FSH receptor binding inhibitor depresses carcinogenesis of ovarian cancer via decreasing levels of K-Ras, c-Myc and FSHR.

The present study aimed to explore FSH receptor binding inhibitor (FRBI) effects on the levels of c-Myc, K-Ras and VEGF related to ovarian cancer, to evaluate the mRNA and protein levels of FSHR in the cumulus-oocyte complex (COCs). COCs were cultured for 24 h in the in vitro maturation (IVM) media replenished with 0, 10, 20, 30 and 40 µg/mL FRBI. Contents of c-Myc, K-Ras, VEGF, cAMP and IP3 in IVM media were detected with ELISA kits, respectively. The results indicated that the levels of FSHR protein and mRNA were determined with Western blotting. C-Myc contents of four FRBI + FSH-treated groups (COM groups) were reduced after IVM of COCs. C-Myc concentrations of COM-3 group was lower than the FSH group (p < .05). K-Ras and IP3 contents of COM-4 were decreased as compared to FSH group (p < .05). Expression levels of FSHR mRNAs and proteins in COM-4 group were smaller than that of FSH group. This study revealed that FRBI treatment could decrease c-Myc and K-Ras levels in the IVM medium fluids, and depress the FSHR levels of COCs. Expression levels of FSHR mRNAs and proteins of COM-4 group were significantly decreased. FRBI exerted its action via the signal pathway of IP3 and cAMP.

MicroRNAs regulate granulosa cells apoptosis and follicular development - A review.

OBJECTIVE: MicroRNAs (miRNAs) are the most abundant small RNAs. Approximately 2,000 annotated miRNAs genes have been found to be differentially expressed in ovarian follicles during the follicular development (FD). Many miRNAs exert their regulatory effects on the apoptosis of follicular granulosa cells (FGCs) and FD. However, accurate roles and mechanism of miRNAs regulating apoptosis of FGCs remain undetermined. METHODS: In this review, we summarized the regulatory role of each miRNA or miRNA cluster on FGCs apoptosis and FD on the bases of 41 academic articles retrieved from PubMed and web of science and other databases. RESULTS: Total of 30 miRNAs and 4 miRNAs clusters in 41 articles were reviewed and summarized in the present article. Twenty nine documents indicated explicitly that 24 miRNAs and miRNAs clusters in 29 articles promoted or induced FGCs apoptosis through their distinctive target genes. The remaining 10 miRNAs and miRNAs of 12 articles inhibited FGCs apoptosis. MiRNAs exerted modulation actions by at least 77 signal pathways during FGCs apoptosis and FD. CONCLUSION: We concluded that miRNAs or miRNAs clusters could modulate the apoptosis of GCs (including follicular GCs, mural GCs and cumulus cells) by targeting their specific genes. A great majority of miRNAs show a promoting role on apoptosis of FGCs in mammals. But the accurate mechanism of miRNAs and miRNA clusters has not been well understood. It is necessary to ascertain clearly the role and mechanism of each miRNA or miRNA cluster in the future. Understanding precise functions and mechanisms of miRNAs in FGCs apoptosis and FD will be beneficial in developing new diagnostic and treatment strategies for treating infertility and ovarian diseases in humans and animals.

Immunogenicity of adenovirus and DNA vaccines co-expressing P39 and lumazine synthase proteins of Brucella abortus in BALB/c mice.

Brucella poses a great threat to animal and human health. Vaccination is the most promising strategy in the effort to control Brucella abortus (B. abortus) infection, but the currently used live vaccines interfere with diagnostic tests and could potentially result in disease outbreak. Therefore, new subunit vaccines and combined immunization strategies are currently under investigation. In this study, immunogenicity and protection ability of a recombinant adenovirus and plasmid DNA vaccine co-expressing P39 and lumazine synthase proteins of B. abortus were evaluated based on the construction of the two molecular vaccines. Four immunization strategies (single adenovirus, single DNA, adenovirus/DNA, DNA/adenovirus) were investigated. The results showed that the immunization strategy of DNA priming followed by adenovirus boosting induced robust humoral and cellular immune responses, and it significantly reduced the numbers of B. abortus in a mouse model. These results suggest that it could be a potential antigen candidate for development of a new subunit vaccine against B. abortus infection.

