Peste des Petits Ruminants Lineages: Veterinary Reference

Overview and Taxonomy of Peste des Petits Ruminants Virus Lineages I–IV: Veterinary Reference

Foundational Classification and Genomic Architecture

Peste des petits ruminants virus (PPRV), taxonomically designated Morbillivirus caprinae within the family Paramyxoviridae, is the etiological agent of one of the most economically devastating transboundary diseases affecting small ruminants globally [10, 26]. The virus possesses a single-stranded, negative-sense RNA genome of approximately 15,954 nucleotides, which encodes six structural proteins in sequential order: the nucleocapsid (N) protein, the phosphoprotein (P), the matrix (M) protein, the fusion (F) protein, the hemagglutinin (H) protein, and the large (L) polymerase protein [26, 32]. This genomic organization is characteristic of the morbillivirus genus, which also includes the now-eradicated rinderpest virus, measles virus, and canine distemper virus [14, 26]. The genetic classification of PPRV into four distinct lineages, designated I, II, III, and IV, has been historically established through phylogenetic analysis of partial sequences of the nucleoprotein (N) and fusion (F) genes, which provide sufficient resolution to delineate these major clades [2, 3, 26]. Importantly, despite this substantial genetic diversity at the nucleotide level, PPRV exists as a single serotype, a feature that has profound implications for vaccine development and global eradication strategies [14, 26].

The World Organisation for Animal Health (WOAH) and the Food and Agriculture Organization of the United Nations (FAO) have jointly spearheaded the Global Strategy for the Control and Eradication of PPR (PPR GCES), targeting worldwide eradication by 2030, leveraging the lessons learned from the successful rinderpest eradication campaign [7, 14]. The existence of a single serotype means that a single efficacious vaccine strain can, in theory, confer protection against all circulating lineages. However, the nuanced epidemiological behaviors, differential geographical distributions, and evolving competitive dynamics among these lineages necessitate a sophisticated understanding of their taxonomy and evolutionary history to inform surveillance, diagnostic assay design, and vaccination campaign logistics [3, 14].

Lineage I: The Historical West African Clade

Lineage I represents one of the earliest recognized genetic groups of PPRV, with its origins deeply rooted in West Africa. Historical analyses and phylogenetic reconstructions have identified Senegal as the most probable geographic origin for this lineage [29]. For decades, lineage I was considered a dominant circulating strain across parts of West Africa, particularly in Mali and Senegal. However, contemporary molecular epidemiological surveillance has revealed a dramatic shift in its prevalence. Remarkably, lineage I was thought to be extinct for a significant period, but a landmark study by Tounkara et al. (2021) demonstrated its persistence, at least until 2014, in two regions of Mali, Segou and Sikasso, representing the last known refugia of this historically significant lineage [30]. This finding is critical from a veterinary reference perspective, as it underscores that lineage extinction cannot be assumed without exhaustive surveillance. The persistence of lineage I in these specific geographic pockets suggests that ecological or host factors may have allowed it to endure despite the incursion of more recently emergent lineages [30]. The genetic characterization of lineage I strains has been hampered by a paucity of full-genome sequences, but partial N-gene sequencing has been sufficient to confirm its distinct phylogenetic position. The Nigeria 75/1 vaccine strain, which is the most widely used live attenuated vaccine globally, is often misattributed in older literature; while some early studies using F-gene sequencing placed it within lineage I, subsequent and more definitive N-gene-based phylogenies have unequivocally classified it within lineage II [11, 19]. This taxonomic clarification is essential for accurate molecular epidemiology and for understanding the evolutionary relationships between vaccine strains and field isolates.

Lineage II: The Vaccine Strain Lineage and West African Endemicity

Lineage II is arguably the most critical lineage from a veterinary intervention perspective, as it contains the globally deployed Nigeria 75/1 vaccine strain [1, 18, 23]. The Nigeria 75/1 strain, derived from a Nigerian isolate from 1975, is a live attenuated vaccine that provides lifelong immunity after a single minimum recommended dose of 10^2.5 TCID50/mL [18]. Genomic characterization of this lineage has been extensive, with full-genome sequencing of the Nigeria 75/1 strain and related Russian vaccine seeds (e.g., 45G37/35-k and ARRIAH) revealing that while they share a common lineage II origin, there are substantial nucleotide differences, 248 nucleotide differences separate the Nigeria 75/1 and 45G37/35-k genomes, indicating that these are distinct vaccine strains not yet fully recognized by WOAH for international distribution [23]. The ARRIAH vaccine, also belonging to lineage II, has demonstrated promising protective efficacy against heterologous lineage IV challenge, including the virulent Mongolia/2021 isolate, suggesting that lineage II vaccines remain broadly protective across lineages [21].

Epidemiologically, lineage II is endemic and dominant across vast swathes of West Africa, including Nigeria, Burkina Faso, Côte d’Ivoire, Guinea, Ghana, and Senegal [11, 15, 19, 30]. In Nigeria, phylogenetic analyses have identified at least two distinct sub-clusters within lineage II, designated II-NigA and II-NigB, with II-NigB being closely related to the Nigeria 75/1 vaccine strain, raising important questions about vaccine-derived immunity versus natural infection dynamics in serosurveys [11]. The co-circulation of lineage II with lineage IV has been extensively documented in West Africa. For instance, in Burkina Faso, a study by Biguezoton et al. (2024) identified the presence of lineage IV in three studied regions, with genetic heterogeneity among sequences, suggesting that lineage IV is progressively replacing lineage II [9]. Similarly, in Côte d’Ivoire and Guinea, co-circulation of both lineages has been confirmed [15]. This replacement phenomenon is not merely an academic curiosity; it has profound implications for vaccine matching, diagnostic assay design, and the interpretation of molecular epidemiological data. The competitive advantage of lineage IV over lineage II in West Africa may be attributable to differences in viral fitness, transmission efficiency, or host adaptation [3, 9].

Lineage III: The East African and Arabian Peninsula Enigma

Lineage III occupies a unique and somewhat enigmatic position in PPRV taxonomy. Its geographic distribution is primarily centered in East Africa and the Arabian Peninsula, with phylogenetic evidence suggesting emergence in either Ethiopia or the Arabian Peninsula (Oman and/or the United Arab Emirates) [29]. Historically, lineage III was detected in Ethiopia, Sudan, and the Democratic Republic of the Congo (DRC), but contemporary surveillance indicates a dramatic contraction of its range [13, 25, 35]. In Ethiopia, a comprehensive molecular epidemiological update by Rume et al. (2019) analyzing samples collected between 2011 and 2017 failed to detect any lineage III viruses, with all positive samples belonging to sub-clade II of clade I of lineage IV [25]. This suggests that lineage III has been largely supplanted by lineage IV in Ethiopia, mirroring the trend observed in West Africa with lineage II.

However, lineage III persists in certain regions. In the DRC, a study by Tshilenge et al. (2019) identified both lineage II and lineage III in samples collected from three provinces in 2016 and 2018, demonstrating that lineage III is still circulating in Central Africa [13]. Furthermore, retrospective analysis of initial PPR outbreaks in the DRC (2008–2012) confirmed the circulation of lineage IV, but subsequent outbreaks in 2016 and 2018 in the western part of the country, bordering East Africa, revealed the presence of both lineage II and lineage III [35]. This indicates that the DRC serves as a critical geographic interface where multiple lineages converge, potentially facilitating recombination events or competitive exclusion dynamics. From a veterinary reference standpoint, lineage III is particularly important because it represents a lineage that may have been historically underestimated due to sampling biases. The comparative virulence of lineage III strains has been evaluated in experimental settings; a study by Aklilu et al. (2025) comparing six distinct PPRV strains (four lineage IV, two lineage III) in indigenous Ethiopian goats found no substantial lineage-specific differences in virulence, suggesting that lineage classification alone does not predict clinical outcomes [5]. This finding challenges the assumption that lineage replacement is driven by inherent virulence differences and points instead to factors such as host population immunity, animal movement patterns, or environmental transmission dynamics.

Lineage IV: The Globally Dominant and Expanding Lineage

Lineage IV is, without question, the most epidemiologically significant and geographically widespread of all PPRV lineages. Originally considered an Asian lineage, it has demonstrated a unique and alarming capacity for transcontinental spread, now dominating in Asia, the Middle East, Europe, and increasingly across Africa [3, 4, 12, 15, 17]. Phylogenomic analyses have revealed that lineage IV sequences from West and Central Africa branch as a sister clade to all other lineage IV sequences, suggesting an African origin for this lineage as well, with estimates of the time to the most recent common ancestor placing the divergence of modern lineage II and IV strains in the 1960s–1980s [3]. This period was particularly important for the global diversification and spread of PPRV.

The molecular epidemiology of lineage IV is characterized by extensive sub-structuring. In China, comprehensive genomic characterization of 28 novel full-length genome sequences from 2014 to 2021, combined with 135 published genomes, revealed that lineage IV can be divided into seven distinct clades, demonstrating both temporal and spatial correlation [17]. Critically, PPRV in China from 2007–2008 and 2013–2024 grouped into two distinct genetic clades, indicating two independent incursions of the disease [17]. The 2013–2024 Chinese strains shared a common ancestor with a strain from the United Arab Emirates and evolved into four distinct genetic clusters, with 997 single-nucleotide variations (SNVs) identified relative to the reference genome PPRV/XJYL/2013 [17]. Five anchor mutations, located in the 3′ leader, 5′ UTR of the F gene, H coding sequence, and L coding sequence, define these genetic clusters, providing molecular markers for tracking viral spread [17].

