Development of an Indirect ELISA to Distinguish between Porcine Sapelovirus-Infected and -Vaccinated Animals Using the Viral Nonstructural Protein 3AB.

Porcine sapelovirus (PSV) is a new pathogen that negatively impacts the pig industry in China. Affected pigs experience severe diarrhea and even death. Vaccination is used to control disease outbreaks, and sensitive diagnostic methods that can distinguish infected animals from vaccinated animals (DIVA) are essential for monitoring the effectiveness of disease control programs. Tests based on the detection of the nonstructural protein (NSP) 3AB are reliable indicators of viral replication in infected and vaccinated animals. In this study, the recombinant PSV 3AB protein was expressed by a prokaryotic expression system, and an indirect ELISA method was established. Serum samples from healthy animals, immunized animals, and infected animals were evaluated. The ELISA method identified 3AB with high sensitivity (99.78%) and specificity (100.0%), and no cross-reaction was observed with serum antibodies against porcine reproductive and respiratory syndrome virus (PRRSV), infection with classical swine fever virus (CSFV), pseudorabies virus (PRV), bovine viral diarrhea virus (BVDV), porcine epidemic diarrhea virus (PEDV), or foot-and-mouth disease virus (FMDV). The ELISA method described here can effectively distinguish infected and vaccinated animals and is an important inexpensive tool for monitoring serum and controlling PSV.

Coinfection with PEDV and BVDV induces inflammatory bowel disease pathway highly enriched in PK-15 cells.

BACKGROUND: From the 1078 diarrhea stools tested in our survey from 2017 to 2020 in local area of China, PEDV was the key pathogen that was closely related to the death of piglets with diarrhea. In addition, coinfection of PEDV-positive samples with BVDV reached 17.24%. Although BVDV infection in swine is typically subclinical, the effect of PEDV and BVDV coinfection on disease severity and the potential molecular mechanism of coinfection with these two viruses remain unknown. METHODS: In this study, we developed a model of coinfection with porcine epidemic diarrhea virus (PEDV) and bovine viral diarrhea virus (BVDV) in PK15 cells, and a tandem mass tag (TMT) combined with LC-MS/MS proteomic approach was used to identify differential protein expression profiles. Additionally, we performed drug experiments to explore the inflammatory response induced by PEDV or BVDV mono- or coinfection. RESULTS: A total of 1094, 1538, and 1482 differentially expressed proteins (DEPs) were identified upon PEDV monoinfection, BVDV monoinfection and PEDV/BVDV coinfection, respectively. KEGG pathway analysis revealed that PEDV and BVDV coinfection led to a highly significantly enrichment of the inflammatory bowel disease (IBD) pathway. In addition, the NF-κB signaling pathway was more intensively activated by PEDV and BVDV coinfection, which induced higher production of inflammatory cytokines, than PEDV or BVDV monoinfection. CONCLUSIONS: Our study indicated that cattle pathogens might play synergistic roles in the pathogenesis of porcine diarrhea, which might also improve our understanding of the pathogenesis of multiple infections in diarrhea.

Swine influenza virus triggers ferroptosis in A549 cells to enhance virus replication.

BACKGROUND: Recently, Influenza A virus (IAV) has been shown to activate several programmed cell death pathways that play essential roles in host defense. Indeed, cell death caused by viral infection may be mediated by a mixed pattern of cell death instead of a certain single mode. Ferroptosis is a novel form of regulated cell death (RCD) that is mainly mediated by iron-dependent lipid peroxidation. Based on the proteomic data, we wondered whether IAV causes ferroptosis in host cells. METHOD: In this study, a quantitative proteomics approach based on an iTRAQ combined with LC-MS/MS was used to profile proteins expressed in A549 cells infected with H1N1 swine influenza virus (SIV). Meanwhile, we measured the intracellular iron content, reactive oxygen species (ROS) release and lipid peroxidation in response to SIV infection. Finally, a drug experiment was conducted to investigate the effects of ferroptosis on modulating SIV survival. RESULTS: The bioinformatics analysis revealed several proteins closely relevant to iron homeostasis and transport, and the ferroptosis signaling pathway are highly enriched in response to SIV infection. In our experiment, aberrant expression of iron-binding proteins disrupted labile iron uptake and storage after SIV infection. Meanwhile, SIV infection inhibited system the Xc-/GPX4 axis resulting in GSH depletion and the accumulation of lipid peroxidation products. Notably, cell death caused by SIV as a result of iron-dependent lipid peroxidation can be partially rescued by ferroptosis inhibitor. Additionally, blockade of the ferroptotic pathway by ferrostatin-1 (Fer-1) treatment decreased viral titers and inflammatory response. CONCLUSIONS: This study revealed a new mode of cell death induced by IAV infection, and our findings might improve the understanding of the underlying mechanism involved in the interaction of virus and host cells.

