Recombinant porcine interferon-gamma activates in vitro porcine adherent mononuclear cells to produce interleukin 1

The effect of recombinant porcine interferon-gamma (rPoIFN gamma) on in vitro production of interleukin 1 (IL-1) by porcine blood monocytes was studied. Three-day-old cultures of porcine adherent blood mononuclear cells were treated by doses of rPoIFN gamma for 3 or 6 days before lipopolysaccharide (LPS)-induction. While rPoIFN gamma alone had no effect, a combined treatment by rPoIFN gamma and LPS enhanced the IL-1 secretory potential of adherent mononuclear cells and, to a lesser extent, the level of cell-associated IL-1. The IL-1 activity was neutralized by anti porcine IL-1 alpha and beta antisera. These results demonstrate that rPoIFN gamma has immunomodulatory effects in vitro on porcine monokine production.     

Antiviral activity against transmissible gastroenteritis virus, and cytotoxicity, of natural porcine interferons alpha and beta.

Porcine interferon (POIFN)-alpha prepared in primed peripheral blood leukocyte cultures induced with Newcastle disease virus and POIFN-beta from PK-15 cell cultures induced with polyinosinic:polycytidylic acid were partially purified by precipitation with potassium thiocyanate and anion exchange chromatography. Mean purification factors in terms of units of POIFN per mg of protein, of 37 and 12 were obtained for POIFN-alpha and POIFN-beta respectively. In yield reduction assays in swine testis and pig kidney cell cultures, POIFN-alpha and POIFN-beta had greater antiviral activity against vesicular stomatitis virus than against transmissible gastroenteritis virus (TGEV). The antiviral effects were greater at higher concentrations of interferon (IFN), and when the IFN treatments were continued postinfection. Porcine interferon-beta showed greater antiviral activity against TGEV than POIFN-alpha, but this may have been partly due to cytotoxicity. There were no major differences in the antiviral activities of crude and partially purified IFN preparations. Both types of IFN showed antiviral activity against TGEV in yield reduction assays in porcine intestinal explant and intestinal epithelial cell cultures. Crude POIFN-beta was found to be rapidly cytotoxic, especially in porcine cells, and some fractions of partially purified POIFN-beta were also cytotoxic. The cytotoxicity of POIFN-beta was partially neutralized by antibodies against human IFN-beta, but human IFN-beta was not cytotoxic for porcine or bovine cells.     

Serotyping and antigenic comparison of some animal rotaviruses isolated in China.

Eight strains of rotaviruses isolated from diarrheal animals (4 from calves and 4 from piglets) in China were compared by serotyping with reference animal rotavirus strains (bovine NCDV, porcine OSU and simian SA-11 and human rotavirus Wa strain). Two-way cross neutralization test showed no antigenic difference between all 4 local strains of bovine rotavirus (BRV007, BRV014, HN-7 and BRV6555) and reference NCDV, so they belonged to rotavirus serotype 6 (bovine rotavirus serotype 1 or NCDV-serotype). Meanwhile, the four strains of Chinese porcine rotavirus could be determined into 2 different serotypes. One (Li99) was neutralised to a high titer with the antiserum against reference OSU virus and probably related to OSU (serotype 5 or porcine serotype 1). The other three strains (Lin71, Nan86 and Jiang150) were antigenically obviously different from Li99 and did not react with the antiserum against OSU. They were tentatively considered as porcine rotavirus serotype 2. All the strains of bovine and porcine rotavirus did not cross-neutralise with simian SA-11 and human Wa strain. There was also no antigenic relationship between bovine rotaviruses and porcine rotaviruses.     

Effect of recombinant DNA-derived bovine and human interferons on replication of bovine herpesvirus-1, parainfluenza-3, and respiratory syncytial viruses

Antiviral effects of recombinant DNA-derived bovine (Bo) and human (Hu) interferons (IFN) on the replication of bovine herpesvirus-1, parainfluenza-3, and respiratory syncytial viruses were studied. Bovine monolayer cultures were treated with recombinant DNA-produced Bo IFN-alpha 1, Bo IFN-beta 2, Hu IFN-alpha A, or Hu IFN-alpha A/D and then challenge exposed with bovine herpesvirus-1, bovine parainfluenza-3 virus, bovine respiratory syncytial virus, or vesicular stomatitis virus. Treatment with each IFN reduced the viral yield for each of these viruses, compared with that of control cultures.     

