Highly pathogenic PRRSV upregulates IL-13 production through nonstructural protein 9-mediated inhibition of N6-methyladenosine demethylase FTO.

Porcine reproductive and respiratory syndrome virus (PRRSV), a highly infectious virus, causes severe losses in the swine industry by regulating the inflammatory response, inducing tissue damage, suppressing the innate immune response, and promoting persistent infection in hosts. Interleukin-13 (IL-13) is a cytokine that plays a critical role in regulating immune responses and inflammation, particularly in immune-related disorders, certain types of cancer, and numerous bacterial and viral infections; however, the underlying mechanisms of IL-13 regulation during PRRSV infection are not well understood. In this study, we demonstrated that PRRSV infection elevates IL-13 levels in porcine alveolar macrophages. PRRSV enhances m6A-methylated RNA levels while reducing the expression of fat mass and obesity associated protein (FTO, an m6A demethylase), thereby augmenting IL-13 production. PRRSV nonstructural protein 9 (nsp9) was a key factor for this modulation. Furthermore, we found that the residues Asp567, Tyr586, Leu593, and Asp595 were essential for nsp9 to induce IL-13 production via attenuation of FTO expression. These insights delineate PRRSV nsp9's role in FTO-mediated IL-13 release, advancing our understanding of PRRSV's impact on host immune and inflammatory responses.

A nanobody inhibiting porcine reproductive and respiratory syndrome virus replication via blocking self-interaction of viral nucleocapsid protein.

Porcine reproductive and respiratory syndrome (PRRS) is a serious global pig industry disease. Understanding the mechanism of viral replication and developing efficient antiviral strategies are necessary for combating with PRRS virus (PRRSV) infection. Recently, nanobody is considered to be a promising antiviral drug, especially for respiratory viruses. The present study evaluated two nanobodies against PRRSV nucleocapsid (N) protein (PRRSV-N-Nb1 and -Nb2) for their anti-PRRSV activity in vitro and in vivo. The results showed that intracellularly expressed PRRSV-N-Nb1 significantly inhibited PRRSV-2 replication in MARC-145 cells (approximately 100%). Then, the PRRSV-N-Nb1 fused with porcine IgG Fc (Nb1-pFc) as a delivering tag was produced and used to determine its effect on PRRSV-2 replication in porcine alveolar macrophages (PAMs) and pigs. The inhibition rate of Nb1-pFc against PRRSV-2 in PAMs could reach >90%, and it can also inhibit viral replication in vivo. Epitope mapping showed that the motif Serine 105 (S105) in PRRSV-2 N protein was the key amino acid binding to PRRSV-N-Nb1, which is also pivotal for the self-interaction of N protein via binding to Arginine 97. Moreover, viral particles were not successfully rescued when the S105 motif was mutated to Alanine (S105A). Attachment, entry, genome replication, release, docking model analysis, and blocking enzyme-linked immunosorbent assay (ELISA) indicated that the binding of PRRSV-N-Nb1 to N protein could block its self-binding, which prevents the viral replication of PRRSV. PRRSV-N-Nb1 may be a promising drug to counter PRRSV-2 infection. We also provided some new insights into the molecular basis of PRRSV N protein self-binding and assembly of viral particles.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes serious economic losses to the swine industry worldwide, and there are no highly effective strategies for prevention. Nanobodies are considered a promising novel approach for treating diseases because of their ease of production and low costing. Here, we showed that PRRSV-N-Nb1 against PRRSV-N protein significantly inhibited PRRSV-2 replication in vitro and in vivo. Furthermore, we demonstrated that the motif Serine 105 (S105) in PRRSV-N protein was the key amino acid to interact with PRRSV-N-Nb1 and bond to its motif R97, which is important for the self-binding of N protein. The PRRSV-N-Nb1 could block the self-interaction of N protein following viral assembly. These findings not only provide insights into the molecular basis of PRRSV N protein self-binding as a key factor for viral replication for the first time but also highlight a novel target for the development of anti-PRRSV replication drugs.

The 5'UTR of porcine reproductive and respiratory syndrome virus strain JXwn06 harbors a uORF that regulates cellular inflammation.

