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Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV) has caused enormous economic losses to the global swine industry. Due to frequent mutations in the spike (S) gene of PEDV, commercial vaccines used today are gradually losing their protective efficacy against variants. It's significant to monitor the S gene of PEDV variants and understand its evolutionary trend. In this study, we report four novel PEDV strains isolated from Sichuan, Guangdong and Shanxi Provinces and determined their S gene sequences. Phylogenetic analysis showed that they all belong to GII genotype. Amino acid alignment revealed a unique mutation pattern. We also predicted their three-dimensional structures and continuous B-cell epitopes and compared them to those of the vaccine strain. Our study provides references for understanding the evolution of S gene and antigenic change of S protein, which are of great significance for formulating the prevention and control of PEDV.

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Porcine deltacoronavirus (PDCoV) poses a serious threat to pork industry and has the potential for cross-species transmission. Yet, the invasion mechanisms and host factors involved are still unknown. In the present work, using siRNA interference and co-immunoprecipitation, we identified Annexin A2 (ANXA2), Prohibitin-2 (PHB2), or Caveolin-2 (CAV2) as host factors positively regulating the internalization of PDCoV. We further found that Rab11a co-localized with PDCoV S and inhibited PDCoV internalization. Subsequently, a pseudoviral infection model (LV-PDCoV S-GFP) was constructed, and ANXA2 or CAV2 promoted PDCoV invasion by downregulating Rab11a. Our results also indicated that ANXA2, CAV2, and Rab11a interact with the S protein via S-FP, thereby regulating virus-host membrane fusion. Through LV-PDCoV S-GFP infection, we found that Rab11a may act as a host restriction factor, and it could regulate the invasion efficiency of PDCoV by adding of exogenous GTP. These findings revealed that Rab11a was an exciting target to restrict fusion of PDCoV with host cell membranes. AVAILABILITY OF DATA AND MATERIAL: Not applicable.

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Porcine epidemic diarrhea virus (PEDV) causes enormous economic losses to the pork industry, and its extensive cell tropism poses a substantial challenge to public health and safety. However, the invasion mechanisms and relevant host factors of PEDV remain poorly understood. In this study, we identified 422 differentially expressed genes related to PEDV infection through transcriptome analysis. Among these, Annexin A2 (ANXA2), Prohibitin-2 (PHB2), and Caveolin-2 (CAV2) were identified through screening and verifying as having a specific interaction with the PEDV S protein, and positive regulation of PEDV internalization was validated by siRNA and overexpression tests. Subsequently, using host membrane protein interaction networks and co-immunoprecipitation analysis, we found that ANXA2 PHB2 or CAV2 directly interact with Rab11a. Next, we constructed a pseudovirus model (LV-PEDV S-GFP) to further confirm that the downregulation of Rab11a could promote PEDV invasion. In detail, ANXA2, PHB2, or CAV2 promoted PEDV invasion via downregulating Rab11a. Furthermore, we showed that the S-protein fusion peptide (FP) was sufficient for S-protein interaction with ANXA2, PHB2, CAV2, and Rab11a, and the addition of exogenous GTP could regulate the efficiency of PEDV invasion. Collectively, ANXA2, PHB2, or CAV2 influenced the membrane fusion of PEDV with host cells through the host restriction factor Rab11a. This study could be targeted for future research to develop strategies for the control of PEDV.

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African swine fever (ASF), which is casued by African swine fever virus (ASFV), is a fatal infectious disease of pigs that results in significant losses to the breeding industry. Therefore, screening and detection are crucial for the control and prevention of the ASFV. Argonaute is a new detection tool that is being extensively used due to its high specificity and programmability. This study reports on a new nucleic acid assay method, termed REPD, which uses recombinase-aided amplification and restriction endonuclease-assisted Pyrococcus furiosus argonaute (PfAgo) detection. One-pot REPD was developed for the detection of ASFV. The one-pot REPD could detect a single copy of ASFV nucleic acid and showed no cross-reactivity with other pathogens. Detection in clinical samples was 100% consistent with the results of real-time PCR analysis. The results showed that the one-pot REPD assay is convenient, sensitive, specific, and potentially adaptable to the detection of ASFV. In summary, this study highlights a novel method that can be employed for the detection of pathogens.