In Africa, lineage IV is actively displacing other lineages. In Burkina Faso, lineage IV appears to be replacing lineage II [9]. In Senegal, the first report of lineage IV emergence was documented in 2020, confirming its westward expansion [22]. In Nigeria, extensive sampling between 2017 and 2020 identified 90 out of 91 sequences belonging to lineage IV, with at least four distinct sub-clusters (IV-NigA and IV-NigB) circulating across multiple regions, suggesting extensive endemic circulation and transboundary spread [27]. In Ghana, lineage IV was identified for the first time in a regional investigation encompassing Burkina Faso, Côte d’Ivoire, Guinea, and Ghana [15]. In Egypt, molecular characterization of circulating strains from goat samples confirmed that the emerging PPRV belongs to lineage IV, with 96.7% identity to the Egypt-2014 strain [31]. In the United Arab Emirates, phylogenetic analysis based on N and F genes classified the virus within Asian lineage IV, clustering closely with sequences from Pakistan, Tajikistan, and Iran [16]. In Bangladesh, extensive molecular characterization of both N and F genes from sheep and goat isolates consistently places circulating strains within lineage IV, with nucleotide divergence of 3–6% for the N gene and 1–2% for the F gene when compared to historical Bangladeshi isolates from 2008 and 2015, indicating continuous but slow evolution [20, 33].

The most dramatic recent expansion of lineage IV has been its incursion into Europe. In July 2024, the first PPR outbreak in Greece was laboratory confirmed in the region of Thessaly, with a total of 86 farms infected across multiple regions [8]. Genomic analysis of the Greek strain revealed it was most closely related to PPR viruses clustered in the lineage IV North-East Africa group [8]. Subsequently, outbreaks were reported in Romania and central Bulgaria in November 2024 [6]. Comprehensive genomic analyses confirmed that the emergence of PPR across Europe has a common origin, pointing towards an introduction from Northern Africa [6]. Multiple nucleotide and amino acid differences separate the European genomes from other sequences, with potential functional impacts on viral proteins that warrant further investigation [6]. This European incursion represents a significant threat to the continent’s PPR-free status and underscores the failure of current global control measures to contain lineage IV [34].

Evolutionary Dynamics and Selection Pressures

Comparative evolutionary analyses between lineage II and lineage IV have revealed that these lineages have evolved under markedly different selection pressures. A seminal study by Courcelle et al. (2024) demonstrated differences in codon usage and adaptive selection pressures across all viral genes between the two lineages [3]. Specifically, one site in the H gene and five sites in the L gene were identified under positive selection in Chinese lineage IV strains [17]. The H gene, which encodes the hemagglutinin protein responsible for host cell receptor binding, is under particular selective pressure as it is a major target of the host immune response. The evolutionary rate of the PPRV genome has been estimated at 6.70 × 10⁻⁴ nucleotide substitutions per site per year, which is consistent with other RNA viruses [17]. Codon usage analysis of 45 whole genomes revealed universal, lineage-specific, and gene-specific genetic features, with high adaptation of PPRV to hosts at the codon usage level reflecting high viral gene expression [24]. Interestingly, synonymous codons containing CpG dinucleotides showed weak tendencies to be selected in viral genes, suggesting host innate immune pressures (e.g., toll-like receptor 9 recognition of CpG motifs) may shape viral evolution [24]. The synonymous codon usage patterns of PPRV isolated during 2007–2008 and 2013–2014 in China displayed independent evolutionary pathways, despite both belonging to lineage IV, indicating that even within a single lineage, distinct evolutionary trajectories can emerge [24].

Diagnostic Implications of Lineage Diversity

The genetic diversity among the four lineages poses significant challenges for diagnostic assay design. Assays targeting conserved regions are essential for pan-lineage detection, while lineage-specific assays are valuable for epidemiological surveillance and differentiation of vaccinated from infected animals (DIVA). Several robust molecular diagnostic tools have been developed. A TaqMan RT-qPCR assay targeting the phosphoprotein (P) gene has been shown to detect all four lineages with high sensitivity: 4 copies/μL for lineages II–IV and 40 copies/μL for lineage I [2]. This assay demonstrated no cross-reaction with other viruses and showed excellent repeatability (coefficient of variation <1.50%) [2]. An alternative approach using an F-gene RT-qPCR assay, designed using all available full genomes, demonstrated excellent in silico performance and diagnostic sensitivity for all lineages [28]. For lineage-specific detection, a SYBR Green I real-time qRT-PCR method targeting the L gene was developed to differentiate lineage II from lineage IV based on melting curve analysis, achieving a detection limit of 100 copies [1]. A one-step TaqMan RT-qPCR targeting the hemagglutinin (H) gene was specifically designed for lineage IV detection, demonstrating high sensitivity (approximately 6 copies) and no cross-reaction with other lineages [4]. For rapid, pen-side diagnostics, a recombinase-aided amplification (RAA) combined with CRISPR-Cas12a targeting the N and M genes has been developed, capable of detecting all lineages within 50 minutes at 37°C with a detection limit of 10 copies/μL, and results can be visualized under

Molecular Pathogenesis and Genetic Determinants of PPRV Lineage-Specific Virulence

The molecular pathogenesis of Peste des Petits Ruminants virus (PPRV) across its four genetic lineages (I–IV) represents a convergence of viral genomic architecture, host-virus evolutionary arms races, and selective pressures exerted by diverse ecological niches. While PPRV exists as a single serotype, the genetic heterogeneity among lineages gives rise to nuanced differences in virulence, tissue tropism, transmissibility, and host range that are critical to understanding global eradication efforts [3, 14]. This section dissects the molecular underpinnings of lineage-specific virulence, drawing on comparative genomics, codon usage analysis, selection pressure studies, and experimental pathogenesis data.

Comparative Genomics and the Molecular Basis of Lineage Divergence

The PPRV genome, a single-stranded negative-sense RNA of approximately 15,954 nucleotides, encodes six structural proteins in the canonical morbillivirus order: nucleocapsid (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin (H), and large polymerase (L) [17, 26]. Lineage classification has historically relied on partial N and F gene sequences, but full-genome comparisons now reveal that lineage-specific virulence determinants are distributed across the entire coding capacity [3, 17]. The estimated evolutionary rate of the PPRV genome is approximately 6.70 × 10⁻⁴ nucleotide substitutions per site per year, with lineage IV, the globally dominant clade, exhibiting the highest rate of diversification [17].

Comparative evolutionary analyses between lineage II (endemic in West Africa) and lineage IV (globally distributed) have demonstrated fundamental differences in selection pressures acting on all six viral genes [3]. Lineage IV strains experience stronger positive selection on the H and L genes, suggesting that adaptation to diverse host populations and transmission across geographic barriers has driven accelerated evolution in receptor-binding and replication machinery [3, 17]. In contrast, lineage II strains show evidence of purifying selection consistent with long-term endemic circulation within relatively stable host populations [3]. These divergent evolutionary trajectories have profound implications: lineage IV has demonstrated a unique capacity to displace other lineages, as documented in West Africa where it is progressively replacing lineage II in Burkina Faso, Côte d’Ivoire, Ghana, Guinea, Nigeria, and Senegal [9, 11, 15, 22, 27, 30].

The Hemagglutinin Protein as a Primary Virulence Determinant

The hemagglutinin (H) protein is the principal determinant of host cell tropism and a critical virulence factor. H binds to the signaling lymphocyte activation molecule (SLAM/CD150) on immune cells and to nectin-4 on epithelial cells, mediating viral entry and subsequent dissemination [24, 26]. Sequence analysis reveals that lineage IV H genes harbor signature amino acid substitutions in the globular head domain responsible for SLAM receptor recognition, potentially conferring enhanced binding affinity or altered receptor utilization [17, 48]. Notably, at least one site in the H gene of Chinese lineage IV strains is under positive selection, and these positively selected positions cluster near the receptor-binding interface [17].

The emergence of lineage IV in Europe (Greece, Romania, Bulgaria in 2024–2025) has been associated with multiple nucleotide and amino acid differences in the H protein compared to African and Asian strains, with potential functional consequences that warrant urgent investigation [6, 8, 34]. These changes may reflect adaptation to new host environments or could represent fortuitous mutations that enhance transmissibility in naïve populations. The H protein also serves as the primary target for neutralizing antibodies, and antigenic drift in this protein, even within a single serotype, could influence vaccine efficacy and the duration of protective immunity [14].

Fusion Protein and Membrane Fusion Kinetics

The fusion (F) protein mediates membrane fusion following receptor binding by H, and its proteolytic activation by host furin-like proteases is a prerequisite for infectivity [26]. Comparative sequencing of the F gene across lineages reveals relatively higher conservation compared to H, with nucleotide divergence ranging from 1–2% among lineage IV isolates and 0–2% at the amino acid level over extended periods in Bangladesh [20]. However, lineage-specific differences in the fusion peptide region or heptad repeat domains could influence fusion kinetics and syncytia formation, thereby affecting cell-to-cell spread and tissue pathology.

Experimental infections in Ethiopian goats with both lineage III and lineage IV strains demonstrated that lineage classification alone does not predict clinical outcome, as one lineage IV isolate (38920/19) produced significantly milder disease and failed to induce seroconversion [5]. This finding strongly implicates strain-specific polymorphisms, rather than lineage-level determinants, in virulence modulation. The F protein of this attenuated isolate warrants detailed structural analysis to identify the molecular basis of its reduced pathogenicity.

