https://orcid.org/0000-0003-0557-7194
Abstract
Sheeppox and goatpox are transboundary viral diseases of sheep and goats that cause significant economic losses to small and marginal farmers worldwide, including India. Members of the genus Capripoxvirus (CaPV), namely Sheeppox virus (SPPV), Goatpox virus (GTPV), and Lumpy skin disease virus (LSDV), are antigenically similar, and species differentiation can only be accomplished using molecular approaches. The present study aimed to understand the molecular epidemiology and host specificity of SPPV and GTPV circulating in India through sequencing and structural analysis of the RNA polymerase subunit-30 kDa (RPO30) gene. A total of 29 field isolates from sheep (n = 19) and goats (n = 10) belonging to different geographical regions of India during the period: Year 2015 to 2023, were analyzed based on the sequence and structure of the full-length RPO30 gene/protein. Phylogenetically, all the CaPV isolates were separated into three major clusters: SPPV, GTPV, and LSDV. Multiple sequence alignment revealed a highly conserved RPO30 gene, with a stretch of 21 nucleotide deletion in all SPPV isolates. Additionally, the RPO30 gene of the Indian SPPV and GTPV isolates possessed several species-specific conserved signature residues/motifs that could act as genotyping markers. Secondary structure analysis of the RPO30 protein showed four α-helices, two loops, and three turns, similar to that of the E4L protein of vaccinia virus (VACV). All the isolates in the present study exhibited host preferences across different states of India. Therefore, in order to protect vulnerable small ruminants from poxviral infections, it is recommended to take into consideration a homologous vaccination strategy.
1. Introduction
Sheeppox virus (SPPV), Goatpox virus (GTPV), and Lumpy skin disease virus (LSDV) are responsible for causing sheeppox, goatpox, and lumpy skin disease in sheep, goats, and cattle, respectively (Tulman et al. Citation2002). All three viruses belong to the family Poxviridae, subfamily Chordopoxvirinae, and genus Capripoxvirus (CaPV) (King et al. Citation2012). The virions of Capripoxvirus have a brick-shaped structure (Babiuk et al. Citation2008) with an average size of 294 × 273 nm. Their genome is a linear, double-stranded DNA of approximately 150 kb, containing 156 open reading frames (ORFs)/putative genes with a high AT content of 73–75% and 96% nucleotide identity. Among the 156 ORFs, the conserved essential genes for replication, structure, and assembly are located in the middle (ORFs 24–123) and terminal variable regions (ORFs 1–23 and 124–156), which are responsible for virulence and host range functions (Tulman et al. Citation2002).
Goatpox and sheeppox are extremely contagious diseases and are enzootic in many parts of the world, including the Indian subcontinent (Venkatesan et al. Citation2010; Madhavan et al. Citation2016; Chahota et al. Citation2022). In endemic regions, the morbidity rates in sheep and goats can reach 50% in adults and 100% in young animals, and the mortality rate can reach up to 50% (Zhou et al. Citation2012; Saminathan et al. Citation2020). In India, both Sheeppox and goatpox disease outbreaks occur on a regular basis, with an average morbidity and mortality rate of 63.5% and 49.5%, respectively, primarily affecting domestic ruminants. However, there has been a recent report of goatpox virus infection in the wild population of red serows (Capricornis rubidus) in Mizoram, India (Dutta et al. Citation2019). The lack of comprehensive immunization in the field conditions inflicts major economic losses to the sheep and goat farming industries due to decreased weight, lower milk yield, damage to skin, hide, and wool, as well as mortality (Yeruham et al. Citation2007; Babiuk et al. Citation2008; Bhanuprakash et al. Citation2011; Chahota et al. Citation2022).
