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Journal of Virology, January 2001, p. 11-18, Vol. 75, No. 1
International Laboratory of Molecular Biology
for Tropical Disease Agents, Department of Veterinary Pathology,
Microbiology and Immunology, School of Veterinary Medicine,
University of California, Davis, California 95616
Received 23 August 2000/Accepted 3 October 2000
The vaccinia virus (VV) B8R gene encodes a secreted protein
with homology to the gamma interferon (IFN- Vaccinia virus (VV) is the
prototype member of the genus Orthopoxvirus, family
Poxviridae, and it has been used extensively as a vector for
the development of recombinant live vaccines (34). There are currently
two effective recombinant VV (rVV) vaccines, one for rabies and the
other for rinderpest (1, 18, 26, 52, 56). The rabies rVV vaccine has
been used successfully to control fox rabies in Europe and, more
recently, raccoon rabies in the United States (38). The rinderpest rVV
vaccine protected cattle from a challenge with more than 1,000 times
the lethal dose of the virus (18, 56). Not all rVVs, however, have been
as effective as those for rabies and rinderpest. For example, we showed
that rVVs expressing the vesicular stomatitis virus (VSV) G protein (VSV-G) protected against only a minimal experimental challenge dose
(33, 57). Additionally, rhesus macaques immunized with rVVs expressing
genes of the simian immunodeficiency virus (SIV) were not protected
from virus infection, although there was a reduction in virus load and
increased survival time (2, 20, 21, 37). It is clear that there is a
definite need to enhance the efficacy of rVV vaccines against diseases
for which protection is not optimal. However, it is even more critical
that the safety of rVVs be validated before their approval for use as
vaccines. Although VV has not been associated with any disease, it can
cause severe complications in immunodeficient or immunosuppressed
individuals (6, 7, 15, 43). Thus, increasing the safety of rVV vaccines
without compromising their efficacy is an important consideration in
vaccine development.
VV is one of many viruses with genes that code for specific proteins to
suppress host immunity. These immunomodulating genes are usually not
essential for virus growth in vitro. Their products include homologs of
interleukin-1 Cells and viruses.
Human HeLa S3 and A549 cells, African
green monkey kidney BS-C-1 and BS-C-40 cells, hamster BHK-21 cells, and
murine L929 cells were grown at 37°C under 5% CO2 in
Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal
bovine serum (FBS). The Western Reserve strain of VV (WR) (9), obtained
from B. Moss (National Institute of Allergy and Infectious Diseases,
Bethesda, Md.), a WR-derived rVV expressing VSV-G at the thymidine
kinase (TK) site (v50) (33), and their derivatives were propagated in HeLa S3 cells and subjected to titer determination in BS-C-1 cells. Encephalomyocarditis virus (EMCV) was propagated in BHK-21 cells and
subjected to titer determination in L929 cells. The New Jersey serotype
of VSV (55) was propagated and subjected to titer determination in
BHK-21 cells.
Animals.
Male athymic nude BALB/cBy (nu/nu) mice
were purchased from Jackson Laboratory (Bar Harbor, Maine). Female
(BALB/c × C57BL/6) F1 hybrid mice
(CB6F1), male athymic nude Rowett (rnu/rnu)
rats, and female Fischer 344 (F344) rats were purchased from Harlan Sprague-Dawley (Indianapolis, Ind.). All animals were maintained in
accordance with National Institutes of Health guidelines and animal
care protocols approved by the Animal Care Committee at the University
of California, Davis.
Construction of p2B8R.
The strategy used to construct the VV
transfer vector p2B8R is shown in Fig. 1.