FSHR and LHR Expression and Signaling as Well as Maturation and Apoptosis of Cumulus-Oocyte Complexes Following Treatment with FSH Receptor Binding Inhibitor in Sheep.

BACKGROUND/AIMS: Currently, it remains unknown whether FSH receptor binding inhibitor (FRBI) influences follicular development and reproduction functions in humans and animals. The present study aimed to investigate FRBI effects on in vitro maturation (IVM) and apoptosis of cumulus-oocyte complexes (COCs) of sheep, to determine the effect of FRBI on mRNA and protein levels of FSHR and LHR in COCs, and to elucidate the signal pathway of FRBI effects. METHODS: COCs were in vitro cultured for 24h in the IVM media supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40µg/mL) and FSH (10IU/mL). The harvested COCs were observed under an inverted microscope and maturation rates of COCs were determined. Real time RT-PCR and Western blotting were utilized to detect mRNA and protein levels of FSHR and LHR. The concentrations of FSH, LH and caspase-3 were determined using especial ELISA kits for sheep, respectively. RESULTS: Maturation rates of COCs decreased gradually as FRBI concentrations increased from 0 to 40µg/mL, reaching a bottom value of 23.76% of the FRBI-4 group. The maximal apoptosis rate was detected in the FRBI-4 group. IP3 contents of FRBI-3 and FRBI-4 groups were reduced as compared to control group (CG) and FSH groups (P<0.05). Levels of FSHR protein of FRBI-3 and FRBI-4 groups as well as LHR protein of FRBI-4 group were significantly less than that of CG and FSH group. FSH contents of four FRBI treatment groups were gradually decreased along with the supplementation doses of FRBI. Caspase-3 contents of FRBI groups were reduced with a maximum reduction of the FRBI-2 group. CONCLUSION: Our results revealed supplement of FRBI into IVM media could dose-dependently decrease the maturation rate and increase apoptosis rate of sheep COCs. A lower dose of FRBI treatment slightly promoted IP3 production, but a higher dose of FRBI reduced IP3 production. FRBI suppressed the mRNA and protein expression levels of FSHR and LHR in sheep COCs. Our study will help to therapy effectively ovarian diseases, improve ovarian and follicular functions, and further to promote fertility of humans and animals.

Triptorelin and cetrorelix induce immune responses and affect uterine development and expressions of genes and proteins of ESR1, LHR, and FSHR of mice.

