PMC

Measurement of cytopathic effects of SIVMneCL8 and SIVMne170 viruses. Cells were infected with either SIVMneCL8 (black triangles) or SIVMne170 (open circles), and the viability of cells in the cultures was compared to that of noninfected cultures (black squares). The synchronized infection allows direct measurements of cytopathic effects of each virus. All data points are averages and standard deviations of three independent replicates.

High relative fitness of SIVMne170 in rapid turnover systems. Cultures were inoculated with SIVMneCL8- and SIVMne170-infected cells mixed in different ratios. The virus was transferred from infected cultures to uninfected cells by transferring 1/10 of cells (A) or incubating cell-free virus with DC-SIGN-expressing Raji B cells (B) and mixing these cells with uninfected CEMx174 cells. The proportion of SIVMneCL8 RNA in culture medium was determined before the first transfer and after each consecutive transfer. Each line represents an independent competition and shows the reduction in proportion of SIVMneCL8 after each transfer, indicating higher fitness of SIVMne170 in the rapid turnover assays.

The GeneScan-based assay to measure relative levels of SIVMneCL8 and SIVMne170 RNA. The assay was developed for two regions in the viral genome, vif (R1) and env (R2), which each contain a 3-bp insertion in SIVMne170 (A, top). Viral RNA regions amplified by RT-PCR with a 5′-labeled 6-FAM primer (asterisk in the figure) could be distinguished by GeneScan because the product from SIVMne170 is 3 bp longer than the product from SIVMneCL8 (A, middle). The relative amount of RNA of each variant was determined from relative areas of corresponding peaks. In the example shown (numbers in parentheses), the proportion of SIVMneCL8 was 67.4% (A, bottom). (B) The GeneScan assay was validated using plasmids encoding SIVMneCL8 and SIVMne170, mixed in known proportions (1:9, 1:3, 1:1, 3:1, and 9:1). The graph shows the correlation between the expected and the observed proportions of SIVMneCL8.

Overview of different culture conditions tested for effects on viral fitness. (A) Cultures with nonlimited life span of infected cells. Cultures were inoculated with virus at a low MOI (0.005), and the virus was allowed to spread. Cells were maintained at density below 2 × 10⁶ and the medium was replaced every day, but no fresh cells were added. (B) Cell-based rapid turnover system. Cultures were inoculated at a 0.01 MOI. Virus was passaged to an excess of fresh cells every 2 days by transferring 1/10 of cells from infected cultures. (C) Virus-based rapid turnover system. Cultures were inoculated at a 0.01 MOI. Virus was passaged to fresh cells every 2 to 3 days by transferring 1/10 of cell-free virus-containing medium. (D) DC-SIGN-based rapid turnover system. Cultures were inoculated at a 0.01 MOI. Virus was passaged to fresh cells every 2 days by prebinding of cell-free virus to Raji cells expressing DC-SIGN, washing off unbound virus, and mixing these cells with noninfected target cells.

Results of competition in a system with nonlimited life span of cells. (A) The increase in the number of infected cells was observed by measuring SEAP activity in media of cultures infected with mixes of SIVMneCL8 and SIVMne170 viruses. Initial spread of virus through the culture (increase in SEAP activity on days 1 through 4) is followed by exhaustion of available cells (plateau in SEAP activity on days 4 through 7) and expansion of infected cells (additional increase in SEAP activity on day 8). (B) Percentage of SIVMneCL8 RNA in culture medium at different times postinfection measured by GeneScan R1 probe. The relative amount of SIVMneCL8 remained approximately the same during the first 5 days and increased thereafter.