Am. J. Trop. Med. Hyg., 75(6), 2006, pp. 1158–1164
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene
MANIPULATION OF THE YELLOW FEVER VIRUS NON-STRUCTURAL GENES 2A
AND 4B AND THE 3⬘NON-CODING REGION TO EVALUATE GENETIC
DETERMINANTS OF VIRAL DISSEMINATION FROM THE AEDES
AEGYPTI MIDGUT
KATE L. McELROY, KONSTANTIN A. TSETSARKIN, DANA L. VANLANDINGHAM, and STEPHEN HIGGS*
Department of Pathology, University of Texas Medical Branch, Galveston, Texas
Abstract. Although much is known about the ecology, epidemiology, and molecular biology of mosquito-borne
viruses, the viral factors that allow transmission by mosquitoes to humans or animals remain unknown. Using infectious
clones of disseminating (Asibi) and non-disseminating (17D) yellow fever viruses (YFV), we produced chimeric viruses
to evaluate the role of different viral genes in dissemination. Previously, we showed that virus produced from an
infectious clone containing the structural genes of 17D in Asibi disseminated from the mosquito midgut at a rate of 31%,
indicating that some genetic determinants of dissemination must lie within the non-structural (NS) protein genes or 3⬘
non-coding region (NCR). We chose to investigate the roles of NS2A, NS4B, and the 3⬘NCR in YFV dissemination.
Substitution of the 17D NS2A or NS4B into Asibi significantly attenuated YFV dissemination, demonstrating that this
is a multigenic property. There was no difference in dissemination after substitution of the 17D 3⬘NCR.
was used to develop the 17D live attenuated virus vaccine in
use today. Not only is YF-17D highly immunogenic, it has lost
the ability to disseminate in and thus be transmitted by Ae.
aegypti as a result of the attenuation process.14–16 Sequence
comparison of the Asibi and 17D strains used in this study
show nucleotide and amino acid differences throughout the
viral genome with exception of the 5⬘NCR and the capsid
protein gene. Targeted manipulation of chosen genome regions using reverse genetics techniques with an Asibi infectious clone17 and a 17D infectious clone18 forms the basis of
our investigation of the viral molecular determinants of mosquito infection and dissemination.
Consistent with the results of studies with other arboviruses,4–9 we reported that the YFV envelope (E) structural
protein gene, specifically the putative cell-receptor binding
domain III of E, contained important determinants of viral
dissemination from the Ae. aegypti midgut.3 However, the
observation that a chimeric virus containing the M and E
protein genes of the midgut-restricted 17D virus in the Asibi
virus backbone was able to disseminate, even at a low rate
(31%), led us to hypothesize that some determinants of dissemination are located outside of the structural protein gene
region. We therefore investigated the roles of YFV nonstructural protein genes 2A, 4B, and the 3⬘NCR in viral infection and dissemination in Ae. aegypti. These portions of
the YFV genome were targeted based on previous reports
linking them to YFV- or DENV-mosquito infection and/or
dissemination. The non-structural protein gene 2A (NS2A)
was chosen because of its hypothesized role in viral replication and virion assembly and release,19,20 and because mutations in NS2A were associated with a 1998 DENV-4 outbreak
in Puerto Rico.21 NS4B is a small hydrophobic protein of
unknown function that was of interest to us because it contains one of two amino acid substitutions, NS4B-Ile95Met,
shared in common between three attenuated YFV strains17D, French neurotropic vaccine (FNV), and Asibi HeLa p6-,
which do not disseminate in mosquitoes.22,23 Finally, analogous differences in the 3⬘NCR secondary structures between
wild-type/attenuated virus pairs Asibi/17D and French viscerotropic virus/FNV led to the hypothesis that the 3⬘NCR
plays a role in YFV attenuation.24 We investigated whether
3⬘NCR chimerization in our system would have an attenuat-
INTRODUCTION
For a virus to be transmitted by a mosquito, virus taken up
in a blood meal must infect the mosquito midgut epithelium,
replicate, and disseminate from the midgut to the salivary
glands to be deposited in saliva when the mosquito feeds on
a susceptible host.1 The specific viral factors that allow dissemination of virus from the mosquito midgut and thus transmission of virus to vertebrates have been studied for many
years. Information gained from these studies is not only important in defining basic mosquito-virus interactions but
could potentially be used in the rational design of liveattenuated virus vaccine candidates. No treatment exists for
diseases caused by mosquito-borne flaviviruses dengue virus
(DENV), Japanese encephalitis virus (JEV), West Nile virus
(WNV), and yellow fever virus (YFV), making vaccination an
important control measure. A live virus vaccine, YFV-17D, is
currently in use for yellow fever, and other vaccines are in
development for DENV, JEV, and WNV.2 However, the requirement that these vaccine candidates must not be transmitted by mosquitoes necessitates a better understanding of
the viral factors that govern this process. Using monoclonal
antibody–resistant variants and, more recently, reverse genetics techniques, previous studies have largely focused on the
viral structural protein genes as containing the primary determinants of infection and/or dissemination.3–9 However, several flavivirus candidate vaccines use the structural protein
genes of a wild-type virus in an attenuated virus backbone.10–13
We sought to investigate whether dissemination determinants
were localized within the non-structural protein genes or
3⬘NCR.
