First complete genome sequence of lumpy skin disease virus directly from a clinical sample in South India

doi:10.21203/rs.3.rs-2009411/v1

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First complete genome sequence of lumpy skin disease virus directly from a clinical sample in South India

https://doi.org/10.21203/rs.3.rs-2009411/v1

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Lumpy skin disease (LSD), a notifiable disease listed by the World Organization for Animal Health and a fast fast-moving transboundary viral disease infecting cattle and buffaloes, was reported in India in 2019 and has since rapidly spread across the country. This study reports the first complete genome sequence and analysis of a pathogenic LSD virus (LSDV) from India (LSDV/208/PVNRTVU/2020) obtained by direct sequencing of a suspected clinical sample using Illumina and Nanopore sequencing technologies. The complete genome sequence of LSDV/208/PVNRTVU/2020 is 150445 bp long, codes for 156 putative genes and carries identical 2254 bp inverted terminal repeats at either ends. The unique features reported in the LSDV isolates from the recent outbreaks in Asia, namely, the insertions of 12 nucleotides in the viral G-protein coupled receptor (GPCR) and 27 nucleotides leading to duplication of 9 aminoacids in the extracellular enveloped virus-specific (EEV) genes were also conserved in LSDV/208/PVNRTVU/2020. Phylogenetic analysis of the complete genome sequence of LSDV/208/PVNRTVU/2020 revealed its close relation with Kenyan strains and clustered away from vaccine strains. Further analysis showed evidence of strong purifying selection without any recombination events. The data presented in this study could be useful for designing effective strategies such as developing rapid diagnostics and vaccines to control LSD.

lumpy skin disease

lumpy skin disease virus

Capripoxvirus

complete genome sequence

phylogenetic analyses

Lumpy skin disease virus (LSDV), an economically important virus infecting cattle, buffaloes and wild ruminants, belongs to genus Capripoxvirus, along with sheeppox virus (SPPV) and goatpox virus (GTPV) infecting sheep and goat, respectively (Abutarbush, 2017; Davies, 1991b). The virus harbors double-stranded, linear DNA genome (151 kbp) enveloped by lipid bilayer; the genome consists of identical 2.2 to 2.4 kbp-inverted terminal repeats and central region codes for 156 putative genes (Tulman et al., 2001; Tulman et al., 2002). LSD is a notifiable disease as per the World Organization for Animal Health because the virus can spread worldwide and cause huge economic losses. Transmission through haematophagous vectors (Sanz-Bernardo et al., 2021) and experimentally via indirect contact mode are reported (Aleksandr et al., 2020). During LSDV infection, multifocal cutaneous nodules (0.5-5.0 cm sized) are observed throughout the body with pyrexia, nasal discharges and watery eyes. The morbidity rate is high while mortality is low to moderate (Tuppurainen, Alexandrov, & Beltrán-Alcrudo, 2017). The disease severity depends on the virus strain, competence and availability of vectors in a particular geographical region and immune status, age of the infected animal (Tasioudi et al., 2016; Woods, 1988). The direct economic losses are from reduction in milk production, hide damage, abortions, temporary or permanent sterility in male animals and sometimes death, while indirect losses include diagnostic expenses, costs of disinfection, treatment or vaccinating the sick animals and trade restrictions (Casal et al., 2018). Homologous vaccines (live attenuated LSDV) or heterologous vaccines (live attenuated SPPV or GTPV) are used in different countries (Klement et al., 2020). In India, vaccination started in 2019 for LSD as emergency use in cattle and buffaloes with a heterologous live attenuated vaccine (Uttarkashi strain of GTPV) (Sudhakar, Mishra, Kalaiyarasu, Jhade, & Singh, 2022).

