Development and characterization of monoclonal antibodies against FMD virus type Asia-1 and determination of antigenic variations in the field strains

Twelve mouse monoclonal antibodies (MAbs) were developed against an Indian vaccine strain of foot and mouth disease virus (FMDV) type Asia-1 WBN 117/85. The MAbs were tested for their ability to bind to whole virus particle, trypsin-treated 146S (TT-146S) virus particle, sub-viral (12S and disrupted virus) antigens by ELISA and to neutralize virus infectivity in cell culture. Extensive characterization of MAbs revealed the existence of three different groups based on the binding of non-overlapping epitopes. Eight type Asia-1 specific MAbs (RF7, RF8, RD10, RE11, RC11, RC10/O, RB11 and RC10/M), which formed group 1 (G1), were found to bind a neutralizing, trypsin-sensitive (TS) and conformational epitope. Two MAbs (WB8 and WC3) in group 2 (G2) were found to bind a non-neutralizing, trypsin-resistant, conformational and 12S-specific epitope, which was intertypically conserved in all the four serotypes of FMDV (O, A, C and Asia-1) prevalent in India. Two MAbs (KG10 and KF10), which formed group 3 (G3), were found to be against a non-neutralizing, TS and conformational epitope, common to types Asia-1 and A. Members of G1 were IgG2a isotype, while those of G2 and G3 were IgG1 and IgG2b isotypes, respectively. Antigenic analysis of 31 FMDV type Asia-1 field isolates and two vaccine strains, using a panel of type Asia-1-specific MAbs, revealed antigenic similarity of the virus isolates tested and non-existence of neutralization escape mutants. The developed MAbs have practical utility, especially in the manufacture of FMD vaccine, diagnosis and FMDV characterization.     

Study on the porcinophilic foot-and-mouth disease virus I. production and characterization of monoclonal antibodies against VP1

Monoclonal antibodies (MAbs) reported here were produced against the porcinophilic foot-and-mouth disease virus (FMDV) that caused the devastating swine disease on 1997 in Taiwan. A panel (25) of MAbs were found to react with VP1 of O/Taiwan/97 (O/97) by ELISA with various potencies. The biological identities of these VP1 reacting MAbs, such as neutralization activity, isotype and capability to distinguish between two serotype O FMDVs, O/97 and O/Taiwan/KM1/99 (O/99), were further analyzed. Eleven out of the total eighteen O/97 neutralizing MAbs were able to neutralize heterologous O/99. Eight O/97 neutralizing and five non-neutralizing MAbs could differentiate two serotype O FMDVs by immunofluorescence assay (IFA) implied that these thirteen MAbs recognized O/97 specific epitope(s). Furthermore, reactivities of the VP1 reacting MAbs with a 29 amino acids synthetic peptide (P29) representing the betaG-betaH loop of VP1 were analyzed by ELISA and fourteen were found positive. MAb clone Q10E-3 reacting strongest with VP1 and P29, neutralizing both but not differentiating two serotype O viruses suggested that the antibody binding site might involve the RGD motif and its C terminal conserved region on betaG-betaH loop. MAbs with diverse characters presented in this study were the first raised against porcinophilic FMDV. The complete set of MAbs may be used for further studies of vaccine, diagnostic methods, prophylaxis, etiological and immunological researches on FMDV.     

Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979-1981.

Neutralizing epitopes present on field isolates of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were evaluated with a panel of polyclonal and neutralizing monoclonal antibodies (MAbs). A total of 91 field isolates were evaluated, including 15 isolates of BTV-10, 29 isolates of BTV-11, 26 isolates of BTV-13, and 21 isolates of BTV-17. The viruses were isolated from cattle, goats, sheep, elk and deer in Idaho, Louisiana, Nebraska and, predominantly, California, in the years 1979, 1980 and 1981. The isolates were analyzed and compared using a panel of neutralizing MAbs which included five MAbs raised against BTV-2, seven against BTV-10, five against BTV-13, and six against BTV-17. Neutralization patterns obtained with the MAb panel and individual field isolates were compared to those obtained with prototype viruses of each serotype. All field isolates were neutralized by at least some of the MAbs raised against the prototype virus of the same serotype. All field isolates of BTV-10 were neutralized by the seven MAbs raised to BTV-10, whereas the field isolates of BTV-11, BTV-13 and BTV-17 were not consistently neutralized by all of the MAbs raised against the prototype virus of the same serotype. Variation in neutralizing epitopes recognized by the MAb panel was most pronounced amongst the field isolates of BTV-17. A one-way cross neutralization was evident between BTV-10 and BTV-17 as all field isolates of BTV-17 were neutralized by four of the MAbs raised against BTV-10. In contrast, no BTV-10 isolates were neutralized by the MAbs raised against BTV-17. Differences in the MAb neutralization patterns of field isolates of BTV-11, BTV-13 and BTV-17 suggest that the immunogenic domain responsible for their neutralization is plastic, such that individual epitopes within the domain may vary in their significance to the neutralization of different viruses, even of the same serotype. The apparent conservation of neutralizing epitopes on field isolates of BTV-10 suggests that the field isolates may be derived from the modified-live vaccine strain of BTV-10.     

