CHAPTER 3
Experimental Evaluation of Mexican Rodents (Baiomys, Liomys,
Oligoryzomys, Oryzomys and Sigmodon) as Potential Amplifying Hosts
for Venezuelan Equine Encephalitis Virus.
57
ABSTRACT
In 1993, an outbreak of encephalitis in coastal Chiapas, Mexico involving 125
equids with a 50% case fatality rate, was attributed to Venezuelan equine encephalitis
virus (VEEV) subtype IE, which had not previously been associated with equine disease
and mortality. To better understand the ecology of VEEV in coastal Chiapas, I
experimentally infected wild rodents from 5 different species to evaluate their
competence as reservoir and amplifying hosts in the natural transmission cycle. Animals
from four of these species, Liomys salvini, Oligoryzomys fulvescens, Oryzomys couesi
and Sigmodon hispidus, became viremic but survived and developed neutralizing
antibodies, indicating that they may be important in the maintenance of subtype IE VEEV
in the epizootic region. Animals from the fifth species, Baiomys musculus, showed signs
of disease and died by day 8 post inoculation, implying that they may not be a natural
reservoir host of subtype IE VEEV. This is the first time that Liomys salvini,
Oligoryzomys fulvescens and Baiomys musculus have been assessed experimentally for
alphavirus replication in a laboratory setting.
INTRODUCTION
Venezuelan equine encephalitis (VEE) is a potentially fatal, re-emerging disease
in tropical America that can cause major outbreaks involving hundreds-of-thousands of
humans and equines. Most VEEV strains, both epizootic and enzootic, have been
associated with human disease in the past (Oberste, Fraire et al. 1998). During 1993 and
58
1996 2 epizootics involving equids occurred in coastal Chiapas and Oaxaca states in
Mexico. The causative agent was determined to be subtype IE VEEV, which was
previously considered to be equine-avirulent (Oberste, Fraire et al. 1998). The existence
of equine-virulent subtype IE VEEV strains during the 1993 and 1996 epizootics posed a
future threat to both equine and human health in Mexico and in the United States. For
example, in 1971, the subtype IAB VEEV epizootic/epidemic subtype spread through
Mexico and into Texas and caused significant economic losses with a major impact on
the health of both humans and equids (Sudia, Fernandez et al. 1975). The ecological and
biological factors that govern the transmission and maintenance of subtype IE VEEV as
well as other VEEV subtypes in both their enzootic foci and epizootic cycle are not
understood. Furthermore, little is known about the factors that contributed to the
emergence of a virulent strain of subtype IE VEEV. Therefore, further field and
laboratory studies are needed to generate knowledge to develop more effective control
and prevention disease strategies for this virus.
Enzootic strains of VEEV are maintained naturally by transmission between
mosquitoes of the Culex (Melanoconion) subgenus and wild rodents (Weaver, Ferro et al.
2004). These viruses are thought to circulate continuously among mosquitoes and their
principal vertebrate amplifying hosts– horses and humans are considered spillover, deadend hosts that are not required for maintenance of the natural transmission cycle. Several
past studies have shown that terrestrial mammals from 5 different genera (Didelphis,
Oryzomys, Proechimys, Sigmodon, and Zygodontomys) are susceptible to subtype IE
VEEV infection and produce a viremia in sufficient magnitude to infect mosquito
59
vectors, yet survive infection (Young and Johnson 1969; Young, Johnson et al. 1969;
Scherer, Dickerman et al. 1971; Coffey, Carrara et al. 2004; Carrara, Coffey et al. 2007).
The ability of amplification hosts to survive infection is considered a crucial trait for
long-term virus circulation in enzootic foci.
Several field studies in Mexico have reported VEEV-specific antibodies in a
variety of wild vertebrate species. Aguirre et al. found wild mammals from 7 species and
wild birds from 17 different species in the state of Veracruz seropositive for subtype IE
VEEV in 1992(Aguirre, McLean et al. 1992). In the same area from which the animals
for my study were captured, VEEV-neutralizing antibodies were detected in wild S.
