Molecular characterization, phylogenetic analysis and viral load quantification of Lumpy Skin Disease Virus in Cattle

doi:10.21203/rs.3.rs-3220257/v1

Download PDF

Research Article

Molecular characterization, phylogenetic analysis and viral load quantification of Lumpy Skin Disease Virus in Cattle

https://doi.org/10.21203/rs.3.rs-3220257/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Lumpy skin disease is an emerging, remerging and trans-boundary disease caused by lumpy skin disease virus (LSDV) belongs Capripoxvirus genus and Poxviridae family in cattle. The recent episodes of disease outbreak in last 4 years from 2019 to 2023 caused huge socio-economic loss. In the present study, clinical samples from LSD outbreaks were collected from different districts of Karnataka, India for virus isolation and molecular characterization. Preliminary confirmation of LSD virus was performed by targeting envelope protein p32 (192 bp) gene by conventional Polymerase chain reaction (PCR). Real-Time qPCR was developed for absolute quantification of viral copy numbers in the infected samples. The viral copy number Log10 (Mean ± SD) in majority of the representative samples ranged from 4.962 to 12.432. The partial genes of GPCR and P32 were sequenced and submitted to GenBank. Phylogenetic analysis was performed by comparing sequences with other LSDV, Sheep pox virus (SPPV) and Goat pox virus (GPPV). The results indicated that our isolates have similarity with the LSDV sequences from Nepal, Bangladesh, Botswana, Egypt, Kenya Turkey and Saudi Arabia. It was also found that one of our isolate (OQ383668) clustered with the GPPV with 100% similarity. Our results highlight the necessity of ongoing surveillance and molecular characterization of circulating LSDV strains required for perspective development of suitable LSD vaccine to control the disease.

Lumpy Skin Disease

p32 gene

GPCR gene

Real Time qPCR

Phylogenetic analysis

Epidemiology

Lumpy skin disease (LSD) is an emerging and re-emerging, transboundary disease caused by the Lumpy Skin Disease Virus in cattle (LSDV) [1]. Recent LSD outbreaks in India and other countries caused huge socio-economic losses [2]. Due to its potential to spread quickly and its economic importance, LSD has been notified by the World Organisation for Animal Health [3]. LSD was first reported in Zambia in 1929 which later appeared across most African countries [4, 5]. LSD was also reported in other countries of the Middle East and Asian continents [6, 7, 8].

LSD can affect cattle, water buffalo and wild ruminants, however, sheep and goats are not apparently infected by the virus [9, 10]. LSDV survives long time in the environment at ambient temperature, especially in dry scabs. LSDV remains infective from the different skin lesions of affected animals such as necrotic skin nodules, dried scabs and desiccated crusts. The virus can be rendered inactive at temperatures of 65°C for 30 minutes and 55°C for 2 hours [11]. Skin lesions are considered as a primary source of infection. However, the virus was found secreted through nasal and lachrymal secretions, saliva, semen, and milk (REF). According to MacLachlan and Dubovi, 2011[12], blood-sucking insects are the primary vectors for the spread of LSDV and direct virus transmission is uncommon in the early stages of the illness [13, 14].

LSDV belongs to the Capripox genus of Poxviridae family with a genome of double-stranded DNA of around 150 kilobase pairs (kbp). LSDV is related to the sheep pox virus (SPPV) and goat pox virus (GTPV) [15, 16, 17] Although, Capripoxviruses are generally considered to be host-specific, SPPV and GTPV strains can naturally or experimentally cross-infect and cause disease in both host species. In contrast, LSDV can experimentally infect sheep and goats, but no natural infection of sheep and goats with LSDV has been reported [16].

The implementation of LSD eradication and control methods requires an understanding of molecular epidemiology. Critical LSDV data, such as the degree of transboundary circulation, diseases hotspot locations and the origins of LSDV [18], are provided through genetic characterization and phylogenetic study of LSDV during outbreaks. Several investigators have employed the GPCR gene sequences for the characterization and molecular epidemiology of LSDV [18, 19, 20, 21]. According to an analysis in Uganda which employed GPCR gene sequencing, most of the LSDV strains are closely related to the viruses seen in adjacent East African nations and recent outbreaks in Europe. The P32 gene is a conserved gene among CaPVs and not only plays a role in the pathogenicity and diagnosis of LSDV but also in the genetic characterization and the identification of the virus’ origin through sequencing and phylogenetic analysis [22, 23, 24].

The first case of LSD in cattle was reported in India in August 2019 in five coastal districts of Odisha State. Between August and December 2019, LSD outbreaks occurred in various districts of Odisha and West Bengal. Previous research looked at partial gene sequences from a small number of LSDV field strains from India [19]. However, in numerous full-length gene sequences, the genomic characteristics of LSDV isolates from various locations in India during the 2019 outbreaks are not well-characterized. Recent research has revealed that the LSDV implicated in the 2019 outbreaks in Bangladesh and China are phylogenetically different [21]. The genetic analysis of the outbreak's origin LSDV strains provides valuable information about molecular epidemiology and the appropriate selection of the most influential vaccine for disease control.

The present study involved investigation of LSDV outbreaks in the different geographical locations of Karnataka, India. We confirmed the LSDV infection using PCR based amplification of p32 and GPCR gene. Subsequently, viral load in skin scab samples were quantified using Real-time qPCR. The LSDV isolates were subjected to molecular characterization with phylogenetic analysis based on the sequences of p32 and GPCR genes.

Samples collection and Processing

A skin biopsy samples (cutaneous nodules) were collected under local anaesthesia from cattle exhibiting LSD symptoms from different geographical locations of Karnataka, India (Fig 1). The skin nodules were collected aseptically and preserved in viral transport media (VTM) containing 1 % antibiotic and antimycotic solution (Sigma-Aldrich, USA). All the scab materials of LSDV were grounded using sterile mortar and pestle with sterile sand. The suspension (10% w/v) was made in sterile PBS. The homogenated tissue suspension was frozen at -80℃ for 30 minutes and thawed at 37℃ for five times to extrude virus particles from the cells. Subsequently centrifuged at 10,000 rpm for 10 min at +4°C. The clear supernatant fluid was transferred into a sterile microcentrifuge tube and stored at -20 °C till further use.

