The molecular signature & phylogenetic analysis of RNA binding and major essential inclusion body NS2 protein encoding S8 gene of field strain of Bluetongue virus from Central India

Introduction

Blue tongue (BT) is an infectious, non-contagious, and arthropod-borne viral disease of domestic and wild ruminants including sheep, cattle, goats, and wild ungulates like deer caused by Bluetongue virus (BTV), which belongs to the genus Orbivirus, family Reoviridae and subfamily Sedoreoviridae. (Mertens et al., 2009; Singh et al., 2021; Haque et al., 2025) [11,14,3] . There are 29 serotype of Bluetongue virus (BTV) (Yang et al., 2021) [15] . The Bluetongue virus (BTV) is primarily transmitted by Culicoides midges, which act as biological vectors (Singh et al., 2021) [14] . Bluetongue virus (BTV) possesses a segmented double-stranded RNA genome comprising 10 segments that encode seven structural (VP1-VP7) and four nonstructural proteins (NS1, NS2, NS3/NS3a, and NS4) (Mertens et al., 2004) [10] . Among Non structural genes, the non-structural protein NS2, encoded by genome segment 8, plays a crucial role in viral replication and assembly. NS2 is primarily involved in viral RNA packaging and the formation of viral inclusion bodies (VIBs), which serve as sites for viral replication and assembly within infected host cells (Hewat et al., 1994) [4] . It acts as an RNA-binding protein, selectively interacting with viral singlestranded RNA transcripts, thereby facilitating the assortment and packaging of the viral genome segments into progeny virions. Importantly, NS2 is one of the most highly conserved proteins among different BTV serotypes, including conserved regions such as the N-terminal domain. Due to this high level of conservation, NS2 is capable of inducing cross-reactive cellular immune responses across multiple serotypes (Jiménez-Cabello et al., 2025) [5] . However, despite its immunogenicity, NS2 provides only limited protection against homologous BTV serotypes in natural hosts, indicating that it may not be sufficient alone for effective protective immunity. The conserved nature and binding to the RNA genome and a major component of formation of inclusion bodies make it a valuable target for molecular studies, So to understanding BTV biology and pathogenesis, as well functionality of this protein is still unknown hence the molecular signature of this NS2 protein encoding S8 gene need to be decipher by analysing its genome sequence. Hence the present study was carried out to understand its functionality by sequencing the gene encoding NS2 and analysing its phylogenetic relation with global isolates.

Materials and Methods

Collection of clinical samples

The blood samples were collected during an outbreak in sheep at "Umred" Tq. Umred in Dist. Nagpur, central India. Blood samples were collected from sheep via the jugular vein using 18-gauge needles. From suspected sheeps, 2 ml blood sample was collected in EDTA vials and brought to the laboratory on ice-packed container, with the temperature maintained at 4 °C. Blood samples were stored at 4 °C, while the separated serum was preserved at -20 °C.

RNA Isolation, cDNA synthesis

Total RNA was extracted from blood samples using TRI Reagent (Sigma-Aldrich, USA) following standard protocols. Briefly, blood samples were homogenized in TRI Reagent, followed by chloroform extraction and isopropanol precipitation. The RNA pellet was washed with 70% ethanol, air-dried, and resuspended in nuclease-free water. Extracted RNA was stored at -20 °C until further use. The cDNA synthesis was performed using the SuperScript™ IV First-Strand Synthesis System (Invitrogen, Cat. No. 18091050, USA), following the manufacturer's instructions. The reaction was carried out in a total volume of 20 μl, using labeled microcentrifuge tubes and the reagents listed below. The cDNA reaction was comprise of template RNA around 11 μl (approximelty (500 ng), Random hexamer (50ng/μl) 1 μl, 1 μl 10mM dNTP mix (10mM each). The mixture was incubated at 65 °C for 5 minutes in a thermal cycler, followed by immediate chilling on ice for 2 minutes. Subsequently, 4 μl of 5X SSIV Buffer, 1 μl of 100mM DTT, 1 μl of Ribonuclease inhibitor (40U/ μl) and 1 μl of Superscript TM IV Reverse transcriptase(200U/μl)of were added. The cDNA synthesis reaction was carried out in cyclic condition of 23°C for 10 minutes, 50°C for 30 minutes and 80°C for 10 minutes. the reaction was cooled at 4°C and used for the PCR amplification.

