Bovine IFN Research Articles

Biological activity of recombinant bovine IFN-α and inhibitory effect on BVDV in vitro

Type I interferon has great broad-spectrum antiviral ability and immunomodulatory function, and its receptors are expressed in almost all types of cells. Bovine viral diarrhea virus (BVDV) is an important pathogen causing significant economic losses in cattle. In this study, a recombinant expression plasmid carrying bovine interferon-α(BoIFN-α)gene was constructed and transformed into E. coli BL21 (DE3) competent cells. SDS-PAGE and Westernblotting analysis showed that the recombinant BoIFN-α protein (rBoIFN-α) was successfully expressed. It is about 36KD and exists in the form of inclusion body. When denatured, purified and renatured rBoIFN-α protein stimulated MDBK cells, the expression of interferon stimulating genes (ISGs) such as ISG15, OAS1, IFIT1, Mx1 and IFITM1 were significantly up-regulated, and reached the peak at 12 h (P< 0.001). MDBK cells were infected with BVDV with moi of 0.1 and 1.0, respectively. The virus proliferation was observed after pretreatment with rBoIFN-α protein and post-infection treatment. The results showed that the denatured, purified and renatured BoIFN-α protein had good biological activity and could inhibit the replication of BVDV in MDBK cells in vitro, which provided a basis for BoIFN-α as an antiviral drug, immune enhancer and clinical application of BVDV.

Biological Activity of Optimized Codon Bovine Type III Interferon Expressed in Pichia pastoris.

Type III interferons (IFN-λs) exhibit potent antiviral activity and immunomodulatory effects in specific cells. Nucleotide fragments of the bovine ifn-λ (boifn-λ) gene were synthetized after codon optimization. The boifn-λ gene was then amplified by splicing using overlap extension PCR (SOE PCR), resulting in the serendipitous acquisition of the mutated boIFN-λ3V18M. The recombinant plasmid pPICZαA-boIFN-λ3/λ3V18M was constructed, and the corresponding proteins were expressed in Pichia pastoris with a high-level extracellular soluble form. Dominant expression strains of boIFN-λ3/λ3V18M were selected by Western blot and ELISA and cultured on a large scale, and the recombinant proteins purified by ammonium sulfate precipitation and ion exchange chromatography yielded 1.5g/L and 0.3 g/L, with 85% and 92% purity, respectively. The antiviral activity of boIFN-λ3/λ3V18M exceeded 106 U/mg, and they were neutralized with IFN-λ3 polyclonal antibodies, were susceptible to trypsin, and retained stability within defined pH and temperature ranges. Furthermore, boIFN-λ3/λ3V18M exerted antiproliferative effects on MDBK cells without cytotoxicity at 104 U/mL. Overall, boIFN-λ3 and boIFN-λ3V18M did not differ substantially in biological activity, except for reduced glycosylation of the latter. The development of boIFN-λ3 and comparative evaluation with the mutant provide theoretical insights into the antiviral mechanisms of boIFN-λs and provide material for therapeutic development.

Estrous cycle stage-dependent manner of type I interferon-stimulated genes induction in the bovine endometrium.

Interferon tau (IFN-τ) is a ruminant-specific type I IFN secreted by a conceptus before its attachment to the uterus. IFN-τ induces the expression of IFN-stimulated genes (ISGs) via the type I IFN receptor (IFNAR), which is composed of IFNAR1 and IFNAR2 subunits in the endometrium. However, expression patterns of IFNARs during the estrous cycle have not been reported. We hypothesized that the response to a type I IFN changes along with IFNARs and the IFN-regulatory factors (IRFs) driving transcription of IFN signal-related genes and modulating a type I IFN signal during the estrous cycle. We investigated the estrous cycle stage-dependent type I IFN induction of ISGs and expression patterns of IFN signal-related genes in bovine endometrial tissues. Endometrial tissue pieces collected from bovine uteri at each estrous stage (early, mid, and late) were cultured with or without recombinant bovine IFN-α or concentrated pregnant uterine flushing (PUF) on day 18 after confirming the presence of a conceptus. IFN-α and PUF each significantly increased the expression of ISGs in endometrial tissues. The induction levels of the typical ISGs (MX1-a and ISG15) were significantly higher at the mid stage and correlated with high expression of IRFs at the mid stage. The immunostaining of IFNARs showed strong fluorescence intensities in luminal and glandular epithelia at the early and mid stages. Collectively, these results suggest that the endometrium exhibits estrous cycle stage-dependent responsiveness to type I IFN that may be associated with the expression of IFNARs and IRFs for pregnancy recognition.