Our model system, which is based on a comparison of phenotypically distinct YFV strains Asibi and 17D in Aedes aegypti, allows an investigation of sequences present throughout
the viral genome. Asibi virus, the prototype YFV strain that
infects and disseminates in a high proportion of mosquitoes,
* Address correspondence to Stephen Higgs, Department of Pathology, University of Texas Medical Branch, 2.104 Keiller, L20762, 301
University Boulevard, Galveston, TX 77555-0609. E-mail: sthiggs@
utmb.edu
1158
YFV NS2A, NS4B, AND 3⬘NCR IN VIRAL DISSEMINATION
ing effect because deletion of a portion of the DENV-4
3⬘NCR was previously found to attenuate virus for mosquito
infectivity and dissemination.12,13 We report here the studies
of six viruses: Asibi/17D NS2A, Asibi/17D NS4B, Asibi/17D
NS4B-95, Asibi/17D 3⬘NCR, 17D/Asibi NS4B-95, and 17D/
Asibi 3⬘NCR, and the comparison of these viruses to parental
Asibi and 17D strains in vitro and in vivo. Sequence differences between Asibi and 17D in NS2A, NS4B, and 3⬘NCR
are shown in Table 1.
MATERIALS AND METHODS
Construction of YFV cDNA infectious clones. Chimeric
cDNA YFV infectious clones (ic) were constructed using the
previously described YFV 17D and Asibi infectious clones as
backbones.17,18 The sequence of the Asibi strain used to construct the Asibi infectious clone has been deposited in Genbank (Accession no. AY640589). Each YFV chimeric ic was
constructed using techniques described previously.17 Briefly,
polymerase chain reaction (PCR)–based mutagenesis (standard cycling conditions; random hexanucleotide primers
[Promega, Madison, WI], Superscript II [Invitrogen, Carlsbad, CA], and Pfu DNA polymerase [Stratagene, La Jolla,
CA]); restriction digestion at unique sites (enzymes; New England Biolabs, Ipswich, MA), and ligation of DNA fragments
(T4 DNA ligase; Invitrogen) were used to produce and join
DNA fragments, and clones were amplified in Escherichia
coli MC1061 competent cells. The Asibi/17D NS2A ic was
produced by simultaneous ligation and cloning of three DNA
fragments using fusion PCR and restriction digestion, resulting in the insertion of a 958-basepair 17D fragment into the
Asibi ic vector at Mlu I-Sap I restriction sites. Asibi/17D
NS4B ic was constructed by the simultaneous ligation and
cloning of four DNA fragments: the Mlu I-Sph I fragment of
Asibi ic, the Sph I-Bsr GI fragment of 17D ic, and a 1,820basepair Asibi PCR product (nucleotides 6654–8474) digested
with Bsr GI were ligated into the Asibi ic vector digested with
Mlu I and Xho I. Asibi/17D NS4B-95 ic was produced by
simultaneous ligation and cloning of three fragments: a 1,321basepair mutagenized Asibi fusion PCR product was digested
with Eco RV-Sac II, a 2,820-basepair Asibi Sac II-Xba I di-
TABLE 1
Nucleotide and amino acid differences between yellow fever virus
strains Asibi and 17D in NS2A, NS4B, and 3⬘NCR*
Gene
Nucleotide
Amino
acid
Asibi
17D
NS2A
3860
4007
4013
4022
4025
4056
7171
7580
10367
10418
10550
10800
10847
61
110
112
115
116
126
95
232
–
–
–
–
–
Met
Thr
Leu
Thr
Val
Ser
IIe
Tyr
U
U
U
G
A
Val
Ala
Phe
Ala
Met
Phe
Met
His
C
C
C
A
C
NS4B
3⬘NCR
* The sequence of the Asibi strain used in this study has been deposited in Genbank
(AY640589). NS ⳱ non-structural; NCR ⳱ non-coding region.
