Molecular characterization of Capripoxvirus isolates from Moroccan sheep

1 Introduction

Sheep pox (Variola ovina) is a highly contagious infectious disease characterized by a widespread skin eruption caused by the sheep pox virus (SPPV), which is a double-stranded DNA virus belonging to the Capripoxvirus genus [1]. The disease is endemic across much of Africa, the Middle East, and many regions in Asia [2]. In the southern Mediterranean shore, where the farming system is often pastoral, the disease is rambling in Algeria, Tunisia, Morocco, and Egypt causing important consequent economic losses [3,4,5,6]. Nevertheless, no signs of the disease in cattle or in goats have been recorded in Morocco.

Even if this disease is symptomatically recognizable in veterinary practice, the causal agent (SPPV) is hardly indistinguishable from its genetically related viruses causing similar diseases in goats and bovines, namely, the goat pox virus (GTPV) and the lumpy skin disease virus (LSDV) which are also part of the Capripoxvirus group, even though some studies are evocating a kind of host-virus specificity [7,8]. Thus, when frequent SPPV/GTPV cross infections occur in small ruminants, opting for molecular techniques as an accurate tool to characterize the virus strains implicated in such epidemic conditions rises up as an adequate approach. Thus, among reliable genomic markers, the P32 gene encoding for the virus envelope protein is largely employed in alike molecular epidemiology investigations [9,10,11].

As a transboundary disease, the management of this sheep disease in pastoral farming systems requires appropriate control tools, including vaccination, based on field-updated data. With this objective and for any further result investment, the current study aims to characterize the isolated strains from naturally infected and non-vaccinated sheep by sequencing the SPPV P32 gene.

2 Materials and methods

First, nasal swabs were collected from sheep manifesting sheep pox symptoms from two geographical spots of the Souss-Massa area (30.39N–9.20W; 29.70N–9.70W) in Morocco. Viral DNA was extracted from swabs, using the NucleoSpin Virus Mini kit for viral RNA/DNA purification Kit, MACHEREY-NAGEL^® (Düren, Germany). Then, the extracted DNAs were initially analyzed by real-time polymerase chain reaction (PCR) to detect the SPPV genome using the method of Balinsky et al. [12]. Positive samples were submitted to conventional PCR, targeting the P32 gene of the virus envelope protein, using the primers described in the study of Chu et al. [13]. The PCR-positive products of ∼969 bp size were subject to Sanger dideoxy sequencing using the same primers. Eleven sequences were generated and deposited in NCBI Genbank with the given accession numbers: (OK340837–OK340847). Finally, the nucleotide sequences were first blasted using the NCBI-Blastn algorithm searching for similar sequences from where reference sequences were retrieved. Second, sequence alignments were done by BioEdit v7.2.6 software, and phylogenetic computing and tree drawing were done by means of MEGA v7.0 software.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

3 Results and discussion

Preliminary sequence comparisons showed that our sequences belong to the SPPV group. The phylogenetic analyses confirmed that all 11 sequences cluster in the SPPV clade, thus distinguishing them from the other 2 clades of GTPV and LSDV (Figure 1). To sharpen our molecular epidemiology, our SPPV sequences were compared to their SPPV homologous sequences isolated across diverse countries during the last decade showing an overall p-distance of 0.3% (99.7% of similarity). In addition to the sequences previously isolated in Morocco (MG201794–MG201803), the closest sequences (100% similarity) are those isolated in India (MK607135–EU314721), in contrast, the distant one (99.2% similarity) was isolated in Saudi Arabia (MG232385) from sheep (Figure 2).

Figure 1

The phylogenetic condensed tree, dressed by the Neighbor-Joining method based on the Tamura 3-parameter method. The percentages of replicate trees in the bootstrap test (1,000 replicates) are shown next to the branches. The analysis involved 18 nucleotide sequences (11 from the current study and 7 from Genbank). Evolutionary analyses were conducted in MEGA7.

Figure 2

Estimates of evolutionary divergence between sequences and the number of base substitutions per site from between sequences are shown. Analyses were conducted using the Tamura 3-parameter model. The analysis involved 18 nucleotide sequences (2 from the current study and 16 from Genbank). Evolutionary analyses were conducted in MEGA7.

Even though GTPV, LSDV, and SPPV are antigenically related, making clinical and serological-based diagnostic difficult [14], they are genetically different. Consequently, recourse to molecular typing (P32 marker as in the current study) proves to be essential, first to make a precise diagnosis, to characterize genetically the circulating strains, and to choose an adequate vaccine against such a transboundary disease still causing economic losses in endemic regions. However, using a single gene marker has its limitations and the current study should be completed by a multiplex marker approach (e.g., GPCR and RPO30 gene markers).

4 Conclusions

In light of the results obtained above, we confirm that the SPPV is responsible for the disease in investigated sheep from the studied area.