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

Dittmer D, Stoddart C, Renne R, Linquist-Stepps V, Moreno ME, Bare C, McCune JM, Ganem D - J. Exp. Med. (1999)

Bottom Line: Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is a novel human lymphotropic herpesvirus linked to several human neoplasms.KSHV virions were inoculated directly into the implants, and viral DNA and mRNA production was assayed using real-time quantitative polymerase chain reaction.Viral gene expression was most abundant in CD19(+) B lymphocytes, suggesting that this model faithfully mimics the natural tropism of this virus.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of California, San Francisco, California 94143, USA.

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.

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(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.
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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.

Mentions: To test whether KSHV would replicate in the human Thy/Liv implants of SCID-hu mice, we prepared concentrated KSHV virions from induced BCBL-1 cell supernatant 22 and inoculated 50 μl of virus suspension (containing ∼107 genome equivalents) directly into each implant, according to established procedures 32. As an average implant contains ∼108 human cells, the maximal multiplicity of infection is an estimated 0.1 genome equivalents per cell. However, it is likely that this substantially overestimates the true multiplicity of infection, because it is unlikely that all of the input virions are infectious (in many viruses, the particle/pfu ratio can be as high as 102–104). Each cohort included animals injected with UV-inactivated KSHV to serve as negative control; UV irradiation should completely abolish infectivity by cross-linking the double-stranded DNA genome 454647. At various times after inoculation, the mice were killed. To have all cell types represented, the implants were removed in toto, and 2.5 μg of total implant DNA (corresponding to ∼4.5 × 105 cells) was assayed for the presence of viral DNA by quantitative real-time DNA PCR using primers lat-273F, lat-335R, and lat-294T (Table ). Fig. 1 A shows a standard curve for the real-time quantitative DNA PCR to demonstrate the linear range of the assay, and Fig. 1 B shows a time course of infection. KSHV-specific DNA was low or undetectable in most implants 7 d after inoculation, peaked with a mean signal of ∼100 times background at day 14, and then receded to lower levels at days 21 and 28. This increase in KSHV genome copy number between days 7 and 14 after inoculation and subsequent reduction provides evidence for initial viral replication in the implant, as would be expected in a de novo infection. Exposure of KSHV virions to UV irradiation blocked infectivity.


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

Dittmer D, Stoddart C, Renne R, Linquist-Stepps V, Moreno ME, Bare C, McCune JM, Ganem D - J. Exp. Med. (1999)

(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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195708&req=5

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.
Mentions: To test whether KSHV would replicate in the human Thy/Liv implants of SCID-hu mice, we prepared concentrated KSHV virions from induced BCBL-1 cell supernatant 22 and inoculated 50 μl of virus suspension (containing ∼107 genome equivalents) directly into each implant, according to established procedures 32. As an average implant contains ∼108 human cells, the maximal multiplicity of infection is an estimated 0.1 genome equivalents per cell. However, it is likely that this substantially overestimates the true multiplicity of infection, because it is unlikely that all of the input virions are infectious (in many viruses, the particle/pfu ratio can be as high as 102–104). Each cohort included animals injected with UV-inactivated KSHV to serve as negative control; UV irradiation should completely abolish infectivity by cross-linking the double-stranded DNA genome 454647. At various times after inoculation, the mice were killed. To have all cell types represented, the implants were removed in toto, and 2.5 μg of total implant DNA (corresponding to ∼4.5 × 105 cells) was assayed for the presence of viral DNA by quantitative real-time DNA PCR using primers lat-273F, lat-335R, and lat-294T (Table ). Fig. 1 A shows a standard curve for the real-time quantitative DNA PCR to demonstrate the linear range of the assay, and Fig. 1 B shows a time course of infection. KSHV-specific DNA was low or undetectable in most implants 7 d after inoculation, peaked with a mean signal of ∼100 times background at day 14, and then receded to lower levels at days 21 and 28. This increase in KSHV genome copy number between days 7 and 14 after inoculation and subsequent reduction provides evidence for initial viral replication in the implant, as would be expected in a de novo infection. Exposure of KSHV virions to UV irradiation blocked infectivity.

Bottom Line: Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) is a novel human lymphotropic herpesvirus linked to several human neoplasms.KSHV virions were inoculated directly into the implants, and viral DNA and mRNA production was assayed using real-time quantitative polymerase chain reaction.Viral gene expression was most abundant in CD19(+) B lymphocytes, suggesting that this model faithfully mimics the natural tropism of this virus.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of California, San Francisco, California 94143, USA.

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.

Show MeSH
Related in: MedlinePlus