hispidus, Oryzomys alfaroi and Didelphis marsupialis (Estrada-Franco, Navarro-Lopez et
al. 2004). In an extensive field study in southern Mexico during the 1960’s, Scherer et al.,
found wild birds from 29 species, terrestrial mammals from 10 genera (including
Sigmodon and Oryzomys), and bats from 3 genera had serologic evidence of natural
VEEV infection(Scherer, Dickerman et al. 1971). Evidence of similar broad host ranges
of VEEV have been found in coastal Guatemala, where terrestrial mammals from 7
genera (including Sigmodon, Oryzomys and Liomys) and birds from 11 species had
VEEV-specific antibodies (Scherer, Anderson et al. 1976). Following the 1971 Central
American epidemic of subtype IAB VEEV that reached south Texas, extensive field
studies were conducted to determine whether the virus would or could establish a new
enzootic focus (Sudia, Fernandez et al. 1975). In that study, mammals from 10 genera
(including Sigmodon and Liomys) had VEEV-specific antibodies. These seropositivity
60
data implicate these animals as potential amplification hosts, and indicate that at least
some individuals of these species survive VEEV infection.
Mammals are thought to be of primary importance in the natural cycle of VEEV.
However, there is also evidence that wild birds may play a role (Sidwell, Gebhardt et al.
1967). Many species of birds have been found to have VEEV-specific antibodies, and
virus isolates have been made from wild-captured birds and domestic fowl (Galindo,
Srihongse et al. 1966; Sidwell, Gebhardt et al. 1967). Laboratory experiments confirm the
ability of birds from several species to sustain asymptomatic infections that generate
viremias sufficient to infect naïve ornithophilic mosquitoes (Chamberlain, Kissling et al.
1956; Dickerman, Bonacorsa et al. 1976). However, the overall seroprevalence in wild
birds is much lower than that of mammals for most VEEV complex viruses, suggesting
that birds are of lesser importance for maintenance and amplification.
Wild rodents from several species, Sigmodon hispidus, Oryzomys couesi and O.
alfaroi captured in the coastal state of Chiapas, Mexico had VEEV-specific antibody
(Estrada-Franco, Navarro-Lopez et al. 2004). In order to evaluate the potential role of
selected rodents as a reservoir and/or amplifying host for subtype IE VEEV, rodents from
5 genera (Baiomys, Liomys, Oligoryzomys, Oryzomys and Sigmodon) were captured and
transported to the laboratory for experimental infection studies. Previous studies have
been unable to discern with certainty which rodent species in this area may play a role as
amplification hosts. Serosurvey data are minimal and were obtained from a small number
of locations and thus may not represent the region in its entirety. The rodents chosen for
my experimental work, Baoimys musculus, Liomys salvini, Oligoryzomys fulvescens,
61
Oryzomys couesi and Sigmodon hispidus have been found to be abundant and widespread
thus their ability to amplify the virus could contribute to long-term virus circulation.
METHODS
Animals
Wild rodents representing five different species were collected from coastal
Chiapas, Mexico were used in this study: southern pygmy mouse (Baiomys musculus),
Salvin's spiny pocket mouse (Liomys salvini), fulvus pygmy rice rat (Oligoryzomys
fulvescens), Coues’ rice rat (Oryzomys couesi) and hispid cotton rat (Sigmodon hispidus).
All animals were captured during October 2007 from an overgrown field surrounding a
stream in Mapastepec municipality, about 2 kilometers from the Pacific coast
(coordinates N15.413° and W093.070°). Animals were captured between one hour before
dusk and one hour after dawn using live-capture Sherman traps (H.B. Sherman Traps,
Tallahassee, FL) and a bait mixture of whole peanuts, shelled peanuts, whole sunflower
seeds, oats, mushrooms and carrots. Species identification was based initially on
morphology and later confirmed genetically using cytochrome-b gene sequences (Reid
1997; Boakye 1999). All five of the Sigmodon animals best matched the published
cytochrome-b sequence for the S. hirsutus or S. hispidus hirsutus subspecies (Carroll and
Bradley 2005). Animals were housed individually and transported in Taconic Transit
Cages® (Taconic Farms, Inc. Hudson, NY,) to the animal biosafety level 3 facility
(ABSL3) at the University of Texas Medical Branch in Galveston, Texas. Animals were
allowed one week of acclimation before experiments were begun. A baseline serum
62
sample was taken prior to inoculation of animals with VEEV for subsequent antibody
assays. Animals were captured under permit number SGPA/DGVS/03858/07 Julio 2 de
2007, issued to Dr. J.G. Estrada-Franco by the Secretaria de Salud de Mexico and all
studies were approved by the University of Texas Medical Branch Institutional Animal
Care and Use Committee.
