Am. J. Trop. Med. Hyg., 77(5), 2007, pp. 919–924
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene
Yellow Fever 17-D Vaccine Is Neurotropic and Produces Encephalitis in
Immunosuppressed Hamsters
Rosa I. Mateo, Shu-Yuan Xiao, Amelia P. A. Travassos da Rosa, Hao Lei, Hilda Guzman, Liang Lu, and
Robert B. Tesh*
Departments of Pathology and Internal Medicine and Center for Biodefense and Emerging Infectious Diseases, University of Texas
Medical Branch, Galveston, Texas
Abstract. 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 (nonimmunosuppressed) 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.
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.
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.1–3 Rarely,
more severe vaccine-associated adverse events (postvaccinal
encephalitis and viscerotropic disease) occur.1–3 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
MATERIALS AND METHODS
Viruses. Yellow fever vaccine, YF-VAX, lot UE665AA,
expiration date 26 September 2006 (Aventis Pasteur, Swiftwater, 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 leukosisfree 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 ␮L 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
* Address correspondence to Robert B. Tesh, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard,
Galveston, TX 77555-0609. E-mail: [email protected]
919
920
MATEO AND OTHERS
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 ␮L 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 ␮L 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 dose 50 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 ␮L 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 ␮L 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 ␮L 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 hemagglutinationinhibition (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 sucroseacetone 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-␮m 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.
921
YFV-17D ENCEPHALITIS IN IMMUNOSUPPRESSED HAMSTERS
FIGURE 1. Survival of 20 immunosuppressed (Group 2) hamsters
after yellow fever vaccination.
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
TABLE 1
Results of organ and blood cultures done on the 20 immunosuppressed hamsters in Group 2 after yellow fever vaccination
Hamster no.
Subgroup A
138
149
158
167
171
172
174
193
194
195
Subgroup B
139
147
148
150
157
168
169
170
173
196
Day PI*
Brain
Spleen
Liver
Blood
D-32
D-22
D-31
D-31
D-24
D-25
D-19
D-26
D-34
D-30
+
+
+
+
+
+
+
+
+
+
+
0
+
0
0
0
0
0
0
0
+
+
+
0
+
0
+
0
+
+
+
+
+
+
+
+
+
+
+
+
D-48
D-48
D-48
D-48
D-48
D-48
D-48
D-48
D-48
D-48
0
0
+
0
+
0
0
0
+
+
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
* 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.
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
TABLE 2
Comparative sensitivity of Vero E6 and C6/36 cells for isolation of YFV17D from blood and tissue samples of 10 immunosuppressed hamsters
Number of YFV-17D isolates
Sample tested
Vero E6
C6/36
Brain
Liver
Spleen
Blood
10
3
2
4
10
7
2
10
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MATEO AND OTHERS
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).
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.
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.
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
TABLE 3
Hemagglutination-inhibition (HI) antibody titers in Group 1 control
hamsters after yellow fever vaccination (YF-VAX)
HI antibody titer
Hamster number
Day 21*
Day 45
1
2
3
4
5
6
7
1:80
< 1:10
1:40
1:40
1:320
1:40
1:40
1:160
< 1:10
1:320
1:160
1:160
1:40
1:40
* Days after vaccination.
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
YFV-17D ENCEPHALITIS IN IMMUNOSUPPRESSED HAMSTERS
923
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.
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.
924
MATEO AND OTHERS
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 antigennegative. 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.1–3 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 YFV17D 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.
Received November 3, 2006. Accepted for publication July 19, 2007.
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.
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: [email protected].
Reprint requests: Robert B. Tesh, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609,
E-mail: [email protected].
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