Accepted Manuscript

Title: Molecular epidemiology of Dengue, Yellow fever, Zika

and Chikungunya arboviruses: An Update

Authors: Adriana Higuera, Juan David Ramı́rez

PII: S0001-706X(18)31144-6
DOI: https://doi.org/10.1016/j.actatropica.2018.11.010
Reference: ACTROP 4826

To appear in: Acta Tropica

Received date: 10 September 2018

Revised date: 10 November 2018
Accepted date: 10 November 2018

Please cite this article as: Higuera A, Ramı́rez JD, Molecular epidemiology of Dengue,
Yellow fever, Zika and Chikungunya arboviruses: An Update, Acta Tropica (2018),
https://doi.org/10.1016/j.actatropica.2018.11.010

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Molecular epidemiology of Dengue, Yellow fever, Zika and Chikungunya arboviruses: An

Update

Adriana Higuera and Juan David Ramírez*

Grupo de Investigaciones Microbiológicas - UR (GIMUR), Programa de Biología, Facultad

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de Ciencias Naturales y Matemáticas, Universidad del Rosario, Bogotá, Colombia.

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*Corresponding author: Juan David Ramírez, Facultad de Ciencias Naturales y

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Matemáticas, Universidad del Rosario, Bogotá, Colombia. E-mail address:

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juand.ramirez@urosario.edu.co

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Abstract
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Arboviruses are a group of viruses transmitted by arthropods. They are characterized by a

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wide geographic distribution, which is associated with the presence of the vector, and cause
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asymptomatic infections or febrile diseases in humans in both enzootic and urban cycles.

Recent reports of human infections caused by viruses such as dengue, Zika, and
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chikungunya have raised concern regarding public health, and have led to the re-evaluation

of surveillance mechanisms and measures to control the transmission of these arboviruses.

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Viruses such as Mayaro and Usutu are not currently responsible for a high number of

symptomatic infections in humans, but should remain under epidemiological surveillance to

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avoid the emergence of new epidemics, as happened with Zika virus, that are associated

with new or more severe symptoms. Additionally, significant variation has been observed

in these viruses, giving rise to different lineages. Until recently, the emergence of new

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lineages has primarily been related to geographical distribution and dispersion, allowing us

to ascertain the possible origins and direction of expansion of each virus type, and to make

predictions regarding regions where active infections in humans are likely to occur.

Therefore, this review is focused on untangling the molecular epidemiology of Dengue,

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Yellow fever, Zika and Chikungunya due to their recent epidemics in Latinamerica but

provides an update on the geographical distribution globally of these viral variants, and

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outlines the need for further understanding of the genotypes/lineages assignment.

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Keywords: Dengue virus, Yellow fever virus, Zika virus, Chikungunya virus, molecular

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epidemiology.
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1. Background

The term arbovirus encompasses a highly heterogeneous group of approximately 537

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viruses (CDC, 2017a) that are transmitted by hematophagous arthropods. Arboviruses

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infect a wide range of vertebrate hosts, and despite their significant taxonomic diversity,
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their ecology and maintenance mechanisms are similar. Arboviruses require frequent or

occasional horizontal transmission between vertebrate hosts and vectors that maintain an
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adequate level of blood viremia for replication and maintenance in a given population

(Duane J. Gubler, 1996; D. J. Gubler, 2002; S. C. Weaver, 2006). Other routes of

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transmission among vertebrates in the absence of the vector have also been reported,

including mother-to-child transmission (Fritel et al.; Gérardin et al., 2008; P. C. Tan,

Rajasingam, Devi, & Omar, 2008), nosocomial infection (Clark et al., 2012; Wagner et al.,

2004) and as a result of blood transfusion (Tambyah, Koay, Poon, Lin, & Ong, 2008;

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Tomashek & Margolis, 2011) or organ transplantation (F. L.-S. Tan, Loh, & Prabhakaran,

2005) and sexual intercourse (Oster, 2016). Humans are often considered accidental or late

hosts in the transmission cycle of these viruses (Anez, Chancey, Grinev, & Rios, 2012), and

are sporadically infected following intrusion into wild habitats. Human activity can also

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modify the environment to allow contact between vectors and the vertebrates that serve as

reservoirs, increasing vector populations or the level of virus circulation, thereby

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propagating the viruses and generating epidemic outbreaks. The global distribution of

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arboviruses is directly associated with that of the vectors, with changes in environmental

conditions such as temperature, rainfall, humidity, and vegetation affecting transmission

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rates throughout the year (Anez et al., 2012; D. J. Gubler, 2002).

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Initially, the classification of arboviruses was based on serological criteria, but more
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recently, with the advent of molecular testing methods, viral taxonomy has changed and
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viruses can be classified into families, each with specific genera. In South America, the
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most important virus families are Togaviridae and Flaviviridae. Interestingly, most
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arboviruses are RNA viruses. An RNA genome is believed to aid in their replication in both

vertebrate and invertebrate hosts because of the higher genetic plasticity and mutational rate
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of RNA compared with DNA (Domingo & Holland, 2008). This plasticity allows them to

adapt to a variety of hosts, including humans, during vector transmission in urban areas.
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The use of new molecular techniques, along with electron microscopy, has allowed us to

study in greater detail the genomes, structural characteristics, and mechanisms of

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replication and host interaction of arboviruses. These advances have enabled us to

determine the geographic distribution of these viruses, and to conduct phylogenetic

reconstructions that demonstrate their origins and expansion. The identification of specific

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genotypes not only reveals the diversity of these viruses, but can be used to identify their

possible association with geographic regions at risk of outbreaks or different clinical

manifestations of disease, and therefore determine the emergence or reemergence of some

of these pathogenic agents (D. J. Gubler, 2002; C. Klungthong, Putnak, Mammen, Li, &

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Zhang, 2008).

The genotyping schemes proposed for some arboviruses are based on the generation of

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well-supported clusters in phylogenetic analyses that are limited to several specific

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markers, mainly the envelope gene (E) and the non-structural protein (NS5). As such, each

genotype is generally associated with a geographic cluster. For example, in the case of

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dengue virus (DENV) serotype 2, the Cosmopolitan genotype is widely dispersed across
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different geographic regions, including Asia, Africa, and Australia (C. Klungthong et al.,
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2008). In contrast, the Asian lineage of Zika virus (ZIKV) has spread to the Americas,
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generating an internal cluster called the Latin American lineage (Grubaugh et al., 2017),
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while different specific lineages of Usutu virus (USUV) have emerged in both Europe and
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Africa (Engel et al., 2016). In a detailed review of the proposed genotypes for each of these

viruses, we found that each genotype was established based on the emergence of a new
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cluster in the phylogenetic trees and the topologies of the phylogenies depend on the

marker used in the genotyping (C. Klungthong et al., 2008). That means, the addition of
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further markers result in different groupings, changing the topologies of the constructed

phylogenies.
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The lack of congruence in the nomenclature means that a unified and clear criterion to

establish the appearance of new genotypes and standardize the nomenclature used in the

classification of these agents is needed. It is important to highlight that the genotypes

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established to date are based on studies carried out using patient samples, which, although

they have shown the great variation present in these pathogens, have not included samples

of the vectors as in other studies (Costa-da-Silva et al., 2017; Sittivicharpinyo, Wonnapinij,

