Background
Dengue is generally believed to be one of the most hazardous vector-borne diseases,
with over 40% of the world's population at risk of an infection [1]. While in the
past the disease has mainly been observed in the tropical regions, recent studies
suggest that, under the pressure of future climate change, new areas as far north
as Europe may become endangered. In fact, in 2010 the first European cases of autochthonous
dengue since the epidemic outbreak in Greece in the late 1920s [2] were reported from
Croatia [3] and France [4]. Recently, Madeira experienced a severe epidemic of dengue
fever, with about 2,000 cases within two months [5].
When it comes to determining the risk of dengue occurring in a given region, the extrinsic
incubation period (EIP) plays an important role. The EIP is commonly defined as “the
interval between the acquisition of an infectious agent by a vector and the vector's
ability to transmit the agent to other susceptible vertebrate hosts” [6]. In the case
of dengue, after the virus is ingested by a mosquito through a blood meal, some time
is required for the virus to replicate, escape the midgut, and spread through the
mosquito's body until it ultimately reaches the salivary glands (SG), from where it
can be passed on to another host during the next blood meal.
For dengue, the duration of the pathogen's EIP is known to be temperature-dependent,
but very few mechanistic risk models (usually based on the basic reproductive number
R
0, i.e., the number of secondary cases produced by one primary case in a completely
susceptible population [7]) have taken that into account until now. In fact, most
of the models implemented for dengue use fixed values for the duration of the EIP
or rather rough estimates of temperature dependence [8].
This may be due to the fact that experimental studies on this topic are rare, and
their results may appear to some extent inconsistent or even contradictory. However,
the implementation of a realistic, temperature-dependent EIP will greatly improve
mechanistic dengue modeling: since EIP appears as an exponent in the equations used
for the determination of R
0 and vector capacity [7], [9], [10], even small changes in EIP can have a large impact
on the results of mechanistic dengue models that build on the concept of R
0. The practical relevance of this issue has been demonstrated for dengue [9] as well
as other vector-borne diseases such as malaria [11] and bluetongue [12].
In addition, correlative models based on environmental factors and vector distributions
(also referred to as “climate envelope models” or “environmental niche models”) have
to be revised and enhanced. Currently, these models usually focus on the spatial distribution
of vector species. But if temperatures do not support amplification and establishment
of the virus even though the vector is present, risk assessment based solely on vector
distributions leads to an overestimation of areas at risk. Combining such models with
information on temperature requirements for the virus derived from the EIP can reduce
uncertainty [13].
Here, we give a short overview of the few experimental studies that are explicitly
addressing the temperature dependence of the EIP of dengue. We analyze the implications
of these studies and discuss current uncertainties in modeling dengue risk in face
of climate change. We identify methodological challenges and formulate suggestions
for the design of future studies from a spatio-ecological point of view.
What Has Been Done So Far?
In order to assess current knowledge about the temperature dependence of the EIP of
dengue, we conducted an extensive literature search, using the Thomson Reuters Web
of Knowledge research portal (which includes the databases Web of Science, BIOSIS,
Current Contents Connect, MEDLINE, and Journal Citation Reports) as well as Google
Scholar and Google Books. Search terms were built from all possible combinations of
the keywords “dengue,” “DENV,” “extrinsic,” “EIP,” “incubation period,” and “temperature.”
Journal articles and books that were found to provide secondary information on the
topic were scanned for references to experimental studies, and a forward and reverse
literature search was performed for experimental studies.
We found five experimental studies that explicitly addressed the temperature dependence
of the EIP of dengue. The first one was carried out by Blanc and Caminopetros in Greece
during the winter of 1928–1929 [14]. This was followed by two publications by McLean
et al. in the mid-1970s [15], [16] and another article by Watts et al. in 1987 [17].
Rohani et al. revived the topic in 2009 [18]. In addition to these works, we include
two further studies in the dataset that examine the duration of the EIP at a single,
fixed temperature: Salazar et al. [19] studied the spread of dengue virus within the
body of Aedes aegypti at 28°C, and Anderson and Rico-Hesse [20] examined the effect
of viral genotype on the vector capacity of A. aegypti at 30°C.
All experiments have in common that they examined the EIP of dengue virus type 2 in
A. aegypti, with the exception of Blanc and Caminopetros, who did not provide information
about the serotype examined (retrospective studies suggest dengue virus types 1 and
2 occurred during the Greece epidemic [21]), and Rohani et al., who additionally examined
dengue virus type 4. However, the experimental approaches vary considerably in many
respects within and between the studies. An overview of the durations of the EIP as
observed by the different studies is given in Figure 1A; a detailed list can be found
in Table S1.
