# Viral evolution sustains a dengue outbreak of enhanced severity

Catherine Inizan, Marine Minier, Matthieu Prot, Olivia O'Connor, Carole Forfait-Dubuc, Sylvie Laumond, Ingrid Marois, Antoine Biron, Ann-Claire Gourinat, Marie-Amélie Goujart, et al.

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## To cite this version:

Catherine Inizan, Marine Minier, Matthieu Prot, Olivia O'Connor, Carole Forfait-Dubuc, et al. Viral evolution sustains a dengue outbreak of enhanced severity. Emerging microbes & infections, 2021, 10 (1), pp.536 - 544. 10.1080/22221751.2021.1899057. pasteur-03229427

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Submitted on 19 May 2021

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# Viral evolution sustains a dengue outbreak of enhanced severity

Catherine Inizan IDª, Marine Minierª, Matthieu Prot IDᵇ, Olivia O'Connor IDª, Carole Forfaitᶜ, Sylvie Laumondᶜ, Ingrid Maroisᵈ, Antoine Bironᵉ, Ann-Claire Gourinatᵉ, Marie-Amélie Goujartᵉ, Elodie Desclouxᵈ*, Anavaj Sakuntabhaif,g, Arnaud Tarantola IDh, Etienne Simon-Lorière IDᵇ# and Myrielle Dupont-Rouzeyrol IDª#

ªURE Dengue and Arboviruses, Institut Pasteur in New Caledonia, Institut Pasteur International Network, Nouméa, New Caledonia;

ᵇEvolutionary genomics of RNA viruses, Institut Pasteur, Paris, France; ᶜHealth Authorities (DASS), Nouméa, New Caledonia; ᵈInternal

medicine and infectious diseases department, Territorial Hospital Center (CHT), Dumbéa, New Caledonia; ᶠMicrobiology laboratory, Territorial Hospital Center (CHT), Dumbéa, New Caledonia; ᶠFunctional genetics of infectious diseases Unit, Institut Pasteur, Paris, France;

ᶜCNRS UMR2000: Génomique évolutive, modélisation et santé (GEMS), Paris, France;

號URE Epidemiology, Institut Pasteur in New Caledonia, Institut Pasteur International Network, Nouméa, New Caledonia

## ABSTRACT

Compared to the previous 2013–2014 outbreak, dengue 2016–2017 outbreak in New Caledonia was characterized by an increased number of severe forms associated with hepatic presentations. In this study, we assessed the virological factors associated with this enhanced severity. Whole-genome sequences were retrieved from dengue virus (DENV)-1 strains collected in 2013–2014 and from severe and non-severe patients in 2016–2017. Fitness, hepatic tropism and cytopathogenicity of DENV 2016–2017 strains were compared to those of 2013–2014 strains using replication kinetics in the human hepatic cell line HuH7. Whole-genome sequencing identified four amino acid substitutions specific to 2016–2017 strains and absent from 2013–2014 strains. Three of these mutations occurred in predicted T cell epitopes, among which one was also a B cell epitope. Strains retrieved from severe forms did not exhibit specific genetic features. DENV strains from 2016–2017 exhibited a trend towards reduced replicative fitness and cytopathogenicity in vitro compared to strains from 2013–2014. Overall, the 2016–2017 dengue outbreak in New Caledonia was associated with a viral genetic evolution which had limited impact on DENV hepatic tropism and cytopathogenicity. These mutations, however, may have modified DENV strains antigenicity, altering the anti-DENV immune response in some patients, in turn favoring the development of severe forms.

Trial registration: ClinicalTrials.gov identifier: NCT04615364.

**ARTICLE HISTORY** Received 10 January 2021; Revised 18 February 2021; Accepted 1 March 2021

**KEYWORDS** Severe dengue; hepatitis; whole-genome sequencing; viral fitness

## Introduction

Dengue infection is an emerging disease caused by dengue virus (DENV), an arbovirus belonging to the *Flaviviridae* family, genus *Flavivirus*. DENV is transmitted to humans by the bite of infected mosquitoes, mainly from the genus *Aedes*. DENVs are divided into four serotypes (DENV-1 to -4), themselves subdivided in genotypes. Infection with one serotype is thought to provide life-long protection from reinfection with the same serotype but does not prevent secondary infection by another serotype. The spectrum of dengue clinical presentations is broad, ranging from asymptomatic to severe, sometimes fatal, infection. Facilitating and standardizing dengue clinical management [1], dengue fever is categorized by WHO

2009 guidelines into dengue without alert signs, dengue with alert signs, which include abdominal pain, persistent vomiting, clinical fluid accumulation and drop in platelet count, and severe dengue. Severe dengue is characterized by severe plasma leakage, severe bleeding and/or severe organ involvement [2].

