Andes Virus Infection of Cynomolgus Macaques

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1 1706 Andes Virus Infection of Cynomolgus Macaques Anita K. McElroy, 1 Mike Bray, 1,a Douglas S. Reed, 2 and Connie S. Schmaljohn 1 1 Virology Division and 2 Aerobiology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland Andes virus (ANDV), a member of the genus Hantavirus, is a causative agent of hantavirus pulmonary syndrome (HPS) and is the only hantavirus known to be transmissible from person to person. HPS is found in North and South America and is often fatal. To test the virulence of ANDV in nonhuman primates, we exposed cynomolgus macaques to Andes virus, either intravenously or by aerosol. The monkeys did not manifest clinical disease but showed significant lymphocyte decreases between days 8 and 11 postexposure. All monkeys developed (1) both IgM and IgG antibodies against the viral nucleocapsid protein and (2) a neutralizing antibody response. By plaque assay, serum samples were negative for infectious virus, but by nonnested reverse transcriptase polymerase chain reaction, viral S-segment genomes were detected in whole blood from 4 of 6 monkeys. This study is the first demonstration of infection of nonhuman primates by an HPS-causing virus. Hantaviruses are members of the Bunyaviridae family of viruses. Bunyaviruses are small ( nm), spherical enveloped viruses, with genomes consisting of 3 single-stranded, negativesense RNA molecules [1]. Viral RNAs are defined as small (S), medium, and large segments, which code for nucleocapsid protein, glycoproteins, and viral polymerase, respectively. Most members of the Bunyaviridae family are maintained in an arthropod reservoir and cause disease and often death in their animal or plant hosts [2]. In contrast, hantaviruses have no known arthropod reservoir but, instead, maintain persistent apathogenic infections in rodents. Hantaviruses are transmitted to humans by inhalation of aerosolized rodent excreta [3]. Hantavirus infections are associated with 2 severe and sometimes fatal diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS) [4, 5]; and the corresponding hantaviruses themselves can be classified into 2 groups: the Old World hantaviruses (HFRS), found in Europe and Asia, and the New World hantaviruses (HPS), found in the Americas. Almost 30 different hantaviruses have been identified, more than half of which are known to cause disease [2]. Received 19 June 2002; revised 29 August 2002; electronically published 22 November Presented in part: XIIth International Congress of Virology, Paris, France, 27 July 1 August 2002 (poster V1102). The contents of this publication are the views and opinions of the authors and are not necessarily endorsed by the US Department of the Army or the US Department of Defense. a Present affiliation: Office of Clinical Research, Office of the Director, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, Maryland. Reprints or correspondence: Dr. Connie S. Schmaljohn, Virology Division, US Army Medical Research Institute of Infectious Diseases, Ft. Detrick, MD (Connie.Schmaljohn@det.amedd.army.mil ). The Journal of Infectious Diseases 2002;186: This article is in the public domain, and no copyright is claimed /2002/ Hantaviruses are a global public-health concern. In Europe and Asia, they cause as many as 200,000 cases of HFRS per year, with 1% 15% being fatal [6]. The antiviral drug ribavirin has proven effective in the treatment of HFRS. Although several attempts have been made to establish a small-animal model for studies of HFRS, none have been successful. Instead, investigators have relied on a hamster-infection model for vaccine development [7]. A nonhuman-primate model would be more desirable for the development of products designed for human use. To this end, several investigators have performed experimental hantavirus infections of nonhuman primates, using Puumala virus (PUUV), an HFRS-causing hantavirus [8 10]. PUUV is of interest to public-health officials and investigators because it is the most common cause of HFRS in areas of northern and central