Detection and Localization of Aleutian Disease Virus and its Antigens in vivo by Immunoferritin Technique

Tissues from mink infected with aleutian disease virus were examined by the electron microscope for the presence of virus particles. Virus-like particles, measuring 22 nm in diameter, were observed in macrophages of spleen, mesenteric lymph node and in Kupffer cells in liver of mink ten to 13 days after infection. The virus-like particles were usually present in vacuoles inside the cytoplasm of macrophages and Kupffer cells and, occasionally, similar particles were observed inside the nucleus. Cells from uninfected mink did not contain such patricles. To correlate the existence of these virus-like particles with the presence of aleutian disease virus antigen in infected cells, tissues were processed for immunoferritin technique. It was found that aleutian disease virus antigen was present in vacuoles inside the cytoplasm of cells from the infected spleen, lymph node and liver, and that the location was similar to that of the 22 nm virus-like particles. In addition, some viral antigen was also detected as cytoplasmic granular material. The nuclei of some cells also contained aleutian disease virus antigen. The pattern of aleutian disease virus antigen was similar to the distribution of virus-like particles in cells of infected tissue. It is suggested that virus replication occurs inside the nucleus with subsequent accumulation of virus in the vacuoles of the cytoplasm.     

After nasopharyngeal infection,foot-and-mouth disease virus serotype A RNA is shed in bovine milk without associated mastitis

Foot-and-mouth disease (FMD) is a highly contagious aphthoviral infection of cloven-hoofed animals, inducing vesiculopustular stomatitis, pododermatitis, and thelitis. Vesicular fluid represents a major pathway of virus excretion, but bovine milk is another important source of virus shedding. We describe here the time course of FMD virus (FMDV) excretion in the milk and characterize associated lesions in the mammary gland. Three dairy cows were infected by nasopharyngeal instillation of FMDV and monitored over 12 d. Autopsy was performed at the end of the study, and specimens were collected for histopathology, IHC, and RT-qPCR. All 3 cows developed fever, drooling, vesiculopustular stomatitis, interdigital dermatitis, and thelitis. FMDV RNA was detectable in whole milk until the end of the trial, but only transiently in saliva, nasal secretions, and blood serum. Although histology confirmed vesiculopustular lesions in the oral and epidermal specimens, the mammary glands did not have unequivocal evidence of FMDV-induced inflammation. FMDV antigen was detectable in skin and oral mucosa, but not in the mammary gland, and FMDV RNA was detectable in 9 of 29 samples of squamous epithelia but only in 1 of 12 samples of mammary gland.     

Foot-and-mouth disease virus (FMDV) experimental infection: susceptibility and immune response of adult mice

Adult mice are susceptible to foot-and-mouth disease virus (FMDV) infection only under some experimental conditions. This paper report the results of pathogenesis studies on 4 different strains of mice (CF1, C3H, NIH-nude, BALB-c/J) infected with the cloned and uncloned 0(1)C strain of FMDV. High virus titers were detected in blood and pancreas 12-24 h after infection (p.i.); these persisted for up to 48 h p.i. in CF1 and BALB-c/J mice and 72 h p.i. in the two other mouse strains. Virus titers observed in other organs were lower than those found in blood. In pancreas, and occasionally in salivary glands, oropharynx, heart and testicles, viral antigen was detected by direct immunofluorescent assay. Circulating neutralizing antibodies appeared in CF1 and C3H mice at 72 and 96 h p.i. respectively, and their titers remained unchanged during the 30-day experimental period. Antibodies against viral infection-associated antigen (VIA) were detected for a shorter period. In animals irradiated with 1 LD 50 (total body irradiation), viremia persisted up to 14 days p.i. and a low antibody response was observed which began at the end of viremia. No differences in the response of mice to cloned or uncloned FMDV were observed.     

口蹄疫病毒非型特异性检测方法研究进展

口蹄疫病毒非型特异性检测方法避免了口蹄疫不同血清型影响试验的敏感性,可快速简单的诊断口蹄疫病毒,判断免疫效果,检测感染历史等,如口蹄疫非型特异性RT-PCR、口蹄疫3ABC检测、VIA抗原AGID检测等,在口蹄疫防控中有广泛的应用。目前一些新开发的非型特异性检测方法更突出了简便性、快速性的特点,如横流装置(lateral flow device,LFD)可在1min～10min完验,设备只需要一台简单的样品搅拌器,非常适合基层使用。     

Role of apoptosis in the pathogenesis of Asian and South American foot-and-mouth disease viruses in swine