With an estimated 390 million infections each year, dengue has become the most prevalent human arbovirus, affecting 128 countries located mainly in tropical and subtropical countries [3]. In New Caledonia (NC), a French island territory of 280,000 inhabitants located in the South Pacific region, the epidemiology of dengue has evolved over the last decade. Dengue outbreaks are becoming increasingly recurrent [4], concurrent with the introduction and circulation of

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other arboviruses such as Zika and chikungunya viruses [5]. In 2013-2014, NC experienced a massive DENV-1 outbreak of 10,851 reported confirmed or probable cases [6]. In 2016-2017, NC experienced another important DENV outbreak of 5,072 cases [6]. This last dengue outbreak was unusual due to: (i) The co-circulation of DENV-1, -2 and -3 (DENV-1 being responsible for 80.6% of the cases); (ii) Its enhanced severity compared to previous years, leading to 416 hospital admissions (hospital admission rate of 8.2%), and 15 deaths; And (iii) the exceptional rate of hepatic presentations [7]. Major hepatic cytolysis (Aspartate Transminases (AST) and/or Alanine Amino Transferases (ALT) over 1,000 UI/L) was the most frequent severity criterion, accounting for 41.5% of severe cases. Age, comorbidities, presence of at least one alert sign, platelets <30 × 10⁹/L, prothrombin time <60%, AST and/or ALT >10N, previous dengue infection were identified as risk factors to develop severe dengue. Past history of Zika infection was not a risk factor for progression to severe dengue [7].

We compared DENV retrieved during the 2013–2014 and 2016–2017 outbreaks in NC to assess whether any differences in virological factors were associated with the increase in the rate of hepatic presentations.

## Material and methods

### Ethics statement

The study was conducted in compliance with the Declaration of Helsinki principles. Samples collected in 2013–2014 came from patients who did not object to the secondary use of their serum sample for research purposes. This study reusing serum samples received administrative and ethical clearance in France from the “Comité de Protection des Personnes Sud-Est II” (n° ID-RCB 2019-A03114-53, n° CPP 19.12.06.49357) and by the Consultative Ethics Committee of New Caledonia. The study was recorded on Clinicaltrials.gov (ID: NCT04615364). Samples collected in 2016–2017 came from patients who gave their written informed consent as part of this study. This study was approved by the “Comité de Protection des Personnes Ile-de-France I” (n° ID-RCB 2015-A00205-44, CPP n°2015-march-13851 and 2018-january-14793) and by the Consultative Ethics Committee of New Caledonia.

### Patient recruitment and classification

The 2016–2017 sample set was prospectively constituted in the framework of the current study by including consenting patients referring at the Institut Pasteur in New Caledonia or the Territorial Hospital

for dengue diagnosis. Written informed consent was received from participants prior to inclusion in the study. Major hemorrhagic signs, shock, thrombocytopenia/bleeding, acute or fulminant hepatitis, diabetes and obesity were documented. Disease severity in patients was assessed using the WHO 2009 guidelines [2]. Blood samples were taken from patients at first referral, between days 0 and 9 post symptom onset. Patients found positive for DENV by qRT-PCR were included in the study. Methods are described elsewhere [8, 9]. The 2013–2014 control set was constituted by retrospectively including 17 DENV biobanked samples. Samples were randomly selected among DENV+ samples in order to cover the entire 2013–2014 epidemic season. All serum samples were stored at –80°C before analysis.

### Laboratory diagnosis

Infecting dengue serotype was determined using qRT-PCR [10]. IgG against dengue and Zika virus were measured by ELISA method (PanBio®, Brisbane, Australia for dengue and Euroimmun®, Lübeck, Germany for Zika). AST and ALT were titered by spectrophotometry on an Architect C8000 system (Abbott, Lake Forest, USA).

### RNA preparation and generation of RNA libraries

RNA extraction from serum samples was performed using the QiaAmp Viral RNA extraction kit (Qiagen, Hilden, Germany), followed by treatment with Turbo DNase (ThermoFisher, Asnières-sur-Seine, France) to digest contaminating DNA. Host rRNA were depleted from RNA samples using the NEBNext® rRNA Depletion Kit (New England Biolabs, Évry-Courcouronnes, France) as described elsewhere [11]. RNA from selective depletion was used for cDNA synthesis and Illumina library preparation using the Nextera XT kit with dual indexes and sequenced on an Illumina NextSeq500 (75 cycles, paired-end reads) platform.

### RNA sequencing and analysis

Raw paired-end files were processed for removal of Illumina adaptor sequences, trimmed and quality-based filtered using Trimmomatic v0.36 [12]. De novo assembly was performed using metaSPAdes v3.12.0 with default parameters [13]. Scaffolds were queried against the NCBI non-redundant protein database [14] using DIAMOND v 0.9.26 [15]. For each sample, the main scaffold corresponded to DENV and no other viruses were identified. Iterative mapping using CLC-assembly-cell v5.1.0 was used to generate full-length or near full-length genomes.
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Whole-genome sequences are deposited under Gen-Bank Accession Numbers MW315172-MW315195.

