Disease Virus Antigen Research Articles

Multiple-infected diagnostic specimens from foot and mouth disease endemic regions

The current routine diagnostic procedures for foot and mouth disease (FMD) virus antigen detection combine the use of an indirect sandwich enzyme-linked immunosorbent assay (ELISA) with virus isolation and amplification on cell culture. Several field samples recently received by the World Reference Laboratory for Foot and Mouth Disease which were initially diagnosed as containing a single virus type have subsequently been found to contain an additional virus type. Examples are given of the results of ELISAs performed on certain Saudia Arabian samples; these examples illustrate the problem which such multiple-infected samples present for laboratory diagnosis.

A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli

The 3D gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 3D gene from a serotype Cl virus has been cloned and overexpressed in Escherichia coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells.

Precipitation reactions with Newcastle disease virus.

Acid precipitated and detergent treated Newcastle disease virus (NDV) antigen was prepared and characterised using the agar gel precipitation (AGP) test. The detergent treated NDV antigen was used to screen antibodies to NDV and the results compared with the conventional haemagglutination inhibition (HI) test titres. AGP test could detect NDV antibodies in serum samples when the corresponding HI titres were 1:8 and above. Detergent treatment of Newcastle disease virus greatly reduced its haemagglutinating ability. Simultaneous detection of antibodies to NDV and infectious bursal disease virus in AGP test was attempted and found successful.

A recombinant foot-and-mouth disease virus antigen inhibits DNA replication and triggers the SOS response in Escherichia coli.

The 3D gene of foot-and-mouth disease virus encodes the viral RNA dependent RNA polymerase, also called virus infection associated (VIA) antigen, which is the most important serological marker of virus infection. This 3D gene from a serotype C1 virus has been cloned and overexpressed in Escherichia coli under the control of the strong lambda lytic promoters. The resulting 51 kDa recombinant protein has been shown to be immunoreactive with sera from infected animals. After induction of gene expression, an immediate and dramatic arrest of cell DNA synthesis occurs, similar to that produced by genotoxic doses of the drug mitomycin C. This effect does not occur during the production of either a truncated VIA antigen or other related and non-related viral proteins. The inhibition of DNA replication results in a subsequent induction of the host SOS DNA-repair response and in an increase of the mutation frequency in the surviving cells.

Reverse passive haemagglutination test in the diagnosis of infectious bursal disease.

The reverse passive haemagglutination test (RPHA) was used to detect infectious bursal disease (IBD) virus antigen in various organs of experimentally infected chickens and field cases. The results of RPHA were compared with those of agar gel immunodiffusion (AGID) and latex agglutination (LAT) tests. IBD antigen was detected in 86.4%, 80.4% and 80.5% of different organs by RHPA, LAT and AGID respectively. Although the differences are not statistically significant, the LAT is recommended because of cost and speed of obtaining results.

Association between Pathogenicity of Infectious Bursal Disease Virus and Viral Antigen Distribution Detected by Immunohistochemistry

Highly pathogenic infectious bursal disease virus (IBDV) strains (Ehime/91, DV86) and a moderately pathogenic strain (J1) were compared in order to clarify the association between the pathogenicity of IBDV and viral antigen distribution. Virus target cells in the bursa, thymus, spleen, and bone marrow were examined by transmission electron microscopy. Although all strains caused similar bursal atrophy, the highly pathogenic strains brought about a greater decrease in the thymic weight index and more severe lesions in the cecal tonsil, thymus, spleen, and bone marrow. Immunohistochemical detection of IBDV antigen in tissues from chickens infected with Ehime/91 and DV86 strains showed a higher frequency of antigen-positive cells in the spleen and bone marrow. Transmission electron microscopy indicated the presence of viral particles in the cytoplasm of epithelial reticular cells in the thymus and monocytes in the bone marrow. The results show that pathogenicity of field strains of IBDV correlates with lesion production in non-bursal lymphopoietic organs. The results also suggest that pathogenicity of IBDV may be associated with virus antigen distribution in non-bursal lymphopoietic organs.

Occurrence of keratoconjunctivitis apparently caused by Mycoplasma gallisepticum in layer chickens.

