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    Photomicrographs of a kidney from a hamster chronically infected with SLEV. A, Viral antigen positivity in macrophages and tubular epithelium (immunohistochemical stain). B, Higher magnification of the SLEV antigen positive cells in (arrowhead).

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    Immunohistochemical staining for SLEV antigen in the brain of hamster 6092, showing weak focal positivity in large neurons in the hippocampal region (arrowheads). SLEV was also cultured from the brain of this hamster when killed 167 days PI.

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CHRONIC ST. LOUIS ENCEPHALITIS VIRUS INFECTION IN THE GOLDEN HAMSTER (MESOCRICETUS AURATUS)

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  • 1 Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

To further study the phenomenon of flavivirus persistent infection, golden hamsters (Mesocricetus auratus) were inoculated intraperitoneally with a low pathogenicity strain of St. Louis encephalitis virus (SLEV). After inoculation, the animals remained asymptomatic and developed high levels of specific neutralizing antibodies in their sera. However, about one half of the hamsters continued to shed infectious SLEV in their urine for prolonged periods of time. By co-cultivation, SLEV was recovered from selected tissues (kidney, lung, and brain) of some of the animals for up to 185 days after initial infection. Although no specific histopathologic changes were observed in these tissues, SLEV antigen was shown by immunohistochemistry in the interstitium and tubular epithelium of the renal cortex and in a few large neurons of the cerebral cortex. Seventeen SLEV isolates from urine and tissues of the chronically infected hamsters were sequenced. In comparison with the infecting parent SLEV strain, two common mutations and amino acid substitutions were observed in all of the hamster isolates. The findings of this study were very similar to previous descriptions of chronic West Nile, Modoc, and tick-borne encephalitis virus infections in mammals, and they re-emphasize the potential importance of persistent flavivirus infection in vertebrates.

INTRODUCTION

St. Louis encephalitis virus (SLEV) is a neurotropic, mosquito-borne, zoonotic agent that occurs throughout the Americas.1,2 Taxonomically, SLEV is included within the Japanese encephalitis virus group (Flavivirus: Flaviviridae), which includes other important human pathogens such as Japanese encephalitis (JEV), West Nile (WNV), and Murray Valley encephalitis (MVEV) viruses.1,3 In fact, these four flaviviruses have many common ecologic and epidemiologic characteristics.1,2,47

Like JEV and WNV, most human infections with SLEV are subclinical.1 The clinical manifestations of SLEV infection can range from a mild febrile illness to severe neuroinvasive disease such as aseptic meningitis or encephalitis.8 The case-fatality rates in St. Louis encephalitis (SLE) outbreaks range from ~3% to 20%, with the frequency of severe disease being highest in the elderly and the very young.1,2 Although chronic or relapsing SLEV infections have not been shown in humans, a case of apparent post-infectious St. Louis encephalomyelitis was recently reported.9 Clinical studies have shown that patients with SLE also frequently have renal involvement, as manifested by proteinuria, hematuria, pyuria, and/or elevated blood urea nitrogen and serum creatinine.8,10 Luby and others11 reported finding SLEV antigen in uroepithelial cells of four patients with SLE by fluorescent antibody technique and SLEV-like particles in urine of patients by electron microscopy. Collectively, these data suggest that the kidney is another site of SLEV replication and cellular injury.9

In experimental studies, we have shown that adult hamsters infected with WNV develop chronic renal infection and shed infectious virus in their urine for up to 8 months, despite a robust immune response.12,13 Recently a human case of WNV infection was reported in which WNV nucleic acids were detected in the patient’s urine by reverse transcriptase-polymerase chain reaction (RT-PCR), although infectious virus could not be cultured.14

In view of the above findings, a study was undertaken to determine if SLEV might behave similarly in hamsters. Here we report our results that indicate SLEV, like WNV, can produce a persistent infection in hamsters and that some animals develop chronic renal infection and shed infectious virus in their urine for prolonged periods of time.

MATERIALS AND METHODS

Animals.

Six- to 8-week-old female golden hamsters (Mesocricetus auratus) were obtained from Harlan Sprague-Dawley (Indianapolis, IN). The hamsters were housed three or four per cage and cared for in accordance with guidelines of the Committee on Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council, Washington, DC). All experiments were conducted in an animal biosafety level 3 (ABSL-3) facility under a protocol approved by the University of Texas Medical Branch (UTMB) Institutional Animal Care and Use Committee.