CONTEXT: GnRH immunity can reduce the expression of pituitary GnRH levels, and cause the changes in reproductive behaviors. It is unclear whether triptorelin (TRI) and cetrorelix (CET) immunity influences uterine development and expression of follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and estradiol receptor 1 (ERS1) in the uterus. OBJECTIVE: The study investigated the effects of active immunity of GnRH agonist and antagonist on uterine development, microstructures, expression of hormone receptors mRNAs, and proteins in uteri. MATERIALS AND METHODS: One hundred and five mice were assigned into CET, TRI, and control groups (CG). Mice in CET-1, CET-2, and CET-3 (n = 15) were subcutaneously injected with 10, 20, and 40 µg CET antigens for seven days, respectively. Mice in TRI-1, TRI-2, and TRI-3 were injected with 10, 20, and 40 µg TRI antigens for seven days, respectively. The qPCR and Western blot were implemented to determine expressions of ESR1, LHR and FSHR mRNAs, and proteins. RESULTS: Compared with CG, the uterine weights of CET-1, CET-2, and CET-3 increased by 42.86, 62.86, and 10.00% on day 35 (p < 0.05), respectively. Uterine weights of TRI-2, TRI-3 reduced by 28.57% and 11.43% (p < 0.05), respectively. The uterine cavity in CET-1, CET-2, and CET-3 increased; the uterine wall became thick. The cytoplasm of endometrial epithelial cells (EEC) increased slightly. In TRI group, the uterine wall thinned. Uterine cavity became narrow slightly in TRI-1. Numbers of uterine glands reduced. The endometrium epithelial thickness (EET) in CET-1 and CET-2 increased by 68.21% and 79.46% (p < 0.05), respectively. EET in TRI-1 was decreased by 13.69%. Uterine wall thicknesses (UWT) in CET-1 and CET-2 were higher than CG, with the increment of 28.59% and 30.72%. UWT of TRI-1, TRI-2, and TRI-3 reduced by 29.35, 15.36, and 14.41%, respectively. Expressions of ESR1, FSHR, and LHR mRNAs in CET and TRI mice increased. ESR1 and FSHR protein levels increased in all experimental mice (p < 0.05), with a maximum of TRI-3. LHR protein levels of the CET decreased. LHR protein levels of TRI group increased, with a maximum of TRI-3 (p < 0.05). ESR1 protein level had significant negative correlations to mRNA expressions of ESR1, LHR, and FSHR. CONCLUSIONS: CET immunity promoted the uterine development, improved EET and UWT, and also promoted the expressions of ESR1 and FSHR protein levels. It lessened the LHR protein levels. TRI immunity blocked EET and UWT, inhibited uterine growth and development. The efficacy of CET immunity was more obvious than TRI.

Mammalian orthoreovirus capsid protein σ3 antagonizes RLR-mediated antiviral responses by degrading MAVS.

Mammalian orthoreovirus (MRV) outer capsid protein σ3 is a multifunctional protein containing a double-stranded RNA-binding domain, which facilitates viral entry and assembly. We reasoned that σ3 has an innate immune evasion function. Here, we show that σ3 protein localizes in the mitochondria and interacts with mitochondrial antiviral signaling protein (MAVS) to activate the intrinsic mitochondria-mediated apoptotic pathway. Consequently, σ3 protein promotes the degradation of MAVS through the intrinsic caspase-9/caspase-3 apoptotic pathway. Moreover, σ3 protein can also inhibit the expression of the components of the RNA-sensing retinoic acid-inducible gene (RIG)-like receptor (RLR) signaling pathway to block antiviral type I interferon responses. Mechanistically, σ3 inhibits RIG-I and melanoma differentiation-associated gene 5 expression is independent of its inhibitory effect on MAVS. Overall, we demonstrate that the MRV σ3 protein plays a vital role in negatively regulating the RLR signaling pathway to inhibit antiviral responses. This enables MRV to evade host defenses to facilitate its own replication providing a target for the development of effective antiviral drugs against MRV. IMPORTANCE: Mammalian orthoreovirus (MRV) is an important zoonotic pathogen, but the regulatory role of its viral proteins in retinoic acid-inducible gene-like receptor (RLR)-mediated antiviral responses is still poorly understood. Herein, we show that MRV σ3 protein co-localizes with mitochondrial antiviral signaling protein (MAVS) in the mitochondria and promotes the mitochondria-mediated intrinsic apoptotic pathway to cleave and consequently degrade MAVS. Furthermore, tryptophan at position 133 of σ3 protein plays a key role in the degradation of MAVS. Importantly, we show that MRV outer capsid protein σ3 is a key factor in antagonizing RLR-mediated antiviral responses, providing evidence to better unravel the infection and transmission mechanisms of MRV.

miR-21, miR-125b, and let-7b contribute to the involution of atretic follicles and corpus lutea in Tibetan sheep ovaries.

