PBMCs from Cj15-05 and Cj16-05 at day 210 P.I. were used for flow cytometry analysis with anti-CD3, anti-CD20, and anti-HLA-DR to identify T cells, B cells, and activated lymphocytes, respectively. The PBMCs from uninfected marmosets Cj538-03 and Cj537-03 were used as controls. The numbers in the boxes indicate percentages relative to the entire PBMC population.

(A) ELISA using the sera (1∶100 dilution) of infected, FK506-treated common marmosets (Cj333-04 and Cj139-04) against the lysed recombinant virus. The results show the averages of two repeats and standard deviations. These assays were reproduced by two independent occasions. (B) Detection of KSHV DNA from FK506-treated, rKSHV.219-infected common marmosets Cj333-04 and Cj139-04. Genomic DNA (100 ng) purified from the PBMCs of FK506-treated marmosets Cj333-04 and Cj139-04 were used for real-time PCR reactions with LANA-specific primers. Genomic DNA from Vero.rKSHV.219 cells (P) was included as a control and the viral DNA copy number/µg of DNA calculated using a standard curve with pcDNA3-LANA DNA. M, 100-bp DNA molecular markers. The results show the averages of two repeats and standard deviations.

(A) Anti-KSHV ELISA using the sera of Cj10-05 and Cj11-05 (1∶5 dilution) was performed as described in Figure 1A. The results show the averages of two repeats and standard deviations. These assays were reproduced by four independent occasions. (B) Real-time PCR of genomic DNA (100 ng) taken from orally infected marmosets Cj10-05 and Cj11-05 using LANA-specific primers at various time points. Purified genomic DNA from Vero.rKSHV.219 cells (P) was included as a positive control. The viral DNA copy number/µg of DNA was calculated using a standard curve with pcDNA3-LANA DNA. The results show the averages of two repeats with standard deviations. (C) Detection of the LANA protein by confocal microscopy. PBMCs from orally infected marmosets Cj10-05 and Cj11-05 were subjected to immunostaining with an anti-LANA antibody, followed by confocal microscopy. The scale bars represent 2 µm.

(A) ELISA using the sera (1∶100 dilution) of infected marmosets (Cj15-05 and Cj16-05) against the lysed recombinant virus. The results show the averages of four repeats and standard deviations. These assays were reproduced by at least four independent occasions. (B) Immunoblot using the sera of infected marmosets (Cj15-05 and Cj16-05). Sera (1∶500 dilution) taken from naïve control marmosets (Control) and infected marmosets (Cj15-05 and Cj16-05 in the left panel and Cj15-05 in the right panel) at various time points were used to immunoblot 20 µg of purified virion proteins. P.I., days postinfection. (C and D) Real-time PCR analyses of KSHV LANA and ORF9 DNA. Purified genomic DNA (100 ng) from the PBMCs of three naïve controls and two infected (Cj15-05 and Cj16-05) marmosets was used for real time-PCR with primers specific for LANA (c) and ORF9 (D). Purified genomic DNA (100 ng) from Vero.rKSHV.219 cells was included as a control (P) in (C). DNA copy number was calculated using a standard curve with pcDNA3-ORF9 DNA and presented as the viral copy number per µg of DNA. M, 100-bp DNA molecular markers. The results show the averages of four repeats and standard deviations. (E) Detection of the LANA protein by confocal microscopy. The PBMCs of KSHV-infected marmosets were subjected to immunostaining with anti-LANA, followed by confocal microscopy. The arrows indicate LANA-positive cells. The top panels show phase contrast images of the PBMCs. The scale bars represent 20 µm. It should be noted that this picture shows an example of a rare positive field, but not reflecting the overall incidence of LANA positive cells.

(A) Anti-KSHV ELISA with sera from Cj325-04 (1∶100 dilution) was performed as described in Figure 1A. The results show the averages of two repeats and standard deviations. These assays were reproduced by three independent occasions. (B) The PCR products of KSHV DNA from various tissues of infected marmoset Cj325-04 at day 21 P.I. (C) Immunofluorescence and differential interference contrast microscopy [34] of PBMCs from Cj325-04 at day 21 P.I. The scale bars represent 30 µm. It should be noted that this picture shows an example of a rare positive field, but not reflecting the overall incidence of GFP-positive cells. (D) The PBMCs of the KSHV-infected marmoset (day 21 P.I.) were subjected to immunostaining with anti-KSHV LANA. The pictures presented here are representative of multiple immunofluorescence (top panel) and phase contrast (the bottom panel) images of the PBMCs from Cj325-04 at 21 P.I. The scale bars represent 2 µm.

Genomic DNA purified from tissue samples of immune-competent Cj15-05 and Cj16-05 and FK506-treated Cj333-04 and Cj139-04 were used for real-time PCR with LANA-specific primers. Mid, middle; Dis, distal; Prox, proximal; Buccal Muc, buccal mucosa; SubMSG, submandibular salivary gland; ING LN, inguinal lymph node; AxLN, auxiliary lymph node; SubmLN, submandibular lymph node; MesLN, mesenteric lymph node; Parotid SG, parotid salivary gland; ING Skin, inguinal skin. Marker, 100-bp DNA molecular markers. The results show the averages of two repeats and standard deviations.

(A) Skin lesion of rKSHV.219-infected marmoset Cj10-05. (B and C) Histological examination of a marmoset Cj10-05biopsy shows a nonencapsulated dermal mass with characteristics typical of KS lesions, pleomorphic spindle cells along with small blood vessels and extravasated erythrocytes. The spindle cells had a high mitotic index and had infiltrated into the surrounding soft tissues. (D) Real-time PCR of the KS-like skin lesion tissue. Purified genomic DNA (100 ng) from the skin biopsies of two control marmosets and the KS-like lesion of Cj10-05 were used for real time-PCR with LANA-specific primers. Purified genomic DNA from Vero.rKSHV.219 cells was included as a positive control. The viral DNA copy number/µg of DNA was calculated using a standard curve with pcDNA3-LANA DNA. The results show the averages of two repeats with standard deviations. (E and F) Immunohistochemistry of the KS-like skin lesion of Cj10-05 with anti-LANA (E) and anti-vIL-6 (F). The insets in (E) and (F) show the LANA staining of human KS tissue and the vIL-6 staining of KSHV-infected MCD, respectively. The scale bars represent 2 µm (B), 5 µm (C), and 20 µm (E and F). The table on the right is a summary of the results from immunohistochemistry studies of a human KS lesion and the KS-like skin lesion of Cj10-05.