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    Figure 1.

    Survival of 20 immunosuppressed (Group 2) hamsters after yellow fever vaccination.

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    Figure 2.

    Spot slide of C6/36 cells inoculated with brain homogenate of sick hamster (Group 2). YFV-17D antigen appears as yellow-green staining material in cell cytoplasm when examined by indirect fluorescent antibody test (IFAT).

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    Figure 3.

    Photomicrographs showing pathologic changes in the brain of infected immunosuppressed hamster 157. A, Increased cellularity in basal nuclear region, consisting of lymphocytes and reactive glial cells. B, Perivascular infiltration with lymphocytes and macrophages.

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    Figure 4.

    YFV-17D antigen distribution as seen in IHC-stained sections of different regions of the brain of hamster 158 (Group 2). The antigen appears as red stain by this technique; many neurons are positive. A, Deep layers of cerebral cortical region. B, Hippocampus. C, Basal ganglia region. D, Brainstem. Immunohistochemical stain.

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    Figure 5.

    IHC-stained sections of brain and liver from an immunosuppressed hamster infected with YFV-Asibi (Group 3). A, The brain is YFV antigen-negative, whereas the liver (B) is YFV antigen-positive. Antigen staining and microvesicular steatosis can be seen in many hepatocytes.

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Yellow Fever 17-D Vaccine Is Neurotropic and Produces Encephalitis in Immunosuppressed Hamsters

Rosa I. MateoDepartments of Pathology and Internal Medicine and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

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Shu-Yuan XiaoDepartments of Pathology and Internal Medicine and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

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Amelia P. A. Travassos da RosaDepartments of Pathology and Internal Medicine and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

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Hao LeiDepartments of Pathology and Internal Medicine and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

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Hilda GuzmanDepartments of Pathology and Internal Medicine and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

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Liang LuDepartments of Pathology and Internal Medicine and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

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Robert B. TeshDepartments of Pathology and Internal Medicine and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, Texas

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Immunosuppressed (cyclophosphamide) adult golden hamsters inoculated intraperitoneally (IP) with wild-type Asibi yellow fever virus (YFV) developed a rapidly fatal illness. Histopathologic and immunohistochemical studies of tissues from these animals showed typical hepatic changes of severe yellow fever (inflammation, hepatocyte necrosis, and steatosis) without brain involvement. In contrast, 50% of immunosuppressed hamsters receiving the YFV-17D–attenuated vaccine developed a slowly progressive encephalitic-type illness. Brain tissue from these latter animals revealed focal neuronal changes, inflammation, and YFV antigen–positive neurons; however, the liver and spleen appeared normal. YFV was isolated from brain cultures of many of these animals. Immunocompetent (non-immunosuppressed) hamsters inoculated with both viruses developed a subclinical infection. Results of this study indicate that wild-type YFV is hepatotropic in immunosuppressed hamsters, whereas the attenuated YFV-17 is primarily neurotropic. These findings support current recommendations against yellow fever vaccination of immunosuppressed/ immunocompromised people and suggest that this hamster model might be useful for monitoring the safety of other live-attenuated YFV vaccines.

INTRODUCTION

Yellow fever vaccine, a live, attenuated virus preparation made from the 17D yellow fever virus strain (YFV-17D), was developed almost 70 years ago by empirical methods; it is still considered to be one of the safest and most effective virus vaccines.1,2 Furthermore, because yellow fever is still endemic in rural areas of sub-Saharan Africa and tropical South America, the vaccine is widely used in human residents of these regions and for tourists, military personnel, and contract workers entering endemic areas.1,3

Adverse reactions to the YFV-17D vaccine are typically mild and include low-grade fever, myalgia, headache, and in a few cases, hypersensitivity and allergic symptoms.13 Rarely, more severe vaccine-associated adverse events (postvaccinal encephalitis and viscerotropic disease) occur.13 Yellow fever vaccine–associated neurotropic disease (YFVax-AND), formerly known as postvaccinal encephalitis, is the subject of this report.