三种药物对PRRSV的体外抑制试验

选取干扰素、黄芪多糖、利巴韦林作为抗病毒药物,利用MTT法与细胞病变(CPE)抑制试验,通过PRRSV感染Marc-145细胞、病毒在细胞中复制、药物直接和病毒作用三个环节,对药物的作用效果进行评价。结果显示,3种药物体外对PRRSV的都具有抑制作用,干扰素、黄芪多糖体外对PRRSV具有明显的阻断作用,利巴韦林体外对PRRSV具有杀灭作用。因此,这3种药物在细胞水平上均有抑制病毒的作用,为防控PRRS提供了理论依据。     

Contribution of molecular biology to the study of the porcine interferon system

We have performed molecular studies on the pig interferon (IFN) system (i) to analyse the role played by endogenous IFN in neonatal viral enteritis such as transmissible gastroenteritis and possibly to obtain, via recombinant DNA technology, a new anti-infectious and immunomodulatory agent in this species, (ii) to characterize the structure and biological functions of the IFN-like antiviral activity produced by the porcine embryo at the time of implantation in the uterus. By probing porcine genomic libraries with human and porcine IFN-alpha probes to isolate related genes, we have shown that the porcine IFN-alpha multigene family included, like several other mammalian species, two subfamilies of related but distinct genes. Class I subfamily contains at least 11 loci, located on chromosome no. 1, among which nine have been cloned and two (potentially functional) sequenced. Class II subfamily, which is specifically expressed by the embryo of ruminants before implantation, contains at least seven loci among which six have been cloned. One of the sequenced class I loci: PoIFN-alpha 1 encodes a 189 amino acids (AA) preprotein. After removal of the sequence encoding the putative signal peptide (23 N-terminal AA) this gene was inserted into an Escherichia coli bicistronic expression vector allowing intracellular synthesis of mature porcine IFN-alpha 1 (methionyl IFN-alpha 1). Expression of the recombinant protein was optimized by insertion of a seven base pairs long random synthetic sequence in the intercistronic region, followed by cloning in E. coli and immunodetection of clones expressing high amounts of recombinant protein. The E. coli strain obtained produced high levels of a 18,000 Da protein exhibiting the same in vitro overall biological properties as leucocyte derived porcine IFN (LeuIFN). However, it had a stronger antiviral effect on porcine cells than LeuIFN. After immunoaffinity purification to a specific activity of 5-10 x 10(7) International Units (IU)/mg of protein, pharmacokinetic and pharmacological studies were realized to determine the in vivo half life of this rIFN-alpha in the pig. These experiments revealed no major toxic effects in newborn (given 5 x 10(6) IU/kg) or adult (1 X 10(6) IU/kg) pigs. A significant pyrogenic effect (+ 1.5 degrees C) was noted only in the adults.     

Cloning and expression of bovine and porcine interleukin-2 in baculovirus and analysis of species cross-reactivity

The cDNAs encoding bovine and porcine interleukin-2 (IL-2) have been expressed using the baculovirus Autographa californica nuclear polyhedrosis virus as a vector in insect cells. Insect cells infected with recombinant viruses secreted bovine and porcine IL-2 into the culture medium, with biological activities for maintaining the proliferation of homologous cells. When the activities of these two IL-2 proteins and commercially available human IL-2 were tested on heterologous cells differences were found. Recombinant bovine (rb)IL-2 only supported the growth of bovine lymphocytes and was not active on human, mouse or porcine lymphocytes. Recombinant porcine (rp)IL-2 and recombinant human (rh)IL-2 supported the proliferation of human, bovine, porcine and murine cells. However, the proliferative response of human lymphocytes to rpIL-2 was only 50% of that seen with rhIL-2. Sequence differences at the predicted p55 and p75 contact binding sites may explain this.     