Europe [6]. Cynomolgus macaques that have been infected with PUUV have developed a mild proteinuria and have had increased levels of nitric oxide and interleukin-2 and, in 1 study, hematuria [8 10]. Thus, although some aspects of HFRS were seen in the PUUV-infected macaques, only a few indicators of disease have been routinely detected, and the monkeys did not exhibit the severe symptoms that are seen in human patients with HFRS. HFRS does not occur in the Americas. In the United States, Sin Nombre virus (SNV) is the most prevalent disease-causing hantavirus. Since its appearance in 1993, it has caused 322 cases of HPS. Despite the advanced health care that patients receive in the United States, HPS has a 40% 50% case-fatality rate [11]. Andes virus (ANDV) is a related but distinct New World hantavirus that is prevalent in areas of South America [12]. These 2 viruses share 86% homology, as determined on the basis of S-segment nucleotide sequences [13]. SNV and ANDV both cause HPS in humans, with distinct clinical courses that have similar fatality rates in treated cases. However, ANDV is of significant concern to health officials because it is the only

2 JID 2002;186 (15 December) Andes Infection of Macaques 1707 hantavirus that has been documented to be person-to-person transmissible among family members and health-care workers [14 17]. HPS caused by ANDV first presents symptoms including fever, myalgia, nausea, and diarrhea that are similar to those of many viral infections. Patients then progress to the next phase, in which they have trouble breathing and can develop hemorrhagic complications. Respiratory assistance is often necessary in patients with pulmonary edema, whose blood oxygen saturation drops to!90%. The laboratory findings in patients with HPS show elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and creatinine (CRE), as well as the presence of thrombocytopenia, neutrophilia, and a relative lymphopenia [11, 18]. The importance of the increased incidence of hemorrhagic complications that is associated with HPS in South America is underscored by the observation that the severity of bleeding correlates inversely with survival [18]. In addition, HPS caused by ANDV is more likely to have renal involvement and to afflict children than is that caused by SNV. The hemorrhagic complications, the renal involvement, and the respiratory distress combine to make ANDV disease appear to be a hybrid of that caused by SNV in North America and of the hantavirus-associated disease that is seen in Europe and Asia. In contrast to its effect in cases of HFRS, ribavirin has not been effective in the treatment of HPS [19]. These results are disappointing, because the drug has been successful in inhibiting viral growth in cell culture [20, 21]. It should be taken into consideration that the drug has been tested only in patients who have already developed symptoms of HPS, when it might be too late for it to be effective. Since the appearance of HPS in 1993, investigators have extended their studies to the New World hantaviruses. In the process of testing the hamster-infection model for use with New World hantaviruses, a lethal animal model for HPS has been discovered [22]. Syrian hamsters were infected with several Old World and New World hantaviruses, including HTNV, PUUV, SNV, and ANDV, and those infected with ANDV succumbed to an HPS-like disease. The ability to cause disease in hamsters, to be person-to-person transmissible, and to cause symptoms that seem to be a hybrid of HFRS and HPS makes ANDV unique among the hantaviruses. To test the virulence of ANDV in nonhuman primates, we exposed cynomolgus macaques (Macaca fascicularis) to ANDV. In the present study, we report that ANDV-infected macaques had detectable virus in their blood, exhibited hematological changes, and developed antibodies against ANDV but did not develop HPS or other signs of illness. Materials and Methods Virus and animals. ANDV strain Chile (obtained from T. Ksiazek, Centers for Disease Control [CDC], Atlanta) was propagated in Vero E6 cells (cloned by P. J. Price and obtained