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Additional file 2. Distribution of nucleotide and amino acid differences separating the PPR vaccine strains Nigeria/75/1 (Nig75/1) and 45G37/35-k (FRCVM).

Additional file 1. Position and frequency of the 248 nucleotide differences separating the PPR vaccine strains Nigeria/75/1 (Nig75/1) and 45G37/35-k (FRCVM). Nucleotide positions corresponding to mutations associated to the attenuation of Nigeria/75/1 are in bold [14].

International audience; Peste des petits ruminants (PPR) is a highly contagious viral disease that mainly affects goats and sheep in Asia, Africa and the Middle East. The PPR virus (PPRV) can be classified into four genetically distinct lineages (I, II, III and IV). All have been historically present in Africa, except the Asian lineage IV that has been spreading across the globe and across Africa in recent decades. Previous studies have identified the presence of lineage IV in Nigeria since 2010. In the present study, samples were taken from 429 small ruminants with PPR symptoms across Nigeria in 2017-2020 to provide an update on the distribution and genetic diversity of PPRV in the country. Sequences from a portion of the PPRV nucleoprotein (N) gene were obtained from 91 samples, 90 belonging to lineage IV and one to lineage II. Phylogenetic analysis identified at least four lineage IV sub-clusters in Nigeria, grouping samples across multiple regions. Our results suggest extensive endemic circulation of a wide range of PPRV strains across Nigeria and across borders with neighbouring countries, underlining the difficulty involved in controlling the disease in the region.

International audience; Peste des petits ruminants (PPR) is a highly contagious disease of small ruminants. The causal agent, PPR virus (PPRV), is classified into four genetically distinct lineages. Lineage IV, originally from Asia, has shown a unique capacity to spread across Asia, the Middle East and Africa. Recent studies have reported its presence in two West African countries: Nigeria and Niger. Animals are frequently exchanged between Mali and Niger, which could allow the virus to enter and progress in Mali and to other West African countries. Here, PPRV samples were collected from sick goats between 2014 and 2017 in both Mali and in Senegal, on the border with Mali. Partial PPRV nucleoprotein gene was sequenced to identify the genetic lineage of the strains. Our results showed that lineage IV was present in south-eastern Mali in 2017. This is currently the furthest West the lineage has been detected in West Africa. Surprisingly, we identified the persistence at least until 2014 of the supposedly extinct lineage I in two regions of Mali, Segou and Sikasso. Most PPRV sequences obtained in this study belonged to lineage II, which is dominant in West Africa. Phylogenetic analyses showed a close relationship between sequences obtained at the border between Senegal and Mali, supporting the hypothesis of an important movement of the virus between the two countries. Understanding the movement of animals between these countries, where the livestock trade is not fully controlled, is very important in the design of efficient control strategies to combat this devastating disease.

International audience; Peste des Petits Ruminants (PPR) is a viral disease affecting predominantly small ruminants. Due to its transboundary nature, regional coordination of control strategies will be key to the success of the on-going PPR eradication campaign. Here, we aimed at exploring the extent of transboundary movement of PPR in West Africa using phylogenetic analyses based on partial viral gene sequences. We collected samples and obtained partial nucleoprotein gene sequence from PPR-infected small ruminants across countries within West and Central Africa. This new sequence data was combined with publically available data from the region to perform phylogenetic analyses. A total of fifty-five sequences were obtained in a region still poorly sampled. Phylogenetic analyses showed that the majority of virus sequences obtained in this study were placed within genetic clusters regrouping samples from multiple West African and Central African countries. Some of these clusters contained samples from countries sharing borders. In other cases, clusters grouped samples from very distant countries. Our results suggest extensive and recurrent transboundary movements of PPR within West Africa, supporting the need for a regional coordinated strategy for PPR surveillance and control in the region. Simple phylogenetic analyses based on readily available data can provide information on PPR transboundary dynamics and, therefore, could contribute to improve control strategies. On-going and future projects dedicated to PPR should include extensive genetic characterization and phylogenetic analyses of circulating viral strains in their effort to support the campaign for global eradication of the disease.

Peste des petits ruminants (PPR) is an OIE-listed disease of small ruminants, endemic in Africa and Asia, causing high economic impacts. Vaccination is an effective way to control PPR, but hot ambient temperatures associated to possible power cuts in developing countries may alter the vaccine stability during storage and transportation. Thus, making available an effective and thermotolerant vaccine is a priority. Lyophilized form of an experimental vaccine (EXP-VAC), formulated to be thermotolerant, was exposed at 37°C for three days before use, mimicking a cold-chain rupture. Ten PPRV-naïve goats received a dose of well-established PPR-VACTM[1], ten others received a dose of EXP-VAC and another ten remained unvaccinated. Twenty one days later, all goats were infected intranasally with a virulent PPRV (Morocco 2008). Clinical signs and viral ocular excretion (RT-qPCR) were monitored for 14 days. Controls presented PPRV ocular shedding and severe clinical signs related to PPR such as nasal and ocular discharge, mucosa lesions, diarrhea, and weight loss. EXP-VAC-vaccinated goats only presented few mildly swollen lymph nodes. EXP-VAC completely prevented viral ocular excretion. No difference in efficacy was observed between EXP-VAC and PPR-VACTM used according to label. Thus, efficacy of EXP-VAC was demonstrated. This thermotolerant vaccine may be an aid to allow epidemiological control of PPR preventing economic losses in areas where the cold chain cannot be guaranteed.

