Antigenic Characterization of Rabies Virus Isolates from Vaccinated Dogs in Plateau State,Nigeria

Rabies isolates (genotype 1 lyssaviruses) from vaccinated dogs that died of rabies infection in the Plateau area of Nigeria were characterized using monoclonal antibodies (MAbs). The isolates were examined for rabies (genotype 1) and rabies-related (genotypes 2, 3 and 4) viruses by the indirect fluorescent antibody test carried out with MAb 502-2, which recognizes the nucleocapsid protein of all known lyssaviruses, and with MAb 422-5, which identifies only rabies-related viruses. All three isolates showed positive immunofluorescence with MAb 502-2 and were negative with MAb 422-5, indicating that they were all rabies (genotype 1) viruses.Characterization with a panel of 36 anti-nucleocapsid monoclonal antibodies showed that all three isolates reacted positively with 35 of the anti-nucleocapsid MAbs, including MAb 102-27 and MAb 377-7. Characterization using a panel of 44 anti-glycoprotein MAbs differentiated the isolates sharply from LEP Flury and PM vaccine viruses. The pattern of anti-glycoprotein reactivity of the isolates showed them to belong to one distinct viral subtype, except for a minor variation in one isolate that was not neutralized by MAb 1101-3. None of the three isolates was identified as the Flury low egg passage (LEP) vaccine strain used for vaccinating dogs in Nigeria. In fact, all the three isolates had the typical pattern of reactivity of isolates from unvaccinated dogs, including MASS 83, a rabies virus isolated in Nigeria and characterized at the Wister Institute before this study.     

Comparison of rinderpest and peste des petits ruminants viruses using anti-nucleoprotein monoclonal antibodies

Monoclonal antibodies (MAbs) were obtained using a purified preparation of the RBOK strain of a rinderpest vaccine virus. The cytoplasmic immunofluorescent staining test showed that these clones had specificity for the nucleoprotein (N) of the virus. Six clones which immunoprecipitated the N protein corroborated these results. Thirteen anti-N MAbs were used to compare geographically widespread rinderpest viruses (RPV) and peste des petits ruminants viruses (PPRV) to two other morbilliviruses, measles (MV) and canine distemper (CDV). The N protein antigen profiles of the 23 isolates determined by immunofluorescent staining and enzyme linked immunosorbent assay (ELISA) on infected cells enabled us to classify the strains into groups. A differential identification of the morbilliviruses can be made using one MAb or associations of the MAbs. The potential to distinguish between RPV and PPRV and between virulent and avirulent strains of rinderpest is of primary interest.     

The detection of antibody against peste des petits ruminants virus in Sheep,Goats, Cattle and Buffaloes

Monoclonal antibody-based competitive ELISA (C-ELISA) has been used for the specific measurement of antibodies to peste des petits ruminants (PPR) viruses in sheep, goats, cattle and Buffalo. Serum samples from sheep (n = 232), goats (n = 428), cattle (n = 43), buffalo (n = 89) were tested. The animals had not been vaccinated against rinderpest or PPR. Findings suggested that the sero-positive cases were significantly higher in sheep (51.29%) than in goats (39.02%) (P = 0.002). The overall sero-prevalence of PPRV in small ruminants was 43.33%. The PPR antibodies seroprevalence was 67.42% in buffalo and 41.86% in cattle which was significantly higher in buffalo (P = 0.005). The overall sero-prevalence of PPRV in large ruminants was 59.09%. Cattle and buffalo sera showed a high prevalence of antibody against PPR virus which may explain the difficulty experienced in achieving high post-vaccination immunity levels against rinderpest. Because antibodies against PPR virus are both cross-neutralizing and cross-protective against rinderpest virus, further vaccination in the presence of antibodies against PPR virus may be a waste of national resources. It was also suggested that antibodies to PPR virus could prevent an immune response to the rinderpest vaccine. This paper presents serological evidence for the transmission of PPR virus from sheep and goats to cattle and buffalo and highlights the need to include PPR serology in the sero-monitoring programme to give a better indication of national herd immunity of sheep and goats against PPR.     

