Porcine Reproductive And Respiratory Syndrome Virus Exposure Research Articles

Porcine ISG15 fused IFN-λ3 as a novel antiviral agent for treating porcine reproductive and respiratory syndrome virus infection in vivo.

IFN-λs hold promise as therapeutic candidates against mutable respiratory viruses, but their efficacy against porcine reproductive and respiratory syndrome virus (PRRSV) remains unclear. In this study, we expressed a recombinant fusion protein consisting of porcine ISG15 linked porcine IFN-λ3 (ISG15-IFN-λ3) via a rigid protein linker in Escherichia coli (E. coli). In vitro experiments demonstrated that treatment of porcine alveolar macrophage (PAM)-derived CRL-2843 cells with ISG15-IFN-λ3 induced upregulation of several Interferon-stimulated Genes (ISGs) proteins, including ISG15, ISG56, and HERC5. CRL-2843 cells pretreated with ISG15-IFN-λ3 exhibited heightened resistance to Newcastle disease virus infection, while PRRSV-permissive cells treated with ISG15-IFN-λ3 before and during PRRSV exposure showed significantly inhibited PRRSV replication as well. In animal experiments, at 21days post-infection, ISG15-IFN-λ3-treated piglets displayed milder lung tissue pathology and significantly reduced serum PRRSV-RNA, indicating enhanced viral clearance and faster recovery. Additionally, PAMs collected from ISG15-IFN-λ3-treated piglets showed significantly reduced mRNA expression of representative cytokines, chemokines, suggesting that ISG15-IFN-λ3 treatment may mitigate pneumonia severity by reducing the levels of these inflammatory mediators. These findings indicate that recombinant ISG15-IFN-λ3 expressed in E. coli may serve as a novel, effective, and affordable agent for treating severe PRRSV infection in piglets, potentially benefiting the pork industry.

Porcine reproductive and respiratory syndrome virus seroprevalence in Scottish finishing pigs between 2006 and 2018.

Porcine reproductive and respiratory syndrome (PRRS) is a major endemic pig disease worldwide and is associated with considerable economic costs. In Scotland, three abattoir surveys were conducted in 2006 (158 farms), 2012-2013 (94 farms) and 2017-2018 (97 farms) to estimate seroprevalence to PRRS virus (PRRSV) in commercial finishing pigs. These surveys covered around 79%, 59% and 66% of the Quality Meat Scotland assured farms slaughtering pigs in Scotland in 2006, 2012-13 and, 2017-18 respectively. In the 2006 survey, six pigs per farm were sampled and tested using the CIVTEST SUIS PRRS E/S test. In the 2012-2013 and 2017-2018 surveys, 10 pigs per farm were sampled and tested using the IDEXX PRRS X3 Ab test. A farm was considered positive if it had one or more seropositive samples. The prevalence of positive farms was 45.6% (95% CI: 38.0-53.4), 47.8% (95% CI: 38.1-57.9) and 45.4% (95% CI: 35.8-55.3) in the 2006, 2012-2013 and 2017-2018 surveys, respectively, and 70%-75.5% farms did not change their status between sampling periods. The prevalence of PRRSV exposure in Scottish pig herds was high and changed little from 2006 to 2018. These surveys have informed planning for a prospective PRRS control programme in Scotland.

Tonsil scrapings for porcine reproductive and respiratory syndrome virus detection in growing pigs under field conditions

Objective: The main objective of this study was to describe the use and limitations of tonsil scrapings (TS), oral fluids (OF), nasal swabs (NS), and environmental swabs (ES) to detect porcine reproductive and respiratory syndrome virus (PRRSV). Materials and methods: Two PRRSV-positive growing pig farms using different PRRSV control strategies were enrolled in this study. Sampling began approximately 52- and 21-days post PRRSV exposure for farms 1 and 2, respectively, and occurred once a month for four months using fixed spatial sampling. Samples for OF and ES were collected at the pen level and TS and NS samples were collected at the individual level. All samples were tested using reverse transcriptase-polymerase chain reaction (RT-PCR). Results: A total of 192 samples were collected over the study period: 48 TS, 48 OF, 48 NS, and 48 ES. Overall, 20 TS (41.6%), 0 OF (0.0%), 6 NS (12.5%), and 1 ES (2.1%) tested RT-PCR positive for PRRSV throughout this study. Implications: Tonsil scraping samples yielded more positive PRRSV RT-PCR results for longer time periods when compared to OF, NS, and ES for PRRSV detection in growing pigs. Tonsil scraping samples tested RT-PCR positive for PRRSV up to 168 days post exposure. Oral fluids, NS, and ES sampling methods for PRRSV detection in growing pig populations, particularly months after the initial infection or vaccination, should be used with caution given low RT-PCR positive samples found in this study.