The 5' untranslated region (5'UTR) of many positive-stranded RNA viruses contain functional regulatory sequences. Here, we show that the porcine reproductive and respiratory syndrome virus (PRRSV), a member of arteriviruses, harbors small upstream open reading frames (uORFs) in its 5'UTR. Bioinformatics analysis shows that this feature is relatively well conserved among PRRSV strains and Arteriviridae. We also identified a uORF, namely uORF2, in the PRRSV strain JXwn06, that possesses translational activity and exerts a suppressive effect on the expression of the primary ORF evidenced by in vitro reporter assays. We tested its importance via reverse genetics by introducing a point mutation into the PRRSV infectious cDNA clone to inactivate the start codon of uORF2. The recovered mutant virus Mut2 surprisingly replicated to the same level as the wild-type virus (WT), but induced a higher level of inflammatory cytokines (e.g., TNF-α, IL-1ß, and IL-6) both in vitro and in animal experiments, correlating well with more severe lung injury and higher death rate. In line with this, over-expression of uORF2 in transfected cells significantly inhibited poly(I:C)-induced expression of inflammatory cytokines. Together, our data support the idea that uORF2 encodes a novel, functional regulator of PRRSV virulence despite of its short size. IMPORTANCE: PRRSV has remained a major challenge to the world swine industry, but we still do not know much about its biology and pathogenesis. Here, we provide evidence to show that the 5'UTR of PRRSV strain JXwn06 harbors a functional uORF that has the coding capacity and regulates induction of inflammation as demonstrated by in vitro assays and animal experiment. The findings reveal a novel viral factor that regulates cellular inflammation and provide insight into the understanding of PRRSV pathogenesis.

GTPase activity of porcine Mx1 plays a dominant role in inhibiting the N-Nsp9 interaction and thus inhibiting PRRSV replication.

Porcine Mx1 is a type of interferon-induced GTPase that inhibits the replication of certain RNA viruses. However, the antiviral effects and the underlying mechanism of porcine Mx1 for porcine reproductive and respiratory syndrome virus (PRRSV) remain unknown. In this study, we demonstrated that porcine Mx1 could significantly inhibit PRRSV replication in MARC-145 cells. By Mx1 segment analysis, it was indicated that the GTPase domain (68-341aa) was the functional area to inhibit PRRSV replication and that Mx1 interacted with the PRRSV-N protein through the GTPase domain (68-341aa) in the cytoplasm. Amino acid residues K295 and K299 in the G domain of Mx1 were the key sites for Mx1-N interaction while mutant proteins Mx1(K295A) and Mx1(K299A) still partially inhibited PRRSV replication. Furthermore, we found that the GTPase activity of Mx1 was dominant for Mx1 to inhibit PRRSV replication but was not essential for Mx1-N interaction. Finally, mechanistic studies demonstrated that the GTPase activity of Mx1 played a dominant role in inhibiting the N-Nsp9 interaction and that the interaction between Mx1 and N partially inhibited the N-Nsp9 interaction. We propose that the complete anti-PRRSV mechanism of porcine Mx1 contains a two-step process: Mx1 binds to the PRRSV-N protein and subsequently disrupts the N-Nsp9 interaction by a process requiring the GTPase activity of Mx1. Taken together, the results of our experiments describe for the first time a novel mechanism by which porcine Mx1 evolves to inhibit PRRSV replication. IMPORTANCE: Mx1 protein is a key mediator of the interferon-induced antiviral response against a wide range of viruses. How porcine Mx1 affects the replication of porcine reproductive and respiratory syndrome virus (PRRSV) and its biological function has not been studied. Here, we show that Mx1 protein inhibits PRRSV replication by interfering with N-Nsp9 interaction. Furthermore, the GTPase activity of porcine Mx1 plays a dominant role and the Mx1-N interaction plays an assistant role in this interference process. This study uncovers a novel mechanism evolved by porcine Mx1 to exert anti-PRRSV activities.

