MOLECULAR CHARACTERISATION OF LUMPY SKIN DISEASE VIRUS IN CATTLE USING P32 AND RPO30 GENES IN DUHOK

Lumpy skin disease (LSD) is a contagious viral infection of cattle caused by lumpy skin disease virus (LSDV). This infection has a huge economic impact on the cattle industry as a result of skin damage, inflammation of mammary glands and decrease milk production, reduces reproduction and even death as a consequence of secondary bacterial infection. Iraq is considered as an endemic country by OIE that continues breaking throughout different parts of the country. The aims of this research work was to detect and molecular characterization of LSDV in Duhok province for the first time through using a phylogenetic approach of P32 and RPO30 genes. The results showed that LSDV was detected and identified over using PCR and sequencing and the isolate 1 LSDV/Duhok/Kurdistan/2019, isolate 2 LSDV/Duhok/Kurdistan/2019 and isolate 3 LSDV/Duhok/Kurdistan/2019 were clustered in LSDV lineage for both P32 and RPO30 genes. The phylogenetic analysis of these isolates showed a close relationship with the previously published isolates based on P32 gene sequences, while this study is the first study to publish genetic information on RPO30 gene of the Iraqi isolates and compare them to the other publicly available data sets of other countries. To sum up, the findings of this paper could be beneficial to illustrate the spreading nature of LSDV in Iraq and mainly in the Kurdistan region of Iraq. Furthermore, offer the theoretical references for control and prevention of LSDV infections in future.


INTRODUCTION
Lumpy skin disease (LSD) is considered as a viral infection of cattle caused by a lumpy skin disease virus (LSDV) and it is believed that blood-feeding arthropods are the main mechanical carriers of the virus (1,2) .LSDV belongs to the genus Capripoxvirus (CaPV)   in the family of Poxviridae (3) .LSD is characterized by the development of nodules on the skin that appears over all parts of the body along with widespread enlarged lymph nodes (4) .
This infection leads to a huge economic impact as a result of skin damage, inflammation of mammary glands and decreases milk production, reduces reproduction and even death as a consequence of secondary bacterial infection (5) .There is a significant variation in morbidity and mortality rates due to the activity of arthropod carriers, vulnerability and the state of the defence system of the host.Morbidity varies significantly from 2%-85% and some cases are even higher.However, the mortality rate is low 1% -5% and could reach up to 40% in some hosts, specilly if secondary microbial infection occur.The disease was reported for the first time in African countries in 1929 and was detected outside the Africa in 1989 in Israel and afterward was diagnosed in Kuwait, Lebanon, Jordan, Turkey, Bahrain, Yemen and Oman (4,6) .In 2013, the LSDV infection was reported for the first time in Iraq (7) .Iraq was listed by OIE in 2015 in LSD affected countries in 2014 and there has been reports of LSD detection since that time (7,8,9) .
The LSD is identified and diagnosed based on the appearance of a specific clinical disease of the infection, viral isolation, detection by electron microscope, observation of histopathological changes, immunoassay beside the molecular detection (10) .Besides, the genes P32, RPO30 and GPCR that encode for envelope protein, envelope RNA polymerase subunit 30 kD and G-protein, respectively.These are most commonly used genes for the molecular identification and characterization of Capripoxviruses .These genes are greatly conserved in Capripoxviruses.The sequence data of them can be considered for differentiating of Sheeppox virus (SPV), Goatpox virus (GPV) and Lumpyskin disease virus (LSDV), and further presenting the genetic relatedness between varied virus strains (9,11) .The aim of this study work was the detection and molecular characterization of LSDV in Duhok province through using a phylogenetic approach of P32 and RPO30 genes.

Oligonucleotide primers:
In this study, the genes P32 and RPO30 were used for both identification and phylogenetic analysis of LSDV.The primers forward (B68) CTAAAATTAGAGAGCTATACTTCTT and reverse (B69) CGATTTCCATAAACTAAAGTG were used for partial amplification of P32 gene (12).AACCTACATGCATAAACAGAAGC (pair 2) were used to amplify the two overlapping fragments of RPO30 gene in order to generate a full length-sequence (13) .All primers of this study were made by Macrogen (Korea).