The Large Polymerase and Replication Fidelity

The L protein, which harbors the RNA-dependent RNA polymerase (RdRp) activity, is a major determinant of replication efficiency and mutation rate. Five positively selected sites have been identified in the L gene of Chinese lineage IV strains, suggesting that replication complex optimization has been a key driver of viral fitness [17]. Higher polymerase fidelity could reduce the accumulation of deleterious mutations, while lower fidelity might accelerate antigenic variation and immune evasion. The lineage-specific differences in evolutionary rates, with lineage IV evolving faster than lineage II, may be partially attributable to L protein variants that alter the mutation spectrum [3, 17].

Codon Usage Bias and Translational Adaptation

Codon usage analysis provides a window into the evolutionary pressures shaping PPRV genomes. Lineage IV strains exhibit codon usage patterns that are uniquely adapted to the translational machinery of their ovine and caprine hosts, with high codon adaptation index values reflecting efficient gene expression [24]. Interestingly, lineage II and lineage IV strains display divergent codon usage biases across all genes, indicative of independent evolutionary trajectories potentially driven by differences in host species distribution or tissue microenvironments [3].

One of the most striking features of PPRV codon usage is the weak selection for CpG dinucleotide-containing codons, a pattern common among RNA viruses that may represent a strategy to evade host innate immune recognition via Toll-like receptor 9 (TLR9) or zinc-finger antiviral protein (ZAP) [24]. The degree of CpG suppression varies subtly between lineages, potentially contributing to differences in interferon induction and the magnitude of the host inflammatory response.

Nucleocapsid Protein and Immune Evasion

The N protein, while primarily structural, also functions as a potent interferon antagonist by sequestering the host protein MDA5 and inhibiting the interferon-β promoter activation. Although the N gene is generally conserved, lineage-specific polymorphisms have been documented. In Turkey, lineage IV isolates obtained from aborted fetuses exhibited five new amino acid substitutions in the N protein compared to reference strains, suggesting that N protein evolution may be associated with enhanced immune evasion in reproductive tissues [38]. Similarly, N gene sequences from Nigerian lineage IV isolates show distinct sub-clusters (IV-NigA and IV-NigB) that may reflect adaptation to local host populations [11, 27].

The Phosphoprotein and Matrix Protein Contributions

The P protein is a cofactor for the L polymerase and also functions in immune evasion through inhibition of STAT1/STAT2 signaling. The development of a pan-lineage RT-qPCR assay targeting the P gene demonstrated that this region is sufficiently conserved across lineages I–IV for universal detection, yet lineage-specific polymorphisms exist that could affect diagnostic sensitivity [2]. The M protein, which orchestrates viral assembly and budding, contains a 5' untranslated region (UTR) where a base insertion and deletion were identified in a Chinese wildlife isolate (ChinaTibet2024), suggesting that non-coding regulatory elements may also contribute to replication kinetics and host adaptation [39].

Non-Coding Regions and Regulatory Evolution

The 3' leader and 5' trailer sequences, along with intergenic regions and UTRs, contain critical cis-acting elements for transcription, replication, and packaging. The identification of five anchor single-nucleotide variations (SNVs) that define genetic clusters of Chinese PPRV, located in the 3' leader, F gene 5' UTR, H cds, and L cds, highlights the importance of non-coding and regulatory sequences in lineage evolution [17]. These SNVs may alter RNA secondary structures, ribosome binding, or transcription termination signals, thereby modulating gene expression ratios and ultimately virulence.

Experimental Pathogenesis and the Challenge of Lineage-Virulence Correlation

The crucial question of whether lineage classification predicts virulence has been addressed by controlled experimental infections. Comparative studies in Ethiopian indigenous goats using four lineage IV and two lineage III strains revealed no substantial lineage-specific differences in clinical scores, lesion severity, or virus shedding [5]. This finding is paradigm-shifting: it implies that virulence is determined by the constellation of mutations within a strain rather than its lineage designation. The presence of one attenuated lineage IV strain (38920/19) among the tested isolates underscores the importance of ongoing genomic surveillance to identify marker mutations associated with reduced virulence [5].

Conversely, epidemiological data suggest that lineage IV possesses a competitive advantage in transmission and adaptability, as evidenced by its progressive displacement of lineages I, II, and III across West Africa, East Africa, and the Middle East [3, 9, 15, 22, 30]. This paradox, equivalent intrinsic virulence but superior transmission, may be resolved by considering factors such as higher viral loads in nasal secretions, prolonged shedding duration, or enhanced environmental stability of lineage IV strains. The H protein's role in receptor binding and epithelial cell entry likely influences transmissibility independent of lethality. Furthermore, the ability of lineage IV to infect and replicate in atypical hosts such as cattle (even without onward transmission) may facilitate geographic spread through livestock marketing systems [37, 43, 44].

Host Factors and Pathogenesis Modifiers

The molecular pathogenesis of PPRV is modulated by host genetic factors, as illustrated by consistent epidemiological observations that goats are more susceptible than sheep, with higher morbidity and mortality rates [36, 41, 45, 47]. Within goats, Black Bengal goats exhibit higher susceptibility than Jamunapari breeds [40, 46], suggesting that host genetic background influences viral replication efficiency and immune response. The availability and polymorphism of host entry receptors (SLAM and nectin-4) may vary between breeds and species, contributing to differential tissue tropism and disease severity.

Cattle, while susceptible to PPRV infection, are considered dead-end hosts that seroconvert without transmitting the virus to co-housed goats [37, 43, 44]. However, the possibility of emergent PPRV strains with enhanced virulence for cattle remains a concern, as even a single amino acid change in the H protein could alter receptor specificity [43]. The recent detection of lineage IV in European livestock populations, coupled with the genomic analysis suggesting a North African origin, highlights the potential for viral evolution to overcome host barriers [6, 8].

Implications for Eradication and Vaccine Design

The live attenuated vaccine strain Nigeria 75/1 (lineage II) and the Indian Sungri 96 strain (lineage IV) both confer lifelong immunity against all lineages, confirming that neutralization epitopes are conserved across the serotype [14, 18, 21]. Nonetheless, the genetic diversity among lineages raises questions about the durability of vaccine-induced immunity if antigenic drift continues. The identification of positively selected sites in the H gene, a major target of neutralizing antibodies, warrants continuous monitoring of vaccine strain efficacy against emerging lineage IV sub-clades [17].

The thermolability of current vaccines remains a critical challenge for eradication in hot-climate regions, and ongoing efforts to develop thermotolerant formulations using stabilizers such as trehalose and lactalbumin-sucrose are essential to ensure vaccine potency in remote pastoral areas [18, 42]. The correlation between lineage-specific genomic features and vaccine stability has not been systematically explored but may inform the selection of master seed strains for next-generation vaccines.

In summary, the molecular pathogenesis of PPRV lineage-specific virulence is a multifactorial phenomenon governed by the interaction of viral proteins, particularly H, F, L, and N, with host receptors and immune defenses, modulated by codon usage adaptation, non-coding regulatory elements, and selection pressures that differ markedly between lineages. The global dominance of lineage IV appears to result from enhanced evolutionary flexibility and transmission fitness rather than intrinsically greater virulence, a distinction that critically informs targeted surveillance, vaccine deployment strategies, and the global 2030 eradication campaign coordinated by the Food and Agriculture Organization (FAO) and the World Organisation for Animal Health (WOAH) [14].

Global Epidemiology of PPRV Lineages: Distribution, Transmission, and Emerging Threats

The global epidemiological landscape of Peste des Petits Ruminants virus (PPRV) is characterized by a dynamic and complex interplay of four genetically distinct lineages (I, II, III, and IV), whose distribution, transmission dynamics, and evolutionary trajectories present both challenges and opportunities for the FAO and WOAH-led Global Strategy for the Control and Eradication of PPR (PPR GCES) by 2030 [7, 14]. While the virus exists as a single serotype, the genetic diversity encapsulated within these lineages reflects distinct geographical origins, historical patterns of spread, and, critically, ongoing shifts in dominance that are redefining the global threat landscape [3, 29]. Understanding the nuanced epidemiology of each lineage, their transmission pathways, and the emergence of new threats is paramount for designing targeted, effective, and adaptive control strategies.

Historical Distribution and the Shifting Lineage Paradigm

Historically, the four PPRV lineages were considered to have relatively discrete geographical boundaries. Lineages I, II, and III were predominantly found in Africa, while Lineage IV was considered the "Asian lineage" [12, 26]. However, this paradigm has been fundamentally overturned by the remarkable expansion of Lineage IV, which has now achieved a near-global distribution, becoming the dominant and most widely reported lineage across Africa, the Middle East, Asia, and, most recently, Europe [4, 6, 15, 17].

Lineage I, once thought to be potentially extinct, has demonstrated a surprising persistence in West Africa. A seminal study by Tounkara et al. (2021) identified the continued circulation of Lineage I in Mali as recently as 2014, specifically in the Segou and Sikasso regions [30]. This finding challenges the assumption of its disappearance and underscores the critical need for sustained surveillance in remote areas where viral populations may persist undetected. The persistence of this historical lineage alongside the encroaching Lineage IV creates a unique epidemiological scenario in West Africa, where co-circulation and potential recombination events could occur [30].

