Phylogenetic analysis of small ruminant morbilli virus in Tamil Nadu: Lineage IV predominance and variability in gene sequences

Introduction

Among the major diseases affecting small ruminants, Peste des petits ruminants (PPR) poses a global threat to subsistence farming with an annual economic loss due to mortality. The national control program for PPR, 2011 (PPR-CP) is therefore a forerunner to the global PPR-Global Eradication Program (PPR-GEP) founded by the Food and Agriculture Organization of the United Nations (FAO) and the World Organisation for Animal Health (WOAH, formerly OIE) in 2017, but work in consensus towards a singular objective of disease eradication by 2030. Nucleotide sequencing of the causative agent, PPRV, has been an effective tool in disease surveillance, one of the core components of PPR-CP. By providing insights into the evolutionary history of the virus, disease emergence, spread and spillover, it has been critical in decisive strategies towards containment. Comparative analysis of partial gene sequences encoding the viral Fusion (F) protein laid the foundation for differentiating PPRV into four lineages [1] . Lineages I & II are African isolates, lineage III is prevalent in Eastern Africa and parts of Arabian Peninsula, whereas lineage IV is dominant across Asia and the Caucasus region [2,3] . The pattern of lineage clustering with defined geographic distribution is consistent with Nucleocapsid (N), Matrix (M), Haemagglutinin (H) as well as the large (L) Polymerase [4] genes of the virus. However, the highly divergent N gene is considered more reliable in the phylogenetic resolution of closely related strains [5][6][7] . India reported its first case of PPR in 1987 from the Villupuram district of Tamil Nadu. Though initially restricted to the southern state, the epidemic of 1994 was followed by periodic outbreaks across the country establishing an endemic status [8] . With the exception of a lineage III virus reported in 1992, all the isolates from India belong to lineage IV. The outbreaks have specific spatiotemporal characteristics reflective of the major routes of disease expansion, animal movement on account of grazing, migration or trade and transhumance. Agro-climatic factors and variation in animal husbandry practices too have an impact on prevalence and transmission. According to a recent analysis of transmission dynamics, western and northern India have a larger risk of outbreaks and cases whereas risk of epidemic mortality is high in southern states [9] . Though focal and nationwide mass vaccination campaigns could significantly curtail disease occurrence, lacunae in effective surveillance contribute to unprecedented episodes across the country [10] . India is in the 'control-stage' (Stage 2) of the PPR-GCES milestones, a concerted engagement of all stakeholders in the small ruminant value chain is imperative to achieving a PPR free status [11] . We have investigated the presence of PPRV is nonvaccinated, symptomatic ruminants from different Agroclimatic zones of Tamil Nadu, the North Eastern, North Western, western, Cauvery delta, southern, high rainfall, and high-altitude zones. The distinction is made on the basis of ecological, geological as well as social features of the area. Nasal swabs from animals exhibiting classical symptoms of PPR infection were analysed for the presence of live virus as well as its genetic material by PCR using N gene primers. Further, genetic characterization of the samples by sequence comparison with isolates from earlier outbreaks across India, as well as other Asian countries available in GenBank database is described. Apart from being a recent survey, a similar investigation of isolates with an expansive geographic distribution within the state of Tamil Nadu has never been undertaken.

Materials and Methods

Sample collection

Nasal samples were collected from eighty eight PPR disease suspected goats and sheep (Figure 1) from different agroclimatic regions of Tamil Nadu state, sampling details are presented in Table 1. None of the animals had a history of vaccination, and were immunologically naïve to the virus. Swabs were transported in 250 µL virus transport medium (VTM, Diagnostic Care Pvt. Ltd.) maintaining proper cold chain to the laboratory and stored in deep freezer (-80℃) until analysis.

RNA isolation & cDNA synthesis

The nasal swabs were processed for extraction of viral nucleic acid by TRI reagent (RNAiso Plus, TaKaRa). RNA concentration and purity were determined using a Nanodrop by calculating the ratio of optical density at 260/280 nm and 260/230 nm. cDNA was synthesised using iScript cDNA synthesis kit (Bio-Rad) using 1 µg of RNA as per the manufacturer's protocol. cDNA was stored at -20 ℃ until further use. The thermal cycling condition was as follows: initial denaturation at 94 ℃ for 5 min, 40 cycles of denaturation, annealing and extension at 94 ℃ for 30 s, 60 ℃ for 30 s and 72 ℃ for 1 min, respectively followed by a final extension at 72 ℃ for 5 min. The PCR products were analysed on a 1% agarose gel with non-carcinogenic nucleic acid stain (MIDORI Green Advance, NIPPON Genetics). The specific amplicon of 352 bp (Figure 2) was excised from agarose gel and extraction was performed using a commercial kit (Qiagen). Extracted PCR products were sequenced by the Sanger's dideoxy chain termination technique (Eurofins Genomics India Pvt. Ltd.).