Antigenic characterisation of virus isolates from vaccinated dogs dying of rabies

Four rabies virus isolates from dogs that succumbed to rabies infection in Nigeria within one year of anti-rabies vaccination were characterised by monoclonal antibodies (MAbs). The samples were screened for rabies and rabies-related viral antigens by the indirect fluorescent antibody test, performed with MAb 502-2, which recognises the nucleocapsid (NC) protein of all known Lyssaviruses and with MAb 422-5 which identifies African rabies-related viruses. All four canine virus isolates displayed positive fluorescence with MAb 502-2 and were negative with MAb 422-5. In the anti-NC MAb characterisation with a panel of 34 additional MAbs, all isolates displayed positive staining with 32 of the MAbs, were negative with MAb 102-27 and all displayed poor immunofluorescence with MAb 377-7. On the basis of reactivity with a panel of 40 anti-glycoprotein (G) MAbs the isolates were separated into four distinct viral subtypes. None of these canine isolates was identified as the common attenuated Flury LEP rabies strain used for domestic animal vaccination and none resembled other previously characterised rabies viruses from Nigeria.     

Serological studies with peste des petits ruminants and rinderpest viruses in Nigeria

One hundred and ninety-five goat and 67 sheep sera collected from various parts of southern Nigeria were screened for neutralising antibodies to both the peste des petits ruminants (PPR) and rinderpest viruses. Neutralising antibodies against both viruses were found in the sheep and goat sera examined. Parallel titration of samples which neutralised both viruses indicated a primary infection with the PPR virus (PPRV). However, some samples which failed to neutralise PPRV neutralised the rinderpest virus (RV) indicating RV activity in sheep and goats in Nigeria. These findings are discussed in relation to the diagnosis of PPRV infection and the recent reappearance of bovine rinderpest in Nigeria.     

Identification of epitope(s) on the internal virion proteins of rinderpest virus which are absent from peste des petits ruminants virus.

Monoclonal antibodies (MAb) raised against the RBOK vaccine strain of rinderpest virus were characterized by radio-immunoprecipitation (RIPA) and in the indirect ELISA using measles (MV), distemper (CDV), rinderpest (RPV) and peste des petits ruminants viruses (PPRV). Those found to be specific for the matrix (M) protein and the nucleocapsid (N) protein could be classified into different groups on the basis of the anti-morbillivirus MAb classification scheme; a number of these MAb showed a selective recognition of RPV, measles virus and distemper virus, or of different isolates of rinderpest virus, demonstrating that greater inter-isolate variation occurs than was apparent from analyses using polyclonal antisera. One group of anti-F protein MAb (group F1) reacted with all isolates of both RPV and PPRV. A second group of anti-N protein MAb (group N1/A) reacted with all RPV isolates, but not with the PPRV isolates. Furthermore, these group N1/A antibodies reacted strongly with RPV isolates which were upon original isolation of high pathogenicity, but had a weaker reaction against the isolates of this virus which were of low pathogenicity. Thus, MAb against RPV, in particular those against the N protein offered a potential superior to that of molecular analyses for "isolate fingerprinting", the differentiation of RPV from PPRV and the discrimination between rinderpest viruses which had been, upon isolation, of either high or low pathogenicity.     

Attenuation of a strain of rinderpest virus: potential homologous live vaccine

Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants frequently associated with severe mortality in these hosts. In countries where it occurs, PPR represents an important constraint to the improved productivity of sheep and goats. Until now the only way to combat this plague has been the use of heterologous rinderpest vaccine; all attempts to develop a homologous vaccine have ended in failure. The present communication describes the attenuation of the Nigerian strain PPRV Nig 75/1 by serial passage in Vero cells. The avirulent virus obtained has the same characteristics as Plowright and Ferris' rinderpest vaccine. The virus is advanced as a potential homologous vaccine against PPR.     

