Conceived and designed the experiments: V-MC-L CR MA. Performed the experiments: MA AT. Analyzed the data: V-MC-L CR. Contributed reagents/materials/analysis tools: V-MC-L SL ED H-PM DM JA. Wrote the paper: V-MC-L CR SL ED DM JA.
The authors have declared that no competing interests exist.
Infection by dengue virus (DENV) is a major public health concern in hundreds of tropical and subtropical countries. French Polynesia (FP) regularly experiences epidemics that initiate, or are consecutive to, DENV circulation in other South Pacific Island Countries (SPICs). In January 2009, after a decade of serotype 1 (DENV-1) circulation, the first cases of DENV-4 infection were reported in FP. Two months later a new epidemic emerged, occurring about 20 years after the previous circulation of DENV-4 in FP. In this study, we investigated the epidemiological and molecular characteristics of the introduction, spread and genetic microevolution of DENV-4 in FP.
Epidemiological data suggested that recent transmission of DENV-4 in FP started in the Leeward Islands and this serotype quickly displaced DENV-1 throughout FP. Phylogenetic analyses of the nucleotide sequences of the envelope (E) gene of 64 DENV-4 strains collected in FP in the 1980s and in 2009–2010, and some additional strains from other SPICs showed that DENV-4 strains from the SPICs were distributed into genotypes IIa and IIb. Recent FP strains were distributed into two clusters, each comprising viruses from other but distinct SPICs, suggesting that emergence of DENV-4 in FP in 2009 resulted from multiple introductions. Otherwise, we observed that almost all strains collected in the SPICs in the 1980s exhibit an amino acid (aa) substitution V287I within domain I of the E protein, and all recent South Pacific strains exhibit a T365I substitution within domain III.
This study confirmed the cyclic re-emergence and displacement of DENV serotypes in FP. Otherwise, our results showed that specific aa substitutions on the E protein were present on all DENV-4 strains circulating in SPICs. These substitutions probably acquired and subsequently conserved could reflect a founder effect to be associated with epidemiological, geographical, eco-biological and social specificities in SPICs.
Almost all tropical and subtropical regions of the world are concerned by the risk of dengue. Every year, dengue virus (DENV) causes more than 50 million infections, 500 000 hospitalizations and 12 500 deaths, mostly children
In the South Pacific, the earliest dengue epidemics may have occurred during the nineteenth century
Here, we report the epidemiological and molecular characteristics of the introduction, spread and genetic microevolution of DENV-4 in FP.
Laboratory diagnosis of dengue was performed on sera from dengue suspected patients using ELISA tests for the detection of either DENV NS1 (Platelia™ Dengue NS1 Ag, Bio-Rad Laboratories, Marnes-la-Coquette, France) or anti-DENV IgM (Dengue IgM Capture ELISA, Panbio Diagnostics, Brisbane, Australia). A DENV serotype-specific multiplex real-time RT-PCR using primers and probes designed by Johnson
The details of the viruses employed are shown in
ID | Country | Archipelago | Island | District | Year | GenBank |
NC09/060209-1528 | NC | 2009 | JN832498 | |||
WF09/010409-0001 | WF | 2009 | JN832499 | |||