Natural cases of keratoconjunctivitis, apparently caused by Mycoplasma gallisepticum (MG), in layer chickens are described. The disease occurred in a commercial flock consisting of 36,000 pullets (Babcock), first appearing around 30 days of age. Clinically, affected chickens showed unilateral or bilateral swelling of the facial skin and the eyelids, increased lacrimation, congestion of conjunctival vessels, and respiratory rales. Some of the severely affected chickens closed their eyes. The morbidity reached 27.8%, and it was estimated that approximately 10% died from reduced feed intake due to impaired vision. Ten 70-day-old chickens with clinical diseases were examined for lesions. There was acute to subacute keratoconjunctivitis in all of the chickens, and some exhibited laryngitis. Adherence of mycoplasma organisms to epithelial cells of the conjunctiva, cornea, and larynx was frequently observed. These organisms had an ultrastructure characteristic of MG and showed a positive reaction with rabbit polyclonal antibodies against the S6 strain of MG by immunohistochemical analysis. MG was isolated from tissue homogenates of the eyelids and tracheas of the affected chickens. Many of the chickens had atrophic bursae, and infectious bursal disease virus antigens were detected in necrotic bursal follicles by immunostaining. Therefore, immunosuppression due to infectious bursal disease was implicated in the pathogenesis of keratoconjunctivitis in the present cases.

Purification and Biochemical Characteristics of the Rabbit Haemorrhagic Disease Virus

The causative rabbit haemorrhagic disease viral antigen (RHDV) was purified by sucrose and caesium chloride density gradients. The viral fractions were characterised by a haemagglutination test and a dodecylsulfat polyacrilamide gel electrophoresis (SDS-PAGE). Different migration patterns were observed after sucrose and caesium chloride (CsCl) density gradient centrifugations.

Bioluminescent Analysis of β-Galactosidase and Its Application for Immunoassay and DNA-Probes

Abstract Bioluminescent method for β-galactosidase assay is proposed with sensitivity as high as 0.25 fmol/1 with the help of bioluminogenic substrate – firefly luciferin derivative. Method was applied to immunoassay of foot-and-mouth disease viral antigen and biotinylated DNA-probe analysis with the help of β-galactoside labelled conjugates. Methods proved to be very sensitive, rapid and simple in performance. Photographic detection and reprobing of the samples are possible.

Syngeneic lysis of reticuloendotheliosis virus-transformed cell lines transfected with Marek's disease virus genes by virus-specific cytotoxic T cells

Cell-mediated immune responses against Marek's disease virus (MDV) antigens were examined using reticuloendotheliosis virus (REV)-transformed cell lines of two haplotypes ( B 19 B 19 and B 13 B 13). These cell lines were stably transfected with cloned fragments of MDV DNA resulting in the expression of the MDV-specific phosphoprotein pp38. Effector cells were obtained from P2a ( B 19 B 19) and S13 ( B 13 B 13) chickens at 7 days post inoculation with REV, oncogenic or attenuated serotype 1 MDV (JM-16/O and JM-16/A, respectively), serotype 2 MDV (SB-1), or herpesvirus of turkeys (HVT). Transfection of MDV genes did not influence the expression of Class I major histocompatibility complex antigens. The optimal effector to target cell ratio was determined to be 100:1. REV-sensitized effector cells lysed REV cell lines and REV cell lines transfected with MDV DNA in a syngeneic fashion. Effector cells from chickens inoculated with JM-16/O, JM-16/A, SB-1 or HVT lysed only the syngeneic, transfected cell lines, but not the parent REV cell lines. The percentage specific release caused by the MDV-sensitized effector cells was low, but statistically significant.

Distribution of Border disease virus antigen in lymphocyte subpopulations in the peripheral blood of experimentally infected lambs

Ten lambs were experimentally infected with Border disease virus and the distribution of viral antigen in lymphocyte subpopulations studied by flow cytometry. The virus was isolated in culture from the peripheral blood mononuclear cells (MNC) of all experimentally infected lambs for a mean period of 9.8 ± 1.03 days. The peak virus titre of 3.26log 10 TCID 50 per 10 6 MNC was attained Day 9 post-inoculation (pi). Viral antigen was present in peripheral blood lymphocytes of experimentally infected lambs as early as 24 h pi and continued to be detected up to Day 10 pi. The number of lymphocytes expressing viral antigen rose from 12.38 ± 1.22% in samples taken Day 3 pi to 23.21 ± 2.82% on those collected Day 7 pi, dropping gradually thereafter. During the peak period of infection, 12.46 ± 2.09% of B cells, 37.71 ± 10.96% of T cells and 52.33 ± 8.27% of lymphocytes which were neither B nor T lymphocytes expressed viral antigen. Most of the infected lymphocytes expressed the OvCD5 (T cell) molecule. The virus affected all T cell subsets but the suppressor/cytotoxic (OvCD8+) cells appeared to be the main targets. During the peak period of infection, 54.20 ± 6.16% of the infected T cells expressed the OvCD8 molecule, 31.58 ± 7.12% were OvCD4+, and 12.67 ± 6.50% were OvWC1+ (T-19+, γ/ δ).