The production of peste des petits ruminants hyperimmune sera in rabbits and their application in virus diagnosis

Hyperimmune sera were produced by serial inoculation of rabbits with Vero cell-adapted, sucrose gradient-purified Nigerian peste des petits ruminants virus (PPRV) isolate. Two antisera produced, neutralized the homologous PPRV but not the heterologous rinderpest Kabette "O" virus. The antisera gave strong precipitin lines with purified PPRV antigens and were used to detect PPRV and rinderpest virus antigens from ante-mortem secretions and post-mortem tissue homogenates from PPR and rinderpest virus infected goats and cattle by the agar gel precipitation tests (AGPT). The hyperimmune sera gave good titration curves with both purified Nigerian goat and the United Arab Emirate wildlife PPRV isolates in the indirect enzyme linked immunosorbent assay (ELISA). Results of indirect ELISA showed that although there were some cross reactions with the rinderpest, canine-distemper and measles viruses, at 1:100 dilution, the antisera would give a positive signal with only the homologous PPR virus.     

Detection of Rinderpest Virus Using N-Protein Monoclonal Antibodies

A panel of monoclonal antibodies (mAbs) was generated against the RBOK strain of rinderpest virus (RPV). All of them bound to the N protein of RPV. The antigen capture ELISA using the mAbs could detect the virus in crude viral preparations. The mAb 12BF8.1.1 showed higher reactivity with cell-associated (CA) virus, whereas the mAbs 12AD10.1.1, 12BD7.1.1 and 12DG7.1.1 showed higher reactivity with extracellular virus (hereafter referred to as cell-free (CF) virus). The mAbs 12BF8.1.1 and 12AD10.1.1 could detect the virus in infected Vero cell culture supernatants (CCS) as early as 24 h post-cytopathic effect (CPE) initiation. Detergent treatment (Triton X-100) of RPV preparations enhanced the binding of the mAbs to the virus. All the seven mAbs showed specific fluorescence in virus-infected cell cultures. The immunofluorescence (IFA) using mAbs was found to be more sensitive and reliable than the immunoperoxidase test (IPT) for detection of rinderpest.     

Differentiation of peste des petits ruminants and rinderpest viruses by neutralisation indices using hyperimmune rinderpest antiserum

Summary Six field isolates believed to be rinderpest viruses and 2 known strains of peste des petits ruminants (PPR) viruses were titrated in the presence of normal rabbit serum and with hyperimmune rinderpest antiserum prepared in rabbits. The known PPR viruses had indices less than 10 whereas 4 of the suspect field isolates had indices greater than a hundred. Two suspect field isolates had indices less than 20; both were collected from small ruminant wild life and are probably PPR viruses.

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Diferenciacion De Peste De Los Pequenos Rumiantes Y Virus De Rinderpest Mediante Indices De Neutrilizacion Utilizando Antisueros Hiperinmunes De Rinderpest

Resumen Seis aislamientos de campo sospediosos de Rinderpest y dos cepas conocidas de Peste de los pequeños rumiantes (PPR) fueron titulados en titulados en presencia de suero normal de conejo y suero hiperimmune a Rinderpest, prepardo en conjo. Las cepas concidos de PPR dierond titulos menores de 10, mientras que 4 de los aislamientos sospechosos de Rinderpest dieron titulos mayores de 100. Los 2 aislamientos de campo restantes dieron titulos menores de 20. Ambos fueron aislados de pequenños ruminantes silvestras y probablemente sean virus de PPR.

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Differenciation Des Virus De La Peste Bovine Et De La Peste Des Petits Ruminants Par Les Indices De Neutralisation Utilisant Des Antiserums Hyperimmuns De La Peste Bovine

Résumé La titration de 6 isolats recuellis sur le terrain et considérés comme étant du virus de la peste bovine, et 2 souches connues du virus de la peste des petits ruminants (PPR) ont été titrés en présence de sérum normal de lapin et avec un antisérum hyperimmun de la peste bovine préparé sur lapins. Les virus identifiés PPR avaient des indices inférieurs à 10 alors que 4 des isolats suspects présentaient des indices supérieurs à 100. Deux isolats suspects avaient des indices inférieurs à 20 et tous deux provenaient de petits ruminants sauvages et sont probablement des virus de la PPR.

