Antigenic characterization of the internal proteins of Newcastle disease virus by monoclonal antibodies

We have prepared and characterized monoclonal antibodies against the three internal structural proteins, M, P and NP, of Newcastle disease virus. At least two non-overlapping antigenic sites were delineated on the M protein, four on the P, and two on the NP by competitive binding assay. One of the two non-overlapping antigenic sites on the M protein was found to be a cluster of at least three distinct epitopes. Enhancement of antibody binding by the binding of a second antibody was observed with the M protein. The reactivity of these monoclonal antibodies with heterologous strains was studied by enzyme-linked immunosorbent assay. The results indicated that there are both highly conserved antigenic sites and those subject to remarkable change on both M and P proteins. On the other hand, NP appeared to be antigenically more stable.     

Antigenic and phylogenetic studies on a variant Newcastle disease virus using anti-fusion protein monoclonal antibodies and partial sequencing of the fusion protein gene.

A virulent Newcastle disease virus (NDV) isolate, 34/90, reported to show considerable antigenic diversity from more classical strains of NDV, including vaccine strains, was evaluated phylogenetically and for the presence of neutralizing epitopes on the fusion protein. Comparison of a 309 nucleotide sequence of the fusion protein gene of 34/90 with other viruses confirmed the diversity of this virus, placing it in a discrete fifth genetic lineage with an avirulent virus isolated from waterfowl and genetically quite distant from other strains and isolates. The virus-neutralizing mAbs used in the present study were directed against at least seven distinct epitopes on the fusion protein. Of these seven, five are shared by 34/90 and the live vaccine virus Hitchner B1 and these plus an additional epitope are shared by 34/90 and strain Ulster 2C, which is used in inactivated vaccines. Two potential distinct epitopes were also shared by these three viruses. The results suggest that despite the detected antigenic and genetic variation of 34/90, it is unlikely that mutants which fail to be neutralized by antibodies induced by conventional vaccines would arise readily.     

Antigenic analysis of Japanese encephalitis virus by using monoclonal antibodies

Hybridoma cells were produced by fusing P3X63Ag8.653 mouse myeloma cells with spleen cells from BALB/c mice immunized with Japanese encephalitis (JE) virus, Nakayama-RFVL strain. The resulting 26 clones produced hemagglutination inhibition antibodies against the homologous strain. The hemagglutination inhibition reactivity of each clone was tested against six flaviviruses: JE, Murray Valley encephalitis (MVE), Egypt 101 strain of West Nile (WN), St. Louis encephalitis (SLE), Russian spring summer encephalitis, and dengue type 1. The 26 monoclonal antibodies fell into four groups: 14 JE species-specific antibodies, 6 antibodies reactive to JE and MVE viruses, 3 antibodies to three or four viruses in the JE-MVE-WN-SLE subgroup, and 3 antibodies to all six flaviviruses. Furthermore, antigenic comparison of 27 strains of JE virus was carried out by using five JE species-specific monoclonal antibodies. Of these, 24 strains were isolated in various parts of Japan, and 3 strains came from Southeast Asia. In reactivity, the 27 strains were classified into at least four antigenic groups. The results showed that the Nakayama-Yakken strain is a mutant strain which lacks the Nakayama strain-specific antigen and that the recently isolated strains are immunologically different from Nakayama and JaGAr 01 strains. One clone (NARMA 13) produced a JE species-specific antibody which showed almost the same titer against 26 JE virus strains, whereas one clone (NARMA 5) produced a Nakayama strain-specific antibody which reacted only to the Nakayama-RFVL and Nakayama-Yoken strains.     

Antigenic analysis of West Nile virus strains using monoclonal antibodies

Summary Seventeen monoclonal antibodies (MAbs) were prepared against the flavivirus West Nile strain H442. While the majority of these were specific for the major envelope protein, MAbs directed against the NS1 and ns4a nonstructural proteins were also identified. The MAbs were tested by indirect immunofluorescence against 16 southern African West Nile (WN) isolates, representative strains from the two main WN antigenic groups and several viruses from other flavivirus complexes. The MAb reactivities ranged from WN strain-specific to broadly flavivirus-group reactive. Comparison of the local isolates revealed the presence of several different strains, all of which were antigenically distinct from the representative strains of the two WN antigenic groups.     

