Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD). Available commercial vaccines all target PCV2a subtype, although the circulating predominant subtype worldwide is PCV2b, and the emerging PCV2d subtype is also increasingly associated with PCVAD. Here we molecularly bred genetically-divergent strains representing PCV2a, PCV2b, PCV2c, PCV2d, and "divergent PCV2a" subtypes by DNA-shuffling of the capsid genes to produce a chimeric virus representing PCV2 global genetic diversity. When placed in the PCV2a backbone, one chimeric virus (PCV2-3cl14) induced higher neutralizing antibody titers against different PCV2 subtypes. Subsequently, a candidate vaccine (PCV1-3cl14) was produced by cloning the shuffled 3cl14 capsid into the backbone of the non-pathogenic PCV1. A vaccine efficacy study revealed that chimeric virus PCV1-3cl14 induces protective immunity against challenge with PCV2b or PCV2d in pigs. The chimeric PCV1-3cl14 virus is a strong candidate for a novel vaccine in pigs infected with variable PCV2 strains.

Direct functional screening of a cDNA expression library derived from primary porcine alveolar macrophages (PAM) revealed that CD163 is capable of conferring a porcine reproductive and respiratory syndrome virus (PRRSV) -permissive phenotype when introduced into nonpermissive cells. Transient-transfection experiments showed that full-length CD163 cDNAs from PAM, human U937 cells (histiocytic lymphoma), African green monkey kidney cells (MARC-145 and Vero), primary mouse peritoneal macrophages, and canine DH82 (histocytosis) cells encode functional virus receptors. In contrast, CD163 splice variants without the C-terminal transmembrane anchor domain do not provide PRRSV receptor function. We established several stable cell lines expressing CD163 cDNAs from pig, human, and monkey, using porcine kidney (PK 032495), feline kidney (NLFK), or baby hamster kidney (BHK-21) as the parental cell lines. These stable cell lines were susceptible to PRRSV infection and yielded high titers of progeny virus. Cell lines were phenotypically stable over 80 cell passages, and PRRSV could be serially passed at least 60 times, yielding in excess of 10(5) 50% tissue culture infective doses/ml.

Monoclonal antibodies (McAbs) were generated against two tobacco etch virus (TEV)-encoded nonstructural proteins, the 49-kilodalton (kDa) proteinase and the 58-kDa putative RNA-dependent RNA polymerase. This process was facilitated by the fact that these two TEV nonstructural proteins cocrystallize in the nuclei of virus-infected cells to form nuclear inclusion (NI) bodies which can be purified readily. The anti-NI McAbs were shown by Western blot analysis to be specific for either the TEV 49-kDa or the 58-kDa protein. Those McAbs reactive with the 49-kDa proteinase were characterized further with respect to the 49-kDa domain with which they reacted and with respect to their ability to inhibit the autocatalytic or self-processing activity of the 49-kDa proteinase. The 49-kDa antigens were synthesized from a TEV cDNA sequence using cell-free transcription and translation systems. Each anti-49-kDa McAb was used in immunoprecipitation studies with a series of 49-kDa antigens which represented a nested set of 49-kDa proteins with common amino termini but varying in length. Immunoprecipitation results showed that all of the anti-49-kDa proteinase McAbs reacted with one of five binding regions, designated A through E from the carboxy terminus of the proteinase, which were 77, 38, 81, 18, and 61 amino acids long, respectively. The 38-amino-acid binding region B contained the proposed catalytic cysteine 339 residue and was recognized by only one McAb, 4911. McAb 4911 was the only anti 49-kDa McAb capable of inhibiting the self-processing reaction in which the 49-kDa proteinase is released from its 75-kDa polyprotein precursor.