Experimental Infection of Rhesus Macaques with West Nile Virus: Level and Duration of Viremia and Kinetics of the Antibody Response after Infection

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1 MAJOR ARTICLE Experimental Infection of Rhesus Macaques with West Nile Virus: Level and Duration of Viremia and Kinetics of the Antibody Response after Infection Marion S. Ratterree, 1 Robin A. Gutierrez, 2 Amelia P. A. Travassos da Rosa, 3 Bruce J. Dille, 2 David W. C. Beasley, 3 Rudolf P. Bohm, 1 Suresh M. Desai, 2 Peter J. Didier, 1 Larry G. Bikenmeyer, 2 George J. Dawson, 2 Thomas P. Leary, 2 Gerald Schochetman, 2 Katherine Phillippi-Falkenstein, 1 Juan Arroyo, 4,a Alan D. T. Barrett, 3 and Robert B. Tesh 3 1 Division of Veterinary Medicine, Tulane National Primate Research Center, Tulane University, Covington, Louisiana; 2 Hepatitis, Retrovirus, and Infectious Diseases Core Research and Development, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois; 3 Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston; 4 Acambis, Cambridge, Massachusetts Reports of transfusion-associated cases of West Nile virus (WNV) infection indicate the need for sensitive screening methods to identify WNV-infected blood products. We experimentally infected 5 rhesus macaques with WNV, to determine the level and duration of viremia, the kinetics of the humoral immune response, and the sensitivity of various assay systems for detecting WNV in blood. All macaques developed subclinical infections with low levels of viremia; nested reverse-transcription polymerase chain reaction was the most sensitive method for detecting virus or viral RNA in blood. Specific WNV antibodies appeared during the second week of infection; the results of an IgM enzyme-linked immunosorbent assay became positive on the ninth or tenth day after infection, followed in 1 2 days by hemagglutination-inhibiting and neutralizing antibodies. Our results suggest that both nucleic acid and serological testing may be needed to determine exposure to WNV and to identify potentially infected blood donors. Recent reports [1, 2] of transfusion-associated cases of West Nile virus (WNV) infection have focused attention on the level and duration of virus in the blood of infected persons who might serve as donors. Although much has been written about the clinical and pathological manifestations of WNV infection in humans and on the immune response after infection [3 13], surprisingly little information is available on the actual Received 2 July 2003; accepted 25 August 2003; electronically published 4 February Financial support: National Institutes of Health (grants P51RR00163 and RO1 AI and contract NO1-AI25489); Centers for Disease Control and Prevention (contract U50/CCU ); Texas Advanced Research Program; Abbott Laboratories (nonhuman primate transmission studies). a Present affiliation: Dynport Vaccine Company, Frederick, Maryland. Reprints or correspondence: Dr. Robert B. Tesh, Dept. of Pathology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX (rtesh@ utmb.edu). The Journal of Infectious Diseases 2004; 189: by the Infectious Diseases Society of America. All rights reserved /2004/ $15.00 level and duration of viremia or the kinetics of the antibody response during this infection. The latter information is essential for the development and the evaluation of screening methods for the identification of WNV-infected blood products. Serological studies conducted in New York City during 1999 and 2000 indicated that 20% of persons infected with WNV developed West Nile fever and that only 1 in 150 WNV infections resulted in meningitis or encephalitis [12]. These data suggest that the majority ( 80%) of people infected with WNV have a subclinical infection. It is these asymptomatic, infected persons who are of concern as blood donors, because their blood or plasma may contain WNV that probably would not be detected by the screening procedures now in use at most blood collection centers in the United States. As a consequence, there is considerable interest in developing sensitive assay systems to detect infectious WNV or viral RNA in whole blood and blood products. Infection of Macaques with WNV JID 2004:189 (15 February) 669

2 To learn more about the viremia and antibody response during the acute phase of WNV infection, 5 rhesus macaques were experimentally infected with WNV by intradermal inoculation. Blood samples were obtained from the macaques for 14 consecutive days and then at regular intervals. These samples were then assayed for infectious virus and viral RNA, as well as for a variety of antibodies (IgM, IgG, hemagglutination-inhibiting [HI], complement-fixing [CF], and neutralizing). In addition, spinal taps were done on the macaques, and periodic samples of cerebrospinal fluid (CSF) were also tested for infectious virus, viral RNA, and IgM antibodies. The present article summarizes the results of our studies. MATERIALS AND METHODS Animals. The rhesus macaques (Maccaca mulatta) used in the present study