& Surat, 2017) that would allow us to more precisely determine the degree of diversity. It

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would also be useful to understand the distribution of these genotypes in the different non-

human hosts. Despite the availability of complete genome sequences and the small size

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(~11 kb) of the genomes, the use of phylogenomics for these viruses is incipient and the

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majority of arbovirus genotyping studies have focused on the same few genetic markers,

although different primers are used in the various studies. This paradoxically implies that

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there may be variation in these regions, indicating rapid evolution, which suggests that they

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are not suitable for use as high resolution markers to determine stable or fixed genotypes.
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Therefore we propose that the establishment of new genotypes is made from studies with
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complete genomes where the data support is much higher (Volk et al., 2010).
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Therefore, the main objective of this review is to consolidate all current information on
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the geographical global distribution of the different genotypes of DENV, yellow fever

virus (YFV), ZIKV and chikungunya virus (CHIKV), due to their importance in terms of
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public health in Latinamerica, depite of being all of which have triggered major global

epidemics or have the potential for global spread due to their ability to adapt to new hosts,
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particularly mosquito vectors. Herein, we summarize previous studies of some of the

arboviruses of major clinical importance across South America (belonging to the

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Togaviridae and Flaviviridae families). We also showed phylogenetic analyses using the

maximum likelihood method to compile the established and accepted genotypes for each

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viral agent. In the same way, a revision of the genes most frequently used for genotyping

was carried out. The epidemiological consequences will be discussed.

2. Togaviridae

Almost all viruses belonging to the family Togaviridae are arboviruses, with the

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exception of rubeola virus (Rubivirus) and a few alphaviruses that do not have known

vectors (S. C. Weaver, 2006). The alphaviruses constitute the most important genus within

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the family because of their eventual transmission to humans. In general, alphaviruses are

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transmitted by mosquitoes belonging to the genera Aedes, Culex, Anopheles, Haemagogus,

Psorophora, and Culiseta. Birds, rodents, and mammals are the main reservoirs of these

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viruses; however, vector species are very limited as there is no successful adaptation in flies
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and ticks. Alphaviruses are associated with two different patterns of disease: encephalitis,
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such as Venezuelan, Eastern, and Western equine encephalitis viruses, or febrile diseases
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with persistent arthralgias, such as MAYV, Una virus (Ann M Powers et al., 2006), and
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CHIKV.
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2.1. Chikungunya virus (CHIKV)

2.1.1. General features

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The CHIKV virion is characterized by a linear, monopartite, single-stranded RNA

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genome with an icosahedric nucleocapsid and a sheath of approximately 40 nm in diameter.

The genome is approximately 11–12 kb in size, is polyadenylated at the 3′ end, and has a
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CAP 7-methylguanosine at the 5′ end. It contains two open reading frames, the first of

which codes for a non-structural polyprotein that is processed into four non-structural

proteins, while the other codes for a structural polyprotein expressed as a subgenomic RNA

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molecule (E. G. Strauss & Strauss, 1986; J. H. Strauss & Strauss, 1994). Non-structural

protein 4 corresponds to RNA-dependent RNA polymerase (RdRp) (Pietilä, Hellström, &

Ahola; Rubach et al., 2009). Various studies have used partial envelope gene (E1)

sequences or the total genome sequence to establish the genetic diversity of CHIKV. These

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studies found variation in the lengths of the genomes between and within geographically

established lineages (Nyari, Maan, Sharma, Pandey, & Dhole, 2016; Stapleford et al., 2016;

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Tun et al., 2014; Vazeille et al., 2007). The high level of heterogeneity observed in the viral

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genome has been related to the inability of RdRp to correct errors during RNA synthesis,

generating at least one mutation on average per new RNA genome (Coffey, Failloux, &

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Weaver, 2014). The most variable regions of the genome are the untranslated regions

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(UTRs) and the 26S binding region. Occasional indels have been found in the open reading
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frames, and are considered highly conserved. However, it is important to bear in mind that
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these indels in coding regions are associated with negative selection, and that they are

normally compensated by nearby indels that re-establish the reading frames, but that by
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showing divergence in amino acids they would be suggesting adaptive evolution (Williams
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& Wernegreen, 2013), which in the case of CHIKV have been related to a reduced viral
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replication rate and dissemination between rodents and Ae. aegypti (Coffey, Beeharry,

Borderia, Blanc, & Vignuzzi, 2011), showing that such diversity may be associated with
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the ability to survive under selection pressure (Coffey & Vignuzzi, 2011).

Transmission of CHIKV among humans occurs through the vector mosquito, which
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may be involved in epizootic cycles, where vertebrate animals act as viral reservoirs. In

contrast, during active epidemics humans are the reservoirs (Mohan, Kiran, Manohar, &

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Kumar, 2010; Simon, Javelle, Oliver, Leparc-Goffart, & Marimoutou, 2011). Vertical

transmission has also been reported (Robillard et al., 2006).

CHIKV has an incubation period of 3–12 days, and causes symptoms such as sudden

fever, arthralgia, myalgias, and skin rash (Mohan et al., 2010). Studies have shown a

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relationship between CHIKV infection and abortion during the first and last trimesters of

pregnancy in humans (Simon, Savini, & Parola, 2008; Sudeep & Parashar, 2008). In some

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cases, infection triggers symptoms of joint inflammation, paralytic arthritis, and chronic

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arthritis that can last up to 4 months in 33% of infected patients (Fourie & Morrison, 1979).

However, Chikungunya fever received little attention in the medical community until its

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resurgence in 2006, where widespread occurrence of the virus led to increased morbidity in

affected populations.
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2.1.2. Molecular epidemiology
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CHIKV was first described in 1952 following an outbreak of disease amongst the
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Makonde people on the coast of Tanzania (Lumsden, 1955). The name of the virus
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translates as “the disease that bends up the joints” (Ross, 1956). Subsequently, the virus

was identified in Aedes africanus mosquitoes from Uganda to East Africa, where evidence
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of the enzootic wild life cycle was observed (Weinbren, 1958). Later, CHIKV was found in

other regions of Sub-Saharan Africa (Coffey et al., 2014). The first outbreak outside of
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Africa was reported in Bangkok (Hammon, Rudnick, & Sather, 1960), with further
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outbreaks in the 1960s and 1970s in India (Hammon et al., 1960; Myers & Carey, 1967).

The virus then spread across the Asian continent, causing several different epidemics. In

2004, new outbreaks of CHIKV were reported in India, Italy and France, with a total of 1.3

million reported cases in humans (Coffey et al., 2014; J.-H. Huang et al., 2009; Lanciotti et

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al., 2007; Marc et al., 2011; Rezza et al., 2007). Interestingly, an epidemic CHIKV strain

from the French island of Réunion contained an A226V substitution, resulting in a mutated

virus (Vashishtha et al., 1998). This allowed greater efficiency in the biological processes

of the virus, including infectivity and replication, allowing it to be vectored by Ae.

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albopictus without affecting transmission by Ae. aegypti (Tsetsarkin, Vanlandingham,

McGee, & Higgs, 2007; Volk et al., 2010; Scott C. Weaver & Forrester, 2015). The

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adaptation of this new virus genotype may explain its transmission into new areas where

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the main vector is not Ae. aegypti.

