FERNANDO A. OSORIO
This presentation will review the experiments collaboratively conducted between our laboratory and that of Dr. Federico Zuckerman at the University of Illinois – Urbana-Champaign. These joint experiments are focused on obtaining a better understanding of the events that characterize the early and convalescent periods following PRRSV infection in swine. We have studied the infection by PRRSV in groups of gilts, young weaned pigs, and sexually mature pigs using viral isolation and several virus detection methods, including RT/PCR, in situ hybridization (ISH), and immunohistochemistry (IHC). At the same time, the PRRSV-specific humoral (ELISA and SN) and cellular (IFN-gamma prod cells) responses were studied in these animals. Regarding early pathogenesis of PRRSV, we have extensively used the techniques of ISH and IHC to detect the cells and tissues that are targeted during early 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 alevolae can become infected by PRRSV. Our most prominent finding regarding cell tropism is 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 Multinucleated 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. These cells are infected with PRRSV, and 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 affect of PRRSV on the infected cell, our findings suggest the existence of an indirect mechanism for the induction of apoptosis in noninfected, bystander cells. A direct effect of the whole virus or one viral component (i.e., glycoprotein E or p 25) 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 surrounding 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.). Regarding persistence of PRRSV in the body of infected animals beyond acute infection, the overall results seem to indicate—contrary to other known examples of RNA virus persistence—that persistent infection established by PRRSV is finite and seems to involve a low level of productive infection that progressively declines until complete clearance of infectious virus takes place (at around five months PI—with persistence of RNA for a somewhat longer period of time). Immunologically, we found evidence for a selective delay, suppression, or modulation of humoral (neutralizing Abs) and cell-mediated components of the PRRSV-specific immune response, which we postulate may be important for prevention of clearance of infection (thereby explaining the inability of the host to eliminate the virus for long periods of time). This delay in the appearance of the immune response does not seem to affect all of the antigens of the PRRSV, as a vigorous PRRSV antibody immune response can be detected by ELISA serology.
OVERALL OBJECTIVES OF OUR RESEARCH
The objective of this study was to examine the progression of PRRSV infection, the persistence of infectious PRRSV and/or viral RNA during long-term PI periods, and the kinetics of establishment of the PRRSV-specific cellular and humoral immune responses during this process.
In these experiments we used two different pathogenic strains of PRRSV recently isolated in our diagnostic laboratory: the PRRSV 12068-96 strain (Sur, et al., 1996) and the PRRSV 16244B strain (Allende, et al., 1999).
Viremia, isolation of PRRSV from gonadal tissues, and detection of PRRSV in ovaries by ISH and IHC was conducted in six gilts infected with PRRSV 16244B strain and three controls, during early post-infection period, ranging from 4 to 21 days PI.
Early testicular infection events were studied in a group of boars at 5.5 months of age (n =25 principals and 6 controls). The animals were infected with PRRS (12068-96) virus. Twelve of these infected boars were maintained in isolation beyond acute infection in order to study viremia as well as humoral and cellular immune responses to experimental intranasal infection. These 12 boars were monitored for more than 500 days.
Ten 35 day-old pigs were inoculated intranasally with PRRSV 16244B strain. Three uninfected pigs of the same source were used as controls. Animals were kept in isolation for the duration of the experiment (five months). Samples from serum and heparinized blood were taken weekly to monitor humoral and cell mediated immunity. Palatine tonsil biopsies were collected by dermatology punch at monthly intervals and used for the assessment of infectious PRRSV (by conventional viral isolation or by swine bio-assay) and/or viral RNA. All the animals were killed at five month post-infection (mpi). At necropsy, tissue from lung, lung lymph nodes, and tonsils were collected and snap frozen for further PRRSV isolation by cell culture inoculation or bio-assay and/or viral RNA identification.
Serological determinations: PRRSV antibodies were detected in all cases by a commercial ELISA kit (IDEXX, Maine, USA) and/or by virus-serum neutralization using the homologous PRRSV strain as the challenge virus. Cellular immunity: In all studies, the intensity of the cellular immune response was measured utilizing an ELISPOT protocol that enabled enumeration of PRRSV-specific interferon-gamma (IFNg)-producing cells (Zuckermann, 1998). Virus isolation: Virus isolation assays were performed by one passage in cultured Porcine Alveolar Macrophages (PAM) followed by two passages in MARC-145 cells. The assays were conducted on serum and tonsil samples as well as tissue samples taken at necropsy (lung, lymph nodes and serum). In situ detection systems: The ISH and IHC proceures to detect PRRSV in tissue sections was performed as described in Sur, et al., 1997. Bioassay for virus infectivity: We used one- to two-week-old piglets, obtained from an unvaccinated, PRRSV-free, specific-pathogen free herd, as biological substrate to assay with maximum sensitivity for the presence of infectious PRRSV in tissue collected from the principal pigs at long term post-infection periods. RT-PCR for total PRRSV RNA and PRRSV (minus) strand-specific: Reverse Transcription (RT) was conducted as previously described (Allende, et al., 1999). In addition, 10 pg of primer IM-755 5'- GACTGCTTTACGGTCTCTC- 3' (Meng, et al., 1996) was exclusively used in the PRRSV negative strand-specific RT reaction.