Antigenic analysis of tick-borne encephalitis virus group using monoclonal antibodies and immunofluorescence

The study included 18 monoclonal antibodies (MAb) to E- or NS1-antigens tested by immunofluorescence with tick-borne encephalitis (TBE) complex viruses. MAb were induced to 3 strains of TBE virus: the pathogenic 4072 strain isolated from a patient; the Skalica strain of low pathogenicity; and the Neidorf strain isolated from ticks. According to their reactivity to complex viruses, MAb comprised 3 groups: monospecific for TBE virus (T6, T15) which detected tick-borne encephalitis virus alone; widely cross-reactive with 4-6 viruses of the complex (NEK, KEN, T7, T9); and partially complex-reactive (T11, T12, T13, T33/3) and bound to 2-3 viruses of the complex. T13 and T33/3 MAb reacted with the Omsk hemorrhagic fever virus to the same degree or stronger than with TBE virus. The cross-reactivity was more marked in anti-E-than in anti-NS1 MAb. The similarity of the Langat viruses and the Skalica strain was confirmed. Using anti-NS1 MAb in tests with non-fixed cells, the release of NS1-antigen was found to begin at hour 18 (time of observation). The results of the study may be useful for improvement of laboratory diagnosis of TBE and evaluation of the capacity of a vaccine to induce cross immunity to viruses of the TBE complex.     

Antigenic diversity and similarities detected in avian paramyxovirus type 1 (Newcastle disease virus) isolates using monoclonal antibodies.

Newcastle disease (ND) virus (APMV-1) isolates submitted to the International Reference Laboratory for ND were characterised antigenically by their ability to cause binding of mouse monoclonal antibodies (mAbs) to cell cultures infected with the isolate. Since the availability of the mAbs 1526 viruses have been examined using a panel of nine mAbs and 818 with an extended panel of 26 mAbs. Using the nine mAb panel a total of 14 different patterns was seen and viruses grouped by the same pattern showed relationships with each other which were either biological, temporal or geographical or more than one of these. There was a marked tendency of viruses placed in the same group to show similar virulence for chickens. Extension of the panel to 26 mAbs produced 39 distinct patterns, although some of these were seen with only a single virus. Again, viruses inducing similar binding patterns shared similar properties and some binding patterns were specific for viruses causing discrete epizootics. Cluster analysis of the mAb binding patterns did not produce concise, discrete groupings, but did emphasise some relationships between virus properties and antigenicity. Examples of the usefulness of this approach were the ability to link two important outbreaks to the contamination of stored food by infected feral pigeons, and the demonstration of two separate viruses responsible for outbreaks in countries of the European Union during 1991 to 1994 thus preventing erroneous epizootiological tracing.     

Antigenic comparison of foot-and-mouth disease virus serotypes with monoclonal antibodies

The capsid structures of the 7 serotypes of foot-and-mouth disease virus have been compared utilizing a series of neutralizing monoclonal antibodies which were previously shown to recognize at least 4 distinct epitopes on type A12 virus. A radioimmune binding assay using radioactively labeled antigens and the monoclonal antibodies revealed that certain conformation dependent epitopes are conserved among A subtypes, while some continuous epitopes are conserved among A subtypes as well as other FMDV serotypes. On the basis of differential reactivity among other FMDV subtypes and serotypes two additional epitopes have been defined on the A12 particle. Binding and neutralization assays revealed that the presence and function of an epitope are not necessarily correlated.     

Strain analysis and epitope mapping of West Nile virus using monoclonal antibodies.