Comparison of pathogenicity and host responses of emerging porcine reproductive and respiratory syndrome virus variants in piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly variable virus with genetic diversity. This study comparatively examines the pathogenicity and immunological impact of two emergent PRRSV strains, SD53 and HuN4, in piglets. Our results indicate that SD53 strain induces milder clinical syndromes and less severe tissue damage than HuN4, despite similar replication rates. Hematological tests showed less perturbations in peripheral blood cell profiles after SD53 infection, suggesting a less systemic impact. The neutrophil-to-lymphocyte ratio was notably lower in SD53-infected piglets, suggesting a less intense inflammatory reaction. Moreover, SD53 infection led to lower levels of pro-inflammatory cytokines, further supporting a less pronounced inflammatory profile. Both strains induced the production of PRRSV-specific antibodies. However, transcriptomic analysis of lung and lymph node tissues from infected piglets disclosed a more moderate up-regulation of core genes, including ISGs, in the SD53 group. Further analysis indicated that SD53 primarily enhanced immune-related signaling, particularly in T cell response modules, while HuN4 caused a more robust pro-inflammatory reaction and a dampening of T cell functionality. Flow cytometry analyses confirmed these findings, showing higher CD4/CD8 ratios and increased CD4+ T cell percentages in SD53-infected piglets, implying a more robust T cell response. Collectively, these findings broaden our comprehension of PRRSV pathogenesis and may inform the development of future therapeutic or prophylactic strategies for controlling PRRSV infections more effectively. IMPORTANCE: The high mutation rate of porcine reproductive and respiratory syndrome virus (PRRSV) poses significant challenges to its accurate diagnosis and the implementation of effective control measures. This research explores the pathogenic profiles of two emerging PRRSV stains: the NADC30-like strain SD53 and the highly pathogenic strain HuN4. Our investigation reveals that SD53 initiates distinct immunopathological responses in vivo compared with those provoked by HuN4. By conducting a transcriptome analysis of differential gene expression in the lungs and lymph nodes of infected piglets, we unveil the intricate molecular mechanisms underlying the contrasting pathogenicity of these two strains. The comprehensive insights yielded by this study are instrumental in advancing our understanding of the dominant NADC30-like PRRSV strain, which has become increasingly prevalent in China's swine industry.

Lactate-lactylation-HSPA6 axis promotes PRRSV replication by impairing IFN-ß production.

Lactate, traditionally considered a metabolic by-product, has recently been identified as a substrate for the induction of lactylation, a newly identified epigenetic modification that plays an important role in the regulation of host gene expression. Our previous study showed that lactate levels were significantly elevated in cells infected with the porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus that has devastated the swine industry worldwide for over 30 years. However, the role of elevated lactate in PRRSV infections remains unknown. In this study, we found that lactate was required for optimal PRRSV proliferation, and PRRSV infection increased cellular lactylation in a dose-dependent manner. Using the Cleavage Under Targets and Tagmentation (CUT&Tag) combined with RNA sequencing (RNA-seq) to screen the downstream genes regulated by lactylation in PRRSV-infected cells, we found that PRRSV-induced lactylation activated the expression of heat shock 70 kDa protein 6 (HSPA6). Follow-up experiments showed that HSPA6 is important for PRRSV proliferation by negatively modulating interferon (IFN)-ß induction. Mechanistically, HSPA6 impeded the interaction between TNF-receptor-associated factor 3 (TRAF3) and inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKKε), thereby hindering the production of IFN-ß. Taken together, these results indicate that the activated lactate-lactylation-HSPA6 axis promotes viral growth by impairing IFN-ß induction, providing new therapeutic targets for the prevention and control of PRRSV infection. The results presented here also link lactylation to the virus life cycle, improving our understanding of epigenetic regulation in viral infection.IMPORTANCEAs a newly identified epigenetic modification, lactate-induced lactylation has received attentions because it plays important roles in gene expression and contributes to tumorigenesis and the innate immune response. Previous studies showed that many viruses upregulate cellular lactate levels; however, whether virus-elevated lactate induces lactylation and the subsequent biological significance of the modification to viral infection have not been reported. In this study, we demonstrated that porcine reproductive and respiratory syndrome virus (PRRSV) infection induced cellular lactylation, which, in turn, upregulated the expression of HSPA6, an IFN-negative regulator. We also dissected the mechanism by which HSPA6 negatively regulates IFN-ß production. To our knowledge, this is the first report to study virus-induced lactylation and establish the relationship between lactylation and virus infection.

Disruption of pulmonary microvascular endothelial barrier by dysregulated claudin-8 and claudin-4: uncovered mechanisms in porcine reproductive and respiratory syndrome virus infection.

