Dengue Virus 4 (DENV-4) Re-Emerges after 30 Years in Brazil: Cocirculation of DENV-2, DENV-3, and DENV-4 in Bahia

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1 Jpn. J. Infect. Dis., 68, 45 49, 2015 Original Article Dengue Virus 4 (-4) Re-Emerges after 30 Years in Brazil: Cocirculation of -2, -3, and -4 in Bahia Gubio Soares Campos 1, Aryane Cruz Oliveira Pinho 1, Claudio Jose de Freitas Brandãao 2, Antonio Carlos Bandeira 2, and Silvia Ines Sardi 1 * 1 Laboratory of Virology, Department of Biointeraction, Health Science Institute, Federal University of Bahia; and 2 Hospital Aliança, Salvador, Bahia, Brazil SUMMARY: Dengue fever (DF) is a mosquito-borne viral diseaseof great concern in tropicaland subtropical regions of the world. One important cause of the increase in DF is rapid development and urbanization has led to proliferation of the Aedes aegypti mosquito, the vector responsible for transmission of the illness. Surveillance of dengue virus () infection in Brazil shows the predominance of -1, -2, and -3 until This study reports the reappearance of -4 in Brazil for the first time in 30 years.serum samples were collected from individuals (n = 214) exhibiting fever and muscular pain in Bahia, Brazil, during These samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR)/nested PCR, which revealed that 82z ofsampleswere positive for -4; most were older age groups and exhibited a serological pattern consistent with a primary infection. The cocirculation of multiple serotypes within thesamecityplaces thepopulation at risk for a fatal form of the disease. Therefore, with the increasing incidence of severe DF cases, early diagnosis will be a priority for public health efforts in Brazil. INTRODUCTION Dengue fever (DF) is a mosquito-borne viral disease of great concern in tropical and subtropical regions of the world. The World Health Organization estimates that million cases of DF occur annually. Morbidity and mortality due to dengue virus () infections have increased dramatically in recent decades worldwide (1). One important cause of the increase in infections is rapid development and urbanization, which has led to proliferation of the Aedes aegypti mosquito, the vector responsible for transmission (2). DF is clinically characterized as a nonspecific febrile disease with myalgia and arthralgia. The more severe and life-threatening form of DF also involves bleeding, thrombocytopenia, and increased vascular permeability that can result in death (3). belongs to the family Flaviviridae and has 4 serotypes: -1, -2, -3, and -4. Its positive single-stranded RNA genome encodes 10 proteins: 3 structural (C, prm, and E) and 7 nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (4,5). Among these proteins are highlighted protein E and NS1. The E glycoprotein is highly immunogenic, and NS1 protein is expressed on the surface of infected cells and can be detected in the serum (6). DF is an important health problem in Brazil. Every year, the fight against proliferation of the mosquito is Received February 6, Accepted June 11, J-STAGE Advance Publication November 25, DOI: /yoken.JJID *Corresponding author: Mailling address: Instituto de Ciâencias da Sa áude Laborat áorio de Virologia, Universidade Federal da Bahia, Av. Reitor Miguel Calmon s/n Vale do Canela-Salvador, Bahia, Brazil. Tel/Fax: , sissardi@yahoo.com.br intensified, with community workers visiting homes to detect possible reservoirs that facilitate the proliferation of the mosquito. Since 1987, infection has been an important health problem in Bahia (7). In March 2011, the public health authorities in Salvador, Bahia, registered the reappearance of -4 in the country for the first time in 30 years (8). Interestingly, -4 isolates in Salvador are of genotype I, which is derived from Asiatic lines (GenBank JX523699, JX523700, JX523701, and JX ) that had not been detected previously in Brazil. With the increasing incidence of infection, early diagnosis principally during the acute phase of the illness will be a public health priority in Brazil (9,10). Routine diagnostic test for are based on the detection of NS1 viral protein and serum anti- IgM/IgG (11,12). NS1 is a highly conserved protein present in high concentrations in serum from days 1 9 after the onset of fever in infected or reinfected patients (13). During primary viral infection, IgM becomes detectable on days 3 5 after the onset of illness and persists for up to 3 months. IgG appears during the second week of infection and persists for life (14 16). The detection of viral nucleic acid, the gold standard test, can be used to identify circulating serotypes (17). However, this test is not used in routine diagnoses. The aim of this study is to report the appearance of the new -4 serotype circulating in the city of Salvador during and to describe the serological profile of infected patients based on the detection of NS1 and anti- IgM/IgG. MATERIALS AND METHODS Patient serum samples: During , clinicians from Aliança Hospital (Salvador, Bahia) collected sera from individuals (n = 214) with suspected cases of DF 45