Molecular characterization of a porcine sapelovirus strain isolated in China.

Porcine sapelovirus (PSV) infections have been associated with a wide spectrum of symptoms, ranging from asymptomatic infection to clinical signs including diarrhoea, pneumonia, reproductive disorders, and polioencephalomyelitis. Although it has a global distribution, there have been relatively few studies on PSV in domestic animals. We isolated a PSV strain, SHCM2019, from faecal specimens from swine, using PK-15 cells. To investigate its molecular characteristics and pathogenicity, the genomic sequence of strain SHCM2019 was analysed, and clinical manifestations and pathological changes occurring after inoculation of neonatal piglets were observed. The virus isolated using PK-15 cells was identified as PSV using RT-PCR, transmission electron microscopy (TEM), and immunofluorescence assay (IFA). Sequencing results showed that the full-length genome of the SHCM2019 strain was 7,567 nucleotides (nt) in length, including a 27-nucleotide poly(A) tail. Phylogenetic analysis demonstrated that this virus was a PSV isolate belonging to the Chinese strain cluster. Recombination analysis indicated that there might be a recombination breakpoint upstream of the 3D region of the genome. Pathogenicity experiments demonstrated that the virus isolate could cause diarrhoea and pneumonia in piglets. In breif, a recombinant PSV strain, SHCM2019, was isolated and shown to be pathogenic. Our results may provide a reference for future research on the pathogenic mechanism and evolutionary characteristics of PSV.

Development and neutralization analysis of recombinant BVDVs expressing a CSF single chain antibody in vitro.

Although the immunization against swine fever (SF) is compulsory in China, it has still emerged in several areas at times. Herein, this study was conducted to develop an antibody vaccine which can clear the classical swine fever virus (CSFV) immediately after the pathogen invasion. Bovine viral diarrhoea virus (BVDV) infectious cDNA clone pASH28 was used to express a single-chain fragment variable (scFv) antibody against CSFV (CSFV/scFv) by reverse genetic technique. CSFV/scFv was inserted at the N-terminus of the C or Erns gene, generating two rBVDVs (rBVDV/C-CSFV/scFv and rBVDV/Erns-CSFV/scFv). Although both the rBVDVs could stably propagate on MDBK cells, different cellular characteristics existed. Obvious green fluorescence against the CSFV/scFv antibody could be visual on the cytomembrane or outside of the cells infected with rBVDV/Erns-CSFV/scFv, while much weaker fluorescence was observed in rBVDV/C-CSFV/scFv - infected cells. The CSFV/scFv antibodies induced by the two rBVDVs could recognize CSFV, but the rBVDV/Erns-CSFV/scFv induced stronger viral neutralization reaction. It was speculated that the neutralization activity might be associated with the expression location of CSFV/scFv antibody. The datas in this study provide evidence that rBVDV/Erns-CSFV/scFv may be engineered as a new antibody vaccine candidate against CSFV in the future.

Current-Density Regulating Lithium Metal Directional Deposition for Long Cycle-Life Li Metal Batteries.

Uncontrolled dendrite formation in the high energy density of lithium (Li) metal batteries (LMBs) may pose serious safety risks. While numerous studies have attempted to protect separators, these proposed methods fail to effectively inhibit upward dendrite growth that punctures through the separator. Here, we introduce a novel "orientated-growth" strategy that transfers the main depositional interface to the anode/current collector interface from the anode/separator interface. We placed a layer of cellulose/graphene carbon composite aerogel (CCA) between the current collector and the anode (LCL-bottom). This layer works as a charge organizer that induces a high current density and encourages Li to deposit at the anode/current collector interface. Both in situ and ex situ images of the electrode demonstrate that the anode part of the cell has been flipped; with the newly deposited particles facing the current collector and the smooth surface facing the separator. The electrode in half and full cells showed outstanding cyclic stability and rate capability, with the LCL-bottom/LFP full cell capable of maintaining 94 % of its initial capacity after 1000 cycles.