For six decades following its first emergence in Zambia in 1929, the disease was restricted to southern and eastern Africa (Namazi & Khodakaram Tafti, 2021; Woods, 1988). By 1988, LSD spread to Egypt and Israel, possibly due to the movement of infected cattle from neighbouring countries (Davies, 1991a). By 2019, LSD outbreaks expanded into South Asia with reports coming from Viet Nam, China, Bangladesh, Nepal and India (Acharya & Subedi, 2020; Flannery et al., 2022; Mathijs et al., 2021; Sudhakar et al., 2020). The first LSD outbreak in India was in August 2019 from the state of Odisha, with a morbidity rate of 7.1% and no mortality (Sudhakar et al., 2020). Subsequently, between August and December of 2019, the disease was reported in the neighboring state of Jharkhand and later was reported in many other parts of the country (K, B, & K, 2021; N. Kumar et al., 2021).

Previous reports of phylogenetic analyses of the 2019 Indian strains of LSDV based on genes namely, GPCR, RPO30, P32, F, ORF036, and EEV showed a close relationship between the strains and the Kenyan NI-2490/Kenya/KSGP-like field strains (Sudhakar et al., 2020). At the time of submission of this report, severe LSD outbreaks are being mentioned by the media in various parts of India with mortality unlike before. We report here the first complete genome sequence of a pathogenic LSDV directly from a suspected skin biopsy sample collected during an outbreak in 2020 in the Chittoor district of Andhra Pradesh in India.

A skin biopsy sample of a cutaneous nodule was collected under local anaesthesia from a cow exhibiting LSD symptoms in Chittoor district, state of Andhra Pradesh, India, in 2020 and transported on dry ice to the sequencing facility (Genotypic Technology Pvt Ltd, Bengaluru, India).

Genomic DNA was extracted from the skin biopsy and libraries (Illumina and nanopore) were prepared according to the manufacturer’s instructions (Illumina, USA). The hybrid genome assembly for the viral sample was performed using both Illumina and Nanopore data in SPAdes assembler (Antipov, Korobeynikov, McLean, & Pevzner, 2016).

The gene/protein prediction analysis was carried out using Prokka-v1.14, a prokaryotic annotation tool (Seemann, 2014). The predicted proteins were annotated using the DIAMOND BlastP program against Uniprot Viral database (Buchfink, Xie, & Huson, 2015).

The complete genome sequences of 15 reference LSDV strains, two SPPV and two GTPV were mined from NCBI (Table S1). The phylogenetic analyses of the whole genome of LSDV/208/PVNRTVU/2020 was performed by MEGA X software (S. Kumar, Stecher, Li, Knyaz, & Tamura, 2018). To detect recombination event(s), seven recombination detection methods (RDP, GENECONV, Chimera, MaxChi, BootScan, SiScan and 3Seq) were used with Recombination Detection Program (RDP version 4.100) (Martin, Murrell, Golden, Khoosal, & Muhire, 2015). A possible recombination event is indicated if the recombination score was > 0.6. Selection pressures across the genome were determined using DnaSP6 v6.12.03 program (Rozas et al., 2017).

LSD is an endemic disease in African and Middle Eastern countries and has recently spread to other countries (Agianniotaki et al., 2017; Al-Salihi & Hassan, 2015; E. S. M. Tuppurainen et al., 2017). India reported LSD for the first time in 2019 from Odisha (Sudhakar et al., 2020) and later the spread has been rapid to other parts of India. In this study, we analyzed the complete genome sequence of LSDV (LSDV/208/PVNRTVU/2020) from a skin biopsy sample collected from a cow exhibiting typical symptoms of LSD. The complete genome was deposited in GenBank (accession number ON400507). To date, a total of 55 complete genomes of LSDV from across the globe. This is the first complete genome sequence of LSDV of 150 Kbp reported from India, 150445 bp long, with a central coding region of 145937 bp and bounded by two identical 2254 bp long inverted terminal repeats, and an overall GC content of 25.9%. A total of 156 genes were annotated and gene ontology was derived (Supplementary Fig. S1).

