Porcine Reproductive And Respiratory Syndrome Virus Vaccine Research Articles

Acquisition of different transcriptional shear mRNA and biological function of porcine interleukin 18 binding protein in PRRSV infection.

Interleukin-18 binding protein (IL-18BP), a natural regulator molecule of the pro-inflammatory cytokine interleukin-18 (IL-18), plays an important role in regulating the expression of the cellular immunity factor interferon-γ (IFN-γ). In a previous RNA-seq analysis of porcine alveolar macrophages (PAM) infected with the TIM and TJ strains of porcine reproductive and respiratory syndrome virus (PRRSV), we unexpectedly found that the mRNA expression of porcine interleukin 18-binding protein (pIL-18BP) in PAM cells infected with the TJM strain was significantly higher than that infected with the TJ strain. Studies have shown that human interleukin-18 binding protein (hIL-18bp) plays an important role in regulating cellular immunity in the course of the disease. However, there is a research gap on pIL-18BP. At the same time, PRRSV infection in pigs triggers weak cellular immune response problems. To explore the expression and the role of pIL-18BP in the cellular immune response induced by PRRSV, we strived to acquire the pIL-18BP gene from PAM or peripheral blood mononuclear cell (PBMC) with RT-PCR and sequencing. Furthermore, pIL-18BP and pIL-18 were both expressed prokaryotically and eukaryotically. The colocalization and interaction based on recombinant pIL-18BP and pIL-18 on cells were confirmed in vitro. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in pigs with different PRRSV infection states to interpret the biological function of pIL-18BP in vivo. The results showed there were five shear mutants of pIL-18BP. The mutant with the longest coding region was selected for subsequent functional validation. First, it was demonstrated that TJM-induced pIL-18BP mRNA expression was higher than that of TJ. A direct interaction between pIL-18BP and pIL-18 was confirmed through fluorescence colocalization, bimolecular fluorescent complimentary (BIFC), and co-immunoprecipitation (CO-IP). pIL-18BP also can regulate pIFN-γ mRNA expression. Finally, the expression of pIL-18BP, pIL-18, and pIFN-γ was explored in different PRRSV infection states. Surprisingly, both mRNA and protein expression of pIL-18 were suppressed. These findings fill the gap in understanding the roles played by pIL-18BP in PRRSV infection and provide a foundation for further research.IMPORTANCEPRRSV-infected pigs elicit a weak cellular immune response and the mechanisms of cellular immune regulation induced by PRRSV have not yet been fully elucidated. In this study, we investigated the role of pIL-18BP in PRRSV-induced immune response referring to the regulation of human IL-18BP to human interferon-gamma (hIFN-γ). This is expected to be used as a method to enhance the cellular immune response induced by the PRRSV vaccine. Here, we mined five transcripts of the pIL-18BP gene and demonstrated that it interacts with pIL-18 and regulates pIFN-γ mRNA expression. Surprisingly, we also found that both mRNA and protein expression of pIL-18 were suppressed under different PRRSV strains of infection status. These results have led to a renewed understanding of the roles of pIL-18BP and pIL-18 in cellular immunity induced by PRRSV infection, which has important implications for the prevention and control of PRRS.

The Significance of the 98th Amino Acid in GP2a for Porcine Reproductive and Respiratory Syndrome Virus Adaptation in Marc-145 Cells.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the pig industry. Marc-145 cells are widely used for PRRSV isolation, vaccine production, and investigations into virus biological characteristics. Despite their significance in PRRSV research, Marc-145 cells struggle to isolate specific strains of the North American virus genotype (PRRSV-2). The involvement of viral GP2a, GP2b, and GP3 in this phenomenon has been noted. However, the vital amino acids have not yet been identified. In this study, we increased the number of blind passages and successfully isolated two strains that were previously difficult to isolate with Marc-145 cells. Both strains carried an amino acid substitution in GP2a, specifically phenylalanine to leucine at the 98th amino acid position. Through a phylogenetic and epidemiologic analysis of 32 strains, those that were not amenable to isolation widely exhibited this mutation. Then, by using the PRRSV reverse genetics system, IFA, and Western blotting, we identified the mutation that could affect the tropism of PRRSV-2 for Marc-145 cells. Furthermore, an animal experiment was conducted. Through comparisons of clinical signs, mortality rates, and viral load in the organs and sera, we found that mutation did not affect the pathogenicity of PRRSV-2. In conclusion, our study firmly establishes the 98th amino acid in GP2a as a key determinant of PRRSV-2 tropism for Marc-145 cells.

