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Cite this article: Nitatpattana N, Moné Y, Gouilh MAR, Chaiyo K, Joyjinda Y,et al. (2018) Genetic Diversity of Dengue-4 Virus Strains Isolated from Patients During a Single Outbreak of Dengue Fever, Thailand (2011). J Fever 2(1): 1009.

*Corresponding authorJP Gonzalez, Center of Excellence for Emerging & Zoonotic Animal Disease Kansas State University Office Park, 1800 Kimball Ave. Suite 130, Manhattan, KS, 66502, USA, Tel : 1(301) 332-2237; Email: jpgonzalez@vet.k-state.edu

Submitted: 05 March 2018

Accepted: 10 May 2018

Published: 12 May 2018

Copyright© 2018 Gonzalez et al.

OPEN ACCESS

Keywords• Dengue virus• Dengue virus serotype 4• Genetic• Variants• Thailand

Research Article

Genetic Diversity of Dengue-4 Virus Strains Isolated from Patients During a Single Outbreak of Dengue Fever, Thailand (2011)Narong Nitatpattana5, Yves Moné2, Meriadeg AR Gouilh3,4, Kumchol Chaiyo1, Yutthana Joyjinda5, Supoth Ratchakum1, Supaporn Wacharapluesadee5, Sutee Yoksan1, Thiravat Hemachudha5, Francisco Veas2, Tom Vincent6, and Jean-Paul Gonzalez7*1Institute of Molecular Biosciences, Mahidol University at Salaya Putthamonthon, Thailand2Molecular Comparative Immuno-Physiopathology Lab-UMR-Ministry of Defense, Thailand3Environment and Infectious Risks Research and Expertise Unit, Institut Pasteur, 25-28 rue du docteur Roux, France4Normandy University, Groupe de Recherche sur l’Adaptation Microbienne, France5Molecular Biology Laboratory for Neurological Diseases, Chulalongkorn University, Thailand6O’Neill Institute for National and Global Health Law at Georgetown University Law Center, USA7Center of Excellence for Emerging & Zoonotic Animal Disease, Kansas State University, USA

Abstract

Dengue virus, an RNA virus of the Flaviviridae family, is characterized by its exceptional genetic diversity, starting with its four viral serotypes, an intra-serotype genetic diversity illustrated by phenotypic variations (i.e. pathogenicity) and phylogeographic clusters. Cycles within its mosquito vector play an important role in this genetic diversity. After a limited passage of primary virus isolates, we investigated multiple sequences of dengue virus serotype 4 (DENV-4) isolates of naturally, acutely infected patients of the same geographic origin during the 2011 dengue fever outbreak in Thailand. Four patients were sampled as part of routine diagnosis and full virus sequences were generated after two passages in Vero cells and mosquito cell lines. These isolates consistently cluster with contemporaneous old-world DENV-4 clade (Asian), mainly composed of viruses isolated from Thailand after yearly epidemics from 2000-2011, which originated from the lineage first identified in 1977 in Bangkok.

The DENV-4 secondary virus isolates displayed consistent genotypic variations among each patient’s isolates. These genetic variations ranged respectively for nucleotides and amino acid sequences, from a mean diversity of 1.03 - 1.34% of the full nucleotide sequence substitutions and, 0.40 - 0.85% of the amino acid mutations, respectively. Using the epitope predicting method across all four variants, we identified seventeen epitopes exhibiting an amino acid substitution due to a non-synonymous mutation. One of the variants showed two epitope sites in the NS5 gene: one of the methyltransferase domains and the other of the RNA-dependent RNA polymerase domain containing a predicted single amino acid antigenic substitution change in a critical site that could contribute to the generation of antigenic variants. These original findings support our data that DENV-4 potentially exhibits a high rate of genetic diversity within an outbreak, conferring to DENV-4 a high potential for host and environmental adaptability that could potentiate the development of resistance to vaccines and/or antiviral drugs.