Extracellular expression and antiviral activity of a bovine interferon-alpha through codon optimization in Pichia pastoris

Interferons (IFNs) are the primary line of defense against infectious agents. In particular, IFN-α is an important antiviral cytokine and has a wide range of immune-modulating functions. Porcine and human IFN-α have been successfully prepared and play important roles in the prevention and therapy of viral diseases. To date, there has been limited applied research on bovine IFN-α. To achieve high-level expression of recombinant bovine IFN-α (bIFN-α) in Pichia pastoris for large-scale application, the bIFN-α gene was optimized and synthesized on the basis of codon bias of P. pastoris. Optimized bIFN-α (opti-bIFN-α) was successfully expressed in P. pastoris and directly secreted into the culture supernatant. The amount of extracellular soluble opti-bIFN-α was observed to be 200μg/mL in a shake flask. Expression efficiency of opti-bIFN-α was found to be about three times that of wild-type bIFN-α when the expression yield was compared at the same copies of the targeted gene. In addition, both the original cultural supernatant and purified opti-bIFN-α showed strong antiviral activity in MDBK cells (2×106AU/mL and 1×107AU/mg, respectively) and IBRS-2 cells (3×105AU/mL and 1.5×106AU/mg, respectively) against a recombinant vesicular stomatitis virus expressing the green fluorescence protein. In this study, we demonstrated high-level extracellular expression of opti-bIFN-α by P. pastoris. To the best of our knowledge, the opti-bIFN-α yield observed in this study is the highest to be reported to date. Our results demonstrated that the extracellular opti-bIFN-α with strong antiviral activity could be easily prepared and purified at a low cost and that it may be a potential biological therapeutic drug against bovine viral infections.

Type I and III IFNs Produced by Plasmacytoid Dendritic Cells in Response to a Member of the Flaviviridae Suppress Cellular Immune Responses.

The pestivirus noncytopathic bovine viral diarrhea virus (BVDV) can suppress IFN production in the majority of cell types in vitro. However, IFN is detectable in serum during acute infection in vivo for ∼5-7 d, which correlates with a period of leucopoenia and immunosuppression. In this study, we demonstrate that a highly enriched population of bovine plasmacytoid dendritic cells (DCs) produced IFN in response to BVDV in vitro. We further show that the majority of the IFN produced in response to infection both in vitro and in vivo is type III IFN and acid labile. Further, we show IL-28B (IFN-λ3) mRNA is induced in this cell population in vitro. Supernatant from plasmacytoid DCs harvested postinfection with BVDV or recombinant bovine IFN-α or human IL-28B significantly reduced CD4(+) T cell proliferation induced by tubercle bacillus Ag 85-stimulated monocyte-derived DCs. Furthermore, these IFNs induced IFN-stimulated gene expression predominantly in monocyte-derived DCs. IFN-treated immature DCs derived from murine bone marrow also had a reduced capacity to stimulate T cell proliferative responses to tubercle bacillus Ag 85. Immature DCs derived from either source had a reduced capacity for Ag uptake following IFN treatment that is dose dependent. Immunosuppression is a feature of a number of pestivirus infections; our studies suggest type III IFN production plays a key role in the pathogenesis of this family of viruses. Overall, in a natural host, we have demonstrated a link between the induction of type I and III IFN after acute viral infection and transient immunosuppression.

