We previously reported that ANDV was highly lethal in hamsters, whereas SNV was highly infectious but did not cause disease (12
). Moreover, we reported that a reassortant virus having an ANDV M genome segment and SNV S and L genome segments was infectious in hamsters but did not cause disease (26
). Here we performed a serial pathogenesis study to (i) determine when and to what tissues and cell types ANDV and SNV disseminate after challenge, (ii) measure and evaluate hematological changes during the course of ANDV and SNV infections, (iii) determine if blood chemistries in ANDV- or SNV-infected hamsters reveal evidence of organ dysfunction, and (iv) use immunohistochemical techniques and electron microscopy to characterize the inflammation and ultrastructural changes in tissues of hamsters with HPS.
The study consisted of animals in time course groups (8 hamsters injected with ANDV and 8 hamsters injected with SNV) and animals in serial pathology groups (24 hamsters injected with ANDV and 24 hamsters injected with SNV). Animals in the time course groups were weighed, throat swabbed, and bled 1 week before challenge, at the time of challenge, and then every 2 days until day 16. This time course group-based data collection method allowed us to collect samples from the same hamster over time throughout the experiment. Animals in the serial pathology groups were weighed and bled 1 week before challenge and assigned predetermined necropsy dates. On the day of challenge and every 2 days thereafter for 16 days, three ANDV-injected hamsters and three SNV-injected hamsters were removed from the serial pathology groups, and full necropsies were performed (including blood collections and throat swabs for viremia study). In some cases, moribund animals from the time course group were euthanized and necropsies were performed. This was done to ensure that at least three hamsters were necropsied at each of the time points. The fates of the individual animals in each group are shown in Table .
Clinical signs and hematological analysis.
The hamsters infected with ANDV were either found dead, found moribund and euthanized, or killed for necropsy on the indicated day (Table ). Across all groups, there were 14 hamsters that were found dead or were found moribund and euthanized. Two hamsters were suspected to have died due to the blood draw procedure, one on day −7 and one on day 7, and were not included in further analysis. Of the remaining 12 hamsters, all had been injected with ANDV, and the mean time to death was 11 days. None of the SNV-injected hamsters displayed signs of disease, and all survived or were killed for scheduled necropsies. Nevertheless, all of the hamsters injected with SNV were indeed infected because they had anti-nucleocapsid antibody titers as measured by ELISA. ELISA endpoint titers on sera collected on day 28 (terminal bleed) ranged from 200 to 6,400, with a geometric mean titer of 872. All sera collected before day 16 were negative by ELISA. Three of the day 28 sera were further evaluated for neutralizing antibody by PRNT. All were positive, with 80% PRNT titers of 320, 160, and >640. ANDV-injected animals displayed obvious signs of illness within a few hours before death. Signs included tachypnea, staggered gait, cyanosis (bluing of nares and oral cavity), and apparent weakness. There was minor (<6%) weight loss in the ANDV-injected hamsters during the 2 days before death (data not shown).
Blood samples collected from the animals in the serial pathology groups were analyzed by Coulter Counter and manual differential analysis (smears). ANDV-injected animals exhibited an absolute increase in white blood cell counts that was outside of normal reference values (Fig. ). This was likely due to a relative neutrophilia with a peak in segmented neutrophils occurring at the terminal stages of disease (days 12 to 14). We did not observe a significant increase in the percentage of banded neutrophils (data not shown). Interestingly, there was a delayed neutrophilia with SNV, but the increase in segmented neutrophils was not as pronounced as what was seen with ANDV. A relative lymphopenia that was outside normal limits was seen with ANDV on days 12 to 14. Finally, transient thrombocytopenia was observed for both ANDV- and SNV-infected animals, peaking at days 12 and 14, respectively. The small sample size detracted from our ability to assign statistical significance (Wilcoxon rank-sum test) to the observed postinfection alterations in hematological values. A comparison of the ANDV and SNV data for each time point indicated that there were no statistically significant differences between those groups; however, there were significant changes from the prechallenge values (Fig. ).
FIG. 1. Hematological findings. ANDV and SNV serial pathogenesis groups were analyzed for changes in hematological parameters after infection. The area between dotted lines represents the normal reference range for hamsters. ANDV-infected animals exhibited white (more ...) Viremia is not detected in SNV-infected hamsters.
