Andrea Ladinig

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV): Pathogenesis and Interaction with the Immune System

This review addresses important issues of porcine reproductive and respiratory syndrome virus (PRRSV) infection, immunity, pathogenesis, and control. Worldwide, PRRS is the most economically important infectious disease of pigs. We highlight the latest information on viral genome structure, pathogenic mechanisms, and host immunity, with a special focus on immune factors that modulate PRRSV infections during the acute and chronic/persistent disease phases. We address genetic control of host resistance and probe effects of PRRSV infection on reproductive traits. A major goal is to identify cellular/viral targets and pathways for designing more effective vaccines and therapeutics. Based on progress in viral reverse genetics, host transcriptomics and genomics, and vaccinology and adjuvant technologies, we have identified new areas for PRRS control and prevention. Finally, we highlight the gaps in our knowledge base and the need for advanced molecular and immune tools to stimulate PRRS research and field applications.

Phenotypic maturation of porcine NK- and T-cell subsets

Detailed information concerning the development of the immune system in young pigs is still rudimental. In the present study, we analyzed changes in phenotype and absolute numbers of natural killer cells, γδ T cells, T helper cells, regulatory T cells and cytolytic T cells in the blood of pigs from birth to six months of age. For each lymphocyte subpopulation, a combination of lineage and differentiation markers was investigated by six-color flow cytometry. Major findings were: (i) absolute numbers of γδ T cells strongly increased from birth until 19-25 weeks of age, indicating an important role for these cells during adolescence; (ii) phenotype of T helper cells changed over time from CD8α(-)SLA-DR(-)CD27(+) towards CD8α(+)SLA-DR(+)CD27(-) but CD45RC(-) T helper cells were found immediately after birth, therefore questioning the role of this marker for the identification of T-helper memory cells; (iii) for cytolytic T cells, putative phenotypes for early effector (CD3(+)CD8αβ(+)perforin(+)CD27(dim)) and late effector or memory cells (CD3(+)CD8αβ(+)perforin(+)CD27(-)) could be identified.

Emergence of porcine epidemic diarrhea virus in southern Germany

BackgroundOver the last years, porcine epidemic diarrhea virus (PEDV) has caused devastating enteric diseases in the US and several countries in Asia, while outbreaks in Europe have only been reported sporadically since the 1980s. At present, only insufficient information is available on currently circulating PEDV strains in Europe and their impact on the European swine industry. In this case report, we present epidemic outbreaks of porcine epidemic diarrhea in three farms in South-Western Germany.Case presentationEpidemic outbreaks of diarrhea affecting pigs of all age groups were reported in three farms, one fattening farm and two piglet producing farms, in South-Western Germany between May and November 2014. In the fattening farm yellowish, watery diarrhea without evidence of mucus or blood was associated with a massive reduction of feed consumption. Severity of clinical signs and mortality in young suckling pigs varied significantly between the two affected sow farms. While mortality in suckling piglets reached almost 70 % in one sow herd, no increase in suckling piglet mortality was observed in the second sow farm. In all three cases, PEDV was confirmed in feces and small intestines by RT-qPCR. Phylogenetic analyses based on full-length PEDV genomes revealed high identity among strains from all three herds. Moreover, the German strains showed very high nucleotide identity (99.4 %) with a variant of PEDV (OH851) that was isolated in the United States in January 2014. This strain with insertions and deletions in the S-gene (so called INDEL strains) was reported to show lower virulence. Slightly lower identities were found with other strains from the US and Asia.ConclusionPhylogenetic information on the distribution of PEDV strains in Europe is severely lacking. In this case report we demonstrate that acute outbreaks of PEDV occurred in southern Germany in 2014. Current strains were clearly different from isolates found in the 1980s and were closely related to a PEDV variant found in the US in 2014. Moreover, the present case report indicates that variant strains of PEDV, containing insertions and deletions in the S gene, which were reported to be of lower virulence, might be able to cause high mortality in suckling piglets.

