A porcine kidney-derived clonal cell line with clear genetic annotation is highly susceptible to African swine fever virus.

African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.

Generation and Characterization of Monoclonal Antibodies against Swine Acute Diarrhea Syndrome Coronavirus Spike Protein.

Swine acute diarrhea syndrome coronavirus (SADS-CoV), a member of the family Coronaviridae and the genus Alphacoronavirus, primarily affects piglets under 7 days old, causing symptoms such as diarrhea, vomiting, and dehydration. It has the potential to infect human primary and passaged cells in vitro, indicating a potential risk of zoonotic transmission. In this study, we successfully generated and purified six monoclonal antibodies (mAbs) specifically targeting the spike protein of SADS-CoV, whose epitope were demonstrated specificity to the S1A or S1B region by immunofluorescence assay and enzyme-linked immunosorbent assay. Three of these mAbs were capable of neutralizing SADS-CoV infection on HeLa-R19 and A549. Furthermore, we observed that SADS-CoV induced the agglutination of erythrocytes from both humans and rats, and the hemagglutination inhibition capacity and antigen-antibody binding capacity of the antibodies were assessed. Our study reveals that mAbs specifically targeting the S1A domain demonstrated notable efficacy in suppressing the hemagglutination phenomenon induced by SADS-CoV. This finding represents the first instance of narrowing down the protein region responsible for SADS-CoV-mediated hemagglutination to the S1A domain, and reveals that the cell attachment domains S1A and S1B are the main targets of neutralizing antibodies.

Glaesserella parasuis QseBC two-component system senses epinephrine and regulates capD expression.

IMPORTANCE: The key bacterial pathogen Glaesserella parasuis, which can cause Glässer's disease, has caused significant financial losses to the swine industry worldwide. Capsular polysaccharide (CPS) is an important virulence factor for bacteria, providing the ability to avoid recognition and killing by the host immune system. Exploring the alteration of CPS synthesis in G. parasuis in response to epinephrine stimulation can lay the groundwork for revealing the pathogenic mechanism of G. parasuis as well as providing ideas for Glässer's disease control.

Development and Implementation of a Quadruple RT-qPCR Method for the Identification of Porcine Reproductive and Respiratory Syndrome Virus Strains.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) causes porcine reproductive and respiratory syndrome (PRRS), leading to abortion in sows and respiratory distress in breeding pigs. In China, PRRSV1 and PRRSV2 are the two circulating genotypes in swine herds, with distinct virulence. PRRSV2 further consists of classical (C-PRRSV2), highly pathogenic (HP-PRRSV2), and NADC30-Like (N-PRRSV2) subtypes. The diversity of PRRSV poses challenges for control and eradication, necessitating reliable detection assays for differentiating PRRSV genotypes. METHODS: A new TaqMan-based RT-qPCR assay with four sets of primers and probes targeting conserved regions of the ORF7 and NSP2 genes of PRRSV was developed, optimized, and evaluated by us. Reaction conditions such as annealing temperature, primer concentration, and probe concentration were optimized for the assay. Specificity, sensitivity, repeatability, stability, limit of detection (LOD), concordance with the reference method were evaluated for the assay. RESULTS: The assay could detect and type PRRSV1, C-PRRSV2, HP-PRRSV2, and N-PRRSV2 simultaneously with 97.33% specificity, 96.00% sensitivity, 12 copies/µL LOD, 97.00% concordance with reference assays. We applied the assay to 321 clinical samples from swine farms in China. The assay successfully detected and typed 230 PRRSV-positive samples, with 24.78% (57/230) of them further confirmed by ORF5 gene sequencing. The prevalence of PRRSV subtypes among the positive samples was as follows: C-PRRSV2 (15.22%), HP-PRRSV2 (23.48%), and N-PRRSV2 (61.30%). Two samples showed coinfection with different PRRSV subtypes. CONCLUSION: The quadruple RT-qPCR assay is a powerful tool for detecting and typing the currently circulating PRRSV strains in Chinese swine populations. It can assist in the surveillance of PRRSV prevalence and the implementation of prevention and control strategies.

TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

HtrA is involved in stress response and adhesion in Glaesserella parasuis serovar 5 strain Nagasaki.

Glaesserella parasuis is an important pathogen that causes fibrinous polyserositis, peritonitis and meningitis in pigs, leading to considerable economic losses to the swine industry worldwide. It is well established that the serine protease HtrA is closely associated with bacterial virulence, but the role of HtrA in G. parasuis pathogenesis remains largely unknown. To characterize the function of the htrA gene in G. parasuis, a ΔhtrA mutant was constructed. We found that the ΔhtrA mutant showed significant growth inhibition under heat shock and alkaline stress conditions, indicating HtrA is involved in stress tolerance and survival of G. parasuis. In addition, deletion of htrA gene resulted in decreased adherence to PIEC and PK-15 cells and increased phagocytic resistance to 3D4/2 macrophages, suggesting that htrA is essential for adherence of G. parasuis. Scanning electron microscopy revealed morphological surface changes of the ΔhtrA mutant, and transcription analysis confirmed that a number of adhesion-associated genes are downregulated, which corroborated the aforementioned phenomenon. Furthermore, G. parasuis HtrA induced a potent antibody response in piglets with Glässer's disease. These observations confirmed that the htrA gene is related to the survival and pathogenicity of G. parasuis.

Porcine Epidemic Diarrhea Virus and Its nsp14 Suppress ER Stress Induced GRP78.

Porcine epidemic diarrhea virus (PEDV), a member of the α-coronavirus genus, can cause vomiting, diarrhea, and dehydration in piglets. Neonatal piglets infected with PEDV have a mortality rate as high as 100%. PEDV has caused substantial economic losses to the pork industry. Endoplasmic reticulum (ER) stress, which can alleviate the accumulation of unfolded or misfolded proteins in ER, involves in coronavirus infection. Previous studies have indicated that ER stress could inhibit the replication of human coronaviruses, and some human coronaviruses in turn could suppress ER stress-related factors. In this study, we demonstrated that PEDV could interact with ER stress. We determined that ER stress could potently inhibit the replication of GⅠ, GⅡ-a, and GⅡ-b PEDV strains. Moreover, we found that these PEDV strains can dampen the expression of the 78 kDa glucose-regulated protein (GRP78), an ER stress marker, while GRP78 overexpression showed antiviral activity against PEDV. Among different PEDV proteins, PEDV non-structural protein 14 (nsp14) was revealed to play an essential role in the inhibition of GRP78 by PEDV, and its guanine-N7-methyltransferase domain is necessary for this role. Further studies show that both PEDV and its nsp14 negatively regulated host translation, which could account for their inhibitory effects against GRP78. In addition, we found that PEDV nsp14 could inhibit the activity of GRP78 promotor, helping suppress GRP78 transcription. Our results reveal that PEDV possesses the potential to antagonize ER stress, and suggest that ER stress and PEDV nsp14 could be the targets for developing anti-PEDV drugs.

The in vitro antiviral activity of Lacticaseibacillus casei MCJ protein-based metabolites on bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is a ubiquitous immunosuppressive etiological agent which is economically important for a wide host range in the livestock industry. Lactobacillus spp. has widely been using in the field of management and treatment of gastro-enteric disease for both humans and animals. The ability of Lacticaseibacillus casei MCJ protein-based metabolized to suppress BVDV infection in Madin-Darby Bovine Kidney cell line was demonstrated in this study. The protein-based metabolites were extracted from the cultured L. casei to obtain the safest and beneficial form of the probiotic bacteria. It is revealed that LPM have no cytotoxic effect and the cell viability remain more than 80% even after the cells are treated with 3000 µg/mL of LPM. The results of the plaque formation assay showed that LPM can reduce the viral infection rate. To know the mechanism of LPM for anti-BVDV activity, MDBK cells were exposed to LPM before, after and co-incubation of virus infection. The co-treatment of LPM with BVDV revealed the best results. The results suggest that the LPM has a potential anti-BVDV activity which could be a prospective candidate for the prevention and control of BVDV infection in an animal.