Follicular granulosa cells (FGCs) are crucial for ovarian follicle functions, and miRNAs are differentially expressed at various stages of follicular developments. In this study, we confirmed that miR-21, miR-125b, and let-7b were located in FGCs/luteal cells by in situ hybridization experiments. Moreover, miR-21 and miR-125b expressions were upregulated in late corpus lutea (CL) and atretic follicles (AF); let-7b expression was increased in early AF. After transfected with inhibitor or mimic of miRNAs in FGCs, we found that FGCs apoptosis was decreased in the miR-21-mi group but increased in the miR-125b-mi group using flow cytometry. mRNA and protein expression levels were determined for apoptosis-related factors (e.g., Bcl-2 and Bax), the potential target genes of miRNAs (e.g., SMAD7, SP1, and STAT3), hormone receptors (e.g., FSHR and LHR), and genes related to hormone secretion (e.g., CYP19, CYP11, and 3ßHSD). The protein levels of SMAD7 were decreased in the miR-21-mi group but opposite to SP1 and FSHR. In the let-7b-mi group, Bcl-2, SMAD7, and FSHR were suppressed but not Bax, CYP11, and 3ßHSD. However, hormone secretion was not changed in the supernatant of transfected FGCs. This study provides information about ovarian miRNAs to improve the fertility in Tibetan sheep.

Pseudorabies Virus ICP0 Abolishes Tumor Necrosis Factor Alpha-Induced NF-κB Activation by Degrading P65.

Nuclear factor κB (NF-κB) is involved in a wide range of innate immune activities in host cells and serves as an important component of a host's immunity system. To survive in infected cells, viruses have evolved intricate strategies to evade the host immune response. Pseudorabies virus (PRV) is a member of the alpha herpesvirus family and is capable of causing reproductive and neurological dysfunction in pigs. PRV has a large DNA genome and therefore has the ability to encode numerous proteins that modulate host innate immune responses. In the present study, we demonstrated that the PRV-encoded immediate early protein ICP0 inhibits the tumor necrosis factor alpha (TNF-α)-mediated NF-κB signaling pathway. An in-depth study showed that ICP0 protein was able to limit NF-κB activation and decreased the expression of inflammatory cytokines interleukin-6 (IL-6) and interleukin 8 (IL-8). In addition, ICP0 blocked the activation of NF-κB through interacting with p65, degrading its protein expression and limiting its phosphorylation. PRV protein ICP0 is shown for the first time to enable escape from innate immune response through the regulation of NF-κB during PRV infection. These results illustrate that PRV ICP0 is able to block NF-κB activation. This mechanism may represent a critical role in the early events leading to PRV infection.

Corrigendum to "Determine the Role of FSH Receptor Binding Inhibitor in Regulating Ovarian Follicles Development and Expression of FSHR and ERα in Mice".

[This corrects the article DOI: 10.1155/2018/5032875.].

Receptor Binding Inhibitor Suppresses Carcinogenesis of Cervical Cancer by Depressing Levels of FSHR and ERß in Mice.

BACKGROUND: FSH Receptor Binding Inhibitor (FRBI) blocked the binding of FSH to FSHR. Our initial study revealed FRBI reduced the maturation rate, enhanced the apoptosis of sheep Cumulus-Oocyte Complex (COCs). Little is known about whether FRBI modulates ERß and FSHR levels in the normal uterine and cancerous tissues. The present study aimed to evaluate the FRBI effects on the expressions of Estrogen Receptor-beta (ERß) and FSH receptor (FSHR) in the uteri. METHODS: 150 mice were assigned to FRBI+FSH (COM), FSH and control groups (CG). Mice of COM-1, COM-2 and COM-3 groups were simultaneously intramuscularly injected with 500, 750 and 1000 µg FRBI with 10 IU FSH, respectively for five days. Western blotting and qPCR were utilized to determine the expression of ERß and FSHR. RESULTS: In comparison with FSH group, uterine lumen and glands of COM groups became narrow. The uterine wall and endometrial epithelium were thinned, and uterine lumen became narrow. Epithelial cells were decreased. Uterine wall thicknesses of COM-1, COM-2 and COM-3 groups were reduced by 6.49%, 14.89% and 15.69% on day 30 as compared with FSH group. Uterine perimetrium thicknesses of COM-1, COM-2 and COM-3 groups were reduced by 16.17%, 17.93% and 19.92% on day 20 in comparison with FSH group. Levels of FSHR mRNAs and proteins of COM-1, COM-2 and COM-3 groups were less than FSH group on days 20 and 30 (P<0.05). ERß protein of COM-3 group was less than FSH group. Serum estradiol (E2) and FSH concentrations of COM-2 and COM-3 were lower than that of FSH group on day 30. CONCLUSION: FRBI could decrease UWT and UPT, also block the uterine development, decline expression levels of ERß and FSHR protein. Additionally, FRBI reduced the secretion of secretion of FSH and E2. Downregulating expression of FSHR and ERß may be a potential treatment regimen for cervical cancer patients.