PF-NC09/160109-136 | PF (imp. from NC) | Tahiti | Mahina | 2009 | JN832507 | |
PF82/5918 | PF | Windward Islands | Tahiti | 1982 | JN832500 | |
PF83/6074 | Tahiti | 1983 | JN832501 | |||
PF84/131-188 | Tahiti | 1984 | JN832502 | |||
PF85/323-103 | Tahiti | 1985 | JN832503 | |||
PF86/50-70 | Tahiti | 1986 | JN832504 | |||
PF87/19-7 | Tahiti | 1987 | JN832505 | |||
PF09/060309-96 | Tahiti | Papeari | 2009 | JN832514 | ||
PF09/130309-109 | Tahiti | Punaauia | 2009 | JN832519 | ||
PF09/160309-24 | Tahiti | Mahina | 2009 | JN832520 | ||
PF09/230309-126 | Tahiti | Punaauia | 2009 | JN832521 | ||
PF09/310309-162 | Tahiti | Pirae | 2009 | JN832524 | ||
PF09/270409-182 | Tahiti | Faaa | 2009 | JN832530 | ||
PF09/150609-91 |
Tahiti | Punaauia | 2009 | JN832536 | ||
PF09/220709-54 |
Tahiti | Punaauia | 2009 | JN832541 | ||
PF09/220909-50 | Tahiti | Papeete | 2009 | JN832545 | ||
PF09/141009-111 | Tahiti | Papeete | 2009 | JN832546 | ||
PF09/041109-131 | Tahiti | Papara | 2009 | JN832547 | ||
PF09/041209-32 | Tahiti | Arue | 2009 | JN832548 | ||
PF10/260110-67 | Tahiti | Punaauia | 2010 | JN832551 | ||
PF10/050210-117 | Tahiti | Punaauia | 2010 | JN832552 | ||
PF10/120310-31 | Tahiti | Papeete | 2010 | JN832554 | ||
PF10/010410-215 | Tahiti | Mahina | 2010 | JN832555 | ||
PF10/060410-11 | Tahiti | Papeete | 2010 | JN832556 | ||
PF10/300410-48 | Tahiti | Punaauia | 2010 | JN832557 | ||
PF10/170510-92 | Tahiti | 2010 | JN832558 | |||
PF10/170510-191 | Tahiti | Punaauia | 2010 | JN832559 | ||
PF10/170510-192 | Tahiti | Punaauia | 2010 | JN832560 | ||
PF10/150610-28 | Tahiti | Papara | 2010 | JN832561 | ||
PF09/020309-54 | Moorea | 2009 | JN832512 | |||
PF09/190809-58 | Moorea | 2009 | JN832543 | |||
PF10/190110-98 | Moorea | 2010 | JN832550 | |||
PF10/010310-32 | Moorea | 2010 | JN832553 | |||
PF09/090309-122 | Leeward Islands | Raiatea | 2009 | JN832516 | ||
PF09/080409-93 | Raiatea | 2009 | JN832526 | |||
PF09/040509-242 | Raiatea | 2009 | JN832531 | |||
PF09/120609-125 | Raiatea | 2009 | JN832535 | |||
PF09/270709-74 | Raiatea | 2009 | JN832542 | |||
PF09/230209-116 | Bora Bora | 2009 | JN832509 | |||
PF09/100309-172 | Bora Bora | 2009 | JN832517 | |||
PF09/100309-208 | Bora Bora | 2009 | JN832518 | |||
PF09/190509-238 | Bora Bora | 2009 | JN832533 | |||
PF09/200809-131 | Bora Bora | 2009 | JN832544 | |||
PF09/290109-69 | Taha'a | 2009 | JN832508 | |||
PF09/270209-213 | Taha'a | 2009 | JN832511 | |||
PF09/030309-39 | Taha'a | 2009 | JN832513 | |||
PF09/060309-198 | Taha'a | 2009 | JN832515 | |||
PF09/210409-134 | Maupiti | 2009 | JN832527 | |||
PF09/190509-37 | Maupiti | 2009 | JN832532 | |||
PF09/160609-119 | Maupiti | 2009 | JN832537 | |||
PF88/130-74 | Marquesas archipelago | 1988 | JN832506 | |||
PF09/240209-38 | Ua Pou | 2009 | JN832510 | |||
PF09/270309-27 | Ua Pou | 2009 | JN832522 | |||
PF09/270309-31 | Ua Pou | 2009 | JN832523 | |||
PF09/081209-283 | UaPou | 2009 | JN832549 | |||
PF09/290509-25 | Nuku Hiva | 2009 | JN832534 | |||
PF09/070409-227 | Australes archipelago | Rimatara | 2009 | JN832525 | ||
PF09/230409-06 | Rurutu | 2009 | JN832528 | |||
PF09/250609-14 | Rurutu | 2009 | JN832538 | |||
PF09/240409-72 | Tuamotu archipelago | Hao | 2009 | JN832529 | ||
PF09/210709-11 | Hao | 2009 | JN832540 | |||
PF09/080709-23 | Manihi | 2009 | JN832539 |
*patient with severe dengue.
