Download chronic st. louis encephalitis virus infection in the golden hamster

Am. J. Trop. Med. Hyg., 76(2), 2007, pp. 299–306
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene
CHRONIC ST. LOUIS ENCEPHALITIS VIRUS INFECTION IN THE GOLDEN
HAMSTER (MESOCRICETUS AURATUS)
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 and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch,
Galveston, Texas
Abstract. 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.
tected in the patient’s urine by reverse transcriptasepolymerase 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.
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.1,2,4–7
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 de-
MATERIALS AND METHODS
Animals. Six- to 8-week-old female golden hamsters (Mesocricetus auratus) were obtained from Harlan SpragueDawley (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 (Hy-
* Address correspondence to Dr. Robert B. Tesh, Department of
Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0609. E-mail: [email protected]
299
300
SIIRIN AND OTHERS
TABLE 1
Isolation of SLEV from urine and selected tissues of chronically infected hamsters in Experiment 1
Animal
no.
8605
8649
8610
8612
8607
8609
8604
8602
8601
8603
8613
8611
Day(s)* SLEV culture-positive
urine sample collected
D35
D35, D49,
None
None
D81
D53, D82
None
D53, D65,
None
None
D49, D68,
D35, D49,
D53, D62
D81, D89
D81, D93, D116
D65, D78, D124
Co-cultivation results
Day*
tissues
sampled
Spleen
Lung
Kidney
Serum neut.
antibody
titer†
47
62
69
76
81
82
83
89
97
112
116
124
0
0
0
0
0
0
0
0
0
0
0
0
0
+
0
0
+
0
+
0
0
0
0
0
+
+
0
0
0
0
0
0
0
0
+
+
640
1,280
1,280
2,560
2,560
1,280
320
640
1,280
2,560
1,280
1,280
* 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.
drocarbon 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 26gauge 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 ␮L 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 ␮L 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 ␮L 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
TABLE 2
Isolations of SLEV from urine and selected tissues of persistently infected hamsters in Experiment 2
Animal
no.
6092
6090
6091
6089
Day(s)* SLEV culture-positive
urine collected
None
D30
None
D18, D29, D158
Co-cultivation result
Day*
tissues
sampled
Brain
Spleen
Liver
Lung
Kidney
Serum neut.
antibody
titer†
167
168
184
185
+
+
0
+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1,280
320
320
320
* 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.
301
CHRONIC SLE VIRUS INFECTION IN HAMSTERS
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 serumvirus 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 ⱖ 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 ␮m 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.
TABLE 3
Nucleic acid changes in the E-gene of 17 SLEV isolates from chronically infected hamsters compared with the parent strain (BeAr 23379)
Animal
no.
Day
PI
Source
of
isolate
H-8505
H-8649
H-8611
H-8613
H-8649
H-8602
H-8613
H-8611
H-8611
H-6089
H-6089
H-6090
H-6089
H-6089
H-6092
H-6090
H-6089
BeAr
23379
35
35
49
49
62
89
106
124
124
29
58
58
141
158
167
168
185
Urine
Urine
Urine
Urine
Urine
Urine*
Urine
Urine*
Kidney
Urine
Urine
Urine
Urine
Urine
Brain
Brain
Brain
Mutations in nucleotides
1163
1146
1425
1426
1430
1794
1887
2059
2169
2265
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
G
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T
C
C
C
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
T
T
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T
C
C
C
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
G
G
C
C
C
C
C
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
T
C
C
* 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.
Divergence
(%)
0.13
0.13
0.13
0.13
0.13
0.2
0.13
0.13
0.13
0.13
0.13
0.13
0.13
0.13
0.4
0.27
0.2
302
SIIRIN AND OTHERS
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.
H-8505
H-8649
H-8611
H-8613
H-8649
H-8602
H-8613
H-8611
H-8611
H-6089
H-6089
H-6090
H-6089
H-6089
H-6092
H-6090
H-6089
BeAr 23379
Day PI
Source of virus
isolate
35
35
49
49
62
89
106
124
124
29
58
58
141
158
167
168
185
Urine
Urine
Urine
Urine
Urine
Urine*
Urine
Urine*
Kidney
Urine
Urine
Urine
Urine
Urine
Brain
Brain
Brain
67
154
155
156
366
403
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Arg
Tyr
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Phe
Asp
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
Tyr
His
Tyr
Tyr
Tyr
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Phe
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Arg
Gly
Gly
Ser
Ser
Ser
Ser
Ser
Thr
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
Ser
* 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.
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,
CHRONIC SLE VIRUS INFECTION IN HAMSTERS
303
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).
304
SIIRIN AND OTHERS
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.
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.35–37 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
CHRONIC SLE VIRUS INFECTION IN HAMSTERS
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.
Received August 31, 2006. Accepted for publication October 13,
2006.
Acknowledgments: The authors thank Dora Salinas for help in preparing the manuscript.
Financial support: This work was supported by Contracts NO1AI25489 and NO1-AI30027 from the National Institutes of Health.
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.
Reprint requests: Robert B. Tesh, Department of Pathology, University of Texas Medical Branch, 301 University Boulevard, Galveston,
TX 77555-0609. E-mail: [email protected].
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