were born and reared in outdoor corrals at the Tulane National Primate Research Center (TNPRC) in Covington, LA. The experimental animals were male adults (7 13 years old) and were housed singly in stainless steel primate cages in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International and in accordance with the Guide for the Care and Use of Laboratory Animals. All procedures were approved by the TNPRC Institutional Animal Care and Use Committee. The macaques were fed standard monkey chow twice daily, supplemented with fruit 2 3 times weekly. Before infection, a blood sample was obtained from each macaque, and the serum was tested by HI technique against WNV and a variety of other flavivirus antigens, to verify that the macaques had no preexisting flavivirus antibodies. Infection and sampling of macaques. Macaques were anesthetized with ketamine hydrochloride (10 mg/kg) and then were inoculated intradermally with 100,000 pfu of a New York 1999 strain of WNV (WN NY99). This virus was originally isolated from a flamingo at the Bronx Zoo [14] and had been passaged once in C6/36 cells and 3 times in Vero cells. After infection, the macaques were examined a minimum of twice daily for signs of illness. Blood samples were obtained from the anesthetized (with ketamine) macaques daily from the initial day of infection (day 0) until day 14 after infection and then at subsequent intervals of 21, 28, 45, and 63 days. CSF samples were obtained under telazol anesthesia (8 mg/kg), and buprenorphine (0.1 mg/kg) was given after CSF sampling for analgesia. Spinal taps were done on all of the anesthetized macaques on day 0 at the time of initial infection; subsequent spinal taps were staggered so that one of the macaques was sampled almost every day during the first 2 weeks. CSF samples were obtained from macaque J840 on days 0, 6, 11, 14, 21, 45, and at necropsy (day 63); from macaque P659 on days 0, 12, 21, 28, 45, and day 63; from macaque P469 on days 0, 4, 9, 14, 21, 28, 45, and 63; from macaque R470 on days 0, 8, 14, 21, 28, and 45; and from macaque T810 on days 0, 5, 10, 14, 21, 28, 45, and 63. After collection, aliquots of serum and CSF were shipped on dry ice for testing at the University of Texas Medical Branch or at Abbott Laboratories. Complete blood counts and chemistries were obtained before inoculation and on days 4, 7, 14, 21, 28, and 45 after inoculation. Four of the surviving macaques (J840, P469, P659, and T810) were killed and necropsied 63 days after infection. Samples of brain tissue (cerebral cortex, hippocampus, cerebellum, and brain stem) were reserved for culture, polymerase chain reaction (PCR), and histopathologic examination. Tissue specimens for culture were fresh frozen at 70 C, samples for PCR were placed in 5 ml of RNA/ater (Ambion), and specimens for histopathologic examination were fixed in 10% buffered formalin. Virus titration and culture. Titrations of daily serum samples from the infected macaques were done in 24-well tissue culture plates seeded with C6/36 cells, as described elsewhere [15 17]. Serum samples were tested undiluted and at serial 10- fold dilutions from 10 1 to Four wells of a tissue-culture plate were inoculated with 100 ml of each dilution. WNV titers were calculated as the TCID 50 per milliliter of specimen, using the method of Reed and Muench [18]. CSF and brain homogenates (200 ml) were inoculated directly into tissue flasks of Vero cells for virus isolation. Cultures were held for 10 days at 37 C and examined daily for the presence of viral cytopathic effect (CPE). After 10 days, some of the Vero cells were scraped from the flask and examined by indirect fluorescent antibody (IFA) test for the presence of WNV antigen [15 17]. Reverse-transcription (RT) PCR testing of macaque samples. A standard 2-step RT-PCR procedure was used to amplify a 750-bp fragment of the envelope (E) gene using primers WN1751 and WN2504A, as described elsewhere [19]. In brief, viral RNA was extracted directly from 70 ml of macaque serum or CSF using the QiaAmp viral RNA extraction kit (Qiagen). Purified RNA was eluted in 60 ml of sterile distilled water, and a 10-mL aliquot was used as the template for RT using avian myoblastosis virus reverse transcriptase (Roche Diagnostics). A 5-mL aliquot of this RT reaction product was used for PCR amplification of the E gene fragment using Taq DNA polymerase (Roche). Amplified products were visualized by electrophoresis in 2% Tris-acetate EDTA eletrophoresis buffer (TAE)/agarose gels. Nested PCR. RNA was extracted from 50-mL aliquots of serum or CSF, using TRIzol LS Reagent (Invitrogen) according to the manufacturer s instructions. The reverse RNA was transcribed using MuLV reverse transcriptase (Applied Biosystems). In the primary PCR, 5 ml of the RT reaction was used in a 25-mL final reaction volume, with final concentrations of 1 PCR Buffer II, 2.0 mmol/l MgCl 2, 200 mmol/l each dntp, 0.5 mmol/l each oligonucleotide primer, and U of AmpliTaq 670 JID 2004:189 (15 February) Ratterree et al.