from CDC). Cells were maintained in Eagle s minimal essential medium with Earle s salts (EMEM) supplemented with 10% fetal bovine serum (FBS), 10 mm HEPES (ph 7.4), 200 U of penicillin/ml, 200 mg of streptomycin/ml, 1 nonessential amino acids (NEAA), 1.5 mg of amphotericin B/mL, and 50 mg of gentamicin sulfate/ml (cemem), at 37 C ina 5% CO 2 incubator. Blood samples from 2 male and 4 female cynomolgus macaques (M. fascicularis) weighing kg and screened by ELISA for antibodies against nucleocapsid protein were negative for previous exposure to Hantaan virus (HTNV), SNV, and ANDV. Infections, plaque assays, cell counts, and analyses of clinical chemistry were performed under level 4 (i.e., BSL- 4) containment at the US Army Medical Research Institute of Infectious Diseases (Fort Detrick, MD). Serum samples that were used for ELISA and neutralization assays were inactivated by gamma irradiation prior to their removal from the containment suite. Whole-blood samples that were used for RT-PCR were treated with TRIZOL-LS reagent. Intravenous (iv) and aerosol exposures. Cell culture derived virus was diluted in EMEM with 2% FBS. Monkeys were anesthetized by intramuscular injection of Telazol (3 mg/ kg), and then pfu of ANDV was injected into the saphenous vein, followed by a 1-mL flush with sterile saline. Alternatively, anesthetized monkeys were exposed for 10 min to a 1 5 mm mean mass-diameter particle-size aerosol generated by a Collison nebulizer within a head-only plexiglass chamber located inside a class III biological safety cabinet. The viral dose delivered by aerosol was calculated by multiplying the monkey s per-minute volume (i.e., liters of aerosol inhaled per minute) by the duration of the aerosol exposure and the concentration of the virus in the aerosol, as determined by sampling using an all-glass impinger (AGI) [23]. The predicted aerosol concentration was calculated by dividing the plaque-forming units of the input virus by the total volume of air that flowed through the system during the 10-min exposure; the measured aerosol concentration was calculated by multiplying the titer of the virus that was collected in the AGI by the volume in the AGI and then dividing by the total volume of air that flowed through the system during the 10-min exposure. The monkeys were observed daily for signs of disease, and, on days 0, 3, 5, 8, 11, 16, 21, and 29 postexposure, they were anesthetized; then weight, temperature, and blood oxygen saturation were measured, and blood was collected. Blood oxygen saturation was measured in triplicate, by a pulse oximeter (model 3800; OHMEDA). Hematology and clinical chemistries. Total count of white blood cells (WBCs), lymphocyte count, hemoglobin, hematocrit, red-blood-cell indices, and platelet count were measured by a COULTER Ac7T (Beckman Coulter). Blood urea nitrogen (BUN), CRE, creatine kinase (CK), sodium, potassium, am-

3 1708 McElroy et al. JID 2002;186 (15 December) ylase, AST, ALT, LDH, and total bilirubin were measured by a Vitros chemistry analyzer (Ortho-Clinical Diagnostics). Plaque assay. Plaque assay was used to measure virus concentration in serum samples, as has been described elsewhere [24]. In brief, samples were serially diluted in cemem, and 200 ml of each dilution was applied to duplicate wells of 6-well plates containing 7-day-old Vero E6 cells. The plates were incubated for 1hat37 C, with periodic rocking. Cells were then overlaid with 2 ml of 0.6% agarose in EMEM supplemented with 10% FBS, 100 U of penicillin/ml, 100 U of streptomycin/ ml, 1 NEAA, and 1.5 mg of amphotericin B/mL. Cells were incubated at 37 C for 7 days and then were overlaid with the same concentration of agarose but containing % neutral red. Plaques were counted every day for 3 days after the second overlay. Reverse transcriptase polymerase chain reaction (RT-PCR). RNA was purified from 250 ml of whole blood by TRIZOL (Invitrogen). Each blood sample was processed in triplicate, and RT-PCR reactions were performed twice on each RNA sample. Positive-control samples were generated by adding known amounts (plaque-forming units