Histopathology

The skin nodules were collected at different stages of disease development and tested positive according to Conventional PCR. The specimens were fixed in 10% neutral buffered formalin for 7 days. After trimming and embedding in paraffin, 2–3 µm-thick sections were stained with hematoxylin and eosin (H & E) stain

DNA extraction

Genomic DNA of the LSD virus was extracted from scab tissue homogenate supernatant using the DNeasy Blood & Tissue kit (Cat no-69504, QIAGEN, Germany) following the manufacturer’s instructions with some modifications. Briefly, 300µl of scab tissue supernatant was passed through a 0.45μm syringe filter (Millipore) and used as starting material with 10 min of lysis step at 56^oC followed by washing and the DNA was eluted in 50μl of elution buffer included in the kit. The purity and the concentration of the DNA samples were estimated using the micro volume UV spectrophotometer (Eppendorf, USA) at 260 nm and 280 nm with the elution buffer as blank.

Lumpy Skin Disease Virus Confirmation

The presence of LSD virus in outbreak investigated samples were confirmed by using forward primer 5’-TCCGAGCTCTTTCCTGATTTTTCTTACTAT-3’and reverse primer 5’-TATGGTACCTAAATTATATACGTAAATAAC-3’ as per OIE manual (Ireland and Binepal, 1998). To amplify the p32 gene, the PCR reactions were prepared in a reaction volume of 25 μL containing 12.5 μL of 2x DreamTaq green PCR Master Mixture (Thermo scientific, USA), 5.5 μL nuclease free water (NFW), 5.0 μL of the extracted DNA template and 1.0 μL of each primer of 10 μM concentration. Then, the PCR reactions were conducted under the following thermal conditions, including an initial denaturation 95°C for 2 min, followed by 35 cycles of denaturation (95°C for 45 sec), annealing (55°C for 50 sec), elongation (72°C for 60 sec) and final elongation (72°C for 2 min). After that, the PCR products were resolved using 2% agarose gel against 100 bp DNA ladder (Sigma-Aldrich, USA) at 100 V in 1X Tris-Boric acid-EDTA (TBE) buffer containing 0.5 μg/ SYBR Safe DNA gel stain (Invitrogen, USA) for about 45 to 60 min. Further gel images were captured and analysed under GelDoc Go Imaging System, (Bio-Rad, USA).

Amplification of GPCR and P32 genes

Further for phylogenetic molecular characterization of lumpy skin disease virus isolates, new primer were designed for partial amplification of GPCR and P32 genes. GPCR_forward: 5'-TGAGCATAGTCGATATCCCACA-3′, GPCR_reverse; 5′-CTGCAAGGTTGACAAATCGTAA-3': p32 forward 5'-CGTTGGTCGCGAAATTTCAG-3′, p32_reverse: 5′-TGAGCCATCCATTTTCCAACTC-3′ with amplicon sizes of 786 bp and 759 bp of respectively. The PCR was performed for DNA sequencing in a reaction volume of 50 μL containing 25 μL of Ampliqon Red dye (Ampliqon, Denmark), 11 μL nuclease free water (NFW), 10.0 μL of the extracted DNA template and 2.0 μL of each primer of 10 μM concentration. Then, the PCR reactions were conducted under the following thermal conditions, including an initial denaturation 95°C for 5 min, followed by 35 cycles of denaturation (95°C for 45 sec), annealing (58°C for 45 sec), elongation (72 °C for 60 sec) and final elongation (60°C for 60 sec). After that, the PCR products were resolved using 1.5% agarose gel containing 0.5 μg/ SYBR Safe DNA gel stain (Invitrogen, USA) along with 100 bp DNA ladder (Sigma-Aldrich, USA) in 1X Tris-Boric acid-EDTA (TBE) buffer at 100 V for 45 to 60 min. Further gel images were captured and analysed under GelDoc Go Imaging System, (Bio-Rad, USA).

Quantification of viral load using Real-time qPCR

The standard curve was prepared using known quantity of purified product of partial GPCR gene as a template (786 bp). In brief, the purified GPCR gene amplicon was quantified (Nanodrop, Eppendorf) to calculate the copy number. The Linear standard curves (efficiency 95-100%) were then generated by serial diluting purified viral DNA. The tenfold dilutions of the standards were prepared from the known concentration in triplicates to generate a standard curve. For quantification, the new qPCR primers were designed within the region of amplified GPCR gene. (Forward 5’-GTTACAACGCCTTCAACTTATG-3’ and Reverse 5’-GGATATCGACTATGCTCACTTC-3’). The real-time PCR reactions were prepared in a total volume of 20μl containing 10μl of SSO advance universal SYBR green Supermix (Bio-Rad, USA), 0.5μl of each forward and reverse primers, 7.0μl of nuclease‐free water and 1.0μl of template DNA. The real-time PCR reactions were carried out in CFX96 Real-Time PCR System (Bio-Rad, USA) using above mentioned qPCR primers. Thermal cycler conditions were set as follows: 1 cycle at 98°C for 3 minutes, followed by 39 cycles at 98°C for 15 seconds and 57°C for 30 seconds using automatic settings for threshold and baseline. Appropriate positive and negative controls were also included in the assay.

