PRRSV: Study of in vivo cell tropism and virus-induced apoptosis by in situ detection techniquesJ.H. Sura, b, A.R. Dostera, J. Galeotaa, R.W. Willsa and F.A. Osorioa
a Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, 68583-0905, USA
b Current Address: Plum Island Animal Disease Center, USDA/ARS, P.O.B. 848, Greenport, NY 11494, USA
Abstract - We have extensively used the techniques of in situ hybridization and immunohistochemistry to detect the cells and tissues that are targeted during infection with pathogenic strains of PRRSV. We found some important exceptions to the generally accepted concept that macrophages are the main or only type of cell to be infected by PRRSV in vivo. In the lung, we found that pneumocytes type II of the alveolae can become infected by PRRSV. Our most prominent finding regarding cell tropism is the notion that PRRSV can infect important cells other than macrophages during the pathogenesis of testicular infection. The testicular infection by PRRSV centers on two types of cells: (i) epithelial germ cells of the seminiferous tubules, primarily spermatids and spermatocytes, and (ii) macrophages, which are located in the interstitium of the testis. Formation of multi-nucleated giant cells (MGCs) and abundant germ cell depletion and death are observed. Importantly, by use of in situ TUNEL reaction we obtained evidence that such germ cell death occurs by apoptosis. Simultaneously with these testicular alterations, we have observed that there is a significant increase in the number of immature sperm cells (mainly MGCs, spermatids and spermatocytes) in the ejaculates of PPRSV-inoculated boars, that these cells are infected with PRRSV, and that in all likelihood are responsible for the venereal transmission of PRRSV. We observed that the presence of PRRSV in semen ceases when viremia subsides, thus suggesting that the hematogenous route (sp. blood-borne infected macrophages) is the way in which testes are continuously seeded with PRRSV. In the female gonad we found that there is a frank infection by PRRSV in macrophages of the atretic follicles of the ovarium, and some minor involvement of stromal and granulosa cells. Importantly, we did not find any evidence of ova infection and/or perpetuation of PRRSV in these tissues or in the embryo, thus making the female gonad an unlikely site of persistence. In our experience, the lymphoid tissues, including tonsils, have been the most consistent site where the virus could be detected the longest. The apoptosis caused by PRRSV in testes is massive and affects many more cells than those affected by PRRSV infection. We extended our search for apoptosis to the most consistent target sites of PRRSV, lung and lymphoid tissues, using in situ (immunohistochemistry for PRRSV antigens and TUNEL reaction for apoptosis), biochemical (DNA electrophoresis for detection of DNA fragmentation), and electron microscopy for ultra-structural morphologic studies. We confirmed that PRRSV infection resulted in widespread apoptosis in the lungs and lymphoid tissues of infected pigs. While the apoptosis affecting alveolar macrophages in the lungs was easily recognizable, we suspect that apoptosis affects some other cells in the alveolae and also in the germinal cells of the lymphoid tissue. As we had seen previously in testes, virus infection-induced apoptotic cells were more abundant than PRRSV-infected cells in all tissues. Again in this case double-labeling experiments demonstrated that the majority of apoptotic cells did not co-localize with PRRSV-infected cells. Besides the direct apoptogenic effect of PRRSV on the infected cell, our findings suggest the existence of an indirect mechanism for the induction of apoptosis in non-infected, bystander cells. A direct effect of the whole virus or one viral component (i.e. p25) at the level of the cell membrane (without penetrating the cell) can not be ruled out at this time. However, it seems plausible to us that apoptogenic cytokines (i.e. tumor-necrosis-factor, TNF) could be released in the environment that surrounds the testicular seminiferous tubules, alveola of the lung or germinal centers of lymph nodes. A putative source of these cytokines could be PRRSV-infected macrophages, which besides being the most prominent cell in PRRSV-infected tissues, are known to enhance the TNF secretion when infected by viruses.
Corresponding author: F.A. Osorio Tel.: (1) 402 472 7809; fax: (1) 402 472 3094;
© INRA, EDP Sciences 2000