Each PCR fragment contained engineered restriction endonuclease sites
to facilitate vector construction (Table
1). Briefly, a pUC18 PCR fragment was
generated by amplification of plasmid pUC18 (54) with primers pUC18F
and pUC18R, which contained the
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.11-18.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Vaccinia Virus Vectors with an Inactivated Gamma Interferon
Receptor Homolog Gene (B8R) Are Attenuated In Vivo without a
Concomitant Reduction in Immunogenicity
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
) receptor. In vitro, the
B8R protein binds to and neutralizes the antiviral activity of several
species of IFN-, including human and rat IFN-; it does not,
however, bind significantly to murine IFN-. Here we report on the
construction and characterization of recombinant VVs (rVVs) lacking the
B8R gene. While the deletion of this gene had no effect on virus
replication in vitro, rVVs lacking the B8R gene were attenuated for
mice. There was a significant decrease in weight loss and mortality in
normal mice, and nude mice survived significantly longer than did
controls inoculated with parental virus. This is a surprising result
considering the minimal binding of the B8R protein to murine IFN-
and its failure to block the antiviral activity of this cytokine in
vitro. Such reduction in virulence could not be determined in rats,
since they are considerably more resistant to VV infection than are
mice. Finally, deletion of the B8R gene had no detectable effects on
humoral immune responses. Mice and rats vaccinated with the rVVs showed
identical humoral responses to both homologous and heterologous genes
expressed by VV. This study demonstrates that the deletion of the VV
B8R gene leads to enhanced safety without a concomitant reduction in immunogenicity.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
(3, 47) and alpha/beta interferon (IFN-
/)
receptors (14, 49), complement control proteins (23, 28), and serine
protease inhibitors (8, 25, 29, 46). In particular, VV contains a gene
(B8R) that codes for a secreted protein with sequence similarity to the
extracellular domain of the IFN- receptor (50). This protein
neutralizes the antiviral activities of human, rat, rabbit, bovine, and
chicken IFN-, but it binds with significantly lower affinity to
murine IFN- (4, 36, 41, 51). Cytokines such as IFN- play
essential roles in the regulation of the immune system and in defense
against pathogens. Additionally, IFN- exhibits adjuvant activities
when used with a subunit antigen (5), and it has attenuating activities
(up to 106-fold for immunodeficient mice) when expressed by
VV (19, 27). Thus, we hypothesized that the efficacy as well as the
safety of rVVs might be increased by deleting the B8R gene. This
hypothesis was tested in mice and rats inoculated with rVVs with a
deletion of the B8R gene. We found that rVVs with an inactivated B8R
gene are attenuated for normal and nude mice without having a
concomitant reduction in immunogenicity.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-lactamase gene conferring
ampicillin resistance (Ampr) and the origin of replication
(Ori). Next, a B8R left PCR fragment was generated by amplification of
VV WR DNA (22, 45) with primers B8RLF and
B8RLR, which comprised the region to the left of the B8R
gene start codon. This ATG was mutated to an ATA (engineered in the
reverse B8RLR primer [shown in lowercase in Table 1]) to
prevent the expression of a truncated B8R gene. Sequencing of this
fragment (comprising most of the B7R gene) with primers B8RLS1, B8RLS2, B8RLS3, and
B8RLS4 showed that it contains the wild-type sequence. The
pUC18 and B8R left PCR fragments (digested with BglII and
KpnI) were ligated to form plasmid pUCB8RLeft. Next, a B8R
right PCR fragment, generated with primers B8RRF and B8RRR and containing most of the 819-bp B8R protein coding
sequence (nucleotides 157 to 791), was cleaved with BamHI
and BglII and ligated to BglII-cleaved
pUCB8RLeft, producing pUCB8R. Then a pSC11 (12) PCR fragment (generated
with primers pSC11F and pSC11R) was inserted into the BglII
site of pUCB8R, resulting in the transfer vector pB8R, which directs
the insertion of a cassette containing the VV P7.5 promoter
and the lacZ gene (for -galactosidase expression under
the VV P11 promoter) into the B8R genomic region. Finally, the 273-bp SmaI-XbaI fragment of pB8R (containing
the VV P7.5 promoter) was replaced with the 161-bp
SmaI-NheI fragment of pJS5 (13), generating
p2B8R, which contains two back-to-back strong synthetic VV early/late
promoters (dsP).
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FIG. 1.