The pulmonary endothelium is a dynamic and metabolically active monolayer of endothelial cells. Dysfunction of the pulmonary endothelial barrier plays a crucial role in the acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), frequently observed in the context of viral pneumonia. Dysregulation of tight junction proteins can lead to the disruption of the endothelial barrier and subsequent leakage. Here, the highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) served as an ideal model for studying ALI and ARDS. The alveolar lavage fluid of pigs infected with HP-PRRSV, and the supernatant of HP-PRRSV infected pulmonary alveolar macrophages were respectively collected to treat the pulmonary microvascular endothelial cells (PMVECs) in Transwell culture system to explore the mechanism of pulmonary microvascular endothelial barrier leakage caused by viral infection. Cytokine screening, addition and blocking experiments revealed that proinflammatory cytokines IL-1ß and TNF-α, secreted by HP-PRRSV-infected macrophages, disrupt the pulmonary microvascular endothelial barrier by downregulating claudin-8 and upregulating claudin-4 synergistically. Additionally, three transcription factors interleukin enhancer binding factor 2 (ILF2), general transcription factor III C subunit 2 (GTF3C2), and thyroid hormone receptor-associated protein 3 (THRAP3), were identified to accumulate in the nucleus of PMVECs, regulating the transcription of claudin-8 and claudin-4. Meanwhile, the upregulation of ssc-miR-185 was found to suppress claudin-8 expression via post-transcriptional inhibition. This study not only reveals the molecular mechanisms by which HP-PRRSV infection causes endothelial barrier leakage in acute lung injury, but also provides novel insights into the function and regulation of tight junctions in vascular homeostasis.

Multiplex Assay to Determine Acute Phase Proteins in Modified Live PRRSV Vaccinated Pigs.

Acute phase protein (APP) response to vaccine challenges is an attractive alternative to natural infection for identifying pigs with increased disease resilience and monitoring the productive performance. Currently, the methods used for APP quantification are diverse and often based on techniques that use antibodies that are not necessarily pig specific. The objective of this work is the development of a method based on a UPLC-SRM/MS system for simultaneous determination of haptoglobin, apolipoprotein A1, C-reactive protein, pig-major acute protein, and serum amyloid A and its application in pigs to monitor the effect of a vaccine administered against porcine reproductive and respiratory syndrome virus (PRRSV). With the aim of tracing the complete analytical process for each proteotypic peptide, a synthetic QconCat polypeptide construct was designed. It was possible to develop an SRM method including haptoglobin, apolipoprotein A1, pig-MAP, and serum amyloid A1. The PRRSV vaccine only affected haptoglobin. The pigs with positive viremia tended to show higher values than negative pigs, reaching significant differences in the three haptoglobin SRM-detected peptides but not with the data acquired by immunoenzymatic and spectrophotometric assays. These results open the door to the use of SRM to accurately monitor APP changes in experimental pigs.

Epitope screening and vaccine molecule design of PRRSV GP3 and GP5 protein based on immunoinformatics.

Porcine reproductive and respiratory syndrome (PRRS) is a respiratory disease in pigs that causes severe economic losses. Currently, live PRRSV vaccines are commonly used but fail to prevent PRRS outbreaks and reinfection. Inactivated PRRSV vaccines have poor immunogenicity, making PRRSV a significant threat to swine health globally. Therefore, there is an urgent need to develop an effective PRRSV vaccine. This study used immunoinformatics to predict, screen, design and construct a candidate vaccine that fused B-cell epitopes, CTL- and HTL-dominant protective epitopes of PRRSV strain's GP3 and GP5 proteins. The study identified 12 B-cell epitopes, 6 CTL epitopes and 5 HTL epitopes of GP3 and GP5 proteins. The candidate vaccine was constructed with 50S ribosomal protein L7/L1 molecular adjuvant, which has antigenicity, solubility, stability, non-allergenicity and a high affinity for its target receptor, TLR-3. The C-ImmSim immunostimulation results showed significant increases in cellular and humoral responses (B cells and T cells) and production of TGF-ß, IL-2, IL-10, IFN-γ and IL-12. The constructed vaccine was stable and immunogenic, and it can effectively induce strong T-cell and B-cell immune responses against PRRSV. Therefore, it is a promising candidate vaccine for controlling and preventing PRRSV outbreaks.

The Genetic Variation of Porcine Reproductive and Respiratory Syndrome Virus Replicase Protein nsp2 Modulates Viral Virulence and Persistence.

