IAEA scientists develop molecular tools for better understanding of the epidemiology of Capripoxviruses

Transboundary animal diseases (TADs) represent the most important threats to livestock production worldwide. The efficient control of these diseases currently relies primarily on enabling strategies (i.e. rapid recognition of disease incursion, enhanced biosecurity to protect surrounding herds, stamping out and compensation and vaccination) to limit their spread. A prerequisite for the better control of TADs is to rapidly detect and characterize the responsible agent. The accurate identification of pathogens is extremely important in determining which strategy is better suited to limit the spread of the disease, especially when different but closely related strains cause diseases with similar symptoms in the same or different hosts. The diseases caused by capripoxviruses in domestic ruminants are a prime example of this state of affairs. Three viruses, namely, sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) have been identified as the only members of the genus Capripoxvirus (CaPV) of the family Poxviridae. They are responsible for economically important pox diseases of ruminant affecting sheep, goats and cattle respectively.

Sheep pox and goat pox diseases are present in Africa (mainly north of the equator), in the Middle East, in Turkey, in India and other Asian countries from Central Asia to China. Sporadic incursions have also been reported in Greece (Figure 1). The endemic region of lumpy skin disease is limited mainly to African countries (including Madagascar), with sporadic outbreaks occurring in the Middle East (Figure 1).

IAEA scientists develop molecular tools for better understanding of the epidemiology of Capripoxviruses CaPVs are thought to be host specific; however, the host affinity remains complex, particularly in the case of sheep pox and goat pox. Currently, the nomenclature and classification of CaPVs relies specifically on the animal host from which they have been first isolated. However, in the field there are three different scenarios of the occurrence of GTPV and SPPV, (i) outbreaks where only goats are affected, (ii) outbreaks where only sheep are affected, and (iii) outbreaks where both species are affected simultaneously. In addition, there is increasing evidences for wildlife harbouring CaPVs (mainly LSDV). Given the close inter-relationship of LSDV, GTPV and SPPV and that, they possess common immunogenic properties, serological tests cannot be used to undertake a comprehensive epidemiological study of CaPVs.

Fortunately, the analysis of the full genetic profiles of several CaPV isolates has proven that molecular-based methods represent a better alternative to assess their epidemiological relationships. Indeed, it has been shown by using full genetic characterization that these viruses are distinct from each other and can be classified into three distinct groups composed of LSDV, GTPV and SPPV. However, due to the cost and the time required for analyses, the full genome sequencing of each CaPV isolate cannot be systematically undertaken, as CaPVs have a relatively large genome (compared to other viruses). An alternative approach is the characterization of short sequences of the viral genome, consisting of genes or fragments that harbour enough information to enable strain differentiation. This can be accomplished by searching for suitable host-range genes and genotyping targets as a more realistic solution that can be efficiently implemented in several laboratories.

Rapid characterization of CaPV isolates can be of high importance in outbreaks involving wildlife to determine accurately the causative agent, as well as those involving domestic ruminants where it can help in identifying suitable vaccine strains to prevent the further spread of disease.

One of the main pillars of the Animal Production and Health Subprogramme of the joint FAO/IAEA programme in assisting IAEA Member States (MS) to tackle TADs is the promotion of early rapid and accurate detection tools. The Animal Production and Health Laboratory (APHL) within the Animal Production and Health subprogramme, is involved in adaptive research to develop early rapid and accurate detection tools for the control of some selected TADs. Recently, with a view of contributing to a better understanding of CaPVs molecular epidemiology, scientists of the APHL have undertaken collaborative research work to identify genes and genotyping targets carrying species-specific signatures for the development of differential diagnostic tests. This work was supported financially by the French Ministry of Foreign Affairs funded through the FSP-LABOVET project “Strengthening of Five Veterinary Research Laboratories to Monitor and Control Animal Diseases in Africa” that ran from 2005 to 2009. The main collaborators were the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) the institute that, together with the APHL, was one of the two European partners of the FSP-LABOVET project), the Institute for Veterinary Disease Control (Austria), the Onderstepoort Veterinary Institute (South Africa), the Pendik Veterinary Control and Research Institute (Turkey), the Institute for Animal Health, Pirbright Laboratory (UK) and the Institut National de la Médecine Vétérinaire (Algeria).

More than 50 CaPV isolates from different geographical origins were provided by these partners to APHL or CIRAD for sequencing. They included clinical specimens or cell culture-adapted samples from sheep, goats and cattle, as well as from springbok antelope. An important criterion for sample selection in the case of SPPV and GTPV for sequencing was the epidemiological background information. Strains were selected not only from outbreaks where only sheep or only goats were infected, but also from outbreaks where both sheep and goats were infected.

Two genes, the CaPV G-protein coupled chemokine receptor (GPCR) and the 30 kDa RNA polymerase subunit (RPO30) genes were identified and sequenced for all isolates. The sequences obtained were compared together with those of eight other isolates that were retrieved from GenBank.

IAEA scientists develop molecular tools for better understanding of the epidemiology of Capripoxviruses The data revealed that the genetic information within each of these two genes was sufficient to produce phylogenetic trees with similar topologies to the one obtained with the full genome information of CaPVs. For the full genome tree, three clusters composed of LSDV, GTPV and SPPV were found (see example of the RPO30 gene in Figure 2). This suggests that by sequencing each of these genes for a given CaPV strain, it is possible to determine its genotype and assign it to one of the three CaPV groups, using a phylogenetic reconstruction. Given the large number of freely available bioinformatics programmes, (some are web-based) and the development of high throughput sequencing methods, it is feasible for some laboratories in developing IAEA MS to use this approach to better characterize CaPVs. For example, these laboratories could use the primers sets that have been designed by APHL to amplify and sequence one or both of these two genes to characterize virus isolates from disease outbreaks in their own region. Subsequently, the sequencing data could then be used together with the set of CaPV sequences that have been deposited in GenBank by APHL scientists and others to generate phylogenetic trees using freely available software to classify their isolates.

Another important finding was the presence of viral isolates that were located outside the group corresponding to their host of origin. While this provides evidence for cross-infection with CaPVs such as SPPV infecting goats or GTPV infecting sheep, it also shows the weakness of the system for naming and classifying CaPVs that is based solely on the host species from which the virus was first isolated. The results of this collaborative work strengthen the need to use molecular-based tools for CaPVs strain designation and classification. This work has also shown the potential of the species-specific signatures in both of these genes to design simple CaPVs genotyping tools to screen large number of samples during outbreaks without further gene sequencing. The most interesting signatures were the exclusive presence of a 21-nucleotide deletion on the RPO30 gene on all SPPV group members, which can be used to differentiate them from LSDV and GTPV, and the presence of species-specific signatures on the CaPVs GPCR, which represent targets for genotyping with hybridisation probes.

These results have been disseminated to the scientific community through two peer review publications (Journal of General Virology, 2009, 90, 1967-1977 and Veterinary Microbiology (2010, doi:10.1016/j.vetmic.2010.09.038).

The identification of these two genes as potential genotyping targets for CaPVs represents a significant advance in the biology of CaPVs, contribution that will facilitate understanding their epidemiology and assist in rapid decision-making in identifying a suitable vaccine for more efficient control. Certain species-specific signatures that were found within each of these genes were used by the APHL to design simple and cost effective molecular based procedures for CaPVs strain identification, which will not require any sequencing work.