Construction of VV transfer vector p2B8R. p2B8R was
generated by joining pUC18, B8R left (B8RL), B8R right
(B8RR), and pSC11 PCR fragments, as well as a DNA fragment
from plasmid pJS5. The transfer vector p2B8R directs the insertional
inactivation and deletion of the B8R gene of VV by homologous
recombination. It contains the lacZ gene for
-galactosidase expression under the control of the VV
P11 late promoter for screening of rVVs, and two
back-to-back strong synthetic VV promoters (dsP) that are active in
both early and late stages of infection. There are multiple cloning
sites adjacent to each side of the dsP to facilitate the cloning of
heterologous genes (only unique sites are shown).
TABLE 1.
Oligonucleotide primers used in this study
Generation of rVVs and analysis of their genomic DNA, stability,
and purity.
rVVs were generated by standard homologous
recombination using cationic liposome-mediated transfection of BS-C-1
cells infected with the VVs at 0.05 PFU/cell. The rVVs were plaque
purified from transfection supernatants on BS-C-1 cell monolayers by
using 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) to detect the lacZ marker gene (16).
B8R protein bioassay.
B8R protein activity was determined by
its ability to prevent the antiviral activity of human IFN-. HeLa S3
cell suspensions were infected with rVVs at 20 PFU/cell or mock
infected with DMEM for 1 h. The cells were washed twice,
resuspended in DMEM, and incubated for 36 h. Supernatants were
then harvested, and VV particles were removed by centrifugation at
80,000 × g (24,000 rpm in an SW28 rotor) for 75 min at
4°C on a 25% (wt/wt) sucrose cushion. The clarified supernatant was
then concentrated (about 40-fold) with Centriprep-10 concentrators
(10,000 molecular weight cutoff) (Amicon, Beverly, Mass.) and filtered
through 0.2-µm-pore-size filters. Each supernatant was serially
diluted in DMEM-5% FBS. Subsequently, 5 µl (600 U/ml) of
recombinant human IFN- (Genzyme, Cambridge, Mass.) in DMEM-5% FBS
was added to 45 µl of each dilution and incubated at 37°C for
1 h. Mixtures were then transferred to 96-well plates, seeded 4 to
6 h previously with 2 × 104 A549 cells/well in
100 µl of DMEM-5% FBS (final IFN- concentration, 20 U/ml). After
24 h of incubation, cells were challenged with the minimum dose of
EMCV (104 PFU in 50 µl) that gave 100% cytopathic
effects and stained with crystal violet staining solution 1 day later.
Virus growth curves. Virus replication in vitro was determined by generating one-step growth curves (40). Briefly, duplicate monolayers of L929 and A549 cells were infected at 0.01 PFU/cell for 1 h in 12-well plates. The cells were then washed and resuspended in 1 ml of DMEM-2.5% FBS. At each time point, supernatants were collected, centrifuged (to pellet detached cells), and transferred to a new tube (the extracellular virus fraction). Cells in the wells were resuspended in 1 ml of DMEM, scraped, and added to the pellet of detached cells (the intracellular virus fraction). The intracellular fraction was freeze-thawed three times, trypsinized, and sonicated. Duplicates of both virus fractions were subjected to titer determination on BS-C-1 cell monolayers.
Virulence studies in mice and rats. Groups of CB6F1 normal mice and F344 normal rats (8- to 9-week-old females) were challenged intranasally with 10-fold dilutions (in sterile PBS) of each rVV strain in a final volume of 10 µl while under light anesthesia. The animals were examined and weighed daily.
Groups of BALB/cBy nude mice (7- to 8-week-old males) and Rowett nude rats (7-week-old males) were challenged intraperitoneally with 107 PFU of each rVV strain in a final volume of 250 µl of sterile PBS. The animals were examined daily, and the mortality rate for each group was determined.rVV immunogenicity studies. Groups of animals (6- to 7-week-old female CB6F1 mice and F344 rats) were immunized intramuscularly with 106 PFU of each VV strain in a final volume of 50 µl of sterile PBS while under light anesthesia. Serum was collected weekly from each animal, and serum-neutralizing antibody titers to VV and VSV were determined by plaque reduction on BS-C-40 cell monolayers (24) and serum neutralization assays on BHK-21 cell monolayers (33), respectively.