The vast geographical spread and several widespread epidemics have increased the

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global public health significance of CHIKV in recent years (Fig. 2). CHIKV has been
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reported in 45 countries and dependent territories in the Americas, with more than 2.9
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million suspected cases and 296 deaths attributed to the virus (Yactayo, Staples, Millot,
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Cibrelus, & Ramon-Pardo, 2016). However, it is believed that the true number of CHIKV
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infection cases is underestimated as a result of incorrect diagnosis and a lack of reporting.

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Epidemics caused by this virus are characterized by widespread outbreaks that suddenly

appear and then disappear over time, which is likely impacted by different factors such as
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susceptibility of human populations to viral infection, the virus-vector interaction, the

efficiency of the vector for transmission, and environmental conditions that affect the
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biological cycle of the mosquito (Simon et al., 2008).

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Analyses of partial sequences of the CHIKV E1 gene have revealed three genotypes

that are linked to geographical location: the West African lineage, the East, Central, South

African (ECSA) lineage, and the Asian lineage (A. M. Powers et al., 2001; A. M. Powers,

Brault, Tesh, & Weaver, 2000; Volk et al., 2010; Scott C. Weaver & Forrester, 2015). An

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additional epidemic lineage called the Indian Ocean lineage (IOL), whose strains form a

monophyletic group within the ECSA lineage (Figs. 1 and 3), arose during dispersal of

CHIKV across the Asian continent. Until 2013, only the Asian lineage was circulating in

the Americas (J.-H. Huang et al., 2009; Lanciotti & Valadere, 2014). Although the support

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for the ECSA lineage node was not very high, it was possible to visualize all three of the

previously determined major lineages in our phylogenetic analysis. The CHIKV lineages

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have also been confirmed using structural envelope genes (Fig. 1 and Supplementary Table

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S1) (Schuffenecker et al., 2006). These studies showed limited genetic exchange between

the two African lineages, despite the geographical proximity, except for a bat isolation from

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Senegal that grouped with the ECSA lineage, indicating that both lineages were present in

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an enzootic cycle at some point (Volk et al., 2010).
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In 2004, an epidemic associated with the ECSA lineage was reported in Kenya, eastern
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Africa, which then spread into nearby countries and the Indian Ocean Islands (K. C. Chen
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et al., 2013). Towards the end of 2005, this lineage was responsible for outbreaks in India
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and Sri Lanka, as well as east Asian countries including Malaysia, Thailand, and Singapore.

The disease was associated with the presence of Ae. albopictus in rural areas (Vazeille et
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al., 2007; Volk et al., 2010). Phylogenetic analysis of samples from the Indian Ocean

Islands outbreak based on partial E1 gene sequences showed a monophyletic group (IOL)
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with two sublineages, one associated with the Indian Ocean Islands and the other with the

Indian subcontinent (Ng & Hapuarachchi, 2010), apparently generated from different
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ancestors from Kenya (Volk et al., 2010). From the beginning of 2006 until 2010, the

ECSA lineage was the most prevalent lineage in some Asian countries, but this was

subsequently overtaken by the Asian lineage (Masri Sembiring, Ni Ketut, & Nur Ika,

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2015). In 2006, the first case of CHIKV infection was recorded in Italy, reportedly caused

by travelers from India, with subsequent travel-related cases also reported in the American

continent, including the United States (Farnon, 2007; Khan et al., 2014). In Europe, the

vector Ae. albopictus, which is present in more than 20 countries, was responsible for the

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spread of the ECSA genotype. The first case of CHIKV infection on the Caribbean island

of San Martin was reported in 2013 (Coffey et al., 2014; Rodrigues Faria et al., 2016).

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Unexpectedly, the genotype of this strain was associated with the Asian lineage, and

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showed high similarity to circulating strains in Indonesia, China, and the Philippines

(Leparc-Goffart, Nougairede, Cassadou, Prat, & de Lamballerie, 2014). The abundance of

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Ae. aegypti and the constant movement of tourists between the Caribbean Islands and the

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Americas led to further dispersion of CHIKV, causing significant epidemics in Central and
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South America (Camacho et al., 2017). As of 2014, autochthonous cases of CHIKV
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infection had been reported in 45 American countries and dependent territories, with one of

the most important outbreaks being responsible for more than 2.9 million suspected or
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confirmed cases (Wahid, Ali, Rafique, & Idrees, 2017) associated with both the ECSA and
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Asian lineages (Horwood & Buchy, 2015).

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In 2014, the presence of both the Asian (Nunes et al., 2015) and ECSA (Cunha et al.,

2017; Souza et al., 2017) genotypes was confirmed in Brazil. Neither of these lineages had
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previously been detected in the American continent, and the strains appeared to have

originated from Angola (Rodrigues Faria et al., 2016). Phylogenetic studies based on
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CHIKV complete genome sequences and partial E1 gene sequences obtained from samples

from symptomatic individuals who had traveled to the Caribbean showed that they

belonged to the Asian genotype (Conteville et al., 2016). However, differences were

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observed in the topologies of the trees generated using the two different types of sequence

data (complete genome vs. E1 gene). This finding implies that a single marker, such as the

E1 gene, may not be suitable to accurately establish genotypes and phylogenetic

relationships between isolates. As observed by Volk et al. (2010), the use of complete

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genome sequences provides a more robust phylogenetic analysis, and can even show the

absence of some genotypes, in this case, the monophyletic group within the ECSA lineage

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(Sahadeo et al., 2017).

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The natural life cycle of CHIKV was initially recognized to include two transmission

profiles: the African profile, with a mainly enzootic life cycle resulting from primatophilic

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mosquitoes such as Ae. furcifer and Ae. africanus, and the Asian profile, associated with
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urban outbreaks where the main vector is Ae. aegypti (Costa-da-Silva et al., 2017). Recent
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reports also indicate that there is a risk of a wild life cycle being established in Latin
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America (Lourenco-de-Oliveira & Failloux, 2017). However, the appearance of strain

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A226V-CHIKV, with its distinct adaptation to Ae. albopictus, gave rise to the transmission
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of the IOL to countries throughout the Indian Ocean, India, Asia, Africa, and Europe

(Horwood & Buchy, 2015; Kraemer et al., 2015; Schuffenecker et al., 2006; Simon et al.,
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2011; Volk et al., 2010). In 2008, the first CHIKV outbreak was reported in Singapore. The

circulating strains were shown to contain mutations resulting in an ability to be vectored by

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Ae. albopictus, as well as changes in viral replication times compared with African strain

S27 (K. C. Chen et al., 2013). These changes may also affect the rate of transmission
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between hosts. However, different theories have been proposed to explain this difference

between transmission cycles, where higher nucleotide substitution rates have been observed

in epidemic lineages such as the IOL and Asian lineages. These differences are possibly

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affected by factors such as population density, seasonal changes that affect the reproduction

of the insect vectors, the host immune conditions, and the dietary preferences of the vector

(Coffey et al., 2014; Horwood & Buchy, 2015; Volk et al., 2010).

3. Flaviviridae

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The name flavivirus comes from the Latin word “flavus”, meaning yellow, which refers

to the jaundice that is common in yellow fever virus infection. The genus Flavivirus

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comprises 53 species, and includes many of the world’s most clinically important viruses.

