Respiratory Disease In Pigs Research Articles

Characterization of a Mycoplasma hyopneumoniae aerosol infection model in pigs

The purpose of the present study was to develop and characterize an experimental aerosol model for Mycoplasma hyopneumoniae (M. hyopneumoniae) infection and respiratory disease in pigs. The experiment was carried out to determine the pathogenicity, colonization, mucosal immune response, and clinical course of disease of dose-controlled aerosols of M. hyopneumoniae. Four groups of three M. hyopneumoniae-free gilts each were individually exposed to aerosols of diluted lung homogenate containing M. hyopneumoniae strain 232 in a chamber. Each group was exposed to different doses of viable organisms (105 to 106 color changing units/mL during 15-20 or 30-35min in two consecutive days). Nasal, laryngeal, and deep-tracheal secretions were collected from each gilt at 0, 7, 14, 21, and 28 days post-exposure (dpe). Blood samples were collected at 0 and 28 dpe. At necropsy, lung lesions were assessed, and bronchial secretions and bronchoalveolar lavage fluid (BALF) were collected from each lung set. Blood was used to assess seroconversion by means of an indirect ELISA, while BALF, deep-tracheal and nasal secretions were tested by modifying the ELISA to evaluate mucosal IgG and IgA production. Nasal, laryngeal, deep-tracheal, and bronchial secretions were tested by real-time PCR to evaluate bacterial load. Gilts became infected irrespective of the infectious dose, as determined by M. hyopneumoniae detection in deep-tracheal secretions from all gilts at 7 dpe. A specific local humoral immune response starting at 14 dpe was detected in all gilts. While all experimental groups presented gilts with some extent of mycoplasmal pneumonia, only the exposure of gilts to high-dose aerosols consistently reproduced typical clinical signs and severe lung lesions. These results showed that the reproduction of mycoplasmal pneumonia by means of infectious aerosols can be successfully achieved at experimental level, making this model a valuable alternative to evaluate preventive and treatment measures against M. hyopneumoniae.

Effect of vaccination against Mycoplasma hyopneumoniae on divergent pig genetic groups

The bacterium Mycoplasma hyopneumoniae (Mhp) causes a chronic infectious respiratory disease in pigs, leading to important economic losses. This study aimed to compare the immune response of the local Piau breed and a commercial line to Mhp vaccination. For this, two phases were carried out. In the first, gene expression of toll-like receptors (TLR2, TLR4, TLR6, and TLR10) and cytokines (IL2, IL6, IL8, IL10, IL12, IL13, TNFα, and TGFβ) was assessed in porcine blood mononuclear cells (PBMC) from the two genetic groups before and after vaccination. In the second experiment, nitric oxide production, specific antibodies, and gene expression of toll-like receptors and cytokines were evaluated in bronchoalveolar lavage fluid (BALF) cells of vaccinated and unvaccinated pigs. After vaccination against Mhp, TLR2, TLR4, TLR6, TLR10, IL6, TNFα, and TGFβ expression levels were elevated in PBMC from commercial animals, and TLR6, TLR10, and TGFβ expression levels were elevated in PBMC from the Piau group. Vaccination also increased the production of Mhp-specific IgG antibodies in BALF cells in the Piau breed. Comparison of the two genetic groups revealed differences in TNFα and IL10 expression in BALF cells. These results show that Piau pigs have different immune responses to vaccination compared with commercial animals. It is worth noting that these genetic differences between both genetic groups may be related to phenotypic differences in Mhp resistance or susceptibility.

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Structural definition of pseudorabies virus dUTPase reveals a novel folding dimer in the herpesvirus family

The pseudorabies virus (PRV) causes severe and fatal acute respiratory disease in pigs. During PRV proliferation, the enzyme deoxyuridine 5′-triphosphate nucleotide hydrolase (dUTPase) plays a pivotal role in maintaining a low dUTP/dTTP ratio, thereby ensuring the accuracy of viral DNA replication. However, its structure and catalytic mechanisms have not been fully elucidated. Here, we report the crystal structure of PRV dUTPase at a 2.24 Å resolution and demonstrate an unprecedented dimeric architecture, with a conserved enzyme activity center of the herpesvirus family. The enzyme activity center is located in a cavity between the two domains, forming a pocket for binding substrate dUMP and magnesium ions. Remarkably, the exquisite interface of the dimer is primarily composed of four antiparallel β-sheets, which form 11 hydrogen bonds between the residues P33-V36 and R242-A248 to maintain protein stability. To the best of our knowledge, this is the first report demonstrating that dUTPase exists as a dimer in the herpesvirus family. These findings not only present a novel fold dimeric structure but also deepen the scope of our comprehension of structural diversity in dUTPase family.