The tyrosine 73 and serine 83 dephosphorylation of H1N1 swine influenza virus NS1 protein attenuates virus replication and induces high levels of beta interferon.

BACKGROUND: Nonstructural protein 1 (NS1) is a virulence factor encoded by influenza A virus (IAV) that is expressed in the nucleus and cytoplasm of host cells during the earliest stages of infection. NS1 is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. However, to date, the phosphorylation sites of NS1 have not been identified, and the relationship between phosphorylation and protein function has not been thoroughly elucidated. METHOD: In this study, potential phosphorylation sites in the swine influenza virus (SIV) NS1 protein were bioinformatically predicted and determined by Phos-tag SDS-PAGE analysis. To study the role of NS1 phosphorylation sites, we rescued NS1 mutants (Y73F and S83A) of A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability, cytokine production as well as the intracellular localization in cultured cells. Additionally, we used small interfering RNA (siRNA) assay to explore whether changes in the type I IFN response with dephosphorylation at positions 73 and 83 were mediated by the RIG-I pathway. RESULTS: We checked 18 predicted sites in 30 SIV NS1 genes to exclude strain-specific sites, covering H1N1, H1N2 and H3N2 subtypes and identified two phosphorylation sites Y73 and S83 in the H1N1 SIV protein by Phos-tag SDS-PAGE analysis. We found that dephosphorylation at positions 73 and 83 of the NS1 protein attenuated virus replication and reduced the ability of NS1 to antagonize IFN-ß expression but had no effect on nuclear localization. Knockdown of RIG-I dramatically impaired the induction of IFN-ß and ISG56 in NS1 Y73F or S83A mutant-infected cells, indicating that RIG-I plays a role in the IFN-ß response upon rSIV NS1 Y73F and rSIV NS1 S83A infection. CONCLUSION: We first identified two functional phosphorylation sites in the H1N1 SIV protein: Y73 and S83. We found that dephosphorylation at positions 73 and 83 of the NS1 protein affected the antiviral state in the host cells, partly through the RIG-I pathway.

Effects of the S42 residue of the H1N1 swine influenza virus NS1 protein on interferon responses and virus replication.

BACKGROUND: The influenza A virus non-structural protein 1 (NS1) is a multifunctional protein that plays an important role in virus replication, virulence and inhibition of the host antiviral immune response. In the avian influenza virus or human influenza virus, specific amino acids of NS1 have been shown to be important for the virus to antagonize host antiviral defenses and promote viral replication. However, little research has been reported regarding the swine influenza virus (SIV) NS1 protein. METHODS: To study the effects of the key amino acids of NS1, we rescued NS1 mutants (S42P, D92E, and S42P/D92E) of the A/swine/Shanghai/3/2014(H1N1) strain and compared their replication ability and cytokine production as well as the intracellular localization in cultured cells. RESULTS: We found that the S42P and D92E mutation displayed no changes on NS1 nuclear localization. The S42P (but not D92E) mutation suppressed protein synthesis and reduced virus growth properties, and there was an inability to antagonize host cell interferon production and IRF3 activation, which led to high levels of IFN-α and IFN-ß production. CONCLUSION: We conclude that the S42 residue of the NS1 of the A/swine/Shanghai/3/2014(H1N1) strain is the key amino acid in regulating the host IFN response by blocking the activation of IRF3 and thus facilitates virus replication.

Npro His49 and Erns Lys412 mutations in pig bovine viral diarrhea virus type 2 synergistically enhance the cellular antiviral response.

Type I interferons are major components of the innate immune response of hosts, and accordingly, many viruses have evolved mechanisms to modulate the host response during infection. Bovine viral diarrhea virus (BVDV) nonstructural protein Npro and structural protein Erns play important roles in inhibiting type I interferon. The aim of this study was to explore the epistatic effects of amino acid mutations in Npro and Erns in porcine ST cells to characterize the immune response induced by BVDV-2. Plasmids with mutant amino acids His49 (H49), Glu22 (E22) in Npro, and His300 (H300), Lys412 (K412) in Erns which had been changed to Alanine (A) had similar effects on type I interferon production in MDBK and ST cells, but resulted in much greater ISG15, OAS, and Mx production in ST cells. The rescued vASH/NproH49ErnsK412 virus showed the best efficiency with respect to modulating antiviral cytokines, indicating that the amino acids Npro H49 and Erns K412 had highly synergistic effects in abolishing the ability to inhibit type I interferon. These findings have importance practical implications owing to the increasing prevalence of BVDV infections, including persistent infections, in domestic pigs.