Fast evolution in the field of the replicase nsp2 represents a most prominent feature of porcine reproductive and respiratory syndrome virus (PRRSV). Here, we determined its biological significance in viral pathogenesis by constructing interlineage chimeric mutants between the Chinese highly pathogenic PRRSV (HP-PRRSV) strain JXwn06 (lineage 8) and the low-virulent NADC30-like strain CHsx1401 (lineage 1). Replacement with nsp2 from JXwn06 was surprisingly lethal to the backbone virus CHsx1401, but combined substitution with the structural protein-coding region (SP) gave rise to viable virus CHsx1401-SPnsp2JX. Meanwhile, a derivative carrying only the SP region (CHsx1401-SPJX) served as a control. Subsequent animal experiments revealed that acquisition of SP alone (CHsx1401-SPJX) did not allow CHsx1401 to gain much virulence, but additional swapping of HP-PRRSV nsp2 (CHsx1401-SPnsp2JX) enabled CHsx1401 to acquire some properties of HP-PRRSV, exemplified by prolonged high fever, microscopic lung hemorrhage, and a significant increase in proinflammatory cytokines in the acute stage. Consistent with this was the transcriptomic analysis of persistently infected secondary lymphoid tissues that revealed a much stronger induction of host cellular immune responses in this group and identified several core immune genes (e.g., TLR4, IL-1ß, MPO, etc.) regulated by HP-PRRSV nsp2. Interestingly, immune activation status in the individual groups correlated well with the rate of viremia clearance and viral tissue load reduction. Overall, the above results suggest that the Chinese HP-PRRSV nsp2 is a critical virulence regulator and highlight the importance of nsp2 genetic variation in modulating PRRSV virulence and persistence via immune modulation. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) has been a major threat to the world swine industry. In the field, rapid genetic variations (e.g., deletion, mutation, recombination, etc.) within the nsp2 region present an intriguing conundrum to PRRSV biology and pathogenesis. By making chimeric mutants, here, we show that the Chinese highly pathogenic PRRSV (HP-PRRSV) nsp2 is a virulence factor and a much stronger inducer of host immune responses (e.g., inflammation) than its counterpart, currently epidemic, NADC30-like strains. Differences in the ability to modulate host immunity provide insight into the mechanisms of why NADC30-like strains and their derivatives are rising to be the dominant viruses, whereas the Chinese HP-PRRSV strains gradually give away center stage in the field. Our results have important implications in understanding PRRSV evolution, interlineage recombination, and persistence.

Ectopic expression of murine CD163 enables cell-culture isolation of lactate dehydrogenase-elevating virus 63 years after its discovery.

IMPORTANCE: Mouse models of viral infection play an especially large role in virology. In 1960, a mouse virus, lactate dehydrogenase-elevating virus (LDV), was discovered and found to have the peculiar ability to evade clearance by the immune system, enabling it to persistently infect an individual mouse for its entire lifespan without causing overt disease. However, researchers were unable to grow LDV in culture, ultimately resulting in the demise of this system as a model of failed immunity. We solve this problem by identifying the cell-surface molecule CD163 as the critical missing component in cell-culture systems, enabling the growth of LDV in immortalized cell lines for the first time. This advance creates abundant opportunities for further characterizing LDV in order to study both failed immunity and the family of viruses to which LDV belongs, Arteriviridae (aka, arteriviruses).

Antiviral activity and underlying mechanisms of baicalin against porcine reproductive and respiratory syndrome virus in vitro.

Porcine reproductive and respiratory syndrome (PRRS) is a major challenge for the global swine industry, causing huge economic losses worldwide. To date, there are no effective measures to prevent and control the spread of PRRS virus (PRRSV). Baicalin (BA) is a natural flavonoid with various pharmacological effects, including antiviral, anti-inflammatory, antioxidant and immunomodulatory. Here, we demonstrate that BA exhibits potent anti-PRRSV activity in vitro, BA concentrations in the range of 5-20 µg/mL significantly inhibited PRRSV infection in a dose-dependent manner and were independent of PRRSV strain. Mechanistically, BA inhibited PRRSV replication by directly interacting with virions, thereby affecting multiple stages of the virus life cycle. Meanwhile, the preventive effect of BA on PRRSV could be realized by inhibiting CD151 and CD163 expression. Furthermore, BA reduced the PRRSV-induced expression of PAMs cytokines (IFN-α, IL-6, IL-8, and TNF-α), suggesting that BA-induced antiviral cytokines may help BA inhibit PRRSV infection. Taken together, BA can be used as an inhibitor of PRRSV infection in vitro, which provides a theoretical basis for the clinical application of BA and the prevention and control of PRRSV infection, which is worthy of further in vivo studies in swine.