Peste des Petits Ruminants (PPR) is a severe disease of small ruminants and has high economic impacts in developing countries. Endemic in Africa, the Middle East and Asia, the disease is currently progressing with occurrences reported in North Africa, Turkey and in Georgia, and now threatens Europe. Much remains unknown about the infection dynamics, the virulence of the different strains and species/breed susceptibility. Robust experimental challenge models are needed to explore these fields and to confirm the efficacy of currently sold vaccines. We first assessed virulence of two PPR virus strains (CI89 and MA08) in Saanen goats. Whereas the MA08 strain led to classical severe clinical signs of PPR, the CI89 strain appeared to cause a mild disease in Saanen goats, highlighting the difference in virulence between strains in this animal model. We further demonstrated the importance of the inoculation route in the appearance of clinical signs and that ocular excretion is a better choice than blood for viral detection. After developing a robust challenge model, we assessed the efficacy of a vaccine (PPR-VAC , BVI Botswana) against the MA08 strain and demonstrated that this vaccine blocked viral excretion and significantly reduced clinical signs. These results reinforce the paradigm that a strain from one lineage could protect against strains from other lineages.

Peste des Petits Ruminants (PPR) is a highly infectious disease caused by a virus of the Morbillivirus genus. The current PPR eradication effort relies mainly on the implementation of massive vaccination campaigns. One of the most widely used PPR vaccines is the Nigeria 75/1 strain obtained after attenuation by 75 serial passages of the wild type isolate in cell cultures. Here we use high throughput deep sequencing of the historical passages that led to the Nigeria 75/1 attenuated strain to understand the evolution of PPRV attenuation and to assess the risk of reversal in different cell types. Comparison of the consensus sequences of the wild type and vaccine strains showed that only 18 fixed mutations separate the two strains. At the earliest attenuation passage at our disposal (passage 47), 12 out of the 18 mutations were already present at a frequency of 100%. Low-frequency variants were identified along the genome in all passages. Sequencing of passages after the vaccine strain showed evidence of genetic drift during cell passages, especially in cells expressing the SLAM receptor targeted by PPRV. However, 15 out of the 18 mutations related to attenuation remained fixed in the population. In vitro experiments suggest that one mutation in the leader region of the PPRV genome affects virus replication. Our results suggest that only a few mutations can have a serious impact on the pathogenicity of PPRV. Risk of reversion to virulence of the attenuated PPRV strain Nigeria 75/1 during serial passages in cell cultures seems low but limiting the number of passages during vaccine production is recommended.

Background Peste-des-petits ruminants (PPR) is a highly contagious and devastat-ing viral disease affecting sheep, goats, and a large number of spe-cies within the order Artiodactyla. Robust commercial serological and virological diagnostic kits are available to detect PPR infection, but they were mainly developed for domestic small ruminants (goat and sheep) and for high quality, invasive samples sometimes hard to obtain in the field. New tools are needed to detect PPR infection in atypical hosts (e.g. camels, wildlife) and in complex field situations. Here we present adaptation of existing methods and new diagnostic tools to resolve some of these issues. In some regions, farmers are reluctant to have their animals handled and tested for PPR infection. As well, in the case of wildlife survey, animal capture is very costly and demands complicated logistics. Detection of PPR virus in non- invasive samples (feces) can be extremely useful in such cases. Methods and results We adapted methods of RNA extraction, RT-PCR, RT-QPCR and anti-gen capture ELISA to detect PPR viral particles or genetic material from fecal samples. The methods were validated with samples col-lected during an infection experiment. Our protocol was then used with fecal samples obtained in the field in Tanzania in 2015, and compared to results obtained from ocular swab samples taken from the same animals. Another issue is that existing LFD tests used in the field do not allow for direct confirmation by PCR. Here we present a new rapid penside test, produced and distributed by IDvet (France), which can be performed without any lab equipment. Lastly, we also tackled the issue of PPR antibody detection from camelid sera, usually suboptimal because of their particular antibody structure. Results Our results show that virus particles can be detected in fecal sam-ples from 4 days post infection (dpi) until at least 14 dpi. Sensitivity of RT-QPCR from fecal samples was similar to RT-QPCR and lateral flow device (LFD) on ocular swab samples. The penside test was as sensi-tive as the antigen capture ELISA test. Once dried, positive strips can be stored and used later on for confirmation by RT-QPCR or RT-PCR. A simple modification of the protocol of a commercially available competitive ELISA for PPR antibody detection (IDvet) increased the sensitivity of the test on camelid serum. Conclusion These tools will be extremely useful to unravel important questions that still remain about PPR epidemiology, notably the role of atypical host in PPR transmission dynamics.