A preliminary assessment of new bovine and avian tuberculin in cattle by intra-dermal tests and whole blood interferon gamma assay

The present study reports ante-mortem diagnosis of bovine tuberculosis employing tuberculin test using a new tuberculin PPDB (M. bovis, 3/86) and interferon gamma assay in cattle whole blood culture stimulated using a commercial bovine IFN- γ assay kit. Dairy cattle (87) categorized in groups A, B and C were tested using tuberculin test and gamma interferon (IFN-γ). Fresh batches of bovine PPD (from M. bovis 3/86) and avian PPD (M. avium N–17) were prepared and were standardized by conforming to sterility, safety and potency. For tuberculin testing cervical single intradermal test (SIT) and caudal fold test (CFT) were performed using tuberculins at concentration of 1mg/ml and at the concentration of 0.3mg/ ml as a stimulating antigens in IFN-γ test. Out of 80 cattle (group A + B), 44 (55%) were reactor to tuberculin, 21 (26.25%) non reactor and 15 (18.75%) were inconclusive animals and by IFN-γ test 36 (45%) were M. bovis positive, 10 (12.5%) negative, 13 (16.25%) inconclusive and 21 (26.25%) were M. avium positive animals. In group C (not tuberculin tested) only 4 (57.14%) out of 7 cattle were positive. Relative sensitivity of IFN-γ test (63.88%) was higher than the tuberculin test (52.27%). Our study indicated that skin tests (CFT and SIT) and IFN-γ test can be used for successful control and eradication of bovine tuberculosis as IFN-γ test had higher sensitivity.

Molecular cloning and characterization of a novel bovine IFN-ε

A bovine IFN-ε (BoIFN-ε) gene was amplified from bovine liver genomic DNA consisting of a 463bp partial 5′UTR, 582bp complete ORF and 171bp partial 3′UTR, which encodes a protein of 193 amino acids with a 21-amino acid signal peptide and shares 61 to 87% identity with other species IFN-ε. Then BoIFN-ε gene was characterized, and it can be transcribed in EBK cells at a high level after being infected by VSV. Recombinant proteins were expressed in Escherichia coli and the antiviral activity was determined in vitro, which revealed that bovine IFN-ε has less antiviral activity than bovine IFN-α. In addition, an immunofluorescence assay indicated that BoIFN-ε expressed in MDBK cells could be detected by polyclonal antibody against BoIFN-ε. Furthermore, the BoIFN-ε gene can be constitutively expressed in the liver, thymus, kidney, small intestine and testis, but not in the heart. This study revealed that BoIFN-ε has the typical characteristics of type I interferon and can be expressed constitutively in certain tissue, which not only can be a likely candidate for a novel, effective therapeutic agent, but also facilitate further research on the role of bovine IFN system.

Phylogenetic Analysis of Bovine Interferon α

Interferon α is produced by the cells in response to the viral infection and play important role in viral infections. IFN-α coding sequence was amplified from the bovine lymphocytes collected from Bos indicus (Indian cattle breed-Hallikar) and cloned and sequenced. Nucleotide and the deduced amino acid sequence of the bovine IFN-α was compared with the sequences of different animals. The IFN-α nucleotide sequence of B. indicus shows 99.1 % similarity with Bos taurus, with changes at four amino acid positions. Comparison of the nucleotide and deduced amino acid sequence with other species has shown varied structural similarities.

Characterization of the biological activities and physicochemical characteristics of recombinant bovine interferon-α14

A BoIFN-α gene that included signal sequence was amplified from bovine liver genomic DNA. The gene was named BoIFN-α14 according to the position at which the encoded gene of the bovine IFN was located in the bovine genome. The sequence included a 23-amino acid signal peptide and 166-amino acid mature peptide. The structural characteristics and phylogenetic relationships of the BoIFN-α14 gene were analyzed. A recombinant mature BoIFN-α14 (rBoIFN-α14) was expressed in the yeast Pichia pastoris. Antiviral activity, antiproliferation activity and physicochemical characteristics were determined in vitro. Recombinant BoIFN-α14 exhibited a considerable antiviral activity against Vesicular stomatitis virus (VSV), which was neutralized by a rabbit polyclonal anti-rBoIFN-α antibody, and could inhibit cell proliferation. It was also found to be highly sensitive to trypsin and stable at pH 2.0 or 65°C. This study revealed that rBoIFN-α14 has the typical characteristics of IFN-α and can be used for both research and industrial application.

Characterization of bovine interferon α1: expression in yeast Pichia pastoris, biological activities, and physicochemical characteristics.