Invariably, both SPPV and GTPV display similar clinical signs in sheep and goats that are typical of poxviral diseases, including fever, generalized cutaneous lesions or nodules, and the development of bullet-shaped lesions in the lungs (Babiuk et al. Citation2008; Tuppurainen et al. Citation2017). Moreover, it is reported that certain isolates of SPPV and GTPV induce infection in both sheep and goats (Babiuk et al. Citation2009; Tuppurainen et al. Citation2017). Furthermore, the similarity in antigenic properties, provide protection against challenges from heterologous strains but poses a significant challenge in differentiation using various serological methods. Recent experimental cross-protection studies presented contradictory results, where there has been instances of unsuccessful attempts to protect goats (Abd-Elfatah et al. Citation2018) or cattle (Hamdi et al. Citation2020) with the SPPV vaccine, or sheep with the GTPV vaccine (Abd-Elfatah et al. Citation2018). Considering the existing ambiguity regarding the use of either homologous or heterologous vaccines in a particular host, efforts are warranted to characterize the currently circulating CaPV isolates for their lineage and host preferences to formulate suitable vaccination strategies.
Currently, SPPV and GTPV are classified within the genus CaPV based on the animal species from which the viruses are isolated. The limitations of existing conventional tools for differentiating CaPV isolates have led to the use of genome-based approaches. Recently, the differentiation of CaPVs was performed using electrophoretic patterns of viral genome isolates digested with restriction endonucleases (Balinsky et al. Citation2008) and, subsequently, based on whole genome sequencing, P32 gene-based PCR and sequencing (Sumana et al. Citation2020a, Citation2020b), RPO30 gene-based conventional PCR, and G-protein-coupled chemokine receptor (GPCR) gene-based quantitative PCR (Lamien et al. Citation2011; Santhamani et al. Citation2014; Chahota et al. Citation2022). The RPO30 gene, a close homolog of the E4L gene of vaccinia virus (VACV), encodes a 30 kDa DNA-dependent RNA polymerase subunit and it is located in the central region of the SPPV genome (ORF 036), known to play an important role in RNA replication (Lamien et al. Citation2011). In SPPV and GTPV, the RPO30 gene possesses highly species-specific signature residues at both the nucleotide and amino acid levels. Therefore, this gene has been used for the differentiation of SPPV and GTPV (Santhamani et al. Citation2013; Chahota et al. Citation2022) and could potentially assist in designing safer vaccines, antiviral agents and identifying recombinant proteins for the development of diagnostic assays for the detection of SPPV and GTPV.
The current study presents the comprehensive investigation combining the genetic analysis of the sequences, evolutionary relationships with other poxviruses and structural analysis of the RPO30 gene/protein to understand the molecular epidemiology of SPPV and GTPV isolates circulating in India, which would eventually play a crucial role for devising strategies for disease control and prevention.
2. Materials and methods
2.1. Clinical samples
Between 2015 and 2023, the disease outbreak investigations on sheeppox and goatpox was carried out in Jammu and Kashmir in the North, Arunachal Pradesh and Manipur in the North-East, Gujarat in the West, Maharashtra, Karnataka, Andhra Pradesh, and Puducherry in the South and Chhattisgarh in the Central part of India were included in this study (). The samples collected include skin scabs, nasal swabs, ocular swabs from live animals and also postmortem tissue like lung samples from the deceased animals (). A total of 29 Capripoxvirus isolates from sheep (n = 19) and goats (n = 10) are presented in this study.
Figure 1. Details of clinical samples from sheep and goats collected during the investigation of outbreaks occurring between 2015 and 2019 in different states of India.
Table 1. The details of sheeppox virus (SPV) and goatpox virus (GPV) isolates from India and RPO30 gene sequences with GenBank accession numbers used in the study.
2.2. Sample processing
The tissue (scab and lung) samples were triturated to create a 10% suspension in 1x phosphate-buffered saline (PBS, pH 7.2), whereas the nasal and ocular swabs were suspended in 1x PBS (1 ml) and filtered the suspension using a 0.45 μM syringe filter. Following the manufacturer’s instructions, the DNA extraction using the DNeasy Blood and Tissue Extraction Kit (Qiagen, Hilden, Germany) was done. Briefly, 200 μl of the Tissue/swab suspension/was taken, lysis was performed for 20 min at 56 °C, and final elution was done with elution buffer in 30 μl. The DNA concentration was determined using a nano spectrophotometer (Nabi), and subsequently, the DNA was stored at −20 °C until further use.