Development and evaluation of polyclonal antibodies based antigen capture ELISA for detection of porcine rotavirus.

Rotaviruses are rising as zoonotic viruses worldwide, causing the lethal dehydrating diarrhea in children, piglets, and other livestock of economic importance. A simple, swift, cost-effective, highly specific, and sensitive antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) was developed for detection of porcine rotavirus-A (PoRVA) by employing rabbit (capture antibody) and murine polyclonal antibodies (detector antibody) produced against VP6 of PoRVA (RVA/Pig-tc/CHN/TM-a/2009/G9P23). Reactivity of the both polyclonal antibodies was confirmed by using an indirect ELISA, western-blot analysis and indirect fluorescence assay against rVP6 protein and PoRVA. The detection limit of AC-ELISA was found 50 ng/ml of PoRVA protein. The relative sensitivity and specificity of this in-house AC-ELISA were evaluated for detection of PoRVA from 295 porcine diarrhea samples, and results were compared with that of RT-PCR and TaqMan RT-qPCR. The relative sensitivity and specificity of AC-ELISA compared with those of TaqMan RT-qPCR were found as 94.4 and 99.2%, respectively, with the strong agreement (κ -0.58) between these two techniques. Furthermore, AC-ELISA could not detect any cross-reactivity with porcine epidemic diarrhea virus, transmissible gastro-enteritis virus, pseudo rabies virus and porcine circovirus-2. This in-house AC-ELISA efficiently detected PoRVA from clinical samples, which suggests that this technique can be used for large-scale surveillance and timely detection of rotavirus infection in the porcine farms.

In this study, we used a Chinese porcine rotavirus-A (PoRVA) strain containing the I5, a dominant VP6-genotype in pigs, for production of VP6 (most conserved) protein based polyclonal antibodies (pAb) in rabbits (as capture Ab) and mouse (as detector Ab) for development of simple, cost effective, highly specific and sensitive AC-ELISA for detection of PoRVA. Furthermore, there is no any previous published report on application of rabbit and mouse pAb against VP6 for developing an AC-ELISA against PoRVA.

Prevalence of Extended-Spectrum ß-Lactamase-Resistant Genes in Escherichia coli Isolates from Central China during 2016-2019.

The emergence and dissemination of Escherichia coli (E. coli) strains that produce extended-spectrum beta-lactamases (ESBLs) represents a major public health threat. The present study was designed to evaluate the prevalence and characteristics of ESBL-producing Escherichia coli isolates from chickens in central China during 2016-2019. A total of 407 E. coli strains isolated from 581 chicken swabs were identified conventionally and analyzed for various cephalosporin susceptibility by disk-diffusion assay. ESBL-producing strains were screened using the double=disk synergy test and ESBL-encoding genes were carried out by PCR/sequencing. A total of 402 E. coli isolates exhibited strong resistance to first- to fourth-generation cephalosporins and monobactam antibiotics, especially cefazolin (60.69%), cefuroxime (54.05%), cefepime (35.14%), ceftriaxone (54.30%), and aztreonam (40.29%). Piperacillin/tazobactam (1.72%) was the most effective drug against the strains, but the resistance rates increased each year. Among the isolates, 262 were identified as ESBL producers and the isolation rates for the ESBL producers increased from 63.37% to 67.35% over the four years. CTX-M (97.33%) was the most prevalent type, followed by TEM (76.72%) and SHV (3.05%). The most common ESBL genotype combination was blaTEM + blaCTX-M (74.46%), in which the frequency of carriers increased steadily, followed by blaCTX-M + blaSHV (3.05%). In addition, the most predominant specific CTX-M subtypes were CTX-M-55 (48.47%) and CTX-M-1 (17.94%), followed by CTX-M-14 (11.01%), CTX-M-15 (8.02%), CTX-M-9 (6.11%), CTX-M-65 (4.58%), and CTX-M-3 (1.15%). Moreover, a novel multiplex qPCR assay was developed to detect blaCTX-M, blaTEM, and blaSHV, with limits of detection of 2.06 × 101 copies/µL, 1.10 × 101 copies/µL, and 1.86 × 101 copies/µL, respectively, and no cross-reactivity with other ESBL genes and avian pathogens. The assays exhibited 100% sensitivity and specificities of 85%, 100%, and 100% for blaCTX-M, blaTEM, and blaSHV, respectively. In conclusion, our findings indicated that ESBL-producing E.coli strains isolated from chickens in central China were highly resistant to cephalosporins and frequently harbored diversity in ESBL-encoding genes. These isolates can pose a significant public health risk. The novel multiplex qPCR method developed in this study may be a useful tool for molecular epidemiology and surveillance studies of ESBL genes.