Use of combined Shewhart-CUSUM control charts in internal quality control of enzyme-linked immunosorbent assays for the typing of foot and mouth disease virus antigen.

An enzyme-linked immunosorbent assay (ELISA) for the typing of foot and mouth disease virus (FMDV) antigen was employed for the routine laboratory diagnosis of FMD at a regional veterinary laboratory in northern Thailand. An objective procedure was developed to monitor the test performance of the ELISA, using absolute test control limits in a Shewhart-CUSUM (cumulative sum) control chart method. The procedure detected significant data trends and 'beyond control limit' situations for each antigen typing system (types O, A and Asia 1), using an assay variable (gamma i). Retrospective analysis using Shewhart-CUSUM control charts of data from 42 ELISAs demonstrated that control limits were exceeded in two assays for FMDV type A. The Shewhart-CUSUM control chart is a simple and reliable internal quality control method for the detection of significant random and systematic variation in assays.

Border disease virus antigens in lymphocyte subpopulations in the peripheral blood of persistently infected sheep

Biotinylated virus-specific antibodies were used to detect Border disease virus antigen by flow cytometry in the mononuclear cells of the peripheral blood of 15 sheep persistently infected with Border disease virus. The viral antigen was present in 12.86±4.65% (mean±SD) of mononuclear cells (MNC). The percentage of MNC that contained viral antigen was higher in lambs than in adult sheep, with mean rates of 22.68±5.02% and 11.65±4.39%, respectively. Depletion methods were used to estimate the distribution of viral antigen in peripheral blood lymphocyte subsets. The viral antigen was present in T-cells (OvCD5+), B-cells (LCAp220+) and non-T- and non-B-cells. Depletion studies revealed that 5.39±2.47% of the cells expressing the LCAp220 epitope (B-cells), 23.38±11.38% of those expressing the OvCD5 epitope (T-cells) and 55.07±10.93% of those which were neither B- nor T-cells were positive for viral antigen. Most (57.18±5.41%) of the T-cells containing viral antigen were cytotoxic/suppressor (OvCD8), 25.63±2.97% were helper (OvCD4) cells and 12.24±3.21% expressed the γ/ δ (OvWC1) epitope.

Modification of infectious bursal disease virus antigen VP2 for cell surface location fails to enhance immunogenicity

The host protective antigen gene VP2 of infectious bursal disease virus (IBDV) was genetically modified and expressed by recombinant fowlpox viruses (rFPV). To achieve cell surface localization, VP2 was expressed as a hybrid protein with signal sequence and membrane anchors of influenza virus hemagglutinin or neuraminidase. Native VP2 was expressed as VP2 alone or as self-processing VP2-VP4-VP3 polyprotein for coexpression of IBDV structural proteins. VP2 hybrid protein containing the carboxy-terminal membrane anchor sequence of influenza virus hemagglutinin was located on the cell surface and was N-glycosylated. The expression of VP2 fused to the N-terminal signal/anchor sequence of influenza virus neuraminidase led to cell lysis and the VP2 protein remained mainly unglycosylated. Cell surface localization of VP2 reduced immunogenicity (antibody induction) and abolished protection in poultry in comparison with the native VP2 expressed by FPV as VP2 alone or as the self-processing VP2-VP4-VP3. Vaccination of poultry with rFPV expressing native VP2 protein alone provided better protection from IBDV infection than VP2 derived from the VP2-VP4-VP3 polyprotein.

Antibody response to 146S particle, 12S protein subunit and isolated VP1 polypeptide of foot-and-mouth disease virus type Asia-1

The antibody response to foot-and-mouth disease virus (FMDV) antigens of type Asia-1 in guinea-pigs was studied by micro-serum neutralization test (MSNT) and enzyme-linked immunosorbent assay (ELISA). One inoculation of as little as 1 μg of binary ethyleneimine (BEI)-insctivated 146S virus particles in guinea-pigs elicited enough neutralizing antibodies to protect them against challenge with virulent virus. However, one inoculation of live 146S virus particles elicited higher levels of neutralizing antibodies in guinea-pigs than that of inactivated 146S particles. One inoculation of 12S protein subunits in guinea-pigs elicited only non-neutralizing anti-12S antibodies detected by ELISA. Similarly, non-neutralizing anti-VP1 antibodies were detected by ELISA after one inoculation of VP1. However, multiple inoculations of 12S or VP1 elicited measurable neutralizing antibodies in guinea-pigs that protected them against challenge.