1159
gestion product, and Asibi ic vector digested with Eco RVXba I. 17D/Asibi NS4B-95 ic was similarly produced using
17D as the template for mutagenesis and ligation of Ngo
MIV-Sac II, Sac II-Xba I, and Ngo MIV-Xba I fragments.
Asibi/17D 3⬘NCR ic was constructed by the insertion of a
523-basepair 17D fragment that was produced by fusion PCR,
digested with Cla I, and phosphorylated with T4 polynucleotide kinase, into Nru I-Cla I digested Asibi ic. 17D/Asibi
3⬘NCR ic was similarly produced by insertion of the corresponding Asibi fragment into Xho I-digested 17D ic. Maps of
all viruses used in this study are available from the authors
upon request.
Virus production and specific infectivity. Infectious virus
was produced from each YFV cDNA ic as described previously.3,17 Briefly, each ic was linearized with Nru I or Xho I
restriction endonuclease (New England Biolabs), the linear
DNA was purified by phenol-chloroform extraction and ethanol precipitation, and RNA was produced using the SP6
mMessage mMachine capped RNA transcription kit (Ambion, Austin, TX). Purified RNA was then electroporated
into baby hamster kidney (BHK) cells in L-15 medium containing 10% fetal bovine serum, 100 U/mL of penicillin and
100 ␮g/mL of streptomycin using the Gene Pulser Xcell electroporation system (Bio-Rad Laboratories, Hercules, CA).
Electroporated cells were transferred to a T25 cm2 flask and
incubated until cells showed 75% cytopathic effect, at which
time virus was harvested and used for oral infection of Ae.
aegypti or aliquoted and stored at -80C for later use. All
manipulations of live virus were performed under Biosafety
Level 3 conditions. The specific infectivity of viral RNA was
measured on Vero cells post-electroporation as described in
detail previously.3,17 Cells were stained for YFV antigen by
immunohistochemical analysis, and 10 foci per virus were
counted and measured using an Olympus (Center Valley,
PA) IX-71 inverted light microscope. Average focus sizes
were compared by Student’s t-test using SPSS version 11.5
(SPSS, Inc, Chicago, IL).
Oral infection of Ae. aegypti mosquitoes. Aedes aegypti,
RexD colony, were infected as described previously.3,17,25,26
Virus harvested from BHK cells 2–5 days post-electroporation was mixed with an equal volume of defibrinated
sheep blood and supplemented with 3 mM ATP as a phagostimulant. The average virus titers in the artificial blood
meals, expressed as 50% tissue culture infectious dose
(TCID50), are as follows: Asibi ⳱ 6.5 log10TCID50/mL, Asibi/
17D NS2A ⳱ 5.3 log10TCID50/mL, Asibi/17D NS4B ⳱ 6.0
log10TCID50/mL, Asibi/17D NS4B-95 ⳱ 5.7 log10TCID50/
mL, Asibi/17D 3⬘NCR ⳱ 5.8 log10TCID50/mL, 17D/Asibi
3⬘NCR ⳱ 7.0 log10TCID50/mL, 17D/Asibi NS4B-95 ⳱ 6.8
log10TCID50/mL, and 17D ⳱ 7.2 log10TCID50/mL. Artificial
blood meals were given to female Ae. aegypti using a Hemotek membrane feeding system (Discovery Workshops, Accrington, United Kingdom) and hog intestine membrane. After one hour, mosquitoes were chilled, sorted, and engorged
(fed) females were returned to a cage for maintenance. Three
mosquitoes per feed were sampled immediately after feeding
to determine the titer of virus imbibed. Mosquitoes were incubated up to 14 days post-infection (dpi) at 28°C and a humidity of 80% and provided a solution of 10% sucrose ad
libitum.