Polymerase chain reaction
For the primers (B68 and B69), the PCR was performed by using 40 μl total reaction volume that composed of 20 μl of Prime Taq premix 2X Master Mix (GeNet Bio), 2 μl of 10 pmol of each primer (B68 and B69), 2μl of the DNA template, then the reaction was made upto 40 μl by adding 14 μl of DNA-RNA free water.The amplification conditions were: an initial denaturation at 94 °C for 5 min, followed by 40 cycles of a denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and primer extension at this primer set was 390 bp.while for the other two primers sets (CpRPO30-OL1F, CpRPO30-OL1R (pair 1), CpRPO30-OL2F and CpRPO30-OL2R (pair 2) same volume of amplification reaction were used and the quantities of all PCR components were as mentioned in the first reaction.The thermo cycler conditions for these two pairs of primers were: an initial denaturation at 95 °C for 4 min, 40 cycles of denaturation 95 °C for 30 s, annealing 55 °C for 30 s, extension 72 °C for 45 s, and final extension at 72 °C for 7 min.
The expected PCR products for these two primer pairs were: 554 bp (pair 1) and 520 bp (pair 2), respectively.The PCR amplicons of all samples were visualized on a 1.5% agarose gel.
Sequence determination :Three representative samples were considered for sequencing for both partial P32 gene and overlapping CpRPO30 gene.A total of 30 μl of PCR products for positive samples were sent to Macrogen (Korean) for sequencing along with all primer sets used in this study.Moreover, the obtained sequence reads were submitted to the Genbank database.For the RPO30 sequences, the raw reads were assembled through the Staden software (version 2.0.0b8).The accession numbers received from NCBI were MN871848, MN871849, MN871850 for P32 gene, while MN871851, MN871852 and MN871853 for CpRPO30 gene.
Phylogenetic analysis Nucleotide reference sequences of both P32 and CpRPO30 genes of LSD strains were retrieved from NCBI database and multiple sequence alignments were done separately for each gene using bioedit (14) .Phylogenetic and molecular evolutionary analyses were performed by MEGA X (15) .A neighbour-joining phylogenetic tree was created and evaluated with 1000 bootstrap replicates as employed in the program.

Detection of Capripoxvirus
All 20 clinical samples were shown to be positive by detection of the partial P32 gene amplified fragments.The detected amplicon size was 390bp figure 1; the identity of the detected fragments was confirmed over sequencing.Besides, CpRPO30 gene was also successfully amplified in all samples by detecting amplicons of 554 bp and 520 bp for primers sets pair 1 and pair 2 respectively.

Sequence analysis of P32 and
The sequences of both P32 identical by 100% (Figure 5).The analysis of either partial P32 gene and the full length RPO30 gene sequence of the three isolates of this study were clustered in LSDV lineage (Figures 6, 7).The resulted phylogenetic trees were completely agreed with the genotyping results of the conventional analysis.The Multiple sequence alignment was done for all sequences by using bioedit through the ClustalW method as implemented in the program (14) .Phylogenetic tree was constructed by MEGA X program using neighbour-joining method with the maximum composite likelihood nucleotide substitution model and the pairwise deletion options (15) .
The phylogenetic tree was tested for reliability by 1000 bootstrap replicates as employed in the program.The resulted tree for P32 gene, the  7).The three isolates sequence of this study of both genes is marked by black triangle (figure 6, 7).