Rationalizing the use of common parameters and technological tools to follow up Mycoplasma hyopneumoniae infections in pigs

BackgroundMycoplasma (M.) hyopneumoniae is associated with respiratory disease in pigs and is the primary agent of enzootic pneumonia. Quantification of M. hyopneumoniae-related outcome parameters can be difficult, expensive, and time-consuming, in both research and field settings. In addition to well-established methods, technological tools are becoming available to monitor various aspects of relevant animal- and environment-related features, often in real-time. Therefore, this study aimed to assess whether certain parameters, such as animal movement and body temperature using microchips (IMT), correlate with established parameters and whether the currently used parameters can be rationalized.ResultsThe percentage of movement was significantly reduced by M. hyopneumoniae infection in pigs (p < 0.05), where the M. hyopneumoniae-infected group showed a lower percentage of movement (1.9%) when compared to the negative control group (6.9%). On the other hand, macroscopic (MLCL) and microscopic (MLL) lung lesions, respiratory disease score (RDS), M. hyopneumoniae-DNA load, and anti-M. hyopneumoniae antibody levels increased significantly in the M. hyopneumoniae-infected group 28 days post-inoculation (p < 0.05). Moderate (r > 0.30) to very strong correlations (> 0.80) were observed between the abovementioned parameters (p < 0.05), except for IMT. A significant and moderate correlation was reported between IMT and rectal temperature (r = 0.49; p < 0.05). Last, the average daily weight gain and the percentage of air in the lung were not affected by M. hyopneumoniae infection (p > 0.05).ConclusionsM. hyopneumoniae infection significantly reduced the movement of piglets and increased lung lesions, M. hyopneumoniae-DNA load, and anti-M. hyopneumoniae antibody levels; and, good correlations were observed between most parameters, indicating a direct relationship between them. Thus, we suggest that changes in movement might be a reliable indicator of M. hyopneumoniae infection in pigs, and that a selected group of parameters—specifically RDS, MLCL, MLL, M. hyopneumoniae-DNA load, anti-M. hyopneumoniae antibody levels, and movement—are optimal to assess M. hyopneumoniae infection under experimental conditions.

The Reproduction Number of Swine Viral Respiratory Diseases: A Systematic Review.

Diseases in the swine industry can cause significant economic and health impacts. This review examines R0 estimates for respiratory diseases in pigs, assessing variations and comparing transmission risks within and between farms. A literature search of three databases aggregated peer-reviewed research articles on swine viral respiratory diseases' R0 values. The study focused on seven diseases: Aujeszky's disease (AD), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Circovirus, Influenza A (IA), Encephalomyocarditis Virus (EV), Classical Swine Fever (CSF), and African Swine Fever (ASF). R0 values were estimated for transmission within and between herds/farms using various methods, from complex mathematical models to simple calculations. Data primarily came from disease surveillance and laboratory experiments. The median R0 for within-herd and between-herd transmission was 10 and 3.31 for AD, 2.78 and 1.14 for PRRSV, 5.9 and 0.89 for Circovirus, 1.75 and 1.6 for CSF, and 3.94 and 3.15 for ASF. For IA and EV, only within-herd R0 values were estimated at 8.65 and 1.3, respectively. Diseases with high R0 values highlight the need for prompt detection and response to outbreaks. Continuous monitoring and evaluation of pathogen transmissibility are crucial for enhancing disease surveillance and reducing the impact of livestock diseases.

Evaluation of Pulmonary Lesions in Slaughtered Fatteners as Indicators of Respiratory Diseases in Pigs

Evaluation of Pulmonary Lesions in Slaughtered Fatteners as Indicators of Respiratory Diseases in Pigs

Antimicrobial Resistance and Biofilm Formation of Bordetella bronchiseptica in Central China, with Evidence of a Rare Heteroresistance Strain to Gentamicin.

