the experiment, with (*) indicating significant differences between the specified pairs. ADWG was significantly higher in the control group compared with the challenged groups (P<0.05). A significantly lower ADWG was observed in the PCV2b/rBDH-challenged group than in both the PCV2a/rCL- and PCV2b/rJF-challenged groups (P<0.05). No significant difference was seen between the PCV2a/rCL- and PCV2b/rJF-challenged groups (P>0.05).

Figure 3. Quantification and distribution of viral DNA loads.

Detection and quantification of viral DNA loads in lymphoid tissues, as well as spleen and lung, in pigs challenged experimentally with the PCV2a/rCL, PCV2b/rJF or PCV2b/rBDH virus. Group mean logarithm viral genomic copies/gram of tissue (±SD) was calculated as the corresponding value on the x axis for each treatment group. All samples from MEM-challenged pigs were negative. Pairs of treatments with (*) were significantly different (P<0.05).

Figure 4. Histopathological detail of inguinal lymph nodes showing different degrees of severity of lymphocyte depletion.

(A) Inguinal lymph node from PCV2a/rCL-challenged group with mild lymphocyte depletion. (B) Inguinal lymph node from PCV2b/rJF-challenged group with moderate lymphocyte depletion. (C) Inguinal lymph node from PCV2b/rBDH-challenged group with significant lymphocyte depletion. (D) Normal inguinal lymph node from control group with normal lymphocyte count. Hematoxylin & eosin staining (400×).

Figure 5. Proportion of monocytes and granulocytes in leukocyte subpopulations.

The proportion of monocytes and granulocytes in leukocyte subpopulations was significantly higher in the three PCV2 challenge groups compared with that in the control group, but no significant differences were observed among the three PCV2 challenge groups. Pairs of treatments with (*) were significantly different (P<0.05).

Figure 6. Changes of CD4⁺/CD8⁺ cells in peripheral blood of infected piglets.

Ratio of CD3⁺CD4⁺CD8⁻ cells to CD3⁺CD4⁻CD8⁺ cells (CD4⁺/CD8⁺) in PCV2b/rBDH-challenged group was significantly lower than that of the other two challenge groups at 3 and 7 DPC (P<0.05). When evaluated as a whole, all the PCV2 challenge groups showed a significant continuous decrease in the CD4⁺/CD8⁺ ratio, when compared with the control group from 21 DPC. Pairs of treatments with (*) were significantly different (P<0.05).

Figure 7. Schematic diagram of PCV2 strains used in this study.

For the PCV2b/rBDH strain, a shift from TTA to CTT in the genomic sequence resulted in a stop codon mutation (from UAA to AAG) in ORF2 (in the antisense chain of the genomic sequence of PCV2), giving an ORF2 gene of 705 nt with another stop codon, compared with the classical PCV2a and PCV2b genotypes.