Highly selective and efficient imprinted polymers based on carboxyl-functionalized magnetic nanoparticles for the extraction of gallic acid from pomegranate rind.

With the combined surface imprinting technique and immobilized template strategy, molecularly imprinted magnetic nanoparticles were successfully prepared and coupled with high-performance liquid chromatography to selectively separate and determine gallic acid from the pomegranate rind. On the surface of carboxyl-functionalized magnetic nanospheres, thin imprinting shells were formed using dopamine as monomer and crosslinker. The characteristics, polymerization conditions, and adsorption performances of the resultant nanomaterials were investigated in detail. In addition of good crystallinity, satisfactory magnetism, and uniform morphology of the obtained polymers, they had rapid binding kinetics, high adsorption capacity, and favorable reusability. In the mixed solution of four hydroxybenzoic acids, the prepared nanomaterials have an excellent selectivity to gallic acid with an imprinting factor of as high as 17.5. Therefore, the polymers have great potentials in specific extraction and enrichment of gallic acid from the complex natural resources.

Selection and characterization of single-chain recombinant antibodies against phosphoprotein of Newcastle disease virus.

Phosphoprotein (P), involved in virus RNA replication and transcription, had become a new target for the research on treating Newcastle disease virus (NDV). Here we described the cloning and expression of phosphoprotein from NDV, and then screened the anti-P antibodies from the chicken single chain fragment variable (scFv) library, which were generated from chickens immunized with the ND vaccines. As a first step, the recombinant expression vector pET28a-P was successfully constructed. In a following step, two anti-P positive scFv clones from the scFv library were selected by indirect enzyme-linked immunosorbent assay (ELISA) method. The sequence analysis of two positive clones showed that there were more variation in complementary determine region (CDR) of VH and VL, and the CDR3 in VH exhibited a significant change in amino acid number and type. In another experiment, the purified scFv antibodies used in the assay was shown to be specific for NDV-P by western blot. The results indicated that the strategy we used in this experiment proved to be convenient way for screening scFv antibody, which paved a new way for the immunization diagnosis and the exploration of integrated control of NDV.

NLRX1 Mediates the Disruption of Intestinal Mucosal Function Caused by Porcine Astrovirus Infection via the Extracellular Regulated Protein Kinases/Myosin Light-Chain Kinase (ERK/MLCK) Pathway.

Porcine astrovirus (PAstV) has a potential zoonotic risk, with a high proportion of co-infection occurring with porcine epidemic diarrhea virus (PEDV) and other diarrheal pathogens. Despite its high prevalence, the cellular mechanism of PAstV pathogenesis is ill-defined. Previous proteomics analyses have revealed that the differentially expressed protein NOD-like receptor X1 (NLRX1) located in the mitochondria participates in several important antiviral signaling pathways in PAstV-4 infection, which are closely related to mitophagy. In this study, we confirmed that PAstV-4 infection significantly up-regulated NLRX1 and mitophagy in Caco-2 cells, while the silencing of NLRX1 or the treatment of mitophagy inhibitor 3-MA inhibited PAstV-4 replication. Additionally, PAstV-4 infection triggered the activation of the extracellular regulated protein kinases/ myosin light-chain kinase (ERK/MLCK) pathway, followed by the down-regulation of tight-junction proteins (occludin and ZO-1) as well as MUC-2 expression. The silencing of NLRX1 or the treatment of 3-MA inhibited myosin light-chain (MLC) phosphorylation and up-regulated occludin and ZO-1 proteins. Treatment of the ERK inhibitor PD98059 also inhibited MLC phosphorylation, while MLCK inhibitor ML-7 mitigated the down-regulation of mucosa-related protein expression induced by PAstV-4 infection. Yet, adding PD98059 or ML-7 did not affect NLRX1 expression. In summary, this study preliminarily explains that NLRX1 plays an important role in the disruption of intestinal mucosal function triggered by PAstV-4 infection via the ERK/MLC pathway. It will be helpful for further antiviral drug target screening and disease therapy.

Microbial Community and Metabolome Analysis of the Porcine Intestinal Damage Model Induced by the IPEC-J2 Cell Culture-Adapted Porcine Deltacoronavirus (PDCoV) Infection.