## Phylogeny

We downloaded all available complete DENV-1 genomes from the Virus Pathogen Database and Analysis Resource (ViPR) database on 2 November 2019 [16]. We retrieved from GenBank two additional DENV-1 genomes from 2014 generated in the framework of DENV molecular surveillance in NC. Sequence alignment was performed by using MAFFT v7.023 [17]. Maximum-likelihood (ML) phylogenies were inferred using IQ-TREE v1.6.7.2 [18] and branch support was calculated using ultrafast bootstrap approximation with 1,000 replicates [19]. Prior to the tree reconstruction, the ModelFinder application [20], as implemented in IQ-TREE, was used to select the best-fitted nucleotide substitution model.

## Cells and viruses

The *Aedes albopictus* C6/36 cell line was cultured at 28°C in Leibovitz L15 medium (Sigma-Aldrich, Merck, Steinheim, Germany) supplemented with 5% Fetal Calf Serum (FCS, Gibco™, Fisher scientific, Paisley, UK) and 10% Tryptose Phosphate Broth (TPB, Gibco™, Fisher scientific, Paisley, UK). The human hepatocarcinoma HuH7 cell line was cultured at 37° C under 5% CO2 in Dulbecco's Modified Eagle Medium (DMEM, Gibco™, Fisher scientific, Paisley, UK) supplemented with 10% FCS. We recovered DENV from serum samples through infection of C6/36 cells. All final viral stocks were prepared with no more than four passages in C6/36 for 5 days. Supernatants were collected and stored at -80°C.

### Viral titration by immunofluorescent focus assay (IFA)

C6/36 were plated at $2 \times 10^4$ cells per well in 96-well plates. Forty-eight hours after seeding, we inoculated wells with serial 10-fold dilutions of the viral stock in Leibovitz L15 medium supplemented with 2% FCS and 10% TPB. After 2 h adsorption, cells were overlaid with 1.6% CarboxyMethylCellulose (VWR, Leuven, Belgium) in L15 medium supplemented with 5% FCS and 5% TPB. Five days after infection, cells were fixed with 4% Paraformaldehyde (Sigma-Aldrich, Switzerland) and permeabilized using 0.5% Triton X-100 (Biorad, Hercules, USA). Cells were then stained for 1 h with anti-dengue complex antibody clone 2H2 (Millipore, Temecula, USA) at 5 µg/mL in PBS supplemented with 2% FCS, followed by 1 h incubation with secondary anti-mouse IgG antibody coupled to Al488 (Invitrogen, Thermo Fisher Scientific, Eugene, USA) at 2 µg/mL in PBS supplemented with 2% FCS. Fluorescent infection foci

were counted under an inverted fluorescence microscope (DMIL LED, Leica, Wetzlar, Germany).

### Viral replication kinetics in HuH7 hepatic cells

HuH7 were seeded at $2 \times 10^5$ cells per well in 12-well plates. Twenty-four hours later, cells were infected with DENV viral stocks at a multiplicity of infection of one. At 24, 48 and 72 h post-infection, supernatants were collected and stored at -80°C in 0.5 M Sucrose (Acros Organics, Geel, Belgium) and 20 mM Hepes buffer (Gibco, Fisher scientific, Paisley, UK) for subsequent titration. Simultaneously, cells were collected using TrypLE-Express (Gibco, Fisher scientific, Grand Island, USA). Cell mortality was assessed using Fixable Viability Dye eFluor 780 (eBioscence, Thermo Fisher Scientific, Carslbad, USA). Cells were then fixed using 4% Paraformaldehyde and stored at 4°C. Fixed cells were permeabilized at 72 h post-infection using 0.5% Triton X-100 and DENV was stained using anti-dengue complex antibody clone 2H2 at 3 µg/mL and secondary anti-mouse IgG Al488 antibody at 2 µg/mL in PBS supplemented with 2% FCS. Mortality and infection rates were determined by flow cytometry on a BD FACSCantoII (BD Biosciences, San Jose, USA). A DENV-1 strain isolated in 2014 in NC and passaged several times in C6/36 cells was used as a positive control. This viral strain yielded nearly 100% infection of HuH7 cells at day 3 post-infection. Percentages of infected cells were normalized by the percentage of infected cells of the positive control at day 3 post-infection. In brief, relative infection percentages of the tested conditions were expressed as a percentage of the infection rate of the positive control at day 3 post-infection: (infection percentage of the tested condition/infection percentage of the positive control at day 3)*100. Viral titers in the supernatants were determined by IFA.

### Epitope prediction

T cell epitopes were predicted using NetCTL 1.2 server, restricting epitopes to the MHC Class I supertype A24, as HLA A*24:02 allele is reported to be the main MHC Class I supertype in New Caledonia (http://www.allelefrequencies.net/). B cell epitopes were predicted using BepiPred 2.0 server with an epitope threshold of 0.5.