Virus
The VEEV strain MX01-22 (subtype IE) used in these experiments was isolated
in 2001 from a sentinel hamster in coastal Chiapas, Mexico. This strain was chosen
because it is the most recent low-passage isolate of VEEV from the outbreak area and it
had been used in previous studies to evaluate the vector competence of various mosquito
species from my study area in Chiapas, Mexico (Turell, O'Guinn et al. 2003; Brault,
Powers et al. 2004). Additionally, this strain is genetically similar to the equine-virulent
strains that were isolated during the 1993 outbreak and causes encephalitis in horses (R.
Bowen, Colorado State University, personal communication)(Estrada-Franco, NavarroLopez et al. 2004). The virus was passed once in C6/36 mosquito cells to generate a
sufficient volume of high-titered virus for use in animal inoculation.
Inoculation and Husbandry of Rodents
All animals were inoculated subcutaneously in the right thigh with 3.2 log10
plaque-forming units (pfu) of virus, a dose that approximates the maximum amount of
VEEV transmitted by a mosquito bite (Smith, Carrara et al. 2005). All animals were
weighed daily for one week and observed for signs of illness for 2 weeks after
inoculation. Cohort size was based on availability and ranged from 4 animals to 15
63
animals (the cohort of 15 animals was initially 3 cohorts of 5 animals, but all three
cohorts were subsequently determined to belong to the same species).
Animals were examined daily after inoculation and any found to be moribund, as
well as those that died during anesthesia or blood collection were necropsied and tissues
(brain, heart, lung, spleen, liver, kidney) were frozen at -80ºC. Blood was collected daily
for the first 7 days after inoculation, then on days 10, 14, 28, 42 and 66. Animals were
first anesthetized with inhaled isoflurane, then blood was collected from the retro-orbital
sinus in heparinized glass capillary tubes and transferred to 5 volumes of phosphate
buffered saline (PBS). Red blood cells were removed by centrifugation to yield an
approximately 1:10 dilution of plasma, which was stored at -80ºC.
Virus Assays
Viremia titers from plasma were determined by plaque assay on Vero cells
(Beaty, Calisher et al. 1989). Fresh plasma samples were serially diluted in PBS and
inoculated onto confluent monolayers. After adsorption for 60 min at 37ºC an agarose
overlay was added and inoculated monolayers were incubated for 2 days at 37ºC until
formalin fixation and viral plaque visualization.
To detect virus in the tissues approximately 2-10 mg of each tissue was
homogenized in Eagle’s minimal essential medium (MEM) supplemented with 20% fetal
bovine serum (FBS), L-glutamine, penicillin, streptomycin, gentimycin and fungizone
using a TissueLyser® (Qiagen Inc, Valencia, CA). Tissue virus titers were determined by
plaque assay on Vero cells as described above.
Antibody Assays
64
Plasma samples were serially diluted at a ratio of 1:2 in PBS then tested for
subtype IE VEEV-specific antibodies by the hemagglutination inhibition (HI) assay
(Clarke and Casals 1958; Beaty, Calisher et al. 1989). The assays were performed using
antigen derived from the same VEEV strain (MX01-22, isolated in 2001 in Mexico) used
to inoculate the rodents as well as from 3 other arboviruses: Eastern equine encephalitis
virus (TenBroeck strain, isolated in 1933 in the USA), West Nile virus (strain 385-99,
isolated in 1999 in the USA) and St. Louis encephalitis virus (strain TBH28, isolated in
1962 in the USA). Briefly, 4—8 units of acetone extracted hemagglutinin antigen were
reacted with heat-inactivated test plasma in various concentrations in PBS. Failure to
hemagglutinate added goose erythrocytes was considered a positive result. Antibody
titers were confirmed by plaque reduction neutralization tests (PRNT) (Beaty, Calisher et
al. 1989). Briefly, test plasma were serially diluted in PBS and heat-inactivated at 56°C
for one hour, then mixed with approximately 100 pfu of virus in equal volume and
incubated at 37°C for one hour. The mixture was inoculated onto Vero cells and >80%
reduction in the number of plaques in the virus challenge was considered positive, with
titers reported as the reciprocal of the endpoint dilution.
RESULTS
Clinical Responses and Survival
Only B. musculus showed signs of severe disease. These animals began to exhibit
tremor, lethargy, hunching and staggering between days 4 and 6 post-infection. By day 8,
100% of the B. musculus (n = 4) had died or were euthanized after becoming moribund
65
(Figure 3.1A). These animals were also the only ones that consistently lost body weight
after infection (average 22% loss; Figure 3.1B).