Evaluation of infection and dissemination rates in Ae. aegypti. Virus production, infection rates, and dissemination
1160
McELROY AND OTHERS
rates of the YFV chimeras in Ae. aegypti were determined as
described previously.3,17 Virus production was monitored by
removal of mosquitoes from their cages at 1, 2, 3, 7, and 14 dpi
and titration on Vero cells as described previously.3,17,25,26 At
14 dpi, remaining mosquitoes were chilled, and salivary
glands were dissected into phosphate-buffered saline on glass
slides and fixed in cold acetone. Dissemination was evaluated
by staining the salivary glands for YFV antigen by indirect
immunofluorescence assay (IFA) using a YFV-reactive polyclonal antibody MA93. Infection rates were obtained by titration of carcasses on Vero cells in 96-well plates, and the
plates were stained by indirect IFA. Infection is reported as
the number of mosquitoes infected divided by the number of
mosquitoes tested, and dissemination is reported as the number mosquitoes with YFV antigen-positive salivary glands divided by the number of infected mosquitoes. Infection and
dissemination rates for the different viruses were compared
statistically by Fisher’s exact test using SPSS version 11.5
(SPSS Inc., Chicago, IL).
RESULTS
Virus production in vitro. Infectious virus was produced
from each of six chimeric YFV ic: Asibi/17D NS2A, Asibi/
17D NS4B, Asibi/17D NS4B-95, Asibi/17D 3⬘NCR, 17D/
Asibi 3⬘NCR, and 17D/Asibi NS4B-95 (Figure 1 and Table 2).
Specific infectivity values calculated for RNA produced from
each of the YFV ic are shown in Table 2 with values previously reported for Asibi and 17D parental viruses. These values ranged from 2 × 103–3.7 × 104 focus-forming units (FFU)/
␮g of RNA, and there does not appear to be any correlation
between this value and genomic content, linearization site
(Nru I or Xho I), or focus size. Focus sizes for each YFV
chimera were significantly larger than those produced by
Asibi and significantly smaller than those produced by 17D
(P < 0.01). As expected and reported previously, Asibi
formed significantly smaller foci than 17D (P < 0.01).3 This is
most likely because of adaptive mutations accrued by the in
vitro–passaged Asibi virus during 17D virus selection, which
promoted efficient cell-to-cell spread in cell culture. Among
TABLE 2
Specific infectivity values and average focus sizes of yellow fever virus
chimeras used in this study*
Virus
Asibi*
As/17D
As/17D
As/17D
As/17D
As/17D
17D/As
17D/As
17D/As
17D†
M-E†
NS2A
NS4B
NS4B-95
3⬘NCR
3⬘NCR
NS4B-95
M-E†
Specific infectivity
(FFU/␮g)
Average ± SD
focus size (mm)
1.6 × 104
4.7 × 103
4.5 × 103
1.3 × 104
3.2 × 104
2.0 × 103
3.7 × 104
6.7 × 103
1.3 × 104
3.3 × 104
0.191 ± 0.01
0.294 ± 0.02
0.291 ± 0.02
0.267 ± 0.02
0.268 ± 0.02
0.294 ± 0.02
0.257 ± 0.01
0.296 ± 0.02
0.274 ± 0.01
0.380 ± 0.02
* FFU ⳱ focus-forming units. For definitions of abbreviations, see Table 1.
† Adapted from McElroy and others.3
the YFV chimeras, Asibi/17D NS2A and Asibi/17D 3⬘NCR
produced significantly larger foci than Asibi/17D NS4B and
Asibi/17D NS4B-95 (P < 0.01). As expected, Asibi/17D NS4B
and Asibi/17D NS4B-95 formed foci of equivalent size (P ⳱
0.9), which were significantly smaller than those formed by
17D/Asibi NS4B-95 (P < 0.01). Finally, 17D/Asibi NS4B-95
formed significantly larger foci than 17D/Asibi 3⬘NCR (P <
0.01). However, concordant with our findings for the structural protein chimeras, no one sequence element seems to
control focus size of these viruses. There are 67 nucleotide
and 33 amino acid differences between the Asibi and 17D
strains used in this study,3 and it seems likely that a combination of sequence elements throughout the viral genome
control cell-to-cell spread in vitro. This has also been observed in studies of other flavivirus mutants that contained
nucleotide and amino acid substitutions throughout the viral
genome and produced a different plaque phenotype from
their wild-type parent viruses.22,27
Infection of Ae. aegypti. After oral infection of Ae. aegypti
by each of the YFV chimeras, mosquitoes were evaluated for
infection rate by titration of dissected carcasses on Vero cells.
