Acta Tropica 147 (2015) 12–16

Contents lists available at ScienceDirect

Acta Tropica

jo urnal homepage: www.elsevier.com/locate/actatropica

Detection of Mayaro virus infections during a dengue outbreak in

Mato Grosso, Brazil

a a a

Carla Julia da Silva Pessoa Vieira , David José Ferreira da Silva , Eriana Serpa Barreto ,

b c c

Carlos Eduardo Hassegawa Siqueira , Tatiana Elias Colombo , Katia Ozanic ,

d e f

Diane Johnson Schmidt , Betânia Paiva Drumond , Adriano Mondini ,

c a,

Maurício Lacerda Nogueira , Roberta Vieira de Morais Bronzoni ∗

a

Instituto de Ciências da Saúde, Universidade Federal de Mato Grosso, Sinop, MT, Brazil

b

Laboratório Municipal de Análises Clínicas, Sinop, MT, Brazil

c

Faculdade de Medicina de São José do Rio Preto, São José do Rio Preto, SP, Brazil

d

Tufts University, North Grafton, MA, USA

e

Universidade Federal de Juiz de Fora, Juiz de Fora, MG, Brazil

f

Universidade Estadual Paulista, Araraquara, SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Arboviruses are common agents of human febrile illness worldwide. In dengue-endemic areas illness due

Received 19 February 2014

to other arboviruses have been misdiagnosed as dengue based only on clinical–epidemiological data. In

Received in revised form 30 January 2015

this study we investigated the presence of Brazilian arboviruses in sera of 200 patients presenting acute

Accepted 17 March 2015

febrile illness, during a dengue outbreak in Sinop, MT, Brazil. The results showed that 38 samples were

Available online 24 March 2015

positive to Dengue virus (DENV) type 1, two samples to DENV type 4, and six to Mayaro virus. These results

indicate that arboviruses others than DENV are circulating in Sinop and the surrounding region, which are

Keywords:

going undiagnosed. In addition, molecular and evolutionary analyses indicate that two MAYV genotypes

Arboviruses

are co-circulating in Mato Grosso, Brazil. Thus, a strong surveillance program must be implemented to

Febrile illness

RT-PCR evaluate and monitor the distribution and the true importance of non-dengue arboviruses in the etiology

of acute febrile illnesses.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction difficult, especially during the acute phase of illness, where symp-

toms are nonspecific (Calisher, 1994).

Arboviruses are a large group of zoonotic RNA viruses main- Dengue is the most prevalent arboviral disease in Brazil. Over

tained in nature in cycles involving hematophagous arthropod the last 31 years, successive epidemics involving all four dengue

vectors and a broad range of vertebrate hosts (Calisher, 1994). serotypes (DENV-1 to 4) have been occurring in the country

Togaviridae Fla-

Most of these arboviruses belong to the families , (Figueiredo, 2012). There is growing evidences indicating that other

viviridae Bunyaviridae

, and . Mayaro virus (MAYV), dengue virus arboviruses relevant to public health such as YFV (Moreno et al.,

(DENV), yellow fever virus (YFV), Rocio virus (ROCV), Saint Louis 2011), SLEV (Bernardes-Terzian et al., 2011), MAYV (Azevedo et al.,

encephalitis virus (SLEV), and Oropouche virus (OROV) are respon- 2007; Mourão et al., 2012; Zuchi et al., 2014), and OROV (Bernardes-

sible for more than 95% of the human arbovirus diseases cases Terzian et al., 2009; Azevedo et al., 2007; Mourão et al., 2009) are

described in Brazil (Bernardes-Terzian et al., 2009; Mondini et al., present and causing outbreaks in different regions.