3 DNA polymerase. Samples were incubated at 94 C for 2 min and then amplified for 40 cycles (94 C for 20 s, 50 C for 30 s, and 72 C for 30 s), followed by an additional incubation at 72 C for 5 min. A secondary PCR contained 1 PCR Buffer II, 2 mmol/l MgCl 2, 200 mmol/l each dntp, 0.5 mmol/l each oligonucleotide primer, 0.5 U of AmpliTaq DNA polymerase, and 1 ml of the primary PCR amplification product (342 bp) in a final volume of 20 ml. Secondary amplification used the same cycling parameters as the primary amplification, except that 35 cycles were performed. The expected size of the secondary PCR project was 208 bp. Primary and secondary PCR products were visualized on 1.2% TAE/agarose gels. Samples were scored positive if fragments of the appropriate size were detected. The oligonucleotide primers for the primary PCR were WNV F1 5 -GAARGAACTAGGDACYTTGACCAG-3 and WNV R1 5 -ACGTAGACWGRTGAYTTYGTGCACC-3. The oligonucleotide primers for the secondary PCR were WNV F2 5 -TGACC- AGTGCYATYAAYCGSCGGAGC-3 and WNV R2 5 -CTGACA- ATGCAYAGRTTYTTYCCAGC-3 IgM and IgG capture ELISA. Two ELISAs were developed to detect IgM and IgG to WNV; both assays used the capture assay format wherein the solid phase is coated with either antibody directed against human IgM (anti-igm capture) or antibody directed against human IgG (anti-igg capture). Apart from the solid phase, both assays were virtually identical. In brief, goat anti human IgM or goat anti human IgG was coated onto 6.4-mm polystyrene beads. The beads were incubated with 200 ml of serum that had been diluted 1:336 in diluent that contained detergent. After incubation, the beads were washed with deionized water and incubated with 200 ml of a WNVspecific recombinant antigen (CS-2) obtained from the Centers for Disease Control and Prevention (CDC). After incubation and washing, the bound antigen was detected by the addition of 200 ml of horseradish peroxidase labeled flavivirus group specific monoclonal antibody (6B6C-1; CDC). Colorimetric determination was made using 300 ml of 0.3% o-phenylenediamine-2 HC1 in 0.1 mol/l citrate buffer (ph 5.5) that contained 0.02% H 2 O 2 (Abbott Laboratories). The reaction was quenched by the addition of 1 ml of 1 N H 2 SO 4, and the optical density at 492 nm was determined. The absorbance was directly proportional to the amount of WNV-specific antibody bound to the solid phase. Sample-to-negative (S:N) ratios were determined for each macaque: N is the day 0 absorbance value for both the serum and the CSF specimens. An S:N ratio 5.0 was considered to be positive. HI test. HI antibodies to WNV antigen were measured by a standard method [20]. Antigens for the HI and CF tests were prepared from brains of newborn mice injected intracerebrally with WNV; the infected brains were treated by the sucrose-acetone extraction method [18]. Macaque serum samples were tested by HI at serial 2-fold dilutions from 1:20 to 1:5120 at ph 6.6 with 4 U of antigen and a 1:200 dilution of goose erythrocytes [15, 20]. CF test. CF tests were performed by a microtechnique [20] with 2 full units of guinea pig complement and antigen titers 1:32. CF antibody titers were recorded as the reciprocal of the highest serum dilution giving +3 or +4 fixation of complement (scale of 0 4)/antigen dilution. Plaque reduction neutralization (PRN) test. PRN tests on macaque serum were performed by a technique described elsewhere [15] in 24-well microplate cultures of Vero cells, using a fixed virus inoculum ( 100 pfu) against varying serum dilutions (1:10 to 1:20,480). The Egypt 101 strain of WNV was used for PRN tests. Serum samples were diluted in PBS (ph 7.4) that contained 10% fresh guinea pig serum. The virus inoculum was mixed with an equal volume of each serum dilution, and the mixture was incubated overnight at 4 C. The following day, 50 ml of the serum-virus mixture was inoculated into Vero microplate cultures, with 2 wells/dilution. Virus plaques were read 4 days later; 80% plaque reduction was used as the end point. RESULTS Clinical observations. Macaque R470 was killed on day 45 because of scrotal edema, anorexia, weight loss, and possible neurological symptoms (weakness). This macaque developed mild anemia (hemocrit, 27.8%; normal values, 34.8% 55.2%), but it was thought that the anemia was not severe enough to cause the clinical signs. A primary cause of the edema could not be found, and no neurological lesions were observed. There were no abnormal clinical signs noted in any of the other