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Flaviviruses commonly infect humans, and are responsible for major epidemics in different

geographic regions that are associated with illnesses ranging from asymptomatic infections

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to severe encephalitis or hemorrhagic disease, which are sometimes fatal (Bollati et al.,
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2010). Flaviviruses are very diverse, and include viruses such as Japanese encephalitis virus
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(JEV) and tick-borne encephalitis virus (TBEV), as well as other mosquito-borne viruses
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such as DENV, YFV, West Nile virus, and ZIKV, which have high mortality rates in Africa
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and parts of South America(Carrington & Auguste, 2013; Y. J. Huang, Higgs, Horne, &
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Vanlandingham, 2014).

3.1.Dengue Virus (DENV)

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3.1.1. General features

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DENV is a positive-sense single-stranded RNA virus with a genome size of

approximately 11 kb. The genome comprises 10 genes that are directly translated into a
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polyprotein, which is subsequently cut into 10 proteins, including three structural proteins

(capsid (C), membrane (M), and envelope (E)) and seven non-structural proteins (NS1,

NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The genome is flanked at both ends by UTRs

(Y. J. Huang et al., 2014; Idrees & Ashfaq, 2013; Lindenbach & Rice, 2003). The virus has
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a spherical structure with a diameter of 50 nm. Protein C constitutes the nucleocapsid,

which is surrounded by the viral envelope. The envelope is a lipid bilayer derived from the

host that also contains several viral E and M proteins that control the process of penetration

of the virus into human host cells. DENV is transmitted via the blood by female mosquitoes

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of the genus Aedes, mainly Ae. aegypti and Ae. albopictus, which require blood feeding for

oviposition. Vertical transmission has also been reported (Gutierrez-Bugallo et al., 2017).

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These mosquitoes are efficient vectors because of their high affinity for human blood, high

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susceptibility to all virus serotypes, and their adaptation to urban areas necessitated by their

limited flight capacity. This has allowed the domiciliation of the vector, favoring the

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transmission of infection to humans (Murray, Quam, & Wilder-Smith, 2013).

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In humans DENV causes Dengue fever, which is characterized by an asymptomatic,
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subclinical or symptomatic infection that in most cases becomes self-limiting. The main
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symptoms include elevated fever, headache, retroorbital pain, myalgia, joint pain, and in
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some cases, red spots on the skin. These symptoms can be caused by any of the four DENV
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serotypes upon initial infection. However, in epidemic regions where several serotypes are

circulating at the same time, it is possible to acquire a secondary infection caused by a

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strain belonging to a different serotype, which may result in the more severe illnesses

hemorrhagic fever dengue and dengue shock syndrome (Duane J. Gubler, 1998; Scott B.
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Halstead, 2003; Suleman et al., 2017).

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3.1.2. Molecular epidemiology

DENV can be divided into four genetically related but antigenically different groups,

designated DENV-1–4. These serotypes have been further subdivided into genotypes on the

basis of phylogenetic analysis (Holmes & Twiddy, 2003). Sequence analysis of the NS5

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gene indicated divergent and independent evolution of the serotypes, and showed that

viruses involved in the urban and peri-urban cycles come from the wild cycle. Currently, all

four DENV serotypes are circulating in both urban and peri-urban environments.

DENV is considered to be one of the most widely dispersed arboviruses in the world

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(Fig. 2). This is mainly due to human migration, uncontrolled urbanization, armed conflict,

inadequate waste and water management, and unsustainable vector control (Murray et al.,

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2013). This has led to hyperendemicity, where the four DENV serotypes coexist (Vaddadi,

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Gandikota, Jain, Prasad, & Venkataramana, 2017), and genetic variation has allowed the

emergence of several lineages (Figs. 1 and 4). These lineages may frequently appear and

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disappear until stability between the virus and the vector that allows the maintenance of
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lineages is achieved (R. Chen & Vasilakis, 2011; Coffey, Forrester, Tsetsarkin, Vasilakis,
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& Weaver, 2013; Ramos-Castañeda et al., 2017).
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Whether DENV originated in Asia or Africa remains the subject of debate. There are
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several reports that serve as indicators of possible origins in both regions, with findings
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based on initial reports of vector introduction, the transfer of humans by sea voyage, the

reporting of clinical manifestations corresponding with febrile diseases such as dengue,

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circulation of the four serotypes in wild areas, susceptibility of vectors to infection,

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transmission cycles, origin of vectors, and ecological and phylogenetic evidence (R. Chen

& Vasilakis, 2011; D. J. Gubler, 2002; Vasilakis & Weaver, 2008). In the Americas,
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epidemics of illnesses with similar symptoms to dengue disease were first recorded in 1635

and 1699 (Badii, Landeros, Cerna, & Abreu, 2007). Evidence of the association between

DENV and the more severe forms of the disease, hemorrhagic dengue and dengue shock

syndrome, was not obtained until much later, so it was not until the 1950s that these

15
illnesses were linked to DENV (S. B. Halstead, 1980). Increased commercial activity

between the Caribbean and North and South America led to an increased number of

outbreaks in these regions (Tittarelli et al., 2017). Regional initiatives for vector control

began long before the viral agent was identified, with programs similar to those for the

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control of yellow fever transmission. In spite of these initiatives, the occurrence of

outbreaks in countries such as Jamaica, Puerto Rico, the Antilles, and Venezuela, along

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with the deterioration of control measures during the 1960s, allowed a reintroduction and

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geographical expansion of the vector, and therefore of the viral agent. This was the main

cause of the major outbreaks of dengue that have occurred in the Americas since 2000, with

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all four serotypes now circulating in this region (Brathwaite Dick et al., 2012). DENV has

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been reported in more than 100 countries around the world (Fig. 2) (WHO, 2016a), with
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about 100 million people infected per year. Although some reports have estimated that the
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burden of DENV infections per year is at least three times higher than that estimated by the

World Health Organization (Bhatt et al., 2013). The figure 2 implies that approximately 1/3
D

of the global human population is at risk, particularly in tropical and subtropical regions
TE

where conditions are ideal for vector reproduction. Ae. aegypti is the predominant vector in
EP

a domestic setting, while Ae. albopictus and other Aedes species are more common in

peridomestic and wild settings, respectively (Duane J. Gubler, 1998).

CC

RNA sequencing has made it possible to discriminate genotypes with a nucleotide

divergence of 6% within each serotype based on the genomic E/NS1 region (R. Rico-
A

Hesse, 1990). Using this method, DENV-1 contains five different genotypes (R. Chen &

Vasilakis, 2011; Goncalvez et al., 2002; Lewis et al., 1993), DENV-2 has five genotypes

(R. Chen & Vasilakis, 2011; Lewis et al., 1993; Rebeca Rico-Hesse et al., 1997; Twiddy et

16
al., 2002; Vasilakis, 2008; Vasilakis & Weaver, 2008), DENV-3 contains five genotypes

(Chao et al., 2005; R. Chen & Vasilakis, 2011; Lanciotti, Lewis, Gubler, & Trent, 1994;

Trent et al., 1990), and DENV-4 has four genotypes (AbuBakar, Wong, & Chan, 2002; R.

Chen & Vasilakis, 2011; Foster et al., 2003; Chonticha Klungthong, Zhang, Mammen,

PT
Ubol, & Holmes, 2004; Lanciotti, Gubler, & Trent, 1997) (Figs. 1 and 4).

