DNA vaccine for West Nile virus infection in fish crows (Corvus ossifragus).
ABSTRACT A DNA vaccine for West Nile virus (WNV) was evaluated to determine whether its use could protect fish crows (Corvus ossifragus) from fatal WNV infection. Captured adult crows were given 0.5 mg of the DNA vaccine either orally or by intramuscular (IM) inoculation; control crows were inoculated or orally exposed to a placebo. After 6 weeks, crows were challenged subcutaneously with 105 plaque-forming units of WNV (New York 1999 strain). None of the placebo inoculated-placebo challenged birds died. While none of the 9 IM vaccine-inoculated birds died, 5 of 10 placebo-inoculated and 4 of 8 orally vaccinated birds died within 15 days after challenge. Peak viremia titers in birds with fatal WNV infection were substantially higher than those in birds that survived infection. Although oral administration of a single DNA vaccine dose failed to elicit an immune response or protect crows from WNV infection, IM administration of a single dose prevented death and was associated with reduced viremia.
- SourceAvailable from: Michael Lierz[Show abstract] [Hide abstract]
ABSTRACT: West Nile virus (WNV) can lead to fatal diseases in raptor species. Unfortunately, there is no vaccine which has been designed specifically for use in breeding stocks of falcons. Therefore the immunogenicity and protective capacity of two commercially available WNV vaccines, both approved for use in horses, were evaluated in large falcons. One vaccine contained adjuvanted inactivated WNV lineage 1 immunogens, while the second represented a canarypox recombinant live virus vector vaccine. The efficacy of different vaccination regimes for these two vaccines was assessed serologically and by challenging the falcons with a WNV strain of homologous lineage 1. Our studies show that the recombinant vaccine conveys a slightly better protection than the inactivated vaccine, but moderate (recombinant vaccine) or weak (inactivated vaccine) side effects were observed at the injection sites. Using the recommended 2-dose regimen, both vaccines elicited only sub-optimal antibody responses and gave only partial protection following WNV challenge. Better results were obtained for both vaccines after a third dose, i.e. alleviation of clinical signs, absence of fatalities and reduction of virus shedding and viraemia. Therefore the consequences of WNV infections in falcons can be clearly alleviated by vaccination, especially if the amended triple administration scheme is used, although side effects at the vaccination site must be accepted.Veterinary Research 04/2014; 45(1):41. · 3.43 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: West Nile virus (WNV) is maintained in nature in an enzootic transmission cycle between birds and mosquitoes, although it occasionally infects other vertebrates, including humans, in which it may result fatal. To date, no licensed vaccines against WNV infection are available for birds, but its availability would certainly benefit certain populations, as birds grown for restocking, hunting activities, or alimentary purposes, and those confined to wildlife reservations and recreation installations. We have tested the protective capability of WNV envelope recombinant (rE) protein in red-legged partridges (Alectoris rufa). Birds (n=28) were intramuscularly immunized three times at 2-weeks interval with rE and a control group (n=29) was sham-immunized. Except for 5 sham-immunized birds that were not infected and housed as contact controls, partridges were subcutaneously challenged with WNV. Oropharyngeal and cloacal swabs and feather pulps were collected at several days after infection and blood samples were taken during vaccination and after infection. All rE-vaccinated partridges elicited anti-WNV antibodies before challenge and survived to the infection, while 33.3% of the sham-immunized birds succumbed, as did 25% of the contact animals. Most (84%) unvaccinated birds showed viremia 3 d.p.i., but virus was only detected in 14% of the rE vaccinated birds. WNV-RNA was detected in feathers and swabs from sham-immunized partridges from 3 to 7 d.p.i., mainly in birds that succumbed to the infection, but not in rE vaccinated birds. Thus, rE vaccination fully protected partridges against WND and reduced the risk of virus spread.Vaccine 08/2013; · 3.77 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: