TLR4 involved in immune response against Vibrio Parahaemolyticus by MyD88-dependent pathway in Crassostrea hongkongensis.

Vibrio parahaemolyticus (V. parahaemolyticus) is a salt-loving gram-negative bacterium, and is the leading cause of mortality in cultured shellfish in recent years. Toll-like Receptor 4 (TLR4) is a classical pattern recognition receptor (PRRs) that recognizes pathogen-associated molecular patterns (PAMPs) of pathogenic microorganism and activates the immune response. However, the function and signal pathway of TLR4 in oyster are still unknown. In this study, a new TLR4 gene was identified from the Crassostrea hongkongensis (C. hongkongensis). The ChTLR4 contained an open reading frame of 2643 bp, encoding 880 amino acids with seven leucine-rich repeat (LRR) domains and a Toll/IL-1R (TIR) domain. The ChTLR4 shared the highest sequence identity (83.0%) with TLR4 of Crassostrea gigas. Tissue expression analysis revealed that ChTLR4 showed the highest constitutive expression in the gill and hepatopancreas, and was significantly upregulated in immune tissues post V. parahaemolyticus infection, especially in gill and hemocytes. Moreover, TLR4 silencing significantly inhibited the immune-enzyme activities, including SOD, CAT, ACP, AKP in gill and LZM in hemolymph supernatant, and increased MDA content in hemolymph supernatant. Meanwhile, the antimicrobial activities of the hemolymph supernatant were also significantly inhibited by TLR4 silencing. These data demonstrated that the ChTLR4 involved in innate immune response of C. hongkongensis against V. parahaemolyticus challenge. Finally, qRT-PCR analysis showed that ChTLR4 silencing clearly inhibited the expression of genes in TLR4-MyD88 pathway, indicating that MyD88-dependent pathway played a crucial role in ChTLR4-mediated immune response against V. parahaemolyticus.

Epidemiology, molecular characterization, and recombination analysis of chicken anemia virus in Guangdong province, China.

Chicken anemia virus (CAV) causes severe anemia and immunosuppression in young chickens and a compromised immune response in older birds, resulting in great economic losses to the poultry industry worldwide. Here, we report the molecular epidemiology and characterization of CAV circulating in poultry in Guangdong province, China. Ninety-one of 277 chickens collected from 2016 to 2017 were CAV positive. Full-genome sequencing revealed the presence of eight separate strains. Phylogenetic analysis based on the genome sequences obtained in this study and related sequences available in the GenBank database showed that all of the CAV isolates exhibit a close relationship to each other and belong to the same genotypic group. Putative recombination events were also detected in the genomes of the newly isolated CAVs. Collectively, our findings underscore the importance of CAV surveillance and provide information that will lead to a better understanding of the evolution of CAV.

Circular RNA Vav3 sponges gga-miR-375 to promote epithelial-mesenchymal transition.

Circular RNAs (circRNAs) are evolutionarily conserved and widely present, but their functions remain largely unknown. Recent development has highlighted the importance of circRNAs as the sponge of microRNA (miRNA) in cancer. We previously reported that gga-miR-375 was downregulated in the liver tumors of chickens infected with avian leukosis virus subgroup J (ALV-J) by microRNA microarray assay. It can be reasonably assumed in accordance with previous studies that the gga-miR-375 may be related to circRNAs. However, the question as to which circRNA acts as the sponge for gga-miR-375 remains to be answered. In this study, circRNA sequencing results revealed that a circRNA Vav3 termed circ-Vav3 was upregulated in the liver tumors of chickens infected with ALV-J. In addition, RNA immunoprecipitation (RIP), biotinylated RNA pull-down and RNA-fluorescence in situ hybridization (RNA-FISH) experiments were conducted to confirm that circ-Vav3 serves as the sponge of gga-miR-375. Furthermore, we confirmed through dual luciferase reporter assay that YAP1 is the target gene of gga-miR-375. The effect of the sponge function of circ-Vav3 on its downstream genes has been further verified by our conclusion that the sponge function of circ-Vav3 can abrogate gga-miR-375 target gene YAP1 and increase the expression level of YAP1. We further confirmed that the circ-Vav3/gga-miR-375/YAP1 axis induces epithelial-mesenchymal transition (EMT) through influencing EMT markers to promote tumorigenesis. Finally, clinical ALV-J-induced tumor livers were collected to detect core gene expression levels to provide a proof to the concluded tumorigenic mechanism. Together, our results suggest that circ-Vav3/gga-miR-375/YAP1 axis is another regulator of tumorigenesis.

Circular RNA alterations are involved in resistance to avian leukosis virus subgroup-J-induced tumor formation in chickens.

Avian leukosis virus subgroup (ALV-J) is an oncogenic neoplasm-inducing retrovirus that causes significant economic losses in the poultry industry. Recent studies have demonstrated circular RNAs (circRNAs) are implicated in pathogenic processes; however, no research has indicated circRNAs are involved in resistance to disease. In this study, over 1800 circRNAs were detected by circRNA sequencing of liver tissues from ALV-J-resistant (n = 3) and ALV-J-susceptible chickens (n = 3). 32 differentially expressed circRNAs were selected for analyzing including 12 upregulated in ALV-J-resistant chickens and 20 upregulated in ALV-J-susceptible chickens, besides, the top five microRNAs (miRNAs) for 12 upregulated circRNAs in ALV-J-resistant chickens were analyzed. Gene ontology and KEGG pathway analyses were performed for miRNA target genes, the predicted genes were mainly involved in immune pathways. This study provides the first evidence that circRNA alterations are involved in resistance to ALV-J-induced tumor formation. We propose circRNAs may help to mediate tumor induction and development in chickens.

Efficacy of an autophagy-targeted DNA vaccine against avian leukosis virus subgroup J.

Infection with the avian leukosis virus subgroup J (ALV-J) can lead to neoplastic disease in chickens, inflicting significant economic losses to the poultry industry. Recent reports have identified inhibitory effects of ALV-J on autophagy, a process involving in innate and adaptive immunity. Inspired by this connection between autophagy and immunity, we developed a novel DNA vaccine against ALV-J which includes co-administration of rapamycin to stimulate autophagy. To measure the efficacy of the developed prototype vaccine, five experimental groups of seven-day-old chickens was immunized three times at three-week intervals respectively with vector, pVAX1-gp85, pVAX1-gp85-LC3, pVAX1-gp85+rapamycin and pVAX1-gp85-LC3+rapamycin through electroporation. We then tested their antibody titers, cytokine levels and cellular immune responses. The immunoprotective efficacy of the prototype vaccines against the challenge of the ALV-J GD1109 strain was also examined. The results showed that the combination of pVAX1-gp85-LC3 and rapamycin was able to induce the highest antibody titers, and enhance interleukin(IL)-2, IL-10 and interferon (IFN)-γ expression, and the chickens immunized with the combination of pVAX1-gp85-LC3 and rapamycin showed the highest percentage of CD3+CD8+T lymphocytes. Based on our results, we suggest that stimulating autophagy can improve the efficacy of DNA vaccines and that our DNA vaccine shows the potential of being a candidate vaccine against ALV-J. This study provides a novel strategy for developing vaccines against ALV-J.