ARF6 promotes Streptococcus suis suilysin induced apoptosis in HBMECs.

Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.

Inhibition of porcine deltacoronavirus entry and replication by Cepharanthine.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus (CoV) that mainly causes acute diarrhea/vomiting, dehydration, and mortality in piglets, possessing economic losses and public health concerns. However, there are currently no proven effective antiviral agents against PDCoV. Cepharanthine (CEP) is a naturally occurring alkaloid used as a traditional remedy for radiation-induced symptoms, but its underlying mechanism of CEP against PDCoV has remained elusive. The aim of this study was to investigate the anti-PDCoV effects and mechanisms of CEP in LLC-PK1 cells. The results showed that the antiviral activity of CEP was based on direct action on cells, preventing the virus from attaching to host cells and virus replication. Importantly, Surface Plasmon Resonance (SPR) results showed that CEP has a moderate affinity to PDCoV receptor, porcine aminopeptidase N (pAPN) protein. AutoDock predicted that CEP can form hydrogen bonds with amino acid residues (R740, N783, and R790) in the binding regions of PDCoV and pAPN. In addition, RT-PCR results showed that CEP treatment could significantly reduce the transcription of ZBP1, cytokine (IL-1ß and IFN-α) and chemokine genes (CCL-2, CCL-4, CCL-5, CXCL-2, CXCL-8, and CXCL-10) induced by PDCoV. Western blot analysis revealed that CEP could inhibit viral replication by inducing autophagy. In conclusion, our results suggest that the anti-PDCoV activity of CEP is not only relies on competing the virus binding with pAPN, but also affects the proliferation of the virus in vitro by downregulating the excessive immune response caused by the virus and inducing autophagy. CEP emerges as a promising candidate for potential anti-PDCoV therapeutic development.

Hepatoprotective and renoprotective effects of silymarin against salinomycin-induced toxicity in adult rabbits.

Background and Aim: Salinomycin sodium, a licensed coccidiostat in rabbits, is used for fattening at a dose of 20-25 mg/kg. Salinomycin toxicity may arise from many risk factors (e.g., overdosage or use in non-target animal species). Silymarin extracted from milk thistle has antioxidant, anti-inflammatory, and antiviral properties. This study aimed to investigate the adverse impacts of oral administration of salinomycin for 28 consecutive days and how to reduce its risks and side effects by administering silymarin. Materials and Methods: Eighty-four male New Zealand White bucks (1.750-2.000 kg) were randomly divided into seven groups (12 each). Group one was the control. Groups two and three were administered salinomycin orally (doses of 20 and 40 mg/kg ration). Group four was administered salinomycin (20 mg/kg ration) and silymarin (6.5 mg/kg body weight [BW]). Group five received salinomycin (40 mg/kg ration) and silymarin (13 mg/kg BW). Groups six and seven were administered silymarin at doses of 6.5 and 13 mg/kg BW. Rabbits were euthanized and slaughtered on day 29 using the Halal method. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, urea, total proteins, albumin, total cholesterol, and high- and low-density lipoprotein (HDL and LDL) were analyzed in serum. Glutathione (GSH), superoxide dismutase (SOD), catalase, and malondialdehyde (MDA) were estimated in the liver. A histopathological investigation was performed on the liver and kidney. Results: The MDA activity, AST, ALT, total protein, albumin, total cholesterol, triglyceride, LDL, urea, and creatinine values were significantly elevated in groups two and three. The GSH, catalase, SOD, and HDL were significantly lower in these groups than in the control group. There were moderate pathologic changes in the liver and kidney of the third group. However, the results of the fourth and fifth groups improved more than those of the second and third groups. The results of the sixth and seventh groups were nearly the same as those of the control group. Conclusion: Salinomycin toxicity was caused by oxidative damage because of reactive oxygen species formation. Silymarin (6.5 or 13 mg/kg BW) tends to prevent and treat accidental toxicity. However, the high dose of silymarin (13 mg/kg BW) had more renal and hepatoprotective capacities.

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.

The Diversity and Spatiotemporally Evolutionary Dynamic of Atypical Porcine Pestivirus in China.

The presence of congenital tremor (CT) type A-II in newborn piglets, caused by atypical porcine pestivirus (APPV), has been a focus since 2016. However, the source, evolutionary history, and transmission pattern of APPV in China remain poorly understood. In this study, we undertook phylogenetic analyses based on available complete E2 gene sequences along with 98 newly sequenced E2 genes between 2016 and 2020 in China within the context of global genetic diversity. The phylogenies revealed four distinct lineages of APPV, and interestingly, all lineages could be detected in China with the greatest diversity. Bayesian phylogenetic analyses showed that the E2 gene evolves at a mean rate of 1.22 × 10-3 (8.54 × 10-4-1.60 × 10-3) substitutions/site/year. The most recent common ancestor for APPVs is dated to 1886 (1837-1924) CE, somewhat earlier than the documented emergence of CT (1922 CE). Our phylogeographic analyses suggested that the APPV population possibly originated in the Netherlands, a country with developed livestock husbandry, and was introduced into China during the period 1837-2010. Guangdong, as a primary seeding population together with Central and Southwest China as epidemic linkers, was responsible for the dispersal of APPVs in China. The transmission pattern of "China lineages" (lineage 3 and lineage 4) presented a "south to north" movement tendency, which was likely associated with the implementation of strict environmental policy in China since 2000. Reconstruction of demographic history showed that APPV population size experienced multiple changes, which correlated well with the dynamic of the number of pigs in the past decades in China. Besides, positively selected pressure and geography-driven adaptation were supposed to be key factors for the diversification of APPV lineages. Our findings provide comprehensive insights into the diversity and spatiotemporal dynamic of APPV in China.

