Real time PCR method for simultaneous detection, quantitation and differentiation of capripoxviruses.

The genus Capripoxvirus (CaPV) comprises three members namely, sheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affecting sheep, goats and cattle, respectively. CaPV infections produce similar symptoms in sheep and goats, and the three viruses cannot be distinguished serologically. Since there are conflicting opinions regarding the host specificity of CaPVs, particularly for goatpox and sheeppox viruses, the development of rapid genotyping tools will facilitate more accurate disease diagnosis and surveillance for better management of capripox outbreaks. This paper describes a species-specific, real time polymerase chain reaction (PCR), based on unique molecular markers that were found in the G-protein-coupled chemokine receptor (GPCR) gene sequences of CaPVs, that uses dual hybridization probes for their simultaneous detection, quantitation and genotyping. The assay can differentiate between CaPV strains based on differences in the melting point temperature (Tm) obtained after fluorescence melting curve analysis (FMCA). It is highly sensitive and presents low intra- and inter-run variation. This real time PCR assay will make a significant contribution to CaPV diagnosis and to the better understanding of the epidemiology of CaPVs by enabling rapid genotyping and gene-based classification of viral strains and unequivocal identification of isolates.

Monkey CV1 cell line expressing the sheep-goat SLAM protein: a highly sensitive cell line for the isolation of peste des petits ruminants virus from pathological specimens.

Peste des petits ruminants (PPR) is an important economically transboundary disease of sheep and goats caused by a virus which belongs to the genus Morbillivirus. This genus, in the family Paramyxoviridae, also includes the measles virus (MV), canine distemper virus (CDV), rinderpest virus (RPV), and marine mammal viruses. One of the main features of these viruses is the severe transient lymphopaenia and immunosuppression they induce in their respective hosts, thereby favouring secondary bacterial and parasitic infections. This lymphopaenia is probably accounted for by the fact that lymphoid cells are the main targets of the morbilliviruses. In early 2000, it was demonstrated that a transmembrane glycoprotein of the immunoglobulin superfamily which is present on the surface of lymphoid cells, the signalling lymphocyte activation molecule (SLAM), is used as cellular receptor by MV, CDV and RPV. Wild-type strains of these viruses can be isolated and propagated efficiently in non-lymphoid cells expressing this protein. The present study has demonstrated that monkey CV1 cells expressing goat SLAM are also highly efficient for isolating PPRV from pathological samples. This finding suggests that SLAM, as is in the case for MV, CDV and RPV, is also a receptor for PPRV.

Use of the Capripoxvirus 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.

Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are Capripoxviruses (CaPVs) responsible for causing severe poxvirus disease in sheep, goats and cattle, respectively. Serological differentiation of CaPVs is not possible and strain identification has relied on the implicitly accepted hypothesis that the viruses show well defined host specificity. However, it is now known that cross infections can occur and authentication of identity based on the host animal species from which the strain was first isolated, is not valid and should be replaced with molecular techniques to allow unequivocal strain differentiation. To identify a diagnostic target for strain genotyping, the CaPV homologue of the Vaccinia virus E4L gene which encodes the 30 kDa DNA-dependent RNA polymerase subunit, RPO30 was analyzed. Forty-six isolates from different hosts and geographical origins were included. Most CaPVs fit into one of the three different groups according to their host origins: the SPPV, the GTPV and the LSDV group. A unique 21-nucleotide deletion was found in all SPPV isolates which was exploited to develop a RPO30-based classical PCR test to differentiate SPPV from GTPV that will allow rapid differential diagnosis of disease during CaPV outbreaks in small ruminants.