This study was conducted to elucidate the intestinal damage induced by the IPEC-J2 cell culture-passaged PDCoV. The results showed that PDCoV disrupted the intestinal structure and increased intestinal permeability, causing abnormalities in mucosal pathology. Additionally, PDCoV induced an imbalance in the intestinal flora and disturbed its stability. Microbial community profiling revealed bacterial enrichment (e.g., Proteobacteria) and reduction (e.g., Firmicutes and Bacteroidetes) in the PDCoV-inoculated piglet model. In addition, metabolomics analysis indicated that 82 named differential metabolites were successfully quantified, including 37 up-regulated and 45 down-regulated metabolites. Chenodeoxycholic acid, sphingosine, and oleanolic aldehyde levels were reduced in PDCoV-inoculated piglets, while phenylacetylglycine and geranylgeranyl-PP levels were elevated. Correlation analysis indicated a negative correlation between Escherichia-Shigella and choline, succinic acid, creatine, phenyllactate, and hippuric acid. Meanwhile, Escherichia-Shigella was positively correlated with acetylcholine, L-Glutamicacid, and N-Acetylmuramate. Roseburia, Lachnospiraceae_UCG-010, Blautia, and Limosilactobacillus were negatively and positively correlated with sphingosine, respectively. These data suggested PDCoV-inoculated piglets exhibited significant taxonomic perturbations in the gut microbiome, which may result in a significantly altered metabolomic profile.

Genomic Evolution and Selective Pressure Analysis of a Novel Porcine Sapovirus in Shanghai, China.

Porcine sapovirus (PoSaV) is one of the most significant pathogens causing piglet diarrhea, and one with limited genetic characterization. In this study, the prevalence, infection pattern, and genetic evolution of porcine sapovirus were elucidated in detail. The positive rate of PoSaV was 10.1% (20/198), with dual, triple, and quadruple infections of 45%, 40%, and 5%, respectively. To further explore the viral composition in the PoSaV-positive diarrhea feces, metagenomic sequencing was carried out. The results confirmed that RNA viruses accounted for a higher proportion (55.47%), including the two primary viruses of PoSaV (21.78%) and porcine astrovirus (PAstV) (24.54%) in the tested diarrhea feces samples. Afterward, a full-length sequence of the PoSaV isolate was amplified and named SHCM/Mega2023, and also given the identifier of GenBank No. PP388958. Phylogenetic analysis identified the prevalent PoSaV strain SHCM/Mega2023 in the GIII genogroup, involving a recombinant event with MK962338 and KT922089, with the breakpoint at 2969-5132 nucleotides (nt). The time tree revealed that the GIII genogroup exhibits the widest divergence time span, indicating a high likelihood of viral recombination. Moreover, SHCM/Mega2023 had three nucleotide "RPL" insertions at the 151-153 nt site in the VP2 gene, compared to the other GIII strains. Further selective pressure calculations demonstrate that the whole genome of the SHCM/Mega2023 strain was under purifying selection (dN/dS < 1), with seven positively selected sites in the VP1 protein, which might be related to antigenicity. In conclusion, this study presents a novel genomic evolution of PoSaV, offering valuable insights into antigenicity and for vaccine research.

A ferritin nanoparticle vaccine based on the hemagglutinin extracellular domain of swine influenza A (H1N1) virus elicits protective immune responses in mice and pigs.

Introduction: Swine influenza viruses (SIVs) pose significant economic losses to the pig industry and are a burden on global public health systems. The increasing complexity of the distribution and evolution of different serotypes of influenza strains in swine herds escalates the potential for the emergence of novel pandemic viruses, so it is essential to develop new vaccines based on swine influenza. Methods: Here, we constructed a self-assembling ferritin nanoparticle vaccine based on the hemagglutinin (HA) extracellular domain of swine influenza A (H1N1) virus using insect baculovirus expression vector system (IBEVS), and after two immunizations, the immunogenicities and protective efficacies of the HA-Ferritin nanoparticle vaccine against the swine influenza virus H1N1 strain in mice and piglets were evaluated. Results: Our results demonstrated that HA-Ferritin nanoparticle vaccine induced more efficient immunity than traditional swine influenza vaccines. Vaccination with the HA-Ferritin nanoparticle vaccine elicited robust hemagglutinin inhibition titers and antigen-specific IgG antibodies and increased cytokine levels in serum. MF59 adjuvant can significantly promote the humoral immunity of HA-Ferritin nanoparticle vaccine. Furthermore, challenge tests showed that HA-Ferritin nanoparticle vaccine conferred full protection against lethal challenge with H1N1 virus and significantly decreased the severity of virus-associated lung lesions after challenge in both BALB/c mice and piglets. Conclusion: Taken together, these results indicate that the hemagglutinin extracellular-based ferritin nanoparticle vaccine may be a promising vaccine candidate against SIVs infection.