2007; Vasconcelos et al., 1998). Although many arboviral infections In DENV-endemic areas illness due to co-circulating arboviruses

in humans are asymptomatic or result in a mild to moderate febrile have often been misclassified, and frequently are diagnosed as

illness, some arboviruses can cause arthritis, hepatitis, hemorrhagic dengue solely based on clinical–epidemiological data (clinical signs

disease, encephalitis, and death. In most cases, a differential diag- compatible with dengue and occurrence of an outbreak, accord-

nosis between arboviruses based on clinical grounds can be very ing to the Brazilian Ministry of Health). Therefore, it has been

postulated that many febrile cases due to others arbovirus than

DENV are misdiagnosed and the incidences of these infections

∗ Corresponding author. Tel.: +55 66 3531 1663.

are much higher than reported (Figueiredo, 2012; Bernardes-

E-mail addresses: [email protected], [email protected]

(R.V.d.M. Bronzoni). Terzian et al., 2011).

http://dx.doi.org/10.1016/j.actatropica.2015.03.020

0001-706X/© 2015 Elsevier B.V. All rights reserved.

C.J.d.S.P. Vieira et al. / Acta Tropica 147 (2015) 12–16 13

Brazil is a tropical country with more than 50% of the region different set of primers to amplify the S segment (Saeed et al.,

covered by rainforests or other natural ecosystems, which provide 2000; Moreli et al., 2002) and one set to amplify the M segment

an ideal environment for the maintenance of zoonotic arboviruses (Vasconcelos et al., 2011).

with the potential for emergence as significant human pathogens

(Figueiredo, 2007). Both natural and manmade ecological changes

2.4. Nucleotide sequencing and analyses

can have a significant impact on public health due to the emergence

and reemergence of arboviruses, however, little is known about the

Amplicons obtained in the D-RT-PCR were directly sequenced

geographic range, relative impact, and epidemiologic characteris-

using BigDye v3.1 Terminator, and an ABI377 automatic sequencer

tics associated with most arbovirus infections in this region.

(Applied Biosystens, Foster City, CA, USA) using the genus primers.

In this study we have investigated the presence of arboviruses

The nucleotide sequences were subjected to the basic local

in sera of patients presenting with acute febrile illness, during a

alignment search tool (BLAST) (www.ncbi.nlm.nih.gov/blast) and

dengue outbreak in Sinop, in the state of Mato Grosso, Brazil. We

submitted to GenBank.

have used molecular methods based on duplex-RT-PCR to detect

In order to get some insights into the MAYV genotypes circu-

Alphavirus and Flavivirus genuses followed by multiplex-nested-

lating in Sinop-MT, Brazil, nsP1 sequences from MAYV, Una virus

PCR methods to identify the species from each genus. We have

(UNAV), Semliki Forest virus (SFV), Getah virus (GV) and Ross River

detected MAYV in six patients clinically diagnosed with DENV and

virus (RRV) were retrieved from Genbank and aligned using Clustal

demonstrated the co-circulation of two MAYV genotypes in Mato

W, implemented in MEGA 6 (Tamura et al., 2013). The nucleotide

Grosso.

substitution model that best fit the data was chosen using jModel-

Test2 (Darriba et al., 2012). Thus, K80 + G (Kimura, 1980) was used

2. Materials and methods

to reconstruct phylogenetic trees, by Maximum Likelihood method,

with 1000 bootstrap sampling. Sequences from UNAV, SFV, GV and

2.1. Study site

RRV were used as the outgroups. Evolutionary divergence between

and within groups of MAYV strains were estimated by sequence

The study was conducted in Sinop (11◦50′53′′S and

comparisons, using K80 + G model (Kimura, 1980), implemented

55◦38′57′′W), located 503 km from the capital of Cuiabá, and

in MEGA 6 (Tamura et al., 2013). Bayesian analyses were also per-

is considered the main city of the northern state of Mato Grosso,

formed using software from BEAST package v.1.8 (Drummond et al.,

Brazil. The city was founded in 1974 and today has a population of

2002–2014). One hundred million chains were run, eliminating the

120,000 inhabitants with approximately 17% of them living in rural

first 10 million chains. Data and trees were sampled every 5000

areas. Principle economic activities include agriculture, animal

steps. The convergence of parameters was verified with the Tracer

husbandry, logging, and human services, such as healthcare and

v1.6.0 (Rambaut et al., 2003–2013) and the maximum clade credi-

education. It is located in a geographical transition zone between

bility tree was displayed in FigTree v1.4.2 (Rambaut, 2006–2014).

