Purified Newcastle Disease Virus Research Articles

Authorizing of Immunogenicity of Concentrated and Purified Newcastle Disease Virus V4 Strain using Downstream Processing.

Newcastle disease virus (NVD) from the Paramyxoviridae family is a single-stranded negative-sense RNA virus. This infection can affect both domestic poultry and almost all other bird species. It has been considered a very severe difficulty for the poultry industry all over the world. Even though it remains a potential threat to poultry industries, this virus is a powerful oncolytic virus as well. In this study, a process was accomplished to achieve concentrated and highly purified NDV V4 strain particles. Downstream processing of Newcastle virus strain V4 was characterized by amplifying virus in embryonated chicken eggs. Through a sequence of steps, harvesting allantoic fluid, clarification by centrifuge, concentration by ultrafiltration, and size exclusion separation, the reduced volume and pure virus particles were considered for the amount of ovalbumin, hemagglutinin activity, transmission electron microscopy (TEM), electrophoresis, and additionally immunogenicity of prepared antigens. The results presented a high recovery of HA activity in concentrated and purified virus with the removal of ovalbumin and the typical morphology based on TEM. Sepharose CL-4B was determined as the best media among all used resins to purify the virus. Prepared formulations as vaccines demonstrated positive hemagglutinin inhibition for 6 months and stability for 2 years. Strong evidence from organized studies supports the effectiveness of this method in concentrating and purifying intact NDV, which could be valuable in vaccine research, antiserum preparation, or even as an alternative oncotic agent to traditional methods. Despite further studies being conducted, this method can be utilized particularly on a semi-industrial scale to produce various vaccine components.

Concanavalin A- Sandwich-ELISA for the detection of antibodies against Newcastle disease virus (NDV) in chicken

Concanavalin A (Con A), a lectin interacts with carbohydrate moieties of viruses and provide stable and sensitive detection when used as a capture agent. Indirect ELISA methods need purified Newcastle disease virus (NDV) or recombinant antigens for adsorption, whereas use of Con A as capture agent will enable the use of non-purified and non-concentrated virus as antigen replacing costly and time-consuming virus purification step. Con A based sandwich ELISA with non-purified NDV whole virus antigen with single serum dilution format has been developed in this study. The optimum concentrations of the capture agent, Con A and non-purified antigen preparations were determined by checker-board titration. Briefly, microplates were coated with predetermined optimum concentration of ConA (0.5 mg/ml; 50µg per well) and incubated for 18h at 4°C. After washing, allantoic fluid with Newcastle disease virus (NDV) LaSota (HA titre, 210) at a constant predetermined dilution (1:1; 50µl) was coated and incubated for 45 min at 37°C, followed by blocking with 2 % bovine serum albumin for 45 min at 37°C. The antigen coated plates were used in the detection of antibody titre against NDV in serum samples at single serum dilution of 1: 500. Then, wells were added with goat anti-chicken IgG horseradish peroxidase conjugate and incubated for 1h at 37°C, followed by addition of TMB substrate and the plates were read spectrophotometrically at 450 nm. ELISA antibody titres were determined by standard serial dilution of positive sera and endpoints were calculated by a subtraction method. By using positive negative threshold curve (PNT), intercept and slope of the standard curve were calculated. Total of 271 random chicken serum samples were analyzed for antibodies against NDV by Haemagglutination inhibition assay (HI), indirect ELISA and compared with the Con A- S- ELISA developed in this study. The Con A-S-ELISA showed a high coefficient of correlation (r=0.85, n=271, P less than 0.01) and an agreement of ê=0.99 with the commercially available Indirect-ELISA. The relative sensitivity and specificity were 98% and 85% respectively in comparison to HI test. Hence, the Con A-S-ELISA is a simple, easy and effective for monitoring serum antibody levels against NDV.

Proteomic analysis of purified Newcastle disease virus particles

BackgroundNewcastle disease virus (NDV) is an enveloped RNA virus, bearing severe economic losses to the poultry industry worldwide. Previous virion proteomic studies have shown that enveloped viruses carry multiple host cellular proteins both internally and externally during their life cycle. To address whether it also occurred during NDV infection, we performed a comprehensive proteomic analysis of highly purified NDV La Sota strain particles.ResultsIn addition to five viral structural proteins, we detected thirty cellular proteins associated with purified NDV La Sota particles. The identified cellular proteins comprised several functional categories, including cytoskeleton proteins, annexins, molecular chaperones, chromatin modifying proteins, enzymes-binding proteins, calcium-binding proteins and signal transduction-associated proteins. Among these, three host proteins have not been previously reported in virions of other virus families, including two signal transduction-associated proteins (syntenin and Ras small GTPase) and one tumor-associated protein (tumor protein D52). The presence of five selected cellular proteins (i.e., β-actin, tubulin, annexin A2, heat shock protein Hsp90 and ezrin) associated with the purified NDV particles was validated by Western blot or immunogold labeling assays.ConclusionsThe current study presented the first standard proteomic profile of NDV. The results demonstrated the incorporation of cellular proteins in NDV particles, which provides valuable information for elucidating viral infection and pathogenesis.