Evaluation of cytotoxicity and antiviral activity of recombinant human interferon alfa-2a and recombinant human interferon alfa-B/D hybrid against bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, and vesicular stomatitis virus in vitro

OBJECTIVE: To evaluate cytotoxicity and antiviral activity of recombinant human interferon alfa-2a and recombinant human interferon alfa-B/D hybrid against cytopathic and noncytopathic bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBRV), and vesicular stomatitis virus (VSV) in vitro. SAMPLE POPULATION: Primary bovine testicular cells and Mardin Darby bovine kidney cells. PROCEDURES: To evaluate cytotoxicity, cells were added to serial dilutions of each interferon. To evaluate antiviral activity of each interferon, interferons were serially diluted 1:10, and tissue culture cells were added; virus was then added at 3 time points. Prevention of viral infection by interferon was defined as failure to induce cytopathologic effect for VSV, IBRV, and cytopathic BVDV and failure to detect virus immunohistochemically for cytopathic and noncytopathic BVDV. RESULTS: No evidence of cytotoxicity in either cell line was detected after incubation with interferon alfa-2a or interferon alfa-B/D. However, reduced growth rates of tissue culture cells were detected for each interferon when undiluted interferon was tested. Comparable and profound antiviral activities against cytopathic and noncytopathic BVDV were evident for each interferon. Interferon alfa-2a and interferon a-B/D had comparable antiviral activities against VSV. Neither interferon had antiviral activity against IBRV. CONCLUSIONS AND CLINICAL RELEVANCE: The safety and marked in vitro antiviral activity against noncytopathic BVDV, cytopathic BVDV, and VSV suggest that interferons alfa-2a and alfa-B/D may be useful for treatment of natural disease after infection with these viruses.     

Effect of human interferon on vesicular stomatitis virus released from bovine embryonic kidney cells

Human lymphoblastoid interferon (IFN) had an antiviral activity in bovine embryonic kidney cells that resulted in the release of vesicular stomatitis virus (VSV) particles with decreased infectivity. The inhibition was dose dependent and the cells were highly sensitive to human IFN. Examination of the proteins of VSV released from bovine cells after IFN treatment showed a reduction in the glycoprotein. Electron microscopic studies revealed a large number of VSV particles with characteristic spike-like surface projections released from nontreated cells. There was a reduction in the number of mature virions produced in IFN-treated cells and the virions lacked the characteristic surface projections.     

Immune production of interferon by cultured peripheral blood mononuclear cells from calves infected with BHV1 and PI3 viruses

Peripheral blood mononuclear cells (PBMC) from calves infected with bovine herpesvirus type 1 (BHV1) or parainfluenza 3 virus (PI3) were cultured in vitro in the presence of inactivated specific antigen presented on MDBK cells. In the presence of inactivated antigen, PBMC from both BHV1-infected and control calves produced interferon (IFN)-alpha in 24 hour cultures. Altering the culture conditions did not result in the detection of immune-specific IFN produced by mononuclear cells from BHV1-infected calves. However, spontaneous IFN was detected in the absence of antigen in 24 hour cultures from infected animals: this IFN was pH 2 labile and completely neutralised by antiserum to recombinant bovine IFN-gamma. Spontaneous IFN-gamma production was only seen in calves following a second BHV1 inoculation, given four to seven weeks after the primary dose. In contrast PBMC cultures from PI3 virus-infected calves did not produce IFN-gamma spontaneously, but did so in cultures which contained inactivated PI3 antigen. Mononuclear cells from control animals failed to produce either IFN-alpha or -gamma when cultured with inactivated PI3 virus. IFN-gamma was detected in PBMC cultures after the primary infection, with no increase in production occurring following subsequent PI3 virus inoculations. Immunospecific production of IFN-gamma provides a simple method for monitoring cell-mediated immunity in BHV1- and PI3 virus-infected calves and can be used for evaluating the efficacy of vaccines against these viruses.     

Induction and characterization of swine β interferon

Newcastle Disease Virus (NDV) strain “H” and Polyinosinic-Polycytidylic acid (Poly I:C) were used for interferon (IFN) induction in secondary pig kidney cells. A functional IFN system was detected and characterized. A wide similarity with the correspondent human and bovine systems was appreciated, with particular regard to the kinetics of synthesis. A glycosylated protein was essential for activity in bovine cells, but not in swine cells. Poly I:C proved to be a very weak inducer, even in conditions which promote IFN synthesis in other cell substrata. β IFN from secondary pig kidney cells was very effective against Swine Vesicular Disease Virus (SVDV), whereas no activity was detected against porcine Rotavirus; Aujeszky's disease virus, BUK strain, proved to be of intermediate sensitivity. The results of these latter experiments are discussed, with regard to the cells used and to the IFN sensitivity of the tested viruses.     