     

Identification and characterization of fowlpox virus strains using monoclonal antibodies.

The use of 2 monoclonal antibodies (MAbs), P1D9 and P2D4, which recognize different fowlpox virus (FPV) antigens, for the identification and characterization of FPV strains was evaluated. Initially, the MAbs were used in conjunction with a dot blot assay that enabled FPV to be differentiated from the avian herpesvirus, infectious laryngotracheitis virus. Confirmation of the specificity of these MAbs was provided by the demonstration that only FPV antigens were recognized by a combination of both antibodies when used for immunoblotting proteins contained in various avipoxviruses. Later, an antigenic characterization of 11 FPV field isolates, 6 FPV vaccine strains, and 3 pigeonpox virus vaccines was performed by Western blotting with the individual MAbs. Whereas MAb P2D4 consistently recognized a protein with an apparent molecular weight of 60 kD, there was variability in the size of the antigen that was immunoreactive with the other MAb. For example, MAb P1D9 recognized an antigen of apparent molecular weight of 46 kD in all vaccine strains except 2 of FPV origin. In these exceptions, either only a 39-kD or both a 42- and 46-kD protein were immunoreactive. As for the field isolates, a 39-kD antigen was recognized in 8 of them, whereas a 42-kD antigen was detected in the remaining 3. Therefore, the more extensive immunoblotting technique may facilitate FPV strain differentiation, whereas routine diagnosis of fowlpox could be accomplished by using the MAb-based dot blot assay.     

Variation amongst the neutralizing epitopes of bluetongue viruses isolated in the United States in 1979-1981.

Neutralizing epitopes present on field isolates of bluetongue virus (BTV) serotypes 10, 11, 13 and 17 were evaluated with a panel of polyclonal and neutralizing monoclonal antibodies (MAbs). A total of 91 field isolates were evaluated, including 15 isolates of BTV-10, 29 isolates of BTV-11, 26 isolates of BTV-13, and 21 isolates of BTV-17. The viruses were isolated from cattle, goats, sheep, elk and deer in Idaho, Louisiana, Nebraska and, predominantly, California, in the years 1979, 1980 and 1981. The isolates were analyzed and compared using a panel of neutralizing MAbs which included five MAbs raised against BTV-2, seven against BTV-10, five against BTV-13, and six against BTV-17. Neutralization patterns obtained with the MAb panel and individual field isolates were compared to those obtained with prototype viruses of each serotype. All field isolates were neutralized by at least some of the MAbs raised against the prototype virus of the same serotype. All field isolates of BTV-10 were neutralized by the seven MAbs raised to BTV-10, whereas the field isolates of BTV-11, BTV-13 and BTV-17 were not consistently neutralized by all of the MAbs raised against the prototype virus of the same serotype. Variation in neutralizing epitopes recognized by the MAb panel was most pronounced amongst the field isolates of BTV-17. A one-way cross neutralization was evident between BTV-10 and BTV-17 as all field isolates of BTV-17 were neutralized by four of the MAbs raised against BTV-10. In contrast, no BTV-10 isolates were neutralized by the MAbs raised against BTV-17. Differences in the MAb neutralization patterns of field isolates of BTV-11, BTV-13 and BTV-17 suggest that the immunogenic domain responsible for their neutralization is plastic, such that individual epitopes within the domain may vary in their significance to the neutralization of different viruses, even of the same serotype. The apparent conservation of neutralizing epitopes on field isolates of BTV-10 suggests that the field isolates may be derived from the modified-live vaccine strain of BTV-10.     