Production and characterization of two serotype independent monoclonal antibodies against foot-and-mouth disease virus

Two foot-and-mouth disease virus (FMDV) monoclonal antibodies (mAbs) were produced from mice immunized with either FMDV serotype A, subunit (12S) or FMDV serotype O, whole virus (140S). Both mAbs (F1412SA and F21140SO) recognized all seven serotypes of FMDV in a double antibody sandwich (DAS) ELISA, suggesting that the binding epitopes of the two mAbs are conserved between serotypes. These mAbs are IgG1 isotype and contain kappa light chains. In order to define the mAb binding epitopes, the reactivity of these mAbs against trypsin-treated and denatured FMDV were examined using an indirect ELISA. The binding site of the mAb, F1412SA is trypsin sensitive and the epitope is linear. Both ELISA and Western blot results suggested that the polypeptide VP2 contributed to the immunodominant site. This mAb showed reactivity to VP2 peptide (DKKTEETTILEDRIL). The mAb, F21140SO, recognized an epitope which is trypsin resistant and discontinuous. This mAb binding to FMDV is dependent on conformational structures of intact viral (140S) or subunit (12S) particle, since it failed to recognize any viral protein in Western blot. This conformational and highly conserved epitope is the first identified epitope among all seven FMDV serotypes. Because the use of mAbs increases the specificity, accuracy and efficiency of diagnostic tests compared to polyclonal antisera, these two mAbs with different specificities are suitable for type-independent diagnosis of FMDV, such as DAS ELISA, or could be adapted to immuno-chromatographic or flow-through rapid test.     

Effect of neutralizing monoclonal antibodies on Hantaan virus infection of the macrophage P388D1 cell line.

The effect of neutralizing monoclonal antibodies (N MAbs) on Hantaan virus infection of macrophages was investigated using P388D1 cells, a murine macrophage cell line. MAbs to the G1 protein (G1b) and the G2 protein (G2a and G2c) neutralized viral infectivity in P388D1 cells. N MAbs to G1b showed much higher neutralizing potency than those to G2a and G2c. With each N MAbs, two distinct effects were observed: neutralization of viral infectivity occurring at high concentrations and enhancement of that at low concentrations. Non-neutralizing MAbs, on the other hand, showed only enhancement of viral infectivity even at high concentrations without any inhibitory effects. The Fab fragments of N MAbs showed neither neutralizing nor enhancing activities. However, when the virus-Fab complexes were reacted with the anti-Fab antibodies, both neutralization and enhancement of viral infectivity were restored depending on the dose of Fab fragments. These results indicate that Hantaan virus infection of P388D1 cells is mediated by the Fc portion of the antibodies and neutralization is dependent on the concentration of N antibodies bound bivalently to the neutralization site on the virion.     

O型、A型及Asia-1型口蹄疫病毒多重RT-PCR检测方法的建立

根据口蹄疫病毒VP1基因序列,利用Primer Premier 5.0软件设计4条特异性引物,通过对退火温度等反应条件进行优化,建立能够同时扩增出O型、A型、Asia-1型口蹄疫病毒的多重RT-PCR检测方法。结果表明,建立的方法最低可以检测出约含1 pg/μL的病毒样品;该方法对猪瘟病毒、猪细小病毒、猪伪狂犬病毒、猪繁殖与呼吸综合征病毒、猪圆环病毒等其他相关病毒的检测结果均为阴性,特异性良好。建立的方法能够对口蹄疫O型、A型、Asia-1型病毒准确定型,可广泛用于口蹄疫病毒3个血清型的快速检测和分子流行病学调查。     

Foot-and-mouth disease virus C3 Resende subtype analysed by means of competition RIA using neutralizing monoclonal antibodies.