savannah and rainforest. The climate is tropical with average tem-

perature of 28 ◦C and high precipitation levels (1900 mm/year),

particularly from January to March. 3. Results and discussion

2.2. Clinical samples From the 200 clinical samples analyzed, arboviruses genomes

were detected in 46 samples (23%). The remaining 154 samples

Patients seen by the Sinop Municipal Emergency Services, are febrile illnesses, which are not related to the arboviruses that

between January 2011 and February 2012, presenting with a clini- were tested herein. DENV-1 sequences were detected in 38 sam-

cal diagnosis of dengue, were selected for this study. Two hundred ples, DENV-4 sequences were detected in two samples, and MAYV

serum samples were collected from patients with acute febrile was detected in six samples. DENV-1, 4 and MAYV each showed

illness, clinical symptoms of headache, diffuse body pain, arthri- greater than 98% sequence identity with DENV or MAYV sequences

tis and retro-orbital pain. Samples were transported on dry ice obtained from GenBank. The NS5 gene partial sequences of DENV-

to the Laboratory of Immunology and Molecular Biology of the 1 and DENV-4, and nsP1 gene partial sequence of MAYV were

Universidade Federal de Mato Grosso (UFMT) campus of Sinop submitted to GenBank, and the accession numbers are KF305670,

and stored at 80 C. Viral RNA was isolated from 140 ␮L of KF305671, and KF305672, respectively. Dengue is considered to be

− ◦

serum using the QIAamp Viral RNA Mini kit (QIAGEN, USA), as per one of the most important and widespread reemerging infectious

the manufacturer’s instructions. The Ethics Committee from Júlio diseases in tropical countries. DENV (serotypes 1–4) is endemic in

Müller University Hospital—UFMT approved this study (128/CEP- many regions of Brazil with 700,000 cases per occurring over the

HUJM/2011). last decade (Figueiredo, 2012). The Aedes aegypti mosquito, with a

range of dissemination across most of Brazil including Mato Grosso,

2.3. RT-PCR assays is the primary vector for DENV transmission. The recent years, Mato

Grosso has suffered successive epidemics of DENV1–3 including:

Initially, duplex-RT-PCR (D-RT-PCR) for Flavivirus and Alphavirus 34,851 cases with 53 deaths in 2010, 4004 cases with 4 deaths

genus detection was performed using specific primers targeting in 2011, and 26,451 cases with 13 deaths in 2012 (Ministério da

the NS5 and nsP1 regions, respectively, followed by multiplex- Saúde, 2013). DENV-4 was re-introduced into Brazil in 2008 in

nested-PCR (M-N-PCR) performed using inner primers to DENV Manaus, the capital of Amazonas state (Figueiredo et al., 2008).

types 1 to 4. DENV negative samples were tested by conventional From there the DENV-4 has spread to other geographic regions,

Nested-PCRs (N-PCR) for YFV, SLEV, ROCV and ILHV identification; including the North (Roraima, Amazonas and Para states), North-

a separate M-N-PCR was performed to identify MAYV, Venezuelan east (Bahia, Pernambuco and Piaui states), and Southeast (Rio de

equine encephalitis virus (VEEV), Eastern equine encephalitis virus Janeiro and São Paulo states) of Brazil. In February 2012, DENV-

(EEEV), Western equine encephalitis virus (WEEV), and Aura virus 4 was reported for the first time in Mato Grosso. Interestingly in

(AURAV) from Flavivirus negative samples. These RT-PCR meth- this study, we have detected DENV-4 in two patients samples from

ods were performed as previously described (Bronzoni et al., 2005, Sinop city collected in February of 2011. It has been shown that

2004). Finally, the Alphavirus and Flavivirus negative samples were an introduction of a new dengue serotype into a susceptible pop-

tested by RT-PCR methods for the presence of OROV using two ulation can cause an increase in disease severity and/or a large

14 C.J.d.S.P. Vieira et al. / Acta Tropica 147 (2015) 12–16

Fig. 1. Bayesian phylogenetic analysis of Mayaro virus (MAYV). The analysis was performed using 21 nucleotide sequences of 106 bp. The maximum clade credibility tree is

shown, summarizing data from 18.000 trees, and posterior probabilities with values 90 are shown in the nodes. The scale represents 0.05 nucleotide substitution/site/year.