A bovine interferon α (BoIFNα) gene that included signal sequence was amplified from bovine liver genomic DNA. The gene was named BoIFN-α1 according to the position at which the encoded gene of the bovine IFN was located in the bovine genome. The sequence included a 23-amino-acid signal peptide and a 166-amino-acid mature peptide. The structural characteristics and phylogenetic relationships of the BoIFN-α1 gene were analyzed. A recombinant mature BoIFN-α1 (rBoIFN-α1) was expressed in the yeast Pichia pastoris. Physicochemical characteristics and antiviral activity were determined in vitro. Recombinant BoIFN-α1 was found to be highly sensitive to trypsin and stable at pH 2.0 or 65°C. It also exhibited antiviral activity, which was neutralized by a rabbit anti-rBoIFNα polyclonal antibody. This study revealed that rBoIFN-α1 has the typical characteristics of IFNα and can be used for both research and industrial application.

ウシ妊娠認識物質インターフェロン（IFN）-τによる黄体・免疫細胞への刺激効果の検証：全身系での早期妊娠認識の可能性

ウシ妊娠認識物質インターフェロン（IFN）-τによる黄体・免疫細胞への刺激効果の検証：全身系での早期妊娠認識の可能性

Water buffalo (Bubalus bubalus)interferon-αgenes: cloning, expression and antiviral activities

AbstractInterferon(IFN)-α genes were cloned from genomic DNA of Fuan and Fuzhong water buffaloes by PCR, and the PCR products were inserted into a pQE30 vector to construct recombinant expression plasmids. Sequence analysis showed that both clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids (aa). They were defined as two new subtypes, with 91.6–94.2% identity at the amino acid level by comparison with eight previously published bovine IFN-α subtypes. Results of SDS-PAGE and Western blotting showed that each of the recombinant proteins was expressed in inclusion bodies inEscherichia coliwith molecular weight of 20 kDa and the recombinant proteins were 25% of the whole proteins. Inclusion bodies were denatured and renatured with urea and the antiviral activities of the recombinant buffalo IFN-α (rBuIFN-α) were 105U/mg and 106U/mg in CEF/VSV and MDBK/VSV cell lines, respectively. Additionally, rBuIFN-α had good effects against challenge byinfectious bovine rhinotracheitis virus. The rBuIFN-α are potential biological agents for the prevention and treatment of various kinds of bovine viral disease.

A comprehensive survey for polymorphisms in the bovine IFN-gamma gene reveals a highly polymorphic intronic DNA sequence allowing improved genotyping of Bovinae.

This study aimed to identify interferon-gamma (IFN-gamma) gene variants in cattle for diagnostic purposes. Therefore, the entire bovine IFN-gamma gene (BoIFNG) and 2605 bp of its promoter DNA were sequenced. The BoIFNG DNA sequence conforms to the published part of Bo-IFN-gamma cDNA. Primer extension experiments show the presence of a 5' extension of exon 1 by 42 nucleotides (nt). One SINE element (Bov-A2) is located in the 5'-region, and two SINE elements (Bov-tA, Bov-B) are contained in the 3'-region of BoIFNG. The variants were detected by comparative sequence analysis of PCR amplicons from different bovine species. Four polymorphic mononucleotide repeats are situated in the promoter and in intron 1. Four distinct series of single nucleotide polymorphisms (SNP) were found in functionally important regions of BoIFNG. The region between the two intron 1 microsatellites contains the highest density of SNPs in Bos taurus breeds. One G-T transversion in the coding region of exon 1 causes a Gly(14) to Val(14) exchange in the BoIFNG signal peptide of different bovine species. A G-A transition in exon 2 encodes a Ser(19) to Asn(19) change in the mature protein of the Tibetan yak. Genotyping of randomly sampled Holstein Friesian cows at selected SNPs and of both intron 1 microsatellites revealed two dominant BoIFNG microhaplotypes. The detected SNPs improve the recently reported genotyping system of cattle.

Respiratory syncytial virus (RSV) nonstructural (NS) proteins as host range determinants: a chimeric bovine RSV with NS genes from human RSV is attenuated in interferon-competent bovine cells.