The lncRNA HCG4 regulates the RIG-I-mediated IFN production to suppress H1N1 swine influenza virus replication.

Influenza A virus (IAV) non-structural protein 1 (NS1) is a virulence factor that allows the virus to replicate efficiently by suppressing host innate immune responses. Previously, we demonstrated that the serine (S) at position 42 of NS1 in H1N1 swine influenza virus (SIV) is a critical residue in interferon (IFN) resistance, thus facilitating viral infections. Here, by lncRNA-seq, a total of 153 differentially expressed lncRNAs were identified, and the lncRNA HCG4 was selected due to its significantly higher expression after infection with the NS1 S42P mutant virus. Overexpression of HCG4 enhanced IFN-ß production and suppressed SIV infection, highlighting the potential antiviral activity of HCG4 against SIV. Further investigation suggested that HCG4 served as a positive feedback mediator for RIG-I signaling. It alleviated the inhibitory effect on RIG-I K63-linked ubiquitination by NS1 protein, thereby resulting in an increase in RIG-I-mediated IFN production. Taken together, our findings demonstrate that HCG4 modulates the innate immune response to SIV infection through K63-linked RIG-I ubiquitination, providing insights into the role of lncRNAs in controlling viral infections.

Development of an indirect ELISA method based on the VP1 protein for detection of IgG antibodies against porcine sapelovirus.

Porcine sapelovirus (PSV) is a newly emerging enterovirus that is widely prevalent in China. Since there is no clinical serological testing for PSV, the objective of this study was to develop an indirect enzyme-linked immunosorbent assay (i-ELISA) for detection of PSV immunoglobulin G (IgG) antibody in pigs. A PSV strain, named SHPD202148, was first isolated from the fecal samples of piglets. Its structural protein, VP1, was prokaryotic-expressed in the pET expression system, followed by purification. Using the recombinant protein with reactogenicity as coating antigen, an i-ELISA, characterized by high sensitivity and specificity, had a detection limit at 1:12 800 dilution with a determined cutoff value of 0.352. Finally, field sera collected from different pig herds were tested in parallel by the serum neutralization (SN) test. The result showed that 126 samples were positive and 36 were negative, with an agreement of 97.0% in both cases. This i-ELISA can be used as an alternative serological test for detecting antibodies against PSV in blood serum.

Le sapelovirus porcin (PSV) est un entérovirus nouvellement émergent largement répandu en Chine. Puisqu'il n'y a pas de test sérologique clinique pour le PSV, l'objectif de cette étude était de développer un test immuno-enzymatique indirect (i-ELISA) pour la détection d'immunoglobuline G (IgG) anti-PSV chez les porcs. Une souche de PSV, nommée SHPD202148, a d'abord été isolée à partir d'échantillons fécaux de porcelets. Sa protéine structurale, VP1, a été exprimée par un procaryote dans le système d'expression pET, suivie d'une purification. Utilisant la protéine recombinante à réactogénicité comme antigène de revêtement, un i-ELISA, caractérisé par une sensibilité et une spécificité élevées, avait une limite de détection à une dilution de 1:12 800 avec une valeur seuil déterminée de 0,352. Enfin, des sérums de terrain collectés dans différents troupeaux de porcs ont été testés en parallèle par le test de neutralisation sérique (SN). Le résultat a montré que 126 échantillons étaient positifs et 36 étaient négatifs, avec un accord de 97,0 % dans les deux cas. Cet i-ELISA peut être utilisé comme test sérologique alternatif pour détecter les anticorps anti-PSV dans le sérum sanguin.(Traduit par Docteur Serge Messier).

The primary use in indirect ELISA of secreted proteins Mb1761c and Mb2277 of M. bovis.