During the last three years Greece is experiencing the emergence of West Nile virus (WNV) epidemics. Within this framework, an integrated surveillance and control programme (MALWEST project) with thirteen associate partners was launched aiming to investigate the disease and suggest appropriate interventions. One out of seven work packages of the project is dedicated to the State of the Art report for WNV. Three expert working groups on humans, animals and mosquitoes were established. Medical databases (PubMed, Scopus) were searched together with websites: e.g., WHO, CDC, ECDC. In total, 1,092 relevant articles were initially identified and 258 of them were finally included as references regarding the current knowledge about WNV, along with 36 additional sources (conference papers, reports, book chapters). The review is divided in three sections according to the fields of interest: (1) WNV in humans (epidemiology, molecular characteristics, transmission, diagnosis, treatment, prevention, surveillance); (2) WNV in animals (epidemiological and transmission characteristics concerning birds, horses, reptiles and other animal species) and (3) WNV in mosquitoes (control, surveillance). Finally, some examples of integrated surveillance programmes are presented. The introduction and establishment of the disease in Greece and other European countries further emphasizes the need for thorough research and broadening of our knowledge on this viral pathogen.International Journal of Environmental Research and Public Health 01/2013; 10(12):6534-610. · 2.00 Impact Factor
A DNA vaccine for West Nile virus (WNV) was evaluat-
ed to determine whether its use could protect fish crows
(Corvus ossifragus) from fatal WNV infection. Captured
adult crows were given 0.5 mg of the DNA vaccine either
orally or by intramuscular (IM) inoculation; control crows
were inoculated or orally exposed to a placebo. After 6
weeks, crows were challenged subcutaneously with 105
plaque-forming units of WNV (New York 1999 strain). None
of the placebo inoculated–placebo challenged birds died.
While none of the 9 IM vaccine–inoculated birds died, 5 of
10 placebo-inoculated and 4 of 8 orally vaccinated birds
died within 15 days after challenge. Peak viremia titers in
birds with fatal WNV infection were substantially higher
than those in birds that survived infection. Although oral
administration of a single DNA vaccine dose failed to elicit
an immune response or protect crows from WNV infection,
IM administration of a single dose prevented death and
was associated with reduced viremia.
Hemisphere during summer 1999 in New York City and
was associated with human, equine, and avian deaths
(1–4). This virus is transmitted by a variety of mosquito
species, mostly in the genus Culex (5–7). The New York
1999 strain of WNV differed genetically from other known
strains of WNV except for an Israeli strain isolated from a
dead goose in Israel in 1998 (1). With the exception of a
est Nile virus (WNV), a mosquito-borne flavivirus,
was recognized for the first time in the Western
laboratory study in Egypt involving hooded crows (Corvus
corone) and house sparrows (Passer domesticus) (8), only
these two nearly identical strains are known to kill birds
(9,10). In 2000, WNV was detected in >4,000 bird carcass-
es in the United States (11), and the overall mortality rate
was considered much greater. Several deaths attributed to
WNV in the United States have occurred in valuable cap-
tive birds in zoologic collections (12). Currently, no treat-
ment or vaccine is available for susceptible birds.
Vaccination may protect birds from lethal WNV infec-
tions. Accordingly, we examined a DNA vaccine devel-
oped for use in horses (13) for its ability to protect crows,
a species known to be highly susceptible to lethal infection
with this virus (8,10).
Materials and Methods
The plasmid DNA, pCBWN, codes for the prM and E
glycoproteins of WNV. The plasmid was purified from
Escherichia coli XL-1 blue cells with EndoFree Plasmid
Giga Kits (QIAGEN, Inc., Santa Clarita, CA) and suspend-
ed in 10 mM Tris buffer, pH 8.5, at a concentration of 10.0
mg/mL. For IM vaccination, the DNAvaccine was formu-
lated in phosphate-buffered saline (PBS), pH 7.5, at a con-
centration of 1.0 mg/mL. For oral exposure, the dry-
microencapsulated DNAwas suspended in PBS, pH 7.5, at
a concentration of 2.0 mg/mL.