Historically, YFVax-AND has been the most common serious adverse event associated with yellow fever vaccines.2,3 Since 1940 and the institution of standardized YFV-17D manufacturing procedures, a total of 25 cases of YFVax-AND have been published.1 Almost all of these cases were in children < 7 months of age.3 In 2002, a case of fatal meningoencephalitis was reported from Thailand in an adult man with undiagnosed HIV infection and low CD4+ cell counts, who received the yellow fever vaccine in preparation to travel.4 Based on these reports, the Centers for Disease Control and Prevention (CDC) now cautions against using the YFV-17D in infants < 9 months of age, pregnant and nursing women, and persons with altered immune status.5 In the case of persons with altered immune status, the current CDC recommendations for yellow fever immunization state the following: “Infection with yellow fever vaccine virus poses a theoretical risk for encephalitis to 1) patients with acquired immunodeficiency syndrome (AIDS); 2) patients who are infected with HIV and have other manifestations of HIV infection; 3) patients with leukemia, lymphoma, generalized malignancy; or 4) those whose immunologic responses are suppressed by corticosteroids, alkylating drugs, antimetabolites, or radiation.”5 Although these hypothetical precautions seem justified, the precise reasons for the increased risk of YFVax-AND in persons with altered immune status are unknown. In an attempt to test the hypothesis and to learn more about the effect of immunosuppression on YFV-17D infection, adult hamsters were maintained on cyclophosphamide (CYP), an alkylating agent with broad immunosuppressive activity,6 and were given the yellow fever vaccine. A second group received YFV-17D but no CYP. A third group of immunosuppressed hamsters received wild-type YFV for comparison. The results of our experiments, which support the current recommendations, are described in this report.

MATERIALS AND METHODS

Viruses

Yellow fever vaccine, YF-VAX, lot UE665AA, expiration date 26 September 2006 (Aventis Pasteur, Swift-water, PA), was the source for YFV-17D. According to the manufacturer’s description, the vaccine was prepared from the 17D-204 substrain of yellow fever virus grown in leukosis-free chick embryos and contained ∼104.7 plaque-forming units of virus per 0.5-mL vial (dose). Two vials of the lyophilized human vaccine, purchased at the university pharmacy, were each reconstituted with 2.4 mL of phosphate-buffered saline, pH 7.4 (PBS). Each hamster in Group 1 received 100 μL of the reconstituted vaccine intraperitoneally (IP) in January 2006. This is ∼1/24 of the usual human vaccine dose.

The second virus used in this study was the wild-type Asibi strain of yellow fever virus (Asibi YFV). The Asibi virus is the prototype strain of YFV; it was originally isolated from the blood of a febrile man in Ghana in 1927.1 The Asibi YFV was the parent of YFV-17D; the YFV-17D attenuated vaccine virus was developed by serial passage of the wild-type Asibi virus in cell cultures prepared from embryonated chicken eggs.1 The Asibi YFV used in our study has been passaged six times in monkeys and three additional times in mosquito cell cultures (C6/36).

Animals

Adult female Syrian golden hamsters (Mesocricetus auratus), 6–9 weeks old, obtained from Harlan Sprague Dawley (Indianapolis, IN) were used in the study. The animals were cared for in accordance with the guidelines of the Committee on Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council) under an animal use protocol approved by the University of Texas Medical Branch. All work with the infected animals was carried out in ABSL-3 facilities.

Immunosuppression of animals

Cyclophosphamide (Cytoxan; Baxter Healthcare Corp., Princetown, NJ) was used as the regimen for immunosuppression.6 Each immunosuppressed animal received CYP, 100 mg/kg given IP, every fourth day as described previously.7 Immunosuppression of the hamsters given YF-VAX (Group 2) was started 10 weeks before vaccination (infection) and was continued for the duration of the experiment. Immunosuppression of the hamsters given the wild-type YFV (Group 3) was started 5 days before infection and repeated every fourth day until death occurred.