per milliliter) of infectious virus to a blood sample from an uninfected macaque prior to RNA extraction. RT was performed by means of Superscript II (Invitrogen) and the following primers: ANDV S RT (5 - TAG TAG TAG ACT CCT TGA GAA GC-3 ) and control primer hgapdh RT (5 -GTC CAC CAC CCT GTT GCT GTA GCC-3 ). After RT, PCR was used to amplify a 509-bp fragment of the ANDV S segment and a 250-bp fragment of the glycerol phosphate dehydrogenase (GAPDH) mrna, for use as a positive, host-rna control. Primers for PCR were as follows: ANDS 135F (5 -AGT GGA GGTT GGA CCC GGA TGA CGT TAA C-3 ), ANDS 643R (5 -TTG TCC TAA ATC GTC CTG GCG TGA TTT C-3 ), hgapdh RT (see sequence provided above), and hgapdh 5 (5 -GGA CCT GAC CTG CCG TCT AGA AAA ACC-3 ). PCR reactions contained 1 Pfx amplification buffer (Invitrogen), 3 mm MgSO 4, 0.2 mm each dntp, 10 ml of the RT reaction, 20 ng of each PCR primer, and 2.5 U of Pfx polymerase (Invitrogen). PCR was performed as follows: 5-min denaturation at 95 C, then 30 cycles each of 30-s denaturation at 95 C and 1-min denaturation at 70 C, and then a 7-min final extension at 72 C. PCR products were analyzed by agarose-gel electrophoresis. Plaque reduction neutralization test (PRNT). For PRNTs, serum samples were serially diluted in EMEM and incubated with 100 pfu of ANDV overnight at 4 C. The following day, virus/antibody mixes were adsorbed to 7-day-old Vero E6 cells and were processed as described above for plaque assays. PRNT 80 is taken to be the serum dilution that is able to neutralize 80% of the plaques, compared with preinfection control serum samples. ELISA. We coated 96-well plates with 20 ng of affinitypurified, recombinant SNV-nucleocapsid protein (a gift from C. Jonsson, New Mexico State University) per well. Plates were incubated with blocking buffer consisting of 5% skim milk (Difco), 5% normal goat serum (Sigma), 3% FBS (Summit Biotechnologies), and 0.5% Tween-20 in phosphate-buffered saline (PBS) (Sigma), for 1hat37 C. Plates were then washed 5 times in PBS containing 0.05% Tween-20 (PBST). Serum samples that previously had been gamma irradiated were serially diluted in blocking buffer, and 100 ml of each dilution was incubated per well, at 37 C for 1 h. Plates were washed 5 times in PBST and incubated with either anti monkey IgG-HRP or anti monkey IgM-HRP (Kirkegaard & Perry Laboratories [KPL]) diluted 1: 15,000 in blocking buffer. Plates were washed 5 times in PBST, and 100 ml of TMB substrate (KPL) was added to each well, for 5 7 min. Stop solution (KPL) was then added, and the plates were read at 450 nm, by a microplate reader (Bio-Rad). Results Aerosol and intravenous (iv) exposure. Of the 6 cynomolgus macaques, 3 were exposed to pfu of ANDV iv, and the other 3 were exposed to ANDV delivered by aerosol. Suspensions containing pfu/ml (the highest titer avail- 6 able) were used in the aerosol exposure, in an attempt to achieve the maximum dose possible. The inhaled dose in the aerosolexposed monkeys was calculated to be , , and pfu, in monkeys , 5486, and 5560, respectively; 5 the predicted aerosol concentration was pfu/liter. The 3 measured aerosol concentration was pfu/liter, depending on the monkey that was exposed. The 100-fold difference between the predicted and measured aerosol concentrations suggests that, under these experimental conditions, the virus was not readily aerosolized. This result could be due to a loss of infectivity of the virus during the procedure or might suggest that collection of the virus by the AGI was not as efficient as had been predicted. Monitoring for signs of disease. On physical examination, we detected no visible signs of illness; the monkeys were neither lethargic nor anorexic. They did not exhibit vomiting or diarrhea, and no evidence of facial flushing was observed. Measurements of body temperature and blood oxygen saturation did not show a pattern of variation that was consistent with viral challenge (data not shown). Laboratory markers of illness. Measurement of WBC count in ANDV-exposed cynomolgus macaques showed a slight decrease in total WBC count, which was most obvious when we examined changes in the total lymphocyte count (figure 1A and data not shown). All animals showed a drop in total lymphocyte count after day 5 postexposure; the mean count declined significantly, from cells/ml on day 5 postexposure to cells/ml on day 8 postexposure ( P p.0038) and to cells/ml on day 11 postexposure ( P p.0001), before returning to baseline on day 16 postexposure. The mean lymphocyte counts of each group on each day were compared