Bordetella bronchiseptica is a significant contributor to respiratory disease in pigs, leading to substantial economic losses in the swine industry worldwide. We isolated 52 B. bronchiseptica strains from 542 samples collected from pigs with atrophic rhinitis and bronchopneumonia in central China. Multi-locus sequence typing identified two prevalent sequence types: ST6 (69.23%) and ST7 (30.77%). PCR-based detection of seven virulence genes (fhaB, prn, cyaA, dnt, bteA, fla, and bfrZ) revealed that six of these genes were present in over 90% of the isolates, with bfrZ being the exception at 59.62%. Antimicrobial susceptibility testing, performed using the K-B method, demonstrated high sensitivity to enrofloxacin, polymyxin, and doxycycline but a notable resistance to tylosin, trimethoprim, tobramycin, ciprofloxacin, and amikacin. Remarkably, 86.54% of the isolates exhibited a multidrug-resistant phenotype. Notably, we successfully screened a strain of B. bronchiseptica with a heteroresistance phenotype to gentamicin using population analysis profiling, which is a rare case. Biofilm-formation assays indicated that 96.15% of the isolates possessed biofilm-forming capabilities. These findings provide crucial insights into the prevalence of B. bronchiseptica in central China, facilitating the development of effective preventive measures to safeguard both animal and human health.

Innate Immune Evasion of PRRSV nsp11 through Degradation of the HDAC2 by Its Endoribonuclease Activity.

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the Arteriviridae family, represents a persistent menace to the global pig industry, causing reproductive failure and respiratory disease in pigs. In this study, we delved into the role of histone deacetylases (HDAC2) during PRRSV infection. Our findings revealed that HDAC2 expression is downregulated upon PRRSV infection. Notably, suppressing HDAC2 activity through specific small interfering RNA led to an increase in virus production, whereas overexpressing HDAC2 effectively inhibited PRRSV replication by boosting the expression of IFN-regulated antiviral molecules. Furthermore, we identified the virus's nonstructural protein 11 (nsp11) as a key player in reducing HDAC2 levels. Mutagenic analyses of PRRSV nsp11 revealed that its antagonistic effect on the antiviral activity of HDAC2 is dependent on its endonuclease activity. In summary, our research uncovered a novel immune evasion mechanism employed by PRRSV, providing crucial insights into the pathogenesis of this virus and guiding the development of innovative prevention strategies against PRRSV infection.

Simultaneous Detection of Porcine Respiratory Coronavirus, Porcine Reproductive and Respiratory Syndrome Virus, Swine Influenza Virus, and Pseudorabies Virus via Quadruplex One-Step RT-qPCR.

Porcine respiratory coronavirus (PRCoV), porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza virus (SIV), and pseudorabies virus (PRV) are significant viruses causing respiratory diseases in pigs. Sick pigs exhibit similar clinical symptoms such as fever, cough, runny nose, and dyspnea, making it very difficult to accurately differentially diagnose these diseases on site. In this study, a quadruplex one-step reverse-transcription real-time quantitative PCR (RT-qPCR) for the detection of PRCoV, PRRSV, SIV, and PRV was established. The assay showed strong specificity, high sensitivity, and good repeatability. It could detect only PRCoV, PRRSV, SIV, and PRV, without cross-reactions with TGEV, PEDV, PRoV, ASFV, FMDV, PCV2, PDCoV, and CSFV. The limits of detection (LODs) for PRCoV, PRRSV, SIV, and PRV were 129.594, 133.205, 139.791, and 136.600 copies/reaction, respectively. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 0.29% to 1.89%. The established quadruplex RT-qPCR was used to test 4909 clinical specimens, which were collected in Guangxi Province, China, from July 2022 to September 2023. PRCoV, PRRSV, SIV, and PRV showed positivity rates of 1.36%, 10.17%, 4.87%, and 0.84%, respectively. In addition, the previously reported RT-qPCR was also used to test these specimens, and the agreement between these methods was higher than 99.43%. The established quadruplex RT-qPCR can accurately detect these four porcine respiratory viruses simultaneously, providing an accurate and reliable detection technique for clinical diagnosis.

Unveiling Shared Immune Responses in Porcine Alveolar Macrophages during ASFV and PRRSV Infection Using Single-Cell RNA-seq.