No animal from the other 4 cohorts exhibited weight loss or outward signs of
illness (e.g. hunching, ruffled fur, lethargy, ataxia) after inoculation. Most of these
rodents survived until the end of the experiment at day 66 post-inoculation. However, 9
animals died during the first 2 weeks after infection without weight loss or signs of
illness: 3 of 5 L. salvini, 1 of 15 adult O. couesi, 4 of 4 O. fulvescens and 1 of 5 S.
hispidus. With the exception of the single O. couesi, which was found dead in its cage on
day 2, the rest of these animals died during anesthesia despite careful regulation and
efforts at resuscitation. To determine whether these animals had died from VEEV
infection or from the stress of anesthesia and blood sampling, necropsy was performed
immediately and organs were frozen for virus assay. Of these 9 animals, only one O.
couesi contained detectable virus in the brain; however this animal did not appear ill and
died on day 2, about 3-5 days before VEEV-associated encephalitis signs typically occur
in rodents (Carrara, Coffey et al. 2007). The levels of virus found in its tissues were less
than the levels found in all 4 B. musculus (Table 1), which supports my suspicion that it
died from the stress of daily manipulations rather than from VEEV infection. Similar
non-infectious mortality in laboratory-manipulated wild rodents has been encountered
previously (Young, Johnson et al. 1969).
66
Percent of Cohort Surviving
100
90
80
70
60
50
40
B. musculus (n=4)
L. salvini (n=5)
O. couesi, adult (n=15)
O. couesi, 2 weeks old (n=3)
Oligoryzomys sp. (n=4)
S. hispidus (n=5)
30
20
10
0
0
5
10
15
20
25
20
25
Day post inoculation
Ratio of final to initial weight
2.5
2.0
1.5
1.0
0.5
0.0
0
5
10
15
Day Post Inoculation
Figure 3.1:
Survival and weight change of wild rodents from Chiapas, Mexico after
experimental infection with 3 log10 pfu of Venezuelan equine encephalitis
virus subtype IE, strain MX01-22. A) Survival: Solid lines represent
animals whose brains yielded live virus after necropsy. Dashed lines
indicate animals whose death was attributed to manipulation and/or stress,
but not to VEEV infection. B) Weight Change: Mean cohort weight
divided by mean cohort starting weight (day 0). Weight gain/loss was used
as an indicator of disease. Only Baiomys musculus showed significant
weight loss during acute infection. Data for days 42 and 66 (not shown)
did not differ significantly from day 28 (Deardorff et. al, 2009).
67
To further address the possibility of a non-infectious cause of death, a sub-cohort
of L. salvini (n = 2) and O. fulvescens (n = 3), the two species that suffered the most
suspected manipulation-related mortality, were inoculated and monitored for survival and
signs of illness with limited handling, anesthesia and blood sampling. These animals all
survived to the end of the experiment (42 days) with no signs of illness and no weight
loss. Blood samples were collected at days 0, 15 and 42 in order to confirm infection by
seroconversion and to determine antibody titers. All 5 animals were found to be
seronegative prior to inoculation, had neutralizing antibodies by day 15 (geometric mean
titer = 488 ± 212), and remained seropositive through day 42 (mean = 976 ± 670).
Table 3.1 Viremia in all animals that died 1—10 days after inoculation with 3.2 log10 pfu
of Venezuelan equine encephalitis virus subtype IE strain MX01-22.
Animal1
Day P-I
Tissue virus content (log10 pfu/gram)2
Brain
Heart
Spleen
Kidney
Liver
Lung
Oryzomys
2
1.8
2.7
3.3
2.0
3.2
0
Oligoryzomys
4
0
0
4.0
0
0
0
6
0
0
3.4
0
0
0
6
3.2
5.0
5.0
4.7
4.9
3.9
7
4.6
3.2
4.2
0
4.3
4.3
7
2.0
2.0
3.0
3.4
2.6
4.1
8
5.0
3.0
5.7
5.0
1.9
5.0
Baiomys
1
Animals not shown are L. salvini (n = 3), O. fulvescens (n = 2) and S. hispidus (n = 1). These animals died
between day 5 and 10 post-infection and showed no detectable virus in any of the organs tested.
2
Limit of detection was one plaque in 150µl homogenate. Tissue sample weights varied between 0.002—
0.01 grams.