### Statistics

Statistical analysis was performed using GraphPad Prism (version 6.1; GraphPad; San Diego, CA; USA). Differences between groups of virus isolates were analysed for statistical significance using a Wilcoxon-Mann-Whitney ranksum test. The *p* value significance threshold was set at 0.05.
<figure><img src="image_10.png" /><figcaption>**Table 1.** Demographic data and clinical and biological parameters of the studied populations.</figcaption></figure>

<table><thead><tr><th></th><th>2013-2014</th><th>2016-2017</th><th>Total</th></tr></thead><tbody><tr><td colspan="4"><strong>All patients</strong></td></tr><tr><td>n</td><td>17</td><td>69</td><td>86</td></tr><tr><td>Days since symptom onset at inclusion (median, IQR)</td><td>2 [1.5; 4] (2 N/A)</td><td>3 [2; 5] (7 N/A)</td><td>3 [2; 5] (9 N/A)</td></tr><tr><td>WHO 2009 classification [2]</td><td></td><td></td><td></td></tr><tr><td>NS</td><td>12/17 (71%)</td><td>51/69 (74%)</td><td>63/86 (73%)</td></tr><tr><td>S</td><td>0</td><td>17/69 (25%)</td><td>17/86 (20%)</td></tr><tr><td>N/A</td><td>5/17 (29%)</td><td>1/69 (1%)</td><td>6/86 (7%)</td></tr><tr><td>Death</td><td>N/A</td><td>2/69 (2.9%)</td><td>2/86 (2.3%)</td></tr><tr><td colspan="4"><strong>Infected serotype</strong></td></tr><tr><td>DENV-1</td><td>14/17 (82%)</td><td>53/69 (77%)</td><td>67/86 (78%)</td></tr><tr><td>DENV-2</td><td>0/17 (0%)</td><td>12/69 (17%)</td><td>12/86 (14%)</td></tr><tr><td>DENV-3</td><td>3/17 (18%)</td><td>4/69 (6%)</td><td>7/86 (8%)</td></tr><tr><td>DENV-4</td><td>0/17 (0%)</td><td>0/69 (0%)</td><td>0/86 (0%)</td></tr><tr><td>Viral load (median, IQR)</td><td>4.35 × 10<sup>5</sup> [5.85 × 10<sup>4</sup> - 1.25 × 10<sup>7</sup>]</td><td>2.37 × 10<sup>5</sup> [1.05 × 10<sup>7</sup>]</td><td>2.80 × 10<sup>5</sup> [3.58 × 10<sup>4</sup> - 1.20 × 10<sup>7</sup>]</td></tr><tr><td>DENV IgG</td><td>1/17 (5.9%)</td><td>24/62 (39%)</td><td>25/79 (32%)</td></tr><tr><td>ZIKV IgG</td><td>0/6 (0%)</td><td>7/60 (12%)</td><td>7/66 (11%)</td></tr><tr><td>DENV&amp;ZIKV IgG</td><td>0/6 (0%)</td><td>5/59 (8.5%)</td><td>5/65 (7.7%)</td></tr><tr><td colspan="4"><strong>Analysis 1: Whole-genome sequencing</strong></td></tr><tr><td>n</td><td>3</td><td>21</td><td>24</td></tr><tr><td>Days since symptom onset at inclusion (median, IQR)</td><td>1 [1; 1] (1 N/A)</td><td>2 [1; 4] (2 N/A)</td><td>2 [1; 4] (3 N/A)</td></tr><tr><td>WHO 2009 classification [2]</td><td></td><td></td><td></td></tr><tr><td>NS</td><td>3/3 (100%)</td><td>15/21 (71%)</td><td>18/24 (75%)</td></tr><tr><td>S</td><td>0</td><td>5/21 (24%)</td><td>5/24 (21%)</td></tr><tr><td>N/A</td><td>0</td><td>1/21 (5%)</td><td>1/24 (4%)</td></tr><tr><td colspan="4"><strong>Infected serotype</strong></td></tr><tr><td>DENV-1</td><td>3/3 (100%)</td><td>21/21 (100%)</td><td>24/24 (100%)</td></tr><tr><td>DENV-2</td><td>0</td><td>0</td><td>0</td></tr><tr><td>DENV-3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>DENV-4</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Viral load (median, IQR)</td><td>1.32 × 10<sup>7</sup> [1.28 × 10<sup>7</sup> - 2.44 × 10<sup>7</sup>]</td><td>2.95 × 10<sup>7</sup> [9.12 × 10<sup>5</sup> - 1.40 × 10<sup>8</sup>]</td><td>2.95 × 10<sup>7</sup> [1.06 × 10<sup>6</sup> - 1.29 × 10<sup>8</sup>]</td></tr><tr><td>DENV IgG</td><td>0/3 (0%)</td><td>3/19 (16%)</td><td>3/22 (14%)</td></tr><tr><td>ZIKV IgG</td><td>0/3 (0%)</td><td>2/19 (11%)</td><td>2/22 (9.1%)</td></tr><tr><td>DENV&amp;ZIKV IgG</td><td>0/3 (0%)</td><td>1/18 (5.6%)</td><td>1/21 (4.8%)</td></tr><tr><td colspan="4"><strong>Analysis 2: Kinetics of replication in human hepatic cells</strong></td></tr><tr><td>n</td><td>6</td><td>7</td><td>13</td></tr><tr><td>Days since symptom onset at inclusion (median, IQR)</td><td>2 [0; 2.5] (2 N/A)</td><td>2 [1.5; 2]</td><td>2 [1; 2] (2 N/A)</td></tr><tr><td>WHO 2009 classification [2]</td><td></td><td></td><td></td></tr><tr><td>NS</td><td>2</td><td>6</td><td>8/13 (62%)</td></tr><tr><td>S</td><td>0</td><td>0</td><td>0/13</td></tr><tr><td>N/A</td><td>4</td><td>1</td><td>5/13 (38%)</td></tr><tr><td colspan="4"><strong>Infected serotype</strong></td></tr><tr><td>DENV-1</td><td>6</td><td>7</td><td>13/13 (100%)</td></tr><tr><td>DENV-2</td><td>0</td><td>0</td><td>0</td></tr><tr><td>DENV-3</td><td>0</td><td>0</td><td>0</td></tr><tr><td>DENV-4</td><td>0</td><td>0</td><td>0</td></tr><tr><td>Viral load (median, IQR)</td><td>2.44 × 10<sup>7</sup> [7.78 × 10<sup>6</sup> - 1.08 × 10<sup>8</sup>]</td><td>6.89 × 10<sup>7</sup> [2.95 × 10<sup>7</sup> - 1.39 × 10<sup>8</sup>]</td><td>3.56 × 10<sup>7</sup> [2.33 × 10<sup>7</sup> - 1.32 × 10<sup>8</sup>]</td></tr><tr><td>DENV IgG</td><td>0/6 (0%)</td><td>1/7 (14%)</td><td>1/13 (7.7%)</td></tr><tr><td>ZIKV IgG</td><td>0/1</td><td>0/7</td><td>0/8</td></tr><tr><td>DENV&amp;ZIKV IgG</td><td>0/1</td><td>0/7</td><td>0/8</td></tr></tbody></table>