Duck TRIM35 Promotes Tembusu Virus Replication by Interfering with RIG-I-Mediated Antiviral Signaling in Duck Embryo Fibroblasts.

In China, the duck industry has been severely impacted by the newly emerging duck Tembusu virus (DTMUV). For DTMUV to successfully infect host cells, it employs several strategies that subvert the host's innate immune response. It has been found that several viral proteins encoded by DTMUV have strategically targeted the crucial molecules of the RIG-I-like Receptor (RLR) signaling pathway to antagonize host antiviral responses. However, it is not well known how the host proteins manipulated by DTMUV contribute to innate immune evasion. The present study reports that duck TRIM35 (duTRIM35) antagonizes DTMUV-induced innate immune responses by targeting duck RIG-I (duRIG-I) in duck embryo fibroblasts. A significant increase in duTRIM35 expression occurred during DTMUV infection. DuTRIM35 overexpression suppressed DTMUV-triggered expression of interferon beta (IFN-ß) and interferon-stimulated genes (ISGs), promoting viral replication, whereas knockdown of duTRIM35 augments the innate immune response, reducing viral replication. Furthermore, duTRIM35 significantly impaired the IFN-ß expression mediated by duRIG-I but not by other RLR signaling molecules. Mechanistically, duTRIM35 interfered with duRIG-I-duTRIM25 interaction and impeded duTRIM25-mediated duRIG-I ubiquitination by interacting with both duRIG-I and duTRIM25. Our findings indicate that duTRIM35 expression induced by DTMUV infection interfered with the duRIG-I-mediated antiviral response, illustrating a novel strategy in which DTMUV can evade the host's innate immunity. IMPORTANCE Duck Tembusu virus (DTMUV), an emerging flavivirus pathogen causing a substantial drop in egg production and severe neurological disorders in duck populations, has led to massive economic losses in the global duck industry. DTMUV has employed various strategies to subvert the host's innate immune response to establish a productive infection in host cells. In this study, we report that duck TRIM35 (duTRIM35) expression was upregulated upon DTMUV infection in vitro and in vivo, and its expression antagonized DTMUV-induced innate immune responses by targeting duck RIG-I (duRIG-I) in duck embryo fibroblasts. Further studies suggest that duTRIM35 interfered with duRIG-I-duTRIM25 interaction and impeded duTRIM25-mediated duRIG-I ubiquitination by interacting with both duRIG-I and duTRIM25. Together, these results revealed that duTRIM35 expression induced by DTMUV infection downregulated duRIG-I-mediated host antiviral response, which elucidated a novel strategy of DTMUV for innate immune evasion.

Epidemiological and genetic characteristics of porcine circovirus 3 in 15 provinces and municipalities of China between 2016 and 2020.

Porcine circovirus 3 (PCV3) is a newly emerging virus and has been found associated with porcine dermatitis and nephropathy syndrome in pigs. Compared with PCV2, research into PCV3 cap gene sequencing is deficient. To investigate the prevalence and genotype distribution of PCV3, we collected 1291 samples from 211 pig farms throughout 15 provinces and municipalities. 312 out of 1291 samples were tested positive by PCR. We further sequenced and analyzed 164 PCR-positive samples. The majority (61.8%) of isolates we sequenced belong to genotype PCV3c. PCV3c is also the dominant genotype in Hubei, Hunan, Hebei province and Chongqing city. We found 3 sites under positive selection and located in predicted epitope peptide, revealing that the pig's immunity may be a reason those sites are undergoing highly positive selection.