Combination of Fe(OH)3 modified diatomaceous earth and qPCR for the enrichment and detection of African swine fever virus in water.

Water is one of the primary vectors for African swine fever virus (ASFV) transmission among swine herds. However, the low concentrations of ASFV in water represent a challenge for the detection of the virus by conventional PCR methods, and enrichment of the virus would increase the test sensitivity. In this study, aiming to enrich ASFV in water quickly and efficiently, a rapid and efficient water-borne virus enrichment system (MDEF, modified diatomaceous earth by ferric hydroxide colloid) was used to enrich ASFV in water. After enrichment by MDEF, conventional real-time PCR (qPCR) was used for ASFV detection. ASFV were inactivated and diluted in 10 L of water, of which 4 mL were collected after 60 min treatment using the MDEF system. Two thousand five hundred times reduction of the sample volume was achieved after enrichment. A high adsorption rate of about 99.99 (±0.01)% and a high recovery rate of 64.01 (±10.20)% to 179.65 (±25.53)% was achieved by using 1g modified diatomaceous earth for 10 L ASFV contaminated water. The limit of qPCR detection of ASFV decreased to 1 × 10-1.11 GU ml-1 (genomic units per milliliter) from 1 × 102.71 GU ml-1 after concentrating the spiked water from 10 L to 4 ml. Preliminary application of MDEF allowed successful detection of African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), and pseudorabies virus (PRV) in sewage. Thus, the combination of modified diatomaceous earth and real-time PCR is a promising strategy for the detection of viruses in water.

The construction and immunogenicity analyses of a recombinant pseudorabies virus with porcine circovirus type 3 capsid protein co-expression.

Porcine circovirus-associated diseases (PCVADs) and pseudorabies (PR) are highly contagious and economically significant diseases of swine in China. Porcine circovirus type 3 (PCV3) is an emerging swine pathogen of PCVAD. Currently, no PCV3 vaccine is commercially available, and the epidemic caused by it is still spreading worldwide. In this study, we used the PRV variant strain HNX as the parental virus to construct recombinant PRV with TK/gE gene deletion and capsid (Cap) protein co-expression, named HNX-ΔTK/ΔgE-ORF2. The results revealed that PCV3 Cap protein can be detected in HNX-ΔTK/ΔgE-ORF2-infected PK-15 cells by both western blotting and immunofluorescence assays. Vaccination with HNX-ΔTK/ΔgE-ORF2 did not cause pruritus, ruffled fur, systemic infection, or inflammation (without high expression of interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) in plasma). Furthermore, HNX-ΔTK/ΔgE-ORF2 immunization induced an anti-Cap specific antibody, activated a PRV-specific cellular immune response, and provided 100 % protection to mice against the challenge of the virulent HNX strain. Thus, HNX-ΔTK/ΔgE-ORF2 appears to be a promising vaccine candidate against PRV and PCV3 for the control of the PRV variant and PCV3.

Untargeted LC-MS based metabolomic profiling of iPAMs to investigate lipid metabolic pathways alternations induced by different Pseudorabies virus strains.

Owing to viral recombination, interspecies transmission, and evolution, variant pseudorabies virus (PRV) strains exhibit different biological characteristics and pathogenicity. To improve the understanding of common and specific metabolic changes that occur upon infection by different PRV strains, we herein describe the comprehensive analysis of metabolites of PRV vaccine strain (Bartha K61), classical strain (EA) and variant strain (HNX) infection in immortalized porcine alveolar macrophage cells. Compared with uninfected cells, cells infected with Bartha K61, EA and HNX had 246, 225, and 272 differing metabolites, respectively. In the three types of PRV-strain-infected cells, lipids and lipid-like molecules accounted for over 50 % of the altered metabolites. As these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of the host metabolism. We analyzed the potential relationship between virus replication and the virus-mediated host metabolism. Our study resulted in the first reconstruction of the major lipid metabolic pathways involved in PRV infection, including those of glycerophospholipids, sphingolipids, glycerolipids, and fatty acyls. In addition, the metabolic perturbations caused by different PRV strain infections are consistent across many species, however, our results also revealed many specific metabolic alterations during HNX infection, such as the enrichment of phosphatidylinositol and 15R-PGE2 methyl ester 15-acetate, and the diminishment of phosphatidylethanolamine, phosphatidic acid, and ceramides. These strain-specific altered metabolites may be linked to the unique biological characteristics and pathogenicity of the HNX strain.

