Porcine circovirus 2 infection induces IFNß expression through increased expression of genes involved in RIG-I and IRF7 signaling pathways.

Porcine circovirus-associated disease (PCVAD), caused by porcine circovirus 2 (PCV2), is characterized by a highly variable pathogenesis that is manifested by various disease syndromes and includes immune evasion. Hence, even though PCVAD is effectively controlled by vaccination, pigs and farms remain infected so that continued vaccination is necessary to control disease. We investigated the molecular interactions of PCV2 and its permissive VR1BL host cell for gene expression signatures that could provide insight into mechanisms leading towards disease. Molecular pathways involved in the innate immune response to PCV2 infection were examined to identify changes in gene expression associated with productive infection of VR1BL cells. RNA profiling from infected and uninfected cells showed that 139 genes were induced by infection and 43 genes were down-regulated, using a p value <0.05 and an absolute fold-change difference>2. A strong type 1 interferon response, including an increase in genes involved in the RIG-I/MDA5 pathway and downstream interferon induced genes, was observed. Key regulators involved in PCV2 infection were identified as IFNß, DDX58 (RIG-I), and IRF7. PCV2 infection induces a strong interferon response which unexpectedly facilitates viral gene expression, perhaps due to the presence of an interferon-sensitive response element in the viral promoter. The findings suggest that PCV2 interventions that attenuate type 1 interferon responses at the cellular level might enhance immunity and eliminate persistent infection.

A longitudinal study of Babesia and Theileria infections in cattle in Sri Lanka.

Throughout the world, infections with the Babesia and Theileria parasites often result in economically significant clinical disease in cattle. We conducted a longitudinal survey of Babesia and Theileria infections in cattle from the Polonnaruwa (n=75; dry zone) and Nuwara Eliya (n=161; wet zone) districts of Sri Lanka. DNA from blood samples collected in June, September, and December 2014 and March 2015 was screened for Babesia bovis, Babesia bigemina, Theileria annulata and Theileria orientalis using specific polymerase chain reactions (PCRs). Additionally, serum samples collected from the animals were screened using enzyme-linked immunosorbent assays (ELISAs) to detect B. bovis- and B. bigemina-specific antibodies. All of the animals surveyed in Polonnaruwa and 150 (93.2%) of the animals surveyed in Nuwara Eliya were PCR-positive for Babesia and/or Theileria at least once during the study period. A greater percentage of the cattle in Polonnaruwa were positive for T. annulata and T. orientalis than B. bovis or B. bigemina at all time points. T. orientalis was the most common infection in Nuwara Eliya. Additionally, more cattle were seropositive for B. bigemina than B. bovis in both districts. Although significant variations were sometimes observed in the rates of animals that were positive for B. bigemina, T. annulata, and T. orientalis at the different sampling time points, the rates of new infections with these parasites (by PCR or ELISA) on second, third, and fourth time points among the parasite-negative samples at the first, second, and third time points, respectively, did not differ between the sampling in either district-suggesting that the parasite species infected cattle at a constant rate in these locations. However, in Polonnaruwa, the rates of new infection with T. annulata were higher than the rates of new infection with T. orientalis. The rates were also higher than those in Nuwara Eliya. In Nuwara Eliya, the rates of new infection with T. orientalis were higher than the rates of new infection with T. annulata. The rates were also higher than those in T. orientalis in Polonnaruwa. These differences might be due to variations in the density and activity of the specific tick vectors within and between the districts. Our findings suggest the need for year-round control measures against bovine Babesia and Theileria infection in Sri Lanka. Further studies to determine the densities of the vector tick species in the different geographical areas of the country are warranted.

Cellular pathogenesis of porcine circovirus type 2 infection.

Porcine circovirus associated disease (PCVAD) and the associated histological lesions are thought to appear due to an increase in the amount of porcine circovirus type 2 (PCV2) present in an infected animal. However, examination of the cellular and molecular pathogenesis of PCVAD is complicated by the lack of a consistent cell culture model that replicates the animal phenotypes of persistent, asymptomatic infection, and acute, pathological disease typified by lymphocyte depletion. The porcine fetal retina cell line, VR1BL, shows a high permissiveness to PCV2 infection, 40 times higher than the alternative PK15 culture model, allowing for high titer viral production, with PCV2b growth higher than PCV2a growth. Cytopathic effect due to apoptosis is observed after challenge with high amounts of PCV2, but at low levels, infection is maintained in passaged cells. Thus, VR1BL cells may be used as a model system to examine both acute viral pathogenesis and cellular innate defense, as well as persistent PCV2 infection.

Diagnostic performance measures of ELISA and quantitative PCR tests for porcine circovirus type 2 exposure using Bayesian latent class analysis.

