Detection of pseudorabies viral DNA in tonsillar epithelial cells of latently infected pigs

Abstract

SUMMARY The Rice strain of pseudorabies virus (prv) was intranasally instilled in pigs that were seronegative to prv. Cells were scraped or brushed from tonsillar surfaces biweekly until pigs were euthanatized at either 10 or 16 weeks after infection. The dna extracted from tonsillar cells or parenchyma were subjected to polymerase chain reaction analysis, using either a single set of oligonucleotide primers or nested primers from the prv gII glycoprotein gene. Pigs became seropositive to prv by 3 weeks after infection. The virus was isolated from the trigeminal ganglia and tonsils of pigs that were euthanatized or died 1 to 2 weeks after infection, but not from pigs that were euthanatized 10 or 16 weeks after infection. The prv gene products were consistently detected in trigeminal ganglia and tonsils of all pigs at 1, 10, and 16 weeks after infection, and sporadically in the nasal mucosa, lymph nodes, and lungs of pigs that were euthanatized or died during the first 2 weeks after infection. Cells collected biweekly from tonsillar surfaces were mostly nucleated, squamous epithelial cells with fewer lymphocytes and neutrophils. Polymerase chain reaction analysis of dna extracted from these cells revealed prv dna in a large proportion of the samples when sufficient cells were collected to provide 1 μg of extracted dna for use in the reaction mixtures. A second group of pigs had prv strain 4892 intranasally instilled. The virus was isolated from tonsillar swab specimens until 3 weeks after infection. Tonsillar brushing specimens were collected biweekly until 14 weeks after infection. Some brushing specimens contained all nucleated, squamous epithelial cells, whereas other specimens contained a mixture of epithelial cells and up to 15% neutrophils, lymphocytes, and small mononuclear cells. Results of polymerase chain reaction analysis of dna extracted from tonsillar cells collected 5,11, and 14 weeks after infection were consistently positive for prv gene products. Intact cells collected from tonsillar surfaces were placed in polymerase chain reaction mixtures with nested oligonucleotide primers from the prv gII glycoprotein gene and were subjected to multiple amplification cycles. Afterward, the specificity of the amplified prv gene products was determined by hybridization procedures, using a virus-specific oligonucleotide probe. Most nucleated, squamous epithelial cells stained positive for prv dna, suggesting that these cells were the primary source of prv gene products in tonsillar brushing specimens.