Research has demonstrated that porcine circovirus type 2 (PCV2) can be shed into boar semen, raising the possibility that artificial insemination may be an important route by which disease associated with PCV2 is transmitted. The objective of this experiment was to determine the effect of vaccination against PCV2 on ejaculate characteristics and PCV2-specific antibody titers in serum of PCV2-positive boars viremia and viral shedding in semen. Semen and blood samples were collected weekly from week 0 to week 8. After collections at week 0, boars were vaccinated with a commercial vaccine against PCV2 (n = 5) (Suvaxyn PCV2 One dose; Fort Dodge Animal Health, Fort Dodge, IA) or served as controls and received 2 ml 0.9% saline (n = 5). Sperm concentrations and characteristics of sperm motility were assessed using a computer-assisted sperm analysis system (Hamilton Thorne Research, Beverly, MA) and sperm morphology was evaluated after staining using light microscopy. The PCV2 antibody titers were determined in serum using an ELISA (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). The genomic copy number of PCV2 DNA in serum and semen was determined by PCR (Iowa State Veterinary Diagnostic Laboratory; Ames, IA). There were no effects of treatment or treatment by week on semen characteristics (P > 0.05). An effect of treatment by week was detected for serum antibody titers (P < 0.01). Compared with controls, antibody titers in vaccinated boars tended to be greater at week 0 (1.13 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P = 0.09) and were greater at week 2 (1.15 ± 0.05 titer/ml vs 1.01 ± 0.05 titer/ml; P < 0.05) but lesser at week 7 (1.01 ± 0.05 titer/ml vs 1.23 ± 0.05 titer/ml; P < 0.01) and tended to be lesser at week 8 (1.05 ± 0.05 titer/ml vs 1.17 ± 0.05 titer/ml; P = 0.07). There were no effects of treatment, week, or treatment by week for serum genomic copy number of PCV2 DNA (P > 0.1). An effect of week was detected for semen genomic copy number of PCV2 DNA (P < 0.04). During week 3, PCV2 genomic copy number was at its greatest numerical value, however, semen PCV2 genomic copy number was at its lowest point. This was followed by an increase in semen PCV2 genomic copy number during week 7. This increase could be related to the increase in viral shedding in the serum. In summary, vaccination against PCV2 can lower antibody titers when given post-infection and has no effect on indicators of semen fertility. Vaccination also can decrease the length of reoccurring infection by decreasing the length of viral shedding in serum. / Master of Science

Porcine circovirus type 2 (PCV2) is the causative agent of porcine circovirus-associated disease (PCVAD). Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV). Both PCV2 and PRRSV have caused devastating diseases in the swine industry worldwide, resulting in immense economic losses. One of the most common co-infections in the swine industry is PCV2 and PRRSV. The aim of this dissertation research is to explore different experimental approaches to develop novel vaccines against the two major pathogens affecting swine production and study the basic mechanisms that may be involved in viral pathogenesis. Two types of porcine circovirus (PCV), PCV1 and PCV2, have been identified thus far. PCV1, first identified as a contaminant of the PK-15 cell line, is non-pathogenic and has a low prevalence in swine herds. PCV2 is highly prevalent in most swine-producing countries and is associated with clinical PCVAD. The non-pathogenic PCV1 shares similar genomic organization with PCV2. Previously, it has been demonstrated that a genetically modified infectious chimeric PCV1-2a virus can tolerate up to a 27 aa insertion in the C-terminus of the ORF2 without affecting infectivity and produce a dual immune response against PCV2cap and the inserted epitope tag. Therefore, we evaluated the use of the non-pathogenic PCV1 wild-type (wt) virus and chimeric PCV1-2a vaccine virus (vs) to express four known B-cell epitopes of PRRSV. Peptide epitopes of PRRSV-VR2385, including GP2II (aa 40–51, ASPSHVGWWSFA), GP3I (aa 61–72, QAAAEAYEPGRS), GP5I (aa 35–46, SSSNLQLIYNLT), and GP5IV (aa 187–200, TPVTRVSAEQWGRP) were inserted in frame into the C-terminus of the ORF2 of PCV1wt as well as the PCV1-2avs. Four PCV1-PRRSVEPI chimeric viruses and four PCV1-2a-PRRSVEPI chimeric viruses were successfully rescued and shown to be infectious in vitro and co-expressed PCV1cap or PCV2cap with each specific PRRSV epitope. Two independent animal studies were conducted to evaluate whether the non-pathogenic PCV1 can serve as a vaccine delivery vector and whether the PCV1-2a vaccine virus can be used to develop a bivalent vaccine against both PCV2 and PRRSV. We demonstrated that three PCV1-PRRSVEPI chimeric viruses and two PCV1-2a-PRRSVEPI chimeric viruses were infectious in pigs. Importantly, we demonstrated that the PCV1-PRRSVEPI and PCV1-2a-PRRSVEPI chimeric viruses not only induced specific PCV1 or PCV2 IgG antibody but also specific anti-PRRSV epitope antibody responses as well. Regardless of the PCV backbone used, we showed that the PCV-PRRSV chimeric viruses elicited neutralizing antibodies against PRRSV-VR2385. These results provided a proof of concept for the potential use of the non-pathogenic PCV1 as a vaccine delivery system for PRRSV or other swine pathogens and the use of PCV1-2a vaccine virus to generate a bivalent vaccine against both PCV2 and PRRSV. PRRSV causes a persistent infection and immunosuppression. Immunomodulation of the host immune system is caused by modulation of numerous interleukins, such as type I interferons, tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-12 (IL-12) in infected pigs. Antigen-presenting cells (APCs) are the first line of defense, and their infection plays an important role in innate-mediated immune regulation during early immune responses. Among the APCs, pulmonary alveolar macrophages (PAMs), pulmonary interstitial macrophages (PIMs), and dendritic cells (DCs) are the main targets for PRRSV replication. The role of PRRSV-DCs interaction is not fully understood, and current research focuses on the production and regulation of interferons through DC-SIGN receptors. In this study, we evaluated the immunomodulation of MoDCs by PRRSV through interactions with the pDC-SIGN receptor, by blocking pDC-SIGN with recombinant hICAM-3-Fc or anti-pDC-SIGN mAb. Our results indicate that recombinant hICAM-3-Fc enhances mRNA expression of proinflammatory cytokines and that anti-pDC-SIGN mAb inhibits mRNA expression of TNF-α and IL-1α and enhances the expression of IL-12 induced by PRRSV in MoDCs. The results will help understand the molecular mechanisms of PRRSV pathogenesis. / Ph. D.