To determine whether the tagged chimeric PCV1-2 viruses are infectious and immunogenic, a total of 12 pigs, 7 weeks old, were randomly assigned to four groups of 3 each. Each group was inoculated intramuscularly with 2 × 103.5
of PCV1-2, PCV1-2-KT3, PCV1-2-GLU, or phosphate-buffered saline (PBS). The HA-tagged viruses were not tested in vivo
, as they are not considered viable MLV candidates in pigs due to potential antibody cross-reactivity in pigs naturally infected with swine influenza viruses. The pigs were each bled prior to inoculation and weekly thereafter until necropsy at 42 days postinoculation (dpi). A nested PCR (first-round primers 5′-TGGAGAAGAAGTTGTTGT-3′ [forward] and 5′-ATGACGTATCCAAGGAGGCGTTACCGCAGAAGAAGACACCGCCCCCGCAG-3′ [reverse] and second-round primers 5′-GGAGGTACCCGAAGGCCGATTTGAAGCAG-3′ [forward] and 5′-CCCTTTGAATACTACAGA-3′ [reverse]) was used to detect viremia in the sera of all pigs. An enzyme-linked immunosorbent assay (ELISA) was used to detect anti-PCV2 antibodies in the weekly sera (19
). Synthetic-peptide-based ELISAs were used to detect anti-GLU and anti-KT3 tag antibodies in the sera. ELISA plates were coated with bovine serum albumin-conjugated GLU or KT3 synthetic peptides (GenScript, Piscataway, NJ). Diluted pig serum was added to each well and incubated 1 h at 37°C, followed by incubation with 1:2,000-diluted, horseradish peroxidase-labeled goat anti-swine IgG antibody (KPL, Gaithersburg, MD). The cutoffs for both the KT3 and GLU ELISAs were determined as the mean optical density at 450 nm (OD450
) of all negative pig sera from 0 to 42 dpi plus 3 standard deviations. To determine if the anti-PCV2 antibodies in the serum of each pig at 42 dpi were neutralizing, an in vitro
serum virus neutralization assay was performed. Serial 2-fold dilutions of each serum were mixed with an equal volume of PCV2a or PCV2b virus stock and incubated at 37°C for 1 h. PK-15 cells at 50% confluence in 96-well plates were infected in duplicate with 50 μl of each reaction mixture. After incubation at 37°C for 72 h, the infected cells were visualized by IFA (8
) and the percentage of virus neutralization was calculated.
Viremia was detected by nested PCR in 8/9 inoculated pigs, thus confirming infection by each chimeric virus (). PCV1-2-GLU had a noticeably lower frequency and duration of viremia than PCV1-2 and PCV1-2-KT3, which were similar. Sequencing and sequence analysis of all positive PCR products confirmed the authenticity and in vivo stability of each epitope-tagged virus (data not shown).
Detection by nested PCR of viremia of tagged and wild-type chimeric PCV1-2 in the sera of infected specific-pathogen-free pigs
Anti-PCV2 capsid antibodies were detected in all infected pigs but not in PBS control pigs (). All pigs infected with PCV1-2 and PCV1-2-KT3 seroconverted to PCV2 antibodies by 28 dpi. One pig in the PCV1-2-GLU group seroconverted by 28 dpi, while the other two had a delayed seroconversion at 35 dpi. Anti-KT3 tag antibodies were detected in all PCV1-2-KT3-infected pigs but not in other groups (). Anti-KT3 tag antibodies were detected in all three pigs by 14 dpi, which remained seropositive at 42 dpi. Similarly, anti-GLU tag antibodies were detected only in PCV1-2-GLU-infected pigs (): two pigs seroconverted at 14 and 28 dpi, while one pig had detectable anti-GLU antibodies only at 28 dpi. Anti-PCV2 neutralizing antibodies were detected in all infected pigs at 42 dpi (). The levels of neutralizing antibodies in the PCV1-2 and PCV1-2-KT3 pigs were similar, while the two PCV1-2-GLU pigs with delayed seroconversion had noticeably lower levels of neutralizing antibodies (). Therefore, the results indicated that PCV1-2-KT3 induced an anti-PCV2 antibody response that was comparable to that induced by PCV1-2 but stronger than that induced by PCV1-2-GLU. The small animal numbers and pig-to-pig variation may play a role in the observed difference in antibody response among groups. Also, insertion of tags may affect the ability of the tagged viruses to replicate in pigs, although the viremia pattern of PCV1-2-KT3 was similar to that of wild-type PCV1-2 (). Importantly, both PCV1-2-KT3 and PCV1-2-GLU elicited anti-PCV2 neutralizing antibodies and anti-epitope tag antibodies.
Fig. 2. Detection of specific anti-GLU and anti-KT3 tag antibodies, as well as anti-PCV2 neutralizing antibodies, in specific-pathogen-free pigs infected with chimeric PCV1-2 containing the inserted KT3 or GLU epitope. Specific antibody responses were examined (more ...)
In summary, we demonstrated that the C terminus of PCV2 capsid protein tolerates insertions of at least 27 aa, whereas short terminal insertions in rep rendered the virus nonviable. We further demonstrated that chimeric PCV1-2 vaccine viruses containing inserted epitopes in the C terminus of the Cap protein are infectious in vitro and in vivo and elicit both anti-epitope tag antibodies and anti-PCV2 neutralizing antibodies. These epitope-tagged PCV1-2 vaccine viruses will allow the serologic differentiation of vaccinated pigs from naturally infected ones and thus could potentially serve as a tractable MLV such as a compliance marker vaccine.