Congenital infection with atypical porcine pestivirus (APPV) is associated with disease and viral persistence

In 2013, several Austrian piglet-producing farms recorded outbreaks of action-related repetitive myoclonia in newborn piglets (“shaking piglets”). Malnutrition was seen in numerous piglets as a complication of this tremor syndrome. Overall piglet mortality was increased and the number of weaned piglets per sow decreased by more than 10% due to this outbreak. Histological examination of the CNS of affected piglets revealed moderate hypomyelination of the white substance in cerebellum and spinal cord. We detected a recently discovered pestivirus, termed atypical porcine pestivirus (APPV) in all these cases by RT-PCR. A genomic sequence and seven partial sequences were determined and revealed a 90% identity to the US APPV sequences and 92% identity to German sequences. In confirmation with previous reports, APPV genomes were identified in different body fluids and tissues including the CNS of diseased piglets. APPV could be isolated from a “shaking piglet”, which was incapable of consuming colostrum, and passaged on different porcine cells at very low titers. To assess the antibody response a blocking ELISA was developed targeting NS3. APPV specific antibodies were identified in sows and in PCR positive piglets affected by congenital tremor (CT). APPV genomes were detected continuously in piglets that gradually recovered from CT, while the antibody titers decreased over a 12-week interval, pointing towards maternally transmitted antibodies. High viral loads were detectable by qRT-PCR in saliva and semen of infected young adults indicating a persistent infection.

Variation in Fetal Outcome, Viral Load and ORF5 Sequence Mutations in a Large Scale Study of Phenotypic Responses to Late Gestation Exposure to Type 2 Porcine Reproductive and Respiratory Syndrome Virus

In spite of extensive research, the mechanisms of reproductive disease associated with Porcine Reproductive and Respiratory Syndrome virus (PRRSv) are still poorly understood. The objectives of this large scale study were to evaluate associations between viral load and fetal preservation, determine the impact of type 2 PRRSv on fetal weights, and investigate changes in ORF5 PRRSv genome in dams and fetuses during a 21-day period following challenge. At gestation day 85 (±1), 114 gilts were experimentally infected with type 2 PRRSv, while 19 gilts served as reference controls. At necropsy, fetuses were categorized according to their preservation status and tissue samples were collected. PRRSv RNA concentrations were measured in gilt serum collected on days 0, 2, 6, and 21 post-infection, as well as in gilt and fetal tissues collected at termination. Fetal mortality was 41±22.8% in PRRS infected litters. Dead fetuses appeared to cluster in some litters but appeared solitary or random in others. Nine percent of surviving piglets were meconium-stained. PRRSv RNA concentration in fetal thymus, fetal serum and endometrium differed significantly across preservation category and was greatest in tissues of meconium-stained fetuses. This, together with the virtual absence of meconium staining in non-infected litters indicates it is an early pathological condition of reproductive PRRS. Viral load in fetal thymus and in fetal serum was positively associated with viral load in endometrium, suggesting the virus exploits dynamic linkages between individual maternal-fetal compartments. Point mutations in ORF5 sequences from gilts and fetuses were randomly located in 20 positions in ORF5, but neither nucleotide nor amino acid substitutions were associated with fetal preservation. PRRSv infection decreased the weights of viable fetuses by approximately 17%. The considerable variation in gilt and fetal outcomes provides tremendous opportunity for more detailed investigations of potential mechanisms and single nucleotide polymorphisms associated with fetal death.