Dengue patients are characterized according to the WHO 2009 criteria [2, 40]. DENV serotype and viral load were determined by qRT-PCR. NS, non-severe; S, severe; N/A, not available.

# Results

## Characteristics of the sample set

In all, samples from 86 DENV patients were included: 17 biobanked serum samples collected during the 2013–2014 outbreak and 69 patients prospectively

included during the 2016–2017 outbreak (Table 1). Blood was collected at a median of two (Interquartile range IQR [1.5; 4]) and three (IQR [2; 5]) days post-symptom onset for patients from 2013–2014 and 2016–2017, respectively. Cases were categorized as severe according to the WHO 2009 classification [2]: no severe cases were included in 2013–2014 and 17/69 (25%) were detected in 2016–2017.

Due to limitations in the amount of biological material available and medical records documentation, biological and clinical characteristics were available for only part of the cohort. Severity criteria were documented in 15/17 (88.2%) severe patients in 2016–2017: the most frequent were thrombocytopenia/bleeding (9/15, 60% severe forms); AST/ALT > 1,000 UI/L (7/15, 47% severe forms); And acute hepatitis (7/15, 47% severe forms) (Supplementary figure 1). Among the latter, three fulminant hepatitis were reported, two of which were fatal. Hepatic dysfunction (ALT/AST > 10 N) was documented in 21/54 (39%) AST/ALT-tested patients in 2016–2017.

DENV-1 was the major infecting serotype, causing 14/17 (82%) and 53/69 (77%) of investigated cases in 2013–2014 and 2016–2017, respectively. Viral loads in 2013–2014 and 2016–2017 samples were comparable, with a median of $4.35 \times 10^5$ RNA copies/mL (IQR [5.85 × 10$^4$ - 1.25 × 10$^7$]) in 2013–2014 and $2.37 \times 10^5$ RNA copies/mL (IQR [0 - 1.05 × 10$^7$]) in 2016–2017 ($p = 0.23$).

Anteriority of dengue or Zika infection was determined by the detection of anti-DENV or ZIKV IgG by ELISA. Secondary infections (presence of anti-DENV IgG) were detected in 1/17 (5.9%) and 24/62 (39%) patients in 2013–2014 and 2016–2017, respectively. Anti-ZIKV IgG were found in 0/17 (0%) and 7/60 (12%) patients from the successive epidemics. Among those anti-ZIKV IgG positive, 5/6 (83.3%) had both anti-ZIKV and DENV IgG.

## Emergence of a new cluster of viruses in 2016–2017

Among the 86 samples, DENV-1 whole-genome could be sequenced for 3/17 (17.6%) samples from 2013 to 2014 and 21/69 (30.4%) from 2016 to 2017. Whole genomes were successfully sequenced for samples with the highest viremia (Table 1). Sequences obtained for 2013–2014 cases were retrieved from non severe cases. In 2016–2017 among the 21 whole-genomes, 15 (71%) cases were non severe, 5 (24%) were severe and one was not documented clinically. To place these new viral sequences in context, we inferred a phylogeny using all near complete genomes retrieved from the ViPR database (Supplementary figure 2). The sequences reported here formed a monophyletic clade when including a sequence from French Polynesia (bootstrap score 100%) and two sequences from strains retrieved
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<figure><img src="image_12.png" /><figcaption>**Figure 1.** Phylogenetic tree of DENV-1 whole-genome sequences retrieved from dengue patients sera from 2013–2014 and 2016–2017. Whole-genome sequences retrieved from severe forms are highlighted in blue. The tree is midpoint rooted and bootstrap values above 80% are indicated. Two DENV-1 genomes from strains retrieved in NC in 2014 are displayed on the tree. The four amino acid substitutions detected in strains from 2016–2017 in comparison to strains from 2013–2014 are indicated.</figcaption></figure>

in NC in 2014, within genotype I. We built another phylogeny using only the sequences reported here, with the two sequences from strains retrieved in NC in 2014 and the closest outgroup (Figure 1).