In figure 1, the genetic diversity within each serotype was clearly evident, as were the

RI
clusters that were established as genotypes in previous studies. Each of the nodes that

SC
determined the genotypes had bootstrap support of >92%, providing solid evidence for each

of the clusters. However, the internal nodes of the tree, which show the relationships among

U
the different genotypes, had low bootstrap support, which implies that this marker does not
N
allow consistent determination of the evolutionary relationships among the different
A
genotypes or lineages established for DENV (Fig. 1). Alternatively, the genotypes may not
M

yet be fixed, as their emergence is very recent and they may still be ongoing changes.
D

Authors such as Holmes and Twiddy (2003) argue that it is important to note that this
TE

marked diversity could be related to the antigenic role of protein E during viral infection, as

selection pressure related to immune response evasion facilitates the emergence of several
EP

genotypes within each serotype. This might explain the occurrence and extinction of

genotypes, and the change from cycles of wild transmission to epizootic cycles. Although
CC

complete genome sequences are now available for each of the DENV serotypes (Yun et al.,

2016), many phylogenetic analyses have relied on the envelope gene alone (Azhar et al.,
A

2015). It is of great relevance to highlight that other studies have used up to 10 different

markers corresponding to coding regions as well as the 3′-UTR. These studies found that

not all of the genes are suitable targets for establishing phylogenetic relationships (C.

17
Klungthong et al., 2008), indicating that there is no marker common to all DENV variants

that can be used to elucidate relationship between genotypes, making the re-evaluation of

its use relevant for the determination of genotypes. There are also inconsistencies between

the studies regarding the location of some strains within the clades depending on the marker

PT
used, making it difficult to obtain clear results and with good support for the genotyping of

DENV.

RI
Further studies based on the complete genome sequences of various DENV strains

SC
showed inter-genotype variations, with evidence of genotype-specific amino acid

substitutions. For example, DENV-1 genotype III had 12 specific amino acid substitutions

U
in comparison with genotype I, of which nine were determinants of genotype III. These
N
variations were found within the NS2A gene sequence, which showed a higher number of
A
substitutions compared with gene E. In addition, it has been observed that from year to
M

year, the number of substitutions in the viral genomes increases, particularly in periods of
D

high epidemic activity (Tang et al., 2010). With the passage of time, many of these
TE

substitutions are fixed, allowing for adaptation to both hosts and vectors. It should be noted

that there has been no association between these mutations and the severity of disease, but
EP

mutations are associated with increases in viral replication and the prevalence of a given

genotype in a geographic region (Sim & Hibberd, 2016). In some DENV-3 strains from
CC

Asia, recombinations have been noted in non-structural genes, whereas in American strains,

recombinations are more common in the UTRs (Waman, Kale, & Kulkarni-Kale, 2017).
A

Interestingly, the American DENV-2 genotype causes a less severe form of the disease than

the Asian DENV-3 genotype. Mutations in the non-structural genes of the Asian strains

will affect the role of these proteins, possibly aiding in the avoidance of the immune

18
response or even changing the secretion levels of non-structural proteins such as NS1,

which induces vascular permeabilization and thus the presentation of the severe form of

dengue (Singh, Anupriya, & Sreekumar, 2017). As each of the genotypes have conserved

unique amino acids, nucleotide substitutions or the introduction of different genotypes into

PT
a population could lead to amino acid differences in both structural and non-structural

proteins, which would be useful in the identification and definition of genotypes (King et

RI
al., 2008). Some studies of DENV-3 strains showed the highest level of diversity in the

SC
capsid gene, followed by the NS2A gene, which is important for viral replication and egress

of viral particles (King et al., 2008). This genetic variation impacts the antigenicity,

U
transmissibility, and response to the disease, and also influences the genotype and intra-

N
genotype typing. The variation may also be responsible for the reported changes in
A
genotypic lineages of DENV (Manakkadan, Joseph, Prasanna, Kailas, & Sreekumar, 2013).
M

High mutation rates and recombination among populations result in variation in the

topologies of phylogenetic trees, suggesting different subclones; however, these differences

D

can be clarified by analyzing the population structure (Waman et al., 2017). Such
TE

diversification is caused by the mixing of populations, which increases the likelihood of the
EP

emergence of new sublineages. On the other hand, selection pressure exerted on some

serotypes has been shown to inversely influence their prevalence, even leading to the
CC

replacement of clades within genotypes (Tang et al., 2010) or other deleterious effects.

However, these findings are limited to phylogenetic analyses based on E gene sequences
A

(King et al., 2008).

At present, DENV-3 strains belonging to genotypes I, II, III, and V are in circulation.

Strains belonging to genotype IV, which were mainly present in Tahiti and Puerto Rico,

19
have not been isolated since 1970. Strains belonging to genotypes I and II have recently

been identified in Asia, while genotype III is distributed in the Caribbean, the Americas,

and Europe (Waman et al., 2017), and genotype V has also been reported in Brazil and

Colombia (Aquino, Amarilla, Alfonso, Batista, & Figueiredo, 2009; Araújo, Bello,

PT
Schatzmayr, Santos, & Nogueira, 2009).

The above findings emphasize that it is necessary to examine other markers in addition

RI
to the E gene to obtain a robust phylogenetic analysis of DENV lineages. Alternatively, the

SC
complete genome sequence can be used for phylogenetic analysis, but should be

fragmented using specific primer sets that allow detection of mutations in different regions

U
of the viral genome (Singh et al., 2017). Finally, further studies using samples from vectors
N
and other hosts, rather than just from human hosts, should be carried out to clarify the
A
genotypes and subtypes present within the DENV serotypes.
M

3.2.Yellow fever virus (YFV)

D

3.2.1. General features

TE

The YFV virion is approximately 50 nm in diameter and has a lipid envelope. The
EP

positive-sense single-stranded RNA genome encodes three structural proteins (C, E, and M)

and seven non-structural proteins (NS) related to viral replicative events or with antagonism
CC

of the host immune system. YFV is maintained in the wild or enzootic cycle by

transmission between non-human primates and mosquitoes, with Aedes spp. recognized as
A

the main vector in Africa (Reiter et al., 1998) and Haemagogus and Sabethes spp. acting as

vectors in the Americas (Cardoso et al., 2010; de Rodaniche & Galindo, 1957; Dutary,

1981). Rural epidemics have been reported in areas where the human population comes into

contact with the wild cycle (T. Monath, 2006), and transovarian transmission among insects

20
has also been reported (Beaty, Tesh, & Aitken, 1980). Transmission of YFV between

humans, known as the urban transmission cycle, occurs as a result of the domiciliation of

Ae. aegypti and the periodic introduction of the vector to urban areas. Therefore, the role of

the Aedes mosquito in the transmission of disease has been crucial in most of the epidemics

PT
reported to date. However, YFV transmission in Argentina involves a new vector, Sabethes

albiprivus (Goenaga et al., 2012), while both Haemagogus leucocelaenus and Aedes

RI
serratus are responsible for transmission in Brazil (Almeida et al., 2014).