Experimental design

A total of 32 hamsters were used in this study. The animals were divided into three groups. Group 1 consisted of seven control hamsters that were inoculated IP with 100 μL of the diluted YFV-17D vaccine (∼103.3 PFU), but were not treated with CYP. Group 2 consisted of 20 adult hamsters that had received CYP for 10 weeks and were inoculated IP with 100 μL of the same lot and dose of YFV-17D vaccine. After infection, all animals were examined daily for a period of 6–7 weeks for evidence of illness or death. During this period of time, immunosuppression with CYP was continued for the animals in Group 2. A random sample of five hamsters each in Groups 1 and 2 was bled retro-orbitally every 7–10 days for serology and virus culture. Seriously ill or moribund animals were killed and exsanguinated. A necropsy was performed, and samples of brain, spinal cord, liver, spleen, kidney, and adrenals were collected from the sick animals and were placed in 10% buffered formalin for 48 hours. These tissues were subsequently transferred to 70% ethanol for storage before processing for histopathology and immunohistochemistry (IHC). Fresh samples of blood, brain, liver, and spleen from the euthanized animals were also taken and stored at −80°C for subsequent virus assay. Between 46 and 48 days after YFV-17D immunization, the surviving hamsters in Group 2 and all hamsters in Group 1 were also killed. At this time, blood (serum) was collected for serology, and a sample of brain was taken for virus assay.

A third group of hamsters (Group 3) received CYP; 5 days after starting the immunosuppressant, they were inoculated IP with 106 tissue culture infectious dose50 units of the wild-type Asibi YFV. After infection, hamsters in Group 3 continued to receive CYP and were examined daily for signs of illness. Seriously ill or moribund animals were killed, a necropsy was performed, and tissues were taken for histopathology and immunohistochemistry, as described above.

Virus assay

Virus assays on selected blood and tissue samples were done simultaneously in cultures of Vero E-6 and C6/36 cells. For blood or serum samples, 200 μL of a 1:10 dilution of the sample were inoculated into a single 12.5-cm2 flask culture of each cell type. Tissue samples were homogenized in 1.0 mL of diluent to prepare an approximate 10% tissue suspension (wt/vol). After centrifugation at 10,000 rpm for 5 minutes, 200 μL of the supernatant was inoculated into culture flasks of Vero and C6/36 cells, as noted above. The diluent consisted of PBS with 10% heat-inactivated fetal bovine serum and antibiotics.

Vero cell cultures were maintained at 37°C and examined daily for evidence of viral cytopathic effect (CPE). The C6/36 (mosquito) cell cultures were kept at 28°C for 6 days. On the sixth day, the mosquito cells were harvested, and 20 μL of the cell suspension were placed on 4 spots of 12-spot glass microscope slides (Cel Line/Erie Scientific, Rochester, NY). After drying and acetone fixation, the C6/36 cells were examined for the presence of YFV antigen by indirect fluorescent antibody test (IFAT), using a YFV-17D mouse immune ascitic fluid and a commercially prepared (Sigma Chemical, St. Louis, MO) fluorescein-conjugated, goat antimouse immunoglobulin.8,9 Vero cell cultures that developed viral CPE were also harvested and examined by IFAT, using the method described above for confirmation. If no CPE was detected after 10 days, some of the Vero cells were scraped from the flask, spotted onto slides, and examined by IFAT, as above.

Antibody detection

Sera from the hamsters were examined for antibodies to YFV by the hemagglutination-inhibition (HI) test.10 Antigen for the HI test was prepared for brains of newborn mice inoculated intracerebrally with YFV-17D; infected brains were treated by the sucrose-acetone extraction method.10 Hamster sera were tested by HI at serial 2-fold dilutions from 1:20 to 1:2,560 at pH 6.4, as previously described.8

Histologic and immunohistochemical examination

After fixation, tissues were processed for routine paraffin embedding. Several 4- to 5-μm sections of each tissue sample were made and stained by the hematoxylin and eosin (H&E) method. Other stained sections were used for IHC and antigen detection, as described previously.11,12

RESULTS

Clinical manifestations and mortality

The seven control hamsters in Group 1 remained well throughout the 6-week observation period. The five immunosuppressed animals in Group 3 that received the wild-type Asibi YFV became ill on Day 7 after infection, and all were dead or moribund by Day 11 (Figure 1). In contrast, the 20 immunosuppressed hamsters in Group 2 remained active and well until ∼18 days after vaccination. At this time, some of the immunosuppressed animals began to sicken and developed progressive signs of lethargy, emaciation, difficulty walking, and limb paralysis. When the animals became severely ill (unable to eat or drink or with marked paralysis), they were killed. Ten (50%) of the immunosuppressed hamsters developed these symptoms and were killed between 19 and 34 days after infection (Figure 1). The other 10 animals in Group 2 did not develop any apparent clinical illness during the post-vaccination period and were killed on Day 48 (Table 1).