2.3. Virus isolation
African green monkey kidney (Vero) cells maintained in growth media consisting of minimum essential medium (MEM) with 10% foetal bovine serum (FBS), available at National Institute of Veterinary Epidemiology and Disease Informatics (ICAR-NIVEDI), Bengaluru, India, were used for virus isolation. A 25 cm2 tissue culture flask with a confluent monolayer of Vero cells were inoculated with the filtered sample suspension, subsequently, the flask was incubated at 37 °C in a 5% CO2 incubator and observed daily for the presence of cytopathic effects (CPE).
2.4. PCR amplification and sequencing of the RPO30 gene
Initially, the DNA extracted from the clinical samples was subjected to a partial P32 gene-based diagnostic PCR assay for preliminary identification of Capripoxvirus, a method previously described (Reddy et al. Citation2015). Subsequently, the SPPV and GTPV isolates obtained through virus isolation [sheep (n = 19) and goat (n = 10)], were subjected for the amplification of the complete coding sequence of RPO30 using the forward and reverse set of primers (Zhou et al. Citation2012). Further, the amplified PCR products were analyzed by agarose gel electrophoresis on a 1.5% agarose gel in 1X Tris-acetate EDTA (TAE) buffer and the products were gel purified using Gel Extraction Kit (Thermo Fisher Scientific, Massachusetts, USA).
Further, the PCR products were cloned into the pGEMT-Easy vector (Promega, Wisconsin, USA) and transformed into TOP10 Escherichia coli cells. Using RPO30 gene-specific primers, the recombinant clones were confirmed through colony PCR and the plasmid DNA was extracted using a GeneJET Plasmid Miniprep kit (Cat No. K0502, Thermo Fisher Scientific). The products were sequenced by sanger-sequencing method at Eurofins Genomics India Private Limited, Bengaluru, India and further analyzed the sequences using Gene tool (Informer Technologies, Inc.). All the complete nucleotide sequences of the RPO30 gene were aligned using BLAST and annotated. Subsequently, all the sequences were submitted to the GenBank database.
2.5. Phylogenetic and multiple sequence analysis
The nucleotide and deduced amino acid sequences of the RPO30 gene of CaPVs as well as other poxviruses from India and other countries, were retrieved from the GenBank database. Multiple sequence alignment was done using the DNASTAR Lasergene software 6.0 version (DNASTAR, Inc., Madison, USA) and the phylogenetic tree was constructed in the MEGA software version 11 using the maximum likelihood method with a bootstrap value of 1000 (Kumar et al. Citation2018). Further, a comparative analysis of the RPO30 and E4L genes of all the poxviruses was performed using existing motif identification methods, as reported earlier (Lamien et al. Citation2011).
2.6. Structural analysis of the RPO30 protein
An automated homology model was constructed using SWISS-MODEL (Swiss Institute of Bioinformatics, BIOZENTRUM, University of Basel, Switzerland) for the consensus RPO30 gene sequences of the SPPV and GTPV isolates. The homology model for all SPPV and GTPV isolates of the RPO30 protein was predicted and proteins were matched from 1M to 204K of the E4L protein of VACV crystallized structure (6RIC_S), which has 259 amino acid residues. Further, the automated structural models were validated using RAMPAGE software (https://www.ccp4.ac.uk/html/rampage.html).
All modeled structures were generated using the X-ray crystallographic structure of the E4L protein (PDB ID: 6RIC_S) of the VACV (1M to 204K) as a reference and visualized using PyMol software version 4.6.0 (Schrödinger, Inc., New York, USA). Structural validation was performed using the Structural Analysis and Verification (SAVES) server (https://saves.mbi.ucla.edu/). Energy minimization and refinement were performed using the ModRefiner server (https://zhanggroup.org/ModRefiner/).