Relationship between weekly porcine reproductive and respiratory syndrome virus exposure in breeding herds and subsequent viral shedding and mortality in the nursery

Objective: Describe the relationship of weekly breeding herd status based on processing fluid (PF) testing for porcine reproductive and respiratory syndrome virus (PRRSV) RNA by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on subsequent viral shedding and cumulative mortality during the nursery phase. Materials and methods: Weekly cohorts (n = 121) of newborn piglets were classified into PRRSV exposure groups according to PRRSV detection in PF: low (quantification cycles [Cq] ≤ 27), medium (27 < Cq ≤ 34), high (34 < Cq ≤ 37), and negative (Cq > 37). At 6 weeks of age, oral fluids (OF) were collected from a subset of 41 cohorts, tested by qRT-PCR, and results used to classify the nursery shedding status into the same aforementioned categories. Cumulative nursery mortality was recorded for all 121 cohorts and compared between the different PRRSV exposure groups. Test agreement was assessed between PF and OF results of 41 cohorts. Moreover, the effect of 4:1 OF pooling on the probability of testing qRT-PCR-positive was evaluated. Results: The nursery mortality for low Cq cohorts was 3.40 percentage points (range, 3.28-3.99) higher than other exposure groups. Overall, Cq values were higher in PF than in OF samples, and fair agreement (κ = 0.2398) between PF and OF was encountered. Compared to individual samples, 4:1 OF pooling resulted in 100% specificity and 76.92% sensitivity. Implications: Weekly PF testing for PRRSV allowed for exposure group classification for each pig batch produced, which was a good predictor of subsequent cumulative nursery mortality.

Potential role of noncommercial swine populations in the epidemiology and control of porcine reproductive and respiratory syndrome virus

To assess the role of noncommercial pigs in the epidemiology of porcine reproductive and respiratory syndrome (PRRS) virus. Seroepidemiologic study and survey study. 661 pigs from which blood samples were collected at slaughter and 32 pigs from which blood samples were collected longitudinally. Spatial databases of commercial farms and 4-H participation were evaluated by use of commercial geographic information systems software. Information on disease knowledge and management methods of 4-H participants was obtained by mail survey and personal interview. Serum samples for antibody testing by PRRS ELISA were obtained from pigs at slaughter or at county fairs and on farms. Participation in 4-H swine programs was geographically associated with commercial swine production in Minnesota, and 39% of 4-H participants reared pigs at locations with commercial pigs. High seroprevalence at fairs (49%; range, 29% to 76%) and seroconversion after fairs indicated that PRRS virus exposure was common in pigs shown by 4-H participants and that transmission could occur at fairs. The small swine population shown by 4-H members (estimated 12,000 pigs) relative to the population of commercial swine in Minnesota (estimated 6.5 million pigs) suggested the former overall was likely of minor importance to PRRS virus epidemiology at present. However, the relative frailty of knowledge of biosecurity practices, evidence that PRRS virus exposure was frequent, common intentions to show pigs at multiple events, and often close interactions with commercial herds suggested that the 4-H community should be involved in regional efforts to control PRRS.

Induction of inducible CD4 +CD25 +Foxp3 + regulatory T lymphocytes by porcine reproductive and respiratory syndrome virus (PRRSV)

