Comparative evolutionary analyses of peste des petits ruminants virus genetic lineages.

Peste des petits ruminants virus (PPRV) causes a highly infectious disease affecting mainly goats and sheep in large parts of Africa, Asia, and the Middle East and has an important impact on the global economy and food security. Full genome sequencing of PPRV strains has proved to be critical to increasing our understanding of PPR epidemiology and to inform the ongoing global efforts for its eradication. However, the number of full PPRV genomes published is still limited and with a heavy bias towards recent samples and genetic Lineage IV (LIV), which is only one of the four existing PPRV lineages. Here, we generated genome sequences for twenty-five recent (2010-6) and seven historical (1972-99) PPRV samples, focusing mainly on Lineage II (LII) in West Africa. This provided the first opportunity to compare the evolutionary pressures and history between the globally dominant PPRV genetic LIV and LII, which is endemic in West Africa. Phylogenomic analysis showed that the relationship between PPRV LII strains was complex and supported the extensive transboundary circulation of the virus within West Africa. In contrast, LIV sequences were clearly separated per region, with strains from West and Central Africa branched as a sister clade to all other LIV sequences, suggesting that this lineage also has an African origin. Estimates of the time to the most recent common ancestor place the divergence of modern LII and LIV strains in the 1960s-80s, suggesting that this period was particularly important for the diversification and spread of PPRV globally. Phylogenetic relationships among historical samples from LI, LII, and LIII and with more recent samples point towards a high genetic diversity for all these lineages in Africa until the 1970s-80s and possible bottleneck events shaping PPRV's evolution during this period. Molecular evolution analyses show that strains belonging to LII and LIV have evolved under different selection pressures. Differences in codon usage and adaptative selection pressures were observed in all viral genes between the two lineages. Our results confirm that comparative genomic analyses can provide new insights into PPRV's evolutionary history and molecular epidemiology. However, PPRV genome sequencing efforts must be ramped up to increase the resolution of such studies for their use in the development of efficient PPR control and surveillance strategies.

The evaluation of five serological assays in determining seroconversion to peste des petits ruminants virus in typical and atypical hosts.

Peste des petits ruminants (PPR) is an infectious viral disease, primarily of small ruminants such as sheep and goats, but is also known to infect a wide range of wild and domestic Artiodactyls including African buffalo, gazelle, saiga and camels. The livestock-wildlife interface, where free-ranging animals can interact with captive flocks, is the subject of scrutiny as its role in the maintenance and spread of PPR virus (PPRV) is poorly understood. As seroconversion to PPRV indicates previous infection and/or vaccination, the availability of validated serological tools for use in both typical (sheep and goat) and atypical species is essential to support future disease surveillance and control strategies. The virus neutralisation test (VNT) and enzyme-linked immunosorbent assay (ELISA) have been validated using sera from typical host species. Still, the performance of these assays in detecting antibodies from atypical species remains unclear. We examined a large panel of sera (n = 793) from a range of species from multiple countries (sourced 2015-2022) using three tests: VNT, ID VET N-ELISA and AU-PANVAC H-ELISA. A sub-panel (n = 30) was also distributed to two laboratories and tested using the luciferase immunoprecipitation system (LIPS) and a pseudotyped virus neutralisation assay (PVNA). We demonstrate a 75.0-88.0% agreement of positive results for detecting PPRV antibodies in sera from typical species between the VNT and commercial ELISAs, however this decreased to 44.4-62.3% in sera from atypical species, with an inter-species variation. The LIPS and PVNA strongly correlate with the VNT and ELISAs for typical species but vary when testing sera from atypical species.

Genomic characterization of peste des petits ruminants vaccine seed "45G37/35-k", Russia.

Production of peste des petits ruminants (PPR) vaccines in Russia is based on two attenuated virus strains ("45G37/35-k" and "ARRIAH") of common origin. Here, the identity of the strain PPRV/45G37/35-k was investigated using a full genome, Illumina deep sequencing approach. Phylogenomic analysis showed that PPRV/45G37/35-k belongs to the same lineage as the widely used PPRV vaccine strain Nigeria/75/1 (lineage II). However, 248 nucleotide differences separate the genomes of these vaccine strains, indicating that the PPRV vaccine strains produced in Russia are new strains not yet recognised by the World Organization for Animal Health (WOAH). Detailed information on the safety and efficacy of these vaccines should be provided to the WOAH before further national and international distribution.