Author Statement: Although dengue virus serotypes are antigenically closely related to one another, all dengue virus strains possess exceptional genetic diversity--including viral serotypes, intra-serotype phenotypic variations, and phylogeographic genetic clusters; such diversity appears as punctual but multiple mutations. Moreover, as an RNA virus, dengue virus has an error-prone polymerase that produces complex and variable virus populations that increase with multiple passages within human and vector hosts during an outbreak. Due to the challenges associated with the development of a live attenuated tetravalent vaccine, it is critical to understand any potential for genetic or phenotypic change with each dengue virus serotype during their multiple replication phases. DENV-4 is responsible for a few extended outbreaks, but is a subject of only a limited number of studies, despite the virus playing the same role in terms of pathogenicity and immune response as the other dengue serotypes. Our study provides some insight on the genetic plasticity of the DENV-4 serotype during the intense replication phases of an outbreak in humans.

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INTRODUCTIONDengue virus (DENV) is one of the most geographically

widespread mosquito-borne viruses affecting humans, with a worldwide distribution especially within the inter-tropical zone. It is the causative agent of acute dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue hemorrhagic fever with shock syndrome (DHS) [1]. Globally, it is estimated that there are 390 million new DENV infections per year and more than 3.9 billion people are at-risk of infection, while neither vaccine nor treatment is available [2]. DENV is a member of the Flaviviridae family presenting four distinct viral serotypes, including DENV-1, DENV-2, DENV-3, and DENV-4. Moreover, such diversity can be observed among each serotype; intra-serotype genetic diversity is displayed by phenotypic variations (i.e. pathogenicity) [3,4] and phylogeographic clusters [5]. The mosquito vector also plays an important role in genetic diversity by selecting virus genotypes of the transmitted DENV serotype [6,7]. Also, as with most RNA viruses, DENV polymerases lack the ability to proofread, and as such, DENV replicates with high mutation rates, demonstrating significant genetic diversity as viral quasispecies when applied to a single infected host showing a multitude of DENV mutant [8,9]. Understanding the role of viral genetic diversity below the species level is important for understanding DENV evolution, as genetic diversity during acute DF infection remains unclear and poorly documented [10]. Altogether, these genetic changes could eventually contribute to the production of new potent viral particles that are highly adapted to their host or their environment. Since the first detection of DENV-4 serotype in Brazil in 1982 [11], DENV-4 appears to re-emerge and extend, in 2010, DENV-4 after more than 2 decades of absence [12] moreover, unusual re-emergences were described in Brazil dues to several re-introduction of the serotype from Asia [11]. Although Malaysia-Thailand region appears the most likely ancestral area of DENV-4, its spread and circulation appear relatively limited and eventually and eventually due to a competition with other serotypes [13].

Our objective was to document the DENV-4 genetic diversity, including genetics and prediction of phenotypic characteristics based on genetic diversity during a unique outbreak that need to be considered for the purpose of any vaccine development or therapy. Within the research framework of DF surveillance and dengue vaccine development, this study investigated groups of sequences of DENV serotype 4 (DENV-4) isolated from naturally-infected patients with similar disease phenotypes during the 2011 DF epidemic in Thailand. Virus isolates from the different patients were sequenced for genetic analysis, after a limited number of passages on mosquito and human cell for virus isolation and amplification [14].

METHODSStudy sites and sample origin: The serum samples were

originally collected from four different locations (Tambon) during the 2011 DF outbreak in Thailand (Figure 1), from four acute severely ill patients which demonstrated with any clinical presentation (s) corresponding to a DHF syndrome case definition following the literature of dengue infection severity Score of DHF including acute fever for 2-7 days, bleeding episode

(e.g. positive tourniquet), thrombocytopenia <100,000/mm3, and plasma leakage (e.g. hemoconcentration, pleura effusion) [15].