Type I and II interferons, transcription factors and major histocompatibility complexes were enhanced by knocking down the PRRSV-induced transforming growth factor beta in monocytes co-cultured with peripheral blood lymphocytes.

The innate and adaptive immune responses elicited by porcine reproductive and respiratory syndrome virus (PRRSV) infection are known to be poor. This study investigates the impact of PRRSV-induced transforming growth factor beta 1 (TGFβ1) on the expressions of type I and II interferons (IFNs), transcription factors, major histocompatibility complexes (MHC), anti-inflammatory and pro-inflammatory cytokines in PRRSV-infected co-cultures of monocytes and peripheral blood lymphocytes (PBL). Phosphorothioate-modified antisense oligodeoxynucleotide (AS ODN) specific to the AUG region of porcine TGFβ1 mRNA was synthesized and successfully knocked down TGFβ1 mRNA expression and protein translation. Monocytes transfected with TGFβAS1 ODN, then simultaneously co-cultured with PBL and inoculated with either classical PRRSV-2 (cPRRSV-2) or highly pathogenic PRRSV-2 (HP-PRRSV-2) showed a significant reduction in TGFβ1 mRNA expression and a significant increase in the mRNA expressions of IFNα, IFNγ, MHC-I, MHC-II, signal transducer and activator of transcription 1 (STAT1), and STAT2. Additionally, transfection of TGFβAS1 ODN in the monocyte and PBL co-culture inoculated with cPRRSV-2 significantly increased the mRNA expression of interleukin-12p40 (IL-12p40). PRRSV-2 RNA copy numbers were significantly reduced in monocytes and PBL co-culture transfected with TGFβAS1 ODN compared to the untransfected control. The yields of PRRSV-2 RNA copy numbers in PRRSV-2-inoculated monocytes and PBL co-culture were sustained and reduced by porcine TGFβ1 (rTGFβ1) and recombinant porcine IFNα (rIFNα), respectively. These findings highlight the strategy employed by PRRSV to suppress the innate immune response through the induction of TGFβ expression. The inclusion of TGFβ as a parameter for future PRRSV vaccine and vaccine adjuvant candidates is recommended.

PRRSV-Vaccinated, Seronegative Sows and Maternally Derived Antibodies (II): Impact on PRRSV-1 Vaccine Effectiveness and Challenge Outcomes in Piglets.

Vaccination against the Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is widely practiced in both sows and piglets. However, it has been shown that multivaccinated sows sometimes lack a detectable antibody response, testing seronegative in ELISA (non-responders). Moreover, PRRSV-vaccinated piglets can remain seronegative as well, which is mainly attributed to the interference of maternally derived antibodies (MDAs). The current study investigated the impact of the sow's immune status on the PRRSV vaccine effectiveness in the progeny. The experimental trial included forty-eight piglets (n = 48) originating from a commercial Belgian breeding herd, with twenty-four piglets born from PRRSV vaccinated responder sows (E+ piglets) and twenty-four piglets born from PRRSV vaccinated non-responder sows (E- piglets). Eight piglets in each group were either non-vaccinated (NoVac piglets; n = 8), intramuscularly vaccinated (IM piglets; n = 8), or intradermally vaccinated (ID piglets; n = 8), with the same PRRSV-1 vaccine as used in the sow population. Vaccination was performed at weaning at three weeks of age, and all study piglets were challenged with a high dose of the PRRSV-1 07V063 strain at 6 weeks of age. A clear interference of MDAs was observed in the E+ piglets: 66.7% of the vaccinated E+ piglets lacked an antibody response at 3 weeks post-vaccination (non-responders). Consequently, post-challenge, only the responding E+ piglets had a significantly reduced serum viremia compared to the E+ NoVac piglets. The observed viremia in the non-responding E+ piglets was similar to the viremia of the E+ NoVac piglets. In the vaccinated E- piglets, a lack of antibody response at 3 weeks post-vaccination was observed in 18.8% of the piglets. Interestingly, despite the lack of a vaccine antibody response, the non-responding E- piglets had a significantly reduced serum viremia compared to the NoVac E- piglets. In contrast, the viremia of the responding E- piglets was only numerically reduced compared to the NoVac E- piglets. Finally, some clear differences were observed in both the kinetics of infection and the immune responses post-challenge between the E+ and E- piglets. The results of this study confirm the consequences of the MDA interference on the induced partial protection of PRRSV vaccination in experimentally challenged piglets. More research is warranted to understand the immunological mechanisms behind MDA interference in PRRSV vaccination and to explain the observed differences between E+ and E- piglets.