Rapid immunofiltration assay of Newcastle disease virus using a silicon sensor

A rapid nonradioactive sandwich immunoassay which utilizes biotin-streptavidin mediated filtration capture of immune complexes in conjunction with a silicon sensor was developed for the detection of virus. Using purified Newcastle disease virus as a model, the lower limits of detection (LOD) were determined for a number of immunoassay configurations employing both monoclonal and polyclonal antibodies. The LODs ranged from 1.3 ng/ml (sample volume of 100 μl) for an incubation of 60 min to 400 ng/ml for a 1 min incubation. The sandwich immune complexes were formed from one-step incubation of antibody and antigen. No ‘hook’ effects were observed over a wide range of analyte concentrations. The assays were easy to perform and required a total time equal to the incubation period plus about 5 min. The assay format is suitable for virus, bacteria and protein antigens. New assays can be developed and optimized readily, often within 1 day.

Sensitive fluorogenic enzyme immunoassay on nitrocellulose membranes for quantitation of virus

A highly sensitive fluorogenic enzyme-linked immunosorbent assay (FELISA), which utilizes nitrocellulose membranes as solid phase support, has been developed for the detection and identification of virus in clinical samples. Reagents were standardized and, using purified Newcastle disease virus (NDV) as a model, the theoretical lower limits of test sensitivity of the FELISA were compared, in both “sandwich” and “indirect” formats, to those of a comparable chromogenic enzyme-linked immunosorbent assay (CELISA). Of the systems evaluated, the “sandwich” FELISA exhibited maximum sensitivity and detected 10 fg of purified virus protein per milliliter of test sample (500 ag per test volume). Specificity of the “sandwich” FELISA was evaluated by challenging the system with heterologous strains of NDV and with other serologically related and unrelated viruses. In a clinical trial in which fecal materials from chickens undergoing vaccination with NDV were assayed directly by FELISA, the virus was detected from the first to approximately the tenth day post-vaccination. The test is simple to perform and results can be obtained in approximately 4 h.

Identification of the P proteins and other disulfide-linked and phosphorylated proteins of Newcastle disease virus

A unique abundant protein, designated P by analogy to the putative polymerase proteins of other paramyxoviruses, was identified in purified Newcastle disease virus. Under nonreducing conditions the P proteins could be separated from other viral proteins on sodium dodecyl sulfate-polyacrylamide gels. The P proteins were isolated from detergent-solubilized virions as 53,000- to 55,000-dalton monomers and disulfide-linked trimers. Distinct forms of P having four different isoelectric points and two different electrophoretic mobilities were resolved by two-dimensional electrophoresis. Two forms of P were phosphorylated, as were the nucleocapsid protein and non-glycosylated membrane protein. In addition to disulfide-linked forms of P, dimers of the hemagglutinin-neuraminidase glycoprotein and two disulfide-linked versions of the fusion glycoprotein were identified. Several electrophoretic variants of the nucleocapsid protein that were probably created by intrachain disulfide bonding were also isolated from virions under nonreducing conditions. The locations of the newly identified proteins were determined by detergent-salt fractionation of virions and by surface-selective radioiodination of the viral envelope. The P proteins were associated with nucleocapsids and were not detected at the surface of virions. Both forms of the fusion glycoproteins were on the exterior of the viral envelope. Herein the properties of the P proteins are compared with similar proteins of rhabdoviruses and other paramyxoviruses, and a role for multiple forms of proteins in the genetic economy of newcastle disease virus is discussed.

Use of antibodies to purified Newcastle disease virus glycoproteins for strain comparisons and characterizations.

The H/N and F1 glycoproteins responsible for hemagglutinin/neuraminidase (H/N) activity and fusion (F1) activity were purified from several Newcastle disease virus strains and used to produce antisera. These antisera can be used to clearly distinguish the virus strains by a number of techniques including radioimmunoassay, and so provide a means of classifying new virus isolates. The variations detected between strains are not related to field virulence.

In vitro radiolabeling procedure which labels the proteins of Newcastle disease virions with carbon-14.

A convenient and gentle procedure was used for in vitro radioisotopic labeling the proteins of Newcastle disease virus. This method, which utilizes [14C]formaldehyde and sodium borohydride, labels all the proteins of purified Newcastle disease virus. Virions labeled in this manner retained biological and biopysical integrity

Methylation of messenger RNA of Newcastle disease virus in vitro by a virion-associated enzyme.

Purified Newcastle disease virus contains an enzyme that incorporates the methyl group from S-adenosyl-L-methionine into RNA synthesized in vitro by the virion-associated RNA polymerase (RNA nucleotidyltransferase). Incorporation of radioactivity from S-adenosyl-L-[methyl-3H]methionine was totally dependent upon RNA synthesis. The methylation reaction was completely inhibited by S-adenosyl-L-homocysteine, suggesting the transfer of only the methyl group of S-adenosyl-methionine to RNA products. Velocity sedimentation and hybridization of the in vitro product RNA indicated that both [3H]methyl and [32P]GMP labels resided in single-stranded 18S RNA molecules which were virus specific. Approximately 1 to 2 methyl groups were incorporated per RNA molecule. DEAE-cellulose chromatography of product RNA after alkaline hydrolysis suggested that the 5' terminus was the site of methylation.