≥

Sequences are identified by their GenBank accession number and name. MAYV detected in this study is shown in bold. (gL-E1/E2) and (gD-E2/E1) indicate MAYV strains

previously genotyped based on the analysis of E2/E1 sequences (Powers et al., 2006). Analysis was carried out using BEAST package v.1.8.1, Tracer v.1.6.0 and FigTree v.1.4.2.

outbreak in an urban area (Rico-Hesse, 2003). Detection of viral fever can be. A systematic laboratory-based surveillance for acute

infection through surveillance or diagnosis is a necessary step in febrile illnesses can result in the discovery of non-dengue cases that

responding to and resolving the emergence of epidemics within would likely be missed during large dengue outbreaks. Recently,

a population. However, dengue surveillance may be neglected in during surveillance programs, MAYV (Mourão et al., 2012; Zuchi

many cities in Brazil, particularly in regions with limited economic et al., 2014) and OROV (Mourão et al., 2009) were detected in

development. patients with clinical diagnoses of dengue fever. In 2008, one case of

All 200 patients in the current cohort had been diagnosed with SLEV was reported in a patient from Ribeirão Preto, São Paulo state,

dengue based on clinical and epidemiological criteria, however 160 with an acute febrile illness that was initially diagnosed as dengue

(80%) patients were dengue negative by M-N-PCR assay. It is evi- by positive IgM-ELISA (Maia et al., 2014). In addition, during a 2006

dent that during a large dengue outbreak others febrile illnesses are DENV-3 outbreak in São José do Rio Preto, São Paulo state, SLEV

occurring but being misdiagnosed. Infections, such as poliomyeli- genomes were detected in 12 clinical dengue suspected samples,

tis, rabies, measles, rubella, hepatitis, malaria, leptospirosis, or and it was considered the first SLEV outbreak registered in Brazil

other arboviruses can induce similar symptomatology (Fonseca and (Mondini et al., 2007). In this last incidence, surveillance was car-

Figueiredo, 2005; Vasconcelos et al., 2005). Our detection of six ried out using the same M-N-PCR methodology based on Bronzoni

cases of MAYV would have been missed if not for our more intensive et al. (2005) as used in the present study. This methodology is a

RT-PCR surveillance for other arboviruses. suitable tool for detecting Flavivirus and/or Alphavirus infections,

The MAYV sequence was detected in male and female patient and can be applied when a differential diagnosis is required.

samples between 14 and 62 years of age. The majority of patients Phylogenetic trees reconstructed based on Maximum likelihood

presented with fever (6/6), headache (6/6), nausea and vomiting (data not shown) and Bayesian methods presented the same clus-

(6/6), myalgia (6/6), arthralgia (5/6), ocular pain (4/6), and abdom- tering patterns, demonstrating that MAYV was subdivided into two

inal pain (3/6). Leukocytes and platelet counts were carried out clusters (Fig. 1). Previous studies have demonstrated the exist-

within 5 days of onset of symptoms, and were considered within ence of two MAYV genotypes based on E2/E1 gene sequences.

the normal range in 4/6 patients. Two patients showed low levels of Genotype D contains isolates from a wider geographic area span-

leucocytes and one of the two patients also had low platelet levels ning different countries of South America, while genotype L has

(Table 1). a more limited distribution to the north region of Brazil (Powers

Mayaro fever is a non-fatal acute febrile illness, characterized et al., 2006; Azevedo et al., 2009). Although we have analyzed only

by myalgia, incapacitating arthralgia, headache, epigastric pain, partial nsP1 sequences of MAYV, we have observed similar clus-

maculopapular rash, nausea, and vomiting (Pinheiro and LeDuc, tering when E2/E1 sequences were used. In addition, strains that

1988). In this study, the six MAYV positive patients had similar were formerly identified as genotype L (such as strain BeAr505411)

symptoms, which were typically dengue-like, illustrating how dif- were also grouped together with MAYV MT/SINOP/210/2011. In

ficult clinically differentiating MAYV disease from classical dengue this way, one might infer that MAYV MT/SINOP/210/2011 belongs

C.J.d.S.P. Vieira et al. / Acta Tropica 147 (2015) 12–16 15

to genotype L (cluster 1). Up to now, all the MAYV strains belong- )