The Isolation and Characterization of a Novel Group III-Classified Getah Virus from a Commercial Modified Live Vaccine against PRRSV.

As an epizootic causative agent, the Getah virus (GETV) can cause moderate illness in horses, lethal disease in foxes, and reproductive disorders and fetal death in pigs. Due to the wide range of hosts and multiple routes of transmission, GETV has become a growing potential threat to the global livestock industry, and even to public health. More attention and research on GETV are urgently needed. In this study, we successfully isolated a novel GETV strain, named BJ0304, from a commercial live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) and determined its growth kinetics. Then, genetic and phylogenetic analyses were performed. The results revealed that BJ0304 was clustered into Group III, and it was most related to the GETV-V1 strain based on the complete genome sequence. Furthermore, the pathogenicity of the isolate was assessed and found to be a low virulent strain in mice relative to its closest homolog GETV-V1. Finally, mutation and glycosylation analysis showed that a unique mutation (171 T > I) at one amino acid of E2, which affected the glycosylation of E2, may be associated with viral pathogenicity. In summary, the general characteristic of a novel Group III-classified GETV-BJ0304 isolated from commercial live PRRSV vaccine was defined and then mutation/glycosylation-related potential virulence factor was discussed. This study highlights the complexity of GETV transmission routes in swine and the need for more surveillance on commercial animal vaccines, contributes to the understanding of genetic characterization of clinical isolates, provides possible virulence factors in favor of unveiling the viral pathogenesis, and eventually lays the foundation for the prevention and control of GETV.

The replicase protein nsp2 of Chinese highly pathogenic porcine reproductive and respiratory virus is involved in protective immunity by promoting viral clearance

The genome segment for replicase protein nsp2 represents the fastest evolving region of porcine reproductive and respiratory syndrome virus (PRRSV), and our previous studies have shown that the PRRSV nsp2 genetic variation contributes to poor cross-neutralization. By using in vitro antibody absorption assay, here we show that the papain-like protease 2 (PLP2) domain of nsp2 is a target of neutralizing antibodies. This was further verified by cross-neutralization assay with a series of inter-lineage chimeric mutants between the Chinese highly pathogenic PRRSV (HP-PRRSV) strain JXwn06 and the low virulent NADC30-like strain CHsx1401 (lineage 1). The role of nsp2 in protective immunity was subsequently tested in a one-month SPF piglet model by immunizing the piglets with CHsx1401 or its derivatives carrying JXwn06 structural protein region (SP) alone (CHsx1401-SPJX) or in combination with PLP2 region (CHsx1401-SPplp2JX), or the whole nsp2 region (CHsx1401-SPnsp2JX), followed by challenge with JXwn06 at 42 days post immunization, a time point when the viremia was undetectable. All chimera groups were protected from the challenge by JXwn06, whereas the group CHsx1401 failed to provide beneficial protection. Interestingly, the group CHsx1401-SPnsp2JX, but not CHsx1401-SPplp2JX, showed the lowest lung microscopic lesions and viral tissue load. Significantly, the vaccine virus CHsx1401-SPnsp2JX was undetectable in the examined tissues, and so was for the challenge virus except for one piglet, highlighting an important role of HP-PRRSV nsp2 in promoting viral clearance. The findings provide insight into the mechanisms underlying the protective immunity against PRRSV and have important implications in PRRSV vaccine development.