Investigation and analysis of etiology associated with porcine respiratory disease complex in China from 2017 to 2021.

Porcine respiratory diseases complex (PRDC) is a highly serious threat to the pig industry. In the present study, we investigated and analyzed the etiology associated with PRDC and explored the role of viruses in respiratory bacterial infections. From 2017 to 2021, clinical samples were collected from 1,307 pigs with typical respiratory symptoms in 269 farms in China and screened for pathogens related to PRDC by PCR and bacterial isolation. The results indicated that PRRSV (41.16%, 95%CI: 38.49~43.83%), PCV2 (21.58%,95%CI: 19.35~23.81%), S. suis (63.50%, 95%CI: 60.89~66.11%), and G. parasuis (28.54%, 95%CI: 26.09~30.99%) were the most commonly detected pathogens in pigs with PRDC in China. The dominant epidemic serotypes (serogroups) of S. suis, G. parasuis, and P. multocida were serotype 2, serotype 1, and capsular serogroups D, respectively. Pigs of different ages exhibited different susceptibilities to these pathogens, e.g., PRRSV, PCV2, and G. parasuis had the highest detection rates in nursery pigs, whereas fattening pigs had the highest detection rates of P. multocida and A. pleuropneumoniae. Among the 1,307 pigs, the detection rates of S. suis, G. parasuis, P. multocida, and B. bronchiseptica were higher in virus-positive pigs, especially G. parasuis and P. multocida were significantly (p < 0.01) higher than in virus-negative pigs. In addition, a strong positive correlation was found between coinfection by PRRSV and G. parasuis (OR = 2.33, 95%CI: 1.12~2.14), PRRSV and P. multocida (OR = 1.55, 95%CI: 1.12~2.14), PCV2 and P. multocida (OR = 2.27, 95%CI: 1.33~3.87), PRRSV-PCV2 and S. suis (OR = 1.83, 95%CI: 1.29~2.60), PRRSV-PCV2 and G. parasuis (OR = 3.39, 95%CI: 2.42~4.74), and PRRSV-PCV2 and P. multocida (OR = 2.09, 95%CI: 1.46~3.00). In summary, PRRSV, PCV2, S. suis, and G. parasuis were the major pathogens in pigs with PRDC, and coinfections of two or more PRDC-related pathogens with strong positive correlations were common in China, such as PRRSV and G. parasuis, PRRSV and P. multocida, PCV2 and P. multocida, and also PRRSV-PCV2 and G. parasuis and PRRSV-PCV2 and P. multocida.

Low-host double MDA workflow for uncultured ASFV positive blood and serum sample sequencing.

African swine fever (ASF) is a highly lethal and contagious disease caused by African swine fever virus (ASFV). Whole-genome sequencing of ASFV is necessary to study its mutation, recombination, and trace its transmission. Uncultured samples have a considerable amount of background DNA, which causes waste of sequencing throughput, storage space, and computing resources. Sequencing methods attempted for uncultured samples have various drawbacks. In this study, we improved C18 spacer MDA (Multiple Displacement Amplification)-combined host DNA exhaustion strategy to remove background DNA and fit NGS and TGS sequencing. Using this workflow, we successfully sequenced two uncultured ASFV positive samples. The results show that this method can significantly reduce the percentage of background DNA. We also developed software that can perform real-time base call and analyses in set intervals of ASFV TGS sequencing reads on a cloud server.

CpxAR of Actinobacillus pleuropneumoniae Contributes to Heat Stress Response by Repressing Expression of Type IV Pilus Gene apfA.