DISCUSSION
Lumpy skin disease virus is a highly contagious viral infection of cattle leading to considerable economic impact on cattle industry (1,2) .The disease is endemic in many countries like African countries, Israel, Kuwait, Lebanon, Jordan, Turkey, Bahrain, Yemen and Oman (4,6).In Iraq, LSDV was detected for the first time in 2015 and there have been Many subsequent reports of LSDV outbreaks throughout the country (7,8,9) To the extent of our knowledge this is a first study to detect and characterize LSDV virus isolates in Duhok province of Iraq.
The objective of this study was to detect and determine the phylogenetic relationship of circulating LSDV isolate in Duhok province with the other strains worldwide.In this study, the genes P32 and RPO30 that encode for envelope protein and an envelope RNA polymerase subunit 30 KD, respectively were used for either detection or phylogenetic analysis of Duhok LSDV isolates.The sequences of these two genes are very distinguished and are used to differentiate the Sheep pox virus, Goat pox virus and Lumpy skin disease virus as well as the relationship between varied strains (9 11).
The phylogenetic analysis of these two genes can arrange the Capripoxviruses in a three lineages; LSDV, SPV and GPV.All isolates of the present study for both P32 and RPO30 genes were clustered in LSDV lineage (Figures 6, 7) (10) .However, by looking at the phylogenetic tree of P32 gene, the isolate 2 LSDV/Duhok/Kurdistan/2019 and isolate 3 LSDV/Duhok/Kurdistan/2019 were subclustered with a foreign isolates mostly of African origins and other countries (figure 6).
These two later isolates have an exclusive amino acid substitution with Amino acid Histidine (H) at residue position 99 of the sequences, which is obvious by Tyrosine (Y) amino acid in isolate 1 LSDV/Duhok/Kurdistan/2019 as well as other Iraqi isolates (figure 3).
On the other side, the phylogenetic results of RP030 gene sequences of this study showed a homology of 100% with many other LSDV references of the Genbank as all of them are clustered under LSDV lineage (Figure 7).The amino acid sequence of RPO30 gene of all three Duhok/Kurdistan/2019 showed no substitution at the amino acid level that suggests more stability of this gene comparing to the P32 gene (Figure 5).
Furthermore, These findings agreed with an earlier study regardless with the close relationship among the Capv, they have a distinct phylogenetic features (16) and the LSDV signature is clearly noted for both P32 and RP030 gene sequences (Figures 2, 3, 4, 5).
Moreover, the topology of the phylogenetic analysis constructed on either nucleotide or amino acid sequences for both P32 and RPO30 genes showed that LSDV and GPV have a closer genetic relatedness than with SPV (Figures 6, 7).These findings are similar to the results published by Zhu et al., (2013).
In contrast, these results differ from some other previously published studies (11,17) .
Comparing the sequences of both RPO30 and P32 genes, P32 gene is more appropriate for epidemiological studies on Iraqi strains due to its abundant existing published data since this is a first study to report information on RPO30 gene of Iraqi LSDV isolates.However, taking only one gene for phylogenetic analysis and genetic relationship among the CaPV is not sufficient due to their huge size genomes (10) .
To conclude, this study reported and identified three isolates of LSDV in Duhok province of Iraq.Depending on the phylogenetic analysis, this study revealed the genetic relatedness among Iraqi LSDV isolates and comparing them with isolates of other countries.
To sum up, the findings of this paper could be beneficial to illustrate the spreading nature of LSDV, mainly in the Kurdistan region of Iraq.Furthermore, offer the theoretical references for controling and prevention of LSDV infections in future.
: A total of 20 scab samples were collected from LSD suspected cattle and calves from different locations in Duhok province during summer 2019.The scabs of raptured nodules were taken in equal amount of glycerol and phosphatebuffered saline (PBS) solution.The samples were immediately taken to the Duhok research center at the University of Duhok where they were stored below -20 C o for later testing.Sample preparation and DNA extraction:The samples containing scab of skin were homogenized by the tissue homogenizer then centrifuged at 8000 rpm for 2 minutes then 100ml were taken for DNA extraction.The DNA of LSDV was extracted from clinical samples by RIBO-prep nucleic acid extraction kit (REF K2-9-Et-50-CE AmpliSens, Moscow, Rusia) according to the manufacturer's instructions.

Figure 3 :Figure 4 :
Figure 3: sequence alignment of envelope protein sequence (P32 gene) isolates of this study with NCBI references

Figure 5 :
Figure 5: sequence alignment of envelope RNA polymerase subunit 30 kD (RPO30 gene) isolates of this study with NCBI references

Figure 6 :
Figure 6: Neighbour-joining tree illustrating phylogenetic relationships of the LSDV isolates of this study and other Capripoxvirus references based on P32 gene (Envelop protein) nucleotides sequences.

Figure 7 :
Figure 7: Neighbor-joining tree illustrating phylogenetic relationships of the LSDV isolates of this study and other Capripoxvirus references based on RPO30 gene (RNA polymerase subunit 30 kD) nucleotides sequences.