Peste des petits ruminants (PPR) is a highly contagious and devastating viral disease of small ruminants. It represents a serious risk for the economy and food security in regions of Africa, Middle East and Asia where the disease is endemic. Integrated knowledge of evolutionary and epidemiological factors underlying PPR virus (PPRV) emergence, persistence and spread are necessary for better guidance of PPR control strategies and their practical implementation. Efforts are especially needed to better understand the regional dynamics of PPRV evolution and endemic transmission. Here we studied the regional evolutionary dynamics of endemic PPRV in West Africa, focussing on Senegal and neighbouring countries, and assessed the role of livestock trade in explaining the observed viral diversity and phylogenetic patterns. Sheep and goats were sampled in livestock markets and villages across Senegal between 2010 and 2014 and tested for PPRV infection. Other samples were obtained from Mauritania, Mali, and Guinea during the same period. Historical samples (1972-1994) were also collected from the region. In addition, livestock movement data, particularly livestock trade were collected during specific surveys implemented in Mauritania and Senegal. A total of 55 samples collected from 2010 to 2014 were positive for PPRV. Partial sequencing of the N gene showed that 54 belonged to the PPR virus lineage II (PPRV-II) and one to lineage I. We obtained the sequence of the full N and H genes for all PPRV-II samples, and sequenced the full genome for a subset of recent and historical samples. Phylogenetic analyses showed the presence of at least 4 different, geographically delimited, clades within PPRV-II in West Africa. Samples from Mali were distributed across 3 of these clades, suggesting a central position of the country in regional movement of PPR. All PPRV-II samples from Senegal were situated within a single clade, but could be separated in distinct clusters. These clusters pointed to virus movement across long distances within Senegal and between Senegal and its neighbours. Transboundary movements involved mainly major sites for commercial animal movement, but also transhumant movement between Senegal and Mauritania. A statistical model of virus genetic distance was fitted with environnent and animal movement data to assess if the genetic patterns observed can be predicted by commercial connectivity. We discuss how such landscape resistance analyses based on animal movement can be used to predict PPR transmission pathways and control effort within endemic regions. (Texte intégral)

La présente invention concerne l'obtention d'une souche vaccinale marquée du virus de la peste des petits ruminants, par production d'un clone infectieux, et marquage de ce clone par substitution d'un épitope dudit virus par un épitope exogène.

La présente invention concerne l'obtention d'une souche vaccinale marquée du virus de la peste des petits ruminants, par production d'un clone infectieux, et marquage de ce clone par substitution d'un épitope dudit virus par un épitope exogène.

Chemical-based transfection of DNA into cultured cells is routinely used to study for example viral or cellular gene functions involved in virus replication, to analyse cellular defence mechanisms or develop specific strategies to interfere with virus replication. Other applications include rescue of viruses by reverse genetics and/or generation of mutated viruses. A large number of transfection chemicals like calcium phospate, branched organic compounds, liposomes, cationic polymers etc. are available on the market which are used by different laboratories for different cell lines. To obtain an overview on the efficiencies of varying transfection procedures, an interlaboratory ring trial was initiated within EPIZONE theme 5. A total of 15 participitating laboratories from 7 member institutions received RK13 cells, plasmid DNA encoding firefly luciferase under the transcriptional control of the human cytomegalovirus major immediate early promoter, a specially developed lysis buffer and a detailed protocol. Transfected cells were harvested in the laboratories of the participants, frozen and sent to the FLI where both the luciferase activity and protein content of the individual samples were determined to compare transfection efficiency between laboratories with the same protocol and equipment. In addition some laboratories sent samples from cells they are routinely using, transfected with the provided firefly luciferase plasmid, to allow comparison of transfection efficiency between different cell types. About 50 different samples were analysed and the luciferase activity per nanogram total protein (RLU/ng) was determined. The results revealed for RK13 cells a large range of specific luciferase activities between laboratories and, in comparison to RK13 cells, also varying transfection efficacies for other the cell lines. Details will be presented.

La peste bovine et la peste des petits ruminants sont des maladies contagieuses affectant les ruminants domestiques et sauvages et entraînant des taux de mortalités souvent élevés. Elles sont provoquées par deux virus différents mais très apparentés antigéniquement et appartenant au genre Morbillivirus, famille des Paramyxoviridae. Les outils de contrôle effectif de ces deux maladies (vaccins et tests de diagnostic spécifiques) sont disponibles. Ils ont été mis à profit lors du programme mondial d'éradication de la peste bovine, (GREP pour Global Rinderpest Eradication Programme). Grâce à ce programme, il sera certainement possible de déclarer l'éradication de la peste bovine à l'échelle du globe en 2010. Malheureusement une telle campagne mondiale n'existe pas pour la peste des petits ruminants qui est en pleine expansion. Avec l'infection de la Turquie et du Maroc, elle se trouve maintenant aux portes de l'Europe. Pour une lutte plus efficace contre cette maladie, supportable sur le plan économique, la mise au point d'un vaccin marqueur ou DIVA (differentiation of infected and vaccinated animals) et d'un test sérologique discriminant serait un atout supplémentaire. Les outils de génétique inverse permettent désormais d'envisager cette possibilité.