Acute pleuropneumonia in swine, caused by Actinobacillus pleuropneumoniae, is characterized by a high and sustained fever. Fever creates an adverse environment for many bacteria, leading to reduced bacterial proliferation; however, most pathogenic bacteria can tolerate higher temperatures. CpxAR is a two-component regulation system, ubiquitous among Gram-negative bacteria, which senses and responds to envelope alterations that are mostly associated with protein misfolding in the periplasm. Our previous study showed that CpxAR is necessary for the optimal growth of Actinobacillus pleuropneumoniae under heat stress. Here, we showed that mutation of the type IV pilin gene apfA rescued the growth defect of the cpxAR deletion strain under heat stress. RNA sequencing (RNA-seq) analyses revealed that 265 genes were differentially expressed in the ΔcpxAR strains grown at 42°C, including genes involved in type IV pilus biosynthesis. We also demonstrated direct binding of the CpxR protein to the promoter of the apf operon by an electrophoretic mobility shift assay and identified the binding site by a DNase I footprinting assay. In conclusion, our results revealed the important role of CpxAR in A. pleuropneumoniae resistance to heat stress by directly suppressing the expression of ApfA. IMPORTANCE Heat acts as a danger signal for pathogens, especially those infecting mammalian hosts in whom fever indicates infection. However, some bacteria have evolved exquisite mechanisms to survive under heat stress. Studying the mechanism of resistance to heat stress is crucial to understanding the pathogenesis of A. pleuropneumoniae during the acute stage of infection. Our study revealed that CpxAR plays an important role in A. pleuropneumoniae resistance to heat stress by directly suppressing expression of the type IV pilin protein ApfA.

Molecular evolution and genetic characteristics of G3P[3] group A canine rotavirus isolated in Wuhan, China.

Rotaviruses can infect multiple animal species and have the potential for cross-recombination based on the segmented genome characteristics. To study the intra-host recombination and zoonotic potential of group A canine rotavirus (CRV), 438 samples were collected from domestic dogs in six animal hospitals and from stray dogs from October 2019 to May 2021 in Wuhan, China. Seven of the samples were positive (7/438) for group A CRV from which a CRV strain was successfully isolated in MA-104 cells. The genotype of the isolated strain was characterized by whole-genome sequencing showing that the genotype was group A CRV G3P[3]. According to the Rotavirus Classification Working Group (RCWG), the genomic constellation of the isolated CRV was G3-P[3]-I3-R3-C3-M3-A9-N2-T3-E3-H6, which belongs to the AU-1-like group with gene segments of AU-1-like and Cat 97-like strains. Based on the phylogenetic analysis of the 11 gene segments, we found that the different segments of the isolated group A CRV were closely related to several reassortment rotaviruses from different animal sources and bat strains. Based on the analysis of the molecular evolution and genetic characteristics, we concluded that the isolated strain might be a reassortment strain. These data further enrich our understanding of rotavirus molecular evolution and genetic characteristics in China.

TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells.

Glaesserella parasuis is a common bacterium in the porcine upper respiratory tract that causes severe Glasser's disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis. TurboID is an enzyme that mediates the biotinylation of endogenous proteins that can fuse with proteins of interest to label protein interactors and local proteomes. To reveal the host proteins that interact with outer membrane protein P2 (OmpP2) by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage iPAM cells, 0.1 and 2.58 mg/mL His-tagged TurboID-OmpP2 and TurboID recombinant proteins were expressed and purified. By mass spectrometry, we identified 948 and 758 iPAM cell proteins that interacted with His-TurboID-OmpP2 and His-TurboID, respectively. After removal of background proteins through comparison with the TurboID-treated group, 240 unique interacting proteins were identified in the TurboID-OmpP2-treated group. Ultimately, only four membrane proteins were identified, CAV1, ARF6, PPP2R1A, and AP2M1, from these 240 host proteins. Our data indicated that CAV1, ARF6, and PPP2R1A could interact with OmpP2 of G. parasuis, as confirmed by coimmunoprecipitation assay. Finally, we found that CAV1, ARF6, and PPP2R1A were involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells by using overexpression and RNA interference assays. This study provides first-hand information regarding the interaction of the iPAM cell proteomes with G. parasuis OmpP2 protein by using the TurboID proximity labeling system and identifies three novel host membrane proteins involved in the recognition and phagocytosis of G. parasuis by iPAM cells. These results provide new insight for a better understanding of Glasser's disease pathogenesis. IMPORTANCE G. parasuis can cause serious Glasser's disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis in pigs. It can cause high morbidity and mortality in swine herds and major economic losses to the global pig industry. Understanding the mechanism of interactions between alveolar macrophages and pathogenic G. parasuis is essential for developing effective vaccines and targeted drugs against G. parasuis. To reveal the host proteins interacting with OmpP2 by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage (iPAM) cells, we identified 240 unique proteins from iPAM cells that could interact with G. parasuis OmpP2. Among them, only four membrane proteins, CAV1, ARF6, PPP2R1A, and AP2M1, were identified, and further study showed that CAV1, ARF6, and PPP2R1A are involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells. This study provides new insight into proteomic interactions between hosts and pathogenic microorganisms.

