Article Figures & Data
Figures
- FIG 1
Isolation of SRV-4 from Japanese macaques at KUPRI. PCR amplification of genomic DNAs of HEK293T cells inoculated with plasma (A) and cocultured with PBMCs (B) from Japanese macaques JM-a (lane 1), JM-b (lane 2), JM-c (lane 3), JM-d (lane 4), JM-e (lane 5), JM-f (lane 6), JM-g (lane 7), and JM-h (lane 8). (C) PCR amplification of genomic DNAs of TELCeB6 cells inoculated with plasma or stool suspension and cocultured with PBMCs or BM cells from a Japanese macaque exhibiting thrombocytopenia. (D) Confirmation of the infectivity of the virus derived from pSR414, pSR415, pSR416, and pSR424. PCR was conducted to amplify the partial gag gene of SRV-4 from genomic DNA isolated from TELCeB6 cells inoculated with viruses derived from each molecular clone. The results of the LacZ marker rescue assay (LMRA) are shown as negative (−) and positive (+). M, 1-kb ladder marker; N.C., negative control; env, amplicon of partial SRV-4 env; gag, amplicon of partial SRV-4 gag. (E) Unrooted tree of the entire nucleic acid sequences of SRV-4 isolates and other SRVs. Numbers at the nodes indicate the rapid bootstrap values (percentages of 1,000 replicates). MPMV, Mason-Pfizer monkey virus (SRV-3); SERV, simian endogenous retrovirus.
- FIG 2
Experimental infection of four Japanese macaques (JM1, JM2, JM3, and JM4) with SRV-4 strain PRI-172. (A to C) Hematological analyses. Blood was routinely collected, and the numbers of platelets (A), leukocytes (B), and erythrocytes (C) were measured by hematometry. (D) Copy numbers of SRV-4 RNA in plasma samples. SRV-4 viral RNAs were quantified by real-time RT-PCR using a TaqMan probe. Values are means ± standard errors of data from three independent experiments.
- FIG 3
Detection and isolation of SRV-4 in four Japanese macaques (JM1, JM2, JM3, and JM4) experimentally infected with SRV-4 strain PRI-172. (A) Genomic DNAs were isolated from blood and subjected to PCR to amplify the gag region of the SRV-4. Numbers above lanes indicate days postinoculation. (B) Isolation of SRV-4 from Japanese macaques. Data for PCR amplification of genomic DNAs of HEK293T cells inoculated with plasma and those cocultured with PBMCs or BM cells isolated from JM1, JM2, JM3, and JM4 are shown. (C and D) Detection and quantification of SRV-4 proviruses in various tissues by PCR (in JM1) (C) and real-time PCR (in JM1 and JM3) (D) analyses using SRV-4 env-specific primers. Lane M, 1-kb ladder marker; lane N. C., negative control; Ly, lymph node.
- FIG 4
Experimental infection of two Japanese macaques (JM6 and JM7) with SRV-4 clone SR415. (A to C) Hematological analyses. Blood was routinely collected, and the numbers of platelets (A), leukocytes (B), and erythrocytes (C) were measured by hematometry. (D) Copy numbers of SRV-4 RNA in plasma samples. SRV-4 viral RNA was quantified by real-time RT-PCR using a TaqMan probe. Values are means ± standard errors of data from three independent experiments.
- FIG 5
Detection and isolation of SRV-4 in two Japanese macaques (JM6 and JM7) experimentally infected with SRV-4 clone SR415. To prepare SRV-4 inocula, HEK293T cells were transfected with pSR415. Two days after transfection, the culture supernatant was inoculated into TE671 cells, and the cells were further incubated for 2 weeks. Then the culture supernatant was collected and filtered through a 0.45-μm filter unit to make stock virus. To exclude contamination of cellular DNA, the stock virus was treated with DNase I. (A) Genomic DNAs were isolated from blood and subjected to PCR to amplify the Env region of the SRV-4. Numbers above lanes indicate days postinoculation. (B) Quantification of SRV-4 proviral DNA copy numbers in collected blood samples by real-time PCR. The copy numbers of SRV-4 viral DNA were normalized to one copy of GAPDH. Values are the means ± standard errors of data from three independent experiments. (C) Isolation of SRV-4 from Japanese macaques using the PCR assay and the SRV-4 LacZ marker rescue assay (SLMRA). Data for PCR amplification of genomic DNAs of HEK293T cells inoculated with plasma and cocultured with PBMCs or BM cells from JM6 and JM7 are shown. The results of the SLMRA are shown as negative (−) and positive (+). Lane M, 1-kb ladder marker; lane N. C., negative control.
- FIG 6
Immunoblot assay using concentrated SRV-4 as the antigen. (A) Detection of anti-SRV-4 antibodies in plasma samples of JM1, JM2, JM3, and JM4. (B) Detection of anti-SRV-4 antibodies in plasma samples of JM6 and JM7. Numbers shown above the lanes indicate dpi. Rb, rabbit immune serum; CA, rabbit anti-SRV-4 CA antibody; Env, rabbit anti-SRV-4 Env antibody. Dots indicate bands specific to SRV-4. N162, SRV-4-positive serum of a Japanese macaque (KUPRI ID, N162).
- FIG 7
Detection of SRV-4 proviruses, RNA, and proteins in Japanese macaques (JM6 and JM7) infected with SRV-4 clone SR415. (A and B) Detection and quantification of SRV-4 proviruses in various tissues by PCR (A) and real-time PCR analysis (B) using SRV-4 env-specific primers. (C) Quantification of SRV-4 viral RNA copy numbers in various tissues of JM7 by real-time RT-PCR. The copy numbers of SRV-4 RNA were normalized to one copy of GAPDH. Values are means ± standard errors of data from three independent experiments. (D to G) Immunohistochemical staining using anti-SRV-4 CA antibody. Mucosal epithelium on nasal pharynx (D), pharyngeal glands (E), tracheal mucosal epithelium (F), and absorptive epithelium in the small intestine (G) are shown. Ly, lymph node.
- FIG 8
Functional assay of Japanese macaque and cynomolgus macaque ASCT1 and ASCT2 as SRV-4 receptors. (A) Expression of ASCT1-GFP, ASCT2-GFP, and GFP in MDTFs detected by fluorescence microscopy. (B) An SRV-4 infection assay was performed in MDTFs transiently expressing ASCTs from Japanese macaques (JmASCT1 and JmASCT2) and cynomolgus macaques (CyASCT1 and CyASCT2). Infectivity of SRV-4 was quantified by real-time PCR. The copy numbers of SRV-4 DNA were normalized to one copy of mouse Actb. (C) Expression of JmASCT1 in various tissues in JM7. (D) Expression of JmASCT2 in various tissues in JM7. The copy numbers of JmASCT1 and JmASCT2 RNA were normalized to one copy of GAPDH. Values are means ± standard errors of data from three independent experiments. Ly, lymph node.