PCR Amplification and confirmation and sequencing:

The PCR reaction was carried out in using the published primers S5, S8 and S10 gene specific forward primers and revers primers depicted in tables no.1. The PCR reaction was carried out in 50 ul PCR tune containing 5 μl of 10X PCR Buffer; 3 μl of MgCl₂ (50mM); 1 μl of dNTP (10mM); 2μl of forward and reverse primer (10 pmol/μl) each; 0.3 μl of Taq DNA polymerase (5U/μl); 3 μl of cDNA and Nuclease Free Water was added to make the volume 50 μl. For the NS10 gene the cyclic reaction was carried out in Prima-Trio thermal Cycler (Hi-Media, India) of cyclic conditions as initial denaturation 95°C for 2 min, followed by 3 step cycling for 35 cycles at denaturation for 95°C-20 sec; annealing-52°C for 1 min, extension 72°C for 1 min and the final extension at 72°C for 10 min. whereas as for the BTV NS1 and NS2 gene amplification the initial denaturation 94°C for 2 min, followed by 3 step cycling for 10 cycles at Denaturation for 94°C-20 sec; Annealing-45°C for 1 min, Extension 72°C for 1.30 minutes followed by 3 step cycling for 30 cycles of denaturation for 94°C-20 sec; Annealing at 50°C for 1 min, Extension 72°C for 1.30 minutes. the post cyclic final extension of 72°C for 10.00 minutes was carried out. The amplicons were analysed on 1.4% agarose containing the 0.5 ug/ml of ethidium bromide (SIGMA) in 0.5X Tris borate-EDTA buffer (TBE) at 12V/cm of gel in 0.5X TBE running buffer in submarine electrophoresis apparatus (Himedia, India) and power supply (LA960, Electrophoresis power supply, Himedia, India). Bands were visualized under a UV transilluminator and documented using a gel documentation system (BioZen Laboratories, India).

Table1: Primers sequences used in the present study

Primers

Primer sequence Amplicon size Reference BTV-NS1-F1-13 TAG TTG GCA ACC ACC AAA CA 847bp

Gharat et al., 2022 [2] BTV-NS1-R1-860

AAG TGT CGC TGC CAT GCT AT 725bp BTV-S10-F-732 GGG CGC AAC TCT ACC TAC TG

The PCR amplicon of the NS2 encoding gene S8 genes was subjected to bidirectional sequencing using commercial services (Eurofins Genomics, India). The obtained sequences were analyzed using bioinformatics tools. Sequence editing and trimming were performed using Chromas software, followed by homology searches using BLASTn. Consensus sequences were generated and analyzed for open reading frames (ORFs). Phylogenetic analysis was carried out using MEGA software employing Neighbor-Joining and Maximum Likelihood methods with appropriate substitution models and bootstrap analysis. Global BTV sequences retrieved from GenBank were used for comparative analysis to determine genetic relationships and evolutionary patterns.