2 (exhibiting fever and 2 of the following symptoms: frontal headache, retro-orbital pain, myalgias, arthralgias, hemorrhagic manifestations, or rash) (1). The collected samples were stored at -70 C until laboratory processing. This research study was approved by the Ethics Committee for Human Research of Salvador University under protocol number n FR: RNA extraction: The RNA was extracted from 140 ml of serum using the QIAamp viral RNA Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. RNA detection by reverse transcriptionpolymerase chain reaction (RT-PCR) and nested PCR: RNA (8 ml) was reverse transcribed (RT) using the SuperScript II Reverse Transcriptase kit (Invitrogen, Carlsbad, CA, USA), and viral serotyping was performed as reported by Lanciotti et al. (17). Briefly, cdna obtained by RT was amplified using the D1 forward (5 -TCAATATGCTGAAACGCGCGAGAAAC CG-3 ) and D2 reverse (5 -TTGCACCAACAGTCAAT GTCTTCAGGTTC-3 ) primers in PCR Super Mix (Invitrogen). The cycling conditions were 94 C for2min, followed by 35 cycles of 94 C for 30 s, 55 C for1min, and 72 C for 2 min. cdna obtained (5 ml) was used for an additional round (nested PCR) of 95 C for 2 min and 20 cycles of 94 C for 30 s, 55 Cfor1min,and72 Cfor 2 min of amplification using the D1 primer and typespecific internal primers for the C-PrM genomic junction region as follows: TS1--1 reverse, 5 -CGTC TCAGTGATCCGGGGG-3 ; TS2--2 reverse, 5 - CGCCACAAGGGCCATGAACAG-3 ; TS3--3 reverse, 5 -TAACATCATCATGAGACAGAGC-3 ; TS4--4 reverse, 5 -CTCTGTTCTCTTAAACAA GAGA-3. Amplified products of 482 nt (-1), 119 nt (-2), 290 nt (-3), and 392 nt (- 4) were analyzed on ethidium bromide-stained 2z agarose gels. Detection of NS1 antigen, IgM, and IgG: The sera were tested for NS1 protein and anti- IgM/IgG according to the manufacturer's instructions using Dengue Duo Test Bioeasy (Bioeasy Diagnostica, Belo Horizonte, MG, Brazil). Statistical analysis: Sensitivity and specificity values were calculated using RT-PCR as the reference method. The agreement between the tests was calculated accordingtothekappaindex(18). RESULTS Cohort age distribution: The age distribution of included patients (n = 214) is presented in Fig. 1A. This graph shows that 61z of patients were years of age. Children and adolescents represented 29z of patients. Unfortunately, the age of a small number of patients was not registered (8z, 17/214). Detection of anti- IgM antibodies, anti- IgG antibodies, and viral protein NS1: All 214 samples collected between May 2011 andmay 2012 were subjected to the immunochromatographic Dengue Duo Test. Of these samples, 183 (85.5z) were positive and 31 (14.5z) were negative. Fig. 1B shows the distribution of the positive samples according to the 3 serological markers of the test: 147 were positive for viral protein NS1, 54 were positive for anti- IgM, and 32 were positive for anti- IgG. The intersections of the graph represent the simultaneous detection of the serological markers and demonstrate that 11 samples tested positive for all 3 serological markers. Fig. 1B also highlights the high number of samples positive for anti- IgM antibodies (n = 54). However, the presence of anti- IgG antibodies (n = 32) was low compared with that of anti- IgM antibodies and was always combined with one or both of the other markers. Detection and identification of serotypes: Serum samples were analyzed for the viral detection using RT-PCR and nested PCR. Of 214 samples analyzed, 43.4z (n = 93) were -positive and 56.6z (n = 121) were -negative. Of positive samples, serotyping revealed that 82.8z (n = 77) were -4, 14z (n = 13) were -2, and 3.2z (n = 3) were -3. Comparison of viral detection between assays based on NS1 protein and viral genome: Of 214 samples analyzed, 78 samples were positive for both tests, and 52 samples were negative for both tests. However, RT- PCR did not detect in 69 samples that were positive for NS1. Considering that RT-PCR is the gold standard diagnostic test, statistical analysis demonstrated that the detection of NS1 by the Dengue Duo Test had a sensitivity of 53z, a specificity of 77z, anda positive predictive value of 83.3z compared with viral identification by RT-PCR. The Kappa index revealed a reasonable agreement (k, 0.254) between the 2 tests. Simultaneous detection of viral protein NS1, anti- IgM/IgG antibodies, and viral genome by RT- PCR: Table 1 compares the results for virus detection using the 3 serological markers (viral protein NS1, anti- IgM/IgG antibodies) and