Foot-and-mouth disease virus (FMDV) was analysed using 30 monoclonal antibodies (MAbs) obtained from Balb/c mice immunized with FMDV C3 Resende (C3R) subtype 7 and 14 days before fusion No. 15 and 16 respectively. Fourteen MAbs were neutralizing and by means of competition radioimmuno assay it was possible to classify them into four groups. The first group consisted of MAbs specific for three sequential and three conformational epitopes. The second group consisted of MAbs specific for two conformational and for one sequential epitope. The third and the fourth groups consisted only of one MAb each, being specific for conformationally and one sequentially dependent epitope, respectively.     

Monoclonal antibodies against surface antigens of Pasteurella multocida strain P-1059

Four monoclonal antibodies (MAbs) were developed against serotype 3:A, P-1059 strain of Pasteurella multocida. Enzyme-linked immunosorbent assays were used to screen those hybridomas producing antibodies to either a surface protective (2.5 S) or lipopolysaccharide (LPS) antigen. MAbs 6EE11, D7H10, E11E3, and C11H2 were positive against 2.5 S antigen, and two of them, E11E3 and C11H2, were positive for the LPS antigen. MAbs 6EE11 and D7H10 reacted with a major protein band of molecular weight of 35,500, whereas E11E3 and C11H2 recognized a band with a molecular weight of 12,500 of the 2.5 S antigen. Treatment of the 2.5 S antigen with periodic acid abolished epitopes reacting with E11E3 but not with 6EE11. MAb 6EE11 did not recognize any band in Western blot after proteinase K treatment of the 2.5 S antigen, whereas antibody activity of E11E3 did not change. MAb 6EE11 reacted with serotypes 3, 4, 9, 10, 11, 12, and with M-9 strains in the immunofluorescence test. MAb E11E3 was positive only with serotype 3 or 10 strains, excluding M-9 strain. Electron microscopic studies with P-1059 strain indicated that antigens binding to 6EE11 and/or E11E3 were present in the capsule.     

Virus-neutralizing and passively protective monoclonal antibodies to infectious bursal disease virus of chickens.

Murine monoclonal antibodies (MAbs) were produced to assist in the identification and characterization of the virus-neutralizing epitopes of infectious bursal disease virus (IBDV). Only MAbs that reacted in Western blotting with viral protein 2 (VP2) or immunoprecipitated VP2 neutralized the infectivity of the virus in cell culture and passively protected young chickens from infection. Three of the neutralizing MAbs did not react with denatured viral proteins. Additivity enzyme-linked immunosorbent assays indicated that the six virus-neutralizing MAbs recognized two spatially independent epitopes. The ability of two of the virus-neutralizing MAbs to neutralize a variant of IBDV that had escaped neutralization by all the other MAbs confirmed the existence of two distinct neutralizing epitopes. The results support the hypothesis that there are at least two non-overlapping epitopes recognized by the virus-neutralizing MAbs reported in this study, although these may still be within one conformational site on VP2 of IBDV.     

A peptide of foot-and-mouth disease virus serotype Asia1 generating a neutralizing antibody response, and an immunostimulatory peptide

The epitopes of the capsid of foot-and-mouth disease virus (FMDV) play important roles in the construction of highly immunogenic subunit vaccines. However few epitopes have been found for FMDV serotype Asia1. In this study we screened for epitopes of the VP1 and VP2 proteins of FMDV serotype Asia1 isolate, YNBS/58. Fragments consisting of amino acids 133-163 of VP1 and amino acids 1-33 of VP2 contained epitopes, and both induced lymphoproliferation in guinea pigs. Only the VP1 fragment induced neutralizing antibodies but the VP2 peptide dramatically increased the neutralizing antibody response induced by the VP1 peptide.     

Preparation and characterization of monoclonal antibodies against an avian reovirus

Thirteen monoclonal antibodies against avian reovirus strain Uchida were derived. Of the 13 antibodies, three (MAb1, MAb2, and MAb3) had the ability to neutralize the infectivity of the virus. MAb1 neutralized strains Uchida, CS-108, and TS-142 equally. MAb2 neutralized the same three strains, but the activity of neutralization was 10 times higher against Uchida than against CS-108 and TS-142. MAb3 neutralized only strain Uchida. It seems that MAb1 and MAb2 have a rather broad neutralization activity and that MAb3 has a type-specific activity. These data indicate that there are both type-specific and more broadly specific neutralization epitopes in avian reovirus particles, as in mammalian reoviruses. In Western blot analysis, MAb1 bound to a sigma protein of strain Uchida, so it was suggested that this protein carries the epitope of the less specific neutralizing activity.     