Tissues of the infected gilts were collected and examined by virus isolation (VI), ISH, IHC, and double-labeling to identify PRRSV-infected cell types, respectively. We were able to isolate PRRSV from ovaries of 50% of the infected gilts. Initially, PRRSV replication was detected in the follicle at seven days p.i. The PRRSV-positive cells in ovaries were predominantly macrophages. However, double-labeling techniques were used to detect PRRSV antigens in follicles of virusinfected gilts by using a monoclonal antibody specific for the PRRSV nucleocapsid protein, and a monoclonal antibody against proliferating cell nuclear antigen (PCNA). We detected PRRSV in cells that were determined to be granulosa cells in the atretic follicles. In addition, few PRRSV-infected stromal cells were observed in the ovarian cortex by ISH and IHC.
The results of the detection in testicles of PRRSV-infected boars has already been published (Sur, et al., 1997). Regarding the response of the host during persistence of the PRRSV infection, Figure 1 shows the cell-mediated and humoral immunity in 12 sexually mature boars after infection with PRRS (str. 12068-96). Most of these animals cleared PRRSV from blood by one and half months p.i., although in a few cases the viremic phase lasted more than two months p.i. While all the animals had seroconverted by week two p.i., the duration of ELISA antibody response ranged significantly. In some cases, the antibody response was as short as seven months p.i., while in others it lasted for the entire 17+ month p.i. period (Figure 1). Interestingly, the frequency of the PRRS virus-specific, IFN-gamma-producing cells (detected only after three weeks p.i. gradually increased reaching a plateau at 9 to 10 months p.i. The IFN- gamma ELISPOT tested frankly positive at this level in all animals from 318 Days to 522 p.i., even in those animals that had become negative by ELISA.
Upon infection with PRRSV 16244B strain ten young animals developed viremia detected at seven dpi (Table 1). Seroconversion by ELISA took place between 7 dpi and 14 dpi and positive antibody results were maintained during the whole experiment (Table 1). Neutralizing antibodies, instead, did not develop until week five p.i., peaked at 9 to 11 weeks p.i., and remained at a lower (but detectable) level afterwards for the remainder of the experiment (Figure 2). The IFNgamma-producing cell response was detected only after week four p.i. and increased throughout the length of the experiment (21 weeks). It was clear that most of the animals failed to clear infection during the early PI period. Five of the animals still harbored infectious virus at 84 dpi (as detected by bio-assays conducted on tonsillar biopsies) (Table 1). Likewise, two of them still maintained infectious PRRSV at 150 dpi as indicated by the results of a second bioassay experiment (Table 1). The inoculum used for the second bioassay experiment consisted of tissue homogenate suspension, collected at necropsy from the five principal pigs that were positive at 84 dpi. Infectious virus also was recovered in PAM from the tonsil biopsy sample of one pig at 56 dpi (data not shown), from an homogenate of tonsil, and serum from another pig at 84 dpi (Table 1). In vitro virus isolation was not successful from tissue (lung, lymph nodes, tonsils) samples collected at necropsy time (Table 1). Minus strand-specific RT/PCR performed on tonsil samples, collected at 28 dpi, indicated occurrence of negative (replicative) form of PRRSV RNA in most of the infected animals, consistent with active viral replication taking place in those tissues (Table 1). By 119 dpi only one animal (#31) maintained positive (minus) strand signal in tonsil. We then analyzed the same RNAs, using random hexamers for the RT reaction. The RT products were then used as template for the PCR and heminested PCR for PRRSV(+) forms as described before. We were able to amplify PRRSV cDNA from tonsil sample from pig #31 (data not shown).
We can summarize the course of a typical infection of an immune competent pig with a virulent strain of PRRSV (Figure 3). Based on the results of the infectivity, in situ, and RT/PCR assays, we conclude that, after the initial "acute" phase ( ~ three weeks), which is richer in symptoms and lesions—lytic infection and apoptosis detected in gonad, lung, and lymphoid tissues—the level of cells supporting PRRSV replication diminish markedly, although infectivity maintains itself in the tissues. In an initial in situ sequential experiment (Sur, et al., 1996), we had obtained evidence of persistence of PRRSV beyond acute infection in lungs and lymph nodes, by simultaneous use of ISH for PRRSV RNA and IHC for PRRSV antigen in serial tissue sections at 42 and 60 days p.i.—suggesting that the infection still continues in a productive mode at that PI time. Conventional viral isolation, and biological assays conducted in the current experiment, indicated that the level of tissue infectivity decreased markedly over time. We were able to detect low levels of infectious PRRSV in only 2/10 animals by five months p.i. Screening by PCR assays of the tissues from the rest of the necropsied animals was negative for PRRSV sequences, suggesting a complete clearance of PRRSV from the host. As we attempt to illustrate in Figure 3, the long term infection established by PRRSV seems to fit a pattern of progressively decreasing levels of replicating virus that will eventually clear the body (as evidenced by the longer but discrete period of PRRSV RNA detection by PCR). Beyond that the PCR signal also disappears (Wills et al., 1999). This shows an updated status of persistence of PRRSV RNA signal in the body of single animals, up to 251 days PI.
Regarding the PRRSV-specific humoral and cellular responses, we found evidence for a selective delay, suppression, or modulation of humoral (neutralizing Abs) and cell-mediated components of the PRRSV-specific immune response (Figure 3). We postulate that this modulating effect of PRRSV on the specific immune response is important for prevention of clearance of infection, thereby explaining the inability of the host to eliminate the virus for long periods of time. This delay in the appearance of the immune response does not seem to affect all of the antigens of the PRRSV, as a vigorous PRRSV antibody immune response can be detected by ELISA serology (Figures 1 and 3, and Table 1).