3. Results
3.1. Molecular detection and differentiation of CaPV
A total of 29 clinical samples collected from sheep (n = 19) and goat (n = 10) when tested by P32 gene-based diagnostic PCR, all were found to be positive for Capripoxvirus. The positive samples subjected for virus isolation, showed characteristic CPE of rounding and aggregation of the cells after 4–5 blind passages. Subsequently, the full-length RPO30 gene of SPPV (585 bp) and GTPV (606 bp) were successfully amplified and sequenced from all the SPPV and GTPV isolates. The edited sequences of SPPV and GTPV were submitted to the GenBank and the accession numbers obtained for each isolate are mention in .
3.2. Molecular phylogeny of SPPV and GTPV isolates
The evolutionary relationship of SPPV and GTPV isolates obtained in this study to previously described CaPVs based on the RPO30 gene is displayed in . Phylogenetically, the CaPVs formed three major clusters: SPPV, GTPV, and LSDV. The multiple sequence analysis of SPPV and GTPV from different geographical regions revealed considerable sequence identity at both the nucleotide and amino acid levels with other CaPV sequences obtained from the GenBank database.
Figure 2. Phylogenetic tree based on the RPO30 gene sequences of Indian sheeppox virus (SPPV) and goatpox virus (GTPV) isolates: Phylogenetic tree constructed by using RPO30 gene sequences of Indian isolates (n = 22) in this study and previously reported sequences from India and foreign countries (n = 49) of capripoxvirus isolates available in GenBank. The tree was obtained by the maximum likelihood method of MEGA version 10 (bootstrap 1000). The sequences of SPPV from this study are indicted by red-colored triangles and GTPV are indicated by blue-colored circles.
The sequence identity of the RPO30 gene among Indian SPPV isolates showed 98.8–100% and 96.5–99.5% similarity at the nucleotide and amino acid levels, respectively. Whereas, the sequences from other countries showed 92.1–99.8% and 92.6–99.0% similarity at the nucleotide and amino acid levels, respectively. Further, the divergence analysis revealed highest sequence divergence of 1.2 in SPPV-Kanakapura-NI and 3.2 in SPPV-Kolar-1, among the Indian isolates, and 4.8 and 3.7 with other countries (SPPV-GU119932), at nucleotide and amino acid levels, respectively.
Among the GTPV isolates from India, revealed the sequence identity of 99.5–100% and 98.5–99.5%, whereas among the isolates from other countries revealed 93.1–100% and 93.6–99.5% at the nucleotide and amino acid levels, respectively. The highest sequence divergence was 0.3 and 1.0 among Indian isolates (GTPV-Bilgi-204 and GTPV-Chellakere-211), and 3.7 and 2.6 (GTPV-GU11925 and GTPV-GU119928) among other countries isolates, at the nucleotide and amino acid levels, respectively.
3.3. Multiple sequence alignment and motifs in RPO30 gene
Multiple sequence alignment of nucleotide and amino acid sequences revealed a high level of sequence conservation among the CaPVs. Additionally, in the RPO30 gene of all GTPV and LSDV isolates revealed the presence of a unique set of 21 nucleotides (13–33) (corresponding to a seven amino acid sequence), whereas these 21 nts were absent in all SPPV isolates (). Unique signatures were observed among RPO30 of the CaPV isolates. Sequence variations in RPO30 at the nucleotide and amino acid levels between the SPPV and GTPV sequences are listed in . The motifs identified in the RPO30 protein sequence were Transcription elongation factor S-II (TFIIS) motif, GIEYSKD and LRY motif.
Figure 3. Multiple sequence alignment of the RPO30 gene of CaPV isolates: RPO30 gene sequences (13–33 nt) of Indian SPPV and GTPV isolates along with other capripoxvirus sequences available in GenBank were aligned using Clustal-W method of MEGA 10. The conserved regions and deletion are highlighted.
Table 2. The details of nucleotide substitution and amino acid changes present in the RPO30 gene of SPV and GPV isolates.