Virus.

SLEV strain BeAr 23379 was used in the experiments. This strain was originally isolated from a pool of Sabethes belisarioi mosquitoes collected in Para State, Brazil, in 196015; it had received two intracerebral passages in suckling mice and one passage in Vero cells before use.

Experimental design.

In the first experiment (Table 1), a group of 12 hamsters was inoculated intraperitoneally (IP) with about 104 plaque forming units of SLEV strain BeAr 23379 prepared in Vero cells. The animals were observed daily for signs of illness. On day 28 post-inoculation (PI), blood samples were obtained from all animals and assayed for SLEV antibody by hemagglutination-inhibition (HI) test. Starting on day 35 PI, urine samples were collected at irregular intervals from the hamsters for virus culture; the technique used to obtain fresh urine from the live animals was described previously.12 Starting on day 47 PI and at ~1- to 2-week intervals, one hamster was anesthetized with Halothane (Hydrocarbon Laboratories, River Edge, NJ), exsanguinated by cardiac puncture, and necropsied. The blood was allowed to clot, and the serum was saved for antibody studies. At necropsy, the abdomen was opened; any urine present in the bladder was aspirated directly with a 1-mL syringe and a 26-gauge needle for virus assay. Samples of the lung, kidney, and spleen were taken for virus assay by direct culture and by co-cultivation in Vero cells. A piece of each tissue also was placed in 10% buffered formalin for histopathologic and immunohistochemical studies. The last hamster in Experiment 1 was killed and sampled 124 days PI.

In a second experiment (Table 2), four additional hamsters (numbered H-6089, 6090, 6091, and 6092) were each inoculated subcutaneously with 100 μL of a Vero cell culture of SLEV-positive urine, obtained 35 days PI from hamster H-8605 from Experiment 1 (Table 1). The object of the second experiment was to determine if the biologic and genetic properties of the parent virus had changed after persistent infection in a hamster. Beginning on day 18 PI, urine samples were periodically collected from the four hamsters to be assayed for virus. Five to 6 months after inoculation, the four hamsters were killed and necropsied as described above; samples of brain, liver, spleen, kidney, and lung were collected for virus assay and for histopathologic and immunohistochemical examination.

Virus assay.

Direct culture.

Samples (~0.1 g of tissue) of lung, spleen, kidney, liver, and brain from the freshly killed hamsters were homogenized individually in sterile 2 mL Ten Broeck glass tissue grinders that contained a measured 1.0 mL phosphate buffered saline (PBS), pH 7.4, containing 30% heat-inactivated fetal bovine serum (FBS) to yield ~10% (wt/vol) suspensions. After clarification by centrifugation at 10,000g for 5 minutes, 200 μL of each supernatant was inoculated onto confluent monolayers of Vero cells in 12.5-cm2 plastic culture flasks. Freshly voided hamster urine was diluted 5- or 10-fold in PBS; 200 μL of the diluted sample was inoculated directly into flask cultures of Vero cells. Cultures were held at 37°C and observed daily for viral cytopathic effect (CPE). If CPE was observed, a sample of the culture fluid was removed and tested by the VecTest West Nile Virus/St. Louis Encephalitis antigen panel assay (Medical Analysis Systems, Camarillo, CA), according to the manufacturer’s instructions.

Co-cultivation.

Our co-cultivation technique and culture media were described previously.12 Briefly, fresh tissue fragments of each hamster sample were minced and incubated at 37°C for 30–60 minutes in trypsin-EDTA solution to dissociate the cells. After several washes in PBS, the lung, liver, spleen, and kidney cells were resuspended in 3–5 mL of Gibco medium 199 with Earle’s salts, l-glutamine, NaHCO3 (Invitrogen, Carlsbad, CA), 10% FBS, and penicillin-streptomycin. For brain cells, the co-cultivation medium was a 1:1 mixture of Dulbecco modified Eagle medium and F-12 nutrient medium (Gibco) with 10% FBS and gentimicin.12 Equal volumes of the suspended cells from each tissue sample were inoculated into three flasks of monolayer cultures of Vero cells. Co-cultures were maintained at 37°C for 15 days and were examined for evidence of CPE. If CPE was observed, the culture medium was tested for the presence of SLE virus by the VecTest WNV/SLE antigen assay described above.