Development of a monoclonal antibody based competitive-ELISA for detection and titration of antibodies to peste des petits ruminants (PPR) virus

Peste des petits ruminants (PPR) is an acute febrile, viral, disease of small ruminants with great economic importance. A competitive-ELISA (c-ELISA) test was developed for detection of antibodies to PPR virus in the sera samples of goats and sheep. The test uses monoclonal antibody to a neutralizing epitope of haemagglutinin protein of the virus. Based on the distribution of known negative sera samples (n=933) in respect of PPR virus antibodies in the test, a cut-off value was set as 38%. This value was the result of mean of negative population added with two times the standard deviations. A total of 1668 sera samples from goat and sheep and 32 sera from cattle were screened by c-ELISA and virus neutralization test (VNT). Efficacy of c-ELISA compared very well with VNT having high relative specificity (98.4%) and sensitivity (92.4%). The sensitivity of c-ELISA for PPR sero-surveillance could further be increased (95.4%), if the target population is non-vaccinated. c-ELISA test correlated well with VNT (r=0.845) for end-point titration of PPR virus antibody in 64 goat sera samples. It could clearly separate infected population from uninfected in field sera. Using c-ELISA test paired sera samples from 13 goats provided a clear diagnosis of PPR virus infection. Furthermore, antibodies to PPR virus could be successfully detected during 1 year after vaccination in four goats inoculated with an experimental PPR vaccine. Findings suggest that the c-ELISA test developed can easily replace VNT for sero-surveillance, sero-monitoring, diagnosis from paired sera samples and end-point titration of PPR virus antibodies.     

Analysis of glycoprotein I (gI) negative and aberrant pseudorabies viral diagnostic isolates.

Glycoprotein I (gI) phenotypes and genotypes of 4 pseudorabies viral diagnostic isolates were evaluated by use of in vitro DNA amplification, monoclonal antibody binding, gI-specific serodiagnostic responses, and in vivo virulence approaches. Three viruses were avirulent and did not elicit gI-specific serologic responses, react with gI-specific monoclonal antibodies, or contain gI epitope-encoding DNA sequences. The fourth virus was virulent and did elicit a gI-specific serodiagnostic response. Compared with reference virulent pseudorabies viruses, however, the fourth isolate had reduced reactivity with a group of gI monoclonal antibodies and had a single nucleotide sequence substitution with a corresponding putative amino acid change in the epitopically dominant portion of the gI molecule. Presumably, the first 3 isolates represented diagnostic recoveries of viruses derived from gI-deleted modified-live pseudorabies viral vaccines, whereas the fourth isolate was a virulent but gI-aberrant wild-type virus. Thoroughly assessing the gI status of pseudorabies viral diagnostic isolates was considered to be essential in evaluating the epidemiologic importance of these viruses and in monitoring the validity of gI-based vaccine companion tests now used worldwide in pseudorabies control and eradication programs.     

Partial Antigenic Characterization of Buffalopox Virus

The present study was undertaken to antigenically characterize the buffalopox virus (BPV). Six monoclonal antibodies (MAbs) against the BP4 strain of BPV have been produced and characterized. All six MAbs appeared to be specific to BPV, as none of them showed cross-reactivity with other poxviruses in antigen capture ELISA. Only two MAbs (20AB8 and 20CD11) bound significantly with different BPV isolates in antigen capture ELISA, whereas the remaining four MAbs bound weakly with the BPV. In Western blot analysis with purified BPV-BP4, the rabbit hyperimmune serum against purified BPV-BP4 reacted with 15 immunodominant polypeptides (100 kDa to 25 kDa), whereas two MAbs (21CB6, 21DB11) reacted with 42 kDa and 45 kDa polypeptides, respectively. However, three MAbs (20AB8, 20CD11, 21CB5) reacted with three degraded polypeptides (100 kDa, 40 kDa and 87 kDa) of BPV-BP4. In radioimmunoprecipitation assay (RIPA) with the rabbit hyperimmune serum to BPV-BP4, three virus specific polypeptides (69 kDa, 34 kDa, 32 kDa) were recognized in BPV-BP4, whereas two polypeptides (69 kDa, 34 kDa) were recognized in other BPV isolates (BPV-Bly, BPV-Vij96, BPV-Vij97). In virus neutralization test, none of the six MAbs tested showed any significant neutralizing ability to infection with different BPV isolates. However, the hyperimmune serum showed weak neutralizing ability to BPV infection.     

