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Porcine epidemic diarrhea virus (PEDV) causes enteric disease in pigs and spreads rapidly after entering naïve pig populations. The objectives were to (1) compare the disease course following inoculation with PEDV isolate US/Colorado/2013 in naïve 10 day and 8 week-old pigs, and (2) contrast the naïve response to homologous challenge in 8 week-old pigs. Pigs were randomly assigned into group 1 (n = 40, no PEDV exposure), group 2 (n = 43, PEDV inoculation at 10 days of age) and group 3 (n = 48, PEDV inoculation at 8 weeks of age). Thirty-three group 2 pigs received a homologous challenge at 8 weeks of age. Following primary or secondary inoculation, 3–10 pigs were euthanized at days post-inoculation (dpi) 1, 2, 3, 7 or 14. Clinical signs were more pronounced in 10 day-old pigs compared to 8 week-old pigs at dpi 2 and 3, a higher number of 10 day-old pigs shed PEDV RNA in feces compared to 8 week-old pigs. Typical severe atrophic enteritis of PEDV infection was observed at dpi 3 in both age groups, and at dpi 4 and 14 fecal shedding patterns were also similar. While both age groups had seroconverted to PEDV by dpi 14, IgG levels were higher in 8 week-old pigs. PEDV IgA antibodies were detected in feces of approximately 50% of the pigs at dpi 44. In homologous challenged pigs, no clinical signs or lesions were found, and PEDV fecal shedding was restricted to less than 10% of the pigs indicating the existence of homologous protection 44 days after initial PEDV exposure.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in people in many developing countries and is also endemic in many industrialized countries. Mammalian HEV (mHEV) isolates can be divided into at least four recognized major genotypes. Several nucleic acid amplification techniques have been developed for mHEV detection, with great differences in sensitivity. The aim of this study was to compare the performances of two singleplex real-time reverse transcriptase (RT) PCR assays for broad detection of all four mHEV genotypes (assays A and B) and two duplex real-time RT-PCR assays for detection and differentiation of mHEV genotypes 3 and 4 (assays C and D). RNAs extracted from 28 fecal samples from pigs experimentally inoculated with HEV genotype 3 and 186 fecal samples from commercial pigs with unknown HEV exposure were tested by all four assays. In experimental samples, HEV RNA was detected in 96.4% (assay A), 39.2% (assay B), 14.2% (assay C), and 0% (assay D) of the samples. In field samples with unknown HEV exposure, HEV RNA was detected in 67.2% (assay A), 36.4% (assay B), 1.1% (assay C), and 0.5% (assay D) of the samples. The assays showed overall poor agreement (kappa = 0.19 to 0.03), with differences in detection rates between assays (P < 0.01). Assays A and B, which broadly detect HEV genotypes 1 to 4, had significantly higher detection rates for HEV RNA than the duplex assays C and D, which were both designed to detect and differentiate between HEV genotypes 3 and 4.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in people in many developing countries and is also endemic in many industrialized countries. Mammalian HEV (mHEV) isolates can be divided into at least four recognized major genotypes. Several nucleic acid amplification techniques have been developed for mHEV detection, with great differences in sensitivity. The aim of this study was to compare the performances of two singleplex real-time reverse transcriptase (RT) PCR assays for broad detection of all four mHEV genotypes (assays A and B) and two duplex real-time RT-PCR assays for detection and differentiation of mHEV genotypes 3 and 4 (assays C and D). RNAs extracted from 28 fecal samples from pigs experimentally inoculated with HEV genotype 3 and 186 fecal samples from commercial pigs with unknown HEV exposure were tested by all four assays. In experimental samples, HEV RNA was detected in 96.4% (assay A), 39.2% (assay B), 14.2% (assay C), and 0% (assay D) of the samples. In field samples with unknown HEV exposure, HEV RNA was detected in 67.2% (assay A), 36.4% (assay B), 1.1% (assay C), and 0.5% (assay D) of the samples. The assays showed overall poor agreement (kappa = 0.19 to 0.03), with differences in detection rates between assays (P < 0.01). Assays A and B, which broadly detect HEV genotypes 1 to 4, had significantly higher detection rates for HEV RNA than the duplex assays C and D, which were both designed to detect and differentiate between HEV genotypes 3 and 4.

The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) real-time reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days -2, 1, 3, 5, 7, 14, and 21 postinoculation (p.i.) and tested by one of three commercially available real-time RT-PCR assays(VetMax from Applied Biosystems, Foster City, CA [ abbreviated AB]; VetAlert from Tetracore, Rockville, MD[TC]; and AcuPig from AnDiaTec GmbH, Kornwestheim, Germany [ AD]). At day 1 p.i., all assays detected at least one positive sample in each group. The highest detection rates were on days 3 and 5 p.i. Between days 1 and 7 p.i., serum samples had the highest detection rate (90%) with 100% agreement between tests, followed by blood swabs (kappa value of 0.97) and semen (kappa value of 0.80). Oral fluids had the lowest detection rates (AB, 55%; TC, 41%; AD, 46%) and the highest disagreement between kits (kappa value of 0.63). Pools of five samples did not reduce the detection rates if there was one positive sample with a large amount (cycle threshold,< 30) of viral RNA in the pool. Serum and blood swab samples had shorter turnaround times for RNA extraction. The AB assay had a 1.6-times-shorter PCR time. In summary, serum and blood swabs had the best performance with highest detection rates and agreement between assays and the shortest turnaround times.

The aims of this study were to compare three commercial porcine reproductive and respiratory syndrome virus (PRRSV) real-time reverse transcription-PCR (RT-PCR) assays for detection of genetically diverse PRRSV isolates in serum, semen, blood swabs, and oral fluids collected from experimentally infected boars and to evaluate the effects of sample pooling. Six groups of three boars negative for PRRSV were each inoculated with one of six PRRSV isolates (sharing 55 to 99% nucleotide sequence identity in ORF5). Samples were collected on days -2, 1, 3, 5, 7, 14, and 21 postinoculation (p.i.) and tested by one of three commercially available real-time RT-PCR assays(VetMax from Applied Biosystems, Foster City, CA [ abbreviated AB]; VetAlert from Tetracore, Rockville, MD[TC]; and AcuPig from AnDiaTec GmbH, Kornwestheim, Germany [ AD]). At day 1 p.i., all assays detected at least one positive sample in each group. The highest detection rates were on days 3 and 5 p.i. Between days 1 and 7 p.i., serum samples had the highest detection rate (90%) with 100% agreement between tests, followed by blood swabs (kappa value of 0.97) and semen (kappa value of 0.80). Oral fluids had the lowest detection rates (AB, 55%; TC, 41%; AD, 46%) and the highest disagreement between kits (kappa value of 0.63). Pools of five samples did not reduce the detection rates if there was one positive sample with a large amount (cycle threshold,< 30) of viral RNA in the pool. Serum and blood swab samples had shorter turnaround times for RNA extraction. The AB assay had a 1.6-times-shorter PCR time. In summary, serum and blood swabs had the best performance with highest detection rates and agreement between assays and the shortest turnaround times.