Results and Discussion

In the present study, amplification of the S5 (NS1) gene yielded bands of 860bp and 967bp. The S8 (NS2) and S10 (NS3) genes yielded distinct amplicons of 1087bp and 822bp, respectively. These results indicate that the all three nonstructural coding gene of BTV viz S5, S8, and S10 genes were successfully amplified from blood sample, as confirmed by agarose gel electrophoresis (Fig. 1). The particular amplicon sizes of 1087bp which correspond to coding sections of the NS2 (S8) genes, demonstrated the high specificity and efficiency of the chosen primers for BTV genome identification. [9,12] . The curated forward and reverse sequences were assembled to generate a contig, from which a consensus sequence was derived and designated as BTV/S5/NAGPUR/MH/India/sheep/2025(Accession No. PZ163108). Analysis of the sequence revealed that the amplified S8 gene fragment was 1027 bp in length, comprising 54.82% A+T content and 45.18% C+G content, indicating an AT-rich region. The ORF extended from nucleotide position 02 to 1023, with a coding sequence of 1023 bp encoding a protein of 340 amino acids, corresponding to the NS2 protein with an expected molecular weight of approximately 38.627 kDa. Similarly in another study, Seg-8 found to be 1121 bp long encoding the 353 aa of the viral inclusion body (VIB) matrix protein (Maan et al., 2011) [8] . In another study, Segment 8 of BTV2/KM/2003 and BTV12/PT/2003 encodes a conserved 354 aa NS2 protein and shows higher genetic relatedness to eastern BTV strains than to western strains (Yin et al., 2008) [16] . Protein BLASTp analysis demonstrated 97.94% identity with Bluetongue virus serotype 23 (Accession No. AGJ83628.1) and 99.03% to 64.95% identity with complete protein sequences of 100 global BTV isolates representing different serotypes. The phylogenetic analysis of current isolate BTV/NS2/NGP-MS/2025/IND (Accession No. PZ163108) showed, it clustered into Clade IV with other previously reported Bluetongue virus isolates from India and other regions, based on the nucleotide phylogenetic analysis based on the S8 gene (Fig. no. 2). The isolate's S8 sequences of BTV serotypes reported from India, such as KP696658/NS2/BTV9/2009/INDIA and KX302641/NS2/BTV27/GOAT/2014/F, demonstrated close genetic relatedness. The clustering pattern shows that the S8 gene may be genetically conserved among circulating regional strains and that the current isolate has a common evolutionary origin with them. In another study, Phylogenetic analysis places these viruses in a distinct monophyletic group with Asian, Australian, and some Eurasian isolates, clearly separated from African, American, and European BTV strains, supporting their classification within the eastern topotype lineage (Yin et al., 2008) [16] . The strong bootstrap support at this node makes the clustering more reliable (Fig. no. 3). Overall, the phylogenetic analysis confirms that the current research isolate belongs to the Clade IV branch of BTV NS2 gene sequences and demonstrates its genetic relatedness to previously circulating Indian BTV strains. In another study, Seg-8 sequence analysis shows that Indian BTV isolates segregate into distinct eastern and western topotypes. Most Indian BTV serotypes cluster within the eastern topotype, while some isolates, particularly certain BTV-2 and BTV-10 strains, group within the western topotype, displaying very high nucleotide identity with vaccine strains from South Africa and prototype strains from the USA (Maan et al., 2015) [7] . The current isolate BTV/NS2/NAGPUR/MH/2025/India showed a substantial clustering with worldwide reference serotypes, according to phylogenetic analysis based on the inferred amino acid sequences of the NS2 protein (Fig. 3). The investigation specifically showed that the isolate is located in Clade IV and exhibits high commonality with the sheep-derived BTV-16, 9 and 4 strains (namely, ANK78272/NS2/BTV16/SHEEP/2014 from Tamil Nadu, AKP24275/NS2/BTV9/2055/IND from Haryana, and AIK27566/NS2/BTV4/SHEEP/2008/I from Andhra Pradesh), particularly sheep origin isolates, suggesting regional evolutionary continuity and circulation of indigenous BTV lineage. Similar findings have been reported in earlier molecular epidemiological studies, where Indian BTV isolates showed geographical clustering and lineagespecific evolution due to endemic circulation and vector-host adaptation (Maan et al., 2012) [6] . The close relationship of the present isolate with historical Indian strains indicates possible continuous viral evolution within the country rather than recent introduction from external geographical regions (Saminathan et al., 2020) [13] . Previous molecular studies have also demonstrated that Indian BTV isolates often group according to topotype and regional circulation patterns (Maan et al., 2015) [7] . The separation of the study isolate from other serotype clusters such as BTV12, BTV25, and BTV27 further supports serotype-specific genetic divergence within the S8 gene segment. NS2 is known to be relatively conserved but still provides sufficient resolution for studying evolutionary relationships and molecular epidemiology of BTV strains (Butan et al., 2010) [1] . Overall, the findings of the present study are consistent with earlier reports suggesting continuous circulation and evolution of indigenous BTV strains in India, highlighting the importance of molecular surveillance for understanding virus evolution, outbreak tracing, and vaccine strategy planning

Conclusion

The present study concludes that RNA binding and major essential Inclusion body NS 2 protein encoding S8 gene of field strain of Bluetongue virus from Central India is A+T rich, due to its conserved nature and genetic stability, can be effectively utilized for molecular detection, genetic characterization, and AT rich, conserved in nature, can be used for microevolutionary studies.