Porcine circovirus 2 (PCV2) is believed to be a necessary but not sufficient underlying cause of porcine circovirus associated disease (PCVAD) in swine (Opriessnig et al., 2007). Since the potential threat of PCVAD is dependent on the prevalence of PCV2 in swine populations, accurate diagnostic tests are important for epidemiologic surveillance. Therefore, we evaluated the diagnostic sensitivity (Se) and specificity (Sp) of a new indirect ELISA and two quantitative PCR tests for PCV2 in a series of latent class models that used Bayesian estimation procedures. A total of 4140 samples from finisher pigs were tested for evidence of PCV2 by the ELISA and a TaqMan (TM) quantitative PCR, 995 by the ELISA and a SYBR Green (SG) dye-binding PCR, 998 by both PCRs and 993 by all three tests. Overall, the median (95% probability interval) ELISA Se and Sp was 0.85 (0.83-0.87) and 0.74 (0.68-0.79), respectively, when all three tests were analyzed together at an ELISA absorbance (optical density or OD) cutoff of ≥0.3. The TM PCR Se and Sp was 0.86 (0.84-0.88) and 0.94 (0.87-0.97), respectively, and the SG PCR Se and Sp was 0.83 (0.81-0.85) and 0.98 (0.94-1.00), respectively when all three tests were analyzed together at an ELISA OD cutoff of ≥0.3. Sensitivity analysis revealed that Sp estimates in general had less stability than Se estimates, but the SG PCR(Sp) was the most stable. Limited conditional dependence between the two PCR tests was detected. We conclude that the ELISA had the highest diagnostic Se at an absorbance cutoff of ≥0.3, while the SG PCR had the highest diagnostic Sp. The prevalence levels for exposure to PCV2 in finishing swine populations across all analyses ranged from 58 to 100%.

Absence of porcine circovirus type 1 (PCV1) and high prevalence of PCV 2 exposure and infection in swine finisher herds.

Porcine circovirus (PCV) appeared in 1974 as an unidentified, innocuous viral inhabitant of cell cultures and pigs. Today PCV1 is a contaminant of some human vaccines, and PCV2 is a major pathogen of swine. PCV1 is reportedly ubiquitous in swine but nonpathogenic. Since the interplay of PCV1 and PCV2 in swine might explain variable disease results and shed light on the potential for human exposure, we analyzed in depth the prevalence of PCV1 and PCV2 infection and exposure in the U.S. finishing swine herd. Over 82% of sera from 185 farms were positive for PCV2 by PCR, whereas only 2.4% were positive for PCV1. More than 80% of PCV2 DNA-positive swine were also positive for anti-PCV2 antibodies. PCV1 was only rarely present. Exposure of swine, and therefore humans via pigs, to PCV1 is negligible. We conclude that PCV2 causes a persistent infection in pigs and that PCV1 is absent or rare in swine.

Interlaboratory comparison of Porcine circovirus-2 indirect immunofluorescent antibody test and enzyme-linked immunosorbent assay results on experimentally infected pigs.

A blinded interlaboratory assessment of the diagnostic agreement and accuracy of serologic tests for routine detection of antibodies against Porcine circovirus-2 (PCV-2), including indirect fluorescent antibody tests (IFATs) and enzyme-linked immunosorbent assays (ELISAs) was conducted in 7 North American laboratories. Serum samples were collected weekly, on trial days 0, 7, 14, 21, 28, 35, 42, and 49, from the following groups of animals: 1) negative controls (n  =  7), 2) PCV-2a (n  =  8), 3) PCV-2b (n  =  8), 4) PCV-1 (n  =  8), 5) PCV-2 vaccine A (n  =  8; Ingelvac® CircoFLEX™), 6) PCV-2 vaccine B (n  =  8; Circumvent® PCV2), and 7) PCV-2 vaccine C (n  =  8; Suvaxyn® PCV2 One Dose). Results from each laboratory were analyzed by kappa and receiver operating characteristic (ROC) analysis. Kappa analysis indicated that, by trial day 49, IFATs had almost perfect agreement, in-house ELISAs had fair to almost perfect agreement, and commercially available anti-PCV-2 immunoglobulin G ELISAs (I or S) had moderate to substantial agreement. From trial days 14-49, the area under the ROC curve for the 2 laboratories that offered IFATs, the 4 laboratories that offered in-house ELISAs, and the 3 laboratories that used commercially available ELISAs ranged from 0.94 to 1.00, 0.72 to 1.00, and 0.95 to 1.00, respectively. However, test sensitivities varied based on laboratory-specific cutoffs that were used to dichotomize test results.