Evaluation of dynamics of immune responses and protective efficacy in piglets immunized with an inactivated porcine reproductive and respiratory syndrome vaccine candidate

Porcine Reproductive and Respiratory Syndrome (PRRS) is an important viral disease of swine that causes significant mortality in piglets and production losses in adult pigs. In this study, we investigated the protective efficacy of an inactivated PRRS virus vaccine candidate and evaluated the differences in PRRSV specific anamnestic response in piglets when challenged with live PRRSV at two different intervals post-immunization. Six-week-old piglets were immunized intramuscularly with an inactivated, Montanide ISA-206 adjuvanted Indian PRRSV isolate, followed by a booster dose at 21 days post-immunization. Homologous live PRRS virus challenge was done on 60 and 180 days post-booster (dpb). We assessed humoral and cell-mediated immune responses at various intervals post-immunization and after challenge. Viraemia, virus shedding in nasal secretions and lung lesion scores were studied to assess the efficacy of the vaccine candidate. All the immunized pigs developed PRRSV-specific antibodies upon booster dose administration. Neutralizing antibody (NA) titres before challenge, in most animals, ranged between 0 and 4. Potentially protective NA titre of 8 was observed in serum of seven out of the 12 immunized piglets after challenge, across the immunized groups. A significant increase in the mean T-helper, T-cytotoxic, memory or activated T-helper and NK cell populations was observed in immunized piglets challenged at 180 dpb, from 4 to 11 dpc, 5 to 11 dpc, 5 to 7 dpc and 6 to 11 dpc, respectively as compared to the challenge controls. Protective efficacy of the inactivated PRRSV antigen against the homologous virus challenge was evidenced by earlier onset of PRRSV specific virus neutralizing antibodies and cell mediated immune responses, reduced viremia, nasal virus shedding and severity of lung lesions in immunized piglets as compared to unimmunized controls post-challenge. Our results indicated that the inactivated PRRSV antigen elicited better virus specific anamnestic immune responses in piglets when challenged at six months after the single booster dose, due to age related increase in antigen-specific memory T helper cell responses, as compared to those challenged at 2 months post booster.

An attenuated herpesvirus vectored vaccine candidate induces T-cell responses against highly conserved porcine reproductive and respiratory syndrome virus M and NSP5 proteins that are unable to control infection.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a leading cause of economic loss in pig farming worldwide. Existing commercial vaccines, all based on modified live or inactivated PRRSV, fail to provide effective immunity against the highly diverse circulating strains of both PRRSV-1 and PRRSV-2. Therefore, there is an urgent need to develop more effective and broadly active PRRSV vaccines. In the absence of neutralizing antibodies, T cells are thought to play a central role in controlling PRRSV infection. Herpesvirus-based vectors are novel vaccine platforms capable of inducing high levels of T cells against encoded heterologous antigens. Therefore, the aim of this study was to assess the immunogenicity and efficacy of an attenuated herpesvirus-based vector (bovine herpesvirus-4; BoHV-4) expressing a fusion protein comprising two well-characterized PRRSV-1 T-cell antigens (M and NSP5). Prime-boost immunization of pigs with BoHV-4 expressing the M and NSP5 fusion protein (vector designated BoHV-4-M-NSP5) induced strong IFN-γ responses, as assessed by ELISpot assays of peripheral blood mononuclear cells (PBMC) stimulated with a pool of peptides representing PRRSV-1 M and NSP5. The responses were closely mirrored by spontaneous IFN-γ release from unstimulated cells, albeit at lower levels. A lower frequency of M and NSP5 specific IFN-γ responding cells was induced following a single dose of BoHV-4-M-NSP5 vector. Restimulation using M and NSP5 peptides from PRRSV-2 demonstrated a high level of cross-reactivity. Vaccination with BoHV-4-M-NSP5 did not affect viral loads in either the blood or lungs following challenge with the two heterologous PRRSV-1 strains. However, the BoHV-4-M-NSP5 prime-boost vaccination showed a marked trend toward reduced lung pathology following PRRSV-1 challenge. The limited effect of T cells on PRRSV-1 viral load was further examined by analyzing local and circulating T-cell responses using intracellular cytokine staining and proliferation assays. The results from this study suggest that vaccine-primed T-cell responses may have helped in the control of PRRSV-1 associated tissue damage, but had a minimal, if any, effect on controlling PRRSV-1 viral loads. Together, these results indicate that future efforts to develop effective PRRSV vaccines should focus on achieving a balanced T-cell and antibody response.

Research Progress on NSP11 of Porcine Reproductive and Respiratory Syndrome Virus.