The method for microencapsulating DNA was adapted
from procedures previously described for virus and sub-
unit vaccines and isolated proteins (14–16). We performed
all steps with sterile reagents and aseptic technique. Two
10-mg aliquots of WNV cDNA were transferred to sepa-
Emerging Infectious Diseases • Vol. 9, No. 9, September 20031077
DNA Vaccine for West Nile Virus
Infection in Fish Crows
Michael J. Turell,* Michel Bunning,†‡1George V. Ludwig,* Brian Ortman,† Jeff Chang,‡
Tully Speaker,§ Andrew Spielman,¶ Robert McLean,# Nicholas Komar,‡ Robert Gates,‡
Tracey McNamara,** Terry Creekmore,†† Linda Farley,‡‡ and Carl J. Mitchell‡
*U.S. Army Medical Research Institute of Infectious Diseases, Fort
Detrick, Maryland, USA; †U.S. Air Force, Fort Detrick, Maryland,
USA; ‡Centers for Disease Control and Prevention, Fort Collins,
Colorado, USA; §Temple University, Philadelphia, Pennsylvania,
USA; ¶Harvard School of Public Health and the Center for
International Development at Harvard University, Boston,
Massachusetts, USA; #U.S. Department of Agriculture, Fort
Collins, Colorado, USA; **Wildlife Conservation Society, Bronx,
New York, USA; ††Wyoming Department of Health, Laramie,
Wyoming, USA, and ‡‡American Bird Conservancy, Washington,
1Drs. Turell and Bunning are co-lead authors of this article.
rate test tubes with enough water to make 9-mL volumes.
Resulting suspensions were mixed on a clinical rotator
until solution was complete; 1 mL of 0.6% w/v aqueous
sodium alginate (Fluka Chemical Co., Ronkonkoma, NY)
solution was added to each tube, and the contents of each
were gently inverted 20 times. Each DNA/alginate solu-
tion was pumped at 1.2 mL/min through a 76-µm orifice
in a 1-mm internal diameter glass tube against the side of
which a 20-KHz laboratory sonicator probe was firmly
pressed. The emerging train of droplets was directed into
a modified T-tube, through which a recirculated 40 mL of
0.25% w/v neutral aqueous spermine hydrochloride
(Sigma-Aldrich Corp., St. Louis, MO) solution was
pumped at 10 mL/min. A placebo microcapsule formula-
tion was prepared by using alginate reagent without DNA.
Resulting microcapsule suspensions were allowed to
equilibrate for 30 min, pelleted at 500 x g for 20 min, and
washed three times by decanting, suspending, and repel-
leting. Wash liquids were reserved for measuring the
DNA that escaped encapsulation. Placebo and vaccine
formulations and washes were frozen at –20°C and
lyophilized overnight, then suspended in 5 mL of PBS to
produce a final concentration of 2 mg/mL of the encapsu-
Fish crows (C. ossifragus) were captured with a rocket-
propelled net at various locations in Maryland. Birds were
transported to a biosafety level 3 laboratory at the U.S.