FSH receptor binding inhibitor up-regulates ARID1A and PTEN genes associated with ovarian cancers in mice.

Experiments were conducted to determine if the follicle-stimulating hormone (FSH) receptor binding inhibitor (FRBI) impacts the expression levels of AT-rich interactive domain-containing protein 1A (ARID1A) and phosphatase and tensin homolog (PTEN) in ovaries and blood, as well as expressions of follicle-stimulating hormone cognate receptor (FSHR) gene and proteins. Mice in FRBI-10, FRBI-20, FRBI-30, and FRBI-40 groups were intramuscularly injected with 10, 20, 30, and 40 mg FRBI/kg, respectively, for five consecutive days. Western blotting and qRT-PCR were utilized to determine expression levels of ARID1A and PTEN proteins and mRNAs. Serum ARID1A and PTEN concentrations of the FRBI-40 group were higher than the control group (CG) and FSH group (P<0.05). FSHR mRNA levels of FRBI-20, FRBI-30, and FRBI-40 groups were lower than that of CG and FSH groups on day 15 (P<0.05 or P<0.01). Expression levels of FSHR proteins of FRBI-30 and FRBI-40 groups were lower than those of CG and FSH groups (P<0.05). Levels of ARID1A and PTEN proteins of the FRBI-30 group were greater than CG on days 20 and 30 (P<0.05). FRBI doses had significant positive correlations to levels of ARID1A and PTEN proteins. Additionally, ARID1A and PTEN had negative correlations to FSHR mRNAs and proteins. A high dose of FRBI could promote the expression levels of ARID1A and PTEN proteins in ovarian tissues. FRBI increased serum concentrations of ARID1A and PTEN. However, FRBI depressed expression levels of FSHR mRNAs and proteins in mouse ovaries.

Expression Levels of Follicle-Stimulating Hormone Receptor and Implication in Diagnostic and Therapeutic Strategy of Ovarian Cancer.

BACKGROUND: Follicle-stimulating hormone receptor (FSHR) has been shown to be expressed in ovarian cancer. METHODS: Here we have summarized the potential therapeutic and diagnostic implication of FSHR in the ovarian cancers based on a review of the literature. RESULTS: Current research indicates that FSHR comprises several variants: FSHR-1, FSHR-2, FSHR-3 and FSHR-4. Only FSHR-1 and FSHR-3 have biological roles. Although the level of FSHR differs in ovarian cancer tissues, few quantitative correlations have so far been reported on the expression levels of FSHR and carcinogenesis and progression of cancers. CONCLUSION: A comprehensive understanding of the role of FSHR in the ovarian cancers may help the search for novel therapeutic and diagnostic regimens and improve the management of cancer patients.

Determine the Role of FSH Receptor Binding Inhibitor in Regulating Ovarian Follicles Development and Expression of FSHR and ERα in Mice.