3.3.1. TFIIS motif
The N-terminal variable region of the RPO30 gene revealed high divergence between SPPV and GTPV sequences; whereas, the C-terminal domain revealed high sequence identity with the homolog of the eukaryotic transcription elongation factor S-II (TFIIS) domain (158–195), including the C2C2 zinc finger motif (). The TFIIS domain comprises a highly conserved region of two cysteine residues in the SPPV and GTPV isolates. The zinc finger motif showed a high level of conservation among all poxviruses, irrespective of the genus; whereas, the TFIIS domain was highly conserved within the genus Poxviridae (data not shown).
Figure 4. Multiple sequence alignment based on RPO30 protein sequences: the multiple RPO30 protein sequences of Indian SPPV and GTPV isolates were aligned along with selected motifs and their amino acid deletion and variation as indicated by blue- and pink-colored shades, respectively. The conserved motifs and regions are depicted in the alignment.
3.3.2. GIEYSKD and LRY motif
The GIEYSKD and LRY motifs were highly conserved in the RPO30 gene of all CaPV isolates (). Multiple E4L gene sequence alignments revealed a highly conserved GIEYSKD motif in all other poxviruses. The LRY motif was also highly conserved among all poxviruses, whereas residue variations were observed in motifs, such as DPV was changed to FRY and MYXV and RFV were changed to LRH instead of LRY (data not shown).
3.4. Comparative sequence analysis of poxviral E4L protein
Phylogenetic analysis based on E4L homolog protein sequences clustered with the respective members of each genus. Comparison of Capripoxvirus E4L homolog in SPPV showed similarity of 96.8% and 95.4% to GTPV, 96.2%, and 94.9% to LSDV at the nucleotide and amino acid levels, respectively. Furthermore, GTPV showed 98.7% and 97% similarity with LSDV at the nucleotide and amino acid levels, respectively.
Comparison of E4L homolog of SPPV with other poxviruses showed similarity with Suipox virus (70.6% and 68.7%), Yatapoxvirus (70.4% and 67.4%), and Cervidopoxvirus (69.1% and 65.1%) at the nucleotide and amino acid levels, respectively. Additionally, GTPV was similar to other poxviruses, including Suipox virus (71.6% and 67.3%), Cervidopox virus (71.1% and 65.1%), and Yatapoxvirus (70.5% and 67.4%) at the nucleotide and amino acid levels, respectively.
3.5. Structure of the RPO30 protein
The predicted three-dimensional structure of the RPO30 protein of Indian SPPV and GTPV isolates generated based on the template matching sequence (259 residues) is shown in . The model validation results of the RAMPAGE analysis showed 88.8–88.4% of the aa in the favored region, 11.4–9.8% in the allowed region, and 0.8% in the disallowed region, and none of the amino acid residues occupied the generous region of SPPV. Comparison of structural features of the predicted models of both SPPV and GTPV revealed the presence of four α-helices, two loops and three turns ().
Figure 5. Structure of RPO30 protein: a three-dimensional (3D) homology model was generated using the Swiss-model server using a PDB-6RIC_S as template. Panel A: 3D structure of RPO30 protein of SPPV. Panel B: 3D structure of RPO30 protein of GTPV. Panel C. Multiple sequence alignment of RPO30 protein structure of representative SPPV and GTPV isolates with reference to the vaccinia virus E4L protein structure (6RIC_S).
4. Discussion
From an epidemiological perspective, it would be more pertinent to elucidate the function of the genes and proteins in GTPV and SPPV, particularly in light of their sequence diversity. Further, planning diagnostic goals for strain genotyping may aid in the development of appropriate immunization programs. In India, sheeppox and goatpox diseases cause severe annual losses to farmers’ incomes, owing to the enzootic nature of the virus (Saminathan et al. Citation2016). Although the disease can be prevented by vaccination, there is ambiguity regarding the selection of vaccine strains for a particular host species. Currently, in India, homologous live attenuated vaccines, such as the SPPV-Romanian Fenner (RF) vaccine (a foreign isolate from Iraq), is being used to vaccinate both sheep and goats (Yogisharadhya et al. Citation2011). Additionally, the GTPV-Uttarkashi virus vaccine strain is commercially available and is being used to vaccinate goats to control the goatpox disease (Bhanuprakash et al. Citation2012). As SPPV and GTPV are closely related antigenically but genetically distinct, there is a need to differentiate and analyze the diversity of circulating poxvirus isolates among small ruminant populations in India.