Polymerase chain reaction & gel extraction

Result and Discussion

Detection of PPRV in suspected samples by N gene PCR:

All the suspected animals were febrile, presented with oculo-nasal discharges and mouth lesions consistent with the classical symptoms of PPR infection, however out of the 88 samples tested by N gene PCR, only 30 were detected positive for the presence of virus genetic material. All the samples from the Western and High rain fall zone turned out negative for PPR virus, refer Table 1. The importance of nucleic acid amplification tests in confirming clinically suspicious specimens has been highlighted by several researchers when probable mixed infections confound diagnosis [12,13] . PPR is grouped under the Morbillivirus genus of the Paramyxoviridae family along with genetically similar measles virus (MV), rinderpest virus (RPV), canine distemper virus (CDV), and other phocine, cetacean and feline viruses.

The clinical symptoms and progression of PPR, associated with immunosuppression and opportunistic bacterial and parasitic infections, closely resemble and is often confused with those of Rinderpest (RP), or the cattle plague [14] . The absence of differential diagnosis between RP and PPR has therefore been the major cause for delay in identifying PPRV, and the disease itself as a distinct entity. While the first recorded description of the virus is from Ivory Cost in 1942, the time to most recent common ancestor (TMRCA) of PPRV is estimated as the early 20 th century [15] . The eradication of RP and subsequent divergence and spread of PPR called for major strides in analytical techniques for disease diagnosis that detect specific viral antigens, nucleic acid or antibodies, the latter either induced through natural infection or vaccination [16] .

Phylogenetic characterisation of isolates

The nucleocapsid region of the PPR genome, as originally described by Couacy-Hymann et al, 2002 [17] was also used for lineage classification of the outbreak. Aside of surveillance of the source and route of virus circulation, lineage identification is critical for preparation of homologous vaccine [18] . The phylogenetic tree constructed using partial N gene sequences distinguished PPRV into 4 lineages.

Corroborating with earlier reports, isolates from this study also confirmed the predominance of Lineage IV virus in India, similar to other Asian countries. With the exception of a lineage III virus reported in 1992, all the isolates from India belong to lineage IV. Transhumance and animal trade being attributed to this lineage coexistence in a region, it is also known that lineage IV is expanding geographically and outcompeting pre-existing lineages in different endemic zones of the world [19,4,6] . Interestingly, ancestral PPRV to lineage IV was probably located in India [15] . As there has been no evidence for virulence associated positive selection in protein coding regions, unchecked movement of infected animals is speculated to be the reason lineage IV expansion [7] . The dendrogram with Indian isolates alone, (Figure 3) segregated into two major clades, CI and CII, each having 2 subclades, SC I, SCII and SC III, SC IV, respectively. Virus from central, southern, northern, eastern and western parts of India constituted SCI, while SCII comprised of 2 isolates from MP, collected during the year 2004-2005. Tamil Nadu isolates from 2004 and 2011 outbreaks grouped together to form SCIII, whereas those from Tripura, Gujarat, Jhansi and Karnataka formed SCIV. Importantly, the Sungri vaccine strain of North Indian origin was clustered within SCIV. As reported by other researchers, both CI and CII had South Indian and North Indian isolates indicative of disease prevalence sans geographic restrictio n [6] . Unlike the study by the Kumar group (2014), we have utilized a greater number of isolates from South India where a region wise clustering of isolates could be observed among some samples. However, few isolates from north and south India grouped together indicating close relatedness. Clustering of isolates can change based on the target gene as well as geographic distribution of the samples being investigated. We have tried to decipher the phylogenetic relation of the 2023 Tamil Nadu Isolates, from our study, on a global tree by including N gene-Lineage IV GenBank entries from other Asian countries (Figure 4). African isolates were not included as it is well documented by several research groups [6] . The phylogenetic tree, additionally including all the Asian isolates also segregated into 4 lineages. Similarly, the Chengalpattu samples (NE zone), from sheep and goat, appeared to be more similar to those from Northern parts of India. Though N sequences from Caprine samples predominate our analysis, similar to our observation researchers have not observed any species-specific lineages or evolutionary groups amongst Indian isolates of PPRV [6] . Despite a wider geographic presence, the genetic diversity of Lineage IV PPRV is on a gradual decline following effective mass vaccination programmes and other control strategies.

Presence of multiple clusters of N gene haplotypes within the country is reflective of the transboundary nature of PPR and also of re-emergence of virus from multiple bottlenecks [7] .

Conclusion

Given the transboundary nature of PPR, the present isolates reported across Tamil Nadu are probably introduced from recent outbreak episodes in neighbouring states. Distinct from the 2004 and 2011 outbreak strains which were similar to each other, origin of this probably new circulating haplotype cannot be traced without a detailed study. Also, it is to be seen whether the N gene clustering pattern is preserved with F and H gene loci. Though variability in gene sequences of PPRV lineages is not associated with virulence, nucleotide sequencing is important to understand the geographic trajectory of virus evolution and epidemiological dynamics. Further, stringent biosecurity measures in animal trade and movement can be implemented to check disease spread.

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