As previously described, DENV RNA was extracted from cells of a positive viral culture using QIAamp Viral RNA Mini Kit (QIAGEN, Germantown, MD). CDNA was synthesized using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) following manufacturer’s instructions [16]. The RNA extraction was done on an archived sample collection previously isolated from four serum samples labeled TH11/1194, TH11/1373, TH11/1404 and TH11/1666, whereby each was collected from acutely ill patients that presented with severe DHF syndrome during the 2011 DF outbreak in Thailand. The samples were collected within seven days of the onset of illness, and viruses were isolated using C6/36 cell (cell line clone derived from Aedes albopictus mosquito larvae) inoculation. DENV was detected by using Isothiocyanate Fluorescent Antibody Test (IFAT) after one cell passage. Viral isolates were then culture-amplified by a single culture passage in Vero-E6 cells (cell line clone derived from kidney epithelial cells of an African green monkey, Cercopithecus aethiops), as previously described [17]. To characterize the viral genome, we used a random amplification, deep-sequencing approach (454 sequencing) as described elsewhere, where 98% of reads matched with a Flavivirus genome reference sequence [17, 19].

The Basic Local Alignment Search Tool (BLAST) – NCBI program was used against the non-redundant NCBI database (e-value at 1x10–3) to find a known DENV-4 genome sequence that matched sequences of the four DENV-4 isolates (TH11 DENV-4 isolates: /1194, /1373, /1404 and /1666). The genomic sequence of the DENV-4 strain ThD4_0485_01 (accession number AY618992) was chosen as reference. Multiple sequence alignments of the four DENV-4 isolates (TH11 sequences) with the reference sequence were performed using Clustal Omega v1.2.0 [20] with default parameters. The DNA sequence variations between the four TH11 sequences and the reference sequence were analyzed with the DnaSP software (version 5.10.1) to identify synonymous and non-synonymous sites [21]. A total of 255 mutations were identified in the multiple sequence alignment, with 223 synonymous changes and 32 non-synonymous changes, the absolute numbers of changes are presented for each strain in the while some changes are repetitive between strains (Table 1).

Phylogenetic Analysis: Trimmed original sequences were aligned with all sequences of DENV-4 available in GenBank in June 2017 using MAFFT [22]. Preliminary phylogenetic analyses were done using PhyML (maximum likelihood), implemented in Seaview[23,24]. Main phylogenetic analyses were performed on a subset of sequences, representing overall diversity of DENV-4, under a Bayesian statistical framework implemented in BEAST package v. 1.8.4 [25], using the model that fits best to the data, according to the corrected Bayesian Information Criterion (BICc) obtained in Jmodeltest2® [26]. The global time reversible model of substitution was used (PhyML), with a gamma distribution and a proportion of invariant sites (GTR+I+G). The coalescent (constant size) model was specified as tree prior and a relaxed molecular clock with an uncorrelated lognormal distribution was used [27,28]. The MCMC (Markov Chain Monte Carlo) algorithm was launched for 30 E8 iterations to reach significant Effective

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Sampling Size (ESS). All trees were analyzed, after a 10% discard, to produce the maximum clade credibility tree (Figure 2).

To evaluate the significance of the non-synonymous mutations, the hydrophobicity and antigenicity profiles and predicted secondary structure were used to identify any potential of critical amino acid position site. Hydrophobicity profile was determined using the Kyte and Doolittle method[29]. Antigenicity prediction was carried out using the Kolaskar and Tongaonkar method [30]. This prediction was based on a semi-empirical approach, developed on physicochemical properties of amino acid residues, and has the efficiency to detect antigenic peptides with about 75% accuracy. These methods are implemented in the CLC Workbench software (CLC Bio-Qiagen®). The secondary structure was predicted using the JNet algorithm available on the JPred4 server [31]. Furthermore, potentially antigenic regions of the different viral proteins from the four TH11 virus variants were predicted using the Antigenic program (EMBOSS package) [32].

Ethics Statement: The project was modeled after Mahidol University good practices in research and it received ethical approval by Center of Ethical Reinforcement for Human Research, Kingdom of Thailand (MU-IRB 2011/010.1301). All samples were anonymized by the referring hospitals of the Central Region, where samples were drawn and then sent to the laboratory for testing.

