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Misgurnus anguillicaudatus (M. anguillicaudatus) is a widely cultivated fish. However, in M. anguillicaudatus breeding, the frequent cold stress during daily breeding could induce immune suppression and increase the risk of infection, causing serious economic loss. Based on existing findings, CpG Oligonucleotides (CpG-ODNs) may be an ideal protective agent for low temperature fish breeding, performing anti-infective when faced with cold stress with cold shock proteins Y box binding proteins (YBX). Although YBX has pleiotropic functions, its roles in CpG-ODNs-mediated immunity (especially under cold situations) remain largely unexplored. To clarify the relationship among them, we identified the YBX1/YBX2 in M. anguillicaudatus and analyzed using a series of bioinformatics methods. After that, we immunized the fish with 3 types of CpG-ODNs and challenged with Aeromonas hydrophila (A. hydrophila). Here we showed that the best anti-bacterial effect of CpG-B was accompanied by the significant upregulation of YBX1. And the detection of the YBX1 downstream effectors confirmed that CpG-B induced the YBX1-mediated Th1 oriented responses to A. hydrophila by regulation of the NLRP3 (Caspase-A/-B), IL-1β, IL-12 and IFN-γ. Afterwards, we found that under cold stress, CpG-B can activate the NLRP3 and NF-κB pathways through YBX1, a key mediator of anti-A. hydrophila in CpG-B immunization. In this study, we demonstrated CpG-B protection against infection in low temperature, and its interaction with YBX1, expanded the research of CpG-ODN under cold stress, and provided a new CpG-ODN application for low temperature fish farming.

Clostridium butyricum (C. butyricum) is a probiotic that could promote animal growth and protect gut health. So far, current studies mainly keep up with the basic biological functions of C. butyricum, missing the effective strategy to further improve its protective efficiency. A recent report about C. butyricum alleviating intestinal injury through epidermal growth factor receptor (EGFR) inspired us to bridge this gap by porcine epidermal growth factor (EGF) overexpression. Lacking a secretory overexpression system, we constructed the recombinant strains overexpressing pEGF in C. butyricum for the first time and obtained 4 recombinant strains for highly efficient secretion of pEGF (BC/pPD1, BC/pSPP, BC/pGHF, and BC/pDBD). Compared to the wild-type strain, we confirmed that the expression level ranges of the intestinal development-related genes (Claudin-1, GLUT-2, SUC, GLP2R, and EGFR) and anti-inflammation-related gene (IL-10) in IPECs were upregulated under recombinant strain stimulation, and the growth of Staphylococcus aureus and Salmonella typhimurium was significantly inhibited as well. Furthermore, a particular inhibitor (stattic) was used to block STAT3 tyrosine phosphorylation, resulting in the downregulation on antibacterial effect of recombinant strains. This study demonstrated that the secretory overexpression of pEGF in C. butyricum could upregulate the expression level of EGFR, consequently improving the intestinal protective functions of C. butyricum partly following STAT3 signal activation in IPECs and making it a positive loop. These findings on the overexpression strains pointed out a new direction for further development and utilization of C. butyricum. KEY POINTS: • By 12 signal peptide screening in silico, 4 pEGF overexpression strains of C. butyricum/pMTL82151-pEGF for highly efficient secretion of pEGF were generated for the first time. • The secretory overexpression of pEGF promoted the intestinal development, antimicrobial action, and anti-inflammatory function of C. butyricum. • The overexpressed pEGF upregulated the expression level of EGFR and further magnified the gut protective function of recombinant strains which in turn partly depended on STAT3 signal pathway in IPECs.

Multiple recent outbreaks of highly pathogenic H5N8 viruses originating in aquatic birds frequently occurred in most European countries, Russia, South Korea, and Japan during the winter of 2020–21, and one zoonotic event of poultry workers infected with novel H5N8 viruses were reported in Russia. Strikingly, these novel H5N8 viruses had emerged and been co-circulating in wild birds and poultry in multiple provinces of China during 2020–21. In China, the population of aquatic birds has risen significantly in the past twenty years, and China is regarded as the largest reservoir for influenza viruses carried in aquatic birds across the globe. Hence, the co-circulation of these novel H5N8 viruses poses an alarming threat to not only poultry industry but also human health. In this study, we sequenced full-length genomes of these H5N8 viruses circulating in China. Phylogenetic analysis demonstrated that poultry-origin H5N8 viruses in China fell within wild birds-origin clade 2.3.4.4b H5N8 viruses from Europe during 2020–21, and notably, were genetically closely related to human-infecting H5N8 viruses in Russia. Moreover, they possessed several molecular markers associated with mammalian adaption. Bayesian coalescent analysis showed that these H5N8 viruses might have introduced into China during June–September 2020, suggesting that these H5N8 viruses might have introduced via wild bird migration or poultry trade. Besides, we also found that the effective population size of clade 2.3.4.4b H5N8 viruses dramatically increased during the winter season of 2020/21, as is consistent with previous increase of genetic diversity during the winter seasons of 2013/14 and 2016/17, which indicated that the wild bird migration accelerates the genetic diversity of these H5N8 viruses during the winter season of 2020/21. Notably, these novel H5N8 viruses were lethal to chickens and mice, highly transmissible to ducks, and were antigenically distinct from 2.3.4.4h H5 viruses circulating in China, posing considerable threats to public health. Our findings offer novel insights into the evolution and risk assessment of H5N8 viruses during the winter season of 2020–21.

To evaluate the immunity of the adenovirus recombinant rAd-HA-GFP encoding an H3N2 swine influenza virus hemagglutinin.Thee groups of 6-week-old pigs (5 pigs per group) were vaccinated intramuscularly with the recombinant, one group was immunized with 10(-8) TCID50 recombinant adenovirus rAd-HA-GFP, the other groups were vaccinated with 2 x 10(-8) TCID50 and 4 x 10(-8) TCID50. A control groups (5 pigs) were vaccinated with recombinant adenovirus rAd-GFP. Virus-specific hemagglutination-inhibition (HI) antibody was detected by 2-6 weeks post vaccination. Compared the difference of vaccinated intramuscularly, intragastric administration and intranasal inoculation, three groups of different HI antibody pigs and one control group were challenged with a virulent H3N2 field virus.The results showed that pigs in the groups given higher immunizing dose were developed higher levels HI antibody. All of vaccinated intramuscularly, intragastric administration and intranasal inoculation could produce HI antibody, but the titer of vaccinated intramuscularly was higher significantly (P0.01). Three groups of pigs (5 pigs per group) which HI titers were 1:80, 1:160, 1:320 respectively were challenged with a virulent H3N2 field virus, include the negative control group. The immunization efficacy was evaluated by clinical signs, the rate of virus isolation and HI titer. It suggested that immunological response induced by rAd-HA-GFP could resist attack of SIV effectively.The adenovirus vaccines rAd-HA-GFP are efficacious for SIV and have the additional advantage over commercial vaccines that suckling piglets have no pre-existing maternally-derived antibody to block early life vaccination.