In this study, we performed experiments to evaluate the extend of the process of apoptotic cell death by foot-and-mouth disease virus (FMDV). Apoptosis can also occur in some virus-infected cells, and ability of viruses to either inhibit or promote apoptosis may influence the pathologic outcome of infection. In this study, to determine if apoptosis plays a role in the outcome of FMDV infection in swine, we evaluated apoptosis in diseased tissues collected from pigs inoculated with two different stains of FMDV (O1 Campos and O Taiwan). And host cell DNA fragmentation in diseased tissue from animals which were infected with either virus was evaluated by occurrence of a laddering pattern characteristic of apoptosis. Infection of cultured keratinocytes from swine tongue failed to demonstrate apoptosis in the first few hours of infection, suggesting that cell-to-cell correlation between viral antigen and apoptotic changes, e.g. cytokine secretions by immune system cells, could be critical to initiating apoptosis. Consistent with this finding, we were able to detect the pro-inflammatory cytokine TNF-alpha in diseased tissues. A clear difference in the pathogenicity of the two different FMDV isolates to pigs was not demonstrated in our study.     

口蹄疫病毒免疫学研究进展

口蹄疫病毒(FMDV)是小RNA病毒科、口蹄疫病毒属成员,其感染后引起的口蹄疫是一种严重危害畜牧业发展的传染病,并对经济发展、国家声誉及国际关系造成重要影响.论文就FMDV的主要抗原位点、FMDV的细胞受体、机体对FMDV抗原的免疫反应、FMDV抗原的变异与进化等做一综述.     

金丝桃素体外抗口蹄疫病毒与宿主细胞吸附作用研究

对光活化金丝桃素体外抑制口蹄疫病毒与宿主细胞吸附作用进行实验。体外培养的BHK-21细胞加入金丝桃素,日光灯下光活化1h后感染FMDV。通过细胞病变观察、MTT法、双抗体捕获抗原法、扫描电子显微镜进行抑制FMDV与宿主细胞吸附、融合研究。结果表明金丝桃素有明显抑制细胞病变的作用。双抗体捕获抗原法检测金丝桃素有效抑制FMDV和BHK-21细胞的吸附、融合,抑制率可达59．72％。扫描电镜观察,金丝桃素组与病毒对照组相比,BHK-21细胞FMDV颗粒明显减少。提示金丝桃素在体外有显著抑制口蹄疫病毒与宿主细胞的吸附、融合作用。     

Antibodies to foot-and-mouth disease virus infection associated (VIA) antigen: use of a bioengineered VIA protein as antigen in an ELISA

An enzyme-linked immunosorbent assay (ELISA) to detect antibodies to foot-and-mouth disease (FMD) virus infection associated (VIA) antigen (viral RNA polymerase) in cattle sera, was developed using a bioengineered VIA (BioVIA) protein antigen. Compared with the classical immunodiffusion test, with viral RNA polymerase purified from infected cell cultures as antigen, this ELISA was more sensitive. However, depending on the cattle population examined, sera with antibodies to viral RNA polymerase, probably due to infection with other picornaviruses, were detected. Despite these observations, the ELISA using BioVIA provided a rapid answer as to whether or not FMD virus circulated in a given herd of cattle. The main advantage of this ELISA is its absolute safety, since in no step of the antigen production was infectious or uninfectious FMD virus involved. The test can therefore be performed under normal laboratory conditions and no isolation units are needed as they are for the immunodiffusion test.     

Asia I型口蹄疫疫苗效力检验替代方法的初步研究

使用三株针对AsiaⅠ型口蹄疫146S抗原的杂交瘤细胞株(1#G3C2、2#G6B9、10#C2G1)大量生产腹水单克隆抗体,粗提纯化并按一定的比例进行混合。初步建立混合腹水单克隆抗体介导的间接夹心ELISA方法,使用该方法测定AsiaⅠ型病毒灭活抗原、O型病毒灭活抗原、正常BHK21细胞培养液,结果表明该方法特异性好,所测定OD值和AsiaⅠ型抗原稀释倍数呈明显线性关系。用该方法对11批AsiaⅠ型、O型单价或双价成品疫苗破乳抗原进行测定,结果表明不同批次不同企业生产的疫苗完整病毒颗粒抗原含量有差异,进一步完善有望用于AsiaⅠ型口蹄疫成品疫苗中有效颗粒抗原含量的测定,从而发展成一种疫苗效力检验替代方法。     

Application of latex beads agglutination test for the detection of the antibody against virus-infection-associated (VIA) antigen of foot-and-mouth disease (FMD) virus

Latex beads agglutination (LA) for the detection of the antibody against virus infection-associated (VIA) antigen of foot-and-mouth disease (FMD) virus was estimated using experimentally infected animals. The VIA antibody titer by the LA test were compared with the neutralization titer and the titer by agarose gel diffusion (AGD) test, which has been used as a standard method for VIA antibody titration. The latex beads were coated with VIA antigen in carbonate buffer solution (0.5 M, pH 9.6) for the test. The sensitivity of the LA test was clearly higher than that of the AGD test in the results for cattle and swine infected experimentally. The antibody was detected in the bovine serum obtained at the 13th week after inoculation by the LA but not by the AGD test. The LA test appears to be simple, rapid and sensitive for the detection of the antibody of FMD virus in the surveillance of FMD and the FMD quarantine of imported animals.     