Acknowledgement

AcknowledgmentThe authors acknowledged the the Associate Dean, Nagpur Veterinary College, Nagpur & MAFSU, Nagpur for the financial support of PG research grant & infrastructure facility to conduct the research.

References

1. Butan C, Tucker P. Insights into the role of the nonstructural protein 2 (NS2) in bluetongue virus morphogenesis. Virus Research. 2010;151(2):109-117.
2. Gharat DK. Nonstructural gene based molecular characterization of bluetongue virus. Nagpur: MAFSU; 2022.
3. Haque MN, Rahman MH, Naher L, Udoy SAK, Ali MZ, Ahmed S. Comprehensive review of emerging insights into bluetongue virus in small ruminants with emphasis on epidemiology, diagnosis and control in Bangladesh and South Asia. Discover Animals. 2025;2(1):76.
4. Hewat EA, Booth TF, Roy P. Structure of correctly selfassembled bluetongue virus-like particles. Journal of Structural Biology. 1994;112(3):183-191.
5. Jiménez-Cabello L, Utrilla-Trigo S, Illescas-Amo M, Rodríguez-Sabando K, Benavides-Silván J, Calvo-Pinilla E, et al. The MVA-VP2-NS1-2A-NS2-Nt vaccine candidate provides heterologous protection in sheep against bluetongue virus. Frontiers in Immunology. 2025;16:1566225.
6. Maan NS, Maan S, Belaganahalli MN, Ostlund EN, Johnson DJ, Nomikou K, et al. Identification and differentiation of twenty six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2. PLoS One. 2012;7(2):e32601.
7. Maan S, Maan NS, Belaganahalli MN, Rao PP, Singh KP, Hemadri D, et al. Full-genome sequencing as a basis for molecular epidemiology studies of bluetongue virus in India. PLoS One. 2015;10(6):e0131257.
8. Maan S, Maan NS, Nomikou K, Veronesi E, BachanekBankowska K, Belaganahalli MN, et al. Complete genome characterisation of a novel 26th bluetongue virus serotype from Kuwait. PLoS One. 2011;6(10):e26147.
9. Maan S, Maan NS, Samuel AR, Rao S, Attoui H, Mertens PPC. Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes. Journal of General Virology. 2007;88(2):621630.
10. Mertens PP, Diprose J, Maan S, Singh KP, Attoui H, Samuel AR. Bluetongue virus replication, molecular and structural biology. Veterinary Italiana. 2004;40(4):426437.
11. Mertens PP, Ross-Smith NA, Diprose J, Attoui H. The structure of bluetongue virus core and proteins. In: Bluetongue. 2009. p. 101-133.
12. Rao PP, Reddy YV, Meena K, Karunasree N, Susmitha B, Uma M, et al. Genetic characterization of bluetongue virus serotype 9 isolates from India. Virus Genes. 2012;44:286-294.
13. Saminathan M, Singh KP, Khorajiya JH, Dinesh M, Vineetha S, Maity K, et al. An updated review on bluetongue virus: Epidemiology, pathobiology, and advances in diagnosis and control with special reference to India. Veterinary Quarterly. 2020;40(1):258-321.
14. Singh KP, Saminathan M, Dinesh M, Khorajiya JH, Umeshappa CS, Sahoo D, et al. Epidemiology and pathology of bluetongue virus in India: A systematic review. 2021.
15. Yang H, Gu W, Li Z, Zhang L, Liao D, Song J, et al. Novel putative bluetongue virus serotype 29 isolated from inapparently infected goat in Xinjiang of China. Transboundary and Emerging Diseases. 2021;68(4):2543-2555.
16. Yin HQ, Zhang GP, Zhang H, Zhang JG. A pair of novel primers for universal detection of the NS1 gene from various bluetongue virus serotypes. Virologica Sinica. 2008;23(1):68-72.