Sequencing and phylogenetic analysis

Following agarose gel electrophoresis on 1.5% agarose gel, the PCR amplicons were identified as the expected size using a molecular-marker. The PCR amplicon reaction mixture was bulk produced for 50 μL. All the GPCR and p32 gene amplicons were purified by using PCR purification kit (Cat no-28104, QIAGEN, Germany) as per manufacturer’s protocol. Sanger sequencing was performed at M/S Barcode Bioscience Private Limited, Bangalore. The sequences obtained were trimmed based on the quality and assembled into consensus sequences using Molecular Evolutionary Genetics Analysis software (MEGA version 11, Pennsylvania State University, USA) and SerialCloner software version 2.6.1 (Cambridge University, UK). The similarity of the trimmed sequences were then tested with other GPCR and p32 gene sequences of LSDVs in GenBank using the National Center for Biotechnological Information’s (NCBI) and web-based Basic Local Alignment Search Tool (BLAST). LSDV specific of GPCR and p32 nucleotide sequences were also checked by translating the sequences to amino acid sequences using ExPASy online software (SIB, Swiss Institute of Bioinformatics, Switzerland). Multiple sequence alignment was done using MEGA 11 software. The representative of LSDV sequences of South Africa, Kenya, Namibia, Saudi Arabia, Egypt, Turkey, Thailand, China, Bangladesh, Nepal, Vietnam, Taiwan and India from NCBI. Sequences were also selected from established LSDV vaccine strains, as well as those from the goat pox and the sheep pox viruses and vaccine strains. Then, a phylogenetic tree was generated using the Maximum Likelihood method in MEGA 11 and evolutionary distance was calculated [26, 27].

Statistical Analysis

The normality of the Ct values obtained with these methods was tested with Kolmogorov-Smirnov test and the homogeneity of variances with Leven’s test. T-test for paired samples was run to evaluate the significance of differences between the Ct values obtained.

Characteristic symptoms and lesions found in LSDV outbreaks

The disease investigation was done in the districts of Bagalkot, Shimoga, Davanagere, Chitradurga, and Tumkur in the years 2021 and 2022 (Fig. 1). The majority of the cattle that were evaluated displayed the typical clinical symptoms of LSD, including fever, salivation, edema in limb, formation of maggot wound, brisket edema, enlargement in prefemoral lymph node, necrotic skin nodule, formation of generalized skin nodules in the whole body and Animal become weak, develops downer and chronic disease conditions (Fig. 2). Histopathological analysis of nodular skin lesion showed that there were infiltration of mononuclear cells, hyperkeratosis with spongiosis of epithelial cells, higher magnification of C with presence eosinophilic inclusion bodies Epithelial cells undergoing hyperplasia and hydropic degeneration cellular, Hyperkeratosis with hyperplasia of epithelial cells and hair follicular cells, infiltration of mononuclear cells especially macrophages with arterial degeneration.

Histopathology revealed defects in skin epithelial cells.

Histopathological analysis of nodular skin lesion showed that there were infiltration of mononuclear cells, hyperkeratosis with spongiosis of epithelial cells, further in higher magnification the presence eosinophilic inclusion bodies were typically observed. In skin, epithelial cells undergoing hyperplasia and hydropic cellular degeneration, hyperkeratosis with hyperplasia of epithelial cells and hair follicular cells. LSDV infected skin revealed infiltration of mononuclear cells especially macrophages with arterial degeneration were clearly evident (Fig.11).

LSDV confirmed in the outbreak samples

A total of 134 samples were tested by conventional PCR for the presence of LSDV. The samples included 7 blood, 6 nasal swab, and 121 skin scabs. The analysis of results revealed the presence of LSDV in blood (14.28 %), skin sab (95.04 %) and nasal swab (66.66%). The representative positive samples showed for p32 gene and GPCR amplification (Fig. 3 to 5)

Different virus loads detected in LSDV outbreak samples

The LSDV-specific SYBR green based real-time qPCR assay was found more sensitive than the conventional Capripoxvirus generic PCR, In this assay, copy number of virus in infected skin scab materials were estimated and LSDV vial load in terms of copy number depicted in the table 3. The Ct values ranged from 6.81 to 23.23 for LSDV-positive skin scab, 26.01 for LSDV-positive blood, 24.12 for LSDV-positive nasal swab, and skin samples examined for asymptomatic animals 30.16 (n=6) . Subsequently viral load were estimated using linear regression curve through slope y= -3.2678x + 40.588 (R²= 0.9994) with efficiency of 102.5% (Fig. 6). The sample code TS-604 and TS-2showed highest viral copy number Log10 (Mean ± SD) 12.432±0.080 and 11.439±0.005. The sample code A1 show lowest copy number Log10 (Mean ± SD) 4.962±0.000 and majority of the representative samples showed average viral copy number of Log10 (Mean ± SD) 10.341± 0.080.

LSDV showed Molecular evolutionary relationship with SPPV and GTPV

In the present study 20 positive LSDV samples for both GPCR and p32 gene were sequenced and comparatively analysed with other field strains of LSDV vaccine strains of goatpox (GTPV) and sheeppox (SPPV) retrieved from the NCBI data base. Multiple sequence alignments of the GPCR gene revealed that 19 isolates of outbreak strain of the LSD viruses showed single nucleotide mutation in the sample belonging to the Haveri, Chitradurga, and Tumkur districts of Karnataka (sample coded as: D1, D2, D5, D7&8, TS-625, S1, S2, TS-2, TS-6, TS8, TS-9, TS-603, TS-604, TS-605, TS-606 and TS-607) as shown in Fig. 7. In contrast, the P32 gene 15 LSDV outbreak strains were aligned with other LSDV field strains, SPPV and GTPV strains in which single nucleotide mutation was observed from the sample obtained from Haveri, Chitradurga, and Tumkur districts of Karnataka, (sample coded as: D1, D5, TS-604, TS-605 and TS-6) as shown in Fig. 8.

The phylogenetic analysis GPCR gene tree 18 LSD outbreak isolates in Karnataka were closely related to other LSDV sequences from Turkey, Arabia, Bangladesh, Nepal, Tamilnadu, West Bengal, Odisha, Egypt, Kenya and Botswana with homology varied among LSDV, SPPV and GTPV. The Bagalkot isolate clustered (A1) with the Goat pox virus isolate Mukteshwar/1946 and exhibit 100% similarity (Fig. 9). Whereas in the P32 gene tree the LSDV isolates, goatpox and sheeppox viruses formed a separate clades within the Capripoxvirus family (Fig. 10) with 84% similarity. On the other hand LSD outbreak isolates in Karnataka were closely related to other LSDV sequences acquired from Odisha, West Bengal, Egypt, Saudi Arabia, Taiwan, chain, Africa, Vietnam, and Magnolia.