嵌合口蹄疫病毒抗原表位的重组塞内卡病毒A的构建及其鉴定

塞内卡病毒A（SVA）与口蹄疫病毒（FMDV）引起猪的临床症状难以区分,并且以SVA作为载体插入FMDV抗原表位外源基因的研究,目前还没有报道。为了构建一种嵌合FMDV抗原表位的重组SVA毒株并进行鉴定,本研究利用基因克隆技术将O型FMDV的B细胞表位与白蛋白信号肽（human albumin signal peptide,HAS）基因串联插入到SVA基因组中,成功构建了重组质粒,转染细胞后拯救出重组病毒rSVA-HAS-OB。结果显示：RT-PCR扩增与基因测序结果表明目的基因正确插入;细胞间接免疫荧光和Western blot鉴定了嵌合抗原蛋白的有效表达;遗传稳定性分析表明重组病毒在25代次的传代过程中仍稳定存在B细胞表位;病毒蚀斑表型和一步生长曲线结果表明,重组病毒与亲本毒株具有相似的增殖特性。本研究成功构建并拯救出能够表达FMDV抗原表位的重组SVA毒株,为以SVA为基础的嵌合病毒及疫苗的研究提供了理论和实验基础。     

Delayed replication of Marek's disease virus following in ovo inoculation during late stages of embryonal development

Several oncogenic and non-oncogenic isolates of Marek's disease virus (MDV) were inoculated into embryonated eggs on embryonation day (ED) 16 to 18, and embryos or chicks hatching from inoculated eggs were examined for infectious virus and viral internal antigen (VIA) in lymphoid organs. There was no evidence of extensive replication of MDV in any of the embryonic tissues examined. Levels of VIA peaked 4-5 days after chicks hatched. This indicated that MDV remained inactive during embryonation and did not initiate pathogenic events until chicks hatched. Because HVT replicated rapidly in the embryo but MDV did not, in ovo inoculation of HVT simultaneously with oncogenic MDV or several days after MDV resulted in significant protection (P less than 0.025) of hatched chicks against Marek's disease (MD). Little protection was obtained if HVT was given simultaneously with MDV or after MDV to chicks already hatched. The relative susceptibility of the embryo to extensive replication of the vaccine virus but not the challenge virus apparently accounted for protection against MD in chicks hatching from dually infected eggs.     

口蹄疫双佐剂灭活疫苗的研究

在口蹄疫二价灭活疫苗（O型、AsiaⅠ型）常规使用法国SEPPIC公司206佐剂的基础上,设计了一种缓释作用更强的双佐剂成分疫苗。通过MTT法检测淋巴细胞增殖能力、液相阻断ELISA法检测口蹄疫抗体效价及攻毒试验测定PD50来评判该双佐剂疫苗与常规疫苗的效果差异。结果显示,双佐剂疫苗组细胞免疫水平（淋巴细胞刺激指数SI为1.235±0.060）比常规佐剂疫苗组（SI为1.115±0.035）和对照组（SI为1.010±0.045）高,与常规疫苗组相比差异显著（P＜0.05）,与空白对照组相比,差异极显著（P＜0.01）。双佐剂疫苗组O型和AsiaⅠ型抗体与常规佐剂疫苗组相比,全剂量组抗原量较充分,两组抗体水平差距不大;1/3剂量组和1/9剂量组由于抗原量较少和强缓释作用,导致抗体水平明显较低。攻毒保护结果为双佐剂疫苗组略高于常规佐剂疫苗组,前者每头份疫苗AsiaⅠ型为9.0 PD50,O型为11.84 PD50,后者每头份疫苗AsiaⅠ型为9.0 PD50,O型为9.0 PD50。由分析结果可见,双佐剂疫苗可引起较好的细胞免疫应答和缓释作用,达到好的攻毒保护效果。     