Mice of FRBI-1, FRBI-2, and FRBI-3 groups were intramuscularly injected with 20, 30, and 40mg/kg, respectively, for five consecutive days. Ovarian weights of three FRBI groups were reduced in comparison with FSH group. Ovarian cortex thicknesses (OCT) of the FRBI-3 group were less than that of the FSH group (P<0.05). As compared to FSH group, there were fewer numbers of secondary follicles (SFs) and mature follicles (MF) on the ovaries of FRBI-treated mice numbers of primary follicles (PFs) and SFs also decreased. In FRBI-3 mice, we found that the primordial follicles (POF) were scarcer, the follicles developed poorly, and granulosa cells became apoptosis. SF numbers of FRBI-2 and FRBI-3 groups were less than that of the FSH group on day 20 (P<0.05). Maximum longitudinal diameter (MLD) and transverse diameter (MTD) of three FRBI groups became decreased during the experiment. MLD and MTD of the FRBI-3 group were smaller than FSH group. Levels of FSHR mRNA and protein were less than that of CG and FSH group (P<0.05). ERα protein levels of FRBI group and serum concentrations of FSH and estradiol (E2) in the FRBI-treated mice were decreased when compared to CG and FSH group. In conclusion, FSH treatment could increase the numbers of SF and MF, enhance follicle development, reduce the numbers of SF and MF, and depress the follicular development of mice. Furthermore, FRBI declined the mRNA and protein levels of ERα and FSHR in the ovaries and dropped serum concentrations of FSH and E2 of mice.

FSH receptor binding inhibitor impacts K-Ras and c-Myc of ovarian cancer and signal pathway.

The present study aimed to investigate FSHreceptor binding inhibitor (FRBI) effects on relative factors (K-Ras, c-Myc and Vascular endothelial growth factor (VEGF)) to ovarian cancer, and expression levels of FSH receptor (FSHR) mRNAs and proteins in the cumulus-oocyte complex (COCs), to determine changes of protein kinase A (PKA) in sheep granulosa cells, further to elucidate signaling pathway of FRBI action. COCs were cultured in vitro for 24h under supplementation of varying concentrations of FRBI (0, 10, 20, 30 and 40µg/mL) or FSH (10IU/mL). Concentrations of K-Ras, c-Myc, VEGF, cAMP and FSH were detected in IVM media fluids, respectively. The results showed that the concentrations of c-Myc, K-Ras and FSH of FRBI groups were gradually reduced with the increase of FRBI doses. VEGF level of the FRBI-4 group was significantly greater than control group (CG). Expression levels FSHR mRNA and protein and PKA of FRBI-3 and FRBI-4 groups were less than that of CG or FSH group (P<0.05 or P<0.01). Inositol trisphosphate (IP3) concentrations of FRBI-3 and FRBI-4 groups were less than FSH group (P<0.05). FRBI administration doses had significant negative correlations to levels or concentrations of K-Ras, c-Myc, VEGF, FSHR mRNA and protein and PKA protein. K-Ras had significant positive correlations with FSHR mRNA and protein and PKA protein. In conclusion, FRBI could promote the production of VEGF of sheep COCs. Higher doses of FRBI (30 and 40µg/mL) suppressed the production of c-Myc and K-Ras, and declined FSH concentrations in the IVM medium fluid, and decreased the expressions of FSHR at the gene and protein levels, additionally attenuated expression of PKA protein in the granulosa cells.

FSH receptor binding inhibitor up-regulates ARID1A and PTEN genes associated with ovarian cancers in mice