Table: 1 P32 gene sequences of LSDV submission to NCBI (Genbank)

No	Isolate name	Breed type	Age	Symptoms	Accession numbers
A1	LSDV/IND/Karnataka/Ilkal/2021	ND	3 years	Temp-103 ^⃘F, necrotic wounds in and around the edematous region	OQ410643
D1	LSDV/IND/Karnataka/Haveri/Yattinahalli/2022	Local	5 years	Skin nodules, prescapular lymph node	OQ410642
D2	LSDV/IND/Karnataka/Haveri/Agadi 1/2022	HF cross	4 month	Enlargement in prefemoral lymph, salivation	OQ410644
D3	LSDV/IND/Karnataka/Haveri/Yattinahalli 2 /2022	HF cross	2.5 years	Temp-101.6 ^⃘F, maggot wound, Mild lesions	OQ410645
D5	LSDV/IND/Karnataka/Haveri/Agadi 2 /2022	Jersey Cross	3 years	Edema in limb, Fever, sloughing of skin	OQ410646
D7&8	LSDV/IND/Karnataka/Haveri/Yattinahalli 3 /2022	Hallikar	5.5 years	Temp-103.6 ^⃘F, Mild lesions	OQ410647
TS-625	LSDV/IND/Karnataka/Davanagere/Shiramagondanahalli/2022	ND	2 years	Necrotic skin nodule, salivation	OQ599867
S2	LSDV/IND/Karnataka/Tiptur/2022	Hallikar	4.5 years	Temp-104 ^⃘F, Maggot wound, skin nodules	OQ599866
TS2	LSDV/IND/Karnataka/Tumkur/Gubbi/2022	HF cross	3 years	brisket edema, mild lesion	OQ599861
TS5	LSDV/IND/Karnataka/Tumkur/Koratagere 1/2022	Hallikar	4 years	Skin nodules, maggot wound	OQ599862
TS6	LSDV/IND/Karnataka/Tumkur/Hiryur/2022	HF	11 month	Temp 102.6 ^⃘F, brisket edema	OQ599863
TS8	LSDV/IND/Karnataka/Tumkur/Koratagere 2/2022	Local	4 years	Skin nodule, Edema in limb	OQ599864
TS9	LSDV/IND/Karnataka/Tumkur/Turvekere/2022	Amritmahal	ND	maggot wound, Enlargement in prefemoral lymph node	OQ599865
TS-603	LSDV/IND/Karnataka/Chitradurga/Holalkere/Gunderi/2022	Local	3 years	Necrotic skin nodule, maggot wound	OQ599868
TS-604	LSDV/IND/Karnataka/Chitradurga/Holalkere /Bommanakatte/2022	Local	ND	Edema in limb, salivation	OQ599869
TS- 605	LSDV/IND/Karnataka/Chitradurga/Holalkere/Megalakottige /2022	HFX	8 month	Fever, Salivation, Enlargement in prefemoral lymph node	OQ599870
TS-606	LSDV/IND/Karnataka/Chitradurga/Holalkere/Edehalli 1/2022	Hallikar	1.5 year	Edema in limb, salivation, Skin nodule	OQ599871
TS- 607	LSDV/IND/Karnataka/Chitradurga/Holalkere/Edehalli 2/2022	Local	3.5 years	Skin nodule, Fever, salivation	OQ599872
TS- Bagt2	LSDV/IND/Karnataka/Tumkur/Sira/2022	Amritmahal	2 years	Skin nodule, Edema in limb	OQ599873

Table:2 GPCR gene sequences of LSDV submission to NCBI (Genbank)

No	Isolate Name	Breed type	Age	Symptoms	Accession numbers
A1	LSDV/IND/ Karnataka /Ilkal/2021	ND	3 years	Temp-103 ^⃘F, necrotic wounds in and around the edematous region	OQ383668
D1	LSDV/IND/Karnataka/Haveri/Yattinahalli/2022	Local	5 years	Skin nodules, prescapular lymph node	OQ410638
D2	LSDV/IND/Karnataka/Haveri/Agadi 1/2022	HF cross	4 month	Enlargement in prefemoral lymph, salivation	OQ410639
D3	LSDV/IND/Karnataka/Haveri/Yattinahalli 2 /2022	HF cross	2.5 years	Temp-101.6 ^⃘F, maggot wound, Mild lesions	OQ410640
D5	LSDV/IND/Karnataka/Haveri/Agadi 2 /2022	Jersey Cross	3 years	Edema in limb, Fever, sloughing of skin	OQ410641
D7&8	LSDV/IND/Karnataka/Haveri/Yattinahalli 3 /2022	Hallikar	5.5 years	Temp-103.6 ^⃘F, Mild lesions	OQ434749
TS-625	LSDV/IND/Karnataka/Davanagere/Shiramagondanahalli/2022	ND	2 years	Necrotic skin nodule, salivation	OQ434750
S2	LSDV/IND/Karnataka/Tiptur/2022	Hallikar	4.5 years	Temp 104 ^⃘F, maggot wound, skin nodules	OQ512013
TS2	LSDV/IND/Karnataka/Tumkur/Gubbi/2022	HF cross	3 years	brisket edema, mild lesion	OQ512014
TS5	LSDV/IND/Karnataka/Tumkur/Koratagere/2022	Hallikar	4 years	Skin nodules, maggot wound	OQ512015
TS6	LSDV/IND/Karnataka/Tumkur/Hiryur/2022	HF	11 month	Temp 102.6 ^⃘F, brisket edema	OQ512016
TS8	LSDV/IND/Karnataka/Tumkur/Koratagere 2 /2022	Local	4 years	Skin nodule, Edema in limb	OQ512017
TS9	LSDV/IND/Karnataka/Tumkur/Turvekerer/2022	Amritmahal	ND	maggot wound, Enlargement in prefemoral lymph node	OQ512018
TS-603	LSDV/IND/Karnataka/Chitradurga/Holalkere/Gunderi/2022	Local	3 years	Necrotic skin nodule, maggot wound	OQ512019
TS-604	LSDV/IND/Karnataka/Chitradurga/Holalkere/Bommanakatte/2022	Local	ND	Edema in limb, salivation	OQ512020
TS- 605	LSDV/IND/Karnataka/Chitradurga/Holalkere/Megalakottige/2022	HFX	8 month	Fever, Salivation, Enlargement in prefemoral lymph node	OQ512021
TS-606	LSDV/IND/Karnataka/Chitradurga/Holalkere/Edehalli 1/2022	Hallikar	1.5 year	Edema in limb, salivation, Skin nodule	OQ512022
TS- 607	LSDV/IND/Karnataka/Chitradurga/Holalkere/Edehalli 2/2022	Local	3.5 years	Skin nodule, Fever, salivation	OQ512023
TS- Bagt2	LSDV/IND/Karnataka/Tumkur/Sira/2022	Amritmahal	2 years	Enlargement in prefemoral lymph	OQ539685
S1	LSDV/IND/Karnataka/Koduru/2019	HF cross	4 years	Skin nodule, Edema in limb, Fever	OQ512012