口蹄疫146S病毒粒子抗原对微血管内皮细胞分泌IL-1的影响

白介素1（IL-1）是免疫与炎症反应启动的重要调节因子。为了研究微血管内皮细胞（microvascular endothelial cells,MVECs）在口蹄疫病毒（foot and mouth disease virus,FMDV）免疫反应中的作用,本试验采用ELISA方法检测了FMDV 146S抗原对体外培养大鼠心肌膜MVECs分泌IL-1α和IL-1β的影响。研究结果发现,正常情况下大鼠心肌膜MVECs能够低水平的分泌IL-1α和IL-1β,FMDV 146S刺激后其分泌量显著增加。结果表明,MVECs通过上调IL-1分泌,在FMDV抗原的免疫反应中发挥重要的调控作用。     

A replication analysis of foot-and-mouth disease virus in swine lymphoid tissue might indicate a putative carrier stage in pigs

ABSTRACT: Foot-and-mouth disease virus (FMVD), one of the most contagious viruses of cloven-hoofed animals, may cause a prolonged, asymptomatic but persistent infection in ruminants, named the "carrier state". However, it remains an open question whether this carrier state occurs in pigs. Here we present quantitative analyses of the duration of FMDV RNA and infectivity in lymphoid and epithelial tissues in experimentally infected pigs with FMDV C-S8c1. The data indicated that although FMDV RNA remained in blood until day 14 post-infection (pi), viremia was cleared by day 7 pi. However, all tissues tested were positive for FMDV until day 14-17 pi. Interestingly, the specific infectivity of FMDV in these tissues was in some cases even higher than the FMDV C-S8c1. We therefore propose that a "pseudopersistent state" may occur in pigs in which virus replicates in lymphoid tissues for a prolonged period of time, thereby representing a potential source of virus.     

Cattle remain immunocompetent during the acute phase of foot-and-mouth disease virus infection

ABSTRACT: Infection of cattle with foot-and-mouth disease virus (FMDV) results in the development of long-term protective antibody responses. In contrast, inactivated antigen vaccines fail to induce long-term protective immunity. Differences between susceptible species have also been observed during infection with FMDV, with cattle often developing persistent infections whilst pigs develop more severe symptoms and excrete higher levels of virus. This study examined the early immune response to FMDV in na?ve cattle after in-contact challenge. Cattle exposed to FMDV were found to be viraemic and produced neutralising antibody, consistent with previous reports. In contrast to previous studies in pigs these cattle did not develop leucopenia, and the proliferative responses of peripheral blood mononuclear cells to either mitogen or third party antigen were not suppressed. Low levels of type 1 interferon and IL-10 were detected in the circulation. Taken together, these results suggest that there was no generalised immunosuppression during the acute phase of FMDV infection in cattle.     

Indirect immunofluorescence and immunodiffusion tests in the detection of antibodies to foot-and-mouth disease virus

The antibody response detected by indirect immunofluorescence (IIF) as well as that directed against 140 S and virus infection associated antigen (VIA), as detected by agar immunodiffusion, was studied in three mammal species susceptible to Foot and Mouth Disease Virus, after challenge with living virus, immunization and hyperimmunization with inactivated virus, and immunization followed by challenge. By spot indirect immunofluorescence, antibodies were detected only in animals undergoing an active infection, and were not detected in immunized or hyperimmunized animals. This behaviour was similar to that of the anti-VIA antibodies in the same groups of animals and differed from that of anti-140 S antibodies. It appeared that spot indirect immunofluorescence for the detection of VIA antigen is comparable to the immunodiffusion test, but the speed of IIF and the possibility of handling many samples make it more practical.     

Development and standardization of a piezo electric immunobiosensor for foot and mouth disease virus typing

An immunobiosensor using a piezo electric (PZ) crystal was developed and standardized for foot and mouth disease (FMD) diagnosis and virus typing. A 6MHz quartz crystal was used as the frequency determining element. Foot and mouth disease virus (FMDV) type specific antibody raised in rabbits/monoclonal antibody was coated on the crystal surface and the resonance measured. One microlitre of the 10% aqueous suspension of the clinical sample (tongue or foot epithelium) was applied on both surfaces of the crystal and the resonance recorded. A difference in resonance of more than -2.5Hz was obtained in positive samples (homologous antigen and antibody). The test was standardized initially using various dilutions of FMD tissue culture antigen. Repeatability and sensitivity were also tested and it was found that the crystals could be washed and reused eight times. The test could be used for FMDV type specifically and no cross-reaction between FMDV types was observed. The shelf-life of the antibody-coated crystal stored at room temperature was 18 weeks. Application of the biosensor test to the FMDV clinical samples confirmed virus typing results when compared with enzyme-linked immunoabsorbent assay (ELISA) and it could also detect virus in ELISA negative samples and mixed virus infections.