The complete genome sequence was compared with 15 reference genomes of LSDV, two GTPV and two SPPV (Table S1). The current sequence showed 99.99% identity with Neethling 2490 and KSGP 0240 LSDV and 97.73% identity with GTPV isolate from India (GenBank ID MN072620). Alignment of GPCR, RPO30, EEV and P32 genes with other Indian LSDV showed a 100% identity with both RPO30 and P32 genes, whereas, GPCR and EEV proteins showed amino acid percentage identities ranging between 99.2–100% and 99.4–100%, respectively. Alignment with sequences of three LSDV/Cattle/India/2019/Ranchi-1 passaged in Vero cells, P-10 (GenBank ID OK422492; 146171 bp), P-30 (GenBank ID OK422493; 147681 bp) or P-50 (GenBank ID OK422494; 139051 bp) revealed 100%, 99.3% and 99.3% identities, albeit only with a coverage of 97%, 98% and 93%, respectively.

A 12-nucleotide insertion in the GPCR gene, observed in all the recent outbreaks of LSDV in Asia, in vaccine-related field viruses from South Africa, in historial strains of LSDV from Kenya isolated before 1960s and in recently reported recombinant LSDVs, was also noticed in the current sequence (Fig. 1A). It is interesting to note that this 12 nucleotide insertion is not present in the circulating field strains of LSDV from Africa, Europe and the Middle East (Agianniotaki et al., 2017; Badhy et al., 2021; Chibssa et al., 2021; Krotova, Byadovskaya, Shumilova, van Schalkwyk, & Sprygin, 2022; Sprygin et al., 2018; van Schalkwyk et al., 2020). Additionally, we detected a duplicated region of 27 bp (9 aa) in EEV gene of LSDV/208/PVNRTVU/2020 sequence, similar to that found in field/virulent LSDV strains but absent in LSDV Neethling vaccine-like viruses, Chinese LSDV strain and recombinant LSDV strains identified in Russia (Erster et al., 2019; Sudhakar et al., 2022) (Fig. 1B) indicating the current sequence as a virulent field strain.

Phylogenetic analysis confirmed two independent subgroups, cluster 1.1, which includes strains related to the original Neethling strain (vaccine) and cluster 1.2 which includes field strains (Krotova et al., 2022). The LSDV/208/PVNRTVU/2020 sequence belonged to cluster 1.2 (0.6% divergence with cluster 1.2 strains) and showed 10% divergence with cluster 1.1 strains (Table 1). It closely clustered with Kenyan strains and formed a distinct clade from field strains of Bulgaria, Serbia, Kazakhstan and South Africa (Fig. 2). This reaffirms the speculated route of the virus spread to India (N. Kumar et al., 2021; Sudhakar et al., 2020).

The selection pressure on the genome was less than 1, indicating a purifying selection playing a role in the virus evolution (Miyata & Yasunaga, 1980) (Supplementary Table S2). We found no recombination events however, with reports of LSDV from countries sharing land borders with India (Acharya & Subedi, 2020; Badhy et al., 2021; Flannery et al., 2022; Mathijs et al., 2021; Sudhakar et al., 2020), it is important that such genomic approaches are undertaken to identify any recombinant vaccine like LSDV.

LSD is a rapidly spreading transboundary animal disease (https://www.fao.org/europe/resources/transboundary-animal-diseases-leaflets/en/). With a recent study suggesting a higher rate of genetic changes in the rampant spreading monkeypox virus (Isidro et al., 2022), a member of the same virus family as that of LSDV, a routine and large-scale virogenomic survey for LSDV is warranted. It is worth noting that India presents a heterogeneous population of several breeds of cattle and buffaloes, as well as vector populations and weather conditions, all of which could potentially influence virus maintenance, transmission and evolution. Availability of genomic sequences is needed to understand the dynamics of disease outbreaks and to plan effective strategies for mitigating the spread and future epidemics, which otherwise could pose economic threat worldwide.

Ethics approval

This research did not involve any human participants or experiments on live animals. The owner of the subject was informed of the purpose of this study and consent was obtained prior to sample collection.

Conflict of interest

The authors declare no competing interests with respect to the research, authorship, and/or publication of this article.

Author Contributions

KP and MS conceived the study. NH, AS, PLR, VKG designed the analysis. PLR, VKG, DD and SM analyzed the data. PLR, VKG, KP and MS interpreted the data and drafted the manuscript. NH, AS, KP and MS critically reviewed and finalized the manuscript.

