Production and preliminary characterization of monoclonal antibodies to chicken anemia agent

Mice were immunized with partially purified preparations of the Cux-1 isolate of chicken anemia agent (CAA), and their splenocytes were fused with NSO myeloma cells. Three patterns of staining of CAA-infected cells were recognized when the resulting hybridomas were screened by indirect immunofluorescence (IIF). Hybridomas representative of each staining pattern were cloned, and the monoclonal antibodies (MAbs) were characterized. Type 1 staining was indistinguishable from that produced by polyclonal chicken antisera to CAA. Type 2 staining was confined to large nuclear inclusions. Type 3 staining was predominantly nuclear and granular, and differed from type 1 in being more intense and occurring in a higher proportion of nuclei. Three MAbs producing type 1 staining were predominantly Cux-1-specific by IIF; they also reacted to lower titers with the Gifu-1 isolate but not at all with three other CAA isolates. These MAbs had very slight neutralizing activity against Cux-1. Another MAb giving type 1 staining reacted with all CAA isolates tested to high titers in IIF and neutralization tests. MAbs with type 2 and type 3 staining reacted by IIF with all CAA isolates tested but possessed no neutralizing activity. The availability of MABs to CAA should facilitate development of diagnostic tests for the virus.     

Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP.

Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.     

Comparative susceptibility of Marek's disease cell lines to chicken infectious anemia virus

Chicken infectious anemia virus (CIAV) is known to infect and replicate in various Marek's disease chicken cell lines (MDCCs) derived from Marek's disease (MD) tumors. One line, MDCC-MSB1, has been the substrate used in most studies. We compared a total of 26 MDCCs, including two sublines of MDCC-MSB1, MSB1 (L) and MSB1 (S), four other MD tumor-derived lines, and 20 lines derived from MD virus-induced local lesions, for susceptibility to the Cux-1 and CIA-1 strains of CIAV. The cell lines represented six phenotypic groups of T cells based on the expression of CD4, CD8, and TCR-2 and -3 surface markers. Susceptibility was measured by the number of cells positive for viral antigen in immunofluorescence (IF) tests at 3-10 days postinfection. No clear-cut differences were found in susceptibility related to phenotype, although CD4-/8+ lines and CD4-/8- lines might be more susceptible than CD4+/8- lines. However, several individual lines were more susceptible to Cux-1 than the two MSB1 sublines tested. Contrary to an earlier report, cells of MDCC-CU147, a CD8+, TCR3+, local-lesion derived line, were found to be susceptible to CIA-1. In fact, CU147 was distinguished by very high susceptibility to both CIAV strains. In direct comparisons with MSB1, CU147 detected approximately 10-fold lower doses of virus. Also, virus spread was faster (P < 0.05) in CU147 than in MSB1 and other lines. Results from polymerase chain reaction (PCR) tests to detect infection in titrations were in general agreement with IF test results although PCR detected infection in a few terminal dilution cultures that were negative by IF.     

Diagnostic methods for African horsesickness virus using monoclonal antibodies to structural and non-structural proteins.

A panel of 32 hybridoma cell lines secreting monoclonal antibodies (MAbs) reactive with African horsesickness virus serotype 4 (AHSV-4) has been developed. Four of the MAbs recognized the major core antigen VP7, twenty recognized the outer capsid protein VP2 and eight reacted with the non-structural protein NS1. With the VP7-specific MAbs a rapid and sensitive double antibody sandwich immunoassay has been developed to detect viral antigen in infected Vero cells and in spleen tissue from AHSV-infected horses. The sensitivity of the assay is 10 ng viral antigen per 100 microliters. The NS1-specific MAbs allowed visualization by immunofluorescence of tubule-like structures in the cytoplasm of infected Vero cells. This can be very useful as a confirmatory diagnostic procedure. The antigenic map of the outer capsid VP2 protein with MAbs is also reported.     

Studies on the pathogenesis of chicken infectious anaemia virus infection in six-week-old SPF chickens

The pathogenesis of chicken infectious anaemia virus (CAV) infection was studied in 6-week-old and one-day-old SPF chickens inoculated intramuscularly with graded doses of Cux-1 strain (10(6)-10(2) TCID50/chicken). Viraemia, virus shedding, development of virus neutralizing (VN) antibodies and CAV distribution in the thymus were studied by virus isolation, polymerase chain reaction (PCR), immunocytochemistry (IP) and in situ hybridization until postinfection day (PID) 28. In 6-week-old chickens infected with high doses of CAV, viraemia and VN antibodies could be detected 4 PID and onward without virus shedding or contact transmission to sentinel birds. However, virus shedding and contact transmission were demonstrated in one-day-old infected chickens. In the 6-week-old groups infected with lower doses, VN antibodies developed by PID 14, transient viraemia and virus shedding were detected. The thymus cortex of all 1-day-old inoculated chickens stained with VP3-specific mAb. Cells with positive in situ hybridization signal were fewer and scattered throughout the thymus tissue of the one-day-old inoculated chickens as compared to IP-positive cells. These results suggest that early immune response induced by high doses of CAV in 6-week-old chickens curtails viral replication and prevents virus shedding.     