Although DENV-1 from 2016 to 2017 were related to viruses from 2013 to 2014, they formed a distinct cluster compared to 2013–2014 viruses (Figure 1). DENV-1 from 2016 to 2017 all shared 26 synonymous mutations and four mutations resulting in amino acid substitutions in non-structural proteins which were absent from the 2013–2014 viruses (Supplementary table 1). A histidine replaced a tyrosine at position 113 in NS1 (NS1 Y886H). An alanine replaced a valine at position 64 in NS4A (NS4A V2157A). Finally, an arginine replaced a lysine at position 132 (NS5 K2623R) and an alanine replaced a glycine at position 384 (NS5 G2875A) in NS5. Residues 887, 2158 and 2871 are located in T cell epitopes as predicted using NetCTL 1.2 server. Residues 870–894 belong to a B cell epitope as predicted using BepiPred 2.0. Viruses sequenced from severe and non-severe 2016–2017 cases were interspersed in the phylogenetic tree.

**Isolated viruses from 2016 to 2017 tend to be less infectious in human hepatic cells than viruses from 2013 to 2014**

We successfully isolated six DENV-1 from 2013 to 2014 and seven DENV-1 from 2016 to 2017 contained

in patients' sera through inoculation of C6/36 cells (Table 1). Viral fitness of these strains from 2013–2014 and 2016–2017 were compared in kinetics of replication in HuH7. Although differences in the percentage of infected cells at days 1, 2 and 3 post-infection yielded by viruses from 2013–2014 and 2016–2017 were not statistically significant, viral isolates from 2016–2017 showed a twice lower median percentage of infected cells at day 3 post-infection as compared to viral isolates from 2013–2014 (Figure 2A). Differences in infection productivity as measured by the titer of infectious viral particles in the supernatant at day 2 post-infection was not statistically significant between viruses from 2013–2014 and 2016–2017 (Figure 2B).

**Isolated viruses from 2016 to 2017 tend to be less cytopathogenic for human hepatic cells than viruses from 2013 to 2014**

The rate of cell death increased at day 3 post-infection for all DENV strains. Although differences in the percentage of dead cells at days 1, 2 and 3 post-infection yielded by viruses from 2013–2014 and 2016–2017 were not statistically significant, the median percentage of dead cells at day 3 post-infection yielded by viral isolates from 2016 to 2017 was twice lower than the one yielded by viruses from 2013 to 2014 (Figure 2C).
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<figure><img src="image_16.png" /><figcaption>**Figure 2.** Replication kinetics of DENV-1 viral isolates from 2013–2014 and 2016–2017 in the human hepatic cell line **HuH7**. A DENV strain isolated in NC in 2014 passaged several times in C6/36 cells and yielding a high infection rate at day 3 post-infection was used as positive control. Human hepatic HuH7 cells were infected at a MOI of 1 with DENV-1 from 2013–2014 and 2016–2017. A. Percentages of HuH7 cells staining positive for 2H2 antibody measured by flow cytometry at days 1, 2 and 3 post-infection. Percentages of infected cells are normalized by the percentage of infected cells of the positive control at day 3 post-infection. B. Productivity of the infection measured as the viral titer in the supernatant of HuH7 at day 2 post-infection. C. Percentage of dead HuH7 cells staining positive for Viability dye measured by flow cytometry at days 1, 2 and 3 post-infection. NI: non-infected. Each dot is the mean of three independent replicates. Medians and interquartile ranges are indicated. ns: non significant difference in Wilcoxon-Mann-Whitney ranksum test.</figcaption></figure>

Discussion

Dengue 2016–2017 outbreak in New Caledonia was characterized by an increase in the percentage of severe forms reported, associated to an unusually high rate of hepatic dysfunctions.

Our study shows that DENV-1 strains from 2016 to 2017 form a phylogenetic cluster distinct but linked to strains retrieved during the previous outbreak, which occurred in 2013-2014. This suggests continuous DENV-1 circulation associated with a genetic evolution between 2013–2014 and 2016–2017. This DENV-1 lineage was not identified in NC in 2015, suggesting that DENV-1 continuous circulation between 2013–2014 and 2016–2017 may have occurred undetected. Alternatively, DENV-1 circulation in NC in 2016–2017 could result from subsequent introduction from a region where a phylogenetically related DENV-1 strain was circulating. Although recurrent homotypic dengue outbreaks are often associated to genotype/clade/lineage displacements [21–24], persistence of the same DENV lineage over a decade or more has been reported elsewhere [21, 23, 25–27].