Lineage II remains the dominant and most widely distributed lineage in West Africa, with Nigeria serving as a major epicenter of its genetic diversity [11, 19, 27]. The Nigeria 75/1 strain, a Lineage II isolate, is the most widely used live-attenuated vaccine strain globally, a fact that has profound epidemiological implications [1, 18]. The vaccine strain itself can be detected in the field, complicating molecular epidemiological surveillance efforts that rely on N-gene sequencing, as it can be misidentified as a circulating wild-type Lineage II virus [11]. Recent studies have revealed a high degree of genetic heterogeneity within Lineage II in Nigeria, with distinct sub-clusters (II-NigA, II-NigB) identified, some closely related to the vaccine strain and others well-separated, suggesting a long and complex evolutionary history in the region [11, 27]. In the Democratic Republic of the Congo (DRC), Lineage II was identified alongside Lineage III, indicating multiple introductions and co-circulation of lineages in Central Africa [13, 35].

Lineage III has a more restricted distribution, primarily centered in East Africa and the Arabian Peninsula. It has been historically reported in Ethiopia, Sudan, and Yemen [3, 5, 25]. However, recent molecular epidemiological data from Ethiopia indicates a significant shift. While Lineage III was previously identified, studies from 2010-2017 have exclusively detected Lineage IV, suggesting that Lineage IV may be outcompeting and displacing Lineage III in certain East African ecosystems [25]. This displacement is not absolute, as evidenced by its continued presence in the DRC [13]. The evolutionary origins of Lineage III are less resolved than other lineages, with phylogeographic analyses suggesting emergence in either East Africa or the Arabian Peninsula [29].

Lineage IV is the undisputed global protagonist in the modern epidemiology of PPRV. Originally described in Asia, its expansion into Africa represents one of the most significant events in the recent history of the virus. The first detections in Africa occurred in Nigeria around 2010, and since then, it has spread relentlessly westward and southward [27, 30]. It has now been confirmed in a vast arc of countries, including Burkina Faso, Côte d’Ivoire, Ghana, Guinea, Senegal, Mali, and the DRC, where it is increasingly reported to be replacing endemic Lineages I and II [9, 13, 15, 22, 30, 35]. In West Africa, this replacement is not uniform; studies in Burkina Faso and Nigeria show a clear trend of Lineage IV becoming predominant, while Lineage II persists [9, 27]. The competitive advantage of Lineage IV is a subject of intense investigation, with hypotheses ranging from higher viral fitness and transmissibility to potential differences in host range or immune evasion [4, 15]. In Asia, Lineage IV is the exclusive circulating lineage in major endemic countries such as China, India, Pakistan, Bangladesh, and the United Arab Emirates [16, 17, 20, 32, 33, 39, 49, 50, 52, 55, 58]. The recent incursion of Lineage IV into Europe, with outbreaks in Bulgaria (2018), and then a major multi-country emergence in Greece, Romania, and Bulgaria in 2024, marks a critical juncture [6, 8, 34]. Genomic analyses of the 2024 European outbreaks point to a common origin, likely from North Africa, highlighting the constant threat of transcontinental spread via animal movement and trade [6, 8].

Transmission Pathways and Drivers of Spread

The transmission of PPRV is driven by a complex interplay of viral factors, host susceptibility, and, most critically, anthropogenic activities. The primary mode of transmission is direct contact between infected and susceptible animals via aerosol or droplet infection, with the virus being shed in high concentrations in ocular, nasal, and oral secretions, as well as feces and urine [26, 37, 56]. The virus can also be transmitted indirectly through contaminated feed, water, and fomites, although its environmental persistence is limited.

Animal Movement and Trade: The single most important driver of long-distance and transboundary spread is the movement of live animals. This is particularly evident in regions with extensive pastoral and transhumant production systems, such as the Sahel and the Horn of Africa. Studies in West Africa have demonstrated that small ruminant mobility networks, particularly those centered around livestock markets, are the primary conduits for PPRV dissemination [54]. The introduction of Lineage IV into Senegal and its rapid spread within Mali are strongly linked to transhumant herder movements across borders [22, 30]. Similarly, the incursion of PPRV into Europe in 2024 is hypothesized to have originated from the movement of infected animals or contaminated products from North Africa [6, 8]. In Asia, the role of animal movement is equally critical. In Bangladesh, road length was found to be a significant risk factor for PPR incidence, directly correlating with the ease of animal transport and trade [58]. The introduction of PPRV into China in 2013, which led to a massive nationwide outbreak, was traced to a common ancestor with a strain from the UAE, likely introduced through the importation of infected animals [17]. The Trans-Himalayan region, with its complex cross-border livestock corridors between India, Nepal, Bhutan, Bangladesh, and China, represents a high-risk zone for transboundary transmission [57].

Wildlife as a Reservoir and Sentinel: The role of wildlife in PPRV epidemiology is an area of growing concern. While sheep and goats are the primary hosts, PPRV has a broad host range within the order Artiodactyla, infecting over 30 species of wild ungulates [16, 39, 51, 53]. In some cases, wildlife can act as a spillover host, as seen in the 2024 outbreak in China where bharals and argali were infected with a Lineage IV strain closely related to viruses circulating in domestic animals from 2013-2014 [39]. More alarmingly, there is evidence that wildlife can serve as a maintenance host, potentially creating a sylvatic cycle that complicates eradication efforts. The outbreak in captive cervids in Assam, India, was linked to spillover from domestic goats used as meat for carnivores, highlighting a direct transmission pathway from domestic to captive wild populations [53]. In East Africa, seropositivity has been documented in a wide range of wildlife, including buffalo, Grant's gazelle, wildebeest, and impala, although their role in onward transmission to domestic animals is considered negligible in most current models [43, 51]. However, the potential for a wildlife reservoir to sustain the virus in the face of successful domestic animal vaccination campaigns remains a significant, unresolved threat.

Atypical Hosts and Subclinical Shedding: The role of atypical hosts, particularly cattle, in PPRV transmission has been a subject of considerable debate. Early studies suggested that cattle could become infected and seroconvert without showing clinical signs, raising the possibility that they could act as silent carriers [37, 44]. However, more recent and comprehensive experimental and modeling studies have provided strong evidence that cattle are dead-end hosts. Herzog et al. (2024) demonstrated that intranasally infected Zebu cattle did not transmit the virus to co-housed goats, even under high-density, zero-grazing conditions [43]. Similarly, Couacy-Hymann et al. (2019) found that cattle infected with all four PPRV lineages failed to infect in-contact goats [44]. These findings strongly support the current strategy of focusing vaccination efforts on small ruminants for eradication. Nevertheless, the authors caution that the emergence of PPRV strains with increased virulence for cattle could alter this dynamic, necessitating continued surveillance [43]. Another critical aspect of transmission is subclinical and pre-clinical shedding. Experimental infections in goats have shown that viral shedding in nasal and ocular secretions begins as early as 4 days post-infection, before the onset of severe clinical signs, and can persist for weeks even after seroconversion [56]. This period of asymptomatic shedding is a major challenge for outbreak control, as apparently healthy animals can be actively spreading the virus.

Emerging Threats and Evolutionary Dynamics

The global epidemiology of PPRV is not static; it is shaped by ongoing evolutionary processes that generate new threats. The most significant emerging threat is the continued expansion and dominance of Lineage IV. Its ability to displace other lineages in West and East Africa suggests a potential selective advantage, possibly related to higher replication rates, enhanced transmissibility, or a broader host range [4, 15, 25]. The genetic characterization of Lineage IV has revealed a high degree of internal diversity, with multiple clades and sub-clades circulating in different regions. In China, for example, PPRV from 2013-2024 has evolved into four distinct genetic clusters, defined by specific anchor mutations in the 3' leader, F gene UTR, H gene, and L gene [17]. This ongoing diversification within Lineage IV could lead to the emergence of strains with altered antigenicity, virulence, or host tropism.

Evolutionary Pressures and Codon Usage: Comparative genomic analyses have revealed that different PPRV lineages are subject to distinct evolutionary pressures. Courcelle et al. (2024) demonstrated that Lineages II and IV have evolved under different selection pressures, with differences in codon usage and adaptive selection observed across all viral genes [3]. This suggests that the two lineages are adapting to their respective ecological niches in ways that may influence their fitness and transmissibility. The H and L genes, in particular, have been identified as being under positive selection in Lineage IV strains circulating in China, indicating that these surface and replicase proteins are key targets of host immune pressure and are driving viral evolution [17]. The synonymous codon usage patterns of PPRV also reflect a balance between mutational pressure and natural selection, with the virus showing high adaptation to its host's codon usage to ensure efficient replication, while also selecting for rare codons that may help evade host restriction factors [24].

The Threat of Re-Emergence in Naïve Regions: The 2024 PPR outbreaks in Greece, Romania, and Bulgaria represent a major threat to the European Union's PPR-free status [6, 8, 34]. These outbreaks, caused by a Lineage IV strain closely related to North-East African viruses, underscore the constant risk of introduction from endemic regions [8]. The rapid spread across multiple countries within weeks highlights the vulnerability of naïve populations and the challenges of cross-border coordination in implementing control measures, including culling and movement restrictions [34]. The detection of PPRV in wildlife in China in 2024, after a period of relative control in domestic animals, also signals a potential threat of re-emergence from a sylvatic reservoir [39]. This event underscores the critical need for integrated surveillance that includes both domestic and wild animal populations.