3

ing to genotype L were detected in the north region of Brazil

(Para state), from 1955 up to 1991. However we demonstrated Platelets count (/mm

266,000 111,000 173,000 148,000 147,000 139,000

here that genotype L is also circulating in the central region of

the country in more recent years. Additionally, the phylogenetic

analysis also indicated that two MAYV genotypes are sympatric in

) 3

Mato Grosso state (MAYV MT/SINOP/210/2011; MT/VG147/2012,

MT/VG20/2012, MT/CBA9/2012 strains from cluster 1—genotype Leukocytes count (/mm

7460 4020 2480 12,460 12,780 18,300

L, and BR/MT SOR246/2012 and BR/MT CBA322/2012 strains from

cluster 2—genotype D). Nucleotide divergence among sequences

within cluster 1 and 2 presented low and similar values

(2.39 0.09% and 2.21 0.78%, respectively), while it was higher

± ±

(8.98 2.74%) when comparing cluster 1 to cluster 2. These date ±

corroborate with a previous study that suggests distinct transmis-

Vomiting X X X X X X

sion patterns may have played a role in the evolution and current

maintenance of MAYV (Powers et al., 2006).

Brazil is a large tropical country with 54.4% of the territory

covered by rainforests and other ecosystems with a rich flo- pain

ral and faunal biodiversity (Ministério do Meio Ambiente, 2013).

These ecosystems provide ideal conditions for the maintenance of

arboviruses in a variety of zoonotic cycles. To date more than 200

Abdominal X X X arbovirus species have been isolated in Brazil, 40 of which cause

diseases in humans (Rosa et al., 1998). In the last three decades,

extensive native forest areas in Sinop and surrounding regions have

been razed for logging, agriculture, cattle ranching and urbaniza-

tion, resulting in more susceptible people living and working in

closer proximity to the forests, thereby potentially being exposed

Arthralgia X X X X X to numerous arbovirus transmission vectors. Inadequate environ-

mental management can disrupt the forest’s stable state, leading to

increased incidence of emerging or reemerging arboviruses, such

as YFV, MAYV and OROV (Vasconcelos et al., 2001). In addition,

ecological changes produced by man may increase vector preva-

lence, create new reservoirs or induce arboviruses to adapt to new

Myalgia X X X X X X

maintenance cycles (Figueiredo, 2007).

Currently, most MAYV infections in humans are associated with

occupational or recreational exposure in rainforest environments

(Pinheiro and LeDuc, 1988), however, MAYV outbreaks have been

pain reported in South American rural communities including those in

Brazil, Peru, Bolivia, Venezuela, and French Guyana (Azevedo et al.,

Ocular X X X X 2009; Mourão et al., 2012; Zuchi et al., 2014). There is a concern over

the possibility of urban transmission of MAYV since experimental

studies indicate that this virus can also infect and be transmitted

by A. aegypti (Long et al., 2011). Large epidemics of MAYV could

cause temporary incapacitation inability to work in many patients,

which may require hospitalization, thereby significantly impacting

Headache X X X X X X public health.

patients.

4. Conclusions

These results indicate that arboviruses others than DENV are positive Symptoms Fever X X X X X X

circulating in Sinop and the surrounding region, which are going

fever

undiagnosed. It is essential that a strong surveillance program be

implemented using reliable diagnostic methodologies to evaluate

and monitor the distribution and the true importance of non- Mayaro

dengue arboviruses in the etiology of acute febrile illnesses. It was

from

demonstrated the co-circulation of two MAYV genotypes in Mato 14 21 48 19 62 24

Age (years)

Grosso. Therefore, a more thorough epidemiologic and evolution-

ary studies are required to better understand the magnitude of findings

MAYV and other arbovirus infections in Brazil.

Female Female Male Female Male Male Gender Acknowledgement laboratorial

and

The authors gratefully acknowledge the Conselho Nacional de 1

Desenvolvimento Científico e Tecnológico (CNPq) for financial sup-

No 1

2 3 4 5 6

Table Clinical port (478876/2010-6).

16 C.J.d.S.P. Vieira et al. / Acta Tropica 147 (2015) 12–16

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