CD27 expression discriminates porcine T helper cells with functionally distinct properties

Differentiation of porcine T helper cells is still poorly investigated, partly due to a lack of monoclonal antibodies (mAbs) specific for molecules involved in this process. Recently, we identified a mAb specific for porcine CD27 and showed that CD27 is expressed by all naïve CD8α- T helper cells but divides CD8α+ T helper cells into a CD27+ and a CD27- subset. In the present study, detailed phenotypical and functional analyses of these T-helper cell subpopulations were performed. Naïve CD8α-CD27+ T helper cells predominantly resided in various lymph nodes, whereas higher proportions of CD8α+CD27+ and CD8α+CD27- T helper cells were found in blood, spleen and liver. Both CD8α+CD27+ and CD8α+CD27- T helper cells were capable of producing IFN-γ upon in vitro polyclonal stimulation and antigen-specific restimulation. Experiments with sorted CD8α-CD27+, CD8α+CD27+ and CD8α+CD27- T-helper cell subsets following polyclonal stimulation revealed the lowest proliferative response but the highest ability for IFN-γ and TNF-α production in the CD8α+CD27- subset. Therefore, these cells resembled terminally differentiated effector memory cells as described in human. This was supported by analyses of CCR7 and CD62L expression. CD8α+CD27- T helper cells were mostly CCR7- and had considerably reduced CD62L mRNA levels. In contrast, expression of both homing-receptors was increased on CD8α+CD27+ T helper cells, which also had a proliferation rate similar to naïve CD8α-CD27+ T helper cells and showed intermediate levels of cytokine production. Therefore, similar to human, CD8α+CD27+ T helper cells displayed a phenotype and functional properties of central memory cells.

A TaqMan quantitative polymerase chain reaction assay for the detection of Lawsonia intracellularis in fecal and tissue samples from pigs.

In the present study, a TaqMan quantitative polymerase chain reaction (qPCR) assay for detecting the 16S ribosomal RNA gene of Lawsonia intracellularis in porcine native ileal mucosal scrapings, fecal samples, and formalin-fixed and paraffin-embedded (FFPE) ileal samples is described. Samples from 62 pigs were examined. The results of the qPCR were compared with results obtained with conventional detection methods (PCR, immunohistochemistry, in situ hybridization, and silver staining) from a previous study and correlated well. The qPCR assay proved to be very sensitive and specific. In particular, the sensitivity of TaqMan PCR was significantly higher than conventional PCR on FFPE tissues because of a much shorter amplicon. A higher number of copies per gram of sample material was detected in native mucosa and FFPE tissue compared with feces, especially in highly positive animals. The detection limit for the qPCR was at 4 copies per well in native mucosal scrapings and 18 copies per well in feces and FFPE tissue, respectively. Inhibition of the qPCR reaction was checked by simultaneous detection of a recombinant beta-actin plasmid using a second fluorescent probe. A decreased signal from this internal control plasmid revealed inhibition of the PCR reaction in 21% of native mucosal samples and 1.6% of fecal samples. With a 10-fold dilution of template, the inhibition could be overcome.

Cytokine profiles in pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus and relationships with viral load and fetal outcome

In spite of extensive research, immunologic control mechanisms against Porcine Reproductive and Respiratory Syndrome virus (PRRSv) remain poorly understood. Cytokine responses have been exhaustively studied in nursery pigs and show contradictory results. Since no detailed reports on cytokine responses to PRRSv in pregnant females exist, the objectives of this study were to compare host cytokine responses between PRRSv-infected and non-infected pregnant gilts, and to investigate relationships between cytokine levels in infected gilts and viral load or fetal mortality rate. Serum samples and supernatants of peripheral blood mononuclear cells (PBMC) either stimulated with PRRSv or phorbol myristate acetate/Ionomycin (PMA/Iono) were analyzed for cytokines/chemokines: interleukins (IL) 1-beta (IL1β), IL4, IL8, IL10, IL12, chemokine ligand 2 (CCL2), interferon alpha (IFNα) and interferon gamma (IFNγ). Three cytokines (IFNα, CCL2, IFNγ) in gilt serum differed significantly in inoculated versus control gilts over time. In supernatants of PRRSv stimulated PBMC from PRRSv-infected gilts, levels of IFNα were significantly decreased, while IL8 secretion was significantly increased. PRRSv infection altered the secretion of all measured cytokines, with the exception of IFNα, from PBMC after mitogen stimulation, indicating a possible immunomodulatory effect of PRRSv. IFNα, CCL2, and IFNγ in serum, and IFNγ in supernatants of PMA/Iono stimulated PBMC were significantly associated with viral load in tissues, serum or both. However, only IFNα in supernatants of PRRSv stimulated PBMC was significantly associated with fetal mortality rate. We conclude that of the eight cytokines tested in this study IFNα was the best indicator of viral load and severity of reproductive PRRSv infection.