We previously reported that serum viremia (infectious virus) in hamsters injected with ANDV could be detected on day 8 postinfection (12
). Here, we included additional time points to gain further insight into the kinetics of ANDV viremia in the hamster model. In addition, we assayed for the presence of viremia in hamsters injected with SNV. Plaque assays were used to measure infectious virus in whole blood and in serum from the animals in the serial pathology groups. ANDV was first detected in whole blood and serum on day 6 postinfection and peaked by days 8 and 12 postinfection, respectively (Fig. ). In stark contrast, infectious SNV was not detected in the serum or whole blood at any time point. It was possible that SNV virions had disseminated into the blood but were rendered noninfectious (e.g., neutralized by innate immune responses such as complement). To investigate the possibility that noninfectious SNV was present in the blood, we performed real-time PCR to detect viral genomes in PBMCs collected on the days after challenge with ANDV or SNV (Fig. ). ANDV genomes were first detected on day 8 and were detected on all other time points until death. SNV genomes were not detected at any time point. Thus, either SNV did not enter the bloodstream or the numbers of circulating virions were exceedingly low (7 logs lower than the peak level of infectious ANDV in the days preceding disease onset). In addition to looking at viremia in the blood, we performed plaque assays on throat swab samples collected during the course of the study. No infectious ANDV or SNV was detected in throat swab samples at any time point (limit of detection was 50 PFU/ml). For ANDV-infected hamsters, this was unexpected because there were >5 logs of virus per ml of serum on days 8 to 12 postchallenge, and large volumes of fluid, presumably plasma, passed into the alveolar spaces (pulmonary edema) at the later time points.
FIG. 2. Kinetics of viremia. Hantavirus plaque assays were performed on serum (A) and whole blood (B) collected from ANDV- and SNV-infected hamsters. The limit of detection was 50 PFU/ml. (C) RNA was isolated from PBMCs, and real-time PCR was performed following (more ...) Low level of SNV dissemination occurs despite undetectable viremia.
The finding that SNV was not present in the blood and yet was highly infectious, as measured by seroconversion (50% infective dose is 2 PFU [12
]), led us to look for virus in the major organs typically associated with hantavirus infection, including the lungs, liver, spleen, and kidneys. Both postmortem and histological examination indicated that these organs had no significant inflammation or other lesions. Immunohistological examination revealed SNV antigen, albeit rarely, in cells of endothelial origin in multiple organs on days 12 and 14 postinfection (Fig. ). Thus, SNV was not found in the blood of infected hamsters but was occasionally found in endothelial cells in the lungs, heart, kidney, and brain. This low level of infectivity was capable of eliciting a robust immune response but no overt disease.
FIG. 3. Immunohistochemistry of SNV-infected hamsters. An immunoperoxidase-based immunohistochemical assay using anti-nucleocapsid antibody was performed on tissues from SNV-infected animals (Table ). Immunopositive cells (presumably endothelial (more ...) Temporal pathology following injection of ANDV or SNV. (i) Gross examination.
To characterize the temporal pathology associated with ANDV or SNV infection, cohorts of 24 hamsters were injected with either ANDV or SNV, and then on days 0, 2, 4, 6, 8, 10, 12, and 14, three hamsters were euthanized and full necropsies were performed. Because of the number of ANDV-injected animals found dead after day 10, hamsters from the time course group were used to bring the number of necropsied animals to three per time point on days 12 and 14 (Table ). Any animal found moribund was euthanized. Postmortem examination of ANDV-infected hamsters revealed large volumes of yellow or red effusion in the thoracic cavities of animals that succumbed to infection on day 12 (three of three animals [233, 206, and 238]) or 14 (two of three animals [239 and 207]), as previously reported (12
). Large volumes of pleural effusion were not observed on any of the days preceding day 12, although there were small volumes of effusion in some animals as early as day 2 postinfection. One day 14 animal (205) was not viremic and did not have gross lesions consistent with viral infection.
Immunohistochemistry was performed on all organs examined histologically by H&E staining. Viral antigen was detected in the endothelial cells of multiple organs and hepatocytes in ANDV-infected hamsters starting on day 8 postinfection (Table ). There was also rare immunoreactivity in the urothelium of the urinary bladder (one day 12 animal  and two day 14 animals [207 and 239]), the olfactory epithelium (one day 12 animal ), and the squamous epithelium lining the nonglandular stomach (one day 12 animal ) of ANDV-infected hamsters (data not shown). SNV-infected animals on day 12 (two of three animals [256 and 258]) and 14 (three of three animals [259, 260, and 261]) had very rare, scattered immunoreactivity in endothelial cells in multiple organs (kidney, heart, lungs, and brain) as described above. This pattern was so infrequent that as few as one cell to a maximum of only several cells in the entire tissue examined were immunoreactive (Fig. ). Interestingly, one day 14 ANDV-infected animal (animal 205) did not develop viremia but had similar rare immunoreactivities in endothelial cells in multiple organs (data not shown).
Summary of temporal histopathological findings
Pathology in the lungs.