4 JID 2002;186 (15 December) Andes Infection of Macaques 1709 to the baseline mean by a 2-way Student s t test, for which P!.05 is considered to be significant. Monkey had higher numbers of total WBC than did the other monkeys, both before and throughout the experiment, and this was reflected in its total lymphocytes. This monkey was healthy according to both physical examination and other clinical laboratory parameters. Overall, the platelet count did not change significantly. However, monkey did show a 3-fold decrease in platelets between days 5 and 11 postexposure (figure 1B). By day 21 postexposure, this monkey s platelet count had returned to preexposure levels. Human patients with HPS often show increases in the levels of AST, ALT, and LDH. These enzymes are always present, in small quantities, in the blood, but increases can be indicative of injury to the liver, heart, kidney, or muscle [25]. Cynomolgus macaques exposed to ANDV did not have elevated levels of these enzymes in their serum (data not shown). Four monkeys showed elevations of serum CK during the course of the experiment, with peak values occurring on days 5 (in 2 monkeys), 8, and 11 (not shown) postexposure. These elevations do not appear to be related to the development of viremia and likely were due to manipulation and injection trauma. Viremia. RT-PCR specific for the S segment was performed by use of RNA extracted from whole blood. Control samples that were spiked with known amounts of infectious virus were used to demonstrate that this method was effective in detecting as little as 100 pfu/ml (figure 2A). The expected PCR products were a 509-bp fragment of the S segment and a 250-bp fragment of GAPDH. GAPDH was used as an internal control to ensure that the amount of RNA present in the sample was sufficient for amplification to occur. Using this method, we detected viremia (as defined throughout this study as a positive RT-PCR result) in 4 of 6 monkeys (table 1). All monkeys exposed iv were positive for viremia on days 8 and 11 postexposure. One monkey, , had much higher levels on day 11 postexposure, compared with the control samples (figure 2B). Only 1 of the aerosol-exposed monkeys, 5486, was viremic, and this monkey was positive from day 5 to day 16 postexposure, the longest duration detected. Plaque assay of serum samples failed to reveal any infectious virus. This may have been due to the presence of immune complexes, or the virus may have been intracellular and thus would not have been detected in serum. Antibody response. Serum samples were tested for the pres- Figure 1. Hematological changes in 6 cynomolgus macaques exposed to ANDV either by aerosol (dashed lines) or iv (solid lines); blood was collected and values were measured before and after ANDV exposure. In all monkeys, total numbers of lymphocytes decreased on day 11 postexposure (A); in monkey , a 3-fold decrease below the baseline platelet count was seen on day 11 postexposure (B). Figure 2. Results of nonnested RT-PCR of ANDV S segment from whole blood. Whole blood (250 ml) was spiked with the indicated plaque-forming units of ANDV before extraction by TRIZOL LS. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed with primers specific for a 509-bp fragment of ANDV S segment and (as an internal control) for a 250-bp fragment of GAPDH. RT- PCR products were detected on an ethidium bromide stained agarose gel. As little as 100 equivalent pfu/ml was detectable by this method (A). Representative examples of RT-PCR results in blood of ANDVinfected macaques on day 11 postexposure are shown (B); for comparison, a preexposure result (5687 pre) also is shown.

