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One-Step RT-qPCR assay for detection and quantification of equine infectious anemia virus in-vitro

Author(s): B.L. Bueno, F.G. Oliveira, G.K. Lima1, A.A. Fonseca Júnior, T.C. Kassar, R.J.F. Câmara, R.C. Leite and J.K.P. Reis

Equine infectious anemia virus (EIAV) infection often results in an initial febrile response, followed by recurrent cycles of the disease and finally, a prolonged asymptomatic period. These variations in clinical signs are due to a number of factors, including virus strain, equid species and differences in susceptibility among animals. As a consequence of the close relation between viral strain and disease, studies about in-vitro replication and fitness of EIAV in macrophages, which are the target cells of the virus, depend on accurate measurement of viral load throughout the infection period. We developed a method to quantify EIAV in-vitro using a one-step RT-qPCR system from a control RNA synthesized for this purpose. Designed primers amplified a 520 base pair fragment from the gag gene region that was inserted into a pGEM-T Easy Vector plasmid and propagated in Escherichia coli DH5-α.The bacteria with the construct were propagated and sufficient quantities of the template DNA were produced. The RNA was synthesized in-vitro from the plasmid linearization product and was used for standardization of a one-step RT-qPCR system with a minimum detection limit of 10 to 15 molecules. The efficiency of the reaction was 101%, with r2 equal to 0.997. This new method can be used for the determination of virus titer in EIAV replication studies in-vitro.