Molecular detection and genetic characteristics of porcine reproductive and respiratory syndrome virus in central China.

Porcine reproductive and respiratory syndrome caused by porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically devastating viral diseases in the global pork industry. To further clarify the epidemic characteristics of the virus, 365 clinical samples were collected from diseased pigs suffering from abortion and respiratory disease from 2018 to 2023 on 63 pig farms in Henan and Shanxi provinces, and screened for the presence of PRRSV using reverse transcription-polymerase chain reaction (RT-PCR). A total of 62 clinical samples (62/365, 16.99 %) were positive for PRRSV, and subsequently, full-length ORF5 gene sequences of 29 PRRSV strains and the complete genome sequence of one PRRSV HeN-HC isolate were obtained and analyzed. Phylogenetic analysis based on the ORF5 gene showed that 22 of the 29 PRRSV2 strains belonged to sublineage 1.8 (NADC30-like), 5 belonged to sublineage 8.5 (HP-PRRSV), and 2 belonged to sublineage 5.1 (VR-2332-like), indicating that both HP-PRRSV and NADC30-like strains were mainly circulating in Henan and Shanxi provinces. Compared to VR-2332 strain, different types of amino acid mutations were found in the GP5 protein of these 29 strains, and the amino acid deletions were displayed in the Nsp2 protein of the HeN-HC isolate, leading to the variation of protein structures. It is noteworthy that recombination events were identified in the HeN-Ping and HeN-B strains. In addition, a total of 60, 094 pig serum samples from Henan province were collected, and the positive rate of specific antibodies against PRRSV was 86.37 % from 2019 to 2022, and 86.66 %, 84.85 %, 87.54 % and 86.30 % in 2019, 2020, 2021 and 2022, respectively. Overall, this study provides valuable insights into the molecular epidemiology and evolution of PRRSV circulating in central China.

The effect of matrine and glycyrrhizic acid on porcine reproductive and respiratory syndrome virus in Vitro and in vivo.

Porcine reproductive and respiratory syndrome (PRRS) is endemic worldwide, seriously affecting the development of the pig industry, but vaccines have limited protective effects against PRRSV transmission. The aim of this study was to identify potential anti-PRRSV drugs. We examined the cytotoxicity of seven compounds formulated based on the mass ratio of glycyrrhizic acid to matrine and calculated their inhibition rates against PRRSV in vitro. The results showed that the seven compounds all had direct killing and therapeutic effects on PRRSV, and the compounds inhibited PRRSV replication in a time- and dose-dependent manner. The compound with the strongest anti-PRRSV effect was selected for subsequent in vivo experiments. Pigs were divided into a control group and a medication group for the in vivo evaluation. The results showed that pigs treated with the 4:1 compound had 100% morbidity after PRRSV challenge, and the mortality rate reached 75% on the 8th day of the virus challenge. These results suggest that this compound has no practical anti-PRRSV effect in vivo and can actually accelerate the death of infected pigs. Next, we further analyzed the pigs that exhibited semiprotective effects following vaccination with the compound to determine whether the compound can synergize with the vaccine in vivo. The results indicated that pigs treated with the compound had higher mortality rates and more severe clinical reactions after PRRSV infection (p < 0.05). The levels of proinflammatory cytokines (IL-6, IL-8, IL-1ß, IFN-γ, and TNF-α) were significantly greater in the compound-treated pigs than in the positive control-treated pigs (p < 0.05), and there was no synergistic enhancement with the live attenuated PRRSV vaccine (p < 0.05). The compound enhanced the inflammatory response, prompted the body to produce excessive levels of inflammatory cytokines and caused body damage, preventing a therapeutic effect. In conclusion, the present study revealed that the in vitro effectiveness of these agents does not indicate that they are effective in vivo or useful for developing anti-PRRSV drugs. Our findings also showed that, to identify effective anti-PRRSV drugs, comprehensive drug screening is needed, for compounds with solid anti-inflammatory effects both in vitro and in vivo. Our study may aid in the development of new anti-PRRSV drugs.