Army Medical Research Institute of Infectious Diseases,
allocated into four groups, and placed in stainless steel
cages (3–4 birds/cage); blood was collected for evidence
of antibodies against flaviviruses. Birds were provided a
mixture of cat and dog food ad libitum and water. This diet
was supplemented with hardboiled eggs as well as vitamin
Serial 10-fold dilutions of the blood samples from each
crow were made in standard diluent (10% heat-inactivat-
ed fetal bovine serum in medium 199 with Earle’s salts,
NaHCO3, and antibiotics). These samples were tested for
infectious virus by plaque assay on Vero cells in 6-well
plates (Costar, Inc., Cambridge, MA) as previously
described (17), except that the second overlay, containing
neutral red stain, was added 2 or 3 days after the first
Plaque-Reduction Neutralization Assay
Serum samples were assayed for WNV-specific anti-
bodies by using the plaque-reduction neutralization test
(PRNT), as previously described (18). Briefly, each serum
sample was diluted 1:10 in standard diluent (as above) and
mixed with an equal volume of BA1 (composed of Hanks’
M-199 salts, 1% bovine serum albumin, 350 mg/L of sodi-
um bicarbonate, 100 U/mL of penicillin, 100 mg/L of
streptomycin, and 1 mg/L of fungizone in 0.05 M Tris, pH
7.6) containing a suspension of WNV (NY99-4132 strain)
at a concentration of approximately 200 plaque-forming
units (PFU)/0.1 mL, such that the final serum dilution was
1:20 and the final concentration of WNV (the challenge
dose) was approximately 100 PFU/0.1 mL. After 1-h ncu-
bation at 37°C, we added the serum/virus mixtures onto
Vero monolayers in 6-well plates, 0.1 mLper well in dupli-
cate. We determined the mean percentage of neutralization
for each specimen by comparing the number of plaques
that developed (see Plaque Assay section) relative to the
number of plaques in the challenge dose, as determined by
back titration. Preliminary samples were screened for anti-
bodies to WNV in the same manner, as well as for neutral-
izing antibodies to St. Louis encephalitis virus, a closely
related flavivirus that may cross-react serologically with
WNV (19) and may partially protect against WNV infec-
The crows were placed in four groups: 1) those inocu-
lated IM with vaccine, 2) those that had oral vaccine, 3)
positive controls (i.e., those that received placebo inocula-
tion and viral challenge), and 4) room controls (i.e., those
that received placebo inoculation and placebo challenge).
After an acclimatization period of approximately 1 month,
the 10 crows in group 1 (9 fish crows and 1 American crow
[C. brachyrhynchos]) were inoculated IM with 0.5 mg of
the DNA vaccine in a total volume of 0.5 mL (0.25 mL in
each breast). The 9 crows in group 2 (8 fish crows and 1
American crow) were given 0.5 mg of the encapsulated
DNA vaccine orally in 0.25 mL of PBS, and 20 fish crows
(groups 3 and 4) were each inoculated and orally exposed
as above except that a placebo was used in place of the
vaccine. Blood was collected weekly from the jugular vein
and the serum tested for neutralizing antibodies to WNV.
Six weeks after vaccination, all birds in groups 1, 2, and 3
were inoculated subcutaneously with 0.1 mL of a suspen-
sion containing 105PFU (106PFU/mL) of the 397-99
strain of WNV, which had been isolated from the brain of
an American crow that died in New York City during the
fall of 1999 and passaged once in Vero cells before use in
this study. The crows in group 4 were inoculated with 0.1
mLof diluent. Three or four crows in each group were bled
(0.1 mL) from the jugular vein each day; each bird was
bled every third day. Blood samples were added to 0.9 mL
of diluent + 10 U of heparin/mL. Blood samples were
frozen at –70°C until tested for infectious virus by plaque
1078Emerging Infectious Diseases • Vol. 9, No. 9, September 2003
While neutralizing antibodies developed in 5 of the 9
fish crows that received the vaccine by the IM route at the
80% neutralization level for WNV by 14 days after vacci-
nation, neutralizing antibodies to WNV did not develop in
any of the remaining fish crows (8 orally exposed to vac-
cine and 20 placebo-exposed) in the same time period
(Table 1). An antibody response at the 78% level devel-
oped in one of the remaining IM-vaccinated fish crows.
Thus, a serologic response developed in six (67%) of the
nine fish crows that received the vaccine by the IM route.
However, by day 42 after vaccination, none of these crows
retained a response at the 80% neutralization level.