Experiments were conducted to determine if the follicle-stimulating hormone (FSH) receptor binding inhibitor (FRBI) impacts the expression levels of AT-rich interactive domain-containing protein 1A (ARID1A) and phosphatase and tensin homolog (PTEN) in ovaries and blood, as well as expressions of follicle-stimulating hormone cognate receptor (FSHR) gene and proteins. Mice in FRBI-10, FRBI-20, FRBI-30, and FRBI-40 groups were intramuscularly injected with 10, 20, 30, and 40 mg FRBI/kg, respectively, for five consecutive days. Western blotting and qRT-PCR were utilized to determine expression levels of ARID1A and PTEN proteins and mRNAs. Serum ARID1A and PTEN concentrations of the FRBI-40 group were higher than the control group (CG) and FSH group (P<0.05). FSHR mRNA levels of FRBI-20, FRBI-30, and FRBI-40 groups were lower than that of CG and FSH groups on day 15 (P<0.05 or P<0.01). Expression levels of FSHR proteins of FRBI-30 and FRBI-40 groups were lower than those of CG and FSH groups (P<0.05). Levels of ARID1A and PTEN proteins of the FRBI-30 group were greater than CG on days 20 and 30 (P<0.05). FRBI doses had significant positive correlations to levels of ARID1A and PTEN proteins. Additionally, ARID1A and PTEN had negative correlations to FSHR mRNAs and proteins. A high dose of FRBI could promote the expression levels of ARID1A and PTEN proteins in ovarian tissues. FRBI increased serum concentrations of ARID1A and PTEN. However, FRBI depressed expression levels of FSHR mRNAs and proteins in mouse ovaries.

Specific detection of bovine coronavirus N protein with TaqMan probe qRT-PCR

Background: Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infectionmay cause losses to production by reduced weight gain and milk yield of the infected animals. Several methods have beenapplied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRTPCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technicalassay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene.Materials, Methods & Results: The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR)for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collectionand processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from theknown sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimizedincluding concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, therecombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collectedfrom diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCRwere 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assaycould specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL.However, universal PCR could detect only 4.72 × 103 copies/μL...

Specific detection of bovine coronavirus N protein with TaqMan probe qRT-PCR

Background: Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infectionmay cause losses to production by reduced weight gain and milk yield of the infected animals. Several methods have beenapplied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRTPCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technicalassay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene.Materials, Methods & Results: The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR)for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collectionand processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from theknown sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimizedincluding concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, therecombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collectedfrom diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCRwere 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assaycould specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL.However, universal PCR could detect only 4.72 × 103 copies/μL...(AU)

Synchronous detection of BPV and BVDV with duplex Taqman qPCR method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrheain dairy herds. BPV is a member of Bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirusgenus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes accordingto the 5’UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viraldiarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the presentstudy was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV.Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed invitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, andthen used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV.Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probeqRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplificationconditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTRgene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. Forclinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealingtemperature was achieved in 43.2 for duplex BPV and BVDV. Dynamic curves and standard...

Synchronous detection of BPV and BVDV with duplex Taqman qPCR method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrheain dairy herds. BPV is a member of Bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirusgenus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes accordingto the 5UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viraldiarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the presentstudy was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV.Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed invitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, andthen used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV.Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probeqRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplificationconditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTRgene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. Forclinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealingtemperature was achieved in 43.2 for duplex BPV and BVDV. Dynamic curves and standard...(AU)

Genotyping of calves rotavirus in China by reverse transcription polymerase chain reaction.

To investigate the epidemical characteristics and genotype distributions of bovine rotavirus (BRV) in China, 195 fecal samples were collected from calves with diarrhea in China. Fecal samples were detected for rotavirus A antigen using ELISA. The positive samples were screened for VP7 and VP4 by RT-PCR. G serotyping and P genotyping were conducted on 53 VP7 and VP4 positive samples using RT-PCR. The results showed that 82 samples were found positive for BRV. 752bp, 660bp and 285bp bands were amplified for G-typing. 478bp, 375bp and 361bp bands were amplified for P-typing. The G6 and G10 serotypes were 29 (54.7%) and 8 (15.1%) in positive samples for VP7. P[5] and P[11] genotypes were 28 (52.8%) and 10 (18.9%) in the positive samples for VP4. The main combinations of BRV G serotype and P genotype were G6P[5] (28.3%), G6P[5]P[11] (5.7%), G10P[5] (5.7%) and G10G6P[5] (5.7%), respectively. Other combinations (including untypable) of G serotype and P genotype were 54.6%. The dominant G serotype and P genotype were G6 and P[5] respectively. The predominant combination of G and P serotypes was G6P[5]. This has significance for establishing the preventive measures against diarrhea caused by group A rotaviruses in cattle.