RESULTSFour DENV-4 sequences were isolated and identified from

four naturally infected patients during the same DF outbreak. The isolation and identification procedure were carried out at different time during the development of the outbreak and all isolate were originated from different locations within the Central region of Thailand. All four sequences have been submitted to GenBank as polyprotein including GenBank accession numbers: 1/ TH11/1194 (KT026308) 2/ TH11/1373-(KT026309) 3/ TH11/1404 (KT026310); 4/TH11/1666 (KR922405). These four strains consistently clustered with contemporaneous old-world DENV-4 clade known to circulate actively in Asia and particularly in Thailand (Figure 2). This South-East Asian clade was first discovered in the 1950s, separated from the Sri-Lanka cluster between 1960-70, with the first fully sequenced member described in Thailand in 1977 (Figure 2). Phylogenetic analysis integrating all available genetic data showed that this South-East Asian / Thai lineage then followed a gradual evolution, marked with yearly epidemics, that mostly occurred in Thailand since 2000 (Figure 2 & SM2). The four viruses sequenced here nest at the tip of the gradual evolution pattern observed on the tree and represent a subset of the contemporaneous variability of DENV-4.

Selected DENV-4 genomic sequences were aligned with original sequences from this study by using Mafft 7 (http://

Figure 1 Study sites samples, Nakhon Pathom Province, Central Region, Thailand.Caption:The four yellow pinsmark the study sites (i.e. Tambon, administrative subdivision) of Phra Pathon (East: 13° 48’ 46.45”N, 100° 6’ 10.34”E), Yai Hon (South: 13° 43’ 30.53”N, 100° 4’ 30.48”E), Wang Taku (North: 13° 51’ 12.20”N, 100° 1’ 9.07”E) and Nong Din Daeng (West: 13° 47’ 50.97”N, 99° 59’ 8.28”E) where the study samples were taken.Insert right framedisplays the south-central coast of Thailand, showing the relative distances from the study sites tothe Bay of Siam (BS), and Bangkok (B), the capital of Thailand.(Source: Google Earth, 2018)

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Figure 2 Bayesian phylogeny of genomic sequences of DENV-4 isolates from four severely infected patients during the 2011 DF outbreak in Thailand, as compared with worldwide representative DENV-4 sequences.Caption: Selected DENV-4 genomic sequences were aligned with original sequences from this study by using Mafft 7 (http://mafft.cbrc.jp).A Bayesian phylogenetic analysis was performed and statistical support (posterior probability) of nodes are depicted on the maximum clade credibility tree (MCC tree). The Time since Most Recent Common Ancestor (TMRCA) values have been estimated for a number of representative clades and their marginal densities have been depicted to illustrate estimated diversification periods of these clades. As highlighted here, only members of 3 main clades of DENV-4 were reported during the last 5 years (Clade Sri-Lanka, Clade S.E.A., both belonging to the clade D, and new-world members of the clade C). Genbank identification numbers, strain names and main information are written in taxa labels. Viruses detected in this study are indicated by a star.

mafft.cbrc.jp). A bayesian phylogenetic analysis was performed and statistical support (posterior probability) of nodes are depicted. Genbank identification numbers, strain names and main informations are written in taxa labels. Viruses detected in this study are highlighted in grey.

Major variability of the DENV-4 low passage of all four isolates extended from the nucleotide and amino acid sequences respectively with a mean diversity of 1.03 to 1.34% (all mutations), and 0.40 to 0.85% (non-synonymous mutations) (Tables 1 and 2). We identified amino acid variations in the prM, E, NS1, NS2A, NS3, NS4A, NS4B and NS5 gene regions among all four isolates. Variation of DENV-4 genes and corresponding amino acid mutations were observed as charge changes and chain changes among DENV-4/prM (prM = pre-Membrane) protein gene (0 to 1 amino acid change depending on the variant), DENV-4/E (E = Envelop)-structural protein (2 to3 changes), and among six of the seven nonstructural (NS) proteins that play important roles in the pathological aspect of DENV [31], including NS1 (3 to 7 changes), NS2A (0 to 2 changes), NS3 (0 to 4 changes), NS4A (0 to 2 changes), NS4A (1 to 5 changes) and NS5 (4 to 8 changes) (Table 3, SM1).