Table: 3 LSDV viral load quantification in skin scab

Sample code	Viral copy number Log₁₀(Mean ± SD)	Sample code	Viral copy number Log₁₀ (Mean ± SD)
D3	11.182 ±0.020	TS-625	10.445±0.005
D2	11.344±0.020	TS-607	11.982±0.003
A1	4.962±0.000	TS-661	10.129±0.008
D1	6.154±1.097	TS-664	11.067±0.003
D5	9.934±0.014	TS-667	9.335±0.009
TS-2	11.439±0.005	TS-675	10.729±0.002
TS-604	12.432±0.080	TS-651	10.982± 0.012
D7&8	10.824±0.069	TS-657	9.724±0.012
S1	10.878±0.031	TS-658	9.543±0.012
S2	11.305±0.041	TS-659	10.241± 0.003

In recent year the world is witnessing a surge in emerging and re-emerging pandemic diseases in both animals and humans [28]. The COVID-19 pandemic is classical example of newly emerging diseases in humans while monkey pox infection can be considered to be re-emerging disease [29, 30]. Similarly, LSD is emerging, non-zoonotic, vector-borne disease of cattle and buffaloes [31]. The present study focused on the recent outbreaks of LSD in southern part of India. We have investigated and confirmed LSDV in these outbreaks. We presented the details of LSDV molecular characterisation, quantification of viral load in infected tissues and phylogenetic analysis of several different LSDV isolates.

The clinical signs of LSD in suspected cases varied according to severity [32]. Earlier reports revealed classical clinical signs of LSD as fever (102–107℉), salivation, edema of all the limbs, necrotic skin nodule, necrotic wounds in and around the edematous region and sloughing of skin elements in some cases [33, 34, 35]. We observed peculiar lesions such as brisket edema, edema in limb, dewlap oedema, formation of maggot wound, enlargement in prefemoral lymph node and common clinical signs like fever, salivation, necrotic skin nodule, formation of generalized skin nodules in the whole body and animal become weak, develops downer and chronic disease conditions.

The effects of LSDV on production in cattle rise from anorexia dropping milk production and pregnant animals abortion in some cases were reported [36, 37]. The indirect impact of LSD causes inter-state and intra-state border restriction of cattle trade that created a huge socio economic losses to the India’s small dairy farmers [38, 36]. We found that substantial economic hardship among livestock producers as a result of lower animal productivity and disease management expenses. It also disrupt the livestock product and by-product trade and compromise food security due to loss of draught power. Severe economic losses may be incurred due to condemnation of carcass as it damages the hides.

The PCR assay is a specific, sensitive and preferred method for detection of LSDV in clinical samples such as skin scab, nasal swab and blood. [39, 18]. PCR amplification of the p32 gene is the OIE recommended test for LSDV confirmation [25]. In the present study, samples were received from the various districts of Karnataka, India during the outbreaks in the year of 2019 to 2023. The confirmed cases of LSDV revealed 14.28% of positivity in blood, 95.04% of positivity in skin scab and 66.66% of positivity in nasal swab by specific conventional PCR. Similarly the higher percentage of scab samples were found positive for LSDV in this study are in agreement with result of recent reports [40, 41, 42, 43, 44, 27]. Moreover, skin nodule/scab samples are the most preferred sample types for LSD diagnosis and LSDV genome can be detected in skin lesions up to 92 days after infection [40].

Real-time PCR is a fast, sensitive and specific approach for confirming Capripox viruses, including LSDV. One-third of skin scab samples had higher CT values, proving that skin scab material had rich viral load than other samples [45]. All LSDV lineages, including those that are now circulating in Africa, the Middle East, and Asia, could be detected and quantified using the Real-time PCR method. The new assay developed in the present study was simple, sensitive, rapid and affordable to perform similar to the methods reported earlier [45, 46, 45]. We focused to quantify the viral load in infected skin scab materials and the copy number of virus infected in the same were estimated found that majority of the representative samples ranged from 0.367 x10¹⁰ to 3881.15 x 10¹⁰. The qPCR can detect low amounts of viral genomes in skin biopsy, nasal swab and blood samples. Therefore, the SYBR green based viral load quantification assay presented here serves as an easy to detect and quantify LSDV in clinical samples. Real-Time PCR assay could be a new addition to the list of authorised tests provided by the World Animal Health Organisation.