SC
Yellow fever may be a subclinical, non-specific, or cause severe infection. It is

characterized by fever, nausea, vomiting, headaches, myalgia, epigastric pain, and in severe

U
cases, hepatitis with jaundice, renal failure, hemorrhage, shock, and death in 20–60% of
N
cases (Carrington & Auguste, 2013; Y. J. Huang et al., 2014). Although the pathogenesis of
A
the virus is not well understood, it is known that the wild-type strain is viscerotropic,
M

showing a preference for the liver, kidney, spleen, lymph nodes, and heart depending on the
D

virulence of the strain and certain host factors. Some reports have shown that multiple
TE

human mosquito cycles increase virulence in human hosts (T. P. Monath & Vasconcelos,

2015).
EP

3.2.2. Molecular epidemiology

CC

Yellow fever is an acute viral disease of public health importance in both the Americas

and Africa. The disease is considered to be native and endemic to Africa, where it causes
A

large epidemics, with 20–30 times the number of cases compared with the Americas (T. P.

Monath & Vasconcelos, 2015). The risk of infection is greatest in West Africa, with an

incidence of 50 cases per 100,000 inhabitants, while in South America it is endemic, with

an incidence of 5 cases per 100,000 inhabitants (CDC, 2017b). However, the mortality rate

21
in South America is higher than that in Africa, indicating some kind of genetic association

with the host that should be studied in more depth.

Multiple yellow fever epidemics occurred in both the 18th and 20th centuries, but at

present they occur mainly in the tropics. It is hypothesized that YFV arrived in the

PT
Americas 400 years ago along with the slaves from Africa (Bryant, Holmes, & Barrett,

2007). Once in the New World, which has favorable ecological conditions, the virus

RI
became established in the Amazon jungle in an enzootic cycle, which also included

SC
mosquitoes of the genus Haemagogus (D. J. Gubler, 2002). As a result of vaccination

campaigns beginning in 1940 and vector control measures carried out between 1950 and

U
1960 in Central and South America, epidemics caused by YFV, and even DENV, became
N
less common (D. J. Gubler, 2002). However, a lack of surveillance and vaccine coverage of
A
only 80% of the population since 1997 (Carrington & Auguste, 2013) permitted the re-
M

emergence and spread of YFV in 2000 to Brazil, Paraguay, Argentina, Peru, and Colombia
D

(T. P. Monath & Vasconcelos, 2015).

TE

The initial classification of YFV was based on antigen detection by serological tests

that grouped it with other flaviviruses (Gould et al., 1985). Subsequently, oligonucleotide
EP

fingerprinting of strains from Africa and the Americas was used to identify genetic
CC

subtypes and associate them with their geographical distribution (Deubel, Digoutte,

Monath, & Girard, 1986; Deubel et al., 1985). In 1985, the first complete YFV genome
A

sequence was obtained (Rice et al., 1985), which was quickly followed by further YFV

genomic sequences. It was also possible to determine a genetic pattern according to the

geographic region. The phylogenetic analyses showed that virus sequences from West

Africa were in separate clades from those from Central and East Africa (Chang, Cropp,

22
Kinney, Trent, & Gubler, 1995; Mutebi, Wang, Li, Bryant, & Barrett, 2001; Wang et al.,

1996). Studies based on the envelope gene or the complete genome sequence showed that

YFV comes from central and eastern Africa, where three genotypes were identified:

Angola, Central and East Africa, and East Africa. After further westward dispersion across

PT
Africa, two additional genotypes have been identified: West Africa I and II (Mutebi et al.,

2001). The virus was then transported to the Americas via the movement of humans by sea

RI
voyages, and became particularly prevalent in Brazil (Auguste et al., 2010). The South

SC
American strains possibly originated in West Africa (Bryant et al., 2007; Chang et al.,

1995; Wang et al., 1996), with an approximate divergence in the year 1533 (Bryant et al.,

U
2007), and belong to the genotypes South America I and South America II (Fig. 1) (Beck et

N
al., 2013; Bryant et al., 2005; Mutebi et al., 2001; Von Lindern et al., 2006; Wang et al.,
A
1996).
M

As shown in Fig. 1, we confirmed the genotypes established for YFV, but found an
D

inconsistency in the East Africa genotype. The sequences of the two representative strains
TE

used, previously classified within this genotype, were grouped into different clusters.

However, the bootstrap value for this grouping is quite low, which shows that they may not
EP

correspond to the same genotype, and further analyses are therefore required to clearly

establish the existing genotypes.

CC

3.3. Zika virus (ZIKV)

A

3.3.1. General features

ZIKV is an enveloped and spherical virus with a diameter of approximately 50 nm. It

has a single-stranded positive-sense RNA genome of approximately 10,807 nucleotides

(Yun et al., 2016). The genome is linear and non-segmented with a single open reading

23
frame flanked by non-coding regions. The open reading frame codes for a polyprotein that

is cleaved into three structural proteins (capsid, membrane precursor, and envelope) and

seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (Kim et

al., 2015; Kuno & Chang, 2007). The genome has a 5′ cap but lacks a 3′ poly-A tail, and

PT
tends to circularize (Yun & Lee, 2017).

Although the exact mechanism of replication and pathogenesis of ZIKV is still not well

RI
understood, it has the ability to replicate in the intestinal cells and salivary glands of Aedes

SC
spp. and in mammalian cells such as neurons and glial cells from monkeys and skin cells

and dendritic cells from humans in vitro. ZIKV has the capacity to infect different hosts,

U
which helps it to maintain an urban cycle between humans and vectors and an enzootic
N
cycle between non-human primates and mosquitoes. Non-human species that have reported
A
positive for ZIKV in serovigilance studies include orangutans, tamarins, bats, goats,
M

rodents, and sheep (ANDRAL, CASALS, & PANTHIER, 1968; Darwish, Hoogstraal,
D

Roberts, Ahmed, & Omar, 1983). However, these species are not considered reservoirs
TE

because of cross-reactivity with other flaviviruses. ZIKV has been isolated from Ae.

africanus, Ae. aegypti, and Ae. furcifer (Diallo et al., 2014; Marchette, Garcia, & Rudnick,
EP

1969), suggesting that all three species can act as vectors.

CC
A

24
 In the case of infections in humans, it has been reported that most ZIKV infections

are usually asymptomatic, but when clinical manifestations occur, symptoms include fever,

rash, joint pain, and conjunctivitis. Reports from Brazil have also indicated an association

between ZIKV and Guillain-Barré syndrome and with fetal congenital microcephaly when

PT
the virus was acquired during pregnancy (Awadh et al., 2017; Heukelbach, Alencar,

Kelvin, de Oliveira, & de Góes Cavalcanti, 2016). Other manifestations such as peripheral

RI
idiopathic neuropathies, retinopathy, and other neurological birth defects have also been

SC
suggested (Musso & Gubler, 2016). Although cognitive, sensory and motor damage are not

exclusive to this infection, characteristics that are usually unique in congenital Zika

U
syndrome have been specifically determined (Moore et al., 2017).

N
A
3.3.2. Molecular epidemiology
M

ZIKV was discovered in 1947 when cases of yellow fever were reported in Uganda.