Transcriptome analysis of heat resistance regulated by quorum sensing system in Glaesserella parasuis.

The ability of bacteria to resist heat shock allows them to adapt to different environments. In addition, heat shock resistance is known for their virulence. Our previous study showed that the AI-2/luxS quorum sensing system affects the growth characteristics, biofilm formation, and virulence of Glaesserella parasuis. The resistance of quorum sensing system deficient G. parasuis to heat shock was obviously weaker than that of wild type strain. However, the regulatory mechanism of this phenotype remains unclear. To illustrate the regulatory mechanism by which the quorum sensing system provides resistance to heat shock, the transcriptomes of wild type (GPS2), ΔluxS, and luxS complemented (C-luxS) strains were analyzed. Four hundred forty-four differentially expressed genes were identified in quorum sensing system deficient G. parasuis, which participated in multiple regulatory pathways. Furthermore, we found that G. parasuis regulates the expression of rseA, rpoE, rseB, degS, clpP, and htrA genes to resist heat shock via the quorum sensing system. We further confirmed that rseA and rpoE genes exerted an opposite regulatory effect on heat shock resistance. In conclusion, the findings of this study provide a novel insight into how the quorum sensing system affects the transcriptome of G. parasuis and regulates its heat shock resistance property.

Development of a Genetically Engineered Bivalent Vaccine against Porcine Epidemic Diarrhea Virus and Porcine Rotavirus.

Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus that causes acute diarrhea, vomiting, dehydration, and a high mortality rate in neonatal piglets. In recent years, PEDV has been associated with co-infections with other swine enteric viruses, including porcine rotavirus (PoRV), resulting in increased mortality among newborn piglets. In this paper, we developed a bivalent vaccine against PEDV and PoRV by constructing a recombinant PEDV encoding PoRV VP7 (rPEDV-PoRV-VP7). The recombinant virus was constructed by replacing the entire open reading frame 3 (ORF3) in the genome of an attenuated PEDV strain YN150 with the PoRV VP7 gene using reverse genetic systems. Similar plaque morphology and replication kinetics were observed in Vero cells with the recombinant PEDV compared to the wild-type PEDV. It is noteworthy that the VP7 protein could be expressed stably in rPEDV-PoRV-VP7-infected cells. To evaluate the immunogenicity and safety of rPEDV-PoRV-VP7, 10-day-old piglets were vaccinated with the recombinant virus. After inoculation, no piglet displayed clinical symptoms such as vomiting, diarrhea, or anorexia. The PoRV VP7- and PEDV spike-specific IgG in serum and IgA in saliva were detected in piglets after rPEDV-PoRV-VP7 vaccination. Moreover, both PoRV and PEDV neutralizing antibodies were produced simultaneously in the inoculated piglets. Collectively, we engineered a recombinant PEDV expressing PoRV VP7 that could be used as an effective bivalent vaccine against PEDV and PoRV.