Monoclonal antibodies (MAbs) against an Indian strain (804994) and an Egyptian strain (E 101) of West Nile virus (WNV) were prepared in mice. Nine MAbs against the 804994 strain and 5 MAbs against E 101 strain were obtained. All 14 MAbs reacted with the envelope (E) protein of WNV in an immunoblot assay. They were tested by an enzyme-linked immunosorbent assay (ELISA) for their cross-reactivity with WNV, Japanese encephalitis virus (JEV) and Dengue-2 virus (DEN-2), and for their reactivity in haemagglutination-inhibition (HAI) test. Based on these results MAbs were broadly grouped into three groups, namely WNV-specific HAI-positive, WNV-JEV cross-reactive HAI-positive, and WNV-JEV cross-reactive HAI-negative MAbs. The antigenic cross-reactivity between twelve WNV strains isolated from different geographical regions and their respective hosts was assessed using these MAbs in HAI and complement fixation (CF) tests. The strain analysis by CF distinguished Indian from South African strains. However, a similarity between some Indian and South African strains in HAI was observed. E 101 strain appeared to have antigenic similarity with Indian as well as South African strains. Overall it appears that antigenically similar strains of WNV are prevalent in India. A single heterogenous domain was apparent on the epitope map of WNV deduced by ELISA additivity test.     

Antigenic differentiation of strains of turkey rhinotracheitis virus using monoclonal antibodies

Monoclonal antibodies (mAbs) were prepared against one UK isolate of turkey rhinotracheitis virus (TRTV). Those which were virus-neutralizing were selected and used, together with polyclonal antisera raised to each isolate in turkeys, in cross-neutralization tests against TRTV strains isolated in the UK and elsewhere. Whilst the polyclonal antisera showed that there was some diversity between them, all strains examined belonged to one serotype. The TRTV strains isolated in the UK could clearly be differentiated from those isolated elsewhere by some of the mAbs. Isolates of TRTV made in South Africa in 1978 and UK in 1985 were more closely related than were isolates made in UK and France within a few months. TRTV strains isolated from turkeys and chickens could not be differentiated. Some mAbs were found to be group-specific in that they neutralized all TRTV strains examined. All mAbs were of either the IgGl or IgG2a isotype and recognized the surface G glycoprotein.     

Antigenic analysis of avian rotavirus VP6 using monoclonal antibodies

Summary Monoclonal antibodies (MAbs) were prepared to analyze antigens on the major inner capsid protein, VP 6 of avian group A rotavirus. Based on the results of a competitive binding assay using 15 MAbs directed against VP 6 of the PO-13 rotavirus strain, isolated from a pigeon in Japan, it was found that VP 6 of avian rotavirus possesses at least four spatially distinct antigenic sites. Two antigenic sites (I and II) were topologically distinct from the other two (III and IV), which were in close proximity. From the reaction of MAbs in indirect immunofluorescent antibody tests to a series of known rotaviruses, epitopes representing common antigens of all group A rotavirus including avian rotavirus were localized in sites II and III. One epitope in site IV appeared to have a subgroup antigenic specificity that reacted only with rotaviruses belonging to subgroup I. Interestingly, avian rotaviruses isolated from turkeys and chickens in Northern Ireland also reacted only with these subgroup I specific MAbs, but not with subgroup II specific MAb. This indicates that avian rotavirus has subgroup I specific antigen, which is antigenically similar to that of other mammalian rotavirus strains.     

Epitope mapping of the Lassa virus nucleoprotein using monoclonal anti-nucleocapsid antibodies

Summary Monoclonal antibodies with differing specificity were prepared against the Josiah strain of the Lassa virus. All monoclonal antibodies were characterized by subclass determination and the immunofluorescence test against Lassa, LCM (WE & Arm strain), Junin, Machupo, and other arenavirus antigens. In radioimmune precipitation tests using purified Lassa virus antigen all monoclonal antibodies precipitated a single band of 60 kd, specific for the viral nucleoprotein (p 60). Three domains (A, B, C) were identified on the surface of the Lassa virus nucleoprotein using an ELISA-inhibition test. All domains carried different Lassa virus specific epitopes. In addition, the A-domain carried a group specific epitope present within the arenavirus family as a whole as shown by cross-reaction in immunofluorescence tests. The B-domain only carries Lassa virus specific epitopes, whereas the C-domain has a type specific and a subgroup specific (Lassa, LCM) epitope.     