Development and characterization of monoclonal antibodies to subgroup J avian leukosis virus.

In an attempt to develop a specific diagnostic test for avian leukosis virus (ALV) subgroup J (ALV-J) strain Hc1, four monoclonal antibodies (MAbs), JE9, G2, 145, and J47, were generated that are specific for ALV-J envelope glycoprotein, gp85. Polymerase chain reaction (PCR) was used to amplify genomic pro-viral DNA of Avian Disease and Oncology Laboratory (ADOL)-Hc1 and ADOL-4817 envelope genes. Both open reading frames encoding glycoproteins gp85 and gp37 were cloned into baculoviruses. Abundant expression of gp85 and gp37 was detected in the recombinant viruses with specific antibody to Hc1 strain of the ALV-J. The expressed proteins were used for immunization of mice to produce hybridoma cell lines secreting MAbs specific to ALV-J envelope protein. A panel of MAbs was generated by fusing NS1 myeloma cells and spleen cells from mice immunized with the recombinant baculoviruses. With the use of an immunofluorescence assay, three MAbs (JE9, G2, 145) reacted with ALV-J but not with subgroups A, B, C, D, or E of ALV. MAb J47 reacted with all exogenous subgroups of ALV including A, B, C, D, and J but not with endogenous subgroup E viruses. Western blot analysis was performed with all four MAbs against recombinant baculovirus and Hc1-infected chicken embryo fibroblast (CEF) lysates. A major band with a molecular weight about 90 kD corresponding to the size of ALV-J envelope was consistently obtained. With these MAbs, we detected the Hc1 antigen in CEFs infected with several ALV-J viruses isolated in the United States and also in tissue sections from chickens infected with Hc1 strain of ALV-J. These MAbs will be useful reagents for the diagnosis of ALV-J infection because they recognize a common antigenic epitope in six isolates tested thus far.     

鸡贫血病毒VP1和VP2蛋白在家蚕中的联合表达

将鸡贫血病毒VP1和VP2基因分别克隆入转换载体pBacPAK8中，获得重组转移质粒pBac-vp1和pBac-vp2。以上两质粒分别与CvnⅠ酶切线性化的亲本病毒Bm-BacPAK6DNA共转染家蚕细胞，通过蓝白斑筛选，纯化得到重组病毒Bm-vp1和Bm-vp2。PCR分析表明Vp1和Vp2基因已整合进杆状病毒基因组中。将Bm-vp1和Bm-vp2共感染5龄家蚕，通过表达产物免疫SPF鸡产生的抗血清与CAV感染的MDCC－MSB1细胞的间接荧光抗体分析，证明表达产物能诱导鸡产生相应的抗体。该研究表明，表达VP1和VP2蛋白的重组家蚕杆状病毒（recombinant BmNPV）是很有前途的CAV亚单位疫苗的生产系统。     

Temporal appearance of structural and nonstructural bluetongue viral proteins in infected cells, as determined by immunofluorescence staining and flow cytometry

The temporal appearance of 4 viral proteins was detected in bluetongue virus (BTV)-infected Vero cells by indirect immunofluorescence staining with monoclonal antibodies (MAb) to BTV structural proteins VP2 and VP7 and nonstructural proteins NS1 and NS2. Bluetongue viral proteins were detected at distinct intervals after inoculation of Vero cells; VP7 was first detected 3 hours after inoculation, NS1 and NS2 at 5 hours after inoculation, whereas VP2 was not detected until 8 hours after inoculation. Patterns of fluorescence varied with the fixative used, but each MAb induced a distinct pattern of fluorescence in infected cells. Flow cytometry, which was used with each of the 4 MAb, proved to be an accurate and sensitive method of detecting BTV-infected P3 mouse myeloma cells. The temporal appearance of each viral protein in BTV-infected P3 cells was similar to that detected in BTV-infected Vero cells. Advantages of flow cytometry over conventional immunofluorescence staining to detect BTV-infected cells included: (1) enumeration of the proportion of infected cells in a population; (2) further characterization of infected cells, including estimates of their viability; and (3) computer-assisted storage and analysis of data obtained.     