No mutations were recorded in the capsid protein gene. Despite multiple amino acid changes detected, none of these differences seem to affect either the antigenicity or the secondary structure of the viral proteins. The hydrophobicity and antigenicity profiles of the four TH11 virus variants are highly similar [SM2]. Among the predicted antigenic peptides, we selected and studied only those which demonstrating amino acid changes. In TH11/1194, TH11/1373 and TH11/1404 variants, altogether 15 potential epitopes, which contain amino acid changes, were found, while 16 were identified for the TH11/1666 variant [SM3]. Fourteen of these predicted epitopes are the same for the four TH11 variants. In the TH11/1666 variant, one peptide (105-111) was not predicted to be antigenic unlike the three other variants. Two other predicted epitopes (57-63 and 362-367) were found only in the TH11/1666 variant and appear as peptides and therefore are predicted to be antigenic (Table 3) [See Supporting Material 1D ]. These two epitopes of the TH11/1666 variant are in the NS5 region, the first (57-63) in the methyl-transferase (MT) domain, and the second (362-367) in the RNA-dependent RNA polymerase (RdRp) domain. In both cases, a single amino acid substitution change (I57V and R362Q) predicted antigenicity

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and could contribute to generate antigenic variants. These two non-synonymous mutations belong to two functional sites of the predicted epitopes of the N-terminal domain of the viral NS5 protein. Indeed, viral MTases are involved in the mRNA capping process, while RdRp is directly involved in viral replication. Altogether, these mutations occurred in a previously known potential target site for anti-dengue drugs, and therefore are susceptible to interfere with the functionality of this critical site [33-35].

DISCUSSIONStudy samples originated from only four patients, within a

4-month period and among four sites of 13 kilometers equidistant; altogether it is impossible to speculate the origin of the parental strains (i.e. by clonal descent or not; quasi-species significance) in our findings. Although our sampling was as limited as similar, previous studies [36, 37], nevertheless, all samples showed consistent genetic diversity across time and space manners as our study was aimed to demonstrate the genetic diversity of DENV virus which developed during a unique DHF outbreak.

While these four strains consistently clustered with contemporaneous DENV-4 that circulate in Thailand, and two of them shared high similarity, the other strains exhibited significant

genetic variations between each-other (Table 2, Table 3, SM3). Most divergent viruses in our dataset expressed a nucleic acid similarity of 94,82 % for the full genome and 98,4 % on the E gene. Lestrari et al. [38] found (E gene), a mean similarity of nearly 96% in Jakarta, Indonesia. This apparently lower value of diversity is due to the presence in Lestrari’s data, of a divergent group for which the last member was detected in 1977 in Indonesia and that belongs to another clade. More full genome data are still needed to better characterize the phylogenetic relationship between these groups, for which only partial data presently exist in databases. From a phylogenetic point-of-view, the genetic diversity, expressed over the spatial and temporal ranges of our study and together with other sequences commercially available, illustrated by the dynamism of evolution of the DENV-4 South-East Asian clade, known to circulate in Thailand since the seventies (Figure 2). After more than forty years DENV-4 have undergone a gradual evolution and a huge diversification expressed by the South-East Asian / Thai lineage and caused numerous epidemics mostly known from Thailand since 2000 (Figure 2, SM3). Based on this South-East Asian clade and the epidemiological data, time and root-to-tip distance correlates from the first record of this lineage in Thailand in 1977, to the most recent sequences available. Our data represent a subset of the contemporaneous diversity of DENV-4, provided by intense epidemic circulation

Table 1: DENV-4 nucleotide sequence diversity of virus strain isolated from four DHF-patients with severe infection during a dengue fever outbreak (Thailand, 2011).SID (1) NR NS TN % Mutation frequency (2)

TH11/1666 176,298 12 / 110 10,164 1.20

TH11/1194 81,602 18 / 115 10,164 1.30

TH11/1373 53,723 19 / 117 10,164 1.34

TH11/1404 66,475 14 / 91 10,142 1.03

Mean+-std 1.25+-0.13Legend: (1) Strain Identification Number; NR = Number of Reads; NS = Nucleotide Substitutions (non-synonymous/ synonymous); TN = Total Number (i.e. length of the coding DNA sequence);(2)Mutation frequency = mutations rate (as compared to the reference) calculated by dividing the total number of mutations (non-silent plus silent nucleotide substitutions) by the total number of nucleotides sequenced for the four combined samples.