Bovine respiratory syncytial virus (BRSV) escapes from cellular responses to alpha/beta interferon (IFN-alpha/beta) by a concerted action of the two viral nonstructural proteins, NS1 and NS2. Here we show that the NS proteins of human RSV (HRSV) are also able to counteract IFN responses and that they have the capacity to protect replication of an unrelated rhabdovirus. Even combinations of BRSV and HRSV NS proteins showed a protective activity, suggesting common mechanisms and cellular targets of HRSV and BRSV NS proteins. Although able to cooperate, NS proteins from BRSV and HRSV showed differential protection capacity in cells from different hosts. A chimeric BRSV with HRSV NS genes (BRSV h1/2) was severely attenuated in bovine IFN competent MDBK and Klu cells, whereas it replicated like BRSV in IFN-incompetent Vero cells or in IFN-competent human HEp-2 cells. After challenge with exogenous IFN-alpha, BRSV h1/2 was better protected than wild-type BRSV in human HEp-2 cells. In contrast, in cells of bovine origin, BRSV h1/2 was much less resistant to exogenous IFN than wild-type BRSV. These data demonstrate that RSV NS1 and NS2 proteins are major determinants of host range. The differential IFN escape capacity of RSV NS proteins in cells from different hosts provides a basis for rational development of attenuated live RSV vaccines.

Detection of [formula omitted] -2 and [formula omitted] -γ m [formula omitted] expression in bovine milk cells at the late stage of the lactation period with [formula omitted] - [formula omitted

The expression of bovine interferon-gamma (IFN -γ) and interleukin-2 (IL -2) in the late stage of the lactation period in milk cells was detected with reverse transcriptase-polymerase chain reaction (RT - PCR). IFN -γ was detected with all primer concentrations, 0·2, 0·5 and 1μM.IL -2 however, was faintly detected only with 0·5μM primer concentration. It was shown thatIFN -γ m RNA expression can be demonstrated clearly with RT - PCR at the late stage of the lactation period. In contrast, IL -2 was weakly expressed at this stage of the lactation period. The work is important for substantiating the therapeutic use of recombinant bovine IFN -γ and IL -2 and for helping to reveal the activity of cytokine in the mammary gland at the late stage of the lactation period.

Development of chicken antibodies to bovine interferon α

We have developed chicken polyclonal antibody to bovine interferon α (IFNα). Five hundred μg of recombinant bovine IFNα suspended with complete Freund' s adjuvant was used in the first immunization round. A suspension of the same amount of IFNα and incomplete Freund's adjuvant was used for all subsequent boosters. The antibody was purified from egg yolks using polyethylene glycol precipitation. The first reactive antibody appeared several weeks after the first immunization. The antibody is specific for IFNα in immunoblotting, it is also useful in ELISA and immunohistochemistry. This method provides a fast, cheap and efficient alternative to development of monoclonal antibodies to conserved mammalian antigens.

IFN gamma and IL-10 production by hepatic lymph node and peripheral blood lymphocytes in Fasciola hepatica infected sheep.

The present study was designed to determine whether IFN gamma and IL-10 were secreted by peripheral blood mononuclear cells (PBMC) and hepatic lymph node mononuclear cells (HLNMC) from 17 Fasciola hepatica infected sheep after in vitro stimulation by Fasciola hepatica excretory-secretory products (FhESP) during the first six weeks of two different infections. The IFN gamma concentration in mononuclear cells (MC) culture supernatants was determined by sandwich ELISA. The presence of IL-10 in MC culture supernatants was determined by the capacity of these to inhibit, with or without monoclonal antibodies to IL-10, the NO production of recombinant bovine IFN gamma (rBoIFN gamma) activated monocytes. FhESP-stimulated PBMC and HLNMC produced IFN gamma in culture supernatants from DPI 7 (day post-infestation 7) to DPI 14. Culture supernatants of PBMC stimulated with FhESP first induced an increase in the NO production of rBoIFN gamma activated monocytes. After 2 or 3 weeks of infection, MC culture supernatant reduced the NO production of rBoIFN gamma activated monocytes. From DPI 35, anti-IL-10 antibodies addition restored or enhanced NO production. During sheep fasciolosis, IFN gamma production increased only until the first two weeks post infection. IL-10 could be secreted throughout the first six weeks of infection and could inhibit monocytes from DPI 35.