Data Availability

The complete genome sequence reported in this study is submitted to GenBank. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Funding

Institutional core research funds were utilized for this work.

Acknowledgements

We thank the P V Narsimha Rao Telangana Veterinary University, Hyderabad and the National Institute of Animal Biotechnology, Hyderabad for providing a platform for this work. We express our gratitude to the farmer who participated to provide the clinical sample and the veterinary assistant surgeon who helped us with the sample collection.

Abutarbush, S. M. (2017). Lumpy Skin Disease (Knopvelsiekte, Pseudo-Urticaria, Neethling Virus Disease, Exanthema Nodularis Bovis). Switzerland Springer, Cham.
Acharya, K. P., & Subedi, D. (2020). First outbreak of lumpy skin disease in Nepal. Transbound Emerg Dis, 67(6), 2280–2281. doi:10.1111/tbed.13815
Agianniotaki, E. I., Tasioudi, K. E., Chaintoutis, S. C., Iliadou, P., Mangana-Vougiouka, O., Kirtzalidou, A.,.. . Chondrokouki, E. (2017). Lumpy skin disease outbreaks in Greece during 2015-16, implementation of emergency immunization and genetic differentiation between field isolates and vaccine virus strains. Vet Microbiol, 201, 78–84. doi:10.1016/j.vetmic.2016.12.037
Al-Salihi, K. A., & Hassan, I. Q. (2015). Lumpy Skin Disease in Iraq: Study of the Disease Emergence. Transbound Emerg Dis, 62(5), 457–462. doi:10.1111/tbed.12386
Aleksandr, K., Olga, B., David, W. B., Pavel, P., Yana, P., Svetlana, K.,.. . Alexander, S. (2020). Non-vector-borne transmission of lumpy skin disease virus. Sci Rep, 10(1), 7436. doi:10.1038/s41598-020-64029-w
Antipov, D., Korobeynikov, A., McLean, J. S., & Pevzner, P. A. (2016). hybridSPAdes: an algorithm for hybrid assembly of short and long reads. Bioinformatics, 32(7), 1009–1015. doi:10.1093/bioinformatics/btv688
Badhy, S. C., Chowdhury, M. G. A., Settypalli, T. B. K., Cattoli, G., Lamien, C. E., Fakir, M. A. U.,.. . Sadekuzzaman, M. (2021). Molecular characterization of lumpy skin disease virus (LSDV) emerged in Bangladesh reveals unique genetic features compared to contemporary field strains. BMC Vet Res, 17(1), 61. doi:10.1186/s12917-021-02751-x
Buchfink, B., Xie, C., & Huson, D. H. (2015). Fast and sensitive protein alignment using DIAMOND. Nat Methods, 12(1), 59–60. doi:10.1038/nmeth.3176
Casal, J., Allepuz, A., Miteva, A., Pite, L., Tabakovsky, B., Terzievski, D.,.. . Beltran-Alcrudo, D. (2018). Economic cost of lumpy skin disease outbreaks in three Balkan countries: Albania, Bulgaria and the Former Yugoslav Republic of Macedonia (2016–2017). Transbound Emerg Dis, 65(6), 1680–1688. doi:10.1111/tbed.12926
Chibssa, T. R., Sombo, M., Lichoti, J. K., Adam, T. I. B., Liu, Y., Elraouf, Y. A.,.. . Lamien, C. E. (2021). Molecular Analysis of East African Lumpy Skin Disease Viruses Reveals a Mixed Isolate with Features of Both Vaccine and Field Isolates. Microorganisms, 9(6). doi:10.3390/microorganisms9061142
Davies, F. G. (1991a). Lumpy skin disease of cattle: A growing problem in Africa and the Near East. Animal genetic resources - Ressources génétiques animales - Recursos genéticos animales – 68 1991/3. Retrieved from http://www.fao.org/3/u4900t/u4900T0d.htm
Davies, F. G. (1991b). Lumpy skin disease, an African capripox virus disease of cattle. Br Vet J, 147(6), 489–503. doi:10.1016/0007-1935(91)90019-J