Maternal and fetal predictors of fetal viral load and death in third trimester, type 2 porcine reproductive and respiratory syndrome virus infected pregnant gilts

Minimal research has focused on understanding mechanisms underlying porcine reproductive and respiratory syndrome virus (PRRSV) induced reproductive failure. We have completed a large-scale project investigating phenotypic and genotypic predictors of reproductive PRRS severity in which numerous clinical, pathological, immunologic and viral responses were characterized in dams and fetuses. The goal was to determine which phenotypic responses were associated with fetal viral load and death after experimental infection of pregnant gilts with type 2 PRRSV, thereby elucidating mechanisms of reproductive PRRS in third trimester pregnant gilts. The presence of fetal infection and increasing RNA concentration at the maternal-fetal interface were strong predictors of the probability of fetal death, while PRRSV RNA concentration in dam sera and systemic tissues were not associated with the odds of fetal death. Fetal infection and death clustered, indicating that the status of adjacent fetuses is crucial for lateral transmission and fetal outcome. Several systemic immune responses of gilts were associated with fetal outcome and viral load: interferon-α contributed to the probability of fetal death, but absolute numbers of T helper cells in early infection, absolute numbers of myeloid cells over time and interleukin 12 levels appeared protective. These results suggest specific immune responses may either contribute to, or protect against, transplacental virus transmission. The WUR10000125 SNP on chromosome 4, associated with PRRS resilience in nursery pigs, was not associated with reproductive outcome. Whereas past research suggested that fetal death results from events occurring at the maternal-fetal interface, we conclude that viral replication within fetuses and spread of PRRSV to adjacent fetuses are pivotal events in the pathogenesis of reproductive PRRS.

Pathogenicity of three type 2 porcine reproductive and respiratory syndrome virus strains in experimentally inoculated pregnant gilts

Mechanisms of reproductive failure resulting from infection with porcine reproductive and respiratory syndrome virus (PRRSV) are still poorly understood. Presented herein are the results of a side-by-side evaluation of the pathogenicity of three type 2 PRRSV strains in a reproductive model, from a pilot study used to develop experimental conditions and laboratory methods for a larger experiment. Pregnant gilts were experimentally infected with PRRSV at gestation day 85 or served as uninfected negative controls. After 21 days, all gilts and fetuses were necropsied. Clinical signs, litter outcome, viral load, cytokine levels, and pathology were compared from samples collected among pigs exposed to the three PRRSV strains. Based on differences in histologic lesions, and fetal weights, and numeric differences in gilt serum cytokine levels, litter outcome and virus replication in fetal tissues KS06-483 appeared less virulent than NVSL 97-7895 and KS06-72109 isolates. Levels of chemokine ligand 2 (CCL2), interferon alpha (IFNα), and interferon gamma (IFNγ) were increased in PPRRSV-infected compared to non-infected gilts (0.01 > P < 0.06). Inoculation with NVSL 97-7895 induced higher levels of all three cytokines. All three PRRSV isolates were able to induce high mean viral load in individual litters, which was closely related to the proportion of PRRSV positive fetuses in the litter. Viral load in fetal samples was also positively associated with viral load at the maternal-fetal interface. All but one dead fetus were positive for PRRSV RNA, and higher concentrations of PRRSV RNA in fetal thymus increased the odds of fetal death. Our results suggest that virus replication in fetal tissues and the maternal-fetal interface, but not in other gilt tissues, are important for the outcome of reproductive PRRS. Additionally, our data indicate that umbilical lesions decreased corresponding to the use of pentobarbital sedation prior to euthanasia of pregnant gilts by captive bolt.