豚鼠动物模型评价牛口蹄疫Asia-1型灭活疫苗效力的研究

以豚鼠为试验动物模型,探索一种应用豚鼠替代牛进行牛口蹄疫Asia-1型灭活疫苗效力检验的方法.豚鼠和牛同步对6批牛口蹄疫Asia-1型灭活疫苗进行PD50效力检验,其中2批进行重复性试验.豚鼠分别在免疫后7、14、21和28天采血检测Asia-1型的中和抗体水平.统计学分析显示,测定的豚鼠PD50和牛PD50之间具有极...     

Production and characterization of monoclonal antibodies against an avian group A rotavirus.

Fifteen monoclonal antibodies (MAbs) against an avian group A rotavirus were cloned and characterized. Eight of the 15 MAbs had neutralizing activity (N-MAbs). Five of the N-MAbs (1G1, 5B8, 4E2, 3G1, 2E3) were VP4-specific by radioimmunoprecipitation assay (RIPA), and two N-MAbs (2D11, 6E8) were possibly VP7-specific (faint bands by RIPA). One N-MAb (4H12) of undefined protein specificity cross-reacted with serotype 3 simian rotaviruses. The other seven N-MAbs did not cross-react with any of the eight distinct serotypes of human and mammalian rotaviruses tested. Of the seven non-neutralizing MAbs, three were VP6-specific (3H10, 4B12, 5F6), two were VP8-specific (6C9, 1D1), one was VP4-specific (4E9), and one was of undefined protein specificity (1B11). Four non-neutralizing MAbs recognized only avian group A rotavirus in cell-culture immunofluorescence tests (6C9, 1D1, 4E9 and 5F6), whereas two MAbs (3H10 and 4B12) cross-reacted with all human and animal rotaviruses tested. The MAb 1B11 did not recognize any human rotavirus serotypes but cross-reacted with all nonhuman animal rotavirus serotypes. The MAbs produced in this study should be useful for the detection and further characterization of avian group A rotaviruses.     

Foot-and-mouth disease: past,present and future

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals including cattle, pigs, sheep and many wildlife species. It can cause enormous economic losses when incursions occur into countries which are normally disease free. In addition, it has long-term effects within countries where the disease is endemic due to reduced animal productivity and the restrictions on international trade in animal products. The disease is caused by infection with foot-and-mouth disease virus (FMDV), a picornavirus. Seven different serotypes (and numerous variants) of FMDV have been identified. Some serotypes have a restricted geographical distribution, e.g. Asia-1, whereas others, notably serotype O, occur in many different regions. There is no cross-protection between serotypes and sometimes protection conferred by vaccines even of the same serotype can be limited. Thus it is important to characterize the viruses that are circulating if vaccination is being used for disease control. This review describes current methods for the detection and characterization of FMDVs. Sequence information is increasingly being used for identifying the source of outbreaks. In addition such information can be used to understand antigenic change within virus strains. The challenges and opportunities for improving the control of the disease within endemic settings, with a focus on Eurasia, are discussed, including the role of the FAO/EuFMD/OIE Progressive Control Pathway. Better control of the disease in endemic areas reduces the risk of incursions into disease-free regions.     

Production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes.

Three panels of monoclonal antibodies (MAbs) were prepared against the spike (S) proteins of infectious bronchitis virus (IBV) strains Arkansas 99, Connecticut 46, and Massachusetts 41. Based on enzyme-linked immunosorbent assay (ELISA), the MAbs were grouped into three categories: 1) group-specific, which reacted with a broad spectrum of homologous and heterologous IBV serotypes; 2) serotype-specific, which reacted only with strains of the homologous serotype; and 3) strain-specific, which reacted "selectively" with only certain strains of homologous and heterologous serotypes. MAbs that displayed serotype specificity were all specific to S1 fractions of the homologous serotype, confirming that epitopes that determine virus serotype are associated with the S1 protein. An excellent correlation was found when the results of IBV serotyping by MAb-based indirect ELISA were compared with those from the conventional virus-neutralization test. This confirms that the MAbs described here will serve as valuable tools in epizootiological studies and serotype-specific diagnosis of IBV infection.     

Detection of foot-and-mouth disease virus by competitive ELISA using a monoclonal antibody specific for the 12S protein subunit from six of the seven serotypes.