All dengue virus strains were obtained from sera initially sampled for dengue diagnostic and surveillance purposes (under medical prescription by a physician), and archived at ILM. The only information provided to the research laboratory was the sample laboratory ID number, the date of collection and the district/island of collection (no personal information on the patient was provided). The use of biological samples and the collection of information were performed in accordance with the French regulations.
Sera or whole blood on filter paper cards (LDA22, France) were used as a source of DENV. RNA was extracted from sera using the QIAamp® Viral RNA Mini Kit (Qiagen, Germany) or the Easymag extraction system (Biomérieux, France) according to manufacturers' instructions. For whole blood, five filter paper spots per patient were incubated for 20 min at room temperature in lysis buffer from the QIAamp® Viral RNA Mini Kit. After centrifugation for 10 min at 8000 rpm, the extraction process was pursued using the supernatant, according to the manufacturer's instructions. Amplification of the E gene was then performed using the OneStep RT-PCR® Kit (Qiagen, Germany) and two manually designed oligonucleotides primer pairs [D4E/777F (
Two patients with DENV-4 infections were detected in January 2009 in Tahiti, Society Islands. The patients were members from the same family recently returned from NC where a DENV-4 outbreak had recently commenced concomitantly with DENV-1 transmission. These were the first cases of infection with DENV-4 detected in FP for more than 20 years. The French Polynesia Public Health and Hygiene Department immediately applied vector control measures by spraying insecticide against adult mosquitoes and destroying breeding sites around the home of these patients. The insecticide treatment was repeated 10 days later. No new DENV-4 cases were detected in Tahiti in the following weeks. However, at the end of February, DENV-4 was detected in three patients living in Taha'a, five in Bora-Bora, one in Tahiti (Society Islands) and one in Ua Pou (Marquesas Islands). None of these patients had traveled out from FP during the two previous weeks. An additional patient from Taha'a with an onset of symptoms at the end of January was found subsequently, by RT-PCR, to also have had a DENV-4 infection. From January to March 2009, the number of patients with DENV-4 infections increased dramatically and DENV-4 had displaced DENV-1 3 months later (
Society archipelago | Marquesas archipelago | Austral archipelago | Tuamotu archipelago | Gambier archipelago | |||
Total | Windward Islands | Leeward Islands | |||||
|
|||||||
DENV-1 | 11 | Tahiti 11 | |||||
DENV-4 | 3 | Tahiti 2 | Tahaa 1 | ||||
|
|||||||
DENV-1 | 29 | Tahiti 29 | |||||
DENV-4 | 10 | Tahiti 1 | Tahaa 3 | Ua Pou 1 | |||
Bora Bora 5 | |||||||
|
|||||||
DENV-1 | 39 | Tahiti 37 | |||||
Moorea 2 | |||||||
DENV-4 | 132 | Tahiti 41 | Tahaa 42 | Ua Pou 3 | |||
Moorea 3 | Bora Bora 33 | ||||||
Raiatea 10 | |||||||
|
|||||||
DENV-1 | 20 | Tahiti 17 | Raiatea 1 | ||||
Moorea 2 | |||||||
DENV-4 | 262 | Tahiti 130 | Tahaa 14 | Ua Pou 8 | Rimatara 1 | Hao 2 | |
Moorea 25 | Bora Bora 55 | Nuku-Hiva 1 | Rurutu 2 | ||||
Raiatea 21 | Fatu-Hiva 1 | ||||||
Maupiti 2 | |||||||
|
|||||||
DENV-1 | 2 | Tahiti 2 | |||||
DENV-4 | 126 | Tahiti 80 | Bora Bora 9 | Ua Pou 4 | Tubuai 1 | ||
Moorea 10 | Raiatea 9 | Nuku-Hiva 6 | |||||