Erster, O., Rubinstein, M. G., Menasherow, S., Ivanova, E., Venter, E., Sekler, M.,.. . Stram, Y. (2019). Importance of the lumpy skin disease virus (LSDV) LSDV126 gene in differential diagnosis and epidemiology and its possible involvement in attenuation. Arch Virol, 164(9), 2285–2295. doi:10.1007/s00705-019-04327-5
Flannery, J., Shih, B., Haga, I. R., Ashby, M., Corla, A., King, S.,.. . Beard, P. M. (2022). A novel strain of lumpy skin disease virus causes clinical disease in cattle in Hong Kong. Transbound Emerg Dis, 69(4), e336-e343. doi:10.1111/tbed.14304
Isidro, J., Borges, V., Pinto, M., Sobral, D., Santos, J. D., Nunes, A.,.. . Gomes, J. P. (2022). Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus. Nat Med. doi:10.1038/s41591-022-01907-y
K, L. K., B, S., & K, R. (2021). Clinico-molecular diagnosis and characterization of bovine lumpy skin disease virus in Andhra Pradesh, India. Trop Anim Health Prod, 53(4), 424. doi:10.1007/s11250-021-02872-3
Klement, E., Broglia, A., Antoniou, S. E., Tsiamadis, V., Plevraki, E., Petrovic, T.,.. . Abrahantes, J. C. (2020). Neethling vaccine proved highly effective in controlling lumpy skin disease epidemics in the Balkans. Prev Vet Med, 181, 104595. doi:10.1016/j.prevetmed.2018.12.001
Krotova, A., Byadovskaya, O., Shumilova, I., van Schalkwyk, A., & Sprygin, A. (2022). An in-depth bioinformatic analysis of the novel recombinant lumpy skin disease virus strains: from unique patterns to established lineage. BMC Genomics, 23(1), 396. doi:10.1186/s12864-022-08639-w
Kumar, N., Chander, Y., Kumar, R., Khandelwal, N., Riyesh, T., Chaudhary, K.,.. . Tripathi, B. N. (2021). Isolation and characterization of lumpy skin disease virus from cattle in India. PLoS One, 16(1), e0241022. doi:10.1371/journal.pone.0241022
Kumar, S., Stecher, G., Li, M., Knyaz, C., & Tamura, K. (2018). MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol, 35(6), 1547–1549. doi:10.1093/molbev/msy096
Martin, D. P., Murrell, B., Golden, M., Khoosal, A., & Muhire, B. (2015). RDP4: Detection and analysis of recombination patterns in virus genomes. Virus Evol, 1(1), vev003. doi:10.1093/ve/vev003
Mathijs, E., Vandenbussche, F., Nguyen, L., Aerts, L., Nguyen, T., De Leeuw, I.,.. . De Clercq, K. (2021). Coding-Complete Sequences of Recombinant Lumpy Skin Disease Viruses Collected in 2020 from Four Outbreaks in Northern Vietnam. Microbiol Resour Announc, 10(48), e0089721. doi:10.1128/MRA.00897-21
Miyata, T., & Yasunaga, T. (1980). Molecular evolution of mRNA: a method for estimating evolutionary rates of synonymous and amino acid substitutions from homologous nucleotide sequences and its application. J Mol Evol, 16(1), 23–36. doi:10.1007/BF01732067
Namazi, F., & Khodakaram Tafti, A. (2021). Lumpy skin disease, an emerging transboundary viral disease: A review. Vet Med Sci, 7(3), 888–896. doi:10.1002/vms3.434
Rozas, J., Ferrer-Mata, A., Sanchez-DelBarrio, J. C., Guirao-Rico, S., Librado, P., Ramos-Onsins, S. E., & Sanchez-Gracia, A. (2017). DnaSP 6: DNA Sequence Polymorphism Analysis of Large Data Sets. Mol Biol Evol, 34(12), 3299–3302. doi:10.1093/molbev/msx248
