Study On The Genome Sequence Of Friend Virus And Experiments On Anti-FV Efficacy And Mechanism Of Resveratrol

1. BackgroundThe family of Retroviridae is divided into seven genera. The virus-host interaction of mostretroviruses is restricted to mammalians causing a variety of symptoms includingoncological, neurological and immunodeficient diseases as well as unapparent courses ofinfection. Retroviruses have a single stranded RNA genome and a double stranded DNAgenome serving as an intermediate product during virus replication and integration into thehost cell genome. All infectious retroviral particles show a similar structure with a diameterof about 100nm. The retroviral genome is differing in sizes from 7000 to 12000 base pairsdepending on the type of virus (HIV: 9000nt, FV: 8300nt). Friend Murine Leukemia Virusis belonging to Gammaretrovirus. When adult mice of susceptible strains are infectedwith FV, their spleens rapidly enlarge because of virus-induced polyclonal proliferation oferythroid precursor cells, and finally the mice collapse due to leukemia. The mostsuccessful animal model for HIV research is SIV model. For retrovirus research the FVmodel is more controllable than SIV model. Research in FV will certain deepen andbroaden our understanding of HIV/AIDS.2. Objectives2.1 Sequence the genome of the Friend Murine Leukemia virus and analyze thepolymorphism of the virus genome.2.2 Replicate the FV model; Replicate the Real Time RT-PCR method to quantify the FVvirus load in serum; Study the anti-FV efficacy and the underlying mechanism ofresveratrol using the FV model.3. Methods3.1 Sequencing the FV genome and analysis: 9 days after infection by FV, the mice weresacrificed and the enlarged spleens were collected. Total RNA was isolated from the spleenusing the Takara RNAiso Reagent. cDNA was synthesized from total RNA using theKit(Fernentas' product. Random primers were used.). Design 6 pairs of specific primersreferred to FV genome sequence in GenBank. Using the cDNA as a template and specificprimers amplify the genome. The PCR products were separated through the gelelectrophoresis and the aimed products were extracted from the gel using the Gel Extraction Kit. Then the purified DNA was ligated to T vector and then transformed to E. coli DH5α, and plated. Transformed cell were screen using the PCR method and the positive cellswere sent to Invitrogen Company for sequencing. The genome sequence was obtained byaligning using the ContigExpress software. Genome sequences were analyzed byClustalx1.83 software, thus the SNPs and insert/absence information were obtained.Phylogeny was obtained by using the PHYLIP software.3.2 Experimental study of resveratrol's anti-FV efficacy and its mechanism3.2.1 Transfer Friend Murine Leukemia virus and measure its viral infection dose (ID₅₀).â‘ Transfer of FV: 5 female Balb/C mice were intraperitoneally injected with 0.5ml DMEMdiluted virus which was diluted 10 times. 13 days after inoculation the mice were sacrificed.The spleens weighting over 1g were collected and grinded. The product was diluted withDMEM to create the 10% spleen cell homogenate and stored in -70℃refrigerator.â‘¡Measure the ID50: the 10% spleen suspension were diluted to 5 concentrations which is10^-1,10^-2,10^-3,10^-4,10^-5. 25 female mice were randomly divided into 6 groups. One grouphaving 5 mice was set the control group; the other 5 groups each having 4 were injectedintraperitoneally with 0.5ml diluted virus respectively as indicated before. The mice weresacrificed 13 days after infection. The body weight and spleen weight were collected andspleen index was calculated by 100 multiplying the ratio of spleen weight versus bodyweight. The positive was set to the one whose spleen index was 3 times of standarddeviation larger than the mean spleen index of the control. The ID50 was calculated byREED-MUENCH method.3.2.2 Resveratrol's inhibitory effect against splenomegalia caused by the FV.61 female Balb/c mice were randomly divided into 5 groups: virus control group 12 mice;AZT group 12 mice; normal control group 12 mice; resveratral large dose group(100mg/kg/day) 13 mice; resveratral small dose group(20mg/kg/day). Every mouse wasinjected intraperitoneally with 100ID₅₀ except the mice in normal control group which wereinjected with 0.5ml DMEM. The drugs were given 1 hour after the injection via oraladministration and then routinely given once a day. The mice were sacrificed 30 days afterthe inoculation. The body weight and spleen weight were collected and the spleen indexwas calculated. Student t test was used and p＜0.05 was defined to be significant. Spleentissues were fixed