Maupiti 4 | Ua Huka 1 | ||||||
Huahine 2 | |||||||
|
|||||||
DENV-1 | 1 | Tahiti 1 | |||||
DENV-4 | 165 | Tahiti 112 | Bora Bora 4 | Nuku-Hiva 1 | Rurutu 6 | Hao 4 | |
Moorea 13 | Raiatea 15 | Raivavae 2 | Fakarava 3 | ||||
Maupiti 2 | Manihi 1 | ||||||
Huahine 1 | Rangiroa 1 | ||||||
|
|||||||
DENV-4 | 35 | Tahiti 23 | Raiatea 1 | Fatu-Hiva 1 | Hao 1 | ||
Moorea 4 | Huahine 1 | Manihi 4 | |||||
|
|||||||
DENV-4 | 7 | Tahiti 3 | Bora Bora 1 | Hao 1 | |||
Moorea 2 | |||||||
|
|||||||
DENV-4 | 9 | Tahiti 5 | Nuku-Hiva 2 | Mangareva 1 | |||
Hiva-Oa 1 | |||||||
|
|||||||
DENV-4 | 5 | Tahiti 4 | Rangiroa 1 | ||||
|
|||||||
DENV-4 | 2 | Tahiti 2 | |||||
|
|||||||
DENV-4 | 7 | Tahiti 6 | Ua Pou 1 |
The 64 E gene sequences generated in the present study were combined with 5 E gene sequences from DENV-4 strains collected in the South Pacific between 2008 and 2009 and a data set of 73 E gene sequences available on GenBank representing the global genetic variability of DENV-4 (
ML original tree derived from 110 DENV-4 E gene sequences. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown for values over 80. The number of strains with identical E gene sequence is indicated in parenthesis (these additional strains could have been collected in a different district or island and at a different date than the strain that appears in the tree).
ML original tree derived from 63 DENV-4 E gene sequences. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches.
Based on the Solomon Island [SB07/EU448462] E gene sequence, 53 sites exhibited a nucleotide (nt) substitution of which 18 were present on more than one strain but didn't lead to an amino acid (aa) substitution (
nt positions | |||||||||||||||||||
Cluster | Strains | 15 | 63 | 213 | 273 | 375 | 498 | 549 | 606 | 657 | 738 | 885 | 957 | 978 | 1146 | 1365 | 1386 | 1446 | 1465 |
|
SB07/EU448462 | G | C | A | T | A | C | C | A | T | G | G | A | T | T | T | A | T | C |
KI08/266 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | |
WS08/73 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | |
|
WF09/010409-0001 | - | - | - | - | T | - | - | - | - | - | - | - | - | - | - | - | - | - |
Punaauia 09 Group (3 strains) | - | - | - | - | T | - | - | - | - | - | - | - | - | - | - | - | - | T | |
PF09/310309-162 Tahiti-Pirae | - | - | - | - | T | - | - | - | - | - | - | - | - | - | - | - | - | T | |
PF09/270409-182 Tahiti-Faaa | - | - | - | - | T | - | - | - | - | - | - | - | - | - | - | - | - | T | |
PF09/190509-238 Bora Bora | - | - | - | - | T | - | - | - | - | - | - | - | - | - | - | - | - | T | |
Punaauia 10a Group (2 strains) | - | - | - | - | T | - | - | - | C | - | - | - | - | - | C | - | - | T | |
PF10/150610-28 Tahiti-Papara | - | - | - | - | T | - | - | - | C | - | - | - | - | - | C | - | - | T | |
|
FJ08/3953 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | G | - | - |
TO08/14 | - | - | - | - | - | - | - | - | - | - | - | - | - | - | - | G | - | - | |
|
VU09/4214 | - | - | - | - | - | - | - | - | - | A | - | - | - | - | - | G | - | - |
PF-NC09/160109-136 Tahiti-Mahina | - | - | - | - | - | - | T | - | - | A | - | - | - | - | - | G | - | - | |
NC09/060209-1528 | - | - | - | - | - | - | T | - | - | A | - | - | - | A | - | G | - | - | |
PF09/10 Group (25 strains) | - | - | - | - | - | - | - | - | - | A | - | - | - | A | - | G | - | - | |
PF09/100309-208 Bora Bora | - | - | - | - | - | - | - | - | - | A | - | - | - | A | - | G | - | - | |