African swine fever virus (ASFV) and porcine reproductive and respiratory syndrome virus (PRRSV) infections lead to severe respiratory diseases in pigs, resulting in significant economic losses for the global swine industry. While numerous studies have focused on specific gene functions or pathway activities during infection, an investigation of shared immune responses in porcine alveolar macrophages (PAMs) after ASFV and PRRSV infections was lacking. In this study, we conducted a comparison using two single-cell transcriptomic datasets generated from PAMs under ASFV and PRRSV infection. Pattern recognition receptors (PRRs) RIG-I (DDX58), MDA5 (IFIH1), and LGP2 (DHX58) were identified as particularly recognizing ASFV and PRRSV, triggering cellular defense responses, including the upregulation of four cytokine families (CCL, CXCL, IL, and TNF) and the induction of pyroptosis. Through weighted gene co-expression network analysis and protein-protein interaction analysis, we identified thirteen gene and protein interactions shared by both scRNA-seq analyses, suggesting the ability to inhibit both ASFV and PRRSV viral replication. We discovered six proteins (PARP12, PARP14, HERC5, DDX60, RSAD2, and MNDA) in PAMs as inhibitors of ASFV and PRRSV replication. Collectively, our findings showed detailed characterizations of the immune responses in PAMs during ASFV and PRRSV infections, which may facilitate the treatments of these viral diseases.

Monitoring using artificial intelligence reveals critical links between housing conditions and respiratory health in pigs

Respiratory disease is the most significant issue in the pig industry and leads to high mortality and reduced growth rates. Housing conditions are crucial for maintaining the health and welfare of animals to optimize growth rates. This study aimed to evaluate the respiratory health status of pigs raised in different housing conditions using artificial intelligence (AI) technology and to determine the relationships between respiratory health and environmental factors. Eighty growing pigs (Largewhite × Landrace) × Duroc) were randomly distributed into two housing conditions: the control group, which had normal housing conditions, and the treatment group, which had poor housing conditions. A two-sample t test was performed to compare the groups, and a cross-correlation analysis was conducted to determine associations between respiratory health and days of exposure to environmental factors. The results show significant (p &lt; 0.001) lower incidence of respiratory health status along with a significant (p &lt; 0.001) greater frequency of coughing in pigs raised under poor housing conditions. Respiratory health is negatively associated with high exposure to temperature, relative humidity, and CO2 at lags 0 (R = -0.873, -0.887, and -0.869, respectively), -1 (R = -0.708, -0.742, and -0.705, respectively), and -2 (R = -0.545, -0.590, and -0.599, respectively). Furthermore, respiratory health was negatively associated with high exposure to NH3 (R = -0.420) at lag 0. In conclusion, AI technology is an effective tool for monitoring respiratory health. However, further evaluation and calibration of the alarm system threshold are recommended. These findings suggest that maintaining proper control of environmental factors can help prevent respiratory diseases in 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-β, 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.

CLONING AND EXPRESSION OF GENE ENCODING P46 ANTIGEN OF MYCOPLASMA HYOPNEUMONIAE IN ESCHERICHIA COLI

Enzootic pneumonia is a highly contagious chronic respiratory disease in pigs caused by Mycoplasma hyopneumoniae (M. hyopneumoniae) worldwide. P46 of M. hyopneumoniae is considered one of the main surface antigen proteins. This study aims to successfully clone and express P46 of M. hyopneumoniae in Escherichia coli M15 cells. The gene encoding P46 of M. hyopneumoniae was isolated from lung samples of infected pigs raised in Thua Thien Hue province, Vietnam, and cloned into vector pGEM®-T Easy. The PCR product digested by KpnI and BamHI enzymes was cloned into the pQE30 and expressed in E. coli M15 cells. The results of sequencing show that the nucleotide sequence of the cloned gene encoding P46 antigen protein has a length of 456 bp, corresponding to 152 amino acid residues and 99.34% similarity to the polypeptide chain of recorded M. hyopneumoniae P46 protein in GenBank (accession number: AAZ53879.1). The results of sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) analysis show that the molecular mass of the expressed protein is approximately 20 kDa, and anti-histag ELISA confirms that the expressed protein is the 6xHis-P46 recombinant protein.

PLK3 facilitates replication of swine influenza virus by phosphorylating viral NP protein

ABSTRACT Swine H1N1/2009 influenza is a highly infectious respiratory disease in pigs, which poses a great threat to pig production and human health. In this study, we investigated the global expression profiling of swine-encoded genes in response to swine H1N1/2009 influenza A virus (SIV-H1N1/2009) in newborn pig trachea (NPTr) cells. In total, 166 genes were found to be differentially expressed (DE) according to the gene microarray. After analyzing the DE genes which might affect the SIV-H1N1/2009 replication, we focused on polo-like kinase 3 (PLK3). PLK3 is a member of the PLK family, which is a highly conserved serine/threonine kinase in eukaryotes and well known for its role in the regulation of cell cycle and cell division. We validated that the expression of PLK3 was upregulated after SIV-H1N1/2009 infection. Additionally, PLK3 was found to interact with viral nucleoprotein (NP), significantly increased NP phosphorylation and oligomerization, and promoted viral ribonucleoprotein assembly and replication. Furthermore, we identified serine 482 (S482) as the phosphorylated residue on NP by PLK3. The phosphorylation of S482 regulated NP oligomerization, viral polymerase activity and growth. Our findings provide further insights for understanding the replication of influenza A virus.