Isolation and genetic characteristics of a neurotropic teschovirus variant belonging to genotype 1 in northeast China.

Porcine teschovirus (PTV) is a causative agent of reproductive disorders, encephalomyelitis, respiratory diseases, and diarrhea in swine, with a worldwide distribution. In this work, we identified PTV-associated nonsuppurative encephalitis as a potential cause of posterior paralysis in neonatal pigs in northeast China. Using indirect immunofluorescence assay, western blot, electron microscopy, and genome sequencing, we identified a neurotropic PTV strain, named CHN-NP1-2016, in the supernatants of pooled cerebrum and cerebellum samples from an affected piglet. Nucleotide sequence alignment revealed that the whole genome of CHN-NP1-2016 shared the highest sequence similarity (86.76% identity) with PTV 1 strain Talfan. A combination of phylogenetic and genetic divergence analysis was applied based on the deduced amino acid sequence of the P1 gene with a cutoff value of the genetic distance (0.102 ± 0.008) for defining PTV genotypes, and this showed that CHN-NP1-2016 is a variant of genotype 1. In total, 16 unique mutations and five mutant clusters were detected in the capsid proteins VP1 and VP2 of CHN-NP1-2016 when compared to other PTV1 isolates. Importantly, we detected three mutant clusters located in the exposed surface loops of the capsid protein, potentially indicating significant differences in major neutralization epitopes. Moreover, a potential recombination event in the P1 region of PTV CHN-NP1-2016 was detected. These findings provide valuable insights into the role of recombination in the evolution of teschoviruses. To our knowledge, this is the first case report of PTV-1-associated encephalitis in northeast China. Future investigations will narrow on the serology and pathogenicity of this novel isolate.

The epidemiological investigation of co-infection of major respiratory bacteria with pseudorabies virus in intensive pig farms in China.

Porcine respiratory disease complex (PRDC), a respiratory disease caused by a variety of factors, is one of the most common problems in the intensive pig farms. To investigate the mixed infection incidence of wild-type pseudorabies virus (WT PRV) and respiratory bacteria, a total of 1,293 clinical samples were collected from pigs with typical respiratory signs from 14 different provinces of China from September 2016 to February 2018. The WT PRV was detected by ELISA targeting gE antibody while the bacteria were detected by bacterial isolation and serotyping by PCR. The results revealed that the detection rate of A. pleuropneumoniae and B. bronchiseptica infection associated with WT PRV infection were 6.30% and 15.99%, respectively, which were significantly higher than those without WT PRV infection (3.41% and 4.41%) at the farm level (p < .05). There were no significant differences in the detection rate of H. parasuis, S. suis or P. multocida between WT PRV positive and negative farms (p > .05). However, the detection rate of attenuated H. parasuis and S. suis strains were 68.19% and 64.75%, respectively, in WT PRV infected farms, which were significantly higher than those (41.56% and 52.25%) in WT PRV free farms (p < .05). The prevalent serotypes of H. parasuis-5/12 and S. suis-2 were also investigated by multiplex PCR. These results indicated that the presence of WT PRV increased the chance of bacterial infection and the number of pathogenic strains in the respiratory system of pigs. Therefore, the eradication of pseudorabies is an effective approach to prevent and control the bacterial respiratory diseases in the intensive pig farms in China.

Establishment of a Blocking ELISA Detection Method for Against African Swine Fever Virus p30 Antibody.

African swine fever (ASF) is a highly lethal hemorrhagic viral disease of domestic pigs caused by African swine fever virus (ASFV). A sensitive and reliable serological diagnostic assay is required, so laboratories can effectively and quickly detect ASFV infection. The p30 protein is abundantly expressed early in cells and has excellent antigenicity. Therefore, this study aimed to produce and characterize p30 monoclonal antibodies with an ultimate goal of developing a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for ASFV antibody detection. Three monoclonal antibodies against p30 protein that were expressed in E. coli were generated, and their characterizations were investigated. Furthermore, a blocking ELISA based on a monoclonal antibody was developed. To evaluate the performance of the assay, 186 sera samples (88 negative and 98 positive samples) were analyzed and a receiver-operating characteristic (ROC) analysis was applied to determine the cutoff value. Based on the ROC analysis, the area under the curve (AUC) was 0.997 (95% confidence interval: 99.2 to 100%). Besides, a diagnostic sensitivity of 97.96% (95% confidence interval: 92.82 to 99.75%) and a specificity of 98.96% (95% confidence interval: 93.83 to 99.97%) were achieved when the cutoff value was set to 38.38%. Moreover, the coefficients of inter- and intra-batches were <10%, indicating the good repeatability of the method. The maximum dilution of positive standard serum detected by this ELISA method was 1:512. The blocking ELISA was able to detect seroconversion in two out of five pigs at 10 Dpi and the p30 response increasing trend through the time course of the study (0-20 Dpi). In conclusion, the p30 mAb-based blocking ELISA developed in this study demonstrated a high repeatability with maximized diagnostic sensitivity and specificity. The assay could be a useful tool for field surveillance and epidemiological studies in swine herd.