Increases in numbers or activities of regulatory T lymphocytes (Tregs) have been linked to the establishments of several persistent infections. It has been previously shown that porcine reproductive and respiratory syndrome virus (PRRSV) can negatively modulate the host immune responses, resulting in persistent infection and secondary immunodeficiency. Recently, the existence of porcine CD4 +CD25 + Tregs has been demonstrated. We investigated the effect of PRRSV on the CD4 +CD25 + Tregs. The CD4 +CD25 +Foxp3 + T lymphocytes in the peripheral blood mononuclear cells (PBMCs) were identified, using the anti-human anti-Foxp3 monoclonal antibody. In vitro culture of porcine PBMC in the presence of PRRSV, but not classical swine fever virus, significantly increased the numbers of Foxp3 + lymphocytes, particularly in the CD4 +CD25 high subpopulation. The time-course study revealed that PRRSV significantly increased the numbers of viral-specific CD4 +CD25 highFoxp3 + subpopulation in the culture starting from 12 h through the end of the observation period. Consistent to the results obtained by flow cytometry, enhanced Foxp3 gene expression was observed in the PBMC cultured with PRRSV in a time-course manner. The presence of monocyte-derived DC in the co-culture significantly enhanced the induction of CD4 +CD25 + Foxp3 + T lymphocytes. The PRRSV-induced CD4 +CD25 high T lymphocytes exhibited suppressive activity when co-cultured with PHA-activated, autologous peripheral blood leukocytes, indicating the suppressive activity of the PRRSV-specific Tregs. In addition, PRRSV exposure significantly increased the numbers of PRRSV-specific CD4 +CD25 +Foxp3 + subpopulation in the PBMC of infected pigs at 10 days post-infection. In summary, the results indicated that PRRSV could increase the numbers of viral-specific, inducible regulatory T lymphocytes in the porcine PBMC, both in vitro and in vivo. The findings suggested the novel immunomodulatory mechanism induced by PRRSV.

Receptor-Determined Susceptibility of Preimplantation Embryos to Pseudorabies Virus and Porcine Reproductive and Respiratory Syndrome Virus

In the present study, the in vitro interaction of embryos with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) was investigated by viral antigen detection and by evaluating the expression of virus receptors, namely, poliovirus receptor-related 1 (PVRL1; formerly known as nectin 1) for PRV and sialoadhesin for PRRSV. Embryonic cells of zona pellucida intact embryos incubated with PRV remained negative for viral antigens. Also, no antigen-positive cells could be detected after PRV incubation of protease-treated embryos, since the protease disrupted the expression of PRVL1. However, starting from the five-cell-stage onwards, viral antigen-positive cells were detected after subzonal microinjection of PRV. At this stage, the first foci of PVRL1, also a known cell adhesion molecule, were expressed. At the expanded blastocyst stage, a lining pattern of PVRL1 in the apicolateral border of trophectoderm cells was present, whereas the expression in the inner cell mass was low. Furthermore, PVRL1-specific monoclonal antibody CK41 significantly blocked PRV infection of trophectoderm cells of hatched blastocysts, while the infection of the inner cell mass was only partly inhibited. Viral antigen-positive cells were never detected after PRRSV exposure of preimplantation embryos up to the hatched blastocyst stage. Also, expression of sialoadhesin in these embryonic stages was not detected. We conclude that the use of protease to investigate the virus embryo interaction can lead to misinterpretation of results. Results also show that blastomeres of five-cell embryos up to the hatched blastocysts can become infected with PRV, but there is no risk of a PRRSV infection.

γδ Lymphocyte Response to Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be one of the most important diseases facing swine industry today. Following PRRSV infection pigs develop both humoral and cell-mediated responses following PRRSV exposure; however, the relative importance in protection and clearance of the virus is not yet completely understood. Swine contain a large percentage of gammadelta T-lymphocytes in peripheral circulation capable of responding to various pathogens in both an innate and specific immune response. The objectives of this study were to determine whether gammadelta lymphocytes functionally respond to PRRSV upon initial exposure and re-exposure. Four month old PRRSV free gilts were intranasally inoculated with a field isolate MN-30100 then assessed at various time points post infection. On day 120, pigs were re-exposed with MN-30100 PRRSV strain and subsequently were bled on days 0, 7, and 14 post re-exposure. Lymphocyte subpopulations, antigen specific proliferation, and IFN-gamma production were evaluated throughout the study. Circulating gammadelta lymphocytes in PRRSV exposed animals expanded between days 14 to 70 (d14-d70, p = 0.016); following antigen stimulation, gammadelta lymphocyte proliferated by day 14 (d0-d14, p = 0.001) continuing through day 60. gammadelta lymphocytes produced IFN-gamma by day 14 pi continuing through day 50 (d0-d50, p = 0.004). Following re-exposure both gammadelta+ and CD4+ lymphocytes increased in IFN-gamma production. These results are not fully conclusive on the role of gammadelta lymphocytes against PRRSV; the data indicate that gammadelta lymphocytes specifically respond to PRRSV.