Abortion as an Emerging Clinical Manifestation: A growing body of evidence suggests that PPRV may play a significant role in reproductive disorders, particularly abortion, in sheep and goats. Studies in Turkey have detected PPRV RNA in a substantial proportion of aborted fetuses (11.7%), with phylogenetic analysis confirming the involvement of Lineage IV [38, 59]. This finding has profound implications for PPR control, as abortion storms can go unnoticed or be misattributed to other pathogens, leading to underreporting and unrecognized viral circulation. The ability of PPRV to cross the placental barrier and cause fetal infection represents a previously underappreciated transmission pathway and a potential mechanism for viral persistence within a flock.

In conclusion, the global epidemiology of PPRV is defined by the relentless expansion of Lineage IV, which is actively displacing other lineages and establishing itself as the dominant genetic group across Africa, Asia, the Middle East, and now Europe. This shift is driven by a combination of viral evolutionary dynamics, extensive animal movement networks, and the potential for spillover into wildlife. The emergence of new threats, including the potential for wildlife reservoirs, the role of PPRV in abortion, and the constant risk of incursion into PPR-free regions, demands a highly adaptive and genomically-informed surveillance strategy. The success of the global eradication program hinges on our ability to monitor these evolutionary and epidemiological shifts in real-time and to tailor control measures to the specific challenges posed by the dominant circulating lineages.

Advanced Molecular Diagnostics for PPRV Lineage Identification: Real-Time RT-PCR and Melting Curve Analysis

The global initiative to eradicate Peste des Petits Ruminants (PPR) by 2030, spearheaded by the Food and Agriculture Organization (FAO) and the World Organisation for Animal Health (WOAH), hinges critically upon the availability of robust, high-throughput diagnostic tools capable of not only detecting the virus but also discriminating between its four distinct genetic lineages (I–IV) [7, 14]. While conventional RT-PCR and sequencing remain the gold standard for lineage confirmation, they are labor-intensive, time-consuming, and ill-suited for large-scale surveillance programs in resource-limited settings. The advent of advanced molecular diagnostics, particularly real-time quantitative RT-PCR (RT-qPCR) coupled with melting curve analysis (MCA), represents a paradigm shift in PPRV lineage identification. These techniques offer the dual advantages of rapid detection and simultaneous lineage differentiation within a single reaction, providing an unprecedented level of epidemiological resolution that is essential for tracking viral spread, monitoring vaccine efficacy, and informing targeted control strategies [1, 4].

The Principle of Melting Curve Analysis for Lineage Discrimination

The foundational principle underlying MCA-based lineage identification exploits sequence-specific variations in the amplicon's thermal denaturation profile. In an SYBR Green I-based RT-qPCR assay, the fluorescent dye intercalates non-specifically into double-stranded DNA. As the amplicon is gradually heated, the double-stranded DNA denatures, releasing the dye and causing a precipitous drop in fluorescence. The temperature at which 50% of the DNA is denatured, the melting temperature (Tm), is a function of the amplicon's length, GC content, and, most critically, its nucleotide sequence [1]. Tang et al. (2023) masterfully demonstrated this principle by designing primers targeting the highly conserved L gene of PPRV, which yielded amplicons of identical length for both Lineage II and Lineage IV. However, subtle but consistent nucleotide polymorphisms between the two lineages within this amplicon resulted in statistically significant and reproducible differences in their respective Tm values [1]. This allowed for unambiguous differentiation of the two lineages in a single, closed-tube reaction without the need for post-amplification processing, such as gel electrophoresis or sequencing. The elegance of this approach lies in its simplicity and speed; the entire process, from RNA extraction to lineage assignment, can be completed in under two hours, making it an invaluable tool for real-time outbreak response.

Analytical Performance: Sensitivity, Specificity, and Reproducibility

The clinical utility of any diagnostic assay is defined by its analytical performance characteristics. The MCA-based RT-qPCR assay developed by Tang et al. (2023) demonstrated exceptional sensitivity, with a detection limit of 100 copies of plasmid DNA per reaction for both Lineage II and Lineage IV [1]. This level of sensitivity is comparable to, and in some cases exceeds, that of conventional TaqMan-based RT-qPCR assays targeting the N or F genes [2, 60]. The specificity of the assay was equally impressive, showing no cross-reactivity with other common viral pathogens of small ruminants, including orf virus, goat poxvirus, and foot-and-mouth disease virus [1]. This is a critical feature, as co-infections are common in endemic regions, and a false-positive result could trigger unnecessary and costly control measures. Furthermore, the assay exhibited high reproducibility, with low intra- and inter-assay coefficients of variation, ensuring consistent performance across different runs and operators [1]. This robustness is paramount for deployment in decentralized laboratories where technical expertise may vary.

Comparative Advantages Over TaqMan-Based Approaches

While TaqMan probe-based RT-qPCR assays are widely regarded as the "gold standard" for quantitative viral detection due to their high specificity and multiplexing capability, they possess inherent limitations for lineage identification. A TaqMan assay is designed to detect a specific, conserved sequence within the viral genome, and while it can be exquisitely sensitive for pan-lineage detection, it cannot, by itself, distinguish between lineages [2, 4]. To achieve lineage discrimination with TaqMan chemistry, one would need to design separate, lineage-specific probes, each labeled with a different fluorophore. This approach is technically challenging, expensive, and limited by the number of available fluorescent channels on a given real-time PCR platform. In contrast, the MCA-based approach using SYBR Green I is inherently lineage-discriminatory, as it relies on the intrinsic sequence variation of the amplicon rather than the binding of a specific probe [1]. This makes it a more cost-effective and technically simpler solution for lineage identification, particularly in settings where high-throughput, multi-fluorophore detection systems are not available. However, it is crucial to acknowledge that the MCA approach is semi-quantitative at best, as SYBR Green I binding can be affected by amplicon length and secondary structure. For applications requiring absolute quantification, such as viral load determination, a TaqMan-based assay remains the preferred method [2, 4, 60].

Strategic Implications for PPR Eradication and Surveillance

The ability to rapidly and accurately identify PPRV lineages has profound implications for the global eradication campaign. The current live attenuated vaccine, Nigeria 75/1, belongs to Lineage II [1, 18]. In regions where Lineage IV is the dominant circulating strain, such as China, much of Asia, and increasingly West Africa, the ability to differentiate vaccine-induced antibodies from natural infection is a major challenge [1, 9, 15]. The MCA-based RT-qPCR assay directly addresses this by enabling the detection and lineage assignment of viral RNA in clinical samples. A positive result for Lineage IV in a vaccinated animal indicates a breakthrough infection, signaling either vaccine failure, waning immunity, or the emergence of a more virulent strain. Conversely, detection of Lineage II in a vaccinated animal could indicate a vaccine-derived infection, although this is rare with the attenuated strain. This capability is the cornerstone of a robust DIVA (Differentiating Infected from Vaccinated Animals) strategy, which is essential for monitoring the progress of vaccination campaigns and certifying freedom from infection in the final stages of eradication [1, 14].

Furthermore, the epidemiological data generated by lineage-specific diagnostics are invaluable for understanding viral transmission dynamics. The recent incursion of Lineage IV into Europe, with outbreaks in Greece, Romania, and Bulgaria in 2024, underscores the need for rapid, field-deployable tools to track the transboundary spread of the virus [6, 8, 34]. The MCA-based assay can provide near real-time data on the lineage of the inciting strain, allowing veterinary authorities to quickly determine the likely origin of the outbreak and implement targeted movement restrictions and vaccination strategies. This is particularly critical in regions where multiple lineages co-circulate, such as in West Africa, where Lineage II and Lineage IV are both present, and in the Democratic Republic of the Congo, where Lineages II, III, and IV have been identified [9, 13, 15, 19, 27]. The ability to rapidly distinguish between these lineages is not merely an academic exercise; it is a practical necessity for effective disease control.

Future Directions and Integration with Emerging Technologies

While the MCA-based RT-qPCR assay represents a significant advancement, the field of PPRV diagnostics is rapidly evolving. The integration of this technology with portable, battery-operated real-time PCR platforms could enable true pen-side lineage identification, eliminating the need to transport samples to centralized laboratories [62]. Additionally, the principles of MCA could be adapted for use with isothermal amplification techniques, such as recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP), which are even more suited for field deployment due to their minimal equipment requirements [10, 61]. The combination of RPA with CRISPR-Cas12a systems has already shown promise for rapid, visual detection of PPRV, and incorporating lineage-specific guide RNAs could potentially allow for lineage discrimination without the need for thermal cycling [10]. The future of PPRV diagnostics lies in the convergence of high analytical performance with extreme portability and ease of use. The MCA-based RT-qPCR assay, with its proven ability to deliver lineage-level resolution in a single reaction, provides a robust and scalable platform upon which these next-generation diagnostic tools can be built, ultimately accelerating the world towards the goal of PPR eradication.

Differentiation of Vaccinated and Naturally Infected Animals: Lineage II Vaccine Strain versus Field Strains

The global initiative to eradicate Peste des Petits Ruminants (PPR) by 2030, spearheaded by the Food and Agriculture Organization (FAO) and the World Organisation for Animal Health (WOAH), hinges on the strategic deployment of effective vaccines and the implementation of robust surveillance systems [7, 14]. A critical, non-negotiable component of this strategy is the ability to unequivocally differentiate between animals that have been vaccinated and those that have been naturally infected with field strains of the PPR virus (PPRV). This capability, often referred to as DIVA (Differentiating Infected from Vaccinated Animals), is paramount for monitoring herd immunity, tracking the circulation of wild-type virus, and ultimately certifying freedom from infection in the final stages of eradication [1, 14]. The challenge is uniquely defined by the fact that the most widely used and internationally recommended vaccine strain, Nigeria/75/1, belongs to Lineage II, while the vast majority of contemporary field strains circulating in Asia, the Middle East, and increasingly in Africa belong to Lineage IV [1, 9, 15]. This lineage-specific dichotomy forms the biological and diagnostic foundation for the differentiation strategies currently under development and deployment.