Cloning and expression of caprine interferon-gamma

Caprine interferon-gamma (IFN- γ) cDNA was cloned from mitogen stimulated peripheral blood mononuclear cell (PBMC) RNA utilizing the reverse transcription-polymerase chain reaction (RT-PCR). The cDNA open reading frame (ORF) is 498 bp, encoding a putative 166 amino acid (aa) protein (19 327 Da). The predicted aa sequence homology of caprine IFN- γ and the corresponding ovine, bovine and cervine cytokine is 98.8%, 95.2% and 92.8%, respectively. IFN- γ cDNA was subcloned and expressed in two different plasmids under the control of either the human cytomegalovirus (CMV) immediate early promoter or the caprine arthritis–encephalitis virus long terminal repeat (CAEV LTR). Recombinant caprine IFN- γ (rCaIFN- γ) secreted by transfected COS-7 cells shared at least two antigenic epitopes with recombinant bovine IFN- γ (rBoIFN- γ) and exhibited biological activity in the vesicular stomatitis virus (VSV) cytopathic effect reduction assay. In-vivo expression of IFN- γ cDNA promoted by the CAEV LTR was confirmed by the intramuscular (IM) injection of Balb/C mice with plasmid followed by Western blot analysis of mouse serum against purified rCaIFN- γ produced in E. coli

Purification of an atypical bovine interferon induced in response to heat shock in bovine cultured cells. Characterization in comparison with the classical α, β and γ interferon

Heat-shock induced factor (HSIF) was excreted by bovine MDBK cells during their recovery period after a heat shock. This factor has the capacity to induce 2′.5′ oligoadenylate synthetase activity, an enzyme generally induced by interferon treatment ( J Biol Chem (1987) 262, 4806–4811). We have observed that an antiviral state was also produced in response to heat shock, HSIF was purified 10 000-fold and different techniques showed a copurification of both activities. Certain properties of HSIF were established, such as its molecular mass (45 kDa) and isoelectric point (6.8). This cytokine was acid-sensitive as IFNγ(type II) and temperature labile contrarily to α, β and γ bovine IFN. Immunoprecipitation and comparative chromatography on lectines or polynucleotides established that HSIF was structurally different from the three classes of bovine IFN. Moreover, two-dimensional electrophoresis and comparative analysis of [ 35S] methionine-labeled proteins induced by HSIF, α, β or γ bovine IFN showed that HSIF induces a specific set of proteins. Taken together, all these results strongly suggest that HSIF is a new atypical bovine interferon induced in response to heat shock.

Role of protein kinase C in the inhibitory action of trophoblast interferons on expression of the oxytocin receptor in sheep endometrium

PhosphoIipid/Ca(2+) -dependent protein kinase C (PKC) and oxytocin receptor were measured in sheep endometrial explants after culture for up to 96 h. Oxytocin receptor binding and PKC activity were reduced by up to 90% in explants exposed to recombinant ovine trophoblast interferon (rolFN-τ), recombinant bovine IFN-α(1) or ovine conceptus secretory proteins (a source of IFN-τ). Inhibition occurred in both caruncular and intercaruncular endometrium taken between days 7 and 10 of the oestrous cycle and in intercaruncular (but not caruncular) endometrium on day 6. Down-regulation of PKC by continued exposure of expiants to 4β-phorbol myristate acetate, or treatment with PKC inhibitors reduced both oxytocin receptor binding and PKC activity by up to 70%. Tyrosine kinase inhibitors were ineffective. Addition of oxytocin or progesterone, which reduce oxytocin receptor bindingin vivo, also lowered oxytocin receptor bindingin vitro in the absence of any effect on PKC. The data indicate that IFN-τ inhibits oxytocin receptor synthesis by a mechanism involving PKC inhibition, but that a non-PKC pathway also operates to control oxytocin receptor binding in non-pregnant animals. These conclusions were supported by measuring PKC activity and oxytocin receptor binding in endometrium without culture. Prolonged exposure of the endometrium to IFN-τin vivo may lead to PKC down regulation by a mechanism analogous to that involved in the action of continuous activation by agonist, and this may represent one function of the prolonged secretion of IFN-τ over a 10-day period in early pregnancy.