viral genome detection by RT-PCR. Of 214 samples, 193 were positive for either a serologic maker or viral genome by RT-PCR. Twenty-one samples were negative for all tests. It is noteworthy that 118 samples (118/214) were positive for viral protein NS1 but negative for anti- antibodies. Ninety-nine of these 118 samples (83.8z) were from patients older than 25 years of age (see also Table 2). Another finding reported in Table 1 is the high number of individuals (n = 33)whotestedpositiveonlyfor anti- IgM (predominantly children aged 0 14 [14/33]) (Table 2) or who tested positive for anti- IgM and IgG (n = 20). The presence of anti- IgG alone was detected in a small number of patients (n = 12). Table 2 shows these test results in relation to the patients' ages. Of patients aged years, 42z had Table 1. Comparison of the results for the detection of viral nucleic acid (RT-PCR), antigen NS1 and anti- (IgM/ IgG) antibodies in sera from patients with suspected infections NS1+/ RT-PCR+ NS1+/ RT-PCR- NS1-/ RT-PCR+ NS1-/ RT-PCR- Total IgM-/IgG IgM+/IgG IgM-/IgG IgM+/IgG Total : positive result. -: negative result. 46

3 Dengue Virus 4 in Brazil Fig. 1. Patient age distribution and serological markers for infection. A: Distribution of patient ages with clinically suspected cases of DF at Hospital Aliança during the study period of in the city of Salvador, Bahia. B: Simultaneous detection of serotype-specific antibodies and NS1 antigen in sera from 183 patients infected with. Each circle and its intersections represent the number of patients positive to NS1 antigen (NS1), anti- IgM (IgM), and/or anti- IgG (IgG) during the viral infection. Table 2. Results for the detection of viral nucleic acid (RT-PCR/nested PCR), and anti- (IgM/IgG) antibodies distributed based on the age of the patients Age RT-PCR (+) IgM- IgG- IgM+ IgG- IgM- IgG+ IgM+ IgG > Not Reg. 1) Total : positive result. -: negative result. 1) :agenotregistered. positive results by RT-PCR (39/93), of which -4 was the most frequently detected serotype (89.7z, 35/39 positive cases). Patients aged years had the second highest rate of positive results by RT-PCR. This age group had 19.4z of positive cases (18/93), of which -4 was the only serotype detected (100z; 18/18 positive cases). In these 2 age groups, the detection of anti- IgM or IgG antibodies was low. -2 was the most frequently detected serotype in children and adolescents (77.7z; 7/9 positive cases). Serological profiles of individuals with only anti- IgG antibodies: A significant percentage of patients with only anti- IgG antibodies were between the ages of 46 and 63 years (66.66z; 8/12) (Table 3). Many of these patients were also NS1-positive (75z; 9/12). This profile is compatible with the acute phase of a secondary infectionor reinfection. We also noted that 3 patients were positive for both -4 and NS1 pro- 47

4 Table 3. Detailed analysis of patients with only anti- IgG antibodies Sample IgG+ (Age) 1) serotype NS1 2) detection 1 (59) -4 Positive 2 (18) -4 Positive 3 (58) -4 Positive 4(20) -2 Negative 5 (56) Negative Positive 6 (52) Negative Positive 7 (27) Negative Positive 8 (48) Negative Positive 9 (63) Negative Positive 10 (52) Negative Positive 11 (13) Negative Negative 12 (46) Negative Negative 1) : Viral detection by RT-PCR. 2) : Dengue Duo Test Bioeasy. tein (patients 1, 2, and 3). Only 2 individuals (patients 11 and 12) who were positive for anti- IgG were also negative for NS1 and the viral genome. DISCUSSION Cocirculation of multiple serotypes within a city places the population at risk for severe disease. During in Bahia, there were 55,460 confirmed cases of ; nearly half of these cases (48z) were concentrated in 10 counties, including Salvador. Within the state, there was also an increase in the incidence of severe forms of the disease, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS); 18 deaths were reported, including 5 in the city of Salvador (10). Citywide monitoring of infection up until 2010 showed -2 and -3 to be predominant; however, the profile of circulating serotypes changed in 2011(8). Accordingly, -4 was the most predominant serotype during the 12 months of this study. This serotype has not circulated in Brazil in over 30 years. The circulation of the 3 serotypes during 1 year was clearly demonstrated by the diagnostic molecular assays. -4 was predominant, with 82z of patients being infected by this new serotype. The dissemination of -4 could have been facilitated by the population being exposed to a serotype never detected before. The individuals infected with -4 tended to older, consistent with observations made by other researchers when a new serotype is introduced into endemic areas. These data show a situation much different than that of other DF outbreaks, with the introduction