Data analysis. Statistical analyses were performed using the statistical software program SAS, release 6.11 (SAS Institute, Cary, N.C.).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Deletion of the B8R gene has no effect on VV replication in vitro. The strategy used for generation of the transfer vectors employed in this study is presented in Fig. 1. rVVs derived from the pB8R transfer vector were unstable, probably due to the presence of P7.5 in the inverted terminal repeat regions of the VV genome as well as in the transfer vector (25). For this reason, p2B8R was developed for generating the two rVVs used in this study. The p2B8R transfer vector directs homologous recombination with the B8R region of the VV genome, leading to inactivation of the gene as a result of partial deletion (154 bp) and insertion of the lacZ gene. In addition, its strong synthetic promoters (dsP) allow increased levels of expression (13; P. H. Verardi, F. H. Aziz, S. Ahmad, and T. D. Yilma, unpublished data) of two heterologous genes.
Two rVVs were generated for the study: (i) v
B8R was derived from the
WR strain of VV, selected because of its high virulence to mice, and
(ii) v50B8R was derived from v50, a WR-derived rVV expressing the
VSV-G gene within the TK locus (33), selected to facilitate the
analysis of the immune response to both homologous (VV) and
heterologous (VSV-G) antigens. The purity and stability of the
final preparations of viruses were confirmed by X-Gal cytochemical staining of plaques after a minimum of four consecutive plaque purifications in cell culture. Restriction analysis
(HindIII digestion) of DNA purified from rVVs confirmed
the insertional inactivation of the B8R and TK regions by the
lacZ and VSV-G genes, respectively (data not shown). To
confirm that the rVVs with a deletion of the B8R gene did not express
any residual IFN- binding activities, HeLa S3 cells were infected
with the viruses at a high multiplicity of infection (MOI) and their
supernatants were harvested, clarified from VV particles, concentrated,
and assayed for human IFN--neutralizing activities. It is clear from
the data that only the supernatants derived from cells infected with
VVs with an intact B8R gene (WR and v50) expressed detectable human
IFN--neutralizing activities (Fig. 2).
These concentrated supernatants at dilutions of 1/32 to 1/64 were able
to neutralize human IFN- at concentrations of about 20 U/ml; IFN-
at 1 U/ml provides 50% protection from cytopathic effects in this
assay. However, there were no detectable activities from concentrated
supernatants of rVVs lacking the B8R gene, even when undiluted (Fig.
2).
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Deletion of the B8R gene decreases VV virulence for normal
mice.
Immunocompetent mice inoculated intranasally with
103 and 104 PFU of either WR or vB8R
exhibited no significant weight loss (data not shown). However, mice
inoculated with 105 PFU of WR had marked weight loss, which
was not observed in vB8R-infected mice (Fig.
4A). This difference was statistically
significant (by analysis of variance [ANOVA]) from days 5 through 21 postinfection (P < 0.05) and highly significant on
days 7, 8, and 9 postinfection (P < 0.001). When
vB8R was used at a dose of 106 PFU, no significant
weight loss was observed, while mice inoculated with 106
PFU of WR showed marked weight loss (data not shown) and one of them
died (20% mortality [Fig. 4B]). Finally, when vB8R was used at a
dose of 107 PFU, the mice had some weight loss (data not
shown) but survived, while three of the mice inoculated with
107 PFU of WR died (60% mortality). This difference in
survival was significant (P < 0.05, log-rank test).
Taken together, these results indicate that inactivation of the B8R
gene decreases VV virulence for mice. On the other hand, rats
inoculated intranasally with doses as high as 107 PFU of
either WR or vB8R did not have significant weight loss or any other
signs of disease and maintained normal growth curves (data not shown).
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Deletion of the B8R gene attenuates VV virulence for nude
mice.