In the present study, the nucleotide sequences of the RPO30 gene from SPPV and GTPV isolates (n = 29) obtained during natural outbreaks in India were used for phylogenetic analysis, along with the available sequences of CaPVs retrieved from GenBank. The resulting phylogenetic tree revealed three distinct clusters. Notably, all Indian SPPV and GTPV isolates were grouped into species-specific clusters, indicating their homologous nature and host species preferences. Multiple sequence analyses identified a deletion of 21 nucleotides (seven amino acid sequence) in all Indian and foreign SPPV isolates. Therefore, the complete coding sequence (CDs) of the RPO30 gene of all SPPV isolates were 195 amino acids, which is consistent with previous studies (Lamien et al. Citation2011; Rouby Citation2018; Lachheb et al. Citation2019). Whereas, all GTPV and LSDV isolates had 202 aa residues. The RPO30 genes of SPPV and GTPV have species-specific signature residues that can be used for disease diagnosis and species differentiation (Santhamani et al. Citation2013; Zeedan et al. Citation2020). Sequence analysis of RPO30 gene showed high sequence identity among SPPV and GTPV isolates from India and other countries, irrespective of geographical region and temporal outbreaks. Two hypotheses could explain these genetic findings: First, there may be similar SPPV and GTPV isolates circulating in India, potentially introduced, and evolved from other countries around the world. Alternatively, all Indian SPPV isolates may be clonal in nature and distributed to other regions, eventually evolving over time.
Phylogenetic analysis of various members of the Poxviridae family within the subfamily Chordopoxvirinae based on the nucleotide sequence of the RPO30 gene, showed clustering of different members under the respective genera, as indicated in the present study. Multiple sequence alignment and comparative analysis showed certain nucleotide and amino acid variations in most members of the genus. The RPO30 gene was conserved among SPPV, GTPV, and LSDV, and showed a high percentage of sequence identity. Multiple sequence analysis of the nucleotide and amino acid of the RPO30 gene/protein revealed that GTPV and LSDV were more closely related to each other than to SPPV, which is consistent with previous reports (Santhamani et al. Citation2014; Lachheb et al. Citation2019). In contrast, Zhou et al. (Citation2012) reported that SPPV and GTPV are more closely related than LSDV based on the RPO30 gene. Similarly, previous studies with phylogenetic analysis of the P32 gene revealed that SPPV is more closely related to LSDV than to GTPV (Rashid et al. Citation2017; Lachheb et al. Citation2019; Sumana et al. Citation2020a, Citation2020b), which differ from our findings.
The E4L gene of VACV encodes a multi-functional protein of 30 kDa, which is an integral subunit of the DNA-dependent RNA polymerase present in all members of the subfamily Chordopoxvirinae and also an intermediate-gene transcription factor that is viral late transcription factor 1 (VLTF1) shares homology with eukaryotic TFIIS (Rosales et al. Citation1994). The VACV RNA polymerase 30 kDa subunit contains no obvious sequence motifs, such as zinc finger, leucine, or glutamic-rich regions, which are frequently found in nucleic acid-binding proteins (Broyles and Pennington Citation1990). However, previous studies have reported that the eukaryotic TFIIS domain, including the C2C2 zinc finger motif, is present in the C-terminal region of the E4L gene of VACV (Lamien et al. Citation2011). In this study, the C-terminal regions of TFIIS and the C2C2 zinc finger motif residues were highly conserved in all SPPV and GTPV sequences of Indian and foreign isolates along with VACV, as reported previously (Lamien et al. Citation2011).