Foot-and-mouth disease (FMD) prevention and control programs are dependent upon rapid, reliable diagnostic procedures. The widely used FMD diagnostic complement fixation (CF) procedures require a specific antiserum for each of the seven FMDV serotypes making the tests both cumbersome and difficult to standardize. An FMD diagnostic, monoclonal antibody based inhibition-ELISA procedure was developed. The test uses a single monoclonal antibody (MAb) that reacts with all European and South American FMDV isolates examined. The procedure detects a highly conserved epitope on the 12S protein subunit of FMDV which appears to be common to all FMDV's with the exception of the South African Territories 2 serotype. The results indicate that the sensitivity of this test is greater than CF and approaches that of virus isolation.     

云南边境O型口蹄疫监测及病毒VP1基因序列分析

口蹄疫是由口蹄疫病毒引起的主要侵袭偶蹄动物的一种急性热性高度接触性传染病。口蹄疫病毒为微RNA病毒科口蹄疫病毒属成员,存在7个不同血清型,病毒VP1蛋白抗原性差异是病毒血清型划分依据,而其编码基因(1D)核苷酸序列差异是同型病毒拓扑型(Topotype)或基因型鉴别依据。采用O/A/C/Asia-1多重RT-PCR技术,对2006年自云南边境地区采集的120份动物组织样品,进行口蹄疫病原监测,检出O型口蹄疫病毒阳性样品15份。对阳性样品中病毒VP1基因全序列进行扩增、纯化后,克隆至pMD18-T载体测序,并与已知代表性毒株进行比对及系统发育分析。结果发现:云南边境O型口蹄疫病毒阳性样品VP1基因核苷酸序列同源性介于77.3%~98.7%,可划分为3个不同的拓扑型或基因型:中东-南亚型(ME-SA)或泛亚型(PAN-Asia)、古典中国型(Cathay)、东南亚型(SEA)。部分样品VP1蛋白表位43位、154位关键性氨基酸位点存在变异。     

Identification of a conformational epitope on the VP1 G-H Loop of type Asia1 foot-and-mouth disease virus defined by a protective monoclonal antibody

Although neutralizing antigenic sites of foot-and-mouth disease virus (FMDV) can be defined by selection of monoclonal antibody (MAb) escape mutants, no conformational neutralizing epitope on the major antigenic site located on the G-H loop of type Asia1 FMDV has been precisely mapped. In this study, we generated a potent neutralizing MAb 3E11, which recognized a conformation-dependent epitope and neutralized FMDV Asia1/YS/CHA/05 in vitro. Importantly, a dose of 5.5 NT(50) of the MAb 3E11 completely protected suckling mice from a dose of 10 LD(50) of homologous virus challenge in vivo. Through a 12-mer random peptide phage display, synthetic peptide analysis and constructing a series of FMDV Asia1/YS/CHA/05 mutants using reverse genetic system, we finely mapped the neutralizing epitope as the 12-amino acid peptide (141)SXRGXLXXLXRR(152). These results provide additional insights into the virus-MAb interaction at the amino acid level and may help in the development of an epitope-based Asia1 FMDV vaccine.     

基因Ⅶ型新城疫病毒HN蛋白单克隆抗体的制备及特性研究

为制备基因Ⅶ型新城疫病毒(NDV)血凝素-神经氨酸酶蛋白(HN)的单克隆抗体(MAb),本研究利用JS/17病毒株的HN重组蛋白和活病毒分别免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞(SP2/0)融合,并通过ELISA、间接免疫荧光和western blot方法筛选,制备了3株特异性识别HN蛋白的单克隆抗体(MAb)。其中,MAb1D4和4D9具有病毒中和活性(VN)及血凝抑制作用(HI),可以识别多种基因型的Ⅱ类NDV,但与Ⅰ类NDV病毒株无反应。MAb 2G8识别线性表位131DYIGGIGKE139,该表位在各病毒株中高度保守。获得的3株MAb可以用于NDV的鉴定及HN蛋白的功能研究。     

A rapid method for detection of Yersinia enterocolitica serotype O:3 in pig feces using monoclonal antibodies.

A simple colony immunoblotting method using monoclonal antibodies (MAbs) was developed to detect Y. enterocolitica serotype O:3 in pig feces. One of the MAbs studied was able to detect single colonies of the organism in the presence of calculated 3.1 x 10(8) heterologous organisms in pig feces. The MAb was found to be specific for the lipopolysaccharide (LPS) O-antigens of Y. enterocolitica serotype O:3. No significant cross-reactivity was found against a variety of closely related serotypes and Gram-negative organisms. The MAb could also be used in a slide agglutination test and an indirect fluorescence antibody assay for rapid identification of Y. enterocolitica serotype O:3.