PF09/120609-125 Raiatea | - | - | - | - | - | - | - | - | - | A | - | - | - | A | - | G | - | - | |
PF09/141009-111 Tahiti-Papeete | - | - | - | - | - | - | - | - | - | A | - | - | - | A | - | G | - | - | |
PF09/041209-32 Tahiti-Arue | - | - | - | - | - | - | - | - | - | A | - | - | - | A | - | G | - | - | |
PF09/081209-283 Marquesas-Ua Pou | - | - | - | - | - | - | - | - | - | A | - | - | - | A | - | G | - | - | |
PF10/050210-117 Tahiti-Punaauia | - | - | - | - | - | - | - | - | - | A | - | - | - | A | - | G | - | - | |
PF10/170510-192 Tahiti-Punaauia | - | - | - | - | - | - | - | - | - | A | - | - | - | A | - | G | - | - | |
Maupiti 09 Group (4 strains) | A | - | - | C | - | - | - | - | - | A | - | - | - | A | - | G | - | - | |
PF09/080409-93 Raiatea | - | - | - | - | - | - | - | - | - | A | - | G | - | A | - | G | - | - | |
PF09/270709-74 Raiatea | - | - | - | - | - | - | - | - | - | A | - | G | - | A | - | G | - | - | |
PF09/240409-72 Tuamotu-Hao | - | - | - | - | - | T | - | - | - | A | - | - | - | A | - | G | - | - | |
PF09/210709-11 Tuamotu-Hao | - | - | - | - | - | T | - | - | - | A | - | - | - | A | - | G | - | - | |
PF09/080709-23 Tuamotu-Manihi | - | T | - | - | - | - | - | - | - | A | - | - | - | A | - | G | - | - | |
Moorea 10 Group (2 strains) | - | T | G | - | - | - | - | - | - | A | A | - | - | A | - | G | C | - | |
Punaauia 10b Group (2 strains) | - | - | - | - | - | - | - | G | - | A | - | - | C | A | - | G | - | - |
- Identical to Solomon.
Clade | Group | Substitutions | Comments |
|
|
M/I34T |
|
I/A46T |
|||
I/T46A |
|
||
L82P |
|
||
S120L | |||
S156P |
|
||
V160M |
|
||
V173I |
|
||
N276S |
|
||
K323Q |
|
||
V335I | |||
T365I |
|
||
I380V |
|
||
D/E384N |
|||
|
|
I/A46T |
|
V/A141I |
|
||
D/S154G |
|
||
T155I | |||
K/E202R | |||
T221A | |||
A/T222V |
|
||
S/L/A227T | |||
V287I | |||
I351V | |||
P356L |
|
||
F357L | |||
D/E384N |
*non conservative aa substitution; strains of interest are in bold characters.
The recent emergence of DENV-4 in the SPICs occurred after a decade of active DENV-1 circulation and about 20 years after the previous circulation of DENV-4 in the region
In order to complement the epidemiological data and get a better understanding of the events that might have led to the introduction and spread of DENV-4 in FP, we conducted a phylogenetic analysis on the complete E gene of DENV-4 strains collected recently and in the 1980s in FP. We also included in the study some strains collected in other SPICs in 2008–2009 and additional sequences available on GenBank (
Our results corroborate previous studies showing that both in the 1980s and in the early year 2000, DENV-4 had been introduced into the region from Southeast Asia
A more global analysis of the aa changes on the E protein based on the consensus aa sequence of 142 DENV-4 strains reveals mutations that are specific to strains collected in the South Pacific. Within genotype IIb, all the strains composing the PF80s group, except [PF79/U18438], exhibited the same substitution V287I within the domain I of the E gene
We are grateful to Myrielle Dupont-Rouzeyrol, Olivia O'Connor and Suzanne Chanteau from the Institut Pasteur de Nouvelle Calédonie (Nouméa, New Caledonia) and Jean-François Yvon from the Laboratoire d'analyses de l'hôpital de Sia (Wallis, Wallis & Futuna) for providing blood samples blotted on filter paper cards from DENV-4 infected patients.