Molecular epidemiology of peste des petits ruminants virus in Nigeria: An update.

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.

Persistence of the historical lineage I of West Africa against the ongoing spread of the Asian lineage of peste des petits ruminants virus.

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.

Combining viral genetic and animal mobility network data to unravel peste des petits ruminants transmission dynamics in West Africa.

Peste des petits ruminants (PPR) is a deadly viral disease that mainly affects small domestic ruminants. This disease threaten global food security and rural economy but its control is complicated notably because of extensive, poorly monitored animal movements in infected regions. Here we combined the largest PPR virus genetic and animal mobility network data ever collected in a single region to improve our understanding of PPR endemic transmission dynamics in West African countries. Phylogenetic analyses identified the presence of multiple PPRV genetic clades that may be considered as part of different transmission networks evolving in parallel in West Africa. A strong correlation was found between virus genetic distance and network-related distances. Viruses sampled within the same mobility communities are significantly more likely to belong to the same genetic clade. These results provide evidence for the importance of animal mobility in PPR transmission in the region. Some nodes of the network were associated with PPRV sequences belonging to different clades, representing potential "hotspots" for PPR circulation. Our results suggest that combining genetic and mobility network data could help identifying sites that are key for virus entrance and spread in specific areas. Such information could enhance our capacity to develop locally adapted control and surveillance strategies, using among other risk factors, information on animal mobility.

Corrigendum: Genetic Evidence for Transboundary Circulation of Peste Des Petits Ruminants Across West Africa.

[This corrects the article DOI: 10.3389/fvets.2019.00275.].

Genetic Evidence for Transboundary Circulation of Peste Des Petits Ruminants Across West Africa.

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 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 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.

Evolution of Attenuation and Risk of Reversal in Peste des Petits Ruminants Vaccine Strain Nigeria 75/1.

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.

Molecular detection and phylogenetic analysis of Peste des petits ruminants virus circulating in small ruminants in eastern Amhara region, Ethiopia.

BACKGROUND: Peste des Petits Ruminants (PPR) is a severe, highly infectious and fatal viral disease of small ruminants. Four lineages of PPR virus have been identified globally based on sequence analysis of the nucleoprotein (N) and fusion (F) gene. The aim of this study was to isolate and genetically characterize recently circulating PPR virus in small ruminants in the eastern Amhara region in Ethiopia. A total of 28 anti-mortem samples (gum debris, nasal and ocular swab) were collected from clinically suspicious animals and examined for the presence of PPRV by a one-step RT-PCR assay. Samples positive with RT-PCR were subjected to isolation of the virus which were subsequently genetically characterized by sequencing of the nucleoprotein (N) gene and phylogenetic analysis of PPR virus (PPRV) strains. RESULTS: Of the 28 clinical samples examined, 46.4% were positive with RT-PCR for viral nucleic acid. The PPRV was successfully isolated on CHS-20 cell line with the ovine signaling lymphocyte activation molecule (SLAM) receptor expressed on the cell surface and confirmed with RT-PCR and IFAT assay. The nucleotide sequence and phylogenetic analysis indicated that the PPRV obtained were clustered genetically with Lineage IV isolates of the virus. CONCLUSION: The successful isolation of the virus and molecular findings of this study confirmed active lineage IV PPRV infections among populations of sheep and goats in eastern Amhara, suggesting risks for potential spread of the disease to currently free areas. Thus, we recommend systematic vaccination to contain outbreaks in affected districts and geographically linked surrounding districts to which the disease could potentially spread due to different epidemiological linkages.

Optimization and evaluation of a non-invasive tool for peste des petits ruminants surveillance and control.