Infection rates at 14 dpi are reported as number of mosquitoes infected/number tested. As described previously, Asibi
FIGURE 1. Schematic representation of yellow fever virus chimeras evaluated in this study. Orange denotes shared sequences, red denotes
Asibi, and yellow denotes 17D, respectively. NCR ⳱ non-coding region; C ⳱ capsid; prM ⳱ premembrane; M ⳱ membrane; E ⳱ envelope;
NS ⳱ non-structural. This figure appears in color at www.ajtmh.org.
YFV NS2A, NS4B, AND 3⬘NCR IN VIRAL DISSEMINATION
infected a higher proportion of Ae. aegypti than 17D, 71%
versus 30% respectively (Table 3). Each of the YFV chimeras
was orally infectious for Ae. aegypti, but the infection rate for
each virus tested was significantly lower than for Asibi (P <
0.02). With only one exception, 17D/Asibi 3⬘NCR (see below), all YFV chimeras had equivalent infection rates to 17D
(P ⳱ 0.38–0.84).
Virus production in Ae. aegypti. Virus production after
oral infection was evaluated by titration of mosquitoes
sampled at 1, 2, 3, 7, and 14 dpi (Figure 2). All viruses displayed a characteristic eclipse phase in the early stage (days
1–3) of infection in which virus titer decreases and the input
virus replicated in the midgut epithelium. The eclipse phase
was monitored to ensure that virus titers measured at days 7
and 14 post-infection were the result of new virus production
rather than persistence of virus present in the infectious blood
meal. Each virus replicated in Ae. aegypti mosquitoes as
shown by titers obtained at 7 and 14 dpi. With the exception
of 17D/Asibi 3⬘NCR (Figure 2C), the eclipse phase was followed by an increase in average titer from 3 to 7 dpi for all
viruses. The average virus titers then remained constant in the
case of Asibi, Asibi/17D NS4B-95, and 17D (Figure 2A); an
increase in the case of Asibi/17D NS2A, Asibi/17D NS4B,
and Asibi/17D 3⬘NCR (Figure 2B); or a decrease in the case
of 17D/Asibi NS4B-95 and 17D/Asibi 3⬘NCR (Figure 2C) to
14 dpi. However, the high variation in whole body titer for
each of the viruses at 7 and/or 14 dpi, the average SD was 0.74
and 0.95 log10TCID50/mosquito, respectively, limited statistical comparisons of average virus titers. Thus, no statistical
difference (P > 0.05) was noted in average whole mosquito
titers between Asibi and any of the YFV chimeras at 7 and 14
dpi.
Dissemination in infected Ae. aegypti. Dissemination rates
determined by YFV antigen detection in dissected salivary
glands at 14 dpi are reported for each virus as the number of
YFV antigen-positive salivary glands/number of infected carcasses (Table 3). The YFV wild-type parental Asibi strain
disseminated at a rate of 82% whereas no dissemination was
detected for 17D (P < 0.01).3 The YFV chimeras segregate
into three groups based on dissemination rate: high dissemination, medium dissemination, and low dissemination. Chimeras with the Asibi virus backbone formed two groups: high
and medium dissemination. The high dissemination group
contained only Asibi/17D 3⬘NCR. This virus disseminated at
TABLE 3
Infection and dissemination rates in Aedes aegypti orally infected
with the yellow fever virus (YFV) chimeras*
Virus
Asibi
As/17D
As/17D
As/17D
As/17D
As/17D
17D/As
17D/As
17D/As
17D
M-E
NS2A
NS4B
NS4B-95
3⬘NCR
3⬘NCR
NS4B-95
M-E
n
No. infected/total (%)
No. disseminated/
no. infected (%)
92
60
45
60
78
66
67
50
76
51
65/92 (71)
16/60 (27)
15/45 (33)
20/60 (33)
19/78 (24)
15/66 (23)
34/67 (51)
11/50 (22)
16/76 (21)
17/56 (30)
53/65 (82)
5/16 (31)
6/15 (40)
7/20 (35)
7/19 (37)
11/15 (73)
2/34 (6)
1/11 (9)
11/16 (69)
0/17 (0)
* Data for YFV strains Asibi, Asibi/17D M-E, 17D/Asibi M-E, 17D (adapted from McElroy and others)3 are provided for comparison. Infection rate is number infected/number
tested, and dissemination rate is number with YFV-antigen positive salivary glands/number
infected. For definitions of abbreviations, see Table 1.
