Geneviève Libeau

Development of a competitive ELISA for detecting antibodies to the peste des petits ruminants virus using a recombinant nucleoprotein.

A competitive ELISA based on the reaction between a monoclonal antibody (mAb) and a recombinant nucleoprotein of the peste des petits ruminants virus (PPRV) was developed. This protein was obtained in large quantities from insect cells infected with a PPR nucleoprotein recombinant baculovirus (N-B). The competitive ELISA was compared with the virus neutralisation test (VNT) for detecting specific antibodies to PPRV in sheep and goats. The time consuming VNT is the only prescribed test that is capable of distinguishing between PPRV and the cross-reactive rinderpest virus (RPV). The competitive ELISA involves the simultaneous addition of the mAb and antibodies present in a positive serum, leading to competition for a specific epitope on the N-B. Optimum conditions were obtained by using serum samples which had positive or negative neutralising activity against PPRV or RPV. A negative cut-off point was determined on PPRV-negative sera from RPV-vaccinated cattle. A threshold value of 48 per cent inhibition, calculated from the mean for this population plus 2.7 standard deviations, was used in routine testing. A total of 683 sera were analysed by the competitive ELISA and the VNT. A good correlation (r = 0.94) was observed between the titres obtained in the two tests, with 80 sera that were from laboratory sources. The agreement between the two tests was determined on 271 field sera (kappa = 0.825). Their relative sensitivity (94.5 per cent) and specificity (99.4 per cent) were assessed on the 148 laboratory sera plus the 271 sera used for the determination of kappa.(ABSTRACT TRUNCATED AT 250 WORDS)

The isolation of peste des petits ruminants virus from Northern India

The aetiological agent responsible for an epizootic of a rinderpest-like disease afflicting sheep and goats in three states of northern India was confirmed as peste des petits ruminants virus. To differentiate the virus from rinderpest a number of diagnostic tests were used, including immunocapture ELISA, specific oligonucleotide primers in a reverse transcriptase polymerase chain reaction, immunofluorescence with virus specific monoclonal antibodies and virus isolation. The virulence profile of one isolate in cattle sheep and goats was established. Infected animals developed specific antibody responses and excreted specific antigen in their lachrymal secretions.

Asian Lineage of Peste des Petits Ruminants Virus, Africa

Interest in peste des petits ruminants virus (PPRV) has been stimulated by recent changes in its host and geographic distribution. For this study, biological specimens were collected from camels, sheep, and goats clinically suspected of having PPRV infection in Sudan during 2000–2009 and from sheep soon after the first reported outbreaks in Morocco in 2008. Reverse transcription PCR analysis confirmed the wide distribution of PPRV throughout Sudan and spread of the virus in Morocco. Molecular typing of 32 samples positive for PPRV provided strong evidence of the introduction and broad spread of Asian lineage IV. This lineage was defined further by 2 subclusters; one consisted of camel and goat isolates and some of the sheep isolates, while the other contained only sheep isolates, a finding with suggests a genetic bias according to the host. This study provides evidence of the recent spread of PPRV lineage IV in Africa.

An outbreak of peste des petits ruminants (PPR) in camels in the Sudan

In mid-August 2004, an outbreak of a previously unknown fatal disease of camels was reported to Kassala State veterinary authorities. Several areas in the state were visited during August-October 2004 to collect epidemiological data and specimens for diagnosis. Clinically the disease was characterized by sudden death of apparently healthy animals and yellowish and later bloody diarrhea and abortion. The disease outbreaks coincided with the seasonal movement of animals towards autumn green pasture. Death was always sudden and proceeded with colic and difficulty in respiration. Mortality rate ranged between 0% and 50% and vary in accordance with the area with a mean of 7.4%. More than 80% of deaths were in pregnant and recently-delivered she-camels. All age, sex and breed groups were affected but more than 50% of deaths were reported in adult animals in comparison to calves and young camels. The main post-mortem findings include lung congestion and consolidation, paleness and fragility of liver, enlarged lymph nodes and congestion and hemorrhage of small intestine and stomach. Agar gel diffusion test (AGDT), RT-PCR and virus isolation in cell culture gave positive results for peste des petits ruminants virus (PPRV), a virus belonging to the Morbillivirus, Genus, member of the family Paramyxoviridae. The effect of this new devastating disease on camel production in the affected area was discussed as well as proposals for future research.