Porcine reproductive and respiratory syndrome (PRRS) is a virulent infectious disease caused by the PRRS virus (PRRSV). The non-structural protein 11 (NSP11) of PRRSV is a nidovirus-specific endonuclease (NendoU), which displays uridine specificity and catalytic functions conserved throughout the entire NendoU family and exerts a wide range of biological effects. This review discusses the genetic evolution of NSP11, its effects on PRRSV replication and virulence, its interaction with other PRRSV and host proteins, its regulation of host immunity, the conserved characteristics of its enzyme activity (NendoU), and its diagnosis, providing an essential theoretical basis for in-depth studies of PRRSV pathogenesis and vaccine design.

Efficacy of a Commercial PRRSV Vaccine on NADC34-Like PRRSV Challenge

NADC34-like porcine reproductive and respiratory syndrome virus (PRRSV) has become endemic in some provinces of China and caused huge economic losses to local pig industry. The increased reports of NADC34-like PRRSV outbreaks in vaccinated pig herds suggest the limited protection of immunization with commercial vaccines. In this study, we evaluated a commercial PRRSV vaccine that has been widely used in China against the challenge of JS2021NADC34 PRRSV, a highly pathogenic Chinese NADC34-like strain isolated in 2021. The vaccinated pigs developed PRRSV-specific antibody responses, as shown by IDEXX ELISA results. After JS2021NADC34 PRRSV challenge, the vaccinated pigs had low level of viremia but suffered pathological lesions in lungs and lymphoid tissues. The viral antigens were also detected in the above tissues of the vaccinated pigs by immunohistochemistry staining. One out of five pigs in vaccinated group died at 13 days postchallenge. The above results suggested that the commercial PRRSV vaccine could not provide complete protection to the NADC34-like PRRSV infection.

Progress in PRRSV Infection and Adaptive Immune Response Mechanisms.

Since its discovery, Porcine reproductive and respiratory syndrome (PRRS) has had a huge impact on the farming industry. The virus that causes PRRS is Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and because of its genetic diversity and the complexity of the immune response, the eradication of PRRS has been a challenge. To provide scientific references for PRRSV control and vaccine development, this study describes the processes of PRRSV-induced infection and escape, as well as the host adaptive immune response to PRRSV. It also discusses the relationship between PRRSV and the adaptive immune response.

A potent CD8 T-cell response may be associated with partial cross-protection conferred by an attenuated Chinese HP-PRRSV vaccine against NADC30-like PRRSV challenge.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating pathogens to the global swine industry. Many commercial PRRSV vaccines, originally designed to provide homologous protection, have shown partial protection against heterologous strains. However, the protective immune mechanisms mediated by these PRRSV vaccines are not fully understood. In this study, we investigated the factors responsible for partial protection conferred by an attenuated Chinese HP-PRRSV vaccine (TJM-F92) against heterologous NADC30-like PRRSV. By analysing peripheral T-cell responses induced by the TJM-F92 vaccine and local and systemic memory responses following challenge with NADC30-like PRRSV (SD17-38 strains) as well as neutralizing antibody response, we found that the TJM-F92 vaccine induced a significant expansion of CD8 T cells but not CD4 T cells or γδ T cells. The expanded CD8 T cells exhibited a phenotype of effector memory T cells and secreted IFN-γ upon restimulation with SD17-38 strains in vitro. In addition, only CD8 T cells in the prior immunized pigs rapidly expanded in the blood and spleen after heterologous challenge, with higher magnitude, compared to the unvaccinated pigs, showing a remarkable memory response. In contrast, no obvious humoral immune response was enhanced in the vaccinated and challenged pigs, and no heterologous neutralizing antibodies were detected throughout the experiment. Our results suggested that CD8 T cells elicited by the TJM-F92 vaccine may be responsible for partial heterologous protection against NADC30-like PRRSV strains and potentially recognize the conserved antigens among PRRSV strains.