Retrospective Analysis of the Detection of Pathogens Associated with the Porcine Respiratory Disease Complex in Routine Diagnostic Samples from Austrian Swine Stocks.

The diagnostic workup of respiratory disease in pigs is complex due to coinfections and non-infectious causes. The detection of pathogens associated with respiratory disease is a pivotal part of the diagnostic workup for respiratory disease. We aimed to report how frequently certain viruses and bacteria were detected in samples from pigs with respiratory symptoms in the course of routine diagnostic procedures. Altogether, 1975 routine diagnostic samples from pigs in Austrian swine stocks between 2016 and 2021 were analysed. PCR was performed to detect various pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) (n = 921), influenza A virus (n = 479), porcine circovirus type 2 (PCV2) (n = 518), Mycoplasma (M.) hyopneumoniae (n = 713), Actinobacillus pleuropneumoniae (n = 198), Glaesserella (G.) parasuis (n = 165) and M. hyorhinis (n = 180). M. hyorhinis (55.1%) had the highest detection rate, followed by PCV2 (38.0%) and Streptococcus (S.) suis (30.6%). PRRSV was detected most frequently in a pool of lung, tonsil and tracheobronchial lymph node (36.2%). G. parasuis was isolated more frequently from samples taken after euthanasia compared to field samples. PRRSV-positive samples were more likely to be positive for PCV2 (p = 0.001), M. hyopneumoniae (p = 0.032) and Pasteurella multocida (p < 0.001). M. hyopneumoniae-positive samples were more likely to be positive for P. multocida (p < 0.001) and S. suis (p = 0.046), but less likely for M. hyorhinis (p = 0.004). In conclusion, our data provide evidence that lung samples that were positive for a primary pathogenic agent were more likely to be positive for a secondary pathogenic agent.

Immunological profile of mice immunized with a polyvalent virosome-based influenza vaccine

BackgroundInfluenza A virus (IAV) causes respiratory disease in pigs and is a major concern for public health. Vaccination of pigs is the most successful measure to mitigate the impact of the disease in the herds. Influenza-based virosome is an effective immunomodulating carrier that replicates the natural antigen presentation pathway and has tolerability profile due to their purity and biocompatibility.MethodsThis study aimed to develop a polyvalent virosome influenza vaccine containing the hemagglutinin and neuraminidase proteins derived from the swine IAVs (swIAVs) H1N1, H1N2 and H3N2 subtypes, and to investigate its effectiveness in mice as a potential vaccine for swine. Mice were immunized with two vaccine doses (1 and 15 days), intramuscularly and intranasally. At 21 days and eight months later after the second vaccine dose, mice were euthanized. The humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with a polyvalent influenza virosomal vaccine were investigated.ResultsOnly intramuscular vaccination induced high hemagglutination inhibition (HI) titers. Seroconversion and seroprotection (> 4-fold rise in HI antibody titers, reaching a titer of ≥ 1:40) were achieved in 80% of mice (intramuscularly vaccinated group) at 21 days after booster immunization. Virus-neutralizing antibody titers against IAV were detected at 8 months after vaccination, indicating long-lasting immunity. Overall, mice immunized with the virosome displayed greater ability for B, effector-T and memory-T cells from the spleen to respond to H1N1, H1N2 and H3N2 antigens.ConclusionsAll findings showed an efficient immune response against IAVs in mice vaccinated with a polyvalent virosome-based influenza vaccine.

