The Methodological Study Of Prototype Foam Virus Titer Detection

Foamy virus（Foamy virus,FV）belongs to the Spumaretrovirinae,Retroviridae,is the only member of the Spumavirus genus.Foamy Virus is so named due to the fact that it can induce host cells to form foam-liked multinucleated cells.FV can infect many many types of cells,without obvious pathogenicity,and possess large gene loading capacity as well as can be integrated into the cell genome to realize the stable expression.These characteristics make it become focal point of vectors for gene therapy.As a candidate carrier for gene therapy,it is necessary to establish a method for detecting the titer of foamy virus.In this paper,a variety of methods for detection of Prototype foamy virus（PFV）were established.It is with great hope to get a effective method to determine the titer of PFV through comparison and analysis.Plaque formation assay is a common method for detection and determination of virus titer,and is applicable to lytic virus.The determination of titer through plaque formation assay is expressed as Plaque Forming Unit（PFU）.In this paper,we tried the plaque formation assay with PFV,but no plaque was observed.The reason might be that HT1080 cells infected with PFV mainly formed syncytium.However,the syncytium stained much more darker than normal cells,so some significant plaques in blue-purple were observed,thus this method can also be used for quantitation of PFV.All retroviruses can express reverse transcriptase,and the reverse transcriptase activity can reflect the amount of virus.Based on this fact,a SYBR Green I-based product-enhanced reverse transcriptase assay（SG-PERT）was established for quantitation of PFV.SG-PERT was with high sensitivity,time-saving and the operation was simple.Reverse transcriptase can be divided into two types according to the divalent cation preference,one kind relies on Mg²⁺,and the other kind relies on Mn²⁺.We explored the preference of divalent cations of PFV and it’s optimum concentration by SG-PERT assay.The genome of Foamy virus is composed of a dimer of two linear RNA molecules.The titer of PFV can also be accurately determined by detection the nucleic acid through Real-Time PCR.In this paper,the Real-Time PCR assay for quantitation of PFV was established and based on which the ratio of RNA virus and DNA virus as well as the ratio of fulllength virus and deletion virus were explored.The 5’LTR promoter of Foamy virus can be specifically activated by Tas/Bell,then start the expression of downstream genes.The recombinant plasmids pGL4.17-5’LTR-Luc and pAc-5’LTR-EGFP were constructed in which the luciferase reporter gene and green fluorescent reporter gene were cloned under the downstream of 5’LTR promoter,respectively.Then the plasmids were transfected into BHK-21 separately,and indicator cell lines were established by screening with G418 for more than two month which were named as LTR-Luc and LTR-EGFP,respectively.The expression of luciferase or EGFP of indicator cell lines infected with PFV showed positive correlation with the titer of PFV,thus they can be used to quantitate the titer of infectious virus through detecting the chemiluminescence intensity or the expression of EGFP.The results in this study were as follows:1,Established the Plaque Formation assay of PFV.2,Established a SG-PERT assay,the R² and the amplification efficiency with which were 0.99 and 103%,respectively.The detection limit was 103 pU which was equivalent to one virus particle.The assay showed high sensitivity,good repeatability and high accuracy.The PFV RT dependented much more on Mn²⁺ and it showed the highest activity at the concentration of 1 mM.3,Established the quantitative real-time PCR assay for detection of PFV’ nucleic acid.The detection limit was 10 virus particles;the ratio of RNA virus and DNA virus as well as the ratio of fulllength virus and deletion virus were 4.31:1 and 3.18:1,respectively.4,Established the indicator cell line LTR-Luc with which the titer of PFV can be determined sensitively by luciferase system.5,Established the indicator cell line LTR-EGFP with which the titer of PFV can be determined fastly and accurately by flow cytometry.