Recently, it has been shown that approximately 80% of Merkel cell carcinomas harbor a novel polyomavirus named Merkel cell polyomavirus, thought to be a carcinogenic agent. However, it is not fully elucidated whether Merkel cell carcinomas differ with regard to the presence or absence of Merkel cell polyomavirus. To address this, we investigated morphologic differences between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas by morphometry. Using polymerase chain reaction and real-time quantitative polymerase chain reaction, Merkel cell polyomavirus was detected in 20 (77%) of 26 Merkel cell carcinoma cases, including 4 Merkel cell carcinomas combined with squamous cell carcinomas. Interestingly, Merkel cell polyomavirus was detected only in ordinary (pure) Merkel cell carcinomas; none of the 4 combined Merkel cell carcinomas + squamous cell carcinomas was positive for Merkel cell polyomavirus (P = .001). Morphometric analyses revealed that Merkel cell polyomavirus-negative Merkel cell carcinomas had more irregular nuclei (P < .001) and more abundant cytoplasm (P = .001) than Merkel cell polyomavirus-positive Merkel cell carcinomas, which had uniform round nuclei and scant cytoplasm. Reliability of the morphometry was confirmed using intraobserver and interobserver reliability tests. These results demonstrated statistically significant differences in tumor cell morphology between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas and reconfirmed the absence of Merkel cell polyomavirus in combined tumors. Furthermore, the results strongly suggest fundamental biological differences between Merkel cell polyomavirus-positive and -negative Merkel cell carcinomas, supporting that Merkel cell polyomavirus plays an important role in the pathogenesis of Merkel cell polyomavirus-positive Merkel cell carcinoma.

Congenital infection by human cytomegalovirus (HCMV) is a major cause of birth defects and developmental abnormalities. Since guinea pig cytomegalovirus (GPCMV) crosses the placenta and causes infection in utero, GPCMV models are useful for studies of the mechanisms of transplacental transmission. During our characterization of a genomic locus required for GPCMV dissemination in animals, we found that the nucleotide sequence in and around the nearby immediate-early genes in our lineage of GPCMV strain 22122 [designated GPCMV (ATCC-P5)] showed clear differences from that reported previously for the same strain [designated GPCMV (UMN)] passaged extensively in vitro. Since in vitro passaging of HCMV is known to result in genetic alterations, especially in the UL128-UL131A locus, and loss of growth ability in particular cell types, in this study we determined the complete genome sequence of GPCMV (ATCC-P5), which grows efficiently in animals. A total of 359 differences were identified between the genome sequences of GPCMV (UMN) and GPCMV (ATCC-P5), and these resulted in structural differences in 29 protein-encoding regions. In addition, some genes predicted from our analysis but not from GPCMV (UMN) are well conserved among cytomegaloviruses. An additional 18 passages of GPCMV (ATCC-P5) in vitro generated no further marked alterations in these genes or in the locus corresponding to the HCMV UL128-UL131A. Our analyses indicate that the published sequence of GPCMV (UMN) contains a substantial number of sequencing errors and, possibly, some mutations resulting from a long history of passaging in vitro. Our re-evaluation of the genetic content of GPCMV will provide a solid foundation for future studies.

No abstract

Epstein-Barr virus (EBV) is spread universally in humans, and it causes infectious mononucleosis and sometimes induces serious EBV-associated disease. The detailed mechanism of primary infection in humans has remained unclear, because it is difficult to examine the dynamics of EBV in vivo. In this study, a natural EBV-infection rabbit model by intranasal or peroral inoculation is described. Ten male rabbits were examined for EBV-DNA or mRNA expression and anti-EBV antibodies in blood. Four of 10 rabbits showed the evidence of EBV infection; detection of EBV-DNA or EBV-related genes mRNA in peripheral blood mononuclear cells, increased EBV antibodies in the plasma, and the presence of lymphocytes expressing EBER1 and EBV-related gene proteins in the lymphoid tissues of a rabbit. Three of four infected rabbits were detected transiently EBV-DNA and/or mRNA of EBV-related genes such as EBNA1, EBNA2, BZLF1, and EA in blood, while in one of four, EBV-DNA and/or mRNA were detected for more than 200 days after viral inoculation. The level of EA-IgG increased and its level was maintained in all infected rabbits, whereas those of VCA-IgM and VCA-IgG increased transiently, and EBNA-IgG was not elevated. Pathological examination of a rabbit infected transiently revealed some scattered lymphocytes expressing EBER1, LMP1, and EBNA2 in the spleen and lymph nodes. EA expression was also observed in the spleen. These findings suggest that EBV can infect the rabbit by the intranasal or peroral route, and that this rabbit model is useful for examining the pathophysiology of natural primary EBV infection in humans.

Graves’ disease is an autoimmune hyperthyroidism caused by thyrotropin receptor antibodies (TRAbs). Because Epstein–Barr virus (EBV) persists in B cells and is occasionally reactivated, we hypothesized that EBV contributes to TRAbs production in Graves’ disease patients by stimulating the TRAbs-producing B cells. In order for EBV to stimulate antibody-producing cells, EBV must be present in those cells but that have not yet been observed. We examined whether EBV-infected (EBV(+)) B cells with TRAbs on their surface (TRAbs(+)) as membrane immunoglobulin were present in peripheral blood of Graves’ disease patients. We analyzed cultured or non-cultured peripheral blood mononuclear cells (PBMCs) from 13 patients and 11 healthy controls by flow-cytometry and confocal laser microscopy, and confirmed all cultured PBMCs from 8 patients really had TRAbs(+) EBV(+) double positive cells. We unexpectedly detected TRAbs(+) cells in all healthy controls, and TRAbs(+) EBV(+) double positive cells in all cultured PBMC from eight healthy controls. The frequency of TRAbs(+) cells in cultured PBMCs was significantly higher in patients than in controls (p = 0.021). In this study, we indicated the presence of EBV-infected B lymphocytes with TRAbs on their surface, a possible player of the production of excessive TRAbs, the causative autoantibody for Graves’ disease. This is a basic evidence for our hypothesis that EBV contributes to TRAbs production in Graves’ disease patients. Our results further suggest that healthy controls have the potential for TRAbs production. This gives us an important insight into the pathogenesis of Graves’ disease.