To develop the polymerase chain reaction (PCR) for the detection of type D simian retrovirus (SRV) infection, an oligonucleotide primer pair was designed to hybridize to the sequences within euv gene of SRV subtype 1 (SRV-1). The 3'proximal env sequences annealing to the primers had been rather conserved among three different subtypes of SRV, SRV-1, SRV-2, and SRV-3 (Mason-Pfizer Monkey Virus: MPMV). The PCR using the primer pair targetingan an env region the successfully detected and amplified all three subtypes of SRV with excellent specificity after single round of reaction. The tests with peripheral blood mononuclear cells infected either with simian immunodeficiency virus or simian T-lymphotropic virus type 1, maior immunosuppressive viral agents together with SRV in simian, verified the specificity of the PCR by excluding any cross reactivity. Semiquantitative titration PCR, amplifying serially diluted plasmid DNA of each subtype, was performed to evaluate sensitivity limits of the reaction. Based on molecular weight of each cloned SRV genome, the PCR should be able to detect one SRV-infected cell per more than 5-7x104 uninfected cells after simple ethidium bromide staining of resulting products. The PCR must be very efficient screening sisters with its quickness, certainty, and sensitivity for SRV-infected animals used in human AIDS research model. Second round amplification of the reaction products from the first PCR, or Southern hybridization by radiolabeled probes shall render to compete its efficacy to ELISA which has been the most sensitive technique to screen SRV infection but with frequent ambiguity problem.
Animals ; Betaretrovirus* ; Clone Cells ; DNA* ; Enzyme-Linked Immunosorbent Assay ; Ethidium ; Genome ; Haplorhini ; Humans ; Mason-Pfizer monkey virus ; Mass Screening ; Molecular Weight ; Plasmids ; Polymerase Chain Reaction* ; Retroviruses, Simian ; Sensitivity and Specificity ; Siblings ; Simian Immunodeficiency Virus

OBJECTIVETo develop a safe and effective lentivirus vaccine model and provide insights into the development of other lentivirus vaccines.

METHODSIn this study, a construct of pGPT was made by deleting env gene in the infectious Equine infectious anemia virus (EIAV) molecular clone of WU57. Since the overlaping of EIAV Rev gene with env gene, there was no Rev gene in the construct of pGPT. For compensation of Rev function, the construct of pGPTC was made by inserting 4 copies of constitutive RNA transport elements (CTEs) from Mason-Pfizer monkey virus into the construct of pGPT. In addition, a construct designated pTEB expressing EIAV Env protein was made while env gene-minus viruses were made by co-transfection of pGPT/pTEB or pGPTC/pTEB into 293 cells. Western blot was used to identify the development of recombinant virus particles. Then immunofluorescence assay was used to evaluate the infectivity of recombinant virus particles in vitro.

RESULTSEIAV proteins expression was detected in the supernatant of transfected 293 cells by Western blot within pGPTC/pTEB transfected cells. However, no evidence of EIAV proteins expression was observed within pGPT/pTEB transfected cells. EIAV proteins expression was detected in the first round but not in the second round infected EK cells with EIAV(GPTC) by immunofluorescence assay.

CONCLUSIONRev/RRE was necessary for expression of viral structural proteins; CTEs from Mason-Pfizer monkey virus was functionally interchangeable with EIAV Rev/RRE to help RNAs transportation out of nucleus to express structural proteins and EIAV particles were produced in the transfected 293 cells. A live EIAV recombinant virus with single round infection had been developed.

Animals ; Blotting, Western ; Cells, Cultured ; Fluorescent Antibody Technique ; Genes, rev ; Haplorhini ; Horses ; Humans ; Infectious Anemia Virus, Equine ; genetics ; Lentivirus ; immunology ; Lentivirus Infections ; immunology ; prevention & control ; Mason-Pfizer monkey virus ; genetics ; Transfection ; Vaccines, Synthetic ; Viral Vaccines ; genetics ; immunology