Figure 1.
Phylogenetic relationship of the gammaherpesvirus subfamily.
The DNA polymerase sequences of known human (black highlight) and macaque gammaherpesviruses were analyzed by maximum likelihood. Lymphocryptovirus genus: Human herpesvirus 4/Epstein-Barr virus (HHV4/EBV; YP_401712) and macaque lymphocryptovirus of M. mulatta (MmuLCV; AAK95475), M. nemestrina (MneLCV; unpublished data) and M. fascicularis (MfaLCV; AF534221); Rhadinovirus genus, RV1 lineage of Old World primate rhadinoviruses: human herpesvirus 8/Kaposi's sarcoma-associated herpesvirus (HHV8/KSHV; AAC57086) and macaque retroperitoneal fibromatosis herpesviruses of M. mulatta (RFHVMm; AAC57976), M. nemestrina (RFHVMn; AAF81662), and M. fascicularis (RFHVMf; AAN35122); and the RV2 lineage of Old World primate rhadinoviruses: macaque RV2 rhadinoviruses of M. mulatta (RRV; NP_570750), M. nemestrina (MneRV2; unpublished data), and M. fascicularis (MfaRV2; ABU52895). LCV, RV1 and RV2 gammaherpesviruses have been identified in other New and Old World non-human primates, including chimpanzee and gorilla (not shown). Although the existence of a novel RV2 rhadinovirus in humans (HHV9?) is predicted from an evolutionary perspective [15], [16], the virus has not yet been identified. The phylogenetic clustering of the rhesus and cynomolgus macaque rhadinoviruses reflects the close evolutionary relationship of their primate hosts [51]. The substitutions per site are indicated.
Table 1.
Summary of lymphoma cases.
Figure 2.
Immunohistochemical analysis of nodal and extranodal CD3-positive T-cell lymphomas of pig-tailed macaques.
Metastatic lymphomas from an SRV2-positive/SIV-positive male pig-tailed macaque (T76321) (A–D) and an SRV2-positive female pig-tailed macaque (T76088) (E–G) were analyzed by immunostaining: A) anti-CD3 staining is evident in the lymphoblastic lymphoma (T) of T76321 surrounding a lymph node germinal center (GC). High magnification insert shows strong CD3 staining of large neoplastic cells with abundant cytoplasm, large nucleus and prominent nucleolus; B) anti-CD20 staining of T76321 tumor shown in (A). Strong CD20 staining of the germinal center (GC) cells is evident with a lack of staining in the surrounding tumor cells (T). High magnification insert shows negative staining for CD20 in the tumor cells with occasional CD20-positive cells; C) anti-gB (1508) staining of blastoid tumor cells within the lymph node of T76321. High magnification reveals strong cytoplasmic and paranuclear granular staining of blastoid cells with prominent nucleoli, possibly of the Golgi region; D) anti-ORF59 (425) staining of blastoid tumor cells invading the colon of T76321. The higher magnification reveals strong staining of the blastoid cell nuclei; arrows point to paranuclear and paranucleolar accumulation. Moderate staining of the blastoid cell cytoplasm is also detected (arrowhead). Staining is absent from the nuclei and cytoplasm of the smaller non-blastoid cells (blue nuclei); E) anti-CD3-staining of neoplastic cells within a visceral lymph node of T76088. High magnification insert shows strong CD3 staining of large neoplastic cells with abundant cytoplasm, a large nucleus with vesiculated chromatin and a prominent nucleolus; F) anti-CD20 staining of tumor shown in (E). Staining is absent in the tumor cells; G) anti-gB (1508) staining of tumor shown in (E). High magnification reveals strong cytoplasmic and paranuclear granular staining of blastoid cells with prominent nucleoli, possibly of the Golgi region; H) anti-gB (1508) staining of normal uninfected tissue. Non-specific staining is absent in all cell types. Scale bars = 75 µm (A, B, E, F); 37 µm (C, D, G, H). Panels C and D were autoleveled in Adobe Photoshop.
Figure 3.
Viral loads of gammaherpesviruses in lymphomas from different macaque species.
Viral loads were determined using QPCR assays that detect macaque lymphocryptoviruses (LCV) from rhesus (MmuLCV), pig-tailed (MneLCV) or cynomolgus (MfaLCV) macaques or macaque RV2 rhadinoviruses from the same macaque species (RRV, MneRV2 and MfaRV2, respectively) (see Table 1). The results were normalized to cell number in the tissue sample using a QPCR assay for oncostatin M, a single-copy cellular gene (see Materials and Methods). Viral load is expressed as genome equivalent copies per 106 cells: A) CD3-positive T-cell lymphomas; B) CD20-positive B-cell lymphomas; C) CD3-negative/CD20-negative lymphomas.
Figure 4.
Immunohistochemical analysis of a CD20-positive B-cell lymphoma from the duodenum of a cynomolgus macaque.