Immunodeficient, athymic nude mice inoculated
intraperitoneally with 107 PFU of v50B8R displayed a
significantly greater (P < 0.001, Mann-Whitney-Wilcoxon test) survival rate than did nude mice inoculated with v50 (Fig. 5). While all nude mice
injected with v50 died by 23 days postinfection, mice infected with
v50B8R had a survival rate of 89% on this day (Fig. 5). The median
survival time of nude mice inoculated with v50 and v50B8R was 13 and
29 days, respectively. All nude mice inoculated with v50 and v50B8R
developed typical pox lesions. A group of four control nude mice mock
inoculated with sterile PBS remained healthy throughout the observation
period of 120 days. Nude rats inoculated intraperitoneally with
107 PFU of VV resisted infection, showing no signs of
disease (including pox lesions) even when injected with the highly
virulent WR strain (data not shown).
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Deletion of the B8R gene does not alter the humoral response to
VV.
Groups of normal mice and rats (five animals/group) were
inoculated intramuscularly with 106 PFU of VV. Serum was
collected weekly from each animal, and neutralizing-antibody titers to
VV and VSV were determined by a plaque reduction assay or serum
neutralization, respectively. Although there was a significant increase
in attenuation, no statistical differences were observed in the humoral
immune response to either homologous (VV) or heterologous (VSV-G)
antigens in mice or rats inoculated with rVVs (Fig.
6).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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VV is one of many viruses that code for immunomodulating proteins,
including B8R. This secreted protein has sequence similarity to the
extracellular domain of the IFN- receptor, binding and neutralizing
the antiviral activity of most species of IFN-, including rat
IFN-. However, even at high concentrations, the B8R protein fails to
block the antiviral activity of murine IFN- in vitro (4, 36, 41, 51). IFN- is a pleiotropic cytokine that plays essential roles in
the regulation of the immune system and in the host defense against
pathogens. We and others showed that rVVs expressing IFN- were
attenuated by more than 106-fold in immunodeficient mice
(19, 27) and that IFN- has adjuvant activities when used with a
subunit antigen (e.g., VSV-G) (5). This led us to the hypothesis that
deleting immunomodulating genes (e.g., the B8R gene) in rVVs would
allow the development of safer and more efficacious vaccines.
To test our hypothesis, we constructed two rVVs with deleted B8R genes
to test the role of this gene in virulence and on the humoral immune
response of animals to both homologous (VV) and heterologous (VSV-G)
antigens. Although the B8R protein does not inhibit the antiviral
activity of murine IFN- (4, 36, 41, 51) and has no effect on VV
replication in vitro (Fig. 3), inactivation of the B8R gene reduced the
virulence of VV in a murine intranasal model (as measured by weight
loss and survival rates of normal mice) and in a nude mouse model (as
measured by survival rates of nude mice). Intranasal infection of
normal mice with rVVs has been reported to be an ideal route for
studies of pathogenesis and virulence (53), and CB6F1 mice
have been used extensively to assess the adjuvant activity of IFN-
(39). It has also been reported that rVVs with an insertional
inactivation of the TK gene (i.e., v50 and v50B8R) are greatly
attenuated for normal mice (10); doses as high as 5 × 106 PFU of v50 failed to cause weight loss in
CB6F1 mice (P. H. Verardi, F. H. Aziz, and
T. D. Yilma, unpublished data). For this reason, only WR and
vB8R (with a higher starting virulence) were used in the virulence
studies in immunocompetent mice. Normal mice inoculated intranasally
with WR had significantly higher weight loss and mortality than did
vB8R-infected mice (Fig. 4). These results indicate that the
inactivation of the B8R gene attenuates VV virulence for mice. On the
other hand, such reduction in virulence could not be determined in
normal rats, since they are considerably more resistant to VV infection
than mice (F344 rats infected with either WR or vB8R did not lose
weight or exhibit disease symptoms even when the virus was given at a
dose of 107 PFU). Intraperitoneal inoculation of nude mice
is an established model for disseminated VV infection (17, 19, 42).
Since VVs with an intact TK gene are highly virulent for nude mice,
only v50 and v50B8R (with a lower starting virulence) were used in
the attenuation studies with immunodeficient mice. We demonstrated that
nude mice inoculated with v50B8R had significantly higher survival
rates than did those inoculated with v50 (Fig. 5). The effects of the
B8R protein could not be determined in nude rats due to their greater
resistance to VV infection (44).