A serological comparison of some animal herpesviruses

Bovine herpesvirus 1 (BHV-1) isolates (Cooper-type strain 4975 and Oxford) were compared in neutralization tests with the bovine herpesvirus 4 (BHV-4) isolate (85/16 TV) and the herpesviruses of red deer (D2839/1) and goats (E/CH). Hyperimmune antiserum was prepared in rabbits against the plaque-selected viruses and endpoint and kinetic neutralization test were made. BHV-4 was clearly different from the other four viruses. The closely-related BHV-1 strains were also related in these tests to the red deer herpesvirus. The Oxford strain seemed rather closer antigenically than the Cooper-type strain to the red deer herpesvirus. Antiserum to the caprine herpesvirus failed to neutralize either BHV-1 strain or red deer virus, but antiserum to the Cooper-type and red deer herpesviruses did neutralize caprine virus to a limited extent.     

禽类基因工程重组干扰素研究进展

近年来 ,多种禽类干扰素基因已被克隆和在大肠杆菌中表达。禽类基因工程重组干扰素在抗马立克病毒、劳斯肉瘤病毒、新城疫病毒、传染性法氏囊病病毒、传染性支气管炎病毒和禽流感病毒方面效果显著 ,展现出了广阔的应用前景。文章对禽类基因工程重组干扰素的研究进展作了综述     

重组猪α干扰素冻干保护剂的筛选

为使重组猪仪干扰素（rPoIFN—α）制品更好地应用于生产实践中,采用保守的冻干工艺重点对rPoIFN-α制品的冻干保护剂进行筛选,通过外观检测、水分含量、活性检测对14种冻干保护剂进行了比较和筛选。研究结果表明,终浓度2％、4％和5％的甘露醇的保护效果较好,活性保持率分别为102．8％、95．1％、108．3％。按照活性好的这三个配方再冻干三批制品进行放置40℃加速稳定性实验。结果表明,在40℃放置三个月后,三个保护剂制品效价基本没变化,稳定性良好,有效期暂定为两年。本研究初步探索了rPoIFN一仪的冻干配方,为rPoIFN-α的产业化打下了基础。     

The induction and characterization of natural porcine interferons alpha and beta

The purpose of this study was to define optimum conditions for the production of high concentrations of natural porcine interferon (POIFN)-alpha and POIFN-beta, and to characterize the IFNs which were produced. The inducers used were Newcastle disease virus (NDV), polyinosinic:polycytidylic acid (poly IC), poly IC complexed with diethylaminoethyl dextran (poly IC-DEAEdx) and poly IC complexed with poly-L-lysine and carboxymethylcellulose. The highest yields of POIFN-alpha were obtained from porcine peripheral blood leukocyte (PBL) cultures induced with NDV. The concentrations of both cells and virus were critical for high yields of IFN, which were also enhanced by priming. Poly IC was found to be a relatively poor IFN inducer in PBL, in which low yields were obtained only after priming or in response to poly IC-DEAEdx. POIFN-beta was prepared by induction of the PK-15 cell line with poly IC or poly IC-DEAEdx. The highest yields were obtained from cultures induced 24 h after seeding, although when poly IC-DEAEdx or superinduction was used, the age of the cells was less critical. Priming had little effect on the yields of POIFN-beta. PK-15 cells induced with NDV gave relatively low yields of IFN. Both POIFN-alpha and POIFN-beta were classified as type I IFN on the basis of their resistance or susceptibility to pH 2.0, ultracentrifugation, 56 degrees C and trypsin treatment. Disulphide bonds essential for antiviral activity were demonstrated in both types of IFN by reduction with 2-beta-mercaptoethanol, and anionic exchange chromatography after treatment with dithiothreitol indicated a second disulphide bond in POIFN-alpha which was not essential for antiviral activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of feline infectious peritonitis virus replication by recombinant human leukocyte (alpha) interferon and feline fibroblastic (beta) interferon