A metastatic lymphoma invading the duodenum of a SHIV-positive female cynomolgus macaque (A08044) was analyzed by immunostaining: A) anti-CD20 staining is evident in the lymphoma tumor (T) and in the normal (N) lymphoid follicle in the adjacent duodenum tissue; B) A higher magnification of the lymphoid follicle in (A), circled in red, revealed strong CD20 staining in the normal lymphoid cells; C) A higher magnification of the region in (A), circled in black, revealed strong CD20 staining in the large blastoid cells with prominent nucleoli in the tumor tissue; D) anti-EBNA-2 (LCV) staining is evident in the tumor region (T); E) A higher magnification of the normal follicle region in (D), circled in red, revealed an absence of EBNA-2 staining; F) A higher magnification of the tumor region in (D), circled in black, revealed strong EBNA-2 staining in the nuclei of most blastoid tumor cells. Scale bars = 1 mm (A,D); 100 µm (B,C,E,F).
Figure 5.
Immunohistochemical analysis of a CD20-negative/CD3-negative colonic lymphoma of a rhesus macaque.
A metastatic lymphoma invading the colon of a SIV-positive male rhesus macaque (A01112) was analyzed by immunostaining: A) anti-CD20 staining is evident only in the normal lymphoid follicle (N); B) Higher magnification of the normal follicle in (A), circled in red, revealed strong CD20 staining in the normal lymphoid cells in the aggregate; C) Higher magnification of the tumor (T) region, circled in black, revealed a complete lack of CD-20 staining in the tumor cells; D) anti-gB (1508)(RV2) staining is evident in the tumor region (T); E) Higher magnification of the tumor region of (D), circled in black, revealed strong gB staining in essentially every blastoid tumor cell; F) anti-EBNA-2 (LCV) staining is evident in a few isolated cells in the tumor sample; G) Higher magnification of the tumor (T) region, circled in black, revealed strong nuclear and cytoplasmic EBNA-2 staining of only a few specific cells within the tumor; H) Higher magnification of the normal colonic epithelium in (F), circled in red, revealed discrete nuclear staining of specific cells flanking the epithelial cells in the mucosal crypts. Scale bars = 1 mm (A, D, F); 100 µm (B, C, E, G, H).
Figure 6.
Immunohistochemical analysis of CD20-negative/CD3-negative adrenal lymphoma of a cynomolgus macaque.
A metastatic lymphoma invading the adrenals of a SIV-positive male cynomolgus macaque (99091) was analyzed by immunostaining: A) anti-CD20 staining is evident only in the normal (N) lymphoid region surrounding the tumor (T); B) Higher magnification of the region in (A), circled in red, revealed strong CD20 staining in the normal lymphoid cells; C) Higher magnification of the tumor (T) region in (A), circled in black, revealed a complete lack of CD20 staining in the tumor cells; D) anti-IgM staining is evident in the tumor (T) region; E) Higher magnification of the CD20-positive lymphocyte region in (D), circled in red, revealed a lack of IgM staining in the normal lymphocytes; F) Higher magnification of the tumor region in (D), circled in black, revealed strong IgM staining of the tumor cells; G) anti-gB (1508)(RV2) staining is evident in the tumor region; H) Higher magnification of the CD20-positive lymphocyte region in (G), circled in red, revealed a lack of gB staining in the normal lymphocytes; I) Higher magnification of the tumor (T) region in (G), circled in black, revealed strong gB staining in essentially every blastoid tumor cell. Scale bars = 1 mm (A, D, G); 100 µm (B, C, E, F, H, I).
Figure 7.
Immunohistochemical analysis of CD20-negative/CD3-negative cutaneous and muscle-associated lymphomas from rhesus and cynomolgus macaques.
CD20-negative/CD3-negative lymphomas from a SHIV-positive female cynomolgus macaque (93034; cutaneous) (A,B) and a SIV-positive male rhesus macaque (A01112; abdominal muscle-associated lymphoma)(C,D) were analyzed by immunostaining: A) anti-gB (1508)(RV2) staining is evident in the entire cutaneous tumor of 93034 surrounded by displaced skin; B) High magnification of the region in A circled in black shows widespread expression of gB in the perinuclear area and cytoplasm; C) anti-ORF59 staining is evident in the majority of lymphoma cells in the A01112 lymphoma. High magnification of the boxed region shows strong nuclear, perinuclear and cytoplasmic ORF59 staining (insert); D) anti-gB (1508)(RV2) staining is evident in the majority of lymphoma cells in the A01112 lymphoma. High magnification of the boxed region shows strong staining in the cytoplasm and perinuclear regions, with an aggregate morphology. Figures 7A and C were autoleveled using Adobe Photoshop. Scale bars = 1 mm (A); 75 µm (B); 37 µm (C, D).