68
Viremia Titers
Twenty-two of 35 animals tested, comprising all 5 species, had measurable
viremia during the first week after infection (limit of detection was 1.5 log10 pfu/ml)
(Figure 3.2). Previous studies established that the minimum viremia needed to infect
naïve Culex (Mel.) taeniopus mosquitoes, enzootic VEEV vectors in nearby coastal
Guatemala, is between 2.5 and 3.2 log10 pfu/ml (Scherer, Cupp et al. 1982). All of the L.
salvini, O. fulvescens and B. musculus developed viremia ≥2.7 log10 pfu/ml lasting up to
5, 4 or 8 days, respectively. Conversely, only 60% of the S. hispidus cohort (3/5 animals)
developed detectable viremia lasting up to 4 days, and only 39% of the O. couesi cohort
(7/18 animals) developed detectable viremia lasting a maximum of 2 days. In the L.
salvini, O. fulvescens and O. couesi cohorts, maximum viremia occurred on day 1 postinfection, with mean titers of 3.4 ± 0.6, 3.3 ± 0.2, and 2.5 ± 0.6 log10 pfu/ml respectively
(Figure 3.2). In S. hispidus the cohort peak viremia occurred on day 2 post-infection, with
a mean of 2.9 ± 0.9 log10 pfu/ml; in the B. musculus cohort peak viremia occurred on day
3 with a mean of 5.5 ± 0.4 log10 pfu/ml (Figure 3.2).
Antibody Responses
Of the 40 animals used in this study, only one (S. hispidus) was found to have preexisting VEEV antibodies. This animal had an HI antibody titer of 640 at day 0 and 160
at day 6 when it died during anesthesia and blood collection. This animal did not appear
sick nor did it develop detectable viremia or virus content in any of the organs assayed at
necropsy (brain, heart, lung, spleen, liver, kidney). Mean antibody responses of the 39
69
other rodents by species are shown in figure 3.2. For all 4 of the surviving cohorts,
antibodies were detectable by day 5 and lasted through the end of the experiment. At that
time, S. hispidus had the highest antibody titer with a mean of 3.3 ± 0.1 log10 . L. salvini
and O. fulvescens had mean endpoint titers of 2.7 ± 0.15 and 2.0 ± 0.1 log10
,
respectively, and O. couesi titers plateaued at a peak of 1.8 ± 0.4 log10 .
Liomys salvini
n = 4/4
n=
n = 4/4
Oligoryzomys fulvescens
n = 4/4
n = 6/6
n = 7/7
Log 10 Mean Serum Viremia (PFU/mL)
5
4
3
2
1
0
0
1
2
3
4
5
6
7
0
1
Oryzomys couesi
n = 6/15
6
2
3
4
5
Oryzomys couesi
n = 9/15
6
7
14
28
0
1
2
(juvenile
n = 1/3
3
4
5
6
7
14
28
Sigmodon hispidus
n = 3/5
n = 3/3
n = 3//5
5
4
3
2
Log10 Mean Reciprocal HI Antibody Titer
Baiomys musculus
6
1
0
0
1
2
3
4
5
6
7
14
28
0
1
2
3
4
5
6
7
14
28
0
1
2
3
4
5
6
7
14
28
Day Post Infection
Figure 3.2:
Mean viremia profile (solid lines with square markers) and mean
hemagglutination inhibiting antibody profile (blue dashed lines with
circular markers) of 5 species of wild rodents after experimental infection
with 3 log10 pfu of Venezuelan equine encephalitis virus type-IE, strain
MX01-22. Dashed black lines with no markers indicate approximate
mosquito infection viremia threshold for the enzootic vector Culex (Mel.)
taeniopus. Fractions represent proportion of total cohort that had
measurable response. Data for days 42 and 66 (not shown) did not differ
significantly from day 28.
70
Age Dependence
An unanticipated small cohort of juvenile O. couesi became available during this
study to assess the possible age-dependence on the outcome of VEEV infection. These 3
animals were initially identified as O. fulvescens, a smaller but similarly colored rodent.
Their rapid growth rate during the course of infection made it clear that they were not
adult O. fulvescens, but juvenile O. couesi. This was confirmed with cytochrome-b gene
sequencing (Boakye 1999). The age at infection was determined to be approximately 2
weeks, based on growth of 3 litters of O. couesi born in captivity.