with 10% formalin, sliced and dyed with H.E.3.2.3 Resveratrol's effect on T cell and B cell in FV model61 female Balb/c mice were randomly divided into 5 groups: virus control group 12 mice;AZT group 12 mice; normal control group 12 mice; resveratral large dose group(100mg/kg/day) 13 mice; resveratral small dose group(20mg/kg/day). Every mouse wasinjected intraperitoneally with 100ID50 except the mice in normal control group whichwere injected with 0.5ml DMEM. The drugs were given 1 hour after the injection via oraladministration and then routinely given once a day. Peripheral blood was collected andEDTA was used as an anticoagulant. Fluorescence labeled monoclonal CD3 antibody and CD19 antibody were added to the anticoagulated blood sample. After the standardtreatments the blood sample was measured by Flow Cytometry to determine the CD3+ celland CD19+ cell. Student t test was used and p＜0.05 was set to be significant.3.2.4 Replicate the RNA quantifying method and study the resveratrol's effect on FV virusload in serum.Real-Time RT-PCR for quantifying the FV virus load: the sequencing-proofed FVvirus PCR product was used as a reference DNA. Design a pair of primers and a probeaccording the reference DNA(Sense: 5'-GGACAGAAACTACCGCCCTG-3'; Antisense:5'-ACAACCTCAGACAACGAAGTAAGA-3'; Probe:5'-FAM-TCGCCACCCAGCAGTTTCAGCAGC-TAMRA-3'). The amplified PCRproduct was 133nt. The reference DNA was diluted by 10 folds into 8 gradingconcentrations, and the minimum was supposed as 10^1.3. Set a total 20 ul Real-Time PCRreaction system as: [Master Mix 10ul; Sense (20umol/ml) 0.2ul; Antisense (20umol/ml)0.2ul; Probe 0.8ul; Template DNA 2ul; H₂O 6.8ul]. Run the reaction on ABI 7300Real-Time PCR Machine. The PCR progress was set: 95℃10s; 95℃10s, 60℃30s, 40cycles. Draw the standard cure.61 female Balb/c mice were randomly divided into 5 groups: virus control group 12mice; AZT group 12 mice; normal control group 12 mice; resveratral large dose group(100mg/kg/day) 13 mice; resveratral small dose group(20mg/kg/day). Every mouse wasinjected intraperitoneally with 100ID₅₀ except the mice in normal control group which wereinjected with 0.5ml DMEM. The drugs were given 1 hour after the injection via oraladministration and then routinely given once a day. Peripheral blood was collected and100ul serum was collected by centrifugation. Total RNA was isolated from the 100ul serumand was reverse transferred to cDNA by using commercial kits. cDNA was quantified byReal Time RT-PCR method. The result was checked by student t test. P＜0.05 was set to besignificant.4. Results4.1â‘ 7610nt gcnome sequence was obtained, lacking portion of non-coding region(500ntat 5' end & 150nt at 3' end).â‘¡341 SNP site were found after comparing with the 5 FVgenomes in GenBank. There are 65 SNPs in gag gene, and the variation rate is 4.0%; 161SNPs in pol gene and the variation rate is 4.5%; 97 SNPs in env gene and the variation rateis 4.78%.â‘¢There is only one triallelic variation in 341 SNPs and the vast majority SNPswere biallelic.â‘£80.6% of the SNPs were manifested as transition(A→G or T→C).⑤There is a insert/absence area in nt7480-7490. The insert/absence is up to 84nt.â‘¥Thevariation at nt4849 causes the the premature stop condon occurring at nt4849-4851 not atthe normal site nt5320-5322. It results in the pol coding protein decreased by 157 aminoacids.4.2.1 The ID₅₀ of FV was 10-5.5 10% spleen suspension 0.5ml. If 100 fold ID50 was used to infect the mouse, the injection dose should be 0.5ml 10% spleen suspension diluted 31.6times.4.2.2 Two mice died due to splenomegaly in virus control group (2 out of 12 mice). Themice in two resveratral treated group were all alive after 30 days after inoculation.Resveratral has inhibitory effect on spleen enlargement caused by FV infection. Theinhibitory rate was 32% in 100mg/kg/day resveratral treated mice.4.2.3 The number of T & B lymphocytes was dramatically decreased in FV infected mice.Anti-retrovirus drug AZT can largely increased the T & B lymphocytes in FV infected mice.The intervention of resveratral can improve the T lymphocytes but have no effect on Bcells.4.2.4 AZT can decrease the FV virus load in serum by 86.6% (P＜0.01). resveratral(100mg/kg/day) can decrease the FV virus load in serum by 68.7% (P＜0.05).5. Conclusions5.1 As a member of retroviruses the genome of Friend Murine Leukemia Virus has a highermutation than RNA virus and of course the DNA virus. SNPs are most transitions(A→G orT→C) and mostly are bialletic.5.2 Resveratral can protect the FV infected mice, inhibit the splenomegaly, increase the Tlymphocytes and decrease the virus load. Resveratral have the anti-FV effect.