ZNF283, a Krüppel-associated box zinc finger protein, inhibits RNA synthesis of porcine reproductive and respiratory syndrome virus by interacting with Nsp9 and Nsp10.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a viral pathogen with substantial economic implications for the global swine industry. The existing vaccination strategies and antiviral drugs offer limited protection. Replication of the viral RNA genome encompasses a complex series of steps, wherein a replication complex is assembled from various components derived from both viral and cellular sources, as well as from the viral genomic RNA template. In this study, we found that ZNF283, a Krüppel-associated box (KRAB) containing zinc finger protein, was upregulated in PRRSV-infected Marc-145 cells and porcine alveolar macrophages and that ZNF283 inhibited PRRSV replication and RNA synthesis. We also found that ZNF283 interacts with the viral proteins Nsp9, an RNA-dependent RNA polymerase, and Nsp10, a helicase. The main regions involved in the interaction between ZNF283 and Nsp9 were determined to be the KRAB domain of ZNF283 and amino acids 178-449 of Nsp9. The KRAB domain of ZNF283 plays a role in facilitating Nsp10 binding. In addition, ZNF283 may have an affinity for the 3' untranslated region of PRRSV. These findings suggest that ZNF283 is an antiviral factor that inhibits PRRSV infection and extend our understanding of the interactions between KRAB-containing zinc finger proteins and viruses.

A chimeric strain of porcine reproductive and respiratory syndrome virus 2 derived from HP-PRRSV and NADC30-like PRRSV confers cross-protection against both strains.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine viral infectious diseases worldwide. Vaccination is a key strategy for the control and prevention of PRRS. At present, the NADC30-like PRRSV strain has become the predominant epidemic strain in China, superseding the HP-PRRSV strain. The existing commercial vaccines offer substantial protection against HP-PRRSV, but their efficacy against NADC30-like PRRSV is limited. The development of a novel vaccine that can provide valuable cross-protection against both NADC30-like PRRSV and HP-PRRSV is highly important. In this study, an infectious clone of a commercial MLV vaccine strain, GD (HP-PRRSV), was first generated (named rGD). A recombinant chimeric PRRSV strain, rGD-SX-5U2, was subsequently constructed by using rGD as a backbone and embedding several dominant immune genes, including the NSP2, ORF5, ORF6, and ORF7 genes, from an NADC30-like PRRSV isolate. In vitro experiments demonstrated that chimeric PRRSV rGD-SX-5U2 exhibited high tropism for MARC-145 cells, which is of paramount importance in the production of PRRSV vaccines. Moreover, subsequent in vivo inoculation and challenge experiments demonstrated that rGD-SX-5U2 confers cross-protection against both HP-PRRSV and NADC30-like PRRSV, including an improvement in ADG levels and a reduction in viremia and lung tissue lesions. In conclusion, our research demonstrated that the chimeric PRRSV strain rGD-SX-5U2 is a novel approach that can provide broad-spectrum protection against both HP-PRRSV and NADC30-like PRRSV. This may be a significant improvement over previous MLV vaccinations.

A chimeric porcine reproductive and respiratory syndrome virus 1 strain containing synthetic ORF2-6 genes can trigger T follicular helper cell and heterologous neutralizing antibody responses and confer enhanced cross-protection.

The prevalence of porcine reproductive and respiratory syndrome virus 1 (PRRSV1) isolates has continued to increase in Chinese swine herds in recent years. However, no effective control strategy is available for PRRSV1 infection in China. In this study, we generated the first infectious cDNA clone (rHLJB1) of a Chinese PRRSV1 isolate and subsequently used it as a backbone to construct an ORF2-6 chimeric virus (ORF2-6-CON). This virus contained a synthesized consensus sequence of the PRRSV1 ORF2-6 gene encoding all the envelope proteins. The ORF2-6 consensus sequence shared > 90% nucleotide similarity with four representative strains (Amervac, BJEU06-1, HKEU16 and NMEU09-1) of PRRSV1 in China. ORF2-6-CON had replication efficacy similar to that of the backbone rHLJB1 virus in primary alveolar macrophages (PAMs) and exhibited cell tropism in Marc-145 cells. Piglet inoculation and challenge studies indicated that ORF2-6-CON is not pathogenic to piglets and can induce enhanced cross-protection against a heterologous SD1291 isolate. Notably, ORF2-6-CON inoculation induced higher levels of heterologous neutralizing antibodies (nAbs) against SD1291 than rHLJB1 inoculation, which was concurrent with a higher percentage of T follicular helper (Tfh) cells in tracheobronchial lymph nodes (TBLNs), providing the first clue that porcine Tfh cells are correlated with heterologous PRRSV nAb responses. The number of SD1291-strain-specific IFNγ-secreting cells was similar in ORF2-6-CON-inoculated and rHLJB1-inoculated pigs. Overall, our findings support that the Marc-145-adapted ORF2-6-CON can trigger Tfh cell and heterologous nAb responses to confer improved cross-protection and may serve as a candidate strain for the development of a cross-protective PRRSV1 vaccine.