213 ASSESSMENT OF THE SUSCEPTIBILITY OF PORCINE PRE-IMPLANTATION EMBRYOS TO PSEUDORABIES VIRUS (PRV) AND PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV) USING SUBZONAL MICROINJECTION

It is known that porcine pre-implantation embryos before the morula stage are refractory to infection with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) (Bolin et al. 1981 Am. J. Vet. Res. 42, 1711-1712; Prieto et al. 1996 Theriogenology 46, 687-693, respectively). The effects of PRV and PRRSV on embryonic cells of morulae and blastocysts are unknown. Therefore, the objectives of the present study were to (1) assess the effects of PRV and PRRSV exposure on further embryo development, and (2) determine whether PRV and PRRSV are able to replicate in embryonic cells. Zona pellucida (ZP)-intact morulae (6 days post-insemination, 6 dpi) and early blastocysts (7 dpi) were microinjected subzonally with approximately 3 nL of 109 TCID50/mL PRV (strain 89v87, second passage in swine testicle cells) or 108.6 TCID50/mL PRRSV (Lelystad virus strain, 13th passage in swine alveolar macrophages). Control embryos were microinjected under the same circumstances with phosphate-buffered saline (PBS). Hatched blastocysts (8 dpi) were exposed for 1 h at 39�C to 105 TCID50/mL of PRV or PRRSV of the same strains used for injecting earlier embryonic stages. Control hatched blastocysts were incubated with PBS. Each group of morulae and blastocysts consisted of approximately 20 embryos. Embryonic development was assessed every 12 h. At 48 h post injection, the percentage of infected embryos and the percentage of viral antigen positive cells per embryo were determined by immunofluorescence. Subzonal microinjection of ZP-intact morulae and blastocysts with PRV inhibited in vitro development in comparison to the controls. Moreover, under direct immunofluorescence, PRV antigen-positive cells were detected in association with the embryos. Exposure of hatched blastocysts to PRV inhibited further embryo development; the majority (16/20) of the embryos degenerated 24 h after incubation. Perivitelline microinjection of ZP-intact morulae and blastocysts with PRRSV and incubation of hatched blastocysts with PRRSV did not inhibit in vitro development in comparison to the controls. No PRRSV antigen positive cells were detected in association with the embryos. Based on these results, it can be deduced that embryonic cells of morulae and blastocysts are susceptible to PRV infection but refractory to PRRSV infection. Another argument substantiating insusceptibility of embryos to certain viral pathogens is the demonstration of the lack of virus receptors at a given embryonic cell stage. Therefore, the expression of sialoadhesin, the receptor that mediates the internalization of PRRSV in cells, was investigated in hatched blastocysts (n = 10). By indirect immunofluorescence using monoclonal antibody 41D3 directed against porcine sialoadhesin, no positive signals were detected. The result of this experiment strengthens the statement that embryonic stages up to the hatched blastocyst stage are refractory to PRRSV infection.

Deciphering the involvement of innate immune factors in the development of the host response to PRRSV vaccination

The natural response of pigs to porcine reproductive and respiratory syndrome virus (PRRSV) infections and vaccinations needs to be altered so that better protection is afforded against both homologous and heterologous challenges by this pathogen. To address this problem, real-time gene expression assays were coupled with cytokine Elispot and protein analyses to assess the nature of the anti-PRRSV response of pigs immunized with modified live virus (MLV) vaccine. Although T helper 1 (Th1) immunity was elicited in all vaccinated animals, as evidenced by the genesis of PRRSV-specific interferon-gamma secreting cells (IFNG SC), the overall extent of the memory response was variable and generally weak. Peripheral blood mononuclear cells (PBMC) isolated from these pigs responded to PRRSV exposure with a limited increase in their expression of the Th1 immune markers, IFNG, tumor necrosis factor-alpha and interleukin-15 (IL15), and a reduction in the quantity of mRNAs encoding the innate and inflammatory proteins, IL1B, IL8 and IFNA. Efforts to enhance Th1 immunity, by utilizing an expression plasmid encoding porcine IFNA (pINA) as an adjuvant, resulted in a temporary increase in the frequency of PRRSV-specific IFNG SC but only minor changes overall in the expression of Th1 associated cytokine or innate immune marker mRNA by virus-stimulated PBMC. Administration of pINA, however, did correlate with decreased IL1B secretion by cultured, unstimulated PBMC but had no effect on their ability to release IFNG. Thus, while exogenous addition of IFNA during PRRSV vaccination has an impact on the development of a Th1 immune response, other alterations will be required for substantial boosting of virus-specific protection.