Testable Candidate Immune Correlates of Protection for Porcine Reproductive and Respiratory Syndrome Virus Vaccination.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an on-going problem for the worldwide pig industry. Commercial and experimental vaccinations often demonstrate reduced pathology and improved growth performance; however, specific immune correlates of protection (CoP) for PRRSV vaccination have not been quantified or even definitively postulated: proposing CoP for evaluation during vaccination and challenge studies will benefit our collective efforts towards achieving protective immunity. Applying the breadth of work on human diseases and CoP to PRRSV research, we advocate four hypotheses for peer review and evaluation as appropriate testable CoP: (i) effective class-switching to systemic IgG and mucosal IgA neutralizing antibodies is required for protective immunity; (ii) vaccination should induce virus-specific peripheral blood CD4+ T-cell proliferation and IFN-γ production with central memory and effector memory phenotypes; cytotoxic T-lymphocytes (CTL) proliferation and IFN-γ production with a CCR7- phenotype that should migrate to the lung; (iii) nursery, finishing, and adult pigs will have different CoP; (iv) neutralizing antibodies provide protection and are rather strain specific; T cells confer disease prevention/reduction and possess greater heterologous recognition. We believe proposing these four CoP for PRRSV can direct future vaccine design and improve vaccine candidate evaluation.

Influence of deoxynivalenol-contaminated feed on the immune response of pigs after PRRSV vaccination and infection

The impact of the Fusarium mycotoxin deoxynivalenol (DON) on the immune response against porcine reproductive and respiratory syndrome virus (PRRSV) vaccination and infection was investigated. Forty-two weaned piglets were separated into seven groups and received three different diets: Low DON (1.09 ppm), High DON (2.81 ppm) or No DON. These three treatments were split further into either vaccinated (Ingelvac PRRSFLEX EU) and challenged with PRRSV 28 days post-vaccination, or only infected at day 28. A seventh group received no DON, no vaccination, and no infection. Two weeks after challenge infection, when pigs were euthanized, the number of IFN-γ producing lymphocytes in the blood of vaccinated animals was lower in pigs on High DON compared to animals on Low DON or No DON. Intracellular cytokine staining showed that vaccinated animals fed with the Low DON diet had higher frequencies of TNF-α/IFN-γ co-producing CD4+ T cells than the other two vaccinated groups, particularly in lung tissue. Vaccinated animals on High DON had similar viral loads in the lung as the non-vaccinated groups, but several animals of the Low DON or No DON group receiving vaccination had reduced titers. In these two groups, there was a negative correlation between lung virus titers and vaccine-specific TNF-α/IFN-γ co-producing CD4+ T cells located either in lung tissue or blood. These results indicate that after PRRSV vaccination and infection, high levels of DON negatively influence immune parameters and clearance of the virus, whereas low DON concentrations have immunomodulatory effects.

Follow-Up of PRRSv-Vaccinated Piglets Born from PRRSv-Vaccinated, ELISA-Seropositive and ELISA-Seronegative Sows.

Vaccination against the porcine reproductive and respiratory syndrome virus (PRRSv) is widely used to prevent production losses in the swine industry. In this study, piglets born from both PRRSv-vaccinated ELISA-seropositive sows (E+ piglets) and PRRSv-vaccinated ELISA-seronegative sows (E- piglets) were followed-up pre-vaccination, 3 weeks post-vaccination (wpv) and 8 wpv in two Belgian farrow-to-finish herds. The aim of the study was to analyze the presence of PRRSv-specific maternally-derived antibodies (MDAs) and the PRRSv vaccine response in both groups of piglets. The E- piglets lacked the presence of PRRSv-specific MDAs (0% seropositive), while these were present in the E+ piglets (97% seropositive). Due to this, the E- piglets showed a strong initial vaccine response (72-80% seroconversion) and vaccine viremia (65-75% PCR positive) at 3 wpv. In contrast, the E+ piglets showed only limited initial vaccine responses (25-61% with increased ELISA values) and vaccine viremia (30-31% PCR positive) at 3 wpv. By 8 wpv, the proportion of seropositive E- piglets (78-100%) and seropositive E+ piglets (55-90%) increased in both herds. However, a difference in vaccine viremia duration was observed between both herds at 8 wpv, with a decrease in the proportion of PCR positive piglets in herd 1 (E-: 47%; E+: 25%) and an increase in the proportion of PCR positive piglets in herd 2 (E-: 85%; E+: 92%). This study identified clear differences in the presence of PRRSv-specific maternally-derived antibodies and PRRSv vaccine responses between E- and E+ piglets. Further research is warranted to elicit the biological relevance of these observed differences.