1161
FIGURE 2. Virus production in orally infected Aedes aegypti. Mosquitoes were sampled 0, 1, 2, 3, 7, and 14 days post-infection and
titrated on Vero cells. A, ⽧ ⳱ Asibi; □ ⳱ Asibi/17D NS4B-95; ◊ ⳱
17D. B, 䊉 ⳱ Asibi/17D NS2A; 䊏 ⳱ Asibi/17D NS4B; 䉱 ⳱ Asibi/
17D 3⬘NCR. C, □ ⳱ 17D/Asibi NS4B-95; 䉭 ⳱ 17D/Asibi 3⬘NCR.
Average titer ± SD of eight whole mosquitoes was determined for
each virus at each time point. The dashed lines indicate the limit of
detection of our titration assay. NS ⳱ non-structural; NCR ⳱ noncoding region; TCID50 ⳱ 50% tissue culture infectious dose.
a significantly higher rate than 17D (73% versus 0%; P <
0.01) but this rate was not statistically different from that of
Asibi virus (82%; P ⳱ 0.49).
The second group (medium dissemination) comprised
three viruses, Asibi/17D NS2A, Asibi/17D NS4B, Asibi/17D
NS4B-95, that also disseminated at a significantly higher rate
than 17D (40%, 35%, and 37%, respectively; P < 0.01). Substitution of the 17D NS2A for the NS2A of Asibi significantly
decreased dissemination compared with Asibi virus (40% versus 82%; P < 0.01). Substitution of the 17D NS4B for that of
Asibi (Asibi/17D NS4B) also significantly lowered dissemination compared with Asibi (35% versus 82%; P < 0.01). This
virus contained two amino acid differences from Asibi:
Ile95Met and Tyr232His. The dissemination rate for virus
with a single substitution at position 95 (Asibi/17D NS4B-95),
1162
McELROY AND OTHERS
was not significantly different from that of Asibi/17D NS4B
(37% versus 35%; P ⳱ 1.00). Therefore, within NS4B, the
Ile95Met substitution appears to be sufficient for the attenuation of viral dissemination.
Both viruses with the 17D backbone, 17D/Asibi NS4B-95
and 17D/Asibi 3⬘NCR, formed the third group (low dissemination). The dissemination rates for these viruses (6% and
9%, respectively) were not significantly different from that of
17D (0%; P ⳱ 0.39 and 0.55, respectively) but were significantly lower than for Asibi (82%; P < 0.01). Thus, whereas
substitution of 17D sequences into the Asibi virus backbone
attenuates virus for dissemination, addition of Asibi virus sequences to 17D does not necessarily restore wild-type phenotype.
DISCUSSION
Despite more than 100 years of study on mosquito-virus
interactions, the mechanism by which virus disseminates from
the mosquito midgut is not well understood. Romoser and
others hypothesized that virus escapes the midgut epithelium
through modified basal lamina or productive infection of
muscle or tracheal cells.28 Although midgut escape and infection of secondary tissues may be receptor-mediated, we hypothesize that dissemination dynamics are influenced by the
efficiency of viral replication, virion packaging, and virion
release–events. Since the non-structural proteins and 3⬘NCR
play critical roles in these components of the viral life cycle, it
follows that dissemination may also be influenced by genes
encoding the non-structural proteins and the sequence of the
3⬘NCR. In our previous study when the structural protein
genes of the midgut-restricted 17D were substituted into the
wild-type Asibi backbone, the chimeric virus disseminated in
31% of infected mosquitoes, whilst the complimentary chimera disseminated in 69% of mosquitoes.3 These data suggested that dissemination was not solely determined by the
structural genes. Here we report on experiments designed to
investigate the role of mutations located in regions other than
the structural protein genes, specifically focusing on NS2A,
NS4B, and the 3⬘NCR. Whilst the precise effects of sequence
differences in NS2A, NS4B, and the 3⬘NCR on cell-to-cell
spread in vitro and mosquito infectivity are not conclusive
from these studies, the amino acid substitutions in NS2A and
NS4B clearly had an attenuating effect on viral dissemination
from the mosquito midgut.
