ABSTRACT
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.
Subject(s)
Capripoxvirus/isolation & purification , Cattle Diseases/diagnosis , Goat Diseases/diagnosis , Polymerase Chain Reaction/methods , Poxviridae Infections/veterinary , Sheep Diseases/diagnosis , Virology/methods , Animals , Capripoxvirus/classification , Capripoxvirus/genetics , Cattle , Cattle Diseases/virology , Goat Diseases/virology , Goats , Poxviridae Infections/diagnosis , Poxviridae Infections/virology , Reproducibility of Results , Sensitivity and Specificity , Sheep , Sheep Diseases/virology , Transition TemperatureABSTRACT
Cellular differentiation and programmed cell death are tightly controlled to maintain tissue homeostasis and proper organ function. In a screen for apoptosis specific gene products, we isolated an immediate early apoptosis response gene from myelomonocytic stem cells that appears to play a key regulatory role in a number of cell types and may be of particular importance in cells of the central nervous system. The gene's 28 kDa protein product interacts with the C-terminal ectodomain of the Na+/K+-ATPase (NKA) beta 1 subunit and was therefore named NKIP (NKA Interacting Protein). NKIP is coexpressed with NKA, localizes to lysosomes and the endoplasmic reticulum and is predominantly expressed in excitable tissues including polarized epithelia and the central nervous system. NKIP has been characterized as an endogenous suppressor of the NKA as reduction of NKIP in PC12 cells significantly increases NKA activity. In pluripotent NT2 progenitor cells, NKIP induced rapidly K+-level-dependent cell death. NKIP overexpression induced growth factor-independent neurite outgrowth, which was associated with MEK-independent phosphorylation of the transcription factor ERK1/2. Thus, we have identified NKIP as an important novel protein that interacts to the NKA complex, influencing cellular ion balance, induction of apoptosis and neuronal differentiation.