Characterization of monoclonal antibodies against avian reovirus S1133 protein sigmaA synthesized in Escherichia coli

Monoclonal antibodies (MAbs) were prepared against avian reovirus S1133 protein sigmaA (esigmaA) synthesized in Escherichia coli. MAbs were characterized and used to develop a diagnostic test. Ten MAbs were selected for competitive binding assay following coupling with horseradish peroxidase. The results indicated that these MAbs delineated two epitopes I and II of esigmaA. An immuno-dot binding assay was used to detect the effect of denaturation on antibody recognition of the epitopes. All MAbs bound to esigmaA in its native form. After denaturation by boiling in SDS and 2-mercaptoethanol, the binding of MAbs recognizing epitope I was fully abolished. However, the reactivity of MAbs recognizing epitope II was not affected. MAbs 31 and 32, recognizing epitopes I and II, respectively, were selected for the cross-reactivity to heterologous reovirus strains. The results suggest that the two epitopes are highly conserved among these virus strains. A MAb capture enzyme-linked immunosorbent assay (ELISA) procedure was developed using MAbs 32 and 31 to detect reovirus protein sigmaA in samples from tendon tissues of infected bird and chicken embryo fibroblast (CEF) cell cultures. Avian reovirus sigmaA antigens in tendon specimens were detected from the inoculated birds as early as 2 days post-inoculation (PI), approximated a peak at 7 days PI, and maintained this until 16 days PI, then decreased gradually. A clear difference in absorbance values between the tendon samples of the avian reovirus- and mock-infected birds is obtained. Positive results were also obtained from avian reovirus-infected CEF and from the tendon tissues of naturally infected broilers. These results indicated that the MAb capture ELISA is a useful methods for the detection of avian reovirus from chickens suspected to have avian reovirus infections.     

Downregulation of cellular prion protein inhibited the proliferation and invasion and induced apoptosis of Marek's disease virus-transformed avian T cells

Cellular prion protein (PrP^C) is ubiquitously expressed in the cytomembrane of a considerable number of eukaryotic cells. Although several studies have investigated the functions of PrP^C in cell proliferation, cell apoptosis, and tumorigenesis of mammals, the correlated functions of chicken PrP^C (chPrP^C) remain unknown. In this study, stable chPrP^C-downregulated Marek''s disease (MD) virus-transformed avian T cells (MSB1-SiRNA-3) were established by introducing short interfering RNA (SiRNA) targeting chicken prion protein genes. We found that downregulation of chPrP^C inhibits proliferation, invasion, and migration, and induces G1 cell cycle phase arrest and apoptosis of MSB1-SiRNA-3 cells compared with Marek''s disease virus-transformed avian T cells (MSB1) and negative control cells. To the best of our knowledge, the present study provides the first evidence supporting the positive correlation between the expression level of chPrP^C and the proliferation, migration, and invasion ability of MSB1 cells, but appears to protect MSB1 cells from apoptosis, which suggests it functions in the formation and development of MD tumors. This evidence may contribute to future research into the specific molecular mechanisms of chPrP^C in the formation and development of MD tumors.     

Formation of virus-like particles when the polyprotein gene (segment A) of infectious bursal disease virus is expressed in insect cells.

The baculovirus expression vector system was used to examine the expression of the full-length infectious bursal disease virus (IBDV) segment A cDNA, which encodes the structural proteins in a polyprotein precursor that is autocatalytically cleaved to VPX, VP3, and VP4. No VP2 was observed in lysates of recombinant baculovirus infected cells indicating the lack of processing of VPX to VP2 in this system. Virus-like particles (VLP) were purified from the infected insect cells, and on negative staining electron microscopy, looked very similar to authentic IBDV particles in shape and size, suggesting that processing of VPX to VP2 is not necessary for capsid assembly.     