10.1371/journal.pntd.0002207.g001
Figure 1
Overview of the available data for the temperature dependence of the EIP of dengue.
Each point represents the duration until the first observed transmission or infection
of SG at a given temperature in a single experiment. (A) Complete dataset, divided
by study. (B) Complete dataset, divided by method used to infect the mosquitoes: results
obtained by letting mosquitoes feed on infected mammals or artificial blood meals
versus results obtained via intrathoracic injection of virus solution. (C) Data from
mosquitoes infected via feeding, divided by the amount of virus ingested by mosquitoes.
GE, genome equivalents; LD50, mean lethal dose; PFU, plaque forming units. (D) Data
from mosquitoes infected via feeding, divided by method of demonstration of transmission.
Black circles: Transmission was demonstrated by allowing infected mosquitoes to feed
on mammals. White circles: Tests on mammals yielded negative results, but SG contained
virus. Grey circles: Tests on mammals were not done, but SG contained virus. Xs: Neither
transmission to mammals nor SG were tested.
Differences start with the study material used: the provenance of the mosquitoes used
ranges from recently captured wild animals [14] to colonies that had been held in
the laboratory for more than 30 years [18]. Since populations that have been held
in the laboratory for a longer time may develop adaptions to the artificial environment,
field-relevant mosquitoes are preferred for determining EIP, in order to yield results
that reflect natural processes as closely as possible [19]. This is also true for
viruses that have been maintained in the laboratory for longer periods [19]. Additionally,
it is highly important to cover the whole range of genetic variations that occur in
nature, since it has been demonstrated that different genotypes or strains of the
dengue virus can show significant differences regarding their EIP [10], [15], [20].
Moreover, differences in experimental techniques for infecting the mosquitoes became
obvious: while intrathoracic injection of virus solution provides the opportunity
to exactly determine the amount of virus a mosquito receives, it bypasses the midgut
infection and escape barriers. This drastically shortens the EIP [15], [22], leading
to overestimation in the process of risk assessment. In the case of dengue, this problem
affects about 60% of the data points by McLean et al. [15], [16] (Figure 1B). Hence,
we strongly suggest the use of more natural and realistic feeding techniques that
use viremic vertebrates or artificial blood meals.
Since the duration of the EIP also depends on the amount of virus ingested during
the blood meal, ideally the complete range of virus titers observed in vertebrate
hosts in nature should be considered. The methods and units used for determining and
presenting the amount of virus differ across the experiments, making it difficult
to conduct an adequate comparison (see Figure 1C for an overview and Table S1 for
the details). While a consistent methodology would surely help to make the results
of such experiments more comparable and more accessible for scientists from other
fields, in our eyes the most important issue is to make sure that future experiments
resemble nature as closely as possible.
Furthermore, the method used to test the ability of an infected mosquito to transmit
the virus should be chosen carefully. Allowing the mosquito to take a second blood
meal from uninfected mammals such as mice [15], [16], monkeys [17], or even humans
[14], and then monitoring the mammals for dengue symptoms or virus content may seem
desirable, since it gives rather clear evidence of transmission. However, because
of ethical as well as logistical restraints, in most cases this cannot be considered
as an option anymore today. Consequently, other methods have been developed that focus
on the detection of virus content in the SG of the mosquito. While it is generally
assumed that transmission can occur as soon as the SG are infected, the literature
provides some cases where the SG tested positive for virus content but additional
transmission tests with mammals gave negative results [15]–[17]. A possible explanation
for this may be the existence of a “salivary gland escape barrier,” which has been
shown to exist for other viruses [23] but which is considered controversial for dengue
[24]. However, new techniques exist that circumvent this potential problem by causing
mosquitoes to spill their saliva, which can then be assayed for virus content [22].
Equally, methods that use complete heads or even full bodies to extract virus RNA
are not suitable for the assessment of the EIP. The latter method was used by Rohani
et al. [18], unfortunately making their data unsuitable for real-life modeling approaches
even though the data seem to be consistent with the rest of the dataset. An overview
of the implications of this issue for the dataset is presented in Figure 1D; additional
details are given in Table S1.
Careful preprocessing is crucial in order to gain meaningful results from the data
that are currently available. First, experimental results that were obtained using
intrathoracic injection to infect mosquitoes should be discarded, since their inclusion
would lead to underestimation of the EIP and thus overestimation of areas at risk
(Figure 2A). Then, data points for which verification of transmission does not exist
by either examination of vertebrates bitten during a second blood meal or by examination
of the SG should be discarded, too. Whether one wants to include data points for which
transmission was verified only via examination of the SG may depend on the context
the data are being used in: Figure 2B shows that the inclusion of these points in
general leads to a shorter mean EIP, particularly at the lower end of the temperature
range. Hence, risk maps based on a dataset that includes those points may overestimate
the threat in regions with lower temperatures—which from an ethical point of view
would be preferable to the underestimation that would probably result from the exclusion
of those data. Additionally, data obtained from experiments at low temperatures (<20°C)
are especially scarce, so that further reduction must be carefully weighed for statistical
reasons.