There are several possible explanations to account for the
decreased ability of each YFV chimera to infect Ae. aegypti as
compared with Asibi. Since they are all significantly attenuated for infection of Ae. aegypti, the reduced infectivity does
not appear to be entirely determined by genomic sequence.
Because successful mosquito infection may be titer dependent, it is possible that the range in titers of the artificial blood
meals to which mosquitoes were exposed resulted in the differences in infection rate. The chimeric viral titers in the
blood meals used in our studies ranged from 5.3 to 7.0
log10TCID50/mL, and the infection rates ranged from 22% to
51%. However, higher titers in our study did not consistently
produce higher infection rates. We therefore believe that an
attenuating effect of chimerization between two different viral strains may contribute to the observed lower infection
rates recorded for each of the YFV chimeras, as reported
previously for YFV3 and for DENV chimeras.13,29 An attenuating effect of chimerization might indicate that successful
interactions between proteins encoded by genome sequences,
which have co-evolved within the same virus act synergistically to facilitate efficient virus production, i.e. replication,
translation, packaging, and release. Substitution of heterologous sequences that have been selected for independently (in
this case the addition of 17D sequences into Asibi) decreases
the efficiency of these interactions resulting in less efficient
virus production.
With respect to virus production in infected mosquitoes,
the high variation in titers of individual mosquitoes infected
with the same virus was expected as previously observed with
this and other YFVs3 and with the alphaviruses o’nyongnyong virus (ONNV), chikungunya virus (CHIKV), and
ONNV-CHIKV chimeras.26 This variation most likely reflects the ability of virus not only to infect and produce virus
within one tissue, for example the midgut, but also the ability
of virus to disseminate to other tissues to facilitate virus production.
One interesting overall conclusion of our studies is that
17D sequences attenuate Asibi virus for dissemination in infected Ae. aegypti, but the addition of a limited portion of
Asibi sequence to 17D does not restore wild-type phenotype
to the chimeric virus. This has been observed with YFV chimeras in experimentally infected mosquitoes3 and mice (Engel AR and Barrett ADT, unpublished data). We believe this
is strong evidence for the existence of attenuating mutations
throughout the YFV genome, which must work in concert,
rather than one gene or amino acid alone controlling viral
phenotype. Our observations on the genetic determinants of
viral dissemination outside the M and E structural protein
genes are therefore largely based on data for Asibi, 17D, and
the YFV chimeras Asibi/17D M-E, Asibi/17D NS2A, Asibi/
17D NS4B, Asibi/17D NS4B-95, and Asibi/17D 3⬘NCR.
The role of NS2A in the virulence, attenuation, or vector
competence of YFV has not previously been characterized.
Therefore, it is difficult to speculate how amino acid differences between the NS2A proteins of Asibi and 17D might
alter viral phenotype. There are six amino acid substitutions
between the Asibi and 17D NS2A sequences (Table 1): two of
the changes, at positions 110 and 115 are substitutions of polar
Thr for non-polar Ala residues, and another two, Leu112Phe
and Ser126Phe, result in the substitution of non-polar or polar
for aromatic amino acids. It is possible that the phenotypic
effect we observe in mosquitoes may be due to the structural
differences resulting from these substitutions. Bennett and
others identified three non-polar to polar amino acid residue
substitutions in the NS2A of DENV-4 isolates collected during a 1998 outbreak,21 the implication being that these
changes may have enhanced the infectivity of the DEN-4 isolates for mosquitoes, increasing the efficiency of virus transmission and spread. It is possible that the two polar to nonpolar amino acid substitutions and/or the polar to aromatic
amino acid substitutions from Asibi had the opposite effect
on 17D, i.e., reduced infectivity for mosquitoes.
