Diagnostic sensitivity and specificity of the PRV triplex real-time qPCR
To evaluate the diagnostic sensitivity and specificity of the PRV triplex assay, 114 nasal swab samples were collected from pigs experimentally inoculated with PRV JS-2012 or PRV Bristol at the NCFAD-Winnipeg laboratory animal facility. Severe disease was expected in pigs inoculated with the highly virulent PRV strain JS-2012 (Luoe et al., 2014); therefore, 16 pigs representing two different age groups, 3-weeks and 7-weeks, were used for PRV JS-2012 inoculations. Out of the eight JS- 2012 inoculated 3-week old piglets, six developed fever, depression and respiratory signs (sneezing and nasal discharge) by 3 dpi. Two of the inoculated 3-weeks old piglets were euthanized on 5 dpi; four were euthanized on 7 dpi due to severe neurological signs (tremors and seizers). The remaining two piglets developed mild neurological signs by 14 dpi and were euthanized on 16 dpi. The JS-2012 inoculated 7-week old pigs survived the infection until the end of the experiment and were euthanized on 14 dpi. The majority of those pigs (6 of 8) developed fever by 4 dpi, as well as mild respiratory (sneezing, nasal discharge) and neurological signs (lameness, prostration); but by 11 dpi, they all had recovered. The three 3-week old piglets inoculated with PRV Bristol developed clinical signs (fever, lack of appetite) by 3 dpi; and they were euthanized on 7 dpi as they had developed severe neurological signs.
Both the PRV gB assay and the triplex assay detected PRV nucleic acid in nasal swabs from 3-week old piglets as early as 4 dpi and starting at 6 dpi in 7-week old pigs (Table 3). Low levels of PRV nucleic acid were detected earlier in some animals by both assays (on 4 dpi in some 7-week old pigs and on 2 dpi in some 3-week old piglets). Of the 102 samples tested, 62 samples were positive, 36 negative, and 4 were suspect (Ct >35.99 were determined as suspect) on the triplex assay for PRV nucleic acids. The PRV gB assay identified 57 samples as a positive, 35 samples as negative, and 10 samples as suspect. While the gB PCR as published did not define a suspect range, for this project, we designated Ct greater than 35.99 as suspect (Table 4).
Virus isolation was performed on a subset of samples representing different Ct values and sample types (Table 5). PRV-like cytopathic effect (rounding up of cells and occasional formation of syncytia) and positive staining of cells was observed for the positive control wells, and no staining was observed in negative wells, as expected. The Ct values of the triplex assay and the gB assay were comparable for all the tissue samples from PRV JS-2012 and PRV Bristol infected animals. No cross-reactivity was observed on the triplex assay. Virus isolation was successful from all samples with Ct values less than 30 on both the triplex and gB assays.