of - 4 altering the profile of infection to older inhabitans. This age distribution is contrary to the usual profile of infection, which state health officials claim affects mostly children and adolescents (8,10,19). Despite not having the patients' clinical histories, our results suggest that 70z ofpatientswithwerein the acute phase of infection and were on days 1 9 of the febrile state. This hypothesis is strengthened by data demonstrating NS1 and the circulating virus in the serum with the absence of immunoglobulins (9,14,12,20). The sensitivity of the test used in the hospital was fundamental to the early diagnosis of infection in these patients. Our data confirm what some researchers have demonstrated regarding this immunochromatographic test that it has greater sensitivity for the detection of antigen NS1 than do other tests based on enzyme linked immunosorbent assay (ELISA) methods (11,12). The immune response of patients infected with is easily monitored with the commercially available serological tests. Anti- IgM appears on days 3 5 after the onset of fever, immediately followed by the presence of anti- IgG (14,15). The detection of viral protein NS1 is inversely associated with the presence of antibodies because the formation of immunocomplexes lowers the circulation NS1 in the blood (21,22). Therefore, the presence of antibodies makes it difficult to detect NS1. This phenomenon was clearly observed in our study patients. We observed an inverse relationship between the detection of NS1 protein and anti- antibodies. The immunochromatographic test used in this study showed that the presence of anti- IgM was greater than the presence of anti- IgG, the latter being an immunoglobulin with little relevance in patients studied, as it is an indicator of recurrent infection/convalescence. This profile (high occurrence of -4, increased anti- IgM, low anti- IgG) reinforced the hypothesis that individuals in the acute phase of the disease were undergoing their first exposure to the virus (19,23,24). This response profile was detected in the vast majority of patients in this study. Secondary infections or reinfections are characterized by a transient IgM and IgG response and a rapid decrease in the detection of the circulating virus or NS1 (13,15). This response profile was observed in a group of patients with the presence of anti- IgG alone. In these individuals, the detection of NS1 antigen and/or the virus in the serum suggested a secondary infection during the acute phase. These results confirm findings from secondary infections demonstrating that anti- IgG is higher than that in primary cases, particularly in the early stages of the illness (16,25 28). We also show that RT-PCR is a reliable test for detecting secondary infections when serum samples are collected in the febrile period during the early, acute phase of infection. The probability of reinfection in a city with the endemic virus increases with the age of the individual. Interestingly, 75z of patients in the group with secondary infections were between 40 and 63 years of age. These data contrast with the observation that 50z of patients with anti- IgM antibodies alone were children and adolescents aged 14 years and below. This finding demonstrates that in an endemic population, exposure to the virus begins in early childhood (19,24). Our findings show that infection is a great challenge for the Brazilian public health authorities despite a well-established Aedes mosquito control program. Other factors must be involved in the emergence or re-emergence of this illness, such as population growth, increased tourism, migration, and international commerce, particularly in a heavily visited city such as Salvador. The introduction of the new serotype 48

5 Dengue Virus 4 in Brazil -4 in the endemic population of Salvador distinguishes this city from other areas in Brazil, as all 4 serotypes are circulating. With the spread of all 4 serotypes, the need for rapid, early diagnosis increases, as severe DF cases requiring early clinical treatment will be seen more frequently. Our study demonstrates that a rapid test with high sensitivity (such as the immunochromatographic Dengue Duo Test) that simultaneously detects the serologic markers NS1 and anti- IgM/IgG would be of great utility for early detection and rapid treatment of infection. Acknowledgments We thank the clinicians from Hospital Aliança, Salvador, Bahia and FAPESB for financial support. Conflict of interest None to declare. REFERENCES 1. World Health Organization (WHO). Dengue Guidelines for Diagnosis, Treatment, Prevention and Control. Geneva: WHO and the Special Programme for Research and Training in Tropical Diseases Guzman MG, Halstead SB, Artsob H, et al. 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