Measles Virus (MV) Nucleoprotein Binds to a Novel Cell Surface Receptor Distinct from FcγRII via Its C-Terminal Domain: Role in MV-Induced Immunosuppression

ABSTRACT During acute measles virus (MV) infection, an efficient immune response occurs, followed by a transient but profound immunosuppression. MV nucleoprotein (MV-N) has been reported to induce both cellular and humoral immune responses and paradoxically to account for immunosuppression. Thus far, this latter activity has been attributed to MV-N binding to human and murine FcγRII. Here, we show that apoptosis of MV-infected human thymic epithelial cells (TEC) allows the release of MV-N in the extracellular compartment. This extracellular N is then able to bind either to MV-infected or uninfected TEC. We show that recombinant MV-N specifically binds to a membrane protein receptor, different from FcγRII, highly expressed on the cell surface of TEC. This new receptor is referred to as nucleoprotein receptor (NR). In addition, different Ns from other MV-related morbilliviruses can also bind to FcγRII and/or NR. We show that the region of MV-N responsible for binding to NR maps to the C-terminal fragment (NTAIL). Binding of MV-N to NR on TEC triggers sustained calcium influx and inhibits spontaneous cell proliferation by arresting cells in the G0 and G1 phases of the cell cycle. Finally, MV-N binds to both constitutively expressed NR on a large spectrum of cells from different species and to human activated T cells, leading to suppression of their proliferation. These results provide evidence that MV-N, after release in the extracellular compartment, binds to NR and thereby plays a role in MV-induced immunosuppression.

A competitive ELISA using anti-N monoclonal antibodies for specific detection of rinderpest antibodies in cattle and small ruminants

A competitive ELISA (C-ELISA) using monoclonal antibodies (mAbs) which bind to the nucleo-protein (NP) of rinderpest virus (RPV) for detection of RPV antibodies in cattle and small ruminant sera is described. Unlike virus neutralisation test (VNT), this test using mAb IVB2-4, can detect specific RPV antibodies without showing a cross-reaction with antibodies to peste-des-petits ruminants-virus (PPRV); by contrast, when mAb VE4-1 is used the test detects both RPV and PPRV antibodies, including low levels of antibodies that can be found in sera containing maternal antibodies. Although antibodies to the PPRV 75-1 strain are also detected with mAb 51-5-6, the test is suitable for assessing the immune status of cattle against the Rinderpest Old Kabete (RBOK) strain. The results from a panel of sera with a known status of vaccination provide evidence for a highly significant correlation between C-ELISA and VNT. This test may be a useful tool for a standardized and accurate determination of the immunity status of both cattle and small ruminants.

Peste des petits ruminants, the next eradicated animal disease?

Peste des Petits Ruminants (PPR) is a widespread viral disease caused by a Morbillivirus (Paramyxoviridae). There is a single serotype of PPR virus, but four distinct genetic lineages. Morbidity and mortality are high when occurring in naive sheep and goats populations. Cattle and African buffaloes (Syncerus caffer) are asymptomatically infected. Other wild ruminants and camels may express clinical signs and mortality. PPR has recently spread in southern and northern Africa, and in central and far-east Asia. More than one billion sheep and goats worldwide are at risk. PPR is also present in Europe through western Turkey. Because of its clinical incidence and the restrictions on animal movements, PPR is a disease of major economic importance. A live attenuated vaccine was developed in the 1980s, and has been widely used in sheep and goats. Current researches aim (i) to make it more thermotolerant for use in countries with limited cold chain, and (ii) to add a DIVA mark to shorten and reduce the cost of final eradication. Rinderpest virus-another Morbillivirus-was the first animal virus to be eradicated from Earth. PPRV has been proposed as the next candidate. Considering its wide distribution and its multiple target host species which have an intense mobility, it will be a long process that cannot exclusively rely on mass vaccination. PPR specific epidemiological features and socio-economic considerations will also have to be taken into account, and sustained international, coordinated, and funded strategy based on a regional approach of PPR control will be the guarantee toward success.