The phylogenetic study of currently circulating LSDV outbreak strains provides important information on the origin of the outbreak, vaccine design and the appropriate selection of the most effective vaccine for disease control [35]. Here, we carried out phylogenetic analysis of twenty LSDV outbreak samples targeting p32 and GPCR gene. The available sequence of other field and vaccine strains of LSDV, SPPV and GTPV were included in the analysis for comparision [32, 47]. The phylogenetic tree constructed for GPCR gene revealed similarity between the isolates obtained from the Haveri Davanagere, Chikkamagaluru and Tumkur. These were also clustered with LSDV isolates from Saudi Arabia, Egypt, Kenya, Botswana, Bangladesh and Turkey. Similar findings were reported for GPCR gene sequence analysis for earlier LSDV/Egypt/2016 isolates from Egypt [32]. According to this data, the conserved nature of the LSDV genome is changing and leading to variations in the sequences. This may exacerbate the severity of the disease in cattle, as evidenced by recent outbreaks in the Indian state of Karnataka. The LSDV isolates from Bagalkot districts (A1) exhibit 100% similarity with the Goat pox virus isolate of Mukteshwar/1946, India.

The phylogenetic tree analysis of the LSDV strain based on the p32 gene also showed that they shared significant similarities with earlier LSDV strains from such as Egypt and Saudi Arabia. Our LSDV isolates also showed 81% homology with SPPV and GTPV. The study of P32 gene sequencing and phylogeny make it possible to distinguish the local LSDVs and differentiate between the Capripox viruses [32, 47]. Similarly, several recent studies on the p32, RPO30, F, A33 and GPCR gene of LSDV have been reported [47, 48]. It was reported that p32 gene is the most suitable for studying the genetic relationship between different Capripox viruses including LSDV [49, 23, 24, 50].

It is important to note that the field strains and vaccine strains of LSDV, SPPV and GPPV strains were grouped in identical and separate clusters. Similar findings were reported in the earlier studies [51, 52, 32].

In recent outbreak in Karnataka, India evidenced the conserved nature of the LSDV genome is changing and leading to variations in the sequences. This might be contributing increased severity of disease and mortality in cattle. Therefore, focused and vigorous molecular epidemiological research will be required to investigate the genetic variations of LSDV. This will facilitate for better control of the disease by suitable modifications in the vaccine formulations.

Ethics statements

In the present study no animal experiment was conducted as the clinical samples were obtained from the outbreak investigations. Our study does not require animal ethical committee approval from the competent authority.

Authorship contribution statement

Sanganagouda K: Conceptualization, Investigation, Methodology, Software, Writing – original draft, Visualization. Nagraja K: Investigation, Data curation, Basavaraj Sajjanar: Conceptualization, Writing – original draft, Visualization, Writing – review & editing. Sabha Kounin: Methodology, Visualization, Writing. Amitha Rena Gomes: Validation, Data curation. Pavithra B.H: Data Curation. Sanjeevakumar Lalasangi: Validation, Methodology. Sumathi B. R: Funding acquisition. Shivaraj Murag: Writing – review & editing. Shankar B P: Methodology. Anjan Kumar: Software, Methodology. Arun Kharate: Formal analysis. Muniyellappa H.K: Writing – review & editing. M.D Venkatesha: Supervision, Project administration. Raveendra Hegade: Project administration, funding acquisition,

Declaration of Competing Interest

All authors declared no conflict of interest.

Acknowledgments

This study was supported by Karnataka Veterinary, Animal and Fisheries Sciences University, BIDAR and Institute of Animal Health and Veterinary Biologicals (IAH&VB), KVAFSU, Hebbal, Bengaluru.