鸡贫血病病毒VP_2基因在大肠杆菌中的表达及其特性

将鸡贫血病病毒 (chicken anaem ia virus,CAV) VP2 基因克隆入表达性载体 p GEX- 5 X- 3,在大肠杆菌以谷胱甘肽转移酶 (GST)融合蛋白的形式获得了表达。以此表达产物免疫小鼠 ,制备抗 CAV VP2 的多克隆抗体。通过对 CAV感染的 MSB1细胞作间接免疫荧光试验 (IFA)检测 ,结果为阳性 ,这表明表达产物保留了 CAV相关的抗原特性。本研究为进一步探索 CAV VP2 的生物学特性奠定了基础。     

Isolation and preliminary characterization of chicken anemia virus from chickens in Nigeria

Chicken anemia virus (CAV) was isolated for the first time from the Nigerian chicken population. The virus was recovered from necropsied birds from broiler and pullet flocks that suffered disease outbreaks tentatively diagnosed as infectious bursal disease. A sensitive polymerase chain reaction (PCR) assay detected CAV DNA in tissues of necropsied birds. Restriction endonuclease analysis performed with the 733-bp PCR product and the Cfo I enzyme indicated at least two different CAVs were circulating among the Nigerian chicken population. Four isolates were obtained from pooled liver and thymus tissues using the MDCC-MSB1 cell line. These isolates were found to be antigenically closely related to the Cuxhaven-1 (Cux-1) reference strain of CAV when reacted with four monoclonal antibodies prepared against the Cux-1 virus. One of the isolates (isolate A) induced thymus atrophy, bone marrow aplasia, and low hematocrit values when inoculated into 1-day-old specific-pathogen-free chickens. These findings not only demonstrate that CAV is present in Nigeria, but they also likely represent the first cell culture isolation of the virus in Africa.     

鸡传染性贫血病毒VP3蛋白的原核表达及其单克隆抗体的研制

本研究首先利用同源重组一步克隆法将鸡传染性贫血病病毒(CIAV)的VP3基因克隆到pGEX-6P-1原核表达载体上,经IPTG诱导及SDS-PAGE分析成功获得了重组蛋白rGST-VP3的表达.随即以纯化的重组蛋白rGST-VP3免疫Balb/c小鼠,通过脾细胞与SP2/0细胞融合以及间接免疫荧光(IFA)筛选,获得2株稳定分泌CIAV-VP3抗体的杂交瘤细胞株,分别命名为CIAV-VP3-4D7和CIAV-VP3-4G8;亚型鉴定表明,CIAV-VP3-4D7和CIAV-VP3-4G8均为IgG1;效价测定发现,CIAV-VP3-4D7和CIAV-VP3-4G8的腹水间接免疫荧光效价分别为1:102400与1:12800;Western blot进一步证实,CIAV-VP3-4D7和CIAV-VP3-4G8均能识别重组蛋白rGST-VP3.本研究结果为后期研究VP3蛋白在CIAV致病中作用及其诱导凋亡分子机制奠定了坚实的物质基础.     

鸭1型甲肝病毒亚型VP1蛋白单克隆抗体的研制及鉴定

为制备抗鸭1型甲肝病毒亚型（DHAV-1a）结构蛋白VP1的单克隆抗体（MAbs）,本研究以pGEX-VP1重组蛋白免疫BALB/c小鼠,同时以纯化浓缩的全病毒作为包被抗原,建立了筛选抗VP1蛋白阳性杂交瘤细胞株的间接ELISA方法,经融合、筛选制备杂交瘤细胞及鉴定MAbs的稳定性、特异性、腹水效价和中和活性等生物学活性,获得了2株持续且稳定分泌抗体的杂交瘤细胞（1A2和5G3）。MAbs腹水ELISA效价分别为1：3.2×10⁴和1：2.0×10⁶。亚类鉴定均为IgG1/κ型。Western blotting结果显示,2株MAbs均能与DHAV-1a VP1蛋白和DHAV-1a全病毒发生特异性反应。特异性试验结果显示2株MAbs能与鸭1型甲肝病毒（DHAV-1）和DHAV-1a发生交叉反应。中和试验结果显示5G3株具有中和活性。结果表明2株MAbs的ELISA效价高、特异性强、稳定性好,均能与DHAV-1和DHAV-1a全病毒发生特异性反应,其中一株具有中和活性。     

Development and Identification of Monoclonal Antibodies against VP1 Protein of DHAV-1a

To prepare the monoclonal antibodies (MAbs) against DHAV-1a, hybridomas were produced by fusing SP2/0 cells with spleen cells from mouse immunized with DHAV-1a pGEX-VP1 recombinant protein. Two hybridomas stablely secreting MAbs against VP1 protein were identified by indirect ELISA detection with DHAV-1a as coating antigen. The ascetic fluids of MAbs were 1:3.2×10⁴ and 1:2.0×10⁶, respectively. The MAbs were IgG1 with κ chain. Western blotting analysis showed that the MAbs could recognize the recombinant VP1 protein and DHAV-1a. The neutralization tests showed that one MAb (5G3) had better neutralization activity. Therefore, the results showed that the ELISA titers and specialities of two MAbs were very good, with excellent stability. In addition, they all had cross interaction with DHAV-1 and DHAV-1a, one of which had good neutralization activity.     