10.1371/journal.pntd.0002207.g002
Figure 2
Estimated temperature dependence of the EIP of dengue based on the dataset used.
Each point represents the duration until the first observed transmission or infection
of SG at a given temperature in a single experiment. Estimation was done via a simple
linear model in R 2.14.1 [31], using log-transformed values of the duration of the
EIP. (A) Results obtained from experiments with mosquitoes infected via intrathoracic
injection; the solid line depicts the linear model for those data (adjusted R
2 = 0.40, p<0.001). (B) Results obtained from experiments with mosquitoes infected
via feeding. Filled circles: SG tested positive for virus content, but transmission
to mammals was either negative or not tested. Unfilled circles: Transmission to mammals
was observed. Thick solid line: Linear model for cases where either transmission to
mammals was observed or SG tested positive for virus content (adjusted R
2 = 0.34, p<0.001). Thin solid line: Linear model for cases where transmission to
mammals was observed (adjusted R
2 = 0.46, p<0.01) For better comparability, in both panels the dashed line shows the
linear model for all data (injection as well as feeding) combined (adjusted R
2 = 0.32, p<0.00001).
Design of Future Experiments with Respect to Interdisciplinary Research
Apart from the specific problems that arose in analyzing the experiments that have
been done so far, there are some other things that might be worth considering when
it comes to planning future works. Because the EIP varies between single mosquitoes,
usually a batch of mosquitoes is examined for each time point during the experiment.
The EIP can then be estimated as the period of time between the infectious blood meal
and the point in time when (1) for the first time at least one mosquito of the batch
is able to transmit the virus, (2) a given fraction (typically 50%) of the mosquitoes
are transmitting, or (3) all mosquitoes are transmitting. A more advanced approach
has been applied by Paaijmans et al. [25] that considers the fact that even after
long incubation periods not all mosquitoes of a batch are able to transmit the virus.
Here, we decided to use the time until the first observed occurrence of transmission
or infection of the SG for the data shown in Figures 1 and 2 for two reasons. First,
this is the most conservative approach, as it utilizes the shortest possible EIP and
hence is unlikely to underestimate risk. Second, in some cases batches consisted of
only five or fewer mosquitoes [15]–[17], which is too few to derive statistically
meaningful fractions. In order to facilitate the application of advanced statistical
methods, this issue should be taken into account during the design of future experiments:
in our opinion, batches of 20 to 30 mosquitoes, as used by Salazar et al. [19] and
Paaijmans et al. [25], are desirable.
Another important issue to note is that past laboratory studies usually held temperatures
constant over the whole experiment. This neglects the fact that in nature diurnal
temperature is far from constant. Recent studies imply that diurnal fluctuations in
temperature may play a more decisive role for pathogen amplification than previously
thought [26], [27]. Including thermal fluctuations in future experiments and comparing
the results with those from identical experiments with constant temperatures may prove
rewarding.
Furthermore, not only the current main vector of dengue, A. aegypti, deserves attention:
A. albopictus has undergone a vast global spread over the last decades [28] and is
being considered as serving as a potential future main vector of dengue in Europe
[29]. Until recently, knowledge about the EIP of dengue for A. albopictus was scarce
and was mentioned only in a side note in the study by McLean et al. stating that “comparable
results were obtained with…A. albopictus mosquitoes” [16]. In 2012, Richards et al.
compared the vector competence of A. albopictus and A. aegypti for dengue at different
temperatures [30]. Even though the duration of the EIP was not explicitly examined
(a fixed incubation period of 14 days was used), this study can be regarded as a step
in the right direction, since experiments focusing on A. albopictus are urgently needed.
In conclusion, further studies on the EIP of dengue based on experiments with modern
methodology and adequately high resolution in time and temperature may facilitate
risk assessment by improving mechanistic as well as correlative modeling approaches.
Since the lack of knowledge on the temperature dependence of the EIP seems to be even
bigger when it comes to other arthropod-borne viral diseases such as Chikungunya,
the identified challenges and suggestions may turn out to be of relevance beyond the
example of dengue.
Supporting Information
Table S1
Summary of the data obtained from the literature. This table provides information
about the different experimental studies, including study material used and methodological
details. The duration until the first observed transmission or infection of SG at
a given temperature is given for each study.
(XLS)
Click here for additional data file.