In our study, the 5 DENV-1 strains retrieved from severe forms in 2016–2017 did not display characteristic genetic features differentiating them from strains retrieved from 15 non-severe dengue cases. This suggests the absence of genetic specificity for strains causing severe dengue in our sample set. Others

have shown a specific genetic signature for DENV strains according to the related clinical presentation. A study conducted on DENV strains isolated in Cambodia in 2007 found that a DENV strain isolated from a patient experiencing Dengue Shock Syndrome (DSS) differed from DENV strains isolated from patients experiencing Dengue Fever (DF) or Dengue Hemorrhagic Fever (DHF) by six amino acids, with mutations located in the pre-membrane gene, the envelope gene, and the non-structural (NS) 1, NS3 and NS5 genes [28].

In our study, however, the 21 DENV-1 strains from 2016 to 2017 shared 26 synonymous mutations and four mutations resulting in amino acid substitutions undetected in the five DENV-1 strains from 2013 to 2014. These mutations may have affected protein functions and interactions. The replacement of the polar residue tyrosine at position 113 in NS1 by a polar and basic histidine (NS1 Y886H) [29] occurred in the disordered loop of NS1 α/β wing domain [30]. This substitution may have affected both NS1 acid dissociation constant (pKa) and its tridimensional structure, therefore possibly affecting its protein-protein interactions as well as its function. Although NS4A V2157A, NS5 K2623R and NS5 G2875A led to the substitution of amino acids displaying the same biochemical properties as the original amino acids, these mutations could alter protein localization and function, ultimately impacting DENV-1 phenotype.
<figure><img src="image_17.png" /></figure>

Most importantly, these mutations may have
modified the antigenicity of DENV-1 accessory pro-
teins: NS1 Y886H mutation occurred near a T cell epi-
tope as predicted using NetCTL server 1.2 and within
a B cell epitope as predicted using BepiPred 2.0, and
may have affected NS1 antigenicity. NS1 immuniz-
ation leads to immune-mediated liver injury in mice
and passive immunization with anti-NS1 IgG from
DENV patients leads to an increase in ALT/AST levels
in mice [31]. The NS1 Y886H mutation may thus have
modified NS1 antigenicity and contributed to an
enhanced NS1-mediated liver injury. Similarly,
NS4A V2157A and NS5 G2875A occurred in the
vicinity of T cell epitopes. Amino-acid substitutions
in DENV accessory proteins have been reported to
sustain the attenuation of DENV in the South Pacific
region in the 1970s, suggesting that genetic modifi-
cations may condition dengue epidemic severity
[32]. Single amino acid changes have been reported
to lead to drastic phenotypic changes for other viruses:
A single amino acid change E1-A226V affected chi-
kungunya virus vector specificity and is considered
to have modified its epidemic potential [33]. Similarly,
Ebola virus variant A82V in the glycoprotein (GP)
exhibited delays in the replication in insectivorous
bat Tb1.Lu cells [34]. The enhanced severity of
DENV-1 2016-2017 outbreak may thus have been
determined by an altered antigenicity of DENV-1
accessory proteins, affecting the host adaptive immune
response to DENV infection.

The observed mutations shared by 2016–2017
strains did not significantly modify their ability to
replicate in our cellular model of human hepatic cell
line as compared to strains from 2013–2014. Some
mutations, especially occurring in the E gene, have
been associated to an enhanced ability of DENV
strains to replicate either in Monocyte-Derived
Macrophages (MDM) [35] or in dendritic cells (DC)
[36]. Modifications in the secondary structures of
the 5’ and 3’ untranslated region also alter the viral
output in MDM and DC [36]. We observed, however,
a trend towards a less efficient replication of strains
from 2016–2017 as compared to strains from the pre-
vious outbreak. Interestingly, DENV-1 became the
minority serotype in 2018 in NC, being gradually
replaced by DENV-2 [37] and was undetected in
2019-2020. This decrease in viral fitness may have
contributed to DENV-1 decline in 2018.

Despite a trend towards reduced cytotoxicity for
strains retrieved in 2016-2017, the non-synonymous
mutations shared by 2016-2017 strains did not signifi-
cantly alter their ability to trigger cell death in human
hepatic cells HuH7. Other studies have explored the
link between the clinical presentation of DENV infec-
tion and the cytopathogenicity of the corresponding
DENV strain. Tuiskunen et al. [28] found that a
DENV strain isolated from a DSS patient induced

enhanced apoptosis in an *in vitro* model of mosquito
C6/36 cells compared to strains isolated from DF or
DHF patients. Similarly, DENV-2 strains isolated
from non-severe and severe forms of dengue showed
different cytopathic effects in C6/36 cells [38].
Although not observed in our study, DENV cyto-
pathogenicity *in vitro* may therefore contribute to
the severity of dengue clinical presentation.