Viremia Profiles and Survival
All of the mock-challenged crows survived. Similarly,
all nine fish crows that received the IM vaccine survived
(Table 1). However, 5 of 10 fish crows that received the
placebo vaccine and 4 of 8 fish crows that received the oral
vaccine died when challenged with virulent WNV. The dif-
ference in survival rates between the fish crows that
received the IM vaccine and either of the other two groups
was significant (Fisher exact test, p<0.03). A veterinary
pathologist examined all crows that died during these stud-
ies, and signs typical of WNV infection in avian hosts (i.e.,
heart necrosis) were observed in all of these birds. (These
data will be described in a separate article on WNV viral
pathogenesis in fish crows.) Viremias were detected in all
10 crows that received the placebo inoculation, 7 of 8 fish
crows that received the oral vaccine, and 6 of 9 fish crows
that received the vaccine by the IM route (Table 1). Virus
was not detected in any of the crows that received the
placebo challenge. Mean logarithm10peak viremia titers
were significantly lower (T>2.75, df>15, p<0.017) in the
fish crows that received the vaccine by the IM route (mean
+ S.E. = 2.9 + 0.4) than in fish crows that received the
placebo vaccine (mean + S.E. = 4.3 + 0.3) or fish crows
that received vaccine by the oral route (mean + S.E. = 5.2
+ 0.8). The mean peak viremia titers for fish crows that
received the placebo vaccine or the DNA vaccine by the
oral route were not significantly different (T=1.1, df=16,
p=0.287). In both the oral vaccine and placebo groups, fish
crows that died had higher viremia than those that survived
their infection with WNV (Table 2). Because birds were
bled only every third day, accurately determining the dura-
tion of viremia in individual fish crows was not possible.
Viremias were detected on days 1 to 6 after infection, and
9 of 10 birds that were viremic on day 1 were still viremic
on day 4. However, only five of eight birds that were
viremic on day 2 were still viremic on day 5, and only
three of six birds that were viremic on day 3 were still
viremic on day 6. No birds were viremic 7 days after infec-
tion. Thus, most viremias apparently lasted approximately
5 days during this study.
Although the DNAvaccine failed to induce a long-last-
ing immune response, fish crows vaccinated with this vac-
cine by the IM route all survived challenge with virulent
WNV. In contrast, oral administration of this vaccine failed
to elicit an immune response, nor did it protect fish crows
from a lethal challenge with WNV. The death rate in these
crows (4 [50%] of 8), was identical to that observed in the
placebo-vaccinated group (5 [50%] of 10) and in a second
group of unvaccinated fish crows (4 [50%] of 8) tested
later (M.J. Turell and M. Bunning, unpub. data). Although
no deaths occurred in the IM-vaccinated fish crows, low-
level viremia, consistent with that observed in the birds
that survived their WNV infection in the other groups, did
develop in six of the nine crows. Therefore, a single dose
of the DNA vaccine did not elicit complete protection and
sterile immunity to WNV infection. Additional studies
need to be conducted with multiple doses of vaccination
both by the IM as well as by the oral route to determine
whether multiple doses might provide greater protection
against WNV infection.
During the course of these studies, we determined that
we had two American crows mixed in with the fish crows,
Emerging Infectious Diseases • Vol. 9, No. 9, September 20031079
Table 1. Effect of route of administration of a DNA West Nile virus vaccine on the protection of fish crows from challenge with virulent
West Nile virus
Treatmenta,b No. tested % seropositivec
Room control 10 0
IM 9 56
Oral 8 0
Placebo 10 0
aIM, intramuscularly. Crows were inoculated IM with 0.5 mg of the DNA vaccine. Oral, crows were given 0.5 mg of the DNA vaccine orally. Placebo, crows were
inoculated IM with 0.5 mg of nonspecific DNA and given 0.5 mg of nonspecific DNA orally.
bRoom controls were placebo inoculated and then challenged with diluent.
cPercentage of crows whose serum produced >80% neutralization at 1:20 dilution.
dLogarithm10 mean peak viremia in crows bled every third day after challenge (S.E.). No virus was detected in any of the room control birds, and a value of 1.7 was
assigned to birds from which no virus was detected for calculation of mean and S.E. Means followed by the same letter are not significantly different at α = 0.05 by
Student t test.
one in the oral and one in the IM-vaccinated groups. High
viremias (>106PFU/mL of blood) developed in both of
these crows, and they died after challenge with virulent
WNV. These data, based on a single bird in each group,
were not included in the data presented in this report. Both
hooded crows (8) and American crows (10) are highly sus-
ceptible to infection with WNV with nearly 100% case-
fatality rates. In contrast, fish crows appear to be less like-
ly to succumb to the infection.