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Proteus mirabilis can transfer transposons, insertion sequences, and gene cassettes to the chromosomes of other hosts through SXT/R391 integrative and conjugative elements (ICEs), significantly increasing the possibility of antibiotic resistance gene (ARG) evolution and expanding the risk of ARGs transmission among bacteria. A total of 103 strains of P. mirabilis were isolated from 25 farms in China from 2018 to 2020. The positive detection rate of SXT/R391 ICEs was 25.2% (26/103). All SXT/R391 ICEs positive P. mirabilis exhibited a high level of overall drug resistance. Conjugation experiments showed that all 26 SXT/R391 ICEs could efficiently transfer to Escherichia coli EC600 with a frequency of 2.0 × 10-7 to 6.0 × 10-5. The acquired ARGs, genetic structures, homology relationships, and conservation sequences of 26 (19 different subtypes) SXT/R391 ICEs were investigated by high-throughput sequencing, whole-genome typing, and phylogenetic tree construction. ICEPmiChnHBRJC2 carries erm (42), which have never been found within an SXT/R391 ICE in P. mirabilis, and ICEPmiChnSC1111 carries 19 ARGs, including clinically important cfr, blaCTX-M-65, and aac(6')-Ib-cr, making it the ICE with the most ARGs reported to date. Through genetic stability, growth curve, and competition experiments, it was found that the transconjugant of ICEPmiChnSCNNC12 did not have a significant fitness cost on the recipient bacterium EC600 and may have a higher risk of transmission and dissemination. Although the transconjugant of ICEPmiChnSCSZC20 had a relatively obvious fitness cost on EC600, long-term resistance selection pressure may improve bacterial fitness through compensatory adaptation, providing scientific evidence for risk assessment of horizontal transfer and dissemination of SXT/R391 ICEs in P. mirabilis.IMPORTANCEThe spread of antibiotic resistance genes (ARGs) is a major public health concern. The study investigated the prevalence and genetic diversity of integrative and conjugative elements (ICEs) in Proteus mirabilis, which can transfer ARGs to other hosts. The study found that all of the P. mirabilis strains carrying ICEs exhibited a high level of drug resistance and a higher risk of transmission and dissemination of ARGs. The analysis of novel multidrug-resistant ICEs highlighted the potential for the evolution and spread of novel resistance mechanisms. These findings emphasize the importance of monitoring the spread of ICEs carrying ARGs and the urgent need for effective strategies to combat antibiotic resistance. Understanding the genetic diversity and potential for transmission of ARGs among bacteria is crucial for developing targeted interventions to mitigate the threat of antibiotic resistance.

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Autophagy and apoptosis, which are important processes for host immunity, are commonly exploited by viruses to facilitate their survival. However, to the best of our knowledge, very few studies have researched the mechanisms of action of the autophagic and apoptotic signaling pathways following viral infection. Thus, the present study aimed to investigate the mechanisms of action of growth arrest and DNA-damage-inducible ß (GADD45ß), an important resistance gene involved in the host resistance to ALV-J. Both ALV-J infection and the overexpression of GADD45ß inhibited autophagy during the early stages, which prevented the autophagosomes from binding to the lysosomes and resulted in an incomplete autophagic flux. Notably, GADD45ß was discovered to interact with MEKK4 in DF-1 cells. The genetic knockdown of GADD45ß and MEKK4 using small interfering RNA-affected ALV-J infection, which suggested that ALV-J may promote the binding of GADD45ß to MEKK4 to activate the p38MAPK signaling pathway, which subsequently inhibits autophagy. Furthermore, ALV-J was revealed to affect the autophagic pathway prior to affecting the apoptotic pathway. In conclusion, to the best of our knowledge, the present study was the first to investigate the combined effects of ALV-J infection on autophagy and apoptosis, and to suggest that ALV-J inhibits autophagy via the GADD45ß/MEKK4/p38MAPK signaling pathway.

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Yeast nucleotides are a fine functional additive in human and animals. The effects of dietary yeast nucleotides supplementation on intestinal development, expression of intestinal barrier-related genes, intestinal microbiota, and infectious bronchitis virus (IBV) antibody titer of specific pathogen-free (SPF) chickens were investigated. A total of 60 1-d-old chickens were divided into 4 groups, each of which included 3 replicates of 5 chickens. Group 1 served as a control that was fed a basal diet. Groups 2 to 4 were fed the basal diet supplemented with 0.1%, 0.3% and 0.5% yeast nucleotides, respectively. All chickens were inoculated intranasally with inactivated IBV vaccine at day 1 and day 10. At day 17, the intestinal development, expression of intestinal barrier-related genes and microbiota were evaluated. There was a significant increased ileal villus height and villus height to crypt depth ratio in group 2 (P < 0.05). Moreover, group 4 exhibited higher expression of zonula occludens-1 (ZO-1) and Occludin gene in ileum (P < 0.05), whereas groups 2 and 3 exhibited higher expression of Mucin 2 (MUC2) and trefoil factor 2 (TFF2) gene (P < 0.05), group 2 showed lower expression of IFN-α gene (P < 0.05). Dietary yeast nucleotides increased intestinal bacterial diversity (P < 0.05), and the abundance of Lactobacillus (P < 0.05). At day 10, 17, 24, 31, 38, and 45, the serum IBV antibody titers were tested. Group 2 exhibited higher IBV antibody titer at day 17 (P < 0.05), furthermore, groups 2 to 4 reached the effective levels 1 wk earlier than control group. In conclusion, dietary yeast nucleotides supplementation can help birds to mount a faster and stronger antibody response to IBV vaccine. In addition, dietary yeast nucleotides supplementation can also promote the intestinal development and barrier-related genes expression, and diversity and richness of intestinal microbiota.

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