Antigen delivery systems for veterinary vaccine development: Viral-vector based delivery systems

Abstract The recent advances in molecular genetics, pathogenesis and immunology have provided an optimal framework for developing novel approaches in the rational design of vaccines effective against viral epizootic diseases. This paper reviews most of the viral-vector based antigen delivery systems (ADSs) recently developed for vaccine testing in veterinary species, including attenuated virus and DNA and RNA viral vectors. Besides their usefulness in vaccinology, these ADSs constitute invaluable tools to researchers for understanding the nature of protective responses in different species, opening the possibility of modulating or potentiating relevant immune mechanisms involved in protection.

Recent developments in the diagnosis of rinderpest and peste des petits ruminants

Effective implementation of control measures for rinderpest and peste des petits ruminants requires that a proper and rapid diagnosis of the disease is made. Peste de petits ruminants (PPR) can be confused clinically with other infections such as pasteurellosis or contagious ecthyma. Rinderpest, in its classical form, is easy to identify clinically; however, mass vaccination in many countries and also the emergence of mild strains of the virus have made clinical diagnosis more difficult. Clinical observations for both diseases should always be confirmed by a laboratory. Diagnostic techniques used in the past were virus neutralization, agar gel immunodiffusion and virus isolation in cell culture, followed sometimes by reproducing the disease in susceptible animals. All these techniques are either time-consuming, labour intensive, insensitive, or expensive to perform. With the advent of hybridoma and molecular biological techniques, new reagents to assist diagnosis have become available and have led to the development of specific and rapid tests for the diagnosis of each disease. The present article reviews the diagnostic techniques currently available. An indirect ELISA was used successfully to evaluate the status of cattle following the Pan African Rinderpest Campaign. More recently competitive or blocking ELISAs have been developed based on monoclonal antibodies specific for the N or H proteins of the viruses, and which enable differential diagnosis between rinderpest and PPR. This is particularly important in sheep and goats, which may be infected with either virus. In future, improved standardization and reduced costs may be expected with the introduction of ELISAs based on purified antigens expressed in gene vector systems such as baculovirus. ELISA may also be adapted to antigen detection. Nucleic acid technology has also been applied to virus detection procedures. Hybridization probes showed a disappointing sensitivity for diagnostic applications, but more recently the polymerase chain reaction method has shown great promise, providing the potential of high sensitivity combined with specificity.

Quantitative one-step real-time RT-PCR for the fast detection of the four genotypes of PPRV

A one-step real-time Taqman RT-PCR assay (RRT-PCR) for peste des petits ruminants virus (PPRV) was developed to detect the four lineages of PPRV by targeting the nucleoprotein (N) gene of the virus. This new assay was compared to a conventional RT-PCR on reference strains and field materials. Quantitation was performed against a standard based on a synthetic transcript of the NPPR gene for which a minimum of 32 copies per reaction were detected with a corresponding C(t) value of 39. Depending on the lineage involved, the detection limit of RRT-PCR was decreased by one to three log copies relative to the conventional method. The lower stringency occurred with lineage III because of minor nucleotide mismatches within the probe region. The assay did not detect phylogenetically or symptomatically related viruses of ruminants (such as rinderpest, bluetongue, and bovine viral diarrhea viruses). However, it was capable of detecting 20% more positive field samples with low viral RNA loads compared to the conventional PCR method. When compared on a proficiency panel to the method developed by Bao et al. (2008), the sensitivity of the in-house assay was slightly improved on lineage II. It proved significantly faster to perform and hence better adapted for monitoring large numbers of at risk or diseased animals.