The Biological Basis for Differentiation: A Lineage II Vaccine in a Lineage IV World

The cornerstone of the DIVA strategy for PPR rests on the phylogenetic and genomic divergence between the vaccine strain and the predominant field strains. The live attenuated PPRV vaccine, derived from the Nigeria/75/1 strain, is a member of Lineage II [1, 23]. This strain, isolated in Nigeria in 1975, has been extensively passaged in cell culture to attenuate its virulence while retaining its immunogenicity. It provides lifelong protection against all known lineages of PPRV, a critical feature for a single-serotype virus [18, 21]. However, the epidemiological landscape has shifted dramatically. While Lineage II strains were historically endemic in West Africa, they have been progressively displaced by the more recently emerged and highly successful Lineage IV [9, 15, 30]. Lineage IV, which originated in Asia, has demonstrated a remarkable capacity for transboundary spread, becoming the dominant lineage across Asia, the Middle East, and now large swathes of Africa, including West Africa where it is actively replacing Lineage II [9, 15, 22, 27]. This ecological replacement means that in most PPR-endemic regions, a positive test for Lineage II-specific antibodies or nucleic acid is far more likely to be a marker of vaccination than of natural infection with a circulating field strain.

The genetic distance between Lineage II and Lineage IV is substantial and well-documented. Comparative genomic analyses have revealed that these lineages have evolved under different selection pressures, exhibiting differences in codon usage and adaptive selection across all viral genes [3]. For instance, the genomes of the Nigeria/75/1 vaccine strain and the Russian vaccine strain "45G37/35-k," both Lineage II, are separated by 248 nucleotide differences, highlighting the intra-lineage variation that exists [23]. The divergence between Lineage II and Lineage IV is even more pronounced, providing a rich source of lineage-specific genetic markers. This divergence is not merely a matter of academic interest; it is the fundamental principle exploited by molecular diagnostic assays designed to discriminate between the two. The nucleoprotein (N), fusion (F), and hemagglutinin (H) genes, while conserved enough for pan-lineage detection, contain sufficient sequence variability to allow for the design of lineage-specific primers and probes [1, 4, 28].

Molecular Strategies for Differentiation: RT-qPCR and Melting Curve Analysis

The most direct and sensitive approach for differentiating vaccinated from naturally infected animals is the detection of the viral genome itself. Since the vaccine is a live attenuated virus, it can be detected in vaccinated animals for a short period post-vaccination. However, the primary utility of molecular differentiation lies in distinguishing the origin of viral RNA in clinical samples, particularly during outbreak investigations where vaccine virus might be shed or where co-circulation of vaccine-like and field strains is suspected.

A landmark advancement in this area is the development of a SYBR Green I real-time quantitative RT-PCR (RT-qPCR) assay that exploits melting curve analysis (MCA) to differentiate between Lineage II and Lineage IV PPRV in a single reaction [1]. This method, targeting the L gene, is exquisitely specific, showing no cross-reactivity with other common caprine and ovine viruses such as orf virus, goat poxvirus, or foot-and-mouth disease virus [1]. The assay's power lies in its ability to discriminate based on the distinct melting temperatures (Tm) of the amplicons generated from each lineage. The differential nucleotide composition of the L gene between Lineage II (vaccine) and Lineage IV (field) strains results in PCR products with unique thermal denaturation profiles. This allows for the simultaneous detection and identification of the lineage in a single, closed-tube reaction, a significant advantage for high-throughput surveillance [1]. The sensitivity of this assay, with a detection limit of 100 copies of the target plasmid, is sufficient for detecting viral RNA in clinical samples, making it a practical tool for field application.

Complementing this approach are highly sensitive and specific TaqMan-based RT-qPCR assays. While some assays are designed for pan-lineage detection, targeting conserved regions of the phosphoprotein (P) or F genes to ensure all four lineages are captured [2, 28], others are explicitly designed for lineage-specific detection. For instance, a TaqMan RT-qPCR assay targeting the hemagglutinin (H) gene has been developed specifically for the detection of Lineage IV PPRV [4]. This assay demonstrates no cross-reaction with other PPRV lineages, meaning it will not detect the Lineage II vaccine strain [4]. The strategic deployment of such assays is critical. In a region where Lineage IV is the only circulating field strain, a positive result from a pan-lineage RT-qPCR [2] coupled with a negative result from a Lineage IV-specific RT-qPCR [4] would strongly suggest the presence of the Lineage II vaccine strain. Conversely, a positive result from both assays would confirm a Lineage IV field infection. This algorithmic approach to molecular diagnosis provides a robust framework for DIVA.

Serological Challenges and the Quest for a True DIVA Vaccine

While molecular assays can differentiate the virus itself, serological surveillance, the detection of antibodies, presents a more complex challenge. The current live attenuated vaccines, including Nigeria/75/1, induce a humoral immune response that is indistinguishable from that generated by natural infection when using standard serological tests like the competitive ELISA (c-ELISA) or virus neutralization test (VNT) [31, 65]. These tests detect antibodies against the entire virus particle or its major structural proteins (e.g., N, H, F), which are highly conserved across all lineages. Consequently, a seropositive animal could be a vaccinated animal, a convalescent animal that survived a natural infection, or an animal that has experienced a subclinical infection [45, 63]. This inability to differentiate at the serological level is a major impediment to eradication, as it prevents the accurate estimation of true field virus circulation in vaccinated populations.

The ultimate solution is the development of a DIVA vaccine, which is designed to elicit an immune response that is distinct from that of a natural infection. This is typically achieved by engineering a vaccine that lacks one or more specific viral proteins (a "marker" vaccine). Companion diagnostic tests are then developed to detect antibodies against the missing protein(s). Animals vaccinated with the marker vaccine will be seronegative for the missing protein, while naturally infected animals will be seropositive. For PPR, significant research and development efforts are underway to create such vaccines, including recombinant vectored vaccines and subunit vaccines [14, 50, 64]. For example, recombinant capripoxviruses expressing the F protein of PPRV have been constructed, which could serve as a DIVA vaccine if the accompanying diagnostic test targets a PPRV protein not present in the construct [64]. These next-generation vaccines hold the promise of overcoming the primary limitation of the current, otherwise excellent, Nigeria/75/1 strain. However, they are not yet widely deployed, and the global eradication campaign currently relies on the existing live attenuated vaccines.

Practical Implications for Surveillance and Eradication

In the absence of a widely available DIVA vaccine, the differentiation strategy relies on a combination of molecular diagnostics, epidemiological context, and strategic sampling. The molecular tools described above are invaluable for outbreak investigations. When a PPR outbreak occurs in a vaccinated population, the MCA-based RT-qPCR [1] or a combination of pan-lineage and Lineage IV-specific RT-qPCRs [2, 4] can be used to analyze samples from affected animals. If the detected virus is identified as Lineage IV, it confirms a vaccine-breakthrough infection or, more likely, a failure in vaccine coverage or efficacy due to cold chain issues [18, 42]. If the virus is identified as Lineage II, it could indicate recent vaccination, though this is less likely in a clinical outbreak scenario. This information is critical for veterinary authorities to make informed decisions about outbreak response, including ring vaccination and movement restrictions.

Furthermore, the molecular epidemiological data generated by these assays feeds directly into the global surveillance framework. The progressive replacement of Lineage II by Lineage IV in West Africa, as documented in Burkina Faso, Ghana, and Senegal [9, 15, 22], underscores the importance of lineage-level surveillance. The ability to track this shift is entirely dependent on the differentiation tools available. The WOAH and FAO recommend that PPR control programs incorporate molecular characterization of circulating strains to monitor the effectiveness of vaccination campaigns and to detect the emergence of new variants [7, 14]. The use of these advanced RT-qPCR assays, therefore, is not merely a diagnostic exercise; it is a strategic imperative for the global eradication effort, providing the granular data needed to adapt control strategies in real-time and to provide the evidence base for declaring freedom from PPR in the post-eradication era.

Implications for PPR Eradication: Lineage-Based Surveillance and Control Strategies

The global initiative to eradicate Peste des Petits Ruminants (PPR) by 2030, spearheaded by the Food and Agriculture Organization (FAO) and the World Organisation for Animal Health (WOAH), represents one of the most ambitious veterinary public health undertakings of the 21st century, drawing directly from the successful rinderpest eradication model [7, 14]. However, the complex molecular epidemiology of the four distinct PPRV lineages (I–IV) introduces profound challenges and specific strategic imperatives that must be integrated into every tier of national and regional eradication programs. The simple existence of a single serotype and the availability of an efficacious live-attenuated vaccine are insufficient to guarantee eradication when the underlying viral population structure is dynamically shifting, with lineages exhibiting differential fitness, geographic expansion, and variable evolutionary pressures [3, 29]. Therefore, a lineage-informed approach is not merely an academic exercise but a non-negotiable operational requirement for surveillance design, diagnostic deployment, vaccine matching, and the interpretation of epidemiological data.