Virus isolation

Blood and brain samples from the seven control hamsters in Group 1 were cultured when the animals were killed and necropsied 46 days after vaccination. None of these cultures yielded virus. Samples from animals in Group 3 were not cultured.

Table 1 summarizes the results of blood and organ cultures done on the 20 immunosuppressed hamsters in Group 2. Two subgroups, designated A and B, are shown in the table. Subgroup A consists of the 10 hamsters that were killed between the 19th and 34th day after vaccination because of severe illness. Subgroup B includes the 10 immunosuppressed animals that did not manifest any clinical illness and appeared well when killed at the end of the experiment, 48 days after vaccination. YFV was isolated from blood and brain samples taken from all 10 hamsters in subgroup A when they were killed. Virus was also isolated from the liver of 7 of the 10 animals and from the spleen of 2 of the 10 hamsters in subgroup A. When the 10 hamsters in subgroup B were killed 48 days after vaccination, only brain samples were cultured. Despite the absence of any apparent clinical illness in these latter animals, YFV was recovered in culture from brain samples from 3 of the 10 hamsters.

In comparing the sensitivity of the two cell cultures (Vero and C6/36) for isolation of YFV-17D virus, the C6/36 cell line yielded more virus isolates (Table 2; Figure 2). The culture results on blood and tissue samples from the 10 animals in subgroup A are summarized in Table 2. The YFV-17D virus isolation rates in the two cell lines were similar for homogenates of brain and spleen; however, more virus isolates were made in C6/36 cell cultures with blood and liver homogenates.

Antibody response

Table 3 shows the yellow fever HI antibody titers in the seven control animals (Group 1) when bled 21 and 45 days after immunization with the YF-VAX vaccine. One animal in this group (animal 2) failed to develop HI antibodies after vaccination; nonetheless, the hamster remained well, and a culture of its brain at necropsy was negative. The other six animals in Group 1 developed yellow fever HI antibody titers ranging from 1:40 to 1:320. In general, HI antibody titers were higher in the second blood sample taken 45 days after vaccination.

In contrast, none of the animals in Group 2 or 3 had detectable HI antibodies to YFV-17D viral antigen when they were killed (data not shown). Some of the animals in Group 2 were bled repeatedly during the 48-day observation period, and all were sampled at necropsy. This included the 10 survivors in Group B that were killed 48 days after vaccination (Table 1).

Histopathology and immunohistochemistry

Examination of brain tissue from the 10 hamsters in Group A of Group 2 (Table 1) revealed focal neuronal changes mostly in the basal nuclei, which included shrinkage of the cells with deep basophilia of the nuclei. Some of the foci had infiltration of lymphocytes or perivascular lymphocytic infiltration. However, these changes tended to be focal and were not consistent among the 10 animals. The other organs examined from animals in Group A appeared histologically normal.

Brain tissue from the seven hamsters in Group 1 and from the 10 surviving animals in Group 2 (designated Group B) showed no significant histologic abnormalities, except for hamsters 157 and 173 in Group B. Brains of these latter two animals showed increased cellularity (Figure 3A) and perivascular inflammatory infiltration (Figure 3B) in the basal nuclear regions. Brain cultures of these two animals also yielded YFV-17D (Table 1).

IHC staining for YFV antigen done on liver, spleen, and kidney tissue of the 10 hamsters in Group A of Group 2 was negative. In contrast, positive antigen staining of variable intensity was seen in the brains of all 10 animals in Group A (Figure 4). Positive antigen staining was also seen in the spinal cord of some of the animals. None of the surviving hamsters in Group B, including the two with focal histologic abnormalities (157 and 173), showed antigen positivity in brain tissue by IHC.

Immunohistochemical staining was done on tissues of three moribund hamsters in Group 3. The other two animals in this group were found dead and were not examined. Brain, spleen, and kidney tissue from these three animals was negative for YF antigen by IHC (Figure 5A). In contrast, strong antigen-positive straining was seen in many hepatocytes (Figure 5B). Inflammation, moderate hepatocyte necrosis, and microvesicular steatosis (accumulation of fatty acids in small vesicles within hepatocytes) was also observed in the livers of these latter animals.