Antibody determinations.

Serum samples obtained from the hamsters were tested for SLEV antibodies by HI test and plaque reduction neutralization test (PRNT), as described previously.12 The SLEV antigen used in the HI test was prepared from brains of newborn mice infected through the intracerebral route. The infected brains were treated by the sucrose acetone extraction method.16 Sera were tested for antibody at serial 2-fold dilutions ranging from 1:20 to 1:2,560 at pH 6.6 with four units of antigen and a 1:200 dilution of goose erythrocytes, following established protocols.16 The plaque reduction neutralization test was done in 24-well microplate cultures of Vero cells with a constant virus inoculum (~100 PFU) against varying dilutions of hamster serum.16 Hamster sera were prepared in 2-fold dilutions from 1:10 to 1:20,240 in PBS supplemented with 10% FBS. The serum-virus mixtures were incubated overnight at 5°C before inoculation. Two microplate wells were inoculated with each serum dilution. After incubation for 1 hour and addition of a nutrient agar overlay,12 microplate cultures were incubated at 37°C. Plaques were read on the sixth day. The dilution of sera that reduced the virus dose by ≥ 80% was considered the antibody titer and was expressed as the reciprocal of the highest positive dilution.

Histologic and immunohistochemical examinations.

Hamster tissue samples (lung, brain, liver, spleen, kidney) were fixed in 10% buffered formalin for 24 hours and transferred to 70% ethanol for storage and subsequent embedding in paraffin. Sections (4–5 μm thick) were prepared and stained by the hematoxylin and eosin method or immunohistochemically. Immunohistochemical (IHC) straining for SLEV viral antigen was performed as described previously.17,18 A SLEV mouse hyperimmune ascitic fluid was used as the primary antibody at a dilution of 1:100 and incubated overnight at 4°C. An ISO-IHC immunostain kit (Inno-Genex, San Ramon, CA) was used to detect bound primary antibody and to prevent nonspecific binding between species.17,18

Nucleotide sequencing.

The envelope (E) gene from the SLEV parent strain and from 17 selected virus isolates obtained from urine, kidney, and brain of infected hamsters (Tables 3 and 4) was sequenced using F880, F1390, F1990, B1629, B2047, and B2586 primers described by Kramer and Chandler.19 Viral RNA was extracted from culture supernatants of virus-infected Vero cells by a Trizol/chloroform procedure described previously.20 For cDNA synthesis, the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA) was used, following the manufacturer’s protocol. cDNA products were amplified using the single-reaction Titan RT-PCR kit (Roche Applied Science, Indianapolis, IN), according to the manufacturer’s recommendations. PCR products were purified by QIAquick Gel Purification Kit (Qiagen, Santa Clarita, CA) and were sequenced in both directions to generate consensus sequences. All sequencing reactions were performed at the UTMB Protein Chemistry Core Laboratory and run on an ABI Prism model 3100 DNA sequencer (Applied Biosytems, Foster City, CA) Sequences were assembled and analyzed using the SeqMan suite of the DNAStar program (DNASTAR, Madison, WI).

Cloning of PCR product.

To evaluate variability of the mutation site in the parent strain, ~149-bp PCR products from F880 and B1629 primers were extracted with the QIAquick Gel Purification Kit (Qiagen) and cloned into the pGEM-T vector (Promega, Madison, WI), according the manufacturer’s protocol. Ten white colonies were screened by PCR to verify the inserted fragment and sequenced using T7 and Sp 6 primers. The sequences were analyzed using DNAStar programs.

RESULTS

Clinical observations.

The 12 hamsters inoculated initially with the BeAr 23379 parent strain of SLEV (Experiment 1) and the 4 hamsters inoculated with the virus isolate obtained from urine of animal H-8605 (Experiment 2) all remained active and showed no signs of illness.

Serologic results.

The hamsters in Experiment 1 all had HI antibodies to SLEV antigen when tested 28 days PI. Serum antibody titers of these animals ranged from 1:160 to 1:320 (data not shown). In addition, all of these hamsters had SLEV neutralizing antibodies in their sera when bled at the time of necropsy (Table 1).