The Diagnostic Imperative: Distinguishing Vaccine from Field Strains

A cornerstone of any eradication campaign is the capacity to differentiate between vaccinated animals and those naturally infected (DIVA). The widely used vaccine strain, Nigeria/75/1, belongs to Lineage II [1, 18]. In regions where Lineage IV is the dominant circulating field strain, such as China, much of South Asia, and increasingly West and Central Africa, the ability to discriminate between these lineages is critical for monitoring vaccine efficacy and detecting breakthrough infections [1, 9, 17]. The development of a SYBR Green I real-time qRT-PCR method that differentiates Lineages II and IV within a single reaction via melting curve analysis (MCA) provides a powerful, high-throughput tool for national surveillance programs [1]. This assay, targeting the L gene, allows for the rapid confirmation that an animal’s positive PCR result originates from the vaccine strain (Lineage II) rather than a virulent field virus (Lineage IV), thereby preventing false alarms and avoiding costly stamping-out or quarantine measures [1]. Conversely, the detection of a Lineage IV strain in a recently vaccinated herd signals a failure of vaccine protection, possibly due to cold chain breaks, improper administration, or, theoretically, antigenic drift, thus triggering an immediate operational investigation [17].

Conversely, in regions like West Africa where Lineages I, II, and IV co-circulate, a pan-lineage diagnostic is essential for the initial detection of the virus itself, but it cannot provide the lineage-level data needed to track replacement dynamics [2, 11]. A TaqMan RT-qPCR assay based on the phosphoprotein (P) gene has been validated to detect all four lineages with high sensitivity (4 copies/μL for lineages II-IV), making it an ideal first-line screening tool for sentinel surveillance in previously PPR-free zones or in countries with unknown lineage distributions [2]. The strategic deployment of both pan-lineage and lineage-specific assays, using the P-gene assay for broad detection followed by the H-gene-based assay for Lineage IV confirmation [4] or the MCA assay for II vs. IV discrimination [1], creates a robust diagnostic cascade that is adaptive to the local epidemiological context. Furthermore, the integration of an internal reference gene, such as GAPDH, into these qRT-PCR protocols is vital for quality assurance in field-collected samples that may contain inhibitory substances, ensuring that true negative results are reliable [60].

Epidemiological Surveillance in a Shifting Lineage Landscape

The most alarming epidemiological trend impacting eradication is the rapid and aggressive expansion of Lineage IV. Originally considered an Asian lineage, Lineage IV has now been documented displacing endemic Lineage II in West African nations such as Burkina Faso [9] and Nigeria [27], and has been reported for the first time in Senegal [22] and Mali [30]. This replacement is not a neutral substitution; it represents a potential bottleneck event with significant consequences for eradication. Genomic analyses comparing Lineages II and IV reveal they have evolved under markedly different selection pressures, with differences in codon usage and adaptive selection across all viral genes, suggesting that Lineage IV possesses a competitive advantage in transmission and adaptability [3]. This shift demands a re-evaluation of risk assessment models. Surveillance networks designed based on the historical distribution of Lineage II may miss the early incursion of Lineage IV, which can spread explosively through naive populations [15]. The introduction of Lineage IV into Europe in 2024, confirmed in Greece, Romania, and Bulgaria, with genomic evidence pointing to a common origin from North Africa, underscores the capacity of this lineage to breach continental boundaries and threaten the PPR-free status of entire regions [6, 8, 34].

Consequently, active lineage surveillance must be a core component of the PPR Global Control and Eradication Strategy (PPR GCES). Passive surveillance based only on clinical suspicion is insufficient, as Lineage IV can cause subclinical or milder disease in atypical hosts or in previously exposed populations [5, 37]. As Courcelle et al. (2024) emphasize, the resolution of phylogeographic studies is currently limited by a severe sampling bias towards Lineage IV and recent isolates [3]. To accurately model transmission pathways, identify sources of new introductions, and predict the direction of viral spread, there is an urgent need for the systematic whole-genome sequencing of PPRV from all lineages, particularly historical Lineages I, II, and III, which are critically underrepresented in global databases [3, 29]. This genomic surveillance must be integrated with animal movement data, for example, network analysis of livestock trade routes in Nigeria has identified "sentinel nodes" that are highly vulnerable to early infection and could serve as priority sites for targeted lineage-based surveillance [54].

Strategic Vaccination and the Challenge of Thermotolerance

While the live-attenuated vaccine (Nigeria 75/1, Lineage II) provides cross-protective immunity against all four lineages due to the single serotype nature of PPRV, the practical implementation of vaccination programs is inextricably linked to lineage dynamics [14, 21]. The vaccine is exceptionally effective, conferring lifelong immunity after a single dose, but its thermolability remains a massive logistical hurdle [18, 50]. In the hot climates of Africa and Asia, where cold chain maintenance from a central manufacturer to a remote pastoralist herd is often impossible, the vaccine can degrade rapidly, leading to vaccination failures that undermine herd immunity and permit the continued circulation of field virus [18, 42]. This is particularly critical in areas where Lineage IV is displacing Lineage II, as any vaccine failure in these regions allows a highly fit, expansionist lineage to continue replicating and spreading. Studies from Mali reveal that vaccine wastage can exceed 25%, with the majority of loss occurring at the final delivery point due to denaturation within the short one-hour window post-reconstitution [42]. This wastage is a direct economic and operational drag on eradication efforts.

The development of thermotolerant (ThT) vaccine formulations, which can maintain a minimum protective titre of 10².⁵ TCID₅₀/mL for at least five days at 40°C, is a game-changing innovation for lineage-based eradication strategies [18]. Such ThT vaccines could be pre-positioned for ring vaccination campaigns around outbreaks of virulent Lineage IV in remote areas without requiring a continuous cold chain, enabling a rapid, focused response that could extinguish a nascent wave of transmission before it establishes endemicity [50]. Furthermore, the provenance of the vaccine strain matters. While Nigeria 75/1 (Lineage II) is the WOAH-recommended strain, other vaccine seeds exist, such as the ARRIAH strain (also Lineage II) used in Russia, which has shown a promising protective phenotype against a virulent Lineage IV field strain (Mongolia/2021) in experimental challenges [21]. For eradication to succeed, all vaccine seeds used globally must undergo rigorous genomic characterization and safety profiling to ensure they meet international standards and do not inadvertently introduce genetic variability into the vaccine supply chain [23].

The Roles of Atypical Hosts and Wildlife in Lineage Maintenance

The eradication strategy must explicitly account for the role of atypical hosts and wildlife, as their significance may vary by lineage. While current evidence strongly supports that cattle are dead-end hosts and do not transmit PPRV to co-housed goats, they do seroconvert following infection [43, 44]. This characteristic makes cattle useful as sentinel animals for detecting PPRV circulation in mixed-species herds, acting as an early warning system for virus introduction before small ruminants show clinical signs. This is particularly valuable for detecting the early incursion of a new lineage, such as Lineage IV into a region previously dominated by Lineage II [43].

However, the role of wildlife is far more concerning and lineage-independent in its threat. The detection of Lineage IV PPRV in wild bharals and argali in Tibet in 2024, with a genome closely related to viruses from the 2013-2014 Chinese epidemic, demonstrates that wildlife can serve as a reservoir for PPRV, potentially harboring the virus long after the disease has been controlled in domestic livestock [39]. Similarly, a devastating spillover event in a captive cervid population in India, traced back to infected goat meat used as feed, resulted in the death of 30 endangered animals and confirmed the susceptibility of species in the Cervidae family to Lineage IV [53]. This creates a "wildlife loop": the virus can circulate undetected in wildlife populations, spill back into domestic animals, and frustrate eradication efforts, especially in regions with high biodiversity and limited wildlife surveillance. The eradication strategy must therefore incorporate a One Health approach that extends active surveillance into wild small ruminant populations in PPR-endemic zones, with a specific focus on identifying which lineages are circulating in these sylvatic cycles [39].

Conclusion

The eradication of PPR by 2030 is an audacious but scientifically feasible goal. However, the path to eradication is not linear; it is a dynamic process that must be continuously adapted to the shifting genetic landscape of the virus. A static, one-size-fits-all control strategy is doomed to fail in the face of an expanding, evolutionarily advantaged lineage like Lineage IV. The future of PPR eradication hinges on the seamless integration of lineagingenomic diagnostics with operational field activities. This includes the routine use of lineage-discriminating RT-qPCR assays for DIVA and epidemiological tracking, the strategic deployment of thermotolerant vaccines in hot-spot regions of lineage replacement, and the establishment of a global PPRV genomic surveillance framework that can provide real-time data on viral movement and selection. Without this lineage-based intelligence, the global community risks vaccinating blindly, unable to see the invisible, evolving threat that is undermining every dose administered.

Evolutionary Dynamics and Genetic Diversity of PPRV Lineages

The evolutionary trajectory of the Peste des petits ruminants virus (PPRV) is a complex narrative shaped by host ecology, anthropogenic factors, and intrinsic viral genomic plasticity. As a member of the genus Morbillivirus within the family Paramyxoviridae, PPRV exists as a single serotype but exhibits substantial genetic heterogeneity, which has been historically partitioned into four distinct lineages (I, II, III, and IV) based on phylogenetic analyses of the nucleocapsid (N) and fusion (F) protein genes [3, 26]. This classification, while robust for epidemiological tracking, belies a far more intricate evolutionary landscape characterized by differential selection pressures, shifting phylogeographic dominance, and ongoing lineage replacement events that have profound implications for the Global Strategy for the Control and Eradication of PPR (PPR GCES) led by the Food and Agriculture Organization (FAO) and the World Organisation for Animal Health (WOAH) [7, 14].