DISCUSSION

Previous studies13,14 have shown that the Asibi YFV (non–hamster-adapted) has a wild-type phenotype and causes a subclinical infection with mild viremia and minor histopathologic changes when inoculated IP into immunocompetent adult hamsters. As shown by hamsters in Group 1 of this study, IP inoculation of YFV-17D also produces a subclinical, immunizing infection in adult hamsters. No virus was cultured from blood or brain samples from the seven animals in Group 1 when they were examined 48 days after infection.

However, the pathogenesis of the two viruses in the immunosuppressed animals (Groups 2 and 3) was quite different. When immunosuppressed hamsters were infected with the wild-type Asibi YFV (Group 3), the animals developed a fulminant illness and all were moribund or dead within 11 days. Histopathologic examination of their tissues showed inflammation, hepatocyte necrosis, and microvesicular steatosis, similar to the pathology observed in severe human cases of yellow fever,15,16 experimentally infected rhesus monkeys,16 and in hamsters infected with hamster-adapted YFV strains.11,13,14,17,18 No pathology or YFV antigen was detectable in brain tissue of the Group 3 animals, indicating that the wild-type Asibi YFV was primarily hepatotropic in the immunosuppressed hamsters.

Infection of immunosuppressed hamsters with YFV-17D (Group 2) gave different and more variable results. One half of the immunosuppressed animals in Group 2 developed progressive symptoms of neurologic disease within 5 weeks after immunization and were killed for humane reasons. One assumes that they would have shortly died of the disease. YFV was isolated from the blood and brain samples from all 10 of these hamsters (Group A) at necropsy. The virus was also isolated from liver and spleen of some of these animals (Table 1), but this may have been the result of residual blood in these tissues rather than to actual infection of the organs, because the liver and spleen were not perfused before culture. Histopathologic changes and IHC evidence of yellow fever viral antigen were only observed in brain tissue of the Group A animals and not in liver, spleen, or kidney.

None of the hamsters in Group B of Group 2 (N = 10) developed clinical signs of illness during the 48 days after immunization, but none of them had detectable HI antibodies to YFV-17D when killed 7 weeks later. YFV-17 virus was isolated from brain tissue of 3 of the 10 hamsters in Group B at necropsy, indicating that the virus infected and persisted in some of the animals. Because the experiment was terminated after 7 weeks, it is unknown whether these three animals eventually would have developed neurologic symptoms, as did the hamsters in Group A. However, it is noteworthy that only two of the Group B animals showed any significant histologic changes in their brains and that all of the brain samples from the Group B hamsters were IHC antigen-negative. This suggests that some of the immunosuppressed animals were able to control or to eliminate YFV-17D virus infection, despite their poor humoral antibody response. The point to be made here is that, in the immunosuppressed animals, the pathogenesis of YFV-17D virus was neurotropic rather than hepatotropic.

As noted before, YFVax AND has been the most common serious adverse event associated with yellow fever vaccination.13 Most of the YFVax-AND cases have been in children < 7 months of age, whereas post-vaccination viscerotropic disease has been seen in adults. In another unpublished experiment, we (RBT and SYX) compared the pathogenesis of YFV-Asibi and YFV-17D in newborn hamsters. Two-day-old hamsters were inoculated IP with YFV-Asibi (N = 12) or YFV-17D (N = 9). All of the pups were dead or moribund by the fifth day. IHC staining of tissues from the YFV-Asibi–infected pups showed strongly positive antigen staining in the brain, liver (hepatocytes), and acinar cells of pancreas. In contrast, the YFV-17D–infected pups only showed strong positive staining in the brain, whereas other organs (i.e., liver and spleen) were negative. These findings suggest that YFV-17D is predominantly neurotropic in very young hamsters as well. In summary, the results of our experiments in hamsters support the recommendations against using YFV-17D in infants and in immunosuppressed adults. Our data also suggest that these hamster models could be useful for monitoring the safety of live-attenuated yellow fever vaccines.