Replication of feline infectious peritonitis virus (FIPV) in feline cell cultures was inhibited after incubation of cells with either human recombinant leukocyte (alpha) interferon (IFN) or feline fibroblastic (beta) IFN for 18 to 24 hours before viral challenge exposure. Compared with virus control cultures, FIPV yields were reduced by ranges of 0.1 to 2.7 log10 or 2 to 5.2 log10 TCID50 in cultures treated with human alpha- or feline beta-IFN, respectively; yield reductions were IFN dose dependent. Sensitivity to the antiviral activities of IFN varied with cell type; feline embryo cells had greater FIPV yield reductions than did similarly treated feline kidney or feline lung cells. Comparison of the virus growth curves in IFN-treated and virus control cultures indicated marked reduction in intracellular and extracellular FIPV in IFN-treated cultures. Compared with virus control cultures, intracellular and extracellular infectivity in IFN-treated cultures was delayed in onset by 12 and 30 hours, respectively, and FIPV titers subsequently were reduced by 3 to 3.5 and 5 log10 TCID50, respectively. Frequently, immunofluorescent and electron microscopy of IFN-treated cells or cell culture fluids did not reveal virus; however, even in cultures without viral cytopathic changes, small amounts of virus occasionally persisted in cells.     

Greater sensitivity of caprine synovial membrane cells to human interferon-alpha than human or bovine cells

The sensitivity of caprine synovial membrane cells to the antiviral effects of natural and recombinant DNA-derived human interferons (HuIFN) was compared with that of human foreskin fibroblast (FS7), ovine choroid plexus, and bovine turbinate cells. Caprine cells were found to be more sensitive (P less than 0.01) to natural HuIFN-alpha than human, ovine, and bovine cells. The sensitivity of caprine cells to recombinant DNA-derived HuIFN-alpha was equivalent to that of ovine cells, but greater than human or bovine cells. The sensitivity of caprine cells to natural and recombinant DNA-derived HuIFN-beta was equivalent to human cells, but less than that of ovine cells.     

Kinetics of large-scale production of bovine leukocyte interferon, using three viral inducers

Kinetics of large-scale production of naturally derived bovine leukocyte interferon (IFN) was investigated using Sendai virus, Newcastle disease virus, and infectious bovine rhinotracheitis virus inducers. Cultures were tested for IFN production every 6 hours for 66 hours. The effect of varying the priming dose of Sendai virus from 0 to 50% of total virus dose and the effect of varying the priming time from 0 to 4 hours before induction also were investigated. Other factors explored were effects of varying the fetal bovine serum concentration (from 0 to 8%) and individual cow donors on bovine IFN titers. Highest bovine leukocyte IFN titers (15,314 U/ml) were obtained using Sendai virus (priming dose, 60 hemagglutinating units/ml; inducing dose, 240 hemagglutinating units/ml) and incubating for 12 hours. Up to 24 L (over 360 million U) of naturally derived leukocyte IFN were produced at one time.     

从江香猪源干扰素γ植物表达载体构建及在生菜中瞬时表达

为构建从江香猪源干扰素γ(IFNγ)植物表达载体,通过农杆菌真空渗透法在新鲜生菜中实现瞬时表达。参考GenBank登录的猪IFNγ基因序列设计1对特异性IFNγ引物,采用RT-PCR方法从贵州从江香猪源肝脏组织扩增IFNγ基因序列,将其克隆至植物表达载体PBI121,转化根癌农杆菌LBA4404,筛选阳性菌株提取质粒,对重组质粒进行PCR扩增、双酶切及测序鉴定;携带重组质粒根癌农杆菌真空渗透法感染生菜,GUS染色检测感染的生菜叶片,ELISA检测感染生菜叶片中从江香猪源IFNγ蛋白,分析重组质粒在生菜中表达效果;采用细胞病变抑制实验检测生菜中从江香猪源IFNγ蛋白抗病毒活性。结果表明:本研究成功地从贵州从江香猪源肝脏组织扩增获得IFNγ基因序列并构建了从江香猪源IFNγ植物表达载体PBI121-IFNγ;GUS染色感染的生菜叶片可见蓝色物质;ELISA检测感染生菜叶片中从江香猪源IFNγ蛋白含量为169.397、170.657、170.37 pg/mL;生菜中表达从江香猪源IFNγ经4^-6稀释在Marc145细胞上仍能够抑制100 TCID50的PRRSV的致细胞病变作用。本实验成功构建从江香猪源IFNγ植物表达载体PBI121-IFNγ,生菜中瞬时表达蛋白具有免疫反应性和抗病毒活性,为从江香猪源IFNγ药用植物蛋白的研究开发提供参考依据。