The continued spread of WNV infection across the
United States and reported deaths in raptors and rare cap-
tive birds in zoologic parks indicate the need to develop an
effective avian vaccine for WNV. To break the transmis-
sion cycle, the vaccine must be able to substantially reduce
the level of viremia below the level needed to infect a feed-
ing mosquito, which is about 105PFU/mL of blood (21).
By this standard, the vaccine performed reasonably well,
with no vaccinated fish crow having a recorded viremia
>104.7. In contrast, 3 of 10 placebo-vaccinated fish crows
had viremias >105PFU/mL of blood, and 5 of 10 had a
peak viremia >104.8PFU/mL of blood. However, because
the crows were bled only every third day, determining the
actual peak viremias in these birds was not possible. If the
goal of the vaccine is to protect rare and endangered avian
species from death, rather than to prevent transmission,
then the occurrence of a low-level viremia is not critical.
We thank R. Lind, P. Rico, and S. Duniho for their excellent
assistance in caring for the crows; R. Schoepp for his assistance
in bleeding the crows; D. Dohm and J. Velez for technical assis-
tance; J. Blow, R. Schoepp, C. Mores, P. Schneider, and K.
Kenyon for editorial assistance; E. Peterson for administrative
support; D. Rohrback, S. Bittner, K. Musser, B. Riechard, M.
Castle, C. Dade, G. Timko, and B. Davenport for their work in
capturing the crows; and the staff at Umberger Farm for allowing
us to use their property for the fieldwork.
This work was supported by a grant from The American Bird
Conservancy, Pesticides and Birds Campaign, Washington, D.C.
Research was conducted in compliance with the Animal
Welfare Act and other Federal statutes and regulations relating to
animals and experiments involving animals and adheres to prin-
ciples stated in the Guide for the Care and Use of Laboratory
Animals, National Research Council, 1996. The facility where
this research was conducted is fully accredited by the Association
for Assessment and Accreditation of Laboratory Animal Care,
Dr. Turell is a research entomologist at the United States
Army Medical Research Institute of Infectious Diseases, Fort
Detrick, Maryland. His research interests focus on factors affect-
ing the ability of mosquitoes and other arthropods to transmit
1. Lanciotti RS, Roehrig JT, Deubel V, Smith J, Parker M, Steele K, et
al. Origin of the West Nile virus responsible for an outbreak of
encephalitis in the Northeastern United States. Science
2. Centers for Disease Control and Prevention. Outbreak of West Nile-
like viral encephalitis—New York, 1999. MMWR Morb Mortal Wkly
3. Trock SC, Meade BJ, Glaser AL, Ostlund EN, Lanciotti RS, Cropp
BC, et al. West Nile virus outbreak among horses in New York State,
1999 and 2000. Emerg Infect Dis 2001;7:745–7.
4. Eidson M, Komar N, Sorhage F, Nelson R, Talbot T, Mostashari F, et
al. Crow deaths as a sentinel surveillance system for West Nile virus
in the northeastern United States, 1999. Emerg Infect Dis
5. Hayes C. West Nile fever. In: Monath TP, editor. The arboviruses:
epidemiology and ecology. Volume V. Boca Raton (FL): CRC Press;
1989. p. 59–88.
6. Turell MJ, Sardelis MR, O’Guinn ML, Dohm DJ. Potential vectors of
West Nile virus in North America. In: Mackenzie JS, Barrett ADT,
Deubel V, editors. Japanese encephalitis and West Nile viruses, vol-
ume 267. Current topics in microbiology and immunology. Berlin:
Springer-Verlag; 2002. p. 241–52.
7. Hubalek Z, Halouzka J. West Nile virus—a reemerging mosquito-
borne viral disease in Europe. Emerg Infect Dis 1999;5:643–50.