The structural proteins of Newcastle disease virus: I. Identification of a minor internal protein

Purified Newcastle disease virus (NDV), Miyadera strain, was dissociated and its proteins were analyzed by SDS polyacrylamide gel electrophoresis. Polypeptide VP 9 was consistently found to be the fastest migrating viral protein with an estimated molecular weight of approximately 26,500 when compared to known protein markers. VP 9 comprises approximately 3% of the total viral protein of virions grown in either chick embryo or HeLa cells. VP 9 was also identified by immunological procedures. Hyperimmune Ab-9 serum prepared against the purified VP 9 protein (eluted from polyacrylamide gels), was incapable of neutralizing the hemagglutinin or neuraminidase activities of NDV. In addition, this antibody did not react with the S-antigen (nucleoprotein) of the virus. Complement-fixation (CF) tests revealed that this antibody could react with solubilized NDV, but not intact virions. Immunofluorescent staining of NDV-infected HeLa cells with FITC-labeled Ab-9 resulted in cytoplasmic fluorescence which was not blocked by pretreatment with either unlabeled virus-specific antibody or anti-S antibody. To determine the relative proportion of VP 9 antigen in NDV stocks grown in various cell types, CF tests were performed with detergent-dissociated NDV grown in different cells. If the amount of test virus used in these CF reactions was standardized for S-antigen units, the CF titers for VP 9 antigen were the same for virus preparations grown in various cell types, whereas up to an 8-fold variation in titer was observed if standardization was based on hemagglutinin or neuraminidase units. It is concluded that VP 9 is an internal polypeptide of NDV virions which does not constitute part of the nucleoprotein complex and which does not exhibit significant quantitative variations as influenced by host factors.

Studies on the structural basis of the RNA polymerase activity of newcastle disease virus particles.

SUMMARY Treatment of purified Newcastle disease virus (NDV) with Triton N101 to activate the virus particle RNA polymerase in the in vitro assay removed the glycoproteins and most of the phospholipid from the virus. The subvirus particles produced by the action of Triton N101 on virus had RNA polymerase activity, whereas purified NDV nucleocapsids did not. Apparently, either nucleocapsids did not contain the proteins essential for RNA polymerase activity, or the enzyme was denatured during the preparation of nucleocapsids. Incubation of several purified strains of NDV at 4 °C led to loss of polymerase activity which was regained on incubation at 37 °C. It is suggested that internal configurational changes in the virus particle were the most likely causes of these changes in enzyme activity on incubation at 4 °C and 37 °C.

Subunits of NDV: Hemagglutinin and neuraminidase subunits of Newcastle disease virus

When purified Newcastle disease virus (NDV) was treated with deoxycholate (DOC), two major surface antigens of the virus, hemagglutinin and neuraminidase, were released in biologically active form and the respective antigens could be separated by sucrose gradient centrifugation into two distinct bands. The hemagglutinin subunits obtained by the DOC method were reactive with hemagglutination-inhibiting antibody but did not agglutinate red blood cells even after they were freed from DOC by dialysis. Thus, they appeared to be monovalent. However, they acquired the capacity to agglutinate red blood cells, when they were mixed with the top fraction of the sucrose gradient through which the DOC-disrupted virus had been centrifuged, or by the addition of a phospholipid, phosphatidylethanolamine. Electron microscopic examination of the hemagglutinin subunits revealed filamentous structures associated with several projections on both sides. These results suggested that polymerization of the monomeric subunits occurred, not directly end to end, but mediated through the filament which might be phospholipid in nature.

The structural proteins of Newcastle disease virus

Highly purified Newcastle disease virus has been shown by acrylamide gel electrophoresis to contain three main polypeptides. The probable functional role of each polypeptide has been established by dissociating the radioactively labeled virion with detergents, then separating the resultant subunits by gradient centrifugation, and analysing their composition and function. The nucleocapsid contains a single polypeptide of molecular weight 54,000, which comprises some 45% of the total viral protein. The hemagglutinin and the neuraminidase are composed of polypeptides with molecular weights of 80,000 and 38,000, respectively, which each comprise about 20% of the total viral protein. Other polypeptides make up less than 10% of the total. The purified virion contains little or no host protein.

Physicochemical studies of Newcastle disease virus. 3. The content of virus nucleic acid and its sedimentation pattern.

Chemical analysis was performed on the highly purified Newcastle disease virus (NDV) obtained from infected allantoic fluid. Mean values of RNA content of the virus based on optical density at 260 mμ, phosphorus content and orcinol reaction which were carried out after the fractionation bySchmidt-Thannhauser-Schneider procedure modified byFleck andMunro were 0.72%, 0.99% and 0.92%, respectively. Contents of protein and lipid in the virus were also determined.