Influence of PRRSV-1 vaccination and infection on mononuclear immune cells at the maternal-fetal interface.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating viruses for the global swine industry. Infection during late gestation causes reproductive failure but the local immune response in utero remains poorly understood. In this study, an experimental PRRSV-infection model with two different PRRSV-1 field isolates was used to investigate the immune cell phenotypes at the maternal-fetal interface during late gestation. In addition, phenotypic changes induced by a modified live virus (MLV, ReproCyc® PRRS EU) vaccine were studied. Vaccinated (n = 12) and non-vaccinated pregnant gilts (n = 12) were challenged with either one of the PRRSV-1 field isolates (low vs. high virulent, LV or HV) or sham-inoculated at day 84 of gestation. Twenty-one days post infection all gilts were euthanized and the fetal preservation status for all fetuses per litter was assessed. Leukocytes from the maternal-fetal interface were isolated and PRRSV-induced changes were investigated using ex vivo phenotyping by flow cytometry. PRRSV load in tissue from the maternal endometrium (ME) and fetal placenta (FP) was determined by RT-qPCR. In the ME, a vast increase in CD8β T cells with CD8αposCD27dim early effector phenotype was found for fetuses from the non-vaccinated LV and HV-challenged gilts, compared to non-treated and vaccinated-only controls. HV-challenged fetuses also showed significant increases of lymphocytes with effector phenotypes in the FP, including NKp46pos NK cells, CD8αhigh γδ T cells, as well as CD8αposCD27pos/dim CD4 and CD8 T cells. In vaccinated animals, this common activation of effector phenotypes was more confined and the fetal preservation status significantly improved. Furthermore, a negative correlation between the viral load and CD163highCD169pos mononuclear phagocytic cells was observed in the FP of HV-infected animals. These results suggest that the strong expansion of effector lymphocytes in gilts that were only infected causes immune-pathogenesis rather than protection. In contrast, the attenuated MLV seems to dampen this effect, yet presumably induces memory cells that limit reproductive failure. This work provides valuable insights into changes of local immune cell phenotypes following PRRSV vaccination and infection.

Establishment and Application of a Quantitative PCR Method for E248R Gene of African Swine Fever Virus.

Simple SummaryAfrican swine fever (ASF) is the most serious animal disease that endangers the pig industry in China, causing the heaviest economic loss so far. Effective prevention and control of ASF is necessary for China’s pig industry, and also the focus and hotspot of global swine industry disease prevention and control research. In view of this, rapid, specific and sensitive diagnosis of ASF is of great significance. In this study, ASF virus (ASFV) E248R gene was selected to be the target for establishing a real-time PCR method which did not cross-react with other porcine viruses that could cause similar symptoms. The detection methods can be used for the efficient detection of ASFV infection and the recombinant live-vectored virus-expressing antigen protein of ASFV.ASF has caused huge economic losses to China’s swine industry. As clinical symptoms of ASF were difficult to distinguish from classical swine fever and porcine reproductive and respiratory syndrome (PRRS), rapid and effective differential diagnosis of ASFV seems very important to control the spread of the disease. In this study, the ASFV E248R gene was selected to be the target for establishing a real-time PCR method. TaqMan real-time PCR for the detection of ASFV E248R gene did not cross-react with other porcine viruses that could cause similar symptoms. The results of the repeatability test showed that the coefficients of variation between and within groups were lower than 1.977%. This method can be used for the rapid detection and early diagnosis of ASF. Meanwhile, the recombinant PRRS virus (PRRSV)-expressing E248R gene of ASFV was constructed and rescued by using the reverse genetic platform of live-attenuated PRRSV vaccine. The ASFV E248R gene can be detected by using this real-time PCR detection method, confirming that the ASFV E248R gene could be stably amplified in PRRSV genome at least 20 cell passages. The detection methods can be used for the efficient detection of the ASFV infection and recombinant PRRSV live vector virus-expressing ASFV antigen protein.