5 1710 McElroy et al. JID 2002;186 (15 December) Table 1. Exposure, monkey Comparison of aerosol exposure versus intravenous exposure Decrease ELISA endpoint titer, log 10 Platelets Lymphocytes Viremia, DPE IgM IgG Neutralization titer a Aerosol !4 2! , 8, 11, !4 3!4 320 Intravenous , 11 3! , ! , 11 b NOTE., Yes;, no; DPE, day(s) postexposure when a positive result for viremia was obtained (a dash [ ] denotes that no positive result was observed). a As determined by 80% plaque reduction neutralization test. b An exceptional amount of viremia was observed on day 11 postexposure. ence of antibodies against the viral nucleocapsid protein. A similar IgM response directed against the nucleocapsid was seen by ELISA, in all infected monkeys (figure 3A). The IgM response peaked on day 16 postexposure, with endpoint titers of The IgM response of monkeys infected iv was first seen on day 11 postexposure, whereas the aerosol-infected monkeys did not have an IgM response until day 16 postexposure. The IgG response was first detectable on day 16 postexposure and continued to increase thereafter (figure 3B). It is noteworthy that 1 of the aerosol-exposed monkeys, 5486, had the highest IgG endpoint titer of all the monkeys and that this monkey was also the only aerosol-exposed animal with a detectable viremia (table 1). Antibodies that are able to neutralize infectious particles are generally thought to be the most important correlate of protective immunity against hantaviruses. Serum samples from ANDV-exposed macaques assayed by PRNT revealed that all monkeys developed a neutralizing antibody response. Neutralization (i.e., PRNT 80 ) titers were first detected on day 11 postexposure (figure 3C). At day 29 postexposure, 3 of the 6 monkeys had PRNT 80 titers of 1:640, and the other 3 had titers of 1:320. There was no correlation between route of exposure and neutralizing antibody response. Discussion In this article, we have reported the first example of infection of nonhuman primates by a hantavirus that causes HPS in humans. Monkeys were infected with ANDV, as detected on the basis of circulating viral RNA, a decrease in lymphocytes, and production of antibodies IgM and IgG; however, ANDV did not cause any overt disease in these animals. Hematological markers that suggest the presence of HPS in humans include thrombocytopenia and a WBC-count increase in peripheral blood, with neutrophilia and a relative lymphopenia [11, 26]. All monkeys in our study showed an absolute lymphopenia on day 11 postinfection, and we did not detect the WBC-count increase that is usually seen in human patients with HPS. One monkey in our study, , exhibited a 3- fold decrease in baseline platelet count, coincident with high viremia; however, none of the other monkeys showed any significant changes in platelet count. RT-PCR of whole blood of the 6 monkeys demonstrated that 4 of them including all 3 monkeys exposed iv and 1 of the aerosol-exposed monkeys had detectable levels of viremia (defined as a positive RT-PCR result). Despite the presence of circulating viral RNA in the bloodstream, our attempts at isolating infectious virus from the serum samples were unsuccessful. It has been reported elsewhere that most of the virus in the blood stream is located in peripheral-blood mononuclear cells (PBMCs) [5]; however, the 1 reported case, in the literature, in which ANDV isolation from a human was successful used serum for isolation of virus [27]. We attempted to measure infectious virus in the serum samples from our infected monkeys because of earlier results in hamsters lethally infected with ANDV, in which high levels of serum viremia had been measured [22]. We did not detect virus by plaque assay of serum, even though we did detect virus by RT-PCR of whole blood, and these results are consistent with earlier experiences with human samples, which had indicated that PBMCs are more sensitive to RT-PCR detection of virus RNA than to assay of infectious virus serum [28]. In our study, the failure to isolate virus could also be due to the presence of immune complexes in the serum. However, there were monkeys that had very low neutralizing antibody titers yet were viremic according to RT-PCR. It is likely that the virus detected by RT-PCR was cell associated and, in retrospect, that attempts at isolation of virus should have used whole blood. Analysis of our findings suggests that high levels of viremia were not required for a robust antibody response. The aerosolexposed monkeys received 10-fold less virus than was received by the monkeys exposed iv, and only 1 of them had detectable levels of viremia. Despite the lack of detectable viremia in 2 of the 3 aerosol-exposed monkeys, all 3 of them seroconverted to similar levels. Interestingly, the 1 aerosol-exposed animal that was viremic according to RT-PCR (monkey 5486), had the