Table 1

Results of organ and blood cultures done on the 20 immunosuppressed hamsters in Group 2 after yellow fever vaccination

Hamster no.Day PI*BrainSpleenLiverBlood
* Day PI, day post-infection (vaccination) that hamster was euthanized, and tissue samples taken for culture and histopathology.
+, YF virus isolated; 0, culture negative; NT, not tested.
Subgroup A
    138D-32++++
    149D-22+0++
    158D-31++++
    167D-31+00+
    171D-24+0++
    172D-25+00+
    174D-19+0++
    193D-26+00+
    194D-34+0++
    195D-30+0++
Subgroup B
    139D-480NTNTNT
    147D-480NTNTNT
    148D-48+NTNTNT
    150D-480NTNTNT
    157D-48+NTNTNT
    168D-480NTNTNT
    169D-480NTNTNT
    170D-480NTNTNT
    173D-48+NTNTNT
    196D-48+NTNTNT
Table 2

Comparative sensitivity of Vero E6 and C6/36 cells for isolation of YFV-17D from blood and tissue samples of 10 immunosuppressed hamsters

Number of YFV-17D isolates
Sample testedVero E6C6/36
Brain1010
Liver37
Spleen22
Blood410
Table 3

Hemagglutination-inhibition (HI) antibody titers in Group 1 control hamsters after yellow fever vaccination (YF-VAX)

HI antibody titer
Hamster numberDay 21*Day 45
* Days after vaccination.
11:801:160
2< 1:10< 1:10
31:401:320
41:401:160
51:3201:160
61:401:40
71:401:40
Figure 1.
Figure 1.

Survival of 20 immunosuppressed (Group 2) hamsters after yellow fever vaccination.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 5; 10.4269/ajtmh.2007.77.919

Figure 2.
Figure 2.

Spot slide of C6/36 cells inoculated with brain homogenate of sick hamster (Group 2). YFV-17D antigen appears as yellow-green staining material in cell cytoplasm when examined by indirect fluorescent antibody test (IFAT).

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 5; 10.4269/ajtmh.2007.77.919

Figure 3.
Figure 3.

Photomicrographs showing pathologic changes in the brain of infected immunosuppressed hamster 157. A, Increased cellularity in basal nuclear region, consisting of lymphocytes and reactive glial cells. B, Perivascular infiltration with lymphocytes and macrophages.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 5; 10.4269/ajtmh.2007.77.919

Figure 4.
Figure 4.

YFV-17D antigen distribution as seen in IHC-stained sections of different regions of the brain of hamster 158 (Group 2). The antigen appears as red stain by this technique; many neurons are positive. A, Deep layers of cerebral cortical region. B, Hippocampus. C, Basal ganglia region. D, Brainstem. Immunohistochemical stain.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 5; 10.4269/ajtmh.2007.77.919

Figure 5.
Figure 5.

IHC-stained sections of brain and liver from an immunosuppressed hamster infected with YFV-Asibi (Group 3). A, The brain is YFV antigen-negative, whereas the liver (B) is YFV antigen-positive. Antigen staining and microvesicular steatosis can be seen in many hepatocytes.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 77, 5; 10.4269/ajtmh.2007.77.919

*

Address correspondence to 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: Rosa Mateo, Shu-Yuan Xiao, Amelia P.A. Travassos da Rosa, Hao Lei, Hilda Guzman, Liang Lu, and Robert B. Tesh, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555–0609, Telephone: 409–747–2431, Fax: 409–747–2429, E-mail: rtesh@utmb.edu.

Acknowledgments: The authors thank Dora Salinas for help in preparing the manuscript and Patrick Newman for preparing the histological sections.

Financial support: This work was supported by National Institutes of Health Contracts NO1-AI25489 and NO1-AI 30027. RIM was supported by the James W. McLaughlin Fellowship Fund.

REFERENCES

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    Monath TP, 2004. Yellow fever vaccine. Plotkin SA, Orenstein WA, eds. Vaccines. Fourth edition. Philadelphia: Elsevier, 1095–1176.

  • 2

    Barwick RS, Marfin AA, Cetron MS, 2004. Yellow fever vaccine-associated disease. Scheld WN, Murray BE, Hughes JM, eds. Emerging Infections 6. Washington: ASM Press, 25–34.

  • 3

    Barnett ED, 2007. Yellow fever: epidemiology and prevention. Clin Infect Dis 44 :850–856.

  • 4

    Kengsakul K, Sathirapongsasuti K, Punyagupta S, 2002. Fatal myeloencephalitis following yellow fever vaccination in a case with HIV infection. J Med Assoc Thai 85 :131–134.

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Author Notes

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