PSMB4 Degrades the Porcine Reproductive and Respiratory Syndrome Virus Nsp1α Protein via the Autolysosome Pathway and Induces the Production of Type I Interferon.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory disease in pigs of all ages and reproductive failure in sows, resulting in great economic losses to the swine industry. In this work, we identified the interaction between PSMB4 and PRRSV Nsp1α by yeast two-hybrid screening. The PSMB4-Nsp1α interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pulldown, and laser confocal experiments. The PCPα domain (amino acids 66 to 166) of Nsp1α and the C-terminal domain (amino acids 250 to 264) of PSMB4 were shown to be critical for the PSMB4-Nsp1α interaction. PSMB4 overexpression reduced PRRSV replication, whereas PSMB4 knockdown elicited opposing effects. Mechanistically, PSMB4 targeted K169 in Nsp1α for K63-linked ubiquitination and targeted Nsp1α for autolysosomal degradation by interacting with LC3 to enhance the activation of the lysosomal pathway. Meanwhile, we found that PSMB4 activated the NF-κB signaling pathway to produce type I interferons by downregulating the expression of IκBα and p-IκBα. In conclusion, our data revealed a new mechanism of PSMB4-mediated restriction of PRRSV replication, whereby PSMB4 was found to induce Nsp1α degradation and type I interferon expression, in order to impede the replication of PRRSV. IMPORTANCE In the swine industry, PRRSV is a continuous threat, and the current vaccines are not effective enough to block it. This study determined that PSMB4 plays an antiviral role against PRRSV. PSMB4 was found to interact with PRRSV Nsp1α, mediate K63-linked ubiquitination of Nsp1α at K169, and thus trigger its degradation via the lysosomal pathway. Additionally, PSMB4 activated the NF-κB signaling pathway to produce type I interferons by downregulating the expression of IκBα and p-IκBα. This study extends our understanding of the proteasome subunit PSMB4 against PRRSV replication and will contribute to the development of new antiviral strategies.

Systematic Identification and Comparison of the Expressed Profiles of Exosomal MiRNAs in Pigs Infected with NADC30-like PRRSV Strain.

Exosomes are biological vesicles secreted and released by cells that act as mediators of intercellular communication and play a unique role in virus infection, antigen presentation, and suppression/promotion of body immunity. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most damaging pathogens in the pig industry and can cause reproductive disorders in sows, respiratory diseases in pigs, reduced growth performance, and other diseases leading to pig mortality. In this study, we used the PRRSV NADC30-like CHsx1401 strain to artificially infect 42-day-old pigs and isolate serum exosomes. Based on high-throughput sequencing technology, 305 miRNAs were identified in serum exosomes before and after infection, among which 33 miRNAs were significantly differentially expressed between groups (13 relatively upregulated and 20 relatively downregulated). Sequence conservation analysis of the CHsx1401 genome identified 8 conserved regions, of which a total of 16 differentially expressed (DE) miRNAs were predicted to bind to the conserved region closest to the 3' UTR of the CHsx1401 genome, including 5 DE miRNAs capable of binding to the CHsx1401 3' UTR (ssc-miR-34c, ssc-miR-375, ssc-miR-378, ssc-miR-486, ssc-miR-6529). Further analysis revealed that the target genes of differentially expressed miRNAs were widely involved in exosomal function-related and innate immunity-related signaling pathways, and 18 DE miRNAs (ssc-miR-4331-3p, ssc-miR-744, ssc-miR-320, ssc-miR-10b, ssc-miR-124a, ssc-miR-128, etc.) associated with PRRSV infection and immunity were screened as potential functional molecules involved in the regulation of PRRSV virus infection by exosomes.

Development and laboratory evaluation of a competitive ELISA for serodiagnosis of Nipah and Hendra virus infection using recombinant Nipah glycoproteins and a monoclonal antibody.

Nipah virus (NiV) and Hendra virus (HeV), of the genus Henipavirus, family Paramyxoviridae, are classified as Risk Group 4 (RG4) pathogens that cause respiratory disease in pigs and acute/febrile encephalitis in humans with high mortality. A competitive enzyme-linked immunosorbent assay (cELISA) using a monoclonal antibody (mAb) and recombinant NiV glycoprotein (G) was developed and laboratory evaluated using sera from experimental pigs, mini pigs and nonhuman primates. The test depends on competition between specific antibodies in positive sera and a virus-specific mAb for binding to NiV-G. Based on 1,199 negative and 71 NiV positive serum test results, the cutoff value was determined as 35% inhibition. The diagnostic sensitivity and specificity of the NiV cELISA was 98.58 and 99.92%, respectively. When testing sera from animals experimentally infected with NiV Malaysia, the cELISA detected antibodies from 14 days post-infection (dpi) and remained positive until the end of the experiment (28 dpi). Comparisons using the Kappa coefficient showed strong agreement (100%) between the cELISA and a plaque reduction neutralization test (PRNT). Because our cELISA is simpler, faster, and gives comparable or better results than PRNT, it would be an adequate screening test for suspect NiV and HeV cases, and it would also be useful for epidemiological surveillance of Henipavirus infections in different animal species without changing reagents.