Risk Factors and Spatial-Temporal Analysis of Porcine Reproductive and Respiratory Syndrome Seroprevalence in China Before and After African Swine Fever Outbreak.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious viral disease that causes great harm to the pig industry. PRRS virus (PRRSV), the causative agent of PRRS, is characterized by severe reproductive failure and respiratory confusion. This study performed a cross-sectional investigation of PRRSV seroprevalence and collected 14,134 serum samples in pig farms without PRRSV vaccination from 12 provinces and two cities in China from 2017 to 2021 to detect PRRSV antibodies by enzyme-linked immunosorbent assay (ELISA). The apparent and true PRRSV antibody prevalence was estimated and compared based on the Clopper-Pearson method and Pearson chi-square test, respectively. Risk factors associated with the PRRSV serological status of pig farms were analyzed through univariate and multivariable logistic regression analysis. An automatic autoregressive integrated moving average (ARIMA) model procedure was used for time-series analysis for PRRSV seroprevalence. Spatial clusters of high PRRSV seroprevalence were detected by SaTScan software. The total true PRRSV seroprevalence of the animal level was 62.56% (95% confidence interval [CI]: 61.74–63.37%). Additionally, 286 out of 316 pig farms were positive for PRRSV antibodies at the herd level. Pig farms without pseudorabies virus (PRV) infection were 5.413 (95% CI: 1.977–17.435) times more likely to be PRRSV antibody positive than those with PRV. Identically, the possibility of pig farms being PRRSV antibody positive before an African swine fever (ASF) outbreak was 3.104 (95% CI: 1.122–10.326) times more than after ASF. The odd ratio values of medium and large pig farms with PRRSV infection are 3.076 (95% CI: 1.005–9.498) and 6.098 (95% CI: 1.814–21.290). A fluctuant decline pattern for PRRSV prevalence was observed in the temporal analysis. Three significant clusters of high PRRSV seroprevalence were first detected in China, covering a time frame from January 2018 to September 2018, which reveals high PRRSV prevalence before the outbreak of ASF. These findings show the epidemic situation and spatial-temporal distribution of PRRSV infection in China in recent years and could help develop reasonable measures to prevent PRRSV infection.

Differences in Humoral Immune Response against the Type 2 Porcine Reproductive and Respiratory Syndrome Virus via Different Immune Pathways.

The intramuscular vaccine is the principal strategy to protect pigs from porcine reproductive and respiratory syndrome virus (PRRSV), However, it is still difficult to control PRRSV effectively. This study infected piglets with PRRSV through intramuscular and intranasal inoculation. Subsequently, viral loads, anti-PRRSV antibody levels, and neutralizing antibodies (NAs) titers in both serum and saliva were monitored for 43 days. Meanwhile, tissues were obtained through necropsy at 43 days post-inoculation (dpi) to detect viral loads. The results indicated that viremia lasted from 3 to 31 dpi in both the inoculation groups, but the viruses survived in the lungs and lymph nodes after viremia clearance. The antibody response was detected from 11 dpi, but the response of NAs was delayed until 3–4 weeks. Furthermore, intranasal inoculation induced lower viral load levels than injection inoculation. In addition, positive SIgA and NAs levels were produced early, with higher levels through intranasal inoculation. Therefore, our data indicated that a more robust antibody response and lower virus loads could be induced by intranasal inoculation, and mucosal inoculation could be a suitable pathway for PRRSV vaccines.

Characterization of Two Immunodominant Antigenic Peptides in NSP2 of PRRSV-2 and Generation of a Marker PRRSV Strain Based on the Peptides.

Porcine reproductive and respiratory syndrome (PRRS) is a widespread disease with great economic importance in the pig industry. Although vaccines against the PRRS virus (PRRSV) have been employed for more than 20 years, differentiating infected from vaccinated animals remains challenging. In this study, all 907 non-structural protein 2 (NSP2) full-length sequences of PRRSV-2 available from GenBank were aligned. Two peptides, at positions 562–627 (m1B) and 749–813 (m2B) of NSP2, were selected, and their potential for use in differential diagnosis was assessed. Both m1B and m2B were recognized by PRRSV-positive pig serum in peptide-coated enzyme-linked immunosorbent assays. Further epitope identification yielded five overlapping short peptides for the immunodominant regions of m1B and m2B. Using the infectious clone of PRRSV HuN4-F112 as a template, the deletion mutants, rHuN4-F112-m1B, rHuN4-F112-m2B, and rHuN4-F112-C5-m1B-m2B, were generated and successfully rescued in Marc-145 cells. Growth kinetics revealed that the deletion of m1B and m2B did not significantly affect virus replication. Hence, m1B and m2B show potential as molecular markers for developing a PRRSV vaccine.