MiR-339-5p inhibits replication of porcine reproductive and respiratory syndrome virus by targeting viral gene regions.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a variable virus, whose spread cannot be totally stopped by vaccination. PRRSV infection results in abortion and respiratory symptoms in pregnant pigs. One crucial component of the anti-viral infection strategy is microRNA (miRNA), a class of multifunctional small molecules. It is unknown whether miR-339-5p can specifically target the PRRSV gene and prevent the virus from replicating, despite the fact that miR-339-5p is markedly up-regulated during the PRRSV infection. In this pursuit, the present study revealed that the two PRRSV areas targeted by miR-339-5p were PRRSV nsp2-3378 to 3403 and PRRSV nsp2-3112 to 3133 using the miRanda program. Dual luciferase reporter assays showed that the miR-339-5p target region of the PRRSV gene sequence exhibited 100% homology and was highly conserved. Furthermore, the ability of miR-339-5p to target PRRSV gene areas was verified. It was found that the overexpression of miR-339-5p markedly reduced the PRRSV replication through PRRSV infection trials. The precursor sequence of ssc-miR-339-5p was amplified using the DNA of pig lung tissue as a template in order to create a fragment of 402 bp of porcine-derived miR-339-5p precursor sequence, which was then used to produce the eukaryotic expression plasmid of miR-339-5p. In conclusion, miR-339-5p can target the specific PRRSV gene areas and prevent PRRSV replication, offering fresh perspectives for the creation of medications that combat the PRRSV infection.

Quantum dot fluorescent microsphere-based immunochromatographic strip for detecting PRRSV antibodies.

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18-25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening. KEY POINTS: • QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies • Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits • QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus.

Immune response enhancement by dietary supplementation with Caesalpinia sappan extract in weaned pigs challenged with porcine reproductive and respiratory syndrome virus.

BACKGROUND: At present, porcine reproductive and respiratory syndrome (PRRS) caused by the PRRS virus (PRRSV) is one of the most severe epidemics impacting pig farming globally. Despite the fact that a number of studies have been conducted on potential solutions to this problem, none have proven effective. The focus of problem solving is the use of natural ingredients such as plant extracts. Popular throughout Asia, Caesalpinia sappan (CS) is a therapeutic plant that inhibits PRRSV in vitro. Therefore, this study was performed to determine the efficacy of CS extract dietary supplementation on the productive performance, antibody levels, immunological indicators, and lung pathology of PRRSV-challenged weaned pigs. A total of 32 weaned piglets (28 days old) were randomized into 4 groups and kept separately for 14 days. The treatments were organized in a 2 × 2 factorial design involving two factors: PRRSV challenge and supplementation with 1 mg/kg CS extract. The pigs in the PRRSV-challenged groups were intranasally inoculated with 2 mL of PRRSV (VR2332) containing 104 TCID50/mL, while those in the groups not challenged with PRRSV were inoculated with 2 mL of normal saline. RESULTS: In the PRRSV-challenged group (CS + PRRSV), supplementation with CS extract led to an increase in white blood cells (WBCs) on Day 7 post infection (p < 0.05) and particularly in lymphocytes on Days 7 and 14. The antibody titer was significantly greater in the CS + PRRSV group than in the PRRSV-challenged group not administered CS (PRRSV group) on Day 14 postinfection (S/P = 1.19 vs. 0.78). In addition, CS extract administration decreased the prevalence of pulmonary lesions, which were more prevalent in the PRRSV-challenged pigs that did not receive the CS extract. CONCLUSION: The findings of this study suggest that supplementation with CS extract is beneficial for increasing WBC counts, especially lymphocytes, increasing the levels of antibodies and reducing the prevalence of lung lesions in PRRSV-infected pigs.