Table 2: DENV-4 non-synonymous amino acid diversity of virus strain isolated from four DHF patients of the 2011 dengue fever outbreak (Thailand).

Strain ID number

G/R (1) T11/1194 T11/1373 T11/1404 T11/1666 PR(2) Mutation frequency* (10-3)

pM / 166 1 1 1 1 0.60(6.0)

E / 495 2 2 2 2 0.40 (4.0)

NS1 / 352 3 3 3 3 0.85 (8.5)

NS2A / 218 1 1 1 1 0.46 (4.6)

NS3 / 618 4 4 4 4 0.65 (6.5)

NS4A /150 1 1 1 1 0.67 (6.7)

NS4B / 245 1 1 1 1 0.41 (4.1)

NS5 / 900 4 4 4 4 0.44 (0.4)

Total / 3.387 0.50 0.50 0.50 0.50Legend: (1) G/R Gene / amino acidmean number [Klungthong et al. 2004; Zhang C, Mammen MP, Ubol S, Holmes EC. 2004. The molecular epidemiology of DENV-4 in Bangkok, Thailand. Virology. 329:168–179]; (2) PR Percentage Rate ((∑of amino acid mutation /4) / (Totals number of amino acid)) x 100. *=rate of mutations (as compared to the consensus sequence)calculated by dividing the number of mutationby the total number of amino acid sequenced for the four combined samples.

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during a few months only, but as our viral isolates are not closely related so the introduction of previously circulating parental strains cannot be ruled out [39]. This genetic diversity also allows the virus to be able to potentially adapting itself rapidly to environmental dynamics and unfortunately may also develop resistance to vaccines and/or antiviral drugs[40]. With respect to our limited number of patients that uniformly exhibited severe clinical outcomes, one cannot conclude an association between the pathology and the observed virus mutations.

Although mean values of 224 unique synonymous mutations (i.e., nucleotide substitutions) were observed among all four variants that do not alter the overall stability of DENV RNA, the primary nucleotide sequence could play a role, as suspected for the capsid-coding sequence during the intracellular viral events [41]. Indeed, nucleotide substitution could also play a role in DENV geographical diversity [42]. Although more research are needed to study this matter in depth, our finding of repetitive common substitutions across the four strains could eventually lead to an understanding of genetic markers in time and space manners.

It has been suggested by Leitmeyer et al. [43], that key mutations which occur during the transmission of DENV and are structured as quasispecies could change disease pathogenic profiles, as demonstrated for DENV-2. Altogether, our findings showed a consistent rate of mutations among DENV-4 populations, thus stressing their importance when considering any strategy for the development of efficiently protective DENV vaccines.

Consequently, given the high potential of DENV to generate virus variants, a live-attenuated vaccine will certainly require detail studies of variation of medicinal-related fields on such genetic diversity for the development of a safe and stable vaccine [44]. In addition, these new variants can potentially enter a newly infected host/vector (mosquito) and subsequently develop viral subpopulations with a dominant phenotype, potentially leading to an increase or decrease of disease severity, and/or a epidemic pattern [45]. Ultimately, as suggested by others [46-50], beside the expected selection pressure done on structural proteins (i.e.: E, pM), more research needs to done on NS protein genes, particularly from our observation of a high level of NS2A dN/dS rate.

ACKNOWLEDGEMENTSThe authors are grateful to the staffs of the Center for

Vaccine Development (Institute of Molecular Biosciences, Mahidol University, Salaya, Thailand), and the Molecular Biology Laboratory for Neurological Diseases (Thai Red Cross Society, Faculty of Medicine, Chulalongkorn University, Thailand).

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Nitatpattana N, Moné Y, Gouilh MAR, Chaiyo K, Joyjinda Y,et al. (2018) Genetic Diversity of Dengue-4 Virus Strains Isolated from Patients During a Single Out-break of Dengue Fever, Thailand (2011). J Fever 2(1): 1009.

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