Immune escape of bovine parvovirus by VP1 inhibiting IFN-β production through the RIG-I-like receptor pathway / Escape inmunológico del parvovirus bovino mediante la inhibición de VP1 de la producción de IFN-β a través de la vía del receptor tipo RIG-I

Objective: The present study aimed to explore if bovine parvovirus (BPV) impacts beta interferon (IFN-β) production and to reveal further molecular mechanism of BPV immune escape. Method: The pCMV-Myc-BPV-VP1 recombinant plasmid was verified with both double-enzyme digestion and sequence. HEK 293 T cells were transfected with this recombinant protein and then infected with the vesicular stomatitis virus (VSV). Expression levels of IFN-β mRNA were detected using qPCR. Results: The expression level of BPV VP1 mRNA in the pCMV-Myc-BPV-VP1 group was significantly higher than those of the untreated group (UT) and pCMV-Myc vector group. BPV virus copies in bovine turbinate (BT) cells of the BPV-VP1 group were raised (P < 0.05) with an increment of 5.8 × 104. Expression levels of IFN-β mRNA of the BPV VP1 group in HEK 293 T cells were decreased (P < 0.01). Following treatment of TBK1 and IRF3(5D), IFN-β expression levels in HEK 293 T cells were depressed. Additionally, expression levels of TBK1, IRF3(5D), MDA5, and MAVS were less than those of the flag empty vector, respectively. Conclusion: pCMV-Myc-BPV-VP1 could heighten transcription levels of VP1 protein in BT cells, promote BPV proliferation, and ascend the production of IFN-β. Overexpression of pCMV-Myc-BPV-VP decreased IFN-β mRNA expression in HEK 293 T cells and inhibited IFN-β production induced by TBK1 and IRF3(5D). Furthermore, BPV VP1 obviously declined expression levels of TBK1, IRF3(5D), MDA5, and MAVS in the RIG-I-like receptor (RLR) pathway. Our findings revealed a novel mechanism evolved by BPV VP1 to inhibit type I IFN production and provided a solid scientific basis into the immunosuppression of BPV.(AU)

Immune escape of bovine parvovirus by VP1 inhibiting IFN-ß production through the RIG-I-like receptor pathway.

OBJECTIVE: The present study aimed to explore if bovine parvovirus (BPV) impacts beta interferon (IFN-ß) production and to reveal further molecular mechanism of BPV immune escape. METHOD: The pCMV-Myc-BPV-VP1 recombinant plasmid was verified with both double-enzyme digestion and sequence. HEK 293 T cells were transfected with this recombinant protein and then infected with the vesicular stomatitis virus (VSV). Expression levels of IFN-ß mRNA were detected using qPCR. RESULTS: The expression level of BPV VP1 mRNA in the pCMV-Myc-BPV-VP1 group was significantly higher than those of the untreated group (UT) and pCMV-Myc vector group. BPV virus copies in bovine turbinate (BT) cells of the BPV-VP1 group were raised (P < 0.05) with an increment of 5.8 × 104. Expression levels of IFN-ß mRNA of the BPV VP1 group in HEK 293 T cells were decreased (P < 0.01). Following treatment of TBK1 and IRF3(5D), IFN-ß expression levels in HEK 293 T cells were depressed. Additionally, expression levels of TBK1, IRF3(5D), MDA5, and MAVS were less than those of the flag empty vector, respectively. CONCLUSION: pCMV-Myc-BPV-VP1 could heighten transcription levels of VP1 protein in BT cells, promote BPV proliferation, and ascend the production of IFN-ß. Overexpression of pCMV-Myc-BPV-VP decreased IFN-ß mRNA expression in HEK 293 T cells and inhibited IFN-ß production induced by TBK1 and IRF3(5D). Furthermore, BPV VP1 obviously declined expression levels of TBK1, IRF3(5D), MDA5, and MAVS in the RIG-I-like receptor (RLR) pathway. Our findings revealed a novel mechanism evolved by BPV VP1 to inhibit type I IFN production and provided a solid scientific basis into the immunosuppression of BPV.

Bovine coronavirus nucleocapsid suppresses IFN-ß production by inhibiting RIG-I-like receptors pathway in host cells.