King AMQ, Adams MJ, Carstens EB, Lefkowitz E (2012) Virus Taxonomy-Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press: Amsterdam The Netherlands 291-309.
Anwar A, Na-Lampang K, Preyavichyapugdee N, Punyapornwithaya V (2022) Lumpy Skin Disease Outbreaks in Africa, Europe, and Asia (2005–2022): Multiple Change Point Analysis and Time Series. Forecast Viruses 14(10):2203. doi: 10.3390/v14102203
OIE (2021) Terrestrial Manual: Lumpy skin disease. OIE terrestrial manual 3(4):12.
Macdonald RAS (1930) Pseudourticaria of cattle. Northern Rhodesian Department of Animal Health Annual Report. 20–21.
Wainwright S, El Idrissi A, Mattioli R, Tibbo M, Njeumi F, Raizman E (2013) Emergence of lumpy skin disease in the Eastern Mediterranean Basin countries. FAO Empres Watch 29:1- 6.
Tuppurainen E, Alexandrov T, Beltrán-Alcrudo, D (2017) Lumpy Skin Disease - Field Manual for Veterinarians.
Sohier C, Haegeman A, Mostin L, De Leeuw I, Campe WV, De Vleeschauwer A (2019) Experimental evidence of mechanical lumpy skin disease virus transmission by Stomoxys calcitrans biting flies and Haematopota spp. Horseflies Sci Rep 9 (1):20076. doi: 10.1038/s41598-019-56605-6
Azeem S, Sharma B, Shabir S, Akbar H, Venter E (2022) Lumpy skin disease is expanding its geographic range: A challenge for Asian livestock management and food security. Vet J 279:105785. doi: 10.1016/j.tvjl.2021.105785
EI-Nahas EM, El-Habbaa AS, El-Bagoury GF, Radwan MEI (2011) Isolation and identification of lumpy skin disease virus from naturally infected buffaloes at Kaluobia. Egypt Glob Vet 7(3):234-237.
Lamien CE, Le Goff C, Silber R, Wallace DB, Gulyaz V, Tuppurainen E, Madani H, Caufour P, Adam T, El Harrak M, Luckins AG, Albina E, Diallo A (2011) Use of the Capripox virus homologue of Vaccinia virus 30 kDa RNA polymerase subunit (RPO30) gene as a novel diagnostic and genotyping target: development of a classical PCR method to differentiate Goat poxvirus 'from Sheep poxvirus. Vet Microbiol 149(1-2): 30-9. doi: 10.1016/j.vetmic.2010.09.038.
Mulatu L and Feyisa (2018) Review: Lumpy Skin Disease. J Vet Sci Techno 9(03):1-8. doi:10.4172/2157-7579.1000535
MacLachlan, N. J., and Dubovi, E. J. (2011). Fenner’s veterinary virology, 4th ed. Academic Press.
Magori-Cohen R, Louzoun Y, Herziger Y, Oron E, Arazi A, Tuppurainen E, Shpigel NY, Klement E (2012) Mathematical modelling and evaluation of the different routes of transmission of lumpy skin disease virus. Veterinary bold research 43(1):1-13. doi: 10.1186/1297-9716-43-1
Carn VM, Kitching RP, Hammond JM, Chand P (1994) Use of a recombinant antigen in an indirect ELISA for detecting bovine antibody to capripox virus. Journal of virological methods 49(3):285-294. doi: 10.1016/0166-0934(94)90143-0
Givens MD (2018) Review: Risks of disease transmission through semen in cattle. Animal 12(S1): 165–171. doi: 10.1017/S1751731118000708.
Bhanuprakash V, Indrani BK, Hosamani M, Singh RK (2006) The current status of sheep pox disease. Comparative Immunology Microbiology and Infectious Diseases 29:27–60. doi: 10.1016/j.cimid.2005.12.001.
Buller RM, Arif BM, Black DN, Dumbell KR, Esposito JJ, Lefkowitz EJ, McFadden G, Moss B, Mercer AA, Moyer RW, Skinner MA, Tripathy DN (2005) Family Poxviridae. In Fauquet C. M., Mayo M. A., Maniloff J., Desselberger U., and Ball L. A. (Eds.), Virus taxonomy: Classification and nomenclature of viruses. Eighth Report of the International Committee on Taxonomy of Viruses 117–133.
Ochwo S, Vander Waal K, Munsey A, Nkamwesiga J, Ndekezi C, Auma E, Mwiine FN (2019) Seroprevalence and risk factors for lumpy skin disease virus seropositivity in cattle in Uganda. BMC Vet Res 15(1):236. doi:10.1186/s12917-019-1983-9
Sudhakar SB, Mishra N, Kalaiyarasu S, Jhade SK, Hemadri D, Sood R (2020) Lumpy skin disease (LSD) outbreaks in cattle in Odisha state, India in August 2019: Epidemiological features and molecular studies. Transbound Emerg Dis 67:2408–2422. doi: 10.1111/tbed.13579
Sprygin A, Pestova Y, Bjadovskaya O, Prutnikov P, Zinyakov N, Kononova S, Ruchnova O, Lozovoy D, Chvala I, Kononov A (2020) Evidence of recombination of vaccine strains of lumpy skin disease virus with field strains, causing disease. PLoS ONE 15:0232584. doi.org/10.1371/journal.pone.0232584
Badhy SC, Chowdhury M, Settypalli T, Cattoli G, Lamien CE, Fakir M, Akter S, Osmani MG, Talukdar F, Begum N (2021) Molecular characterization of lumpy skin disease virus (LSDV) emerged in Bangladesh reveals unique genetic features compared to contemporary field strains. BMC Vet Res 17:61. doi: 10.1186/s12917-021-02751-x.
Hosamani M, Mondal B, Tembhurne PA, Bandyopadhyay SK, Singh RK, Rasool TJ (2004) Differentiation of sheep pox and goat poxviruses by sequence analysis and PCR-RFLP of P32 gene. Virus genes 29: 73-80. doi: 10.1023/B:VIRU.0000032790.16751.13.
Mafirakureva P, Saidi B, Mbanga J (2017) Incidence and molecular characterisation of lumpy skin disease virus in Zimbabwe using the P32 gene. Tropical animal health and production 49:47-54. doi: 10.1007/s11250-016-1156-9.
Rashid P, Sheikh, MB, Raheem Z, Marouf A (2017) Molecular characterisation of lumpy skin disease virus and sheeppox virus based on p32 gene. Bulgarian Journal of Veterinary Medicine 20:131-140. doi: 10.15547/bjvm.984
Ireland DC, Binepal YS (1998) Improved detection of capripoxvirus in biopsy samples by PCR. J Virol Methods 74:1–7. doi: 10.1016/s0166-0934(98)00035-4.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular biology and evolution, 30(12):2725–2729. doi: 10.1093/molbev/mst197
Singhla T, Boonsri K, Kreausukon K, Modethed W, Pringproa K, Sthitmatee N, Punyapornwithaya V, Vinitchaikul P (2022) Molecular Characterization and Phylogenetic Analysis of Lumpy Skin Disease Virus Collected from Outbreaks in Northern Thailand in 2021. Veterinary Sciences 9(4):194.
Morens DM, Fauci AS (2013) Emerging infectious diseases: threats to human health and global stability. PLoS Pathog 9:1003467. doi: 10.1371/journal.ppat.1003467