The direct inactivation of the antiviral activity of murine IFN- by
the B8R protein cannot explain the higher virulence of rVVs with an
intact B8R gene, since the B8R protein does not bind significantly to
murine IFN- in vitro. Knockout mice with an inactivated IFN-
receptor gene were found to be more susceptible to infection with VV or
herpesvirus than were mice with an inactivated IFN- gene (11),
although one would assume that the two groups of mice would be equally
susceptible. This suggests that there may be more than one ligand for
the IFN- receptor and its poxvirus homologs. Indeed, the myxoma
virus homolog of the B8R protein (M-T7), which was shown to be a
virulence factor in rabbits (35), has a number of ligands including
rabbit IFN- and human interleukin-8; it also inhibits the biological
activity of the chemokine RANTES (31). In addition, VVs expressing
human IFN- are attenuated for nude mice (19). This is surprising
since human IFN- has no detectable antiviral activity in mouse
cells. Thus, it is reasonable to deduce that the B8R protein may bind
to other immunoregulatory proteins or may have immunomodulating
properties in vivo that affect VV virulence by a mechanism other than
direct interference with the antiviral activity of IFN-.
B8R-deleted rVVs elicited humoral immune responses to both homologous
(VV) and heterologous (VSV-G) antigens that did not differ
significantly from those of the respective parental strains (Fig. 6).
One could speculate that the deletion of the B8R gene would augment the
immune response by eliminating immunomodulating functions of the B8R
protein (e.g., binding IFN- to neutralize its antiviral activity),
just as the deletion of serine protease inhibitor homolog genes of VV
increases antibody response to a heterologous antigen (58). However, we
have shown here that the B8R gene also acts as a virulence factor; its
deletion attenuates the vector, which should result in a decreased
immune response. The two competing actions could effectively cancel
each other, accounting for the experimental observation that humoral
immune responses elicited by both B8R+ and
B8R
viruses are essentially the same. In future studies,
it will be of great interest to determine the effect of the B8R gene in cell-mediated immune responses, which are essential to vaccine efficacy.
Two highly effective rVV vaccines have been developed to date: one for
protection against rinderpest and the other for the eradication of
rabies in wildlife (1). However, other rVVs have not been as
efficacious. Also, the safety of rVVs is a major concern, especially
for immunocompromised individuals. Strategies to increase safety
include the use of modified VV Ankara (MVA), a highly attenuated and
host cell-restricted vector (37, 48). Here we show that the B8R protein
is a VV virulence factor in mice despite the lack of detectable
inhibition of the antiviral activity of murine IFN- by the B8R
protein in vitro. In addition, deletion of the B8R gene did not alter
the immune responses to homologous (VV) or heterologous (VSV-G)
antigens. Consequently, the B8R locus emerges as an excellent site for
heterologous gene expression in rVV vaccine development; inactivation
of the B8R gene attenuates VV (increasing the safety of the vector)
without compromising the effectiveness of the immune response elicited
by the recombinant vaccine. All of these features are highly desirable
for the development of new, effective rVV vaccines.
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ACKNOWLEDGMENTS |
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This work was supported by an NIH grant awarded to T.D.Y. (AI37182) and a grant awarded to L.A.J. by The Harold Wetterberg Foundation. Other support included NIH grants AI29207 and AI36197 and USA-DAMD contract 17-95-C-5054N to T.D.Y. P.H.V. received support from Conselho Nacional de Desenvolvimento Científico Tecnológico (CNPq), Brazil.
We are grateful to Lauretta Turin, Ian Crossley, and Kartik Nettar for critical advice and assistance. We thank Sally Owens for helpful discussions and review of the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: International Laboratory of Molecular Biology for Tropical Disease Agents, Department of Veterinary Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616. Phone: (530) 752-8306. Fax: (530) 752-1354. E-mail: tdyilma{at}ucdavis.edu.
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Materials and Methods
Results
Discussion
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Journal of Virology, January 2001, p. 11-18, Vol. 75, No. 1
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.1.11-18.2001
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