Antigenic and cellular localisation analysis of the severe acute respiratory syndrome coronavirus nucleocapsid protein using monoclonal antibodies

A member of the family of coronaviruses has previously been identified as the cause of the severe acute respiratory syndrome (SARS). In this study, several monoclonal antibodies against the nucleocapsid protein have been generated to examine distribution of the nucleocapsid in virus-infected cells and to study antigenic regions of the protein. Confocal microscopic analysis identified nucleocapsids packaged in vesicles in the perinuclear area indicating viral synthesis at the endoplasmic reticulum and Golgi apparatus. The monoclonal antibodies bound to the central and carboxyterminal half of the nucleocapsid protein indicating prominent exposure and immunogenicity of this part of the protein. Antibodies recognised both linear and conformational epitopes. Predictions of antigenicity using mathematical modelling based on hydrophobicity analysis of SARS nucleoprotein could not be confirmed fully. Antibody binding to discontinuous peptides provides evidence that amino acids 274-283 and 373-382 assemble to a structural unit particularly rich in basic amino acids. In addition, amino acids 286-295, 316-325 and 361-367 that represent the epitope recognised by monoclonal antibody 6D11C1 converge indicating a well-structured C-terminal region of the SARS virus nucleocapsid protein and functional relationship of the peptide regions involved. Alternatively, dimerisation of the nucleocapsid protein may result in juxtaposition of the amino acid sequences 316-325 and 361-367 on one nucleoprotein molecule to amino acid 286-295 on the second peptide. The monoclonal antibodies will be available to assess antigenicity and immunological variabilities between different SARS CoV strains.     

Antigenic differences between strains of Newcastle disease virus

Summary Comparison of strains of Newcastle disease virus by kinetic neutralization tests and immunodiffusion tests (after virion disruption by ether and Tween 80) enabled antigenic differences to be demonstrated between four out of five strains tested. No correlation was demonstrated between the antigenic structure and virulence of these strains.With 3 Figures     

Structure of the Newcastle disease virus F protein in the post-fusion conformation

The paramyxovirus F protein is a class I viral membrane fusion protein which undergoes a significant refolding transition during virus entry. Previous studies of the Newcastle disease virus, human parainfluenza virus 3 and parainfluenza virus 5 F proteins revealed differences in the pre- and post-fusion structures. The NDV Queensland (Q) F structure lacked structural elements observed in the other two structures, which are key to the refolding and fusogenic activity of F. Here we present the NDV Australia-Victoria (AV) F protein post-fusion structure and provide EM evidence for its folding to a pre-fusion form. The NDV AV F structure contains heptad repeat elements missing in the previous NDV Q F structure, forming a post-fusion six-helix bundle (6HB) similar to the post-fusion hPIV3 F structure. Electrostatic and temperature factor analysis of the F structures points to regions of these proteins that may be functionally important in their membrane fusion activity.     

Epitope mapping of dengue 1 virus E glycoprotein using monoclonal antibodies

Summary Ten monoclonal antibodies (MAbs) were raised against dengue 1 (DEN 1, Hawaii) virus E glycoprotein. Specificity of the MAbs was tested by ELISA and immunofluoresence. Eight were DEN 1 type-specific, one was DEN group-reactive (DGR) and one was flavivirus cross-reactive (FCR). Two of these type specific MAbs exhibited haemagglutination-inhibition (HI) and neutralized (N) DEN 1 virus in vivo (HS). These two MAbs showed 100% protection against a challenge of 100 LD₅₀ of DEN 1 virus in adult Swiss albino mice. The remaining six MAbs were HI negative, N negative and non-protective against challenge (NHS). Of these only three were reactive in the CF test. The DGR, FCR and one of the NHS MAbs (NHS-3) did not react with DEN 1 virus grown in Vero cells, whereas they reacted with DEN 1 virus grown in LLC-MK2 and C6/36 cells in immunofluorescence, probably indicating a difference in the synthesis/processing of viral proteins in these different cell lines. An epitope map of the E gp was drawn using a computer programme based on the additivity index values. The epitope map delineated five domains, a) S-I representing type-specific, HI positive, N positive and protecting MAbs. b) S-II representing type-specific, HI negative, N negative MAbs. c) S-III representing type-specific HI/N negative MAb, but distinct from S-II. d) DGR representing HI/N negative DEN group reactive MAb. e) FCR representing HI/N negative flavivirus cross-reactive MAb. Epitope analysis of a number of different DEN 1 strains isolated in India over a period of 30 years showed that the domains S-II and S-III which react with HI negative, DEN-1 specific MAbs were variable. The DGR domain and the S-I domains were conserved.     