Since the exact function of NS4B is unknown, we cannot be
certain of the precise mechanism underlying attenuation that
results from manipulation of this protein. However, we hypothesize that mutations within NS4B must be involved in
attenuation of YFV phenotype in mosquitoes because an
identical substitution, Ile95Met, is the only common mutation
YFV NS2A, NS4B, AND 3⬘NCR IN VIRAL DISSEMINATION
shared by three attenuated YFV strains, 17D, FNV, and Asibi
HeLa p6,22,23 and none of these viruses are able to disseminate in infected mosquitoes. Further evidence to suggest a
role of NS4B in mosquito infection with flaviviruses includes
data that a Pro101Leu substitution in the NS4B of DENV-4
abolished infectivity of the virus for orally infected Ae. aegypti, and no disseminated infections were observed in intrathoracically inoculated Toxorhynchites splendens mosquitoes.30 Muñoz-Jordán and others implicated the DENV
NS4B residues 77–125 in the inhibition of interferon-␣/␤ signaling in vitro and found that this antagonism is conserved for
both YFV and WNV,31,32 and this may explain the attenuation of vertebrate virulence as it relates to NS4B.
Based on our data, we conclude that the 3⬘NCR plays no
role in viral dissemination from the midgut in our system.
There are five nucleotide differences between the Asibi and
17D 3⬘NCR, but the resulting predicted difference in secondary structure is relatively minor (Rijnbrand RCA, McElroy
KL, and Higgs S, unpublished data) and apparently insufficient to affect dissemination of virus from the mosquito midgut.24 Our findings are in contrast to other studies published
on the attenuating effects of manipulating the DENV 3⬘NCR.
Those viruses were attenuated in both mosquitoes and vertebrates. However, the viruses used in these studies did contain
a relatively large deletion of 30 nucleotides rather than a
precise alteration of nucleotide sequence as used in our study,
which may account for the difference in outcomes.12,13,32 This
large deletion would alter the secondary structure of this region and therefore attenuate the ability of these viruses to
replicate compared with their wild-type parent viruses.
Our model system used the existence of sequence differences between a wild-type, disseminating YFV strain (Asibi)
and the attenuated, midgut-restricted YFV strain derived
from it (17D). This system was useful in elucidating genetic
determinants of YFV dissemination from the Ae. aegypti midgut. These determinants localized to domain III of the envelope protein gene,3 NS2A, and NS4B-95. The viruses characterized or referenced in this study that contain mutations in
NS2A, NS4B, or the 3⬘NCR have altered phenotypes in vitro
in vertebrates and/or mosquitoes. Therefore, it is difficult to
distinguish if individual mutations only affect vector competence, only affect virus virulence, or if some or all of the
changes affect both properties by separate or common
mechanisms. Although we cannot extrapolate our data to determine if the viral genetic determinants of YFV dissemination of YFV in Ae. aegypti midgut have analogues in other
mosquito-borne flaviviruses, our data provide a basis on
which these criteria may be established. This information will
be useful for the rational design of additional live attenuated
virus vaccine candidates that can neither infect nor disseminate in mosquito vectors.
Received June 28, 2006. Accepted for publication July 28, 2006.
Acknowledgments: We thank Jing H. Huang for expert technical assistance; the Protein Chemistry Laboratory of the University of Texas
Medical Branch for sequencing; and Alan Barrett, Amber Engel, and
Peter Mason for helpful discussions during the execution of this work.
Financial support: This work was in part supported by a Defense
Advanced Research Projects Agency grant cooperative agreement
N00178-02-2-9002 with the Chemical Biological Radiological Sciences and Technology Defense Branch of the Naval Surface Warfare
Center and by Acambis, Inc. (Cambridge, MA). K. L. McElroy was
1163
supported by the Centers for Disease Control and Prevention Fellowship Training Program in Vector-Borne Infectious Diseases (T01/
CCT622892).
Authors’ address: Kate L. McElroy, Konstantin A. Tsetsarkin, Dana
L. Vanlandingham, and Stephen Higgs, Department of Pathology,
University of Texas Medical Branch, 2.104 Keiller, L20762, 301 University Boulevard, Galveston, TX 77555-0609, Telephone: 409-7472426, Fax: 409-747-2437, E-mail: [email protected]. Current address
for Kate L. McElroy, Centers for Disease Control and Prevention
Dengue Branch, 1324 Calle Cãnada, San Juan, PR 00920.
Reprint requests: Stephen Higgs, Department of Pathology, Keiller
2.104, 301 University Boulevard, Galveston, TX 77555-0609.
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