The present study aimed to explore if bovine coronavirus nucleocapsid (BCoV N) impacts IFN-ß production in the host cells and to reveal further molecular mechanism of BCoV pathogenesis. Human embryonic kidney (HEK) 293 T cells were transiently transfected with pMyc-BCoV-N recombinant plasmids, then infected with the vesicular stomatitis virus (VSV). Expression levels of beta interferon (IFN-ß) mRNA were detected using RT-qPCR. The results showed that BCoV N gene was 1347 bp that was consistent with the expected size. pMyc-BCoV-N recombinant protein was 1347 bp which was successfully transcribed and overexpressed in HEK 293 T cells. BCoV-N recombinant protein inhibited dose-dependently VSV-induced IFN-ß production (p < 0.01). MDA5, MAVS, TBK1 and IRF3 could promote transcription levels of IFN-ß mRNA. But, BCoV-N protein demoted IFN-ß transcription levels induced by MDA5, MAVS, TBK1 and IRF3. Furthermore, expression levels of MDA5, MAVS, TBK1 and IRF3 mRNAs were reduced in RIG-I-like receptor (RLR) pathway. In conclusion, BCoV-N reduced IFN-ß levels in RIG-I-like receptor (RLR) pathway in HEK 293 T cells which were induced by MDA5, MAVS, TBK1 and IRF3(5D). BCoV-N protein inhibited IFN-ß production and activation of RIG-I-like receptors (RLRs) signal pathway. Our findings demonstrated BCoV N protein is an IFN-ß antagonist through inhibition of MDA5, MAVS, TBK1 and IRF3(5D) in RLRs pathway, also revealed a new mechanism of BCoV N protein to evade host innate immune response by inhibiting type I IFN production, which is beneficial to developing novel prevention strategy for BCoV disease in the animals and humans.

FSH receptor binding inhibitor restrains follicular development and possibly attenuates carcinogenesis of ovarian cancer through down-regulating expression levels of FSHR and ERß in normal ovarian tissues.

OBJECTIVES: The current study aimed to investigate FSH receptor binding inhibitor (FRBI) effects in the expressions of FSH receptor (FSHR) and estrogen receptor-beta (ERß) in the mice ovaries at the gene and protein levels, also to find the potential efficacy of FRBI on suppressing ovarian cancer through down-regulating over-expression of FSHR and ERß in the normal ovarian tissues. METHODS: 180 female mice were randomized into six groups (n = 30). Mice of FRBI-1, FRBI-2 and FRBI-3, FRBI-4 were intramuscularly injected with FRBI of 10, 20, 30 and 40 mg/kg, respectively, for five consecutive days. The qPCR and Western blotting were used to determine expression levels of FSHR and ERß mRNAs and proteins in mouse ovaries. RESULTS: The ovarian cortex thickness (OCT) of the FRBI-4 group were less than that FSH group on day 30 (P < 0.05). The numbers of secondary follicles (SF) and the maximum transverse diameters (MTD) of secondary follicles of FRBI-3 and FRBI-4 groups were decreased as compared to FSH group (P < 0.05 or P < 0.01) by 24.11% and 27.47% on day 20 based on the control group (CG) levels. On day 15, the reductions of FSHR mRNA levels in FRBI-2, FRBI-3 and FRBI-4 were 27.78%, 29.37% and 43.65% (P < 0.05 or P < 0.01), respectively in comparison with CG. ERß and FSHR protein levels of FRBI-treated mice were gradually decreased as compared to and CG and FSH group. ERß protein level of FRBI-4 was less than that of CG on day 20 (P < 0.05). On days 15 and 20, estradiol (E2) concentrations of FRBI-2, FRBI-3 and FRBI-4 groups were lower than those of the CG and FSH group (P < 0.05 or P < 0.01). CONCLUSIONS: FRBI could reduce OCT and follicle numbers. A high dose of FRBI (30 mg/kg to 40 mg/kg) could suppress ovarian and follicular development, and attenuate expression levels of ERß and FSHR mRNAs and proteins in the ovaries, additionally inhibit E2 production. Therefore, FRBI will possibly be utilized to restrain the carcinogenesis of ovarian cancer by down-regulating overexpression of FSHR and ERß in the ovaries.

FSH receptor binding inhibitor influences estrogen production, receptor expression and signal pathway during in vitro maturation of sheep COCs.

Follicle-stimulating hormone (FSH) promotes secretion of follicle fluid and follicle development. FSH acts via cognate FSH receptor (FSHR). It remains unknown whether the supplement of FSH-receptor binding inhibitor (FRBI) into the in vitro maturation (IVM)medium influence the estrogen receptor expression and signal pathway of oocytes in sheep. The present study aimed to investigate FRBI effects on inositol trisphosphate (IP3) of oocytes and protein kinase A (PKA) of sheep granulosa cells, further to elucidate the signal pathway of FRBI effects. Cumulus-oocyte complexes (COCs) were recovered from antral follicles. COCs were cultured for 24 h in the IVM medium supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40 µg/mL) and FSH (10IU/mL). ELISA was used to measure the concentrations of estradiol (E2) and IP3 in the IVM medium. Western blotting was utilized to detect protein expression of ERß of COCs and protein kinase A (PKA) of granulosa cells. The results showed IP3 concentrations of FRBI-3 and FRBI-4 groups were less than that of CG and FSH groups at 22 h and 24 h (P < 0.05). PKA levels of FRBI-3 and FRBI-4 groups were significantly less than that of CG and FSH group (P < 0.05 or P < 0.01). Expression levels of ERß mRNA and protein of FRBI-treated groups were gradually decreased in comparison to CG and FSH group. The minimum value was detected in the FRBI-4 group. ERß protein level of the FRBI-4 group was significantly less than that of FSH group (P < 0.05). E2 concentrations of FRBI-treated groups were elevated as compared to CG, with the highest increment of FRBI-2 group (P < 0.05). Our results revealed a higher dose of FRBI reduced IP3 production. FRBI could suppress slightly expression levels of ERß mRNA and protein of COCs and PKA of granulosa cells, additionally increased E2 production of sheep COCs.