Multiple sequence alignment of the amino acid sequences of the VACV E4L with Capripoxvirus RPO30 homolog showed varying levels of conservation of amino acids in the protein sequence. The E4L protein of VACV has more than 54 amino acids in comparison to CaPV RPO30 gene homologs, which is consistent with earlier studies (Lamien et al. Citation2011; Santhamani et al. Citation2014). The N-terminal region (amino acids 1–54 of the SPPV reference sequence) showed high divergence, whereas the C-terminal region was conserved between the two poxvirus genera. The seven amino acid deletions in the N-terminal variable region were observed only in the SPPV group and led to the loss of serine, threonine, and tyrosine phosphorylation sites. Notably, amino acid deletion was also observed in the N-terminal region of the GPCR homolog of SPPV isolates (Lamien et al. Citation2011). This loss may be due to host species (sheep) and viral interactions, which require further investigation. Despite the shorter C-terminal region in CaPV RPO30 compared to that in VACV E4L, the transcription elongation factor domain (GIEYSKD and LRY) with the C2C2 zinc finger motif was conserved. The evolutionary conservation of the various motifs of the RPO30 protein plays a role in its interaction with other viral polymerase subunits, indicating its essential role during RNA elongation and transcription. The low number of single nucleotide polymorphisms and divergence in the N-terminal region of CaPV RPO30 in comparison with other genes (P32 and GPCR) make it a reliable and suitable marker gene for genotyping CaPVs.
The complete CDs of the VACV 30 kDa RNA polymerase subunit contains 260 amino acid residues, including proline and acidic residues at the C-terminal, which have been shown to function as activator domains in these proteins. The RNA polymerase 30 kDa subunit gene is estimated to have 45 C-terminal amino acids, containing 15 acidic residues and 13 proline residues (Broyles and Pennington Citation1990). Whereas, a C-terminal region containing four acidic residues and seven proline residues were observed in all the SPPV and GTPV isolates in this study.
Secondary structure analysis of the RPO30 protein of SPPV and GTPV (approximately 259 amino acid residues) matched with the 30 kDa RNA polymerase subunit of the E4L protein of VACV. Furthermore, the predicted models validated with a Ramachandran plot showed 88.8%–88.4% amino acids in the favored region, 11.4%–9.8% in the allowed region, and 0.8% in the disallowed region, and none occupied the generous region. Based on these results, the predicted homology model was highly stable and reliable. The RPO30 proteins of SPPV and GTPV possess a structure similar to that of the VACV E4L protein. Theoretically, the allowed regions of the Ramachandran plot showed values of Φ/Ψ (Phi/Psi) angles that could possibly form a tripeptide. Practically, the distribution of the Φ/Ψ values observed in a protein structure can be used for validation of the structure (Ramakrishnan et al. Citation2007). An automated homology model of the RPO30 protein of representative SPPV and GTPV revealed the presence of α-helices and β-sheets in a pattern similar to that of the VACV E4L protein (PDB: 6RIC_S) (Hillen et al. Citation2019).
In conclusion, the N-terminal heterogeneity observed between SPPV and GTPV helps to resolve the problem of classifying viruses within the Capripoxvirus genus using sequence analysis of the RPO30 protein. The CaPV RPO30 gene showed species-specific signature clusters along with various signature residues that could be used for genotyping. Based on sequence analysis, RPO30 can be used as a diagnostic marker for the differentiation of viruses among CaPVs at the species level. Furthermore, the sequence analysis revealed that homologous viral species infected their respective host species in India. All SPPV and GTPV isolates in this study were similar to other Indian CaPV isolates, indicating that common CaPV isolates are circulating in India. In addition, for the first time, the structural features of the RPO30 protein were studied for SPPV and GTPV, which revealed highly conserved motifs along with common structural features among all the CaPVs, which were similar to the E4L gene of VACV. The findings of this work may be useful in developing new antiviral treatments for goatpox and sheeppox in India, as well as in creating a safer and more effective homologous vaccination and/or recombinant protein-based diagnostic assay.
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The authors acknowledge the support rendered by the present and past Directors of ICAR-National Institute of Veterinary Epidemiology and Disease Informatics (NIVEDI), Bengaluru, Karnataka, for providing the facilities available in the Institute. The authors are also grateful to the field veterinary doctors and animal owners for their tremendous help during the clinical samples collection and outbreak investigations.
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