Sanz-Bernardo, B., Haga, I. R., Wijesiriwardana, N., Basu, S., Larner, W., Diaz, A. V.,.. . Beard, P. M. (2021). Quantifying and Modeling the Acquisition and Retention of Lumpy Skin Disease Virus by Hematophagus Insects Reveals Clinically but Not Subclinically Affected Cattle Are Promoters of Viral Transmission and Key Targets for Control of Disease Outbreaks. J Virol, 95(9). doi:10.1128/JVI.02239-20
Seemann, T. (2014). Prokka: rapid prokaryotic genome annotation. Bioinformatics, 30(14), 2068–2069. doi:10.1093/bioinformatics/btu153
Sprygin, A., Babin, Y., Pestova, Y., Kononova, S., Wallace, D. B., Van Schalkwyk, A.,.. . Kononov, A. (2018). Analysis and insights into recombination signals in lumpy skin disease virus recovered in the field. PLoS One, 13(12), e0207480. doi:10.1371/journal.pone.0207480
Sudhakar, S. B., Mishra, N., Kalaiyarasu, S., Jhade, S. K., Hemadri, D., Sood, R.,.. . Singh, V. P. (2020). Lumpy skin disease (LSD) outbreaks in cattle in Odisha state, India in August 2019: Epidemiological features and molecular studies. Transbound Emerg Dis, 67(6), 2408–2422. doi:10.1111/tbed.13579
Sudhakar, S. B., Mishra, N., Kalaiyarasu, S., Jhade, S. K., & Singh, V. P. (2022). Genetic and phylogenetic analysis of lumpy skin disease viruses (LSDV) isolated from the first and subsequent field outbreaks in India during 2019 reveals close proximity with unique signatures of historical Kenyan NI-2490/Kenya/KSGP-like field strains. Transbound Emerg Dis, 69(4), e451-e462. doi:10.1111/tbed.14322
Tasioudi, K. E., Antoniou, S. E., Iliadou, P., Sachpatzidis, A., Plevraki, E., Agianniotaki, E. I.,.. . Dile, C. (2016). Emergence of Lumpy Skin Disease in Greece, 2015. Transbound Emerg Dis, 63(3), 260–265. doi:10.1111/tbed.12497
Tulman, E. R., Afonso, C. L., Lu, Z., Zsak, L., Kutish, G. F., & Rock, D. L. (2001). Genome of lumpy skin disease virus. J Virol, 75(15), 7122–7130. doi:10.1128/JVI.75.15.7122-7130.2001
Tulman, E. R., Afonso, C. L., Lu, Z., Zsak, L., Sur, J. H., Sandybaev, N. T.,.. . Rock, D. L. (2002). The genomes of sheeppox and goatpox viruses. J Virol, 76(12), 6054–6061. doi:10.1128/jvi.76.12.6054-6061.2002
Tuppurainen, E., Alexandrov, T., & Beltrán-Alcrudo, D. (2017). Lumpy skin disease field manual –A manual for veterinarians. FAO Animal Production and Health Manual No. 20. (pp. 60).
Tuppurainen, E. S. M., Venter, E. H., Shisler, J. L., Gari, G., Mekonnen, G. A., Juleff, N.,.. . Babiuk, L. A. (2017). Review: Capripoxvirus Diseases: Current Status and Opportunities for Control. Transbound Emerg Dis, 64(3), 729–745. doi:10.1111/tbed.12444
van Schalkwyk, A., Kara, P., Ebersohn, K., Mather, A., Annandale, C. H., Venter, E. H., & Wallace, D. B. (2020). Potential link of single nucleotide polymorphisms to virulence of vaccine-associated field strains of lumpy skin disease virus in South Africa. Transbound Emerg Dis, 67(6), 2946–2960. doi:10.1111/tbed.13670
Woods, J. A. (1988). Lumpy skin disease–a review. Trop Anim Health Prod, 20(1), 11–17. doi:10.1007/BF02239636

Table 1 is available in the Supplementary Files section.

No competing interests reported.

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Editorial decision: Accepted
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First complete genome sequence of lumpy skin disease virus directly from a clinical sample in South India

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