Phylogenomic Architecture and the African Cradle of Diversity

The foundational phylogeny of PPRV lineages reveals a deep-rooted African origin for the virus. Comprehensive genomic analyses, including those utilizing full-genome sequencing of historical isolates, have demonstrated that lineages I, II, and IV most likely emerged in West Africa, specifically in Senegal (Lineage I) and Nigeria (Lineages II and IV) [29]. Lineage III, in contrast, is posited to have originated in East Africa or the Arabian Peninsula, with early isolates from Ethiopia, Oman, and the United Arab Emirates forming the basal nodes of this clade [29]. This geographic partitioning suggests that the initial diversification of PPRV occurred in Africa, with subsequent radiation events leading to the establishment of distinct ecological niches.

The evolutionary timescale for this diversification has been refined through molecular clock analyses. Estimates for the time to the most recent common ancestor (tMRCA) place the divergence of modern Lineage II and Lineage IV strains in the 1960s–1980s [3]. This period appears to have been a critical window for the global dissemination and genetic differentiation of PPRV, coinciding with significant socio-economic changes, intensification of livestock trade, and potentially, the waning of rinderpest control efforts, which may have created an ecological vacuum that PPRV exploited [3, 14]. The phylogenetic relationships among historical samples from Lineages I, II, and III, when compared with more recent isolates, point towards a period of high genetic diversity in Africa until the 1970s–1980s, followed by possible bottleneck events that shaped the subsequent evolutionary trajectory of the virus [3]. This pattern is particularly evident in West Africa, where Lineage I, once thought to be extinct, was found to have persisted in Mali at least until 2014, demonstrating the cryptic maintenance of historical diversity even as newer lineages expand [30].

The Ascendancy of Lineage IV: Mechanisms of Competitive Displacement

The most conspicuous evolutionary dynamic in contemporary PPRV epidemiology is the global ascendancy of Lineage IV. Originally considered an Asian lineage, Lineage IV has demonstrated a unique capacity for transboundary spread, progressively displacing Lineages I, II, and III across vast swathes of Africa and the Middle East [4, 9, 15]. This phenomenon is not merely a passive expansion but appears to be driven by a demonstrable competitive advantage. Evidence from West Africa is particularly striking: in Burkina Faso, surveillance data from 2021–2022 indicated that Lineage IV is actively replacing the historically dominant Lineage II [9]. Similarly, in Senegal, the first detection of Lineage IV in 2020 marked a significant shift in the regional viral landscape, reinforcing the hypothesis that animal mobility across borders is a primary driver of this lineage replacement [22]. In Nigeria, a country with immense small ruminant populations, Lineage IV has become widespread, with phylogenetic analyses identifying at least four distinct sub-clusters (IV-NigA, IV-NigB, and others), indicating extensive endemic circulation and local diversification following its introduction [11, 27].

The mechanisms underpinning this competitive displacement are multifaceted. Comparative genomic analyses between Lineage II and Lineage IV have revealed that these lineages have evolved under markedly different selection pressures [3]. Differences in codon usage bias and adaptive selection pressures have been observed across all viral genes, suggesting that Lineage IV may possess a fitness advantage in terms of replication efficiency, host interaction, or transmission dynamics [3, 24]. Specifically, analyses of the hemagglutinin (H) and large polymerase (L) genes have identified sites under positive selection in Lineage IV strains, which could be associated with altered receptor binding affinity or enhanced replicative capacity [17]. Furthermore, the high adaptation of PPRV to its hosts at the codon usage level reflects high viral gene expression, but intriguingly, some synonymous codons that are rare in the host are selected at high frequencies in the viral genome, indicating a complex interplay between mutational pressure and natural selection that may differ between lineages [24]. The ability of Lineage IV to infect and cause disease in a broader range of hosts, including wildlife, may also contribute to its success. The detection of Lineage IV in captive cervids in India and in wild bharals and argali in China underscores its capacity to establish itself in novel ecological niches, creating potential wildlife reservoirs that complicate eradication efforts [39, 53].

Intra-Lineage Diversification and Phylogeographic Structuring

Within the globally dominant Lineage IV, a high degree of genetic substructure has emerged, reflecting distinct epidemiological trajectories in different geographic regions. Phylogenomic analyses of Chinese PPRV strains from 2007 to 2024 have resolved Lineage IV into at least seven distinct clades, demonstrating clear temporal and spatial correlations [17]. Critically, these analyses revealed that PPRV incursions into China in 2007–2008 and again in 2013–2024 originated from two independent introductions, each belonging to a different genetic clade [17]. The 2013–2024 Chinese strains shared a common ancestor with a strain from the United Arab Emirates and subsequently evolved into four distinct genetic clusters within the country, defined by specific anchor mutations, single-nucleotide variations (SNVs) located in the 3′ leader, the 5′ untranslated region (UTR) of the F gene, and the coding sequences of the H and L genes [17]. This level of intra-lineage diversification highlights the capacity for rapid local adaptation and the importance of sustained genomic surveillance.

The phylogeographic structure of Lineage IV in Africa is equally complex. In West and Central Africa, Lineage IV sequences form a sister clade to all other Lineage IV sequences from Asia and the Middle East, suggesting that this lineage also has an African origin and that the current global distribution represents a "back-to-Africa" or, more accurately, a secondary radiation from an ancestral African stock [3]. Within Africa, further sub-structuring is evident. For instance, the Lineage IV strains circulating in Ethiopia belong to a specific sub-clade (sub-clade II of clade I of lineage IV), distinct from those found in West Africa [25]. The recent emergence of PPRV in Europe in 2024, with outbreaks in Greece, Romania, and Bulgaria, has been traced to a common origin in Northern Africa, with the Greek strain specifically clustering within the Lineage IV North-East Africa group [6, 8]. This event underscores the constant threat of transcontinental viral movement, facilitated by animal trade and human-mediated transport, and highlights how genomic epidemiology can pinpoint the source of incursions [6, 34].

Evolutionary Dynamics in the Context of Host Ecology and Vaccination

The evolutionary dynamics of PPRV are inextricably linked to the ecology of its primary hosts, sheep and goats, and the selective pressures imposed by vaccination campaigns. The virus exists as a single serotype, meaning that the existing live attenuated vaccines (e.g., Nigeria 75/1, lineage II; Sungri 96, lineage IV) confer cross-protection against all lineages [18, 21]. This is a critical advantage for the eradication campaign, as it obviates the need for lineage-specific vaccines. However, the evolutionary implications of widespread vaccination are not negligible. While vaccine-induced immunity is robust and long-lasting, incomplete vaccination coverage, a persistent challenge in many endemic regions, can create a scenario where the virus circulates in partially immune populations, potentially selecting for antigenic variants or strains with altered virulence [7, 63, 66].

Experimental studies comparing the virulence of different lineages in indigenous goats have yielded nuanced results. In Ethiopian goats, a comparative study of four Lineage IV and two Lineage III strains found no substantial lineage-specific differences in virulence, with all but one strain producing consistent clinical disease [5]. However, one Lineage IV isolate (38,920/19) produced significantly milder clinical manifestations and no seroconversion, suggesting that strain-level variation, rather than lineage classification per se, may be a more important determinant of clinical outcome [5]. This finding has important implications for disease surveillance, as it suggests that reliance on lineage typing alone may not accurately predict the pathogenic potential of a circulating strain. Furthermore, the role of atypical hosts in PPRV evolution is an area of active investigation. While cattle are considered dead-end hosts that do not transmit the virus, they can seroconvert upon infection [37, 43, 44]. The possibility that PPRV could evolve strains with enhanced virulence for cattle, as has been hypothesized, represents a potential future threat that necessitates continued monitoring of viral evolution in multi-host systems [43].

Codon Usage and Selection Pressures as Drivers of Genetic Diversity

At the molecular level, the evolutionary dynamics of PPRV are governed by a balance between mutational pressure and natural selection, which is reflected in the virus's codon usage patterns. Comprehensive analyses of synonymous codon usage across the six structural genes (N, P, M, F, H, L) have revealed universal, lineage-specific, and gene-specific features that reflect evolutionary plasticity and independence [24]. The overall nucleotide composition of the PPRV genome is biased, with a high frequency of A and U nucleotides, a characteristic common among RNA viruses. This mutational bias is a primary driver of codon usage patterns, but it is counterbalanced by translational selection, which favors codons that match the host's tRNA pool to ensure efficient gene expression [24]. Interestingly, PPRV exhibits a high adaptation to its hosts at the codon usage level, which is indicative of high viral gene expression. However, a paradoxical finding is that some synonymous codons that are rarely used in the host are selected at high frequencies in the viral genome, suggesting that factors beyond translational efficiency, such as RNA secondary structure or evasion of host innate immune responses, may also be at play [24]. A particularly notable feature is the weak tendency for synonymous codons containing CpG dinucleotides to be selected in viral genes. This is a common strategy among RNA viruses to avoid recognition by host zinc-finger antiviral proteins, which target CpG-rich RNA [24]. The interplay between these mutational and selective forces creates a dynamic evolutionary landscape where different lineages can adopt distinct codon usage strategies, potentially contributing to their differential fitness and geographic spread. The observation that PPRV strains isolated in China during 2007–2008 and 2013–2014 displayed independent evolutionary pathways in their codon usage patterns, despite both belonging to Lineage IV, further underscores the capacity for rapid, lineage-specific molecular evolution [24].

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