Finally, the occurrence of severe dengue in 2016–
2017 may have been conditioned by host-related fac-
tors as well. Consistent with the study by Marois
et al. [7], secondary dengue infection showed a ten-
dency to be associated to a higher risk of developing
severe dengue (OR 2.847, CI95 [0.88; 9.2]).

Our study suffers from biases and limitations.

First, a limitation to our study is the missing clin-
ico-epidemiological data for some patients whose
samples were included. Documented data, however,
are consistent with the data collected by Marois et al.
[7], strengthening their reliability.

Second is the low number of whole-genome
sequences and viral isolates retrieved from our initial
sample set of 86 serum samples. Such low yield results
from the comparatively low viral load in these serum
samples, which can be due to viral and RNA degra-
dation occurring between sample collection and bio-
banking. Such sample degradation unfortunately
often occurs in studies dealing with human samples
as the workload of diagnosis laboratories inexorably
increases during dengue outbreaks. Nonetheless, the
number of strains challenged in our *in vitro* replica-
tion model is comparable to other studies [39].
Further, the trends we observed may have been
confirmed would a higher number of strains have
been sequenced and isolated.

Third, although HuH7 cells have been used by
others in similar *in vitro* replication studies, this cell
line may not represent the best cellular model for
our study as it may have affected the measured infec-
tivity and cytopathogenicity of isolated strains. The
effects we measured could be challenged in other
human hepatic cellular models such as HepG2 cells.

Fourth, although no specific genetic features were
characteristic of viruses sequenced from severe
forms, we were unable to isolate and test viruses
from severe forms in our cellular model. Challenging
the replicative fitness and cytopathogenicity of viruses
isolated from severe forms would have been
informative.

Fifth and finally, we decided to focus our study on
DENV-1, which was the major circulating serotype in
2016-2017. Severe forms, hepatic presentations and
death occurred in DENV-2 and DENV-3 cases as
well although to a lesser extent. Although severity
seemed to be independent from the incriminated ser-
otype [7], most severe cases were DENV-1 + . Further,
the DENV-2 strain circulating in NC in 2017 as
<figure><img src="image_18.png" /></figure>

minority serotype pursued its circulation in 2018–2019 [37]. The lethality in 2018 was however lower in 2018 as compared to 2017 (0.1% in 2018 VS 0.3% in 2017) [6] and the rate of hepatic presentations decreased in 2018 according to clinicians.

In conclusion, with a hospital admission rate of 8.2%, an important rate of hepatic presentations and 15 deaths, dengue 2016–2017 outbreak in NC was of enhanced severity as compared to previous years. Although DENV strains involved in severe forms did not display a specific genetic signature, dengue 2016–2017 outbreak in NC was characterized by the evolution of the DENV-1 strain as compared to the strain circulating during the previous 2013–2014 outbreak. This viral evolution was not associated to a statistically significant modification in virus replication or cytopathogenicity in our cellular model of human hepatic cells, perhaps due to low number of strains tested. The increase in the number of severe cases during dengue 2016–2017 outbreak in NC is probably not determined by the virus only but rather by host-related factors, history of previous arboviral infection and virus-host interactions. Finally, the contribution of DENV-1 molecular evolution in 2016–2017 on DENV-1 extinction in 2018 in NC remains to be investigated.

## Acknowledgments

We warmly thank the Clinical Research Department of the Centre for Translational Research at Institut Pasteur in Paris for their support in ethic procedures. We thank Fabiana Gámbaro and Deborah Delaune for their contribution to whole-genome sequencing. We thank Ludivine Grzelak for her support in the implementation of *in vitro* replication kinetics. CI, ESL and MDR designed the research study; CI, MM, ESL, MP and OOC conducted the experiments; IM, CC, EKF, MS, AM, AB, ACG and ED collected and acquired data on patients; CF, AP, AT, CI, MM, ESL, MDR analysed the data; CI, wrote the manuscript; ESL and MDR edited the manuscript; all authors carefully revised the manuscript.

## Disclosure statement

No potential conflict of interest was reported by the author(s).

## Funding

The current work was supported by an internal seed-funding from the Institut Pasteur in New Caledonia as well as the Arbo-VIRTUESS project funded by the Actions Concernées Interpasteuriennes (project number ACIP 2014-053). ESL acknowledges funding from the French Agence Nationale de la Recherche (INCEPTION program, Investissements d'Avenir grant ANR-16-CONV-0005). This study has received funding from the French Agence Nationale de la Recherche, Investissement d'Avenir program for the

Laboratoire d'Excellence "Integrative Biology of Emerging Infectious Diseases" (grant n°ANR-10-LABX-62-IBEID).

## ORCID

Catherine Inizan https://orcid.org/0000-0003-1933-1515

Matthieu Prot https://orcid.org/0000-0003-2337-2491

Olivia O'Connor https://orcid.org/0000-0002-8108-8408

Arnaud Tarantola https://orcid.org/0000-0002-6946-7958

Etienne Simon-Lorière https://orcid.org/0000-0001-8420-7743

Myrielle Dupont-Rouzeyrol https://orcid.org/0000-0002-6991-489

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