Antigenic analysis of Punta Toro virus and identification of protective determinants with monoclonal antibodies

Hybridomas producing monoclonal antibodies to the three major structural proteins of Punta Toro virus (PTV) were established by fusion of spleen cells with Sp2/0-Ag-14 mouse plasmacytoma cells. Thirty-six independently derived monoclonal antibodies were evaluated in neutralization, hemagglutination inhibition, and ELISA assays and the isotype, antigen specificities, and cross-reactivities were determined. These antibodies were also assessed for their ability to provide protection in a murine model. Both G1- and G2-specific antibodies were obtained which neutralized virus infectivity in vitro and inhibited hemagglutination, whereas nucleocapsid-specific antibodies exhibited neither activity. All of the anti-G1 antibodies were PTV-specific, whereas anti-G2 and anti-nucleocapsid antibodies exhibited varying patterns of cross-reactivity with heterologous phleboviruses. All of the G1-reactive monoclonal antibodies, which bound to epitopes in two distinct topological sites as determined by competitive binding assays, provided efficient protection to both immunocompetent and immunosuppressed mice. In contrast, of the 23 G2-reactive antibodies, only 8 were able to protect immunocompetent mice and only one was able to protect immunosuppressed animals. The degree of protection achieved in vivo did not correlate directly with the neutralization titers determined in vitro.     

Monoclonal antibodies against avian paramyxovirus-3: antigenic mapping and functional analysis of the haemagglutinin-neuraminidase protein and the characterization of nonspecific monoclonal antibodies

Summary Sixteen monoclonal antibodies (Mabs) which immunoprecipitated the haemagglutinin neuraminidase (HN) of chorio-allantoic membrane-grown avian paramyxovirus-3 (PMV-3) of British turkeys were produced. Thirteen were PMV-3 specific. Three were nonspecific because they also bound to other viral proteins and to bovine kidney cells treated with neuraminidase enzyme or infected with influenza virus.The thirteen specific Mab defined four antigenic regions A-D by competition and variant selection assays. Region A was subdivided into five epitopes and region B into two epitopes. The thirteen Mab neutralized and were active in haemagglutination inhibition (HI) and twelve were active in haemolysis inhibition (HLI) tests. Neuraminidase inhibition (NI) was epitope-dependent.Mabs to five of the epitopes A₁, A₂, A₃, A₅, and C bound to the 1981 British turkey isolates but not the 1968 American turkey isolate. The Mab to epitope A₄ bound to both viruses. The Mabs to epitopes B₁, B₂, and D also bound to a parakeet isolate of PMV-3 which was the third PMV-3 tested. The Mab to B₂ gave identical titres to all three viruses and had HI, HLI, and NI activities. This made it a potential diagnostic reagent for avian PMV-3 viruses.One of the nonspecific Mabs bound to lactose-like moeities as reported on influenza virus and one to maltose-like moeities as on retroviruses. Immunoglobulin from all three nonspecific Mab had some HI activity.     

Antigenic analysis of the surface glycoproteins of a Venezuelan equine encephalomyelitis virus (TC-83) using monoclonal antibodies

Splenic lymphocytes from BALB/c mice immunized with the TC-83 vaccine strain of Venezuelan equine encephalomyelitis virus (VEE) were fused to the nonsecreting myeloma cell line Sp2/0-Ag 14. Fusion products were screened for antibody synthesis using an enzyme-linked immunosorbent assay (ELISA) with purified TC-83 virus. Antigenic specificity of cloned hybridoma cells was determined by ELISA or radioimmune precipitation using purified E2 (gp56), El (gp50), and capsid. Antibodies were charcterized by chromatography on protein A-Sepharose and by isoelectric focusing. Analysis of antigenic epitopes by cross-reactivity panels using closely related VEE strains and competitive binding assays indicated the presence of three epitopes on the gp56 and four epitopes on the gp50. Close spatial arrangement of gp56 and gp50 in the native virion could be demonstrated. The biologic functions of hemagglutination and virus neutralization were primarily associated with only one antigenic epitope present on the gp56.