Experimental transmission of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) to SCID-hu Thy/Liv mice

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is a novel human lymphotropic herpesvirus linked to several human neoplasms. To date, no animal model for infection by this virus has been described. We have examined the susceptibility of C.B-17 scid/scid mice implanted with human fetal thymus and liver grafts (SCID-hu Thy/Liv mice) to KSHV infection. KSHV virions were inoculated directly into the implants, and viral DNA and mRNA production was assayed using real-time quantitative polymerase chain reaction. This revealed a biphasic infection, with an early phase of lytic replication accompanied and followed by sustained latency. Ultraviolet irradiation of the inoculum abolished all DNA- and mRNA-derived signals, and infection was inhibited by ganciclovir. Viral gene expression was most abundant in CD19(+) B lymphocytes, suggesting that this model faithfully mimics the natural tropism of this virus. Short-term coinfection with HIV-1 did not alter the course of KSHV replication, nor did KSHV alter levels of HIV-1 p24 during the acute phase of the infection. Although no disease was evident in infected animals, SCID-hu Thy/Liv mice should allow the detailed study of KSHV tropism, latency, and drug susceptibility.

Figure 2

Primer design and control reaction for the detection of latent messages. (A) The schematic outlines the genomic organization and message structure as determined previously 17. Arrows indicate a given primer identified by its number. SD and SA denote the splice donor and splice acceptor sites, respectively. 71, 72, and 73 refer to the latent orfs of KSHV. (B) The autoradiograph of a Southern hybridization of an RT-PCR reaction using forward primer #7209 and the different primers indicated directly above each lane, which were then hybridized with a latency-specific probe. The reactions used BCBL-1–derived RNA in either the absence (RT−) or presence (RT+) of RT or with no template (water). MW, 100-bp molecular mass markers with the hybridized marker.

Figure 2

Primer design and control reaction for the detection of latent messages. (A) The schematic outlines the genomic organization and message structure as determined previously 17. Arrows indicate a given primer identified by its number. SD and SA denote the splice donor and splice acceptor sites, respectively. 71, 72, and 73 refer to the latent orfs of KSHV. (B) The autoradiograph of a Southern hybridization of an RT-PCR reaction using forward primer #7209 and the different primers indicated directly above each lane, which were then hybridized with a latency-specific probe. The reactions used BCBL-1–derived RNA in either the absence (RT−) or presence (RT+) of RT or with no template (water). MW, 100-bp molecular mass markers with the hybridized marker.

Figure 1

(A) Standard curve for quantitation of DNA copy number. The vertical axis shows copies of target DNA per reaction. The horizontal axis shows the ct values as determined by real-time quantitative PCR. (B) Time course of infection. Implants are measured in duplicates (except at day 28). Each mark (○) shows the copy number (logarithmic scale) per 2.5 μg of total implant DNA. The horizontal axis shows times after inoculation. The column labeled UV presents the signal obtained from implants that received UV-inactivated virus. The median of each group is indicated by the dashed line.

Figure 1

(A) Standard curve for quantitation of DNA copy number. The vertical axis shows copies of target DNA per reaction. The horizontal axis shows the ct values as determined by real-time quantitative PCR. (B) Time course of infection. Implants are measured in duplicates (except at day 28). Each mark (○) shows the copy number (logarithmic scale) per 2.5 μg of total implant DNA. The horizontal axis shows times after inoculation. The column labeled UV presents the signal obtained from implants that received UV-inactivated virus. The median of each group is indicated by the dashed line.

Figure 3

Time course of infection as determined by RT-PCR for the presence of latent (filled bars) or lytic (hatched bars) messages. Depicted is the percentage of implants giving a positive RT-PCR signal at indicated days after inoculation; n denotes the number of mice analyzed at each time point. The column with UV represents the signal obtained from implants that received UV-inactivated virus.

Figure 4

Primer design for quantitative RT-PCR. (A) The schematic outlines the genomic organization and message structure for orfs 71, 72, and 73 as determined previously 17. Arrows indicate given primers. (B) The schematic outlines the genomic organization and message structure of orf29 as determined previously 27. Arrows indicate given primers. C and D show detection levels for latent (C, •) and lytic (D, ♦) probes compared with c-myc (□). The number of latent BCBL-1 cells per reaction is shown on the vertical axis, and the corresponding ct value as determined by real-time quantitative RT-PCR is shown on the horizontal axis.

Figure 4

Primer design for quantitative RT-PCR. (A) The schematic outlines the genomic organization and message structure for orfs 71, 72, and 73 as determined previously 17. Arrows indicate given primers. (B) The schematic outlines the genomic organization and message structure of orf29 as determined previously 27. Arrows indicate given primers. C and D show detection levels for latent (C, •) and lytic (D, ♦) probes compared with c-myc (□). The number of latent BCBL-1 cells per reaction is shown on the vertical axis, and the corresponding ct value as determined by real-time quantitative RT-PCR is shown on the horizontal axis.