Excler JL, Saville M, Berkley S (2021) Vaccine development for emerging infectious diseases. Nat Med 27:591–600. doi:10.1038/s41591-021-01301-0
Kipkorir V, Dhali A, Srichawla B, Kutikuppala S, Cox M, Ochieng D (2022) The re-emerging monkeypox disease. Trop Med Int Health 27(11):961– 969. doi:10.1111/tmi.13821
Hamdi J, Boumart Z, Daouam S, El Arkam A, Bamouh Z, Jazouli M, Tadlaoui KO, Fihri OF, Gavrilov B, El Harrak M (2020) Development and Evaluation of an Inactivated Lumpy Skin Disease Vaccine for Cattle. Vet Microbiol 245:108689. doi: 10.1016/j.vetmic.2020.108689.
Selim Abdelfattah, Eman Manaa, Hanem Khater (2021) Molecular characterization and phylogenetic analysis of lumpy skin disease in Egypt. Comparative Immunology, Microbiology and Infectious Diseases 79. doi: 10.1016/j.cimid.2021.101699.
El-Sherif A, Samir S, Azam R, Roby SR (2010) Epizootiology of lumpy skin disease outbreak in cattle in middle of Egypt, 2006. J Vet Med Res 20(1):364–373. doi:10.21608/jvmr.2020.77640
Elmohsen Abd M, Selim A, Abd Elmoneim AE (2019) Prevalence and molecular characterization of Lumpy Skin Disease in cattle during period 2016-2017. Benha Veterinary Medical Journal 37(1):172-175. doi:10.21608/bvmj.2019.18293.1118
Ochwo S, VanderWaal K, Ndekezi C, Nkamwesiga J, Munsey A, Witto SG, Nantima N, Mayanja F, Okurut A, Atuhaire DK (2020) Molecular detection and phylogenetic analysis of lumpy skin disease virus from outbreaks in Uganda 2017–2018. BMC Vet Res 16:66. doi: 10.1186/s12917-020-02288-5.
Kumar N, Barua S, Kumar R, Khandelwal N, Kumar A, Verma A, Singh L, Godara B, Chander Y, Kumar G, Riyesh T, Sharma DK, Pathak A, Kumar S, Dedar RK, Mehta V, Gaur M, Bhardwaj B, Vyas V, Chaudhary S, Tripathi BN. (2023) Evaluation of the safety, immunogenicity and efficacy of a new live-attenuated lumpy skin disease vaccine in India. Virulence 14(1):2190647. doi: 10.1080/21505594.2023.2190647
CFSPH. The Center for Food Security and Public Health. Ames, IA, USA: Iowa State University, College of Veterinary Medicine (2008)
Elhaig MM, Selim A, Mahmoud M (2017) Lumpy skin disease in cattle: frequency of occurrence in a dairy farm and a preliminary assessment of its possible impact on Egyptian buffaloes. Onderstepoort J Vet Res 84(1):1–6. doi: 10.4102/ojvr.v84i1.1393.
Babiuk S (2018) Clinical Signs, Lumpy Skin Disease. Springer 65–69.
Tuppurainen ESM, Venter EH, Coetzer JAW (2005) The detection of lumpy skin disease virus in samples of experimentally infected cattle using different diagnostic techniques. Onderstepoort Journal of Veterinary Research 72:153–164. doi: 10.4102/ojvr.v72i2.213.
Awad WS, Ibrahim AK, Mahran K, Fararh KM, Abdel Moniem MI (2010) Evaluation of different diagnostic methods for diagnosis of Lumpy skin disease in cows. Tropical Animal Health and Production 42 777–783. doi: 10.1007/s11250-009-9486-5
Adedeji A, Adole J, Dogonyaro B, Kujul N, Tekki I, Asala O, Chima N, Dyek Y, Maguda A, Aba-Adulugba E (2017) Recurrent outbreaks of lumpy skin disease and its economic impact on a dairy farm in Jos Plateau State Nigeria Niger. Vet J 38(2):151–158.
Sprygin, A, Pestova, Y, Prutnikov, P, Kononov A (2018) Detection of vaccine-like lumpy skin disease virus in cattle and Musca domestica L. flies in an outbreak of lumpy skin disease in Russia in 2017. Transboundary and Emerging Diseases 65:1137–1144. doi: 10.1111/tbed.12897
Li L, Qi C, Li J, Nan W, Wang Y, Chang X, Chi T, Gong M, Ha D, De J, Ma L and Wu X (2022) Quantitative real-time PCR detection and analysis of a lumpy skin disease outbreak in Inner Mongolia Autonomous Region, China. Front Vet Sci 9:936581. doi:10.3389/fvets.2022.936581
Agianniotaki EI, Chaintoutis SC, Haegeman A, Tasioudi KE, De Leeuw I, Katsoulos PD, Sachpatzidis A, De Clercq K, Alexandropoulos T, Polizopoulou ZS, Chondrokouki ED, Dovas CI (2017) Development and validation of a TaqMan probe-based real-time PCR method for the differentiation of wild type lumpy skin disease virus from vaccine virus strains. J Virol Methods 249:48-57. doi: 10.1186/s13104-019-4412-z
Agianniotaki EI, Chaintoutis SC, Haegeman A, De Clercq K, Chondrokouki E, Dovas CI (2021) A TaqMan probe-based multiplex real-time PCR method for the specific detection of wild type lumpy skin disease virus with beta-actin as internal amplification control. Mol Cell Probes 60:101778. https://doi.org/10.1016/j.mcp.2021.101778.
Elhaig MM, Almeer R, Abdel-Daim MM (2021) Lumpy skin disease in cattle in Sharkia, Egypt: epidemiological and genetic characterization of the virus. Trop Anim Health Prod 53:287. doi: 10.1007/s11250-021-02711-5
Sudhakar SB, Mishra N, Kalaiyarasu S, Jhade SK, Singh VP (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. Transboundary and Emerging Diseases, 69:451– 462. doi: 10.1111/tbed.14322
Beard P, Sugar S, Bazarragchaa E, Gerelmaa U, Tserendorj S, Tuppurainen E, Sodnomdarjaa R, (2010) A description of two outbreaks of capripoxvirus disease in Mongolia. Veterinary microbiology 142:427-431. doi: 10.1016/j.vetmic.2009.10.018
Yousefi PS, Dalir-Naghadeh B, Mardani K, Jalilzadeh-Amin G (2018) Phylogenetic analysis of the lumpy skin disease viruses in northwest of Iran. Tropical animal health and production 50: 1851-1858. doi: 10.1007/s11250-018-1634-3
Gelaye E, Belay A, Ayelet G, Jenberie S, Yami M, Loitsch A, Tuppurainen E, Grabherr R, Diallo A, Lamien CE (2015) Capripox disease in Ethiopia: genetic differences between field isolates and vaccine strain, and implications for vaccination failure. Antiviral Res 119:28–35. doi: 10.1016/j.antiviral.2015.04.008.
Selim A, Elhaig M, Hoche J, Gaede W (2016) Molecular detection and analysis of Sheeppox and Orf viruses isolated from sheep from Qalubia, Egypt Berl Münch Tierarztl 129:310–317.

No competing interests reported.

Download PDF

Version 1

posted

You are reading this latest preprint version

Molecular characterization, phylogenetic analysis and viral load quantification of Lumpy Skin Disease Virus in Cattle

Status:

Version 1

Abstract

Figures

Introduction

Materials and method

Results

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1