Peste des petits ruminants (PPR) is a highly contagious and devastating viral disease affecting mainly sheep and goats, but also a large number of wild species within the order Artiodactyla. A better understanding of PPR transmission dynamics in multi-host systems is necessary to efficiently control the disease, in particular where wildlife and livestock co-occur. Notably, the role of wildlife in PPR epidemiology is still not clearly understood. Non-invasive strategies to detect PPR infection without the need for animal handling could greatly facilitate research on PPR epidemiology and management of the disease in atypical hosts and in complex field situations. Here, we describe optimized methods for the direct detection of PPR virus genetic material and antigen in fecal samples. We use these methods to determine the detection window of PPR in fecal samples, and compare the sensitivity of these methods to standard invasive sampling and PPR diagnostic methods using field samples collected at a wildlife-livestock interface in Africa. Our results show that quantitative reverse transcription PCR (RT-QPCR) amplification of PPRV from fecal swabs has good sensitivity in comparison to ocular swabs. Animals infected by PPRV could be identified relatively early on and during the whole course of infection based on fecal samples using RT-QPCR. Partial gene sequences could also be retrieved in some cases, from both fecal and ocular samples, providing important information about virus origin and relatedness to other PPRV strains. Non-invasive strategies for PPRV surveillance could provide important data to fill major gaps in our knowledge of the multi-host PPR epidemiology.

Development of a PPRV challenge model in goats and its use to assess the efficacy of a PPR vaccine.

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.

A G-protein-coupled chemokine receptor: A putative insertion site for a multi-pathogen recombinant capripoxvirus vaccine strategy.

Capripoxviruses (CaPVs) have been shown to be ideal viral vectors for the development of recombinant multivalent vaccines to enable delivery of immunogenic genes from ruminant pathogens. So far, the viral thymidine kinase (TK) gene is the only gene used to generate recombinants. A putative non-essential gene encoding a G-protein-coupled chemokine receptor subfamily homologue (GPCR) was targeted as an additional insertion site. Peste des petits ruminants (PPR) was chosen as a disease model. A new recombinant CaPV expressing the viral attachment hemagglutinin (H) of the PPR virus (PPRV) in the GPCR insertion site (rKS1-HPPR-GPCR) was generated in the backbone North African isolate KS1 strain of lumpy skin disease virus (LSDV). Comparison with the recombinant CaPV expressing the H of PPRV in the TK gene (rKS1-HPPR-TK) shown to induce protection against both PPR and LSD in both sheep and goats was assessed. The suitability of the GPCR gene to be a putative additional insertion site in the CaPV genome is evaluated and discussed.

Diverse gammacoronaviruses detected in wild birds from Madagascar.

To date, infectious bronchitis virus (IBV) is potentially found in wild birds of different species. This work reports the survey of coronaviruses in wild birds from Madagascar based on the targeting of a conserved genome sequence among different groups of CoVs. Phylogenetic analyses revealed the presence of gammacoronaviruses in different species of Gruiformes, Passeriformes, Ciconiiformes, Anseriformes, and Charadriiformes. Furthermore, some sequences were related to various IBV strains. Aquatic and migratory birds may play an important role in the maintenance and spread of coronaviruses in nature, highlighting their possible contribution in the emergence of new coronavirus diseases in wild and domestic birds.

Dried fluid spots for peste des petits ruminants virus load evaluation allowing for non-invasive diagnosis and genotyping.

BACKGROUND: Active surveillance of peste des petits ruminants (PPR) should ease prevention and control of this disease widely present across Africa, Middle East, central and southern Asia. PPR is now present in Turkey at the gateway to the European Union. In Bangladesh, the diagnosis and genotyping of PPR virus (PPRV) may be hampered by inadequate infrastructures and by lack of proper clinical material, which is often not preserved under cold chain up to laboratories. It has been shown previously that Whatman® 3MM filter paper (GE Healthcare, France) preserves the nucleic acid of PPRV for at least 3 months at 32°C. RESULTS: In this study, we demonstrate the performances of filter papers for archiving RNA from local PPRV field isolates for further molecular detection and genotyping of PPRV, at -70°C combined with ambient temperature, for periods up to 16 months. PPR-suspected live animals were sampled and their blood and nasal swabs were applied on filter papers then air dried. Immediately after field sampling, RT-PCR amplifying a 448-bp fragment of the F gene appeared positive for both blood and nasal swabs when animals were in febrile stage and only nasal swabs were detected positive in non-febrile stage. Those tested positive were monitored by RT-PCR up to 10 months by storage at -70°C. At 16 months, using real time RT-PCR adapted to amplify the N gene from filter paper, high viral loads could still be detected (~2 x 10(7) copy numbers), essentially from nasal samples. The material was successfully sequenced and a Bayesian phylogenetic reconstruction achieved adequate resolution to establish temporal relationships within or between the geographical clusters of the PPRV strains. CONCLUSIONS: This clearly reveals the excellent capacity of filter papers to store genetic material that can be sampled using a non-invasive approach.