There was no apparent difference between these juvenile animals and the adult O.
couesi in survival, viremia or antibody responses (Figures 3.1 and 3.2). One of 3 juvenile
O. couesi and 6 of 15 adults developed detectable viremia (33% and 40% respectively).
Mean maximum viremia was 2.3 log10 pfu/ml for the juveniles and 2.6 ± 0.6 log10 pfu/ml
for adults. No viremia was detected after day one for either juveniles or adults, with the
exception of one adult that had a viremia titer of 2.6 log10 pfu/ml on day 2.
The single juvenile O. couesi that had measurable viremia was the first to develop
VEEV-specific antibodies on day 7 (160); it also had the most robust and prolonged
response, with a titer of 1280 at every point measured between day 28 and the end of the
experiment at day 66. The 2 other juveniles had different antibody responses; one
developed a weak response of 20 on day 14, which increased to 40 by day 28 and was
<20 thereafter. The other animal showed no antibody response at all until day 66, at
which point it measured 160. Four of the adult animals showed similarly weak antibody
responses of short duration and/or delayed onset following a non-detectable viremia.
71
DISCUSSION
Reservoir Status and Potential
Rodents from 5 species were evaluated in this study and only S. hispidus has been
included in previously published experimental VEEV infection studies. Sigmodon
hispidus is considered both a competent reservoir host for and largely resistant to disease
caused by sympatric VEEV complex alphaviruses in Panama and Florida(Young,
Johnson et al. 1969; Coffey, Carrara et al. 2004; Carrara, Coffey et al. 2007). Carrara et
al. (2007) infected 3 geographically distinct populations of S. hispidus with 2 different
enzootic VEEV strains, and found that only the population from a VEE complex
alphavirus-endemic region (Florida) routinely survived infection; cohorts from the 2 nonendemic populations succumbed to disease(Carrara, Coffey et al. 2007). For this reason,
it was important that we used a VEEV strain and rodents matched on geographic locality.
In addition to S. hispidus, Proechimys semispinosus, Zygodontomys microtinus
and Oryzomys capito (closely related to O. couesi) produce viremia sufficient to infect at
least some mosquito vectors and survive after infection with sympatric strains of VEEV
(Young and Johnson 1969; Young, Johnson et al. 1969; Carrara, Gonzales et al. 2005).
My results further support the hypothesis that enzootic VEEV selects for resistance to
disease in its sympatric reservoir host populations (Young, Johnson et al. 1969).
Viremia and Immunologic Response
The goal of my study was to evaluate the role of various rodent species in
maintenance of subtype IE VEEV and to help interpret the patchwork of seroprevalence
data gathered in the field. Animals from all 5 species produced viremias sufficient to
72
infect the proven enzootic mosquito vector Cx. taeniopus. Of these 5 species, the lowest
and shortest lasting viremias were found in O. couesi; of the 7 animals that developed
viremia, 3 measured >2.5 log10 pfu/ml, a dose that infects 100% of Cx. taeniopus
(Scherer, Cupp et al. 1982). However, all 7 viremic O. couesi animals exhibited titers
above 2.0 log10 pfu/ml, which is still high enough to infect some Cx. taeniopus (Scherer,
Cupp et al. 1982). The other 4 cohorts all exhibited viremia well above the minimum
infection threshold for this vector. Therefore, assuming that they are bitten by Cx.
taeniopus, which are known to be catholic feeders, all the animals we studied should be
able to infect this mosquito (Cupp, Scherer et al. 1986).
The uniform susceptibility of B. musculus to VEE disease was an unexpected
result and appeared to contradict the hypothesis that VEEV circulation selects for
resistance to disease in wild rodents. A potential explanation is the relatedness of this
rodent to the others examined. Four species, B. musculus, O. couesi, O. fulvescens and S.
hispidus, belong to the rodent family Cricetidae (two sub families: Neotominae and
Sigmodontinae) (Figure 3.3). Liomys salvini, is the outlier and belongs to the family
Heteromyidae. However, L. salvini showed survival and viremia comparable to three of
the other four closely related species. Thus, the disparities in infection outcome between
B. musculus (subfamily Neotominae) and the other rodents are probably not explained by
their relationships. It could be that B. musculus is a relative newcomer to the geographic
area, evolutionarily speaking, and has not yet had the chance to develop resistance to
VEE disease. However, previous studies have shown geographic populations of the same
73
species have different responses to VEEV infection, suggesting that the selective pressure
to evolve resistance to disease is a recent phenomenon.