The first isolation was from rhesus monkeys in the Zika forest, and later from an Ae.
D

africanus mosquito (Dick, Kitchen, & Haddow, 1952). The first isolation from humans
TE

occurred in 1952 (Sirohi et al., 2016). Initially, only sporadic cases of ZIKV infection were
EP

reported, with distribution centered around equatorial Africa and parts of Asia. It wasn’t

until 2000 that ZIKV became important, as before this the virus was only associated with a
CC

self-limiting febrile illness in endemic regions (Hayes, 2009). In 2007, 73% of the

population of Micronesia became infected with ZIKV, which then began to spread amongst
A

the Pacific Islands, where a second major outbreak involving 28,000 suspected cases

occurred in 2013 (Besnard, Lastere, Teissier, Cao-Lormeau, & Musso, 2014). This

outbreak was followed by an increase in the proportion of cases of Guillain-Barré

25
syndrome (Cao-Lormeau et al., 2016). Subsequently, ZIKV was detected in Central and

South America (Yun & Lee, 2017), primarily in Chile in 2014 and in Brazil in 2015

(Lessler et al., 2016), with a report of suspected cases in the latter. Between 440,000 and

1,300,000 cases of ZIKV infection occurred in these two outbreaks (Hennessey, Fischer, &

PT
Staples, 2016), and were associated with an increase in cases of Guillain-Barré syndrome

and microcephaly in newborns (Cauchemez et al., 2016; Schuler-Faccini, 2016). In 2016,

RI
the World Health Organization announced that ZIKV is an international emergency (WHO,

SC
2016b). To date, cases of ZIKV have been reported in 48 American countries and

dependent territories (Metsky et al., 2017; PAHO, 2016), including imported cases in the

U
United States, Canada, and Europe.

N
Through phylogenetic analyses, the origin of ZIKV has been traced to East Africa
A
(Gatherer & Kohl, 2016). Further analysis based on the sequences of gene E and the NS5
M

gene have classified ZIKV into two lineages. The first is from Africa, and contains two
D

sublineages (East Africa and West Africa), while the second is from Asia, and includes
TE

three sublineages (Southeast Asian, Pacific, and American Islands) (Fig. 1) (Faye et al.,

2014; Haddow et al., 2012; Lanciotti et al., 2008). These lineages correlate with the areas of
EP

dispersion of the virus after the reports of the three main outbreaks (Zhu et al., 2016).
CC

Phylogenomic analyses based on the total ZIKV polyprotein sequence produced results

consistent with previously established lineages identified using the nonstructural genes NS5
A

and NS3. These studies showed that strains belonging to the Asian and African lineages

were more closely related to each other than to those obtained in the Americas (May &

Relich, 2016). Fig. 1 clearly shows two groups, the Asian and African groups,

corresponding to the Asian and West African and East African lineages, respectively. The

26
African group has low bootstrap support, and, unlike the tree generated by May & Relich

(2016), the African lineages group more closely with each other than with the Asian

lineage.

As many cases of infection have historically been asymptomatic, it was not until 2007

PT
that ZIKV became recognized as a significant public health concern. At this time, there was

an increase in the dispersion and number of cases of ZIKV infection in the Pacific caused

RI
by the Asian genotype. In this region, ZIKV caused symptoms similar to DENV, including

SC
neurological disorders, which had not been seen in the endemic regions of Thailand and

Southeast Asia. However, the reasons for the increase in disease prevalence are unclear,

U
leading some researchers to compare the genomic sequences of strains before and after
N
epidemics. Results showed that epidemic strains were more similar to the Asian lineage
A
than to the African lineage (Zhu et al., 2016). Although phylogenetic analyses of different
M

regions of the genome have produced trees with similar topologies, it is important to keep
D

in mind that some regions may show variability, indicating different evolutionary histories.
TE

Phylogenetic analysis of strains circulating in 2013 showed that strains from South

America were more closely related to those from French Polynesia than to those from
EP

Southeast Asia, indicating that the South American strains were not directly descended

from the South East Asian strains (Ramaiah et al., 2016). Different events that cause
CC

sequence variation have been reported in ZIKV, including recombination with other viruses

and substitutions in specific amino acids, both of which would allow differentiation
A

between lineages (Zhu et al., 2016).

Epidemic strains from islands in the Pacific and South America grouped within the

Asian lineage (Zhu et al., 2016). Amino acid substitutions were only found in the South

27
America strains, located in the E, PrM, and NS1 proteins, and may be involved in the

pathogenesis and reproductive success of the virus (Kochakarn et al., 2016; Metsky et al.,

2017). However, other studies suggest that the substitutions occurred prior to the

dissemination of the virus into the different regions (Ross, 1956). It has also been found

PT
that the strain diversity dating from before the arrival of the virus in the Americas is much

greater than that in the established American lineage, implying that ZIKV circulated for a

RI
long period of time in Asia and the Pacific, and that the diversity in the American lineage is

SC
likely to increase (Woodall, 1964). Analysis of the outbreak strains from Brazil in 2015,

where the first cases of ZIKV associated with neurological diseases and malformations in

U
infants were reported, suggested that the neurotropism may be the result of variations

N
between the strains from the two continents, or even differences in host-virus interactions in
A
South America (Kochakarn et al., 2016). Analysis of the complete genome sequences of the
M

strains showed that the variant proteins had a low dN/dS ratio, indicating that they were not

under positive selection. However, structural analysis revealed selection at specific residues
D

related to functional domains and catalytic sites (Kochakarn et al., 2016; Zhu et al., 2016).
TE

Non-synonymous mutations have a low probability of selection because they may affect the
EP

replicative processes. As such, the epidemic ZIKV proteome has been found to be

conserved by negative selection of amino acid substitutions (Kochakarn et al., 2016;

CC

Ramaiah et al., 2016). Another study showed that the 3′ UTR is less conserved in some

parts, possibly because of its relation to host adaptation and pathogenesis (Villordo,
A

Filomatori, Sanchez-Vargas, Blair, & Gamarnik, 2015). Changes have also been reported in

the secondary RNA structure depending on the host (Ramaiah et al., 2016).

28
 Several complete ZIKV genome sequences have been reported, including three

historically important and spatiotemporally different viruses reported as strain MR766 (Yun

et al., 2016). Comparison of the genomic sequences of the MR766 strain isolates from the

African and Asian lineages with that of DENV-2 showed high amino acid identity,

PT
probably as a result of the high level of conservation of elements critical for replication.

Previous studies have found evidence of events of selection, recombination, and

RI
glycosylation pressure in ZIKV strains present prior to the Pacific outbreak (Ramaiah et al.,

SC
2016). ZIKV appears to have dispersed into the Western Hemisphere between 2013 and

2014, but it was almost a year before the first case of ZIKV infection was reported in

U
Brazil, with similar lags reported for each country in the American continent. A different

N
window of introduction has been deduced for each region, and multiple introductions have
A
occurred in areas such as Puerto Rico, Honduras, Colombia, and the Caribbean Islands
M

(Metsky et al., 2017). Four ZIKV subgroups have been reported in human samples and

vectors from Florida in the United States (Worobey, 2017), with strains likely to have
D

originated from the Caribbean (Grubaugh et al., 2017). The first report of complications
TE

from ZIKV infection in the Americas occurred in 2015, and epidemiological studies
EP

showed that the strain corresponded to the Asian lineage and came from Micronesia, where

it expanded to the Pacific and Central Islands before entering South America (Shrinet,
CC

Agrawal, Bhatnagar, & Sujatha Sunil, 2016). Northeastern Brazil showed a greater number

of ZIKV infection cases along with a higher confirmed rate of microcephaly. This is
A

possibly because the region is highly populated and has ideal conditions for vectorial

transmission, which facilitated the dispersal of the American lineage to other regions of the

Americas (Faria et al., 2017; Metsky et al., 2017). Analysis of strains responsible for the

2015–2016 outbreak revealed variations in both coding and non-coding regions of the
29
genome, with a high mutation rate when compared with the Malaysian strains of the 1966

epidemic. Structural proteins showed greater variation than non-structural proteins, which

were more conserved, and a greater number of recombination events were found in African

isolates (Shrinet et al., 2016).

