induced strong resistance to subsequent infection of EIAV
in eMDM. (A) Comparison of intracellular viral levels in the early phase of infection. The same infectious dose (1 × 103 TCID50/well) of the attenuated EIAV strain EIAVFDDV13 and the pathogenic strain EIAVUK3 was used to infect eMDM in 96-well microplates. At 3 hpi, the copy numbers of intracellular EIAV RNA were measured with qPCR. (B) Co-infection of eMDM with EIAVFDDV13 and EIAVUK3. Cultivated eMDM were simultaneously infected with equivalent TCID50 of EIAVFDDV13 and EIAVUK3. Intracellular viruses were detected with ViewRNA in situ hybridization at 48 hpi and are indicated as fluorescently labeled granules (red for EIAVFDDV13 and green for EIAVUK3). (C) Comparison of the replication kinetics of EIAVFDDV13 and EIAVUK3 in eMDM. Stocks of these two viruses with an equal TCID50 or equal RNA copy numbers were used to infect eMDM as indicated. Viruses in the culture medium were quantified as viral RNA copy numbers at various time points up to 7 dpi. (D) Quantitative analysis of the restriction of EIAVUK3 or EIAVFDDV13 infection by prior EIAVFDDV13 or EIAVUK3 infection. Cultivated eMDM were pre-infected by 1 × 103 TCID50 of EIAVFDDV13 or EIAVUK3 in 96-well plates. The culture medium was changed after 2 h of infection. The cells were washed three times with PBS at 6, 12, 24, 36 and 48 hpi and were infected with equivalent infectious amounts of EIAVUK3 or EIAVFDDV13 diluted in the culture medium. After another 48 h, the culture supernatant was collected and the viral loads of the subsequently infected virus in the medium were measured as the copy numbers of EIAV RNA. The Y-axis of the graph represents the ratio of viral RNA copies to the number of viral RNA copies in cells without pre-infection with EIAV.