macaques. Serum chemistry results on all animals were within normal limits. Level and duration of infectious virus in blood. Figure 1 shows the results of virus cultures, nested RT-PCR, and ELISA assays for IgM and IgG done on serial serum samples from the 5 macaques. All of the macaques developed a transient and low level ( 100 TCID 50 /ml) of infectious WNV in their blood after infection. Macaque R470 had infectious virus present in its serum on the first day after infection, but the remainder did not have detectable infectious virus until day 2. The duration of infectious WNV varied from 1 to 5 days. Duration of viral RNA in blood. Two different PCR techniques (RT-PCR and nested RT-PCR) were used to detect the presence of WNV RNA in serum samples from infected macaques, but only the results of the nested RT-PCR are shown in table 1. Viral RNA was detected in all macaques on the day after inoculation and lasted for 4 9 days after inoculation, according to the results of 1 or both PCR techniques. The RT- PCR detected 13 positive specimens, whereas the nested RT- PCR detected 28 specimens as positive. Furthermore, 26 of 28 Infection of Macaques with WNV JID 2004:189 (15 February) 671

4 Figure 1. Temporal relationship between infectious virus and viral RNA and the appearance of IgM and IgG antibodies in the serum of 5 rhesus macaques after inoculation with West Nile virus. RT-PCR, reverse-transcription polymerase chain reaction. S/N, sample:negative ratio.

5 Figure 1. (Continued.) samples that tested positive in the nested RT-PCR yielded detectable product after the first round of PCR, which suggests that the larger quantity of extracted RNA sample used in the RT step (see Materials and Methods) was a critical factor for the increased sensitivity of this procedure. Thus, nested RT- PCR detected all of the specimens that were detected by either culture or by RT-PCR and extended the virus detection in all macaques for several days longer than the latter 2 techniques. Antibody determinations. Table 1 summarizes results of IgM and IgG ELISAs, as well as HI, CF, and PRN tests done on serial serum samples from the infected macaques. The IgM ELISA was the first test to provide a positive result; IgM against WNV appeared in the serum between the ninth and twelfth days. HI and PRN antibodies were initially detected between the tenth and thirteenth days after infection; and the median titers of these 2 antibodies continued to increase over the next several weeks. CF antibodies appeared days after infection in 4 of the macaques; the fifth macaque (R470) tested negative on day 14 but positive when next tested on day 21. Like the IgM ELISA ratios, the CF antibody titers generally began to decrease days after infection. The IgG ELISA was the last test to become positive; none of the macaques had detectable IgG ELISA antibodies when tested 14 days after infection, but 4 of 5 macaques tested positive at 21 days. Overlap of antibody detection and viral RNA detection. Four of 5 macaques tested negative for both viral RNA and infectious virus for 3 5 days prior to generating a detectable antibody response (figure 1). For these macaques, there was a clear distinction between the end of viremia (lack of either viral RNA or infectious virus) and the first detection of the antibody response. For 1 macaque (T810), 1 specimen (day 10 after inoculation) tested positive both for viral RNA (nested RT-PCR) and for antibodies (IgM ELISA and PRN test). The amount of virus present in macaque T810 on day 10 was apparently very low, because both culture and RT-PCR results were negative, and nested RT-PCR results were positive on one test and negative in a retest. Subsequent bleed dates were negative for the presence of virus and positive for antibodies. IgM and IgG ELISA results on CSF. IgM and IgG ELISAs were performed on a total of 35 CSF samples obtained from the infected macaques between days 0 and 63. The frequency of and intervals between the spinal taps on each macaque were described above. One macaque (T810) had a positive signal on Infection of Macaques with WNV JID 2004:189 (15 February) 673