PT
Studies have shown that the adaptability and infectivity of ZIKV for its vectors has

increased over time, which has contributed to its dispersion. A study by Liu et al. (2017)

RI
showed that infection of the vector with post-epidemic strains of the Asian lineage resulted

SC
in greater production of NS1 in serum, which is related to increases in infectivity,

prevalence, and transmissibility between hosts and vectors. The increased virulence of the

U
post-epidemic strains was apparently caused by an A188V substitution in the ZIKV
N
genome (Liu et al., 2017). It is important to bear in mind that at present it is difficult to
A
diagnose ZIKV because mutations in the genome can influence the effectiveness of the
M

diagnostic tests (Metsky et al., 2017). In addition, some of the primers used for detection of
D

ZIKV also amplify DENV genomic sequence. Mutations also prevent the differentiation of
TE

ZIKV genotypes, which is crucial for establishing the geographical origin and distribution

of epidemic strains, and may interfere with the development of a vaccine and the
EP

management of the disease (Kochakarn et al., 2016).

CC

4. Conclusions
A

Epidemiological surveillance of these arboviruses is of great importance, as their

dispersion and variability are increasing, and is dependent on factors such as climate,

ecology, and human migration. The emergence and spread of novel pathogens into new

areas as the case of Mayaro virus (MAYV) (Acosta-Ampudia et al., 2018) and Oropuche

30
(Romero-Alvarez & Escobar, 2018), and the reemergence of unattended pathogens with

alterations in their virulence, seems to be imminent, as in the case of ZIKV and the

Madariaga virus in Central and South America (Blohm et al., 2018; Luciani et al., 2015).

New outbreaks in different parts of the world can initiate epidemics with more severe

PT
manifestations of disease than were initially observed. Therefore, it is necessary to continue

with research that clarifies the mechanisms of pathogenicity, virulence factors, and

RI
transmission mechanisms of these viruses. Perhaps even more importantly, studies that

SC
directly examine the vector will allow us to establish the role of these mosquitoes in the

selection of pathogenic viruses or in the variability of genotypes that cause infections in

U
humans.

N
In addition, we should note that studies carried out with the aim of genotyping these
A
arboviruses are greatly important as they seek to establish relationships between genotype
M

and dispersion, clinical severity, and/or cycles of transmission to improve the mechanisms
D

of prevention and control, and to anticipate possible new epidemics. Although, the majority
TE

of studies already conducted are concordant regarding the genotypes defined for each virus,

some inconsistencies among different markers are evident, and it is vitally important to
EP

ensure that the genotypes have sufficient support and agreement between the different

algorithmic methods. Therefore, there are several factors that should be actioned as we
CC

move forward with this vital research: 1) specifically defining the statistical and

phylogenetic requirements for establishing, or ruling out, a new genotype; 2) establishing a

A

consensus on the markers that should be used for virus genotyping, which is particularly

important considering that currently, several studies can report different primers that

amplify the same regions; and 3) considering the increasing access to whole genome

31
sequencing technology, the small sizes of viral genomes, and that there are discordances

between the sequences of unique markers for typing, genomic sequences should be used as

the basis of robust genotyping. Studies should also include samples from different sources

to provide an accurate panorama of the molecular epidemiology of these viral agents. This

PT
is relevant since the already existance of publica database as the NIAID Virus Pathogen

Database and Analysis Resource (ViPR) (www.viprbrc.org) among others that would

RI
improve the genotyping of viral agents.

SC
Finally, it is clear that it is necessary to better understand the genotyping criteria for

these viruses, and it is imperative to consider phylogenomics as an alternative tool that

U
could help to solve many of the established doubts, not only with regard to nomenclature
N
and typing, but also other aspects of scientific interest. Therefore, in light of the current
A
published data, an international consensus is needed to revise the current nomenclature for
M

genotype assignment of infectious viral agents. Here we have only investigated some of the
D

more prevalent arboviruses, but future studies should also consider the true genotype
TE

assignment for other viral agents.

EP

Acknowledgments
CC

We thank Tamsin Sheen, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a

draft of this manuscript.

A

32
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Figure legends

Fig. 1. Molecular phylogenetic analysis based on the Env gene sequences of dengue virus

(DENV), yellow fever virus (YFV), Zika virus (ZIKV), chikungunya virus (CHIKV),

Mayaro virus (MAYV), and Usutu virus (USUV).

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All sequences were obtained from GenBank. The evolutionary history was inferred using

the maximum likelihood (ML) method based on the Tamura-Nei model with 1000

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bootstrap replicates. The percentage of trees in which the associated taxa clustered together

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is shown next to the branches. The tree is drawn to scale, with branch lengths measured in

the number of substitutions per site. The analysis involved 86 nucleotide sequences and

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was conducted in MEGA7.
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A
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CC
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Fig. 2. Distribution of dengue virus, yellow fever virus, Zika virus, chikungunya virus,

Mayaro virus, and Usutu virus according to reported human cases.

The colors and figures on the map correspond to each of the viruses, and data are based on

human infection reports obtained by the Centers for Disease Control and other references

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(Anez et al., 2012; Calzolari et al., 2010; Calzolari et al., 2013; Engel et al., 2016; Halsey et

al., 2013; Pecorari et al., 2008; Savini et al., 2011; Talarmin et al., 1998). The map was

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designed using ArcGIS v10.3.

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Fig. 3. Location and distribution of chikungunya virus (CHIKV) genotypes.

The colors and figures show the location of each CHIKV lineage. Data were obtained from

previously published studies (Mowatt & Jackson, 2014; Nunes et al., 2015;

Pulmanausahakul, Roytrakul, Auewarakul, & Smith, 2011; Volk et al., 2010). The map was

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designed using ArcGIS v10.3.

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Fig. 4. Approximation of the genotype location by dengue virus (DENV) serotype.

The figures on each map indicate the serotype location of the DENV strain, as well as the

genotype of each (dotted lines). In some cases, no data were obtained on the specific

genotype (Unknown). Serotype and genotype data were obtained from previously published

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studies (Badii et al., 2007; Brathwaite Dick et al., 2012; R. Chen & Vasilakis, 2011; Coffey

et al., 2013; Goncalvez et al., 2002; Duane J. Gubler, 1998; Guzman & Kouri, 2003; S. B.

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Halstead, 1980; Scott B. Halstead, 2003; Holmes & Twiddy, 2003; Karbaat, Jonkers, &

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Spence, 1964; Murray et al., 2013; Ramos-Castañeda et al., 2017; Suleman et al., 2017;

Vasilakis & Weaver, 2008; WHO, 2016a). The map was designed using ArcGIS v10.3.

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