The Inactivated gE/TK Gene-Deleted Vaccine Against Pseudorabies Virus Type II Confers Effective Protection in Mice and Pigs.

The highly virulent and antigenic variant of Pseudorabies virus (PRV) that emerged from classical Bartha-K61-vaccinated pig herds has caused substantial economic losses to the swine industry in China since 2011. A safe and more effective vaccine is most desirable. In this study, a gE/TK gene-deficient PRV, namely, HD/c, was constructed based on a PRV type II DX strain isolated from a commercial vaccine-immunized farm and the HD/c-based inactivated vaccine was formulated and evaluated for its safety, immunogenicity, and protective efficacy in mice and piglets. The resulting PRV HD/c strain has a similar growth curve to the parental DX strain. After vaccination, the inactivated HD/c vaccine did not cause any visible gross pathological or histopathological changes in the tissues of mice and piglets and provided rapid and potent protection against the challenge of the classical and variant PRVs at day 21 post-vaccination in mice. A single immunization of 108.5TCID50 inactivated PRV HD/c strain-elicited robust immunity with high titer of neutralizing antibody and provided complete protection from the lethal challenge of PRV DX strain in piglets. These results indicated that the inactivated PRV HD/c vaccine with the deletion of gE/TK genes was a safe and effective PRV vaccine candidate for the control of PRV.

Anti-inflammatory effect of Bifidobacterium animalis subsp. lactis A6 on DSS-induced colitis in mice.

AIMS: This study aimed to investigate the protective effect of Bifidobacterium animalis subsp. lactis A6 on dextran sodium sulphate (DSS)-induced colitis in C57BL/6J mice. METHODS AND RESULTS: Mice were randomly divided into three groups (n = 8 per group). Each group was administered with PBS (Control and DSS group) or B. lactis A6 with a dosage of ~4.0 × 109  CFU day-1 (DSS + A6 group) for 21 consecutive days. The DSS and DSS + A6 group mice were ad libitum drinking 2.5% DSS water during day 15-21, while the Control group mice were given normal water. The administration of B. lactis A6 significantly inhibited DSS-induced bodyweight loss and colon shortening (p < 0.001), but showed no significant influence on the spleen enlargement (p > 0.05). The intestinal barrier integrity was improved by reducing colonic damage, recovering mucus layer loss and enhancing tight junction expression including ZO-1, occludin and claudin-1. In addition, B. lactis A6 attenuated the oxidative stress by decreasing MDA and increasing SOD and GSH levels in colon tissues. Moreover, B. lactis A6 suppressed DSS-induced inflammatory responses via downregulating TNF-α, IL-1ß and IL-6 levels and upregulating IL-10 level in colon tissues. CONCLUSION: B. lactis A6 effectively alleviated DSS-induced colitis by maintaining intestinal barrier integrity, reducing oxidative stress and inhibiting inflammatory responses. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that B. lactis A6 could act as a candidate probiotic for UC treatment.

Transcriptome analysis revealed growth phase-associated changes of a centenarian-originated probiotic Bifidobacterium animalis subsp. lactis A6.

BACKGROUND: The physiology and application characteristics of probiotics are closely associated with the growth phase. Bifidobacterium animalis subsp. lactis A6 is a promising probiotic strain isolated from the feces of a healthy centenarian in China. In this study, RNA-seq was carried out to investigate the metabolic mechanism between the exponential and the stationary phase in B. lactis A6. RESULTS: Differential expression analysis showed that a total of 815 genes were significantly changed in the stationary phase compared to the exponential phase, which consisted of 399 up-regulated and 416 down-regulated genes. The results showed that the transport and metabolism of cellobiose, xylooligosaccharides and raffinose were enhanced at the stationary phase, which expanded carbon source utilizing profile to confront with glucose consumption. Meanwhile, genes involved in cysteine-cystathionine-cycle (CCC) pathway, glutamate dehydrogenase, branched-chain amino acids (BCAAs) biosynthesis, and Clp protease were all up-regulated in the stationary phase, which may enhance the acid tolerance of B. lactis A6 during stationary phase. Acid tolerance assay indicated that the survival rate of stationary phase cells was 51.07% after treatment by pH 3.0 for 2h, which was 730-fold higher than that of 0.07% with log phase cells. In addition, peptidoglycan biosynthesis was significantly repressed, which is comparable with the decreased growth rate during the stationary phase. Remarkably, a putative gene cluster encoding Tad pili was up-regulated by 6.5 to 12.1-fold, which is consistent with the significantly increased adhesion rate to mucin from 2.38% to 4.90% during the transition from the exponential phase to the stationary phase. CONCLUSIONS: This study reported growth phase-associated changes of B. lactis A6 during fermentation, including expanded carbon source utilizing profile, enhanced acid tolerance, and up-regulated Tad pili gene cluster responsible for bacterial adhesion in the stationary phase. These findings provide a novel insight into the growth phase associated characteristics in B. lactis A6 and provide valuable information for further application in the food industry.

iTRAQ-based quantitative subcellular proteomic analysis of Avibirnavirus-infected cells.

Infectious bursal disease virus (IBDV) enters the host cells via endocytic pathway to achieve viral replication in the cytoplasm. Here, we performed LC-MS/MS coupled with isobaric tags for relative and absolute quantification labeling of differentially abundant proteins of IBDV-infected cells using a subcellular fractionation strategy. We show that the viral infection regulates the abundance and/or subcellular localization of 3211 proteins during early infection. In total, 23 cellular proteins in the cytoplasmic proteome and 34 in the nuclear proteome were significantly altered after virus infection. These differentially abundant proteins are involved in such biological processes as immune response, signal transduction, RNA processing, macromolecular biosynthesis, energy metabolism, virus binding, and cellular apoptosis. Moreover, transcriptional profiles of the 25 genes corresponding to the identified proteins were analyzed by quantitative real-time RT-PCR. Ingenuity Pathway Analysis clustered the differentially abundant proteins primarily into the mTOR pathway, PI3K/Akt pathway, and interferon-ß signaling cascades. Confocal microscopy showed colocalization of the viral protein VP3 with host proteins heterogeneous nuclear ribonucleoprotein H1, nuclear factor 45, apoptosis inhibitor 5, nuclear protein localization protein 4 and DEAD-box RNA helicase 42 during the virus infection. Together, these identified subcellular constituents provide important information for understanding host-IBDV interactions and underlying mechanisms of IBDV infection and pathogenesis.

Binding of the pathogen receptor HSP90AA1 to avibirnavirus VP2 induces autophagy by inactivating the AKT-MTOR pathway.

Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa α [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon viral recognition, a direct connection between HSP90AA1 and the AKT-MTOR pathway trigger autophagy, a critical step for controlling infection.