Disease monitoring on bovine tuberculosis (BTB) has been proven to be a capable strategy to control this disease, which has significantly progressed in developed countries. However, such strategies require a prompt, accurate live animal test, which has thus far been lacking in developing countries, such as China. The two secreted proteins, Mb1761c and Mb2277, were selected from the complete genome of M. bovis by using a bioinformatics method. After having been proven by the reactogenicity by Western blot, the two expressed prokaryotic proteins served as coat antigens in ELISA plates to evaluate their ability to detect the antibodies against M. bovis. The results showed that the sensitivity of the indirect ELISA method coated with Mb2277 as antigen was as same as that coated with Mb1761c. To explore the possibility of increasing the sensitivity of the ELISA test, the two proteins were further mixed in a different ratio and coated as antigen in ELISA. The results showed that the sensitivity of the combination of the coated reconstructive proteins (1:1) was stronger than that of single coated protein.

Genomic Divergence Characterization and Quantitative Proteomics Exploration of Type 4 Porcine Astrovirus.

Porcine astrovirus (PAstV) has been identified as an important diarrheic pathogen with a broad global distribution. The PAstV is a potential pathogen to human beings and plays a role in public health. Until now, the divergence characteristics and pathogenesis of the PAstV are still not well known. In this study, the PAstV-4 strain PAstV/CH/2022/CM1 was isolated from the diarrheal feces of a piglet in Shanghai, which was identified to be a recombination of PAstV4/JPN (LC201612) and PAstV4/CHN (JX060808). A time tree based on the ORF2 protein of the astrovirus demonstrated that type 2-5 PAstV (PAstV-2 to 5) diverged from type 1 PAstV (PAstV-1) at a point from 1992 to 2000. To better understand the molecular basis of the virus, we sought to explore the host cell response to the PAstV/CH/2022/CM1 infection using proteomics. The results demonstrate that viral infection elicits global protein changes, and that the mitochondria seems to be a primary and an important target in viral infection. Importantly, there was crosstalk between autophagy and apoptosis, in which ATG7 might be the key mediator. In addition, the NOD-like receptor X1 (NLRX1) in the mitochondria was activated and participated in several important antiviral signaling pathways after the PAstV/CH/2022/CM1 infection, which was closely related to mitophagy. The NLRX1 may be a crucial protein for antagonizing a viral infection through autophagy, but this has yet to be validated. In conclusion, the data in this study provides more information for understanding the virus genomic characterization and the potential antiviral targets in a PAstV infection.

The Complex Co-infections of Multiple Porcine Diarrhea Viruses in Local Area Based on the Luminex xTAG Multiplex Detection Method.

The large-scale outbreaks of severe diarrhea caused by viruses have occurred in pigs since 2010, resulting in great damage to the pig industry. However, multiple infections have contributed to the outbreak of the disease and also resulted in great difficulties in diagnosis and control of the disease. Thus, a Luminex xTAG multiplex detection method, which was more sensitive and specific than general multiplex PCR method, was developed for the detection of 11 viral diarrhea pathogens, including PKoV, PAstV, PEDV, PSaV, PSV, PTV, PDCoV, TGEV, BVDV, PoRV, and PToV. To investigate the prevalence of diarrhea-associated viruses responsible for the outbreaks, a total of 753 porcine stool specimens collected from 9 pig farms in Shanghai during 2015-2018 were tested and the pathogen spectrums and co-infections were analyzed. As a result, PKoV, PAstV and PEDV were most commonly detected viruses in diarrheal pigs with the rate of 38.65% (291/753), 20.32% (153/753), and 15.54% (117/753), respectively. Furthermore, multiple infections were commonly seen, with positive rate of 28.42%. Infection pattern of the viral diarrhea pathogens in a specific farm was changing, and different farms had the various diarrhea infection patterns. A longitudinal investigation showed that PEDV was the key pathogen which was closely related to the death of diarrhea piglets. Other pathogens might play synergistic roles in the pathogenesis of diarrhea disease. Furthermore, the surveillance confirmed that variant enteropathogenic viruses were leading etiologic agents of porcine diarrhea, either mono-infection or co-infections of PKoV were common in pigs in Shanghai, but PEDV was still the key pathogen and multiple pathogens synergistically complicated the infection status, suggesting that controlling porcine diarrhea might be more complex than previously thought. The study provides a better understanding of diarrhea viruses in piglets, which will aid in better preventing and controlling epidemics of viral porcine diarrhea.