Likewise, the four hamsters in Experiment 2 had SLEV neutralizing antibodies in their sera when they were killed and cultured (Table 2).

Viruria.

As shown in Table 1, SLEV was detected in urine of 7 of the 12 hamsters in Experiment 1. Urine samples were collected from these animals at irregular intervals. Also, if the animal’s bladder was empty at the time of collection, no sample was obtained. Nonetheless, infected urine samples were obtained at various times between 35 and 124 days PI.

As shown in Table 2, infectious urine samples were also obtained from two of the four hamsters in Experiment 2. A positive urine sample was obtained from one hamster (6089) in this group 150 days after infection.

Results of organ cultures.

Tissue samples from the hamsters were assayed for SLEV by two methods: direct culture and co-cultivation. As observed previously with hamsters chronically infected with WNV,12 co-cultivation of tissues yielded more virus isolates that direct culture of tissue homogenates. Results of co-cultivation of spleen, lung, and kidney from hamsters in Experiment 1 are summarized in Table 1. By this technique, SLEV was isolated from the lung and/or kidney of 6 of the 12 infected animals, when necropsied 47 to 124 days after initial infection. Brain samples from this group of hamsters were not assayed. The six hamsters with positive organ cultures also shed SLEV in their urine. Virus assay of organs of these same animals by direct culture of tissue homogenates yielded only two virus isolates (lung of hamster 8607 on day 81 and kidney of hamster 8611 on day 124).

Table 2 summarizes co-cultivation results on the four hamsters from Experiment 2. When killed 167 to 185 days after infection, SLEV was recovered by co-cultivation of brain tissue from three of the four animals. None of these brain samples yielded SLEV by direct culture, again confirming the increased sensitivity of the co-cultivation technique. Two of the three culture-positive animals (hamsters 6089 and 6090) also had one or more SLEV-positive urine cultures, although virus was not recovered by co-cultivation or by direct culture of the kidney or other organs of the fourth hamster in Experiment 2.

Histologic and immunohistochemical examinations.

No significant histopathologic changes were observed in spleen, lung, brain, or liver of any of the experimentally infected hamsters when killed. Occasionally, dilation of the renal tubules, with atrophy and flattening of the tubular epithelia, mostly in the cortical regions, was observed in renal tissue. Rare foci of microcalcification were also seen in kidneys of the animals; these changes were more prominent in kidneys of hamsters that were killed later in the experiments. However, these changes were likely non-specific and unrelated to the chronic SLEV infection. Progressive glomerulonephropathy and tubular atrophy are commonly observed in older (normal) hamsters, especially females.21,22

Immunohistochemical examination of kidneys of hamsters from the first experiment (Table 1) revealed SLEV antigen in the interstitium (mainly in macrophages) and tubular epithelium of the renal cortex of hamsters 8603, 8611, and 8649 (Figure 1A and B). Although less intense, the distribution and pattern of SLEV antigen in kidneys of the chronically infected hamsters were similar to that observed in hamsters persistently infected with WNV.12 SLEV viral antigen was also detected in large neurons of the cerebral cortex of hamsters 6091 and 6092 from Experiment 2 with relatively weaker staining intensity (Figure 2).

Genotypic changes in SLEV during chronic infection.

The flavivirus envelope glycoprotein is a major antigen that elicits neutralizing antibodies during the immune response and is responsible for host cell binding and tissue tropism.23,24 To evaluate genetic changes that might have occurred during SLEV persistence, we determined the complete nucleotide sequences of the envelope gene of the parent SLEV strain (BeAr 23379) and of 17 SLEV isolates obtained from infected hamsters. In comparison with the parent BeAr 23379 strain, nine nucleotide changes were found in the viral isolates obtained from the infected hamsters (Table 3). Mutations at position 1425 and 1430 were consensus for all of the hamster isolates and resulted in two amino acid substitutions (Table 4). The cloning of 10 PCR fragments containing this site did not reveal the presence of these mutations in parent BeAr 23379 strain. Nucleotide sequences of the E-gene of all urine and kidney isolates were conserved. Only one additional coding nucleotide substitution was found in a SLEV urine isolate from hamster 8602. In contrast, seven unique mutations were found in the three brain isolates. Four of those mutations were silent, and three resulted in amino acid substitutions (Table 4). Mutation rates of the E-protein gene for the 16 urine and brain isolates were 0.13–0.2 and 0.2–0.04, respectively.

