Molecular detection and phylogenetic analysis of lumpy skin disease virus from outbreaks in Uganda 2017-2018

Abstract

Background: Lumpy skin disease (LSD) is an infectious viral disease of cattle caused by a Capripoxvirus. LSD has substantial economic implications, with infection resulting in permanent damage to the skin of affected animals which lowers their commercial value. In Uganda, LSD is endemic and cases of the disease are frequently reported to government authorities. This study was undertaken to molecularly characterize lumpy skin disease virus (LSDV) strains that have been circulating in Uganda between 2017 and 2018. Secondly, the study aimed to determine the phylogenetic relatedness of Ugandan LSDV sequences with published sequences, available in GenBank.

Results: A total of 7 blood samples and 16 skin nodule biopsies were screened for LSDV using PCR to confirm presence of LSDV nucleic acids. PCR positive samples were then characterised by amplifying the GPCR gene. These amplified genes were sequenced and phylogenetic trees were constructed. Out of the 23 samples analysed, 15 were positive for LSDV by PCR (65.2%). The LSDV GPCR sequences analysed contained the unique signatures of LSDV (A11, T12, T34, S99, and P199) which further confirmed their identity. Sequence comparison with vaccine strains revealed a 12 bp deletion unique to Ugandan outbreak strains. Phylogenetic analysis indicated that the LSDV sequences from this study clustered closely with sequences from neighboring East African countries and with LSDV strains from recent outbreaks in Europe. It was noted that the sequence diversity amongst LSDV strains from Africa was higher than diversity from Eurasia.

Conclusion: The LSDV strains circulating in Uganda were closely related with sequences from neighboring African countries and from Eurasia. Comparison of the GPCR gene showed that outbreak strains differed from vaccine strains. This information is necessary to understand LSDV molecular epidemiology and to contribute knowledge towards the development of control strategies by the Government of Uganda.

Keywords: GPCR; Lumpy skin disease; Molecular detection; Phylogenetic analysis; Uganda.

Fig. 1

Lumpy skin disease virus, observed clinical signs and molecular (PCR) confirmation results: Cattle showing characteristic LSDV clinical signs; nodular skin lesions covering the whole body; and lacrimal discharge (panel A shows a cow with nodular skin lesions covering the whole body, panel B shows skin nodules on the neck and fore body and panel C shows skin nodules covering the whole body and lacrimal discharge). Panel D; PCR results showing a 192 bp fragment of the LSDV P32 gene, Lane M is a 100 bp molecular ladder (GeneDireX Inc., UK), lane N is a negative control, lane P a positive control. Lane 2 is a negative sample, while lanes 1 and 3 are samples positive for LSDV. All PCR products were run in 1.5% agarose gel

Fig. 2

Multiple sequence alignment of GPCR sequences from Ugandan isolates and LSDV vaccine strains, showing positions of LSDV signature amino acid sequences A11, T12, T34, S99 and P199. Locations of the signature sequences are marked in a black horizontal rectanglar shape

Fig. 3

Phylogenetic tree showing the relationship between LSDV GPCR gene sequences from Uganda, marked with red square, with other Capripoxvirus GPCR gene sequences from GenBank. A homologous gene sequence from Deerpox virus retrieved from GenBank was used as out-group to root the tree

Fig. 4

Multiple sequence alignment of GPCR gene sequences of Ugandan LSDV field isolates, vaccine strains, Sheeppox and Goatpox virus. A 12 bp nucleotide (position 94 to 105) deletion unique to only LSDV from this study is shown. Sequences from Uganda are marked with a red rectangle, vaccine strains in blue, Sheeppox in yellow and Goatpox in purple

Fig. 5

Location of study area. Districts where outbreaks occurred are shown in grey with a bold dark boundary, coordinates of sampled sites are marked in a red cross. (The image depicted in Figure 5 is our own)