6 Table 1. Median antibody titers obtained on serum of 5 rhesus macaques at various times after West Nile virus infection, as determined by IgM and IgG ELISAs and hemagglutination-inhibition (HI), complement-fixation (CF), and plaque reduction neutralization (PRN) tests. Day after infection IgM ELISA Median antibody titer IgG ELISA HI CF PRN :20 0 1: :40 0 1: :80 8: 32 1: :160 16: 32 1: :320 16: 32 1: :320 32: 32 1: :160 64: :160 16: 32 1: :160 16: 32 1:640 NOTE. ELISA results are expressed as the sample-to-negative (S:N) ratio, where N is the day 0 median absorbance on the 5 macaques serum samples. S:N 5.0 was considered to be positive. HI and PRN titers are expressed as the highest median serum dilution, giving a positive result. CF titers are expressed as the reciprocal of the median positive serum dilution:antigendilution. 0, negative result day 63 in the IgM ELISA. All other CSF samples tested negative in the 2 ELISAs. PCR results on CSF and brain samples. The same 35 CSF samples were also examined for WNV RNA by the nested PCR technique. A single CSF sample from macaque T810, obtained on day 5 after infection, provided a positive result in the nested PCR. This result is supported by the long duration of viral RNA detection the macaque s serum; virus was detected by nested RT-PCR on days 1 7, 9, and 10. However, this particular CSF sample contained red blood cells, which suggests that the positive result may have been due to a contaminated (bloody) CSF preparation. All other CSF samples from this macaque and the other macaques gave negative results. RT-PCR was done on 11 different samples of brain taken at necropsy from macaques J840, P659, P469, and T810; all were negative. Culture research on CSF and brain samples. The 35 CSF samples and the 11 brain homogenates noted above were cultured individually in flasks of Vero cells. No viral CPE was detected in any of the cultures, and spot slides made of the cells on day 10 were negative for WNV antigen in an IFA. Histopathologic examination of brain tissue. Hematoxylin-eosin stained sections of cerebral cortex, hippocampus, cerebellum, and brain stem, taken at necropsy from 4 of the macaques (J840, P659, P469, and T810) were examined microscopically. A single area of perivascular cuffing was noted in 1 brain section from macaque T810; all of the other histological sections appeared normal. As noted before, macaque R470 was killed on day 45; no tissue samples were obtained for histopathologic testing. DISCUSSION The primary objective of the present study was to obtain information on the level and duration of viremia and the kinetics of the humoral immune response in primates after infection with WNV. Little is known about this aspect of WNV infection in humans, because most of the recognized cases in the United States have been in patients with encephalitis or meningitis [12, 21]. The available data from animal experiments [15, 22, 23] and from induced or accidental human infections [1, 2, 6, 25, 26], where the precise time of WNV infection was known, have suggested that central nervous system involvement occurs during the second or third week after the initial virus infection. In fact, many patients with symptoms of West Nile encephalitis already have WNV-specific IgM antibodies in their serum and/ or CSF when they are first admitted to the hospital [10, 12, 24]. This is probably one reason why it is uncommon to isolate WNV from blood or CSF of West Nile encephalitis cases at the time of hospitalization. The most common clinical form of WNV infection in humans is West Nile fever [12]. This illness has a sudden onset and is characterized by fever, malaise, anorexia, headache, eye pain, nausea, vomiting, arthralgia, and rash of 3 6 days duration. West Nile fever is generally a self-limited disease in otherwise healthy younger people. Detailed studies of West Nile fever among Israeli settlers during the 1950s [3, 4] demonstrated that a brief period of viremia occurs during the acute febrile phase of the illness. Titrations of blood collected from patients with West Nile fever at various intervals during their illness showed the presence of virus for up to 4 days after the onset on symptoms, although the titers were relatively low and were rarely suckling mouse LD 50 /ml [4]. These studies further demonstrated that CF and neutralizing antibodies appeared in the serum of convalescent patients with West Nile fever within 2 3 weeks after infection. A similar pattern was recently reported in 2 laboratory workers who accidentally contracted WNV infection [25]. There is also increasing evidence that WNV infection in persons with severe underlying disease or immunosuppression may result in a more virulent or fatal illness [1, 2, 6, 26 30]. During the 1950s, WNV was tested as a potential antineoplastic agent 674 JID 2004:189 (15 February) Ratterree et al.