The lungs from ANDV-infected hamsters appeared normal through day 6. Mild changes, including thickening of alveolar septal walls, were first observed on day 8 (Table ). The damage to the lungs increased over time and culminated with severe pulmonary edema, mononuclear cell infiltration, and hemorrhage by day 14. Representative images are shown in Fig. . The inflammatory component consisted primarily of lymphocytes, plasma cells, and fewer neutrophils that expanded the alveolar, perivascular, and peribronchiolar interstitia. H&E-stained lungs from SNV-infected hamsters were unremarkable at all time points and resembled the lungs of the negative control animals (data not shown).
FIG. 4. Lung pathology in ANDV-infected hamsters. H&E staining of lungs collected from normal hamsters (day −7) (266) (A) or from ANDV-infected hamsters on day 4 (222), 6 (225), 8 (228), 12 (233), or 14 (239) (B to F, respectively). H&E-stained (more ...)
The most striking ultrastructural changes were seen in the lungs. The first signs of focal pulmonary edema were noticed at day 6. No significant changes in alveolar epithelial cells or capillary endothelial cells were detected at that time. By day 8, alveolar edema became diffuse and was often accompanied by fibrin precipitation and an accumulation of extracellular surfactant (Fig. ). Some endothelial cells contained granular and granulofilamentous inclusions (Fig. ), which is typical for hantavirus infection, as early as day 8. The number and size of these inclusions increased with time. Similar inclusions were found in endothelial cells of kidneys, spleen, heart, and liver (data not shown; also see Fig. ). Intracellular edema of type 1 pneumocytes with disruption of cellular membranes and thinning of the basement membranes was also frequently observed starting on day 8 (Fig. ). Starting on day 4, apoptotic lymphocytes (Fig. ) and, less frequently, polymorphonuclear leukocytes (data not shown) were observed in lung capillaries. The numbers of apoptotic lymphocytes and polymorphonuclear leukocytes increased with time.
FIG. 5. Ultrastructural changes to ANDV-infected hamster lungs. Thin-section transmission electron microscopy was performed on ANDV-infected hamster lungs. (A) Fibrin deposition (arrowheads), alveolar airspace edema, and apoptotic lymphocytes were observed for (more ...)
FIG. 7. Effects of hantavirus infection on the liver. An alkaline phosphatase immunohistochemical assay using anti-nucleocapsid antibody was performed on liver samples from ANDV-infected (animal 207) (A) or SNV-infected (animal 259) (B) animals at 14 days postinfection. (more ...) Pathology in the spleen.
The spleens of two ANDV-infected animals (one day 12 animal  and one day 14 animal ) (Table ) had a diffuse mononuclear cell infiltrate that expanded the splenic red pulp and obscured lymphoid architecture compared to what was seen for control and SNV-infected animals (Fig. ). The spleen was evaluated by electron microscopy, and the majority of these cells were identified as plasma cells and lymphoblasts (Fig. ). ANDV antigen was detected in the spleens of infected but not control animals (Fig. ).
FIG. 6. Mononuclear cell infiltrate of the spleen. Mononuclear infiltrate was noted in the spleens of some ANDV-infected animals. (A) Normal appearance of spleen from an SNV-injected hamster (animal 265, day −7). (B) Mononuclear cell infiltrate in the (more ...) Pathology in the liver.
Changes in the liver ranged from mild to moderate hepatitis and hepatocellular necrosis in ANDV-infected hamsters, also beginning on day 8 postinfection and remaining static through the endpoint of the study (day 14) (Table ). Immunohistochemistry revealed antigen in endothelial cells and hepatocytes in the livers from ANDV- but not SNV-infected hamsters starting on day 8 (Fig. ). The inflammatory component in the liver was distributed both periportally and randomly and was composed primarily of lymphocytes and plasma cells, with fewer macrophages. Electron microscopy revealed intracytoplasmic inclusions in endothelial cells (data not shown) and in hepatocytes (Fig. ). Immunoelectron microscopy confirmed that these inclusions were of hantavirus origin (Fig. ). In spite of the large size of some of these inclusions, endothelial cells and hepatocytes did not appear damaged. Moreover, blood chemistry analysis indicated normal liver function during the course of the experiment (Fig. ).
Pathology in other tissues.
All other tissues from ANDV-injected hamsters, including the heart, thymus, trachea, thyroid gland, gastrointestinal tract, pancreas, kidneys, adrenal glands, urinary bladder, uterus, ovaries, skin, submandibular salivary gland, multiple lymph nodes, and multiple sections through the head, including brain, pituitary gland, eyes, and nasal turbinates, did not have lesions attributable to ANDV infection (data not shown). All tissues evaluated histologically in SNV-infected animals had no significant lesions attributed to viral infection. There were a variety of other lesions present in both the ANDV-infected hamsters and SNV-infected hamsters, all of which were considered age-related changes, background changes, or incidental findings.