DISCUSSION

Although persistent SLEV infection has been shown in birds,25 this is the first clear evidence that chronic SLEV infection can occur in a mammalian host. Our results also confirm previous observations that the BeAr 23379 strain of SLEV is of relatively low virulence and that it induces a robust immune response in experimental animals without producing clinical signs of illness.26,27 However, despite the high level of SLEV neutralizing antibodies, the hamsters still developed chronic SLEV infection and shed virus in their urine. In 1943, Slavin28 reported that chronic SLEV brain infection occasionally occurred in passively immunized young mice, born of SLEV-immune mothers, after intranasal challenge with the virus. Although the work of Slavin was not confirmed and has largely been forgotten, the results of these experiments suggest that his observations were probably valid.

The persistence of SLEV in the lung, kidney, and brain of experimentally infected hamsters (Tables 1 and 2) is compatible with reports of persistence infection with other flaviviruses in mammals. As noted before, persistent WNV infection has been shown in hamsters12,13 and rhesus monkeys.29 Persistent JEV infection has been reported in mice30 and humans.31,32 Chronic infection of rhesus monkeys33 and of humans34 has been reported with the Far Eastern subtype of tick-borne encephalitis virus. Chronic renal and pulmonary infection and viruria have been shown with Modoc virus in deer mice and hamsters.3537 Less well-documented reports also have suggested persistent infection of bats with Rio Bravo virus38 and of muskrats with Omsk hemorrhagic fever virus.39 Collectively, these reports suggest that persistent infection can occur with a variety of flaviviruses in vertebrates and that it is not an uncommon phenomenon or laboratory curiosity. Its potential clinical and epidemiologic significance for humans remains to be determined; but it seems to warrant further study and cannot be ignored.

Another intriguing finding of this study was the genetic changes in the E-gene of the hamster isolates of SLEV (Tables 3 and 4). It seems remarkable that the same two mutations at amino acid positions 154 and 156 were found in all 17 virus isolates obtained from the infected hamsters. It is unlikely that these mutations occurred during virus isolation in Vero cells, because sequencing of viral RNA isolated directly from hamster urine (samples H-8602 and H-8611), without passing in Vero cells, also revealed the same mutations. This result could be explained by adaptive selection of these mutations by spontaneous mutagenesis of SLEV during persistence in hamster tissues or because the BeAr 23379 parent strain contained a pool of pre-existing quasispecies. Although the cloning experiment did not reveal divergence at positions 154 and 156 in the parent strain, the possibility of the presence of these mutations at low level in the parent strain cannot be ruled out. It is also noteworthy that asymptomatic persistence of SLEV in the brains of the hamsters H-6089, H-6090, and H-6092 also resulted in a higher mutation rate in the brain isolates compared with the urine and kidney isolates.

The presence of the two mutations at amino acid positions 154 and 156 are of interest and could be related to persistence. However, further experiments to test this hypothesis are needed. The experiment reported in this communication is mainly descriptive; experiments to study the relationship of specific mutations to SLEV persistence are beyond the scope of this paper.

Table 1

Isolation of SLEV from urine and selected tissues of chronically infected hamsters in Experiment 1

Co-cultivation results
Animal no.Day(s)* SLEV culture-positive urine sample collectedDay* tissues sampledSpleenLungKidneySerum neut. antibody titer†
* Day after initial experimental infection (PI).
† Reciprocal of SLEV serum neutralizing antibody titer on day the animal was killed and tissues cultured.
0, culture negative; +, culture positive for SLEV.
8605D354700+640
8649D35, D49, D53, D62620++1,280
8610None690001,280
8612None760002,560
8607D81810+02,560
8609D53, D82820001,280
8604None830+0320
8602D53, D65, D81, D8989000640
8601None970001,280
8603None1120002,560
8613D49, D68, D81, D93, D11611600+1,280
8611D35, D49, D65, D78, D12412400+1,280
Table 2

Isolations of SLEV from urine and selected tissues of persistently infected hamsters in Experiment 2