6 JID 2002;186 (15 December) Andes Infection of Macaques 1711 Figure 3. Antibody response in cynomolgus macaques exposed to ANDV either by aerosol (dashed lines) or iv (solid lines). All ANDV-infected monkeys developed an IgM (A) and IgG (B) antibody response against the nucleocapsid protein. All monkeys had 80% PRNT titers 320 by day 29 postexposure (C). highest IgG titer of all 6 monkeys and had a relatively high IgM and neutralizing antibody titer as well. There were no remarkable factors that would distinguish this monkey from the other monkeys; its age, weight, sex, and exposure dose were all similar to those of monkey Clearly, the aerosol-exposed monkeys had a delayed IgM response, compared with the monkeys exposed iv. Aerosol-exposed monkeys did not show peak levels of IgM until day 16 postexposure, whereas monkeys exposed iv had high levels of IgM on day 11 postexposure. All monkeys exposed iv had detectable viremia and robust antibody responses. The animal with the highest levels of viremia detected by RT-PCR (monkey ) had antibody responses similar to those of the other animals, suggesting that more virus does not necessarily produce an increased antibody response. It is unclear why the monkeys in our study did not develop disease. In human patients with HPS, very high virus-copy numbers have been detected by RT-PCR: virus-copies/ ml of blood [29]. The number of virus RNA copies is positively correlated with disease severity and is inversely proportional to platelet count [29]. It has been suggested that increased viremia indicates high levels of viral antigen in the pulmonary endothelial cells, which could be responsible for the immunopathology (e.g., pulmonary edema) [30]. Therefore, for HPS to develop, a high viral load may be necessary. This is consistent with the data from the hamster model, because hamsters that had been infected with ANDV developed high levels of viremia and lethal disease [22]. In our study, the 1 monkey that had the highest level of viremia also showed evidence of a decrease in platelets. In light of this, it is tempting to speculate that the ANDV-infected macaques did not develop HPS because they did not achieve a sufficiently high viremia and that this deficiency might be corrected by a higher challenge dose. Alternatively, it is possible that the virus used in our studies, which had been isolated and passed through cultured cells, had altered pathogenicity. However, the virus that was used in establishing the lethal hamster model for HPS was also cell culture derived, and this did not affect its pathogenicity in hamsters. Therefore, our data suggest that disease development is likely to be related to host-specific factors, rather than solely to the infectious dose or passage history of the virus. It is not clear what factors dictate an asymptomatic infection versus fulminant HPS, in any host. One model suggests that HPS is caused by endothelial-cell dysfunction infected pul-

7 1712 McElroy et al. JID 2002;186 (15 December) monary endothelial cells are no longer able to control plasma leakage into the lungs, resulting in HPS. If this model is correct, then infection of endothelial cells may be more efficient in some hosts, or the disruption of endothelial-cell function may be more severe in hosts that develop disease. An alternative theory is that HPS is an immune-modulated disease. In this case, there may be distinct ways in which each host s immune system responds to ANDV infection. A more vigorous immune response in humans and hamsters may account for the severe disease that is seen in these 2 hosts. This concept is supported by the reported increase in cytokines that has been seen in human patients with HPS who are infected with SNV [30]. Therefore, measurements of cytokines and other immune-modulatory factors in ANDV-infected hosts may help to determine whether this is indeed the case. Acknowledgments We thank Larry Sullivan and Krista Maddock, for assistance with clinical chemistries; Josh Shamblin, Diane Negley, Merhl Gibson, and Joan Geisbert, for veterinary assistance; Chuck Rapp, for his cellculture expertise; Kristin Spik, for assisting in biocontainment training; and Drs. Jason Paragas, Jay Hooper, and Mary Guttieri, for critically reading the manuscript. This work was performed while A.K.M. held a National Research Council Research Associateship Award at the US Army Medical Research Institute of Infectious Diseases. References 1. Schmaljohn CS, Dalrymple JM. Analysis of Hantaan virus RNA: evidence for a new genus of bunyaviridae. Virology 1983;131: Nichol ST. Bunyaviruses. In: Fields BN, Knipe DM, Howley PM, eds. Fields virology. New York: Raven Press, 2001: Lee HW, Johnson KM. Laboratory-acquired infections with Hantaan virus, the etiologic agent of Korean hemorrhagic fever. J Infect Dis 1982;146: Lee HW, Baek LJ, Johnson KM. 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