Subunit Vaccine Preparation of Bovine Rotavirus and Its Efficacy in Mice.

BACKGROUND: Rotaviruses (RV) are important viral diarrheal agents in calves. Vaccination is an optimum measure to prevent bovine rotaviruses (BRV) infection. However, little research on BRV VP7 vaccine has been done and currently there is no BRV vaccine. OBJECTIVE: To prepare a subunit vaccine of BRV and investigate its efficacy. METHODS: Total RNA was extracted from MA104 cells infected with bovine rotavirus (BRV) strain GSB01. BRV VP7 gene was amplified using real time fluorescence quantitative PCR (qPCR). The pEASY-T3-VP7 plasmid was digested using HindⅢ and BamHI restriction endonucleases, then recombined into the prokaryotic expression vector pET32a. The pET32a-VP7 and pET32a-VP7-LTB (heat-labile enterotoxin B subunit) were transformed into BL21 (DE3) competent cells of Escherichia coli, respectively, and induced with IPTG, then analyzed using SDS-PAGE. Sixty mice were randomly divided into three groups (n=20). Group A mice was used as His-tag control and mice in group B and C were inoculated with pET32a-VP7 and pET32a-VP7-LTB, respectively. VP7 IgG antibody titers and protection efficiency of pET32a-VP7-LTB were further determined in neonatal mice challenged with GSB01 BRV strain. RESULTS: SDS-PAGE analysis showed that the pET32a-VP7 was highly expressed in the BL21 (DE3) cells. PET32a-VP7 and pET32a-VP7-LTB protein could promote VP7 IgG antibody titer（8.33×103 vs. 17.26×103）in mice. Immunization protection ratios of pET32a-VP7 and pET32a-VP7-LTB proteins in the neonatal mice were 86.4% and 91.7%, respectively. CONCLUSION: The fusion protein of pET32a-VP7-LTB had excellent immunogenicity and protected mice from BRV infection. Our findings can be used for further developing of a high-efficiency subunit vaccine of BRV.

Effects of alarelin active immunity on FSHR expressions and bioinformatics of FSHR in ewes / 中国免疫学杂志

Objective:To investigate the effects of GnRH agonist on the expressions of FSHR proteins in pituitary and ovaries in ewes,also to analyse the bioinformatics characteristics of ovine pituitary FSHR.Methods: Forty-two prepubertal ewes (Ovis aries) were randomly assigned to six groups (n=7).The ewes in experimental group EG-Ⅰ,EG-Ⅱand EG-Ⅲwere subcutaneously injected with 200μg,300μg and 400 μg alarelin antigens twice (at day 0 and 14),respectively.Ewes in EG-Ⅳand EG-Ⅴwere injected sub-cutaneously with 200μg and 300μg alarelin antigen four times (at day 0,7,14 and 21),respectively.Ewes in the control group (CG) were subcutaneously injected with 2.0 ml solvent twice (at day 0 and 14).Fluorescence quantitative RT-PCR was used to detect gene expression of FSHR in pituitary glands.The expression of FSHR protein in the ovaries was detected using Western blot.Results:The 2-ΔΔCt values of GnRHR,FSHR and LHR mRNAs in the pituitary gland of EG were lower than that in control group (CG).The 2-ΔΔCt values in EG-Ⅳ( P<0.05 ) and EG-Ⅴ( P<0.05 ) were lower than those in EG-Ⅰand EG-Ⅱ.Expressions of FSHR proteins in EG ovaries increased.Levels of FSHR proteins in EG-Ⅳand EG-Ⅴ( P<0.05 ) were higher than CG.The length of sheep FSHR sequence of nucleotides were 1 091 bp,the homology of FSHR sequences was 100% with that reported in NCBI.The theory isoelectric point , half-life,unstable index,fat index,hydrophobic average were respectively 1.2 h,5.05,47.75,0.836 and 30.61.Conclusion:Alarelin active immunization can inhibit the expression of FSHR mRNA in the pituitary gland ,but increase FSHR expression in ovarian of ewes.FSHR is an unstable hydrophobic protein.