Co-cultivation result
Animal no.Day(s)* SLEV culture-positive urine collectedDay* tissues sampledBrainSpleenLiverLungKidneySerum neut. antibody titer†
* Day after initial experimental infection (PI).
† Reciprocal of SLEV serum neutralizing antibody on day the animal was euthanized and tissues cultured.
0, culture negative; +, culture positive for SLEV.
6092None167+00001,280
6090D30168+0000320
6091None18400000320
6089D18, D29, D158185+0000320
Table 3

Nucleic acid changes in the E-gene of 17 SLEV isolates from chronically infected hamsters compared with the parent strain (BeAr 23379)

Mutations in nucleotides
Animal no.Day PISource of isolate1163114614251426143017941887205921692265Divergence (%)
* Viral RNA extracted directly from infected urine without passage in Vero cells.
Day PI, day after initial virus infection.
Bold type indicates changes from parent strain.
H-850535UrineCCATCCGGCC0.13
H-864935UrineCCATCCGGCC0.13
H-861149UrineCCATCCGGCC0.13
H-861349UrineCCATCCGGCC0.13
H-864962UrineCCATCCGGCC0.13
H-860289Urine*CCATCCGGTC0.2
H-8613106UrineCCATCCGGCC0.13
H-8611124Urine*CCATCCGGCC0.13
H-8611124KidneyCCATCCGGCC0.13
H-608929UrineCCATCCGGCC0.13
H-608958UrineCCATCCGGCC0.13
H-609058UrineCCATCCGGCC0.13
H-6089141UrineCCATCCGGCC0.13
H-6089158UrineCCATCCGGCC0.13
H-6092167BrainCTACCTAGCC0.4
H-6090168BrainCCATCCGACT0.27
H-6089185BrainGCATCCGGCC0.2
BeAr 23379CCCTTCGGCC
Table 4

Amino acid substitutions in 17 SLEV isolates from chronically infected hamsters compared with the parent strain (BeAr 23379

Amino acid substitutions
Animal no.Day PISource of virus isolate67154155156366403
* Viral RNA isolated directly from urine without passage in Vero cells.
Day PI, day after initial virus infection.
Bold type indicates changes from parent strain.
H-850535UrineTyrPheTyrSerGlySer
H-864935UrineTyrPheTyrSerGlySer
H-861149UrineTyrPheTyrSerGlySer
H-861349UrineTyrPheTyrSerGlySer
H-864962UrineTyrPheTyrSerGlySer
H-860289Urine*TyrPheTyrSerGlyThr
H-8613106UrineTyrPheTyrSerGlySer
H-8611124Urine*TyrPheTyrSerGlySer
H-8611124KidneyTyrPheTyrSerGlySer
H-608929UrineTyrPheTyrSerGlySer
H-608958UrineTyrPheTyrSerGlySer
H-609058UrineTyrPheTyrSerGlySer
H-6089141UrineTyrPheTyrSerGlySer
H-6089158UrineTyrPheTyrSerGlySer
H-6092167BrainTyrPheHisSerGlySer
H-6090168BrainTyrPheTyrSerArgSer
H-6089185BrainArgPheTyrSerGlySer
BeAr 23379TyrAspTyrPheGlySer
Figure 1.
Figure 1.

Photomicrographs of a kidney from a hamster chronically infected with SLEV. A, Viral antigen positivity in macrophages and tubular epithelium (immunohistochemical stain). B, Higher magnification of the SLEV antigen positive cells in (arrowhead).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 2; 10.4269/ajtmh.2007.76.299

Figure 2.
Figure 2.

Immunohistochemical staining for SLEV antigen in the brain of hamster 6092, showing weak focal positivity in large neurons in the hippocampal region (arrowheads). SLEV was also cultured from the brain of this hamster when killed 167 days PI.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 76, 2; 10.4269/ajtmh.2007.76.299

*

Address correspondence to Dr. Robert B. Tesh, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609. E-mail: rtesh@utmb.edu

Authors’ addresses: Marina T. Siirin, Tao Duan, Hao Lei, Hilda Guzman, Amelia P. A. Travassos da Rosa, Douglas M. Watts, Shu-Yuan Xiao, and Robert B. Tesh, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609.

Acknowledgments: The authors thank Dora Salinas for help in preparing the manuscript.

Financial support: This work was supported by Contracts NO1-AI25489 and NO1-AI30027 from the National Institutes of Health.

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Author Notes

Reprint requests: Robert B. Tesh, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609. E-mail: rtesh@utmb.edu.
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