Complete Genome Sequence of a Field Strain of Peste des Petits Ruminants Virus Isolated during 2010-2014 Epidemics in Senegal.

Peste des petits ruminants virus (PPRV) infection is expanding and results in regular epizootic activities in Africa, the Middle East, and Asia. Here, we report the complete genome sequence of a field strain of PPRV isolated in Senegal (SnDk11I13) in 2013.

Protective efficacy of a single immunization with capripoxvirus-vectored recombinant peste des petits ruminants vaccines in presence of pre-existing immunity.

Sheeppox, goatpox and peste des petits ruminants (PPR) are highly contagious ruminant diseases widely distributed in Africa, the Middle East and Asia. Capripoxvirus (CPV)-vectored recombinant PPR vaccines (rCPV-PPR vaccines), which have been developed and shown to protect against both Capripox (CP) and PPR, would be critical tools in the control of these important diseases. In most parts of the world, these disease distributions overlap each other leaving concerns about the potential impact that pre-existing immunity against either disease may have on the protective efficacy of these bivalent rCPV-PPR vaccines. Currently, this question has not been indisputably addressed. Therefore, we undertook this study, under experimental conditions designed for the context of mass vaccination campaigns of small ruminants, using the two CPV recombinants (Kenya sheep-1 (KS-1) strain-based constructs) developed previously in our laboratory. Pre-existing immunity was first induced by immunization either with an attenuated CPV vaccine strain (KS-1) or the attenuated PPRV vaccine strain (Nigeria 75/1) and animals were thereafter inoculated once subcutaneously with a mixture of CPV recombinants expressing either the hemagglutinin (H) or the fusion (F) protein gene of PPRV (10(3) TCID50/animal of each). Finally, these animals were challenged with a virulent CPV strain followed by a virulent PPRV strain 3 weeks later. Our study demonstrated full protection against CP for vaccinated animals with prior exposure to PPRV and a partial protection against PPR for vaccinated animals with prior exposure to CPV. The latter animals exhibited a mild clinical form of PPR and did not show any post-challenge anamnestic neutralizing antibody response against PPRV. The implications of these results are discussed herein and suggestions made for future research regarding the development of CPV-vectored vaccines.

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.

The incursion, persistence and spread of peste des petits ruminants in Tanzania: epidemiological patterns and predictions.

Peste des petits ruminants virus, which causes a severe disease in sheep and goats, has only recently been officially declared to be present in Tanzania. An epidemiological study was carried out between September 2008 and October 2010 to investigate the incursion, persistence and spread of the virus in Tanzania. The investigation involved serosurveillance, outbreak investigation and computation of epidemiological indices such as the effective reproductive number, persistence and the threshold level for vaccination. Field and molecular epidemiological techniques were applied to isolate, characterise and trace the origin of the virus in Tanzania. A total of 2182 serum samples from goats and 1296 from sheep from 79 villages across 12 districts were investigated. Village-level prevalence of infection was variable (0.00% ­ 88.00%) and was higher in pastoral than in agro-pastoral villages. The overall antibody response to the virus was 22.10% (CI95% = 20.72% ­ 23.48%). About 68.00% and 73.00% of seropositive goats and sheep, respectively, did not show clinical signs. The proportion of seropositive animals differed significantly (p ≤ 0.001) between age groups, sex and farming practices. Real-time polymerase chain reaction results showed that the isolated strains belong to lineage III, whose origin is in East Africa and the Middle East. This indicates that one of the northern neighbouring countries is most likely the source of infection. The computed overall effective reproductive number, the threshold level of vaccination necessary to eradicate the disease and persistence were 4.75% and 98.00%, respectively. These estimates indicate that achieving elimination of the peste des petits ruminants virus from pastoral flocks will require significant effort and development of highly effective intervention tools.