Figure 4

Primer design for quantitative RT-PCR. (A) The schematic outlines the genomic organization and message structure for orfs 71, 72, and 73 as determined previously 17. Arrows indicate given primers. (B) The schematic outlines the genomic organization and message structure of orf29 as determined previously 27. Arrows indicate given primers. C and D show detection levels for latent (C, •) and lytic (D, ♦) probes compared with c-myc (□). The number of latent BCBL-1 cells per reaction is shown on the vertical axis, and the corresponding ct value as determined by real-time quantitative RT-PCR is shown on the horizontal axis.

Figure 4

Primer design for quantitative RT-PCR. (A) The schematic outlines the genomic organization and message structure for orfs 71, 72, and 73 as determined previously 17. Arrows indicate given primers. (B) The schematic outlines the genomic organization and message structure of orf29 as determined previously 27. Arrows indicate given primers. C and D show detection levels for latent (C, •) and lytic (D, ♦) probes compared with c-myc (□). The number of latent BCBL-1 cells per reaction is shown on the vertical axis, and the corresponding ct value as determined by real-time quantitative RT-PCR is shown on the horizontal axis.

Figure 5

Analysis of transcript levels in infected implants. A and B show latent and lytic transcript levels expressed as the number of latent or lytic cells per 10⁶ cells (logarithmic scale) for implants at day 14 after inoculation that were infected with KSHV (KSHV), infected with KSHV and treated with ganciclovir (KSHV ganc.), or infected with UV-inactivated virus (KSHV UV); BCBL-1 cells are shown as control. The median of each group is indicated by the dashed line. P values were calculated using the nonparametric Mann-Whitney test.

Figure 5

Analysis of transcript levels in infected implants. A and B show latent and lytic transcript levels expressed as the number of latent or lytic cells per 10⁶ cells (logarithmic scale) for implants at day 14 after inoculation that were infected with KSHV (KSHV), infected with KSHV and treated with ganciclovir (KSHV ganc.), or infected with UV-inactivated virus (KSHV UV); BCBL-1 cells are shown as control. The median of each group is indicated by the dashed line. P values were calculated using the nonparametric Mann-Whitney test.

Figure 6

Tissue tropism of KSHV in infected SCID-hu Thy/Liv mice. (A) Representative FACScan™ analysis for CD19 (vertical) or combined CD4⁺, CD8⁺, and CD3⁺ (horizontal) in cells pooled from three KSHV-infected implants harvested 14 d after inoculation: before T cell depletion (implant), after T cell depletion (depleted), or after sorting for CD19⁺ cells of the depleted cell population (sorted). The total number of cells in the sample is also indicated. The percentage of CD19–PE or CD4–, CD8–, CD3–FITC⁺ cells is shown in the upper left and lower right quadrants, respectively. B shows the levels of KSHV-specific latent/orf73 (filled bar) or lytic (hatched bar) transcripts in the indicated cell populations. All samples were assayed in duplicate and normalized for rRNA content. Standard deviations were too small to be visible on this scale. Levels are expressed as fold background (logarithmic scale) from uninfected CD19 cells.

Figure 6

Tissue tropism of KSHV in infected SCID-hu Thy/Liv mice. (A) Representative FACScan™ analysis for CD19 (vertical) or combined CD4⁺, CD8⁺, and CD3⁺ (horizontal) in cells pooled from three KSHV-infected implants harvested 14 d after inoculation: before T cell depletion (implant), after T cell depletion (depleted), or after sorting for CD19⁺ cells of the depleted cell population (sorted). The total number of cells in the sample is also indicated. The percentage of CD19–PE or CD4–, CD8–, CD3–FITC⁺ cells is shown in the upper left and lower right quadrants, respectively. B shows the levels of KSHV-specific latent/orf73 (filled bar) or lytic (hatched bar) transcripts in the indicated cell populations. All samples were assayed in duplicate and normalized for rRNA content. Standard deviations were too small to be visible on this scale. Levels are expressed as fold background (logarithmic scale) from uninfected CD19 cells.

Figure 7

HIV-1 and KSHV viral load in coinfected animals. (A) KSHV genome copy number (○) per 200,000 cells in implants infected with KSHV, HIV-1, HIV-1, and KSHV or UV-inactivated KSHV and HIV-1. The right side depicts a standard curve for dilution series of pDD4 and the corresponding fluorescent threshold (ct). Dashed line indicates the lower limit of detection in this assay. (B) HIV-1 p24 levels in pg per 10⁶ cells (○) in implants 14 d after inoculation infected with KSHV, HIV-1, HIV-1, and KSHV or UV-inactivated KSHV and HIV-1 (mock).

Figure 7

HIV-1 and KSHV viral load in coinfected animals. (A) KSHV genome copy number (○) per 200,000 cells in implants infected with KSHV, HIV-1, HIV-1, and KSHV or UV-inactivated KSHV and HIV-1. The right side depicts a standard curve for dilution series of pDD4 and the corresponding fluorescent threshold (ct). Dashed line indicates the lower limit of detection in this assay. (B) HIV-1 p24 levels in pg per 10⁶ cells (○) in implants 14 d after inoculation infected with KSHV, HIV-1, HIV-1, and KSHV or UV-inactivated KSHV and HIV-1 (mock).