Analysis of properties of monoclonal antibodies to transmissible gastroenteritis virus using TO-163 strain

Nineteen monoclonal antibodies (MAbs), reactive in enzyme-linked immunosorbent assay (ELISA), with porcine transmissible gastroenteritis (TGE) virus TO-163 were obtained. Of these MAbs, 5 showed neutralizing (NT) activity (x 3,200 to 25,600) against TO-163. One of the MAbs which had NT activity showed hemagglutination inhibition activity (x 5,120) too. 14 hybridomas of polypeptide specificity against TO-163 strain were developed from which 11, 2, and 1 were specific for protein E2, N, and E1, respectively. Immunofluorescence staining patterns in TGE virus-infected cells reacted with MAbs were divided into three groups (types I, II and III). The fluorescence staining of E2 specific MAbs having NT activity were limited to the perinuclear area. All MAbs having NT activity showed the same fluorescence staining pattern.     

Replication of infectious bursal disease virus in macrophages and altered tropism of progeny virus

We serially passaged classical infectious bursal disease virus (cIBDV) and antigenic variant IBDV (vIBDV) in an avian macrophage cell line, NCSU cells, referred as mcIBDV and mvIBDV respectively and examined the in vitro and in vivo characteristics of the macrophage-adapted viruses. NCSU adapted viruses caused earlier destruction of NCSU cells than the unadapted viruses. Nitric oxide (NO) was detected earlier in cultures infected with mcIBDV and mvIBDV than in cultures infected with cIBDV and vIBDV. cIBDV and vIBDV were able to infect DF-1 cells, a chicken embryo fibroblast cell line, only after one replication cycle in NCSU cells. The genetic basis of altered tropism of progeny virus from NCSU cells infected cultures was not identified. No aa substitutions were observed in hypervariable region of VP2 of cIBDV and vIBDV passaged 1 time in NCSU cells whereas both mcIBDV and mvIBDV had multiple aa substitutions. To assess protective efficacy of mcIBDV and mvIBDV, embryonated chicken eggs were inoculated with mcIBDV and mvIBDV at embryonation day 18 (ED 18) and challenged with a virulent cIBDV at 3 weeks of age. mcIBDV and mvIBDV were immunogenic and generated antibody responses and provided 100% protection against cIBDV.     

抗蓝舌病病毒VP6和VP7蛋白单克隆抗体的制备及鉴定

为制备针对蓝舌病病毒(BTV)的单克隆抗体(MAb),本研究利用血清型1型BTV(BTV1)免疫BALB/c鼠,将其脾淋巴细胞与SP2/0进行融合,并用BTV1包被ELISA板,通过间接ELISA方法筛选出3株稳定分泌抗BTV1的MAb的杂交瘤细胞株(2B10、3D4和4H8)。利用表达BTV1主要蛋白的真核表达重组质粒转染BHK-21后,对所制备的杂交瘤细胞株上清进行间接免疫荧光(IFA)以及western blot鉴定,结果显示:2B10和4H8与VP7蛋白反应,而3D4与VP6蛋白反应。同时,IFA鉴定结果进一步表明,3株MAb与24个血清型的BTV均可以发生反应。本研究制备的MAb为建立BTV免疫学检测方法和相关病毒蛋白的功能研究奠定了基础。     

Characterization of monoclonal antibodies against fowl poxvirus

Vaccines for the prevention of fowl pox in chickens and turkeys have been available for more than five decades. However, in recent years outbreaks have occurred in several previously vaccinated chicken flocks. Presumably, fowl poxviruses (FPVs) antigenically different from the attenuated vaccine strains are responsible for such occurrences. In support of this concept, we previously detected minor antigenic changes in field isolates based on comparative immunoblotting with polyclonal anti-FPV serum. Realizing the need for antibodies specific against the dominant antigens of FPV, monoclonal antibodies (MAbs) were produced by immunizing mice with either a field strain of FPV or a pigeon poxvirus, currently used for vaccination. Three hybridoma clones producing MAbs reacting with a specific FPV protein were selected from a total of 83 clones. In immunoblots, two of the MAbs, P1D9 and P2H10, recognized an antigen with an apparent molecular weight varying from 39 to 46 kD, depending on the FPV strain. The third MAb, P2D4, reacted with an approximately 80-kD protein, regardless of which FPV isolate was tested. Immunofluorescent staining with P1D9 and P2D4 revealed that these MAbs react with intracytoplasmic antigens in FPV-infected cells.