197SUSCEPTIBILITY OF PORCINE MORULAE AND BLASTOCYST STAGE EMBRYOS TO PSEUDORABIES VIRUS AND PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

Porcine preimplantation embryos are refractory to infection with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) during the 2–4 to 16-cell stage as described by Bolin et al. (1981 Am. J. Vet. Res. 42: 1711–1712) and Prieto et al. (1996 Theriogenology 46: 687–693), respectively. Research on the effects of PRV and PRRSV on embryonic cells of morulae, blastocysts and hatched blastocysts is limited. Therefore, the objectives of the present study were (i) to assess the effects of PRV and PRRSV exposure on further embryonic development, and (ii) to determine whether PRV and PRRSV are able to replicate in embryonic cells of porcine morulae and blastocysts. In vivo produced ZP-intact and ZP-free morulae (6 days post-insemination), early blastocysts (7 days post-insemination), and hatched blastocysts (8 days post-insemination) derived from 22 superovulated sows were exposed to 105 TCID50 PRV (strain 89v87, second passage in swine testicle cells) or to 105 TCID50 PRRSV (Lelystad virus strain, 13th passage in swine alveolar macrophages) for 1h at 39°C. Control embryos were incubated under the same circumstances without viruses. Each group of morulae and blastocysts consisted of approximately 20 embryos. Embryonic development was assessed every 12h and differences in rates of development were analyzed using Chi-square analysis or Fisher’s exact test. At 48h post-incubation, embryos were collected and examined for viral antigen by indirect immunofluorescence. Further embryo development of ZP-intact and ZP-free morulae and blastocysts was not affected by exposure to PRV or PRRSV compared to controls (P<0.05). Moreover, using indirect immunofluorescence, no PRV or PRRSV antigen-positive cells were detected. Exposure of hatched blastocysts to PRV inhibited further embryo development as 100% (n=5) of the embryos degenerated 24h after viral exposure. This was significantly different (P<0.05) from the controls and the PRRSV-incubated hatched blastocysts that did not experience any negative influence on embryo development. Based on these results it can be concluded that embryonic cells are not susceptible to a PRRSV infection up to the hatched blastocyst stage. Embryonic cells of morulae and blastocysts are refractory to PRV, but the virus has a detrimental effect on further embryo development of hatched blastocysts. More experiments are necessary to confirm these results and to investigate whether, or at which preimplantation stage, embryos are susceptible to a PRRSV infection.

Duration of homologous porcine reproductive and respiratory syndrome virus immunity in pregnant swine

The duration of porcine reproductive and respiratory syndrome virus (PRRSV) homologous immunity was tested in this study and found to last for at least 604 days post experimental exposure to field PRRSV. Eleven gilts (group A) received a primary exposure to field PRRSV by either an oronasal ( n = 6) or an intrauterine ( n = 5) route. The gilts were naturally bred at selected times (143 to 514 days) after primary virus exposure. They were oronasally exposed a second time to the same strain of virus on or about gestation day 90. Ten age-matched control sows free of PRRSV-specific antibody from the same source farm (group B) were naturally bred and were oronasally exposed to aliquots of the homologous challenge virus on or about gestation day 90. Nine of the 11 gilts in group A and all animals in group B became pregnant following one breeding cycle. The two nonpregnant gilts in group A were each naturally bred during four additional estrus cycles and neither became pregnant. They were exposed to homologous challenge virus 562 and 604 days post primary exposure, respectively. All animals were necropsied 21 days post homologous challenge. Sera and alveolar macrophages from each dam, and sera from each fetus were tested for virus. Transplacental infection was detected in 0/9 and 8 10 litters in groups A and B, respectively. Virus was detected in 0/11 and 10/10 of the alveolar macrophage samples collected in groups A and B, respectively. Serum was harvested at selected times throughout the experiment and tested for PRRSV-specific antibody by indirect immunofluorescence microscopy. All gilts in group A were seropositive for the duration of the experiment, and all animals in group B seroconverted following exposure to field PRRSV. This study shows that adult swine can produce a homologous protective immunity after PRRSV exposure that may persist for the production life of the animal.