Further studies on rabies virus isolated from healthy dogs in Nigeria

Rabies viruses isolated from healthy dogs, were passaged in mice and adapted to cell culture. After 5-7 passages, isolated viruses were subjected to monoclonal antibody (Mab) characterization with a panel of 36 anti-nucleocapsid (NC) and 40 anti-glycoprotein (G) MAbs. The four viruses showed positive fluorescence with all NC hybridomas except MAb 422-5, confirming them as true rabies virus isolates. The anti-G MAb reactivity pattern was the same in the four isolates indicating that they belong to the same antigenic group, but were antigenically distinct from the Flury LEP rabies vaccine virus which is widely used throughout Nigeria for canine vaccination, and from other previously characterized street lyssaviruses from Nigeria.     

Antigenic comparison of Canadian and US isolates of porcine reproductive and respiratory syndrome virus using monoclonal antibodies to the nucleocapsid protein.

Fifteen Canadian field isolates of porcine reproductive and respiratory syndrome (PRRS) virus from Quebec and Ontario were compared with 5 US PRRS virus (PRRSV) isolates and with the European Lelystad isolate using monoclonal antibodies (MAbs) SDOW17, EP147, and VO17 directed to the 15-kDa nucleocapsid protein of PRRSV. All Canadian and US isolates tested by indirect immunofluorescence were recognized by the 3 MAbs, and individual titers of MAbs towards Canadian and US PRRSV isolates were similar as well. In contrast, the Lelystad virus isolate reacted only with the SDOW17 MAb and showed no reactivity with either EP147 or VO17. The reactivity pattern with these MAbs suggests that the Canadian isolates of PRRSV tested are antigenically similar to US isolates of PRRSV, and that these North American isolates share highly conserved epitopes on the 15-kDa nucleocapsid protein that clearly differentiate them from the European Lelystad virus isolate.     

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.     

Control of peste des petits ruminants and poverty alleviation?

Peste des petits ruminants (PPR) is a contagious and often fatal viral disease of sheep and goats and also wild small ruminants. The PPR virus is distinct from but closely related to rinderpest virus and both belong to the morbivillivirus genus within the family Paramyxoviridae. PPR is a contagious transboundary disease with a significant impact on rural poor farmers. Its control should therefore be considered in programs that aim at alleviating poverty in developing countries.     

Identification of a variable epitope on the Newcastle disease virus hemagglutinin–neuraminidase protein

Fifteen virulent Newcastle disease viruses (NDVs) were isolated from diseased birds in Eastern China in 2005. To investigate the antigenic variation in the epitopes on NDV hemagglutinin–neuraminidase (HN) protein, these isolates, together with six reference strains, were subjected to the hemagglutination inhibition (HI) tests using five HI-positive monoclonal antibodies (MAbs) against velogenic NDV strain ZJ1. The MAbs 2G5, 3A4, 3B5 and 6B1 recognized 12 of the 15 NDV isolates, and exhibited HI activity towards the six reference strains. However, these MAbs did not react with three local isolates, JS-02/05, JS-06/05 and JS-10/05. HN gene sequence analysis of all NDV strains revealed that these MAb-resistant NDV isolates possessed residue K at position 347 of the HN protein, whereas all remaining strains possessed E or G at the same site. To determine the contribution of the residue at position 347 to antigenic epitope formation, we generated by reverse genetics two recombinant viruses, ZJ1HNK with an E347K mutation on ZJ1 HN, and JSHNE with a K347E mutation on JS-06/05 HN. The HI test demonstrated that ZJ1HNK lost reactivity with MAbs 2G5, 3A4, 3B5 and 6B1, whereas JSHNE did react with these MAbs. Further verification by immunofluorescent assay demonstrated that residue 347 was a critical determinant for formation of the antigenic epitope (residues 345–353) on the HN protein.