7 in 96 patients with terminal cancer [6]. Most of the subjects (89%) inoculated with WNV developed nothing more than a febrile illness; virus was detected in their blood within 1 2 days after infection, although, in some patients, viremia persisted for up to 12 or 14 days [6]. Two recent reports [26, 30] of the isolation of WNV from the blood and/or CSF of patients receiving immunosuppressive drugs for other underlying health problems (e.g., leukemia and renal transplant) offered further evidence that such individuals may have a prolonged viremia and more severe disease on infection with WNV We believe that the clinical course, level and duration of viremia, and antibody response observed in our 5 experimentally infected rhesus macaques are similar to what occurs in most uncomplicated human WNV infections. A similar clinical course and antibody response have been observed in monkeys naturally infected with WNV [31]. Thus, our study results give some insight into the problem of identifying asymptomatic WNV-infected blood donors. Of the 3 assay systems used for detecting WNV in the macaques blood (direct culture, RT- PCR, and nested PCR), the nested PCR was the most sensitive. Direct culture is probably not a feasible method for most blood collection centers because of the isolation and biosafety facilities needed and the time required to achieve results ( 7 days). The RT-PCR and nested PCR can be done under less stringent biosafety conditions and can provide a much quicker result. Our results also indicate that, by the time WNV-specific antibodies are present in the serum (on the ninth or tenth day according to the results of the IgM ELISA), blood is probably no longer infectious. However, the serological profile from one of the macaques (T810) was quite distinct from that of the other macaques. First, the duration of viremia extended for 10 days, as determined by nested RT-PCR. Second, IgM class antibodies to WNV and PRN antibodies were detected on day 10, the last day that WNV RNA was detected in serum. Third, there was the possibility of central nervous system involvement in this macaque. A second question that we tried to answer in the present study was whether WNV entered the brain of the infected macaques. In the case of humans, WNV clearly invades the brain in patients with encephalitis, but it is uncertain whether the virus enters the brain in uncomplicated cases of West Nile fever or asymptomatic WNV infection. Our results suggest that WNV probably does not enter the brain in most nonencephalitic cases. Only 1 (macaque T810) of 5 macaques in our study developed WNV-specific IgM in its CSF after infection. IgM class antibodies were detected in the CSF on day 63. A spinal fluid sample taken from the same macaque on the fifth day after infection was also positive for WNV RNA according to nested RT-PCR, and mild histopathologic characteristics (perivascular cuffing) were noted in stained, fixed sections of its brain taken at necropsy 63 days after infection. These findings suggest that WNV entered the brain of macaque T810. However, as noted above, hemorrhagic CSF samples were obtained from macaque T810 on days 5 and 63, so it is unclear whether the blood was present in the CSF before the spinal tap procedure. In contrast, there was no evidence that central nervous system infection occurred in the other 4 macaques. Several observations are noteworthy. First, there were no apparent clinical signs indicative of WNV infection in the macaques, as is also seen in most human WNV infections. Second, the viral titers were relatively low ( 100 TCID 50 /ml) in the blood of the infected macaques. Third, in most macaques, there was no apparent overlap between the detection of viral RNA and the detection of antibodies to WNV. It is unclear whether this apparent gap is associated with the host animal used in our study or is due to the sensitivity of the PCR assay to detect low-level viral RNA in later blood samples or of the immunoassay to detect the IgM response in earlier blood samples. However, viral RNA and IgM were both detected on day 10 in 1 macaque, which suggests that virus may continue to be present at low levels in some subjects after they develop a detectable antibody response to WNV. The present study suggests the need of using both nucleic acid and serological testing to determine exposure to WNV. Finally, studies in small animal models have indicated that the infectious dose of North American WNV strains is probably very low ( 1 pfu or less in National Institutes of Health Swiss mice) [22]. The volumes of blood or plasma associated with transfusion are relatively large, and donor units with even very low levels of live virus could potentially represent a risk for transfusion transmission. The observation that viral RNA could be detected in the macaque serum by nested RT-PCR for several days longer than it was detected by cell culture or standard RT- PCR suggests that this procedure could be advantageous in identifying samples that represent even a low risk of transfusion-related transmission. However, the reliable screening of blood donor units for the presence of very small quantities of infectious WNV may ultimately be limited by the relatively small sample volume used for testing, compared with the much larger volume transfused to the recipient. References 1. Centers for Disease Control and Prevention. 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