Local pathological reactions induced in pigs and cats by adjuvant ISA-70 containing inactivated Newcastle disease virus antigen.

Gross and microscopic changes at the injection site in pigs and cats were investigated for 8 weeks after an intramuscular injection of oil adjuvant ISA-70 emulsion, containing inactivated Newcastle disease virus antigen. In pigs, an egg-sized and discolored lesion with pinpoint to miliary-sized nodules was observed at 2 to 8 weeks post injection (PI). In cats, there was partial thickening of the subcutaneous tissue at 2 to 8 weeks PI. Histopathologic changes in pigs were fundamentally similar to those in cats, however, the local tissue reactions in pigs were severer and more protracted than those in cats. The lesions in pigs were characterized by formation of large-sized cysts and well-developed encapsulation of the cysts with epithelioid cells.

Local Pathological Reactions and Immune Response of Chickens to ISA-70 and Other Adjuvants Containing Newcastle Disease Virus Antigen

Chickens were examined for gross and microscopic changes at the injection site for 16 weeks after an intramuscular injection of the following oil adjuvant emulsions containing inactivated Newcastle disease virus (NDV) antigen: ISA-70, Freund's incomplete adjuvant (FIA), Freund's complete adjuvant (FCA), and aluminum phosphate gel (APG). The reactions induced by ISA-70 emulsion were characterized by proliferation of macrophages, epithelioid cells, and fibroblasts around the small cysts in the muscle. Infiltration of lymphocytes and plasma cells also was present. These changes peaked 2 weeks postinjection and were milder than lesions due to the Freund's adjuvants. FCA induced the most severe granulomatous lesions with abscess formation. The reactions to APG were characterized by proliferation and accumulation of macrophages. The serological results revealed that the enhancing effect of ISA-70 on NDV antigen was the same as that of FIA.

Immunohistochemical Detection of Newcastle Disease Virus in Chickens

An immunoperoxidase histochemical technique utilizing a monoclonal primary antibody was developed for detection of Newcastle disease virus (NDV) antigen in tissues from chickens. The technique was applied to trachea, lung, spleen, Harderian gland, and cecal tonsil harvested from specific-pathogen-free (SPF) chickens at 2, 5, 7, 10, and 14 days postinoculation (PI) with NDV, and to corresponding tissues from commercial broiler chickens representing 30 cases of spontaneous respiratory disease. Positive staining occurred in the cytoplasm of respiratory epithelial cells in the trachea or bronchi of NDV-inoculated SPF chickens at 5 and 7 days PI. Staining also occurred in the respiratory epithelium of the trachea and bronchi of commercial broilers from seven of 30 cases of spontaneous respiratory disease. These results indicate that the immunoperoxidase technique has value as a rapid diagnostic test for Newcastle disease.

Detection of rabbit haemorrhagic disease virus antigen in tissues by immunohistochemistry

Formalin fixed liver, spleen, kidney, heart, lung, duodenum and appendix tissues from nine rabbits, experimentally infected with rabbit haemorrhagic disease virus ( RHDV), were investigated for evidence of RHDV antigen by the direct avidin-biotin peroxidase complex immunohistochemical method. In all the rabbits examined, RHDV antigen was detected in degenerative and necrotic hepatocytes of the liver tissues. The area involved coincided with histopathological lesions on serial liver sections. The RHDV antigen was expressed in the cytoplasm of the hepatocytes, suggesting that RHDV replicated in these cells. RHDV antigen was also detected in the spleen. The results of immunohistochemistry were supported by the demonstration of RHDV protein by Western blot analysis and of RHDV particles by protein A-gold immunoelectron microscopy in the liver homogenate from all the rabbits that were examined.

Immunohistochemical localization of the rabbit haemorrhagic disease viral antigen.

Immunohistochemical investigations were carried out to determine organ and cellular localization of the rabbit haemorrhagic disease viral antigen (RHDVA). It was found in certain parenchymal liver cells near the interlobular septs and in some macrophages and pseudoeosinophils of all studied organs and blood. Whereas in morphologically preserved hepatocytes and macrophages the RHDVA accumulated in the nuclei, in cells with disintegrated nuclei it was distributed throughout the cytoplasm.