8. Work TH, Hurlbut HS, Taylor RM. Indigenous wild birds of the Nile
delta as potential West Nile virus circulating reservoirs. Am J Trop
Med Hyg 1955;4:872–88.
9. Komar N. West Nile viral encephalitis. Rev Sci Tech Off Int Epiz
10. McLean RG, Ubico SR, Docherty DE, Hansen WR, Sileo L,
McNamara T. West Nile virus transmission and ecology in birds. Ann
N YAcad Sci 2001;951:54–7.
11. Marfin AA, Petersen LR, Eidson M, Miller J, Hadley J, Farello C, et
al. Widespread West Nile virus activity, eastern United States, 2000.
Emerg Infect Dis 2001;7:730–5.
12. Steele KE, Linn MJ, Schoepp RJ, Komar N, Geisbert TW, Manduca
RM, et al. Pathology of fatal West Nile virus infections in native and
exotic birds during the 1999 outbreak in New York City, New York.
Vet Pathol 2000;37:208–24.
1080 Emerging Infectious Diseases • Vol. 9, No. 9, September 2003
Table 2. Viremia levels in fish crows that survived or died after challenge with virulent West Nile virus
aIM, intramuscularly. Crows were inoculated IM with 0.5 mg of the DNA vaccine. Oral, crows were given 0.5 mg of the DNA vaccine orally. Placebo, crows were
inoculated IM with 0.5 mg of nonspecific DNA and given 0.5 mg of nonspecific DNA orally.
bLogarithm10 mean peak viremia in crows bled every third day after challenge (S.E.). A value of 1.7 was assigned to birds from which no virus was detected for calculation
of mean and S.E.
13. Chang GJ, Davis BS, Hunt AR, Holmes DA, Kuno G. Flavivirus
DNA vaccines: current status and potential. Ann N Y Acad Sci
14. Moser CA, Speaker TJ, Offit PA. Effect of aqueous based microen-
capsulation on protection against EDIM rotavirus challenge in mice.
J Virol 1998;72:3859–62.
15. Patil RT, Speaker TJ. Water based microsphere delivery system for
proteins. J Pharm Sci 2000;89:9–15.
16. Speaker TJ, Clark HF, Moser CA Offit PA, Campos M, Frenchik PJ.
U.S. Patent 6,270,800 aqueous solvent based encapsulation of a
bovine herpes virus type-1 subunit vaccine. Aug 7, 2001.
17. Gargan TP II, Bailey CL, Higbee GA, Gad A, El Said S. The effect of
laboratory colonization on the vector pathogen interaction of
Egyptian Culex pipiens and Rift Valley fever virus. Am J Trop Med
18. Beaty BJ, Calisher CH, Shope RE. Arboviruses. In: Schmidt NJ,
Emmons RW, editors. Diagnostic procedures for viral, rickettsial and
chlamydial infections. 6th ed. Washington: American Public Health
Association; 1989. p. 797–855.
19. Calisher CH. Antigenic classification and taxonomy of flaviviruses
(family Flaviviridae) emphasizing a universal system for the taxono-
my of viruses causing tick-borne encephalitis. Acta Virol
20. Tesh RB, Travassos da Rosa AP, Guzman H, Araujo TP, Xiao SY.
Immunization with heterologous flaviviruses protective against fatal
West Nile encephalitis. Emerg Infect Dis 2002;8:245–51.
21. Turell MJ, O’Guinn ML, Dohm DJ, Jones JW. Vector competence of
North American mosquitoes (Diptera: Culicidae) for West Nile virus.
J Med Entomol 2001;38:130–4.
Address for correspondence: Michael J. Turell, Department of Vector
Assessment, Virology Division, USAMRIID, 1425 Porter Street, Fort
Detrick, MD 21702-5011, USA; fax: (301) 619-2290; email:
Emerging Infectious Diseases • Vol. 9, No. 9, September 20031081
Search Search p pastast i issuesssues o of f E EID ID a at t w www.cdc.gov/eidww.cdc.gov/eid