FAMILY
Echymyidae
ORDER
Rodentia
FAMILY
Cricetidae
TRIBE
SUBFAMILY
Sigmodontini
Sigmodontinae
TRIBE
Oryzomini
SUBFAMILY
Neotominae
FAMILY
Heteromyidae
Figure 3.3:
GENUS Proechymys
SUBFAMILY
Eumysopinae
GENUS Sigm odon
GENUS Oryzom ys
GENUS Oligoryzom ys
GENUS Zygodontomys
TRIBE
Baiomini
SUBFAMILY
Heteromyinae
GENUS Baiom ys
GENUS Liom ys
Relatedness of seven wild rodent genera that have been experimentally
evaluated for suitability as amplifying hosts in enzootic transmission
cycles of Venezuelan equine encephalitis virus. The five genera included
in this study are presented in bold and the three genera novel to
experimental VEEV infection studies are underlined.
Another hypothesis for B. musculus susceptibility to VEE disease is the lack of
temporal overlap between B. musculus and the enzootic vector, Cx. taeniopus. Baiomys
spp. are diurnally active while Cx. taeniopus is a nocturnal feeder (Galindo, Srihongse et
al. 1966; Cupp, Scherer et al. 1986; Reid 1997). Although they coincide in space, the lack
of a strong temporal association between this rodent and the enzootic VEEV vector may
limit their contact. This may preclude the selection for resistance to VEE that is
manifested in the other 4 groups, which are nocturnal and presumably are regularly
74
exposed to bites from this vector. Experimental infection of other diurnal rodents from
the study area, or similar studies in another VEEV-endemic area, could be used to further
test this hypothesis. Experimentally infected B. musculus animals sustained high viremia
for up to 8 days and were observed to leave their nests and lie fully exposed in their
cages- a condition that would facilitate feeding by mosquitoes. Therefore, although they
do not survive infection, these rodents, if exposed to VEEV infection, could occasionally
play a role in propagating VEEV in nature.
Due to logistical constraints my study ended 66 days post-infection for the
original cohort and 42 days post-infection for the survival sub-cohorts of L. salvini and O.
fulvescens. The antibody responses for all animals that developed measurable viremia
persisted through the end of the experiment. The only exceptions were several O. couesi
animals that did not develop viremia, but did demonstrate brief, low titered (≤40)
antibody responses. Wild rodents have been shown to remain seropositive for up to 6
months following infection with VEEV (Carrara, Coffey et al. 2007). For some rodents
with short life spans in the wild, this is tantamount to life-long immunity offering
protection against re-infection and affording more opportunity for the animal to
reproduce.
Ecological Implications
Although laboratory experimentation has limitations in elucidating natural
processes, the data gathered are sometimes more complete and detailed than field data. In
this study, 5 of the most commonly captured species of rodent in coastal Chiapas, Mexico
were evaluated for their ability to participate in the natural transmission cycle of enzootic
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VEEV IE. Sigmodon hispidus and O. capito (closely related to O. couesi) have previously
been implicated in amplification of other VEEV complex subtypes ID, IE and II but the
other 3 (B. musculus, L. salvini and O. fulvescens) had received little to no study (Young
and Johnson 1969; Carrara, Gonzales et al. 2005; Carrara, Coffey et al. 2007). Animals
from all 5 species developed viremia titers sufficient to infect the enzootic mosquito
vector, Cx. taeniopus. However, only animals from 4 of the 5 species survived infection
with the potential to reproduce, a trait considered critical for true reservoir status in that it
avoids population declines that might jeopardize long-term virus circulation.
History (1971) has shown that an outbreak of highly virulent VEEV in southern
Mexico can rapidly spread into the U.S. Therefore a better understanding of VEEV
ecology in Mexico is essential to assessing the risk for widespread disease. My results
support the conclusions of Scherer et al., (1971) that VEEV has a wide range of
mammalian hosts that may participate in the natural transmission cycle (Scherer,
Dickerman et al. 1971). This may be an adaptive strategy that affords greater population
stability than specialization on one amplifying host that may be subject to more
population instability. The ability to infect rodents from numerous species and produce
adequate viremia for mosquito transmission may aid the long-term persistence of subtype
IE VEEV in nature when weather or environmental conditions affect some but not all
reservoir host populations. This trait could also increase the risk for endemic
establishment as well as amplification when outbreaks spread outside of the endemic
range.
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