Advertisement

Silent Circulation of Ross River Virus in French Polynesia

Open AccessPublished:June 15, 2015DOI:https://doi.org/10.1016/j.ijid.2015.06.005

      Highlights

      • We investigated the prevalence of Ross River virus in French Polynesia
      • Sera of 593 blood donors were collected from July 2011 to October 2013
      • 34.40% of blood donors had immunoglobulin G antibodies against Ross River virus
      • We report silent circulation of Ross River virus in French Polynesia

      Summary

      Objectives

      Ross River is an emerging mosquito-borne disease in the Western Pacific. Ross River virus (RRV) circulation has been sporadically reported in some Pacific Island Countries and Territories but never in French Polynesia. To determine if RRV has circulated locally among the French Polynesian population, we conducted a seroprevalence study on blood donors.

      Methods

      Sera of 593 blood donors were collected from July 2011 to October 2013 and tested by ELISA for the presence of RRV-specific Immunoglobulin G (IgG) antibodies.

      Results

      A total of 204 (34.40%) blood donors were found seropositive for RRV. Among the 132 blood donors that were born in French Polynesia and had never travelled abroad, 56 (42.42%) had RRV-specific IgGs.

      Discussion

      Our results support the existence of autochthonous RRV transmission and suggest that this pathogen has silently circulated in French Polynesia. These findings raise the question of possible undetected circulation of RRV in other Pacific Island Countries and Territories.

      Graphical abstract

      Keywords

      1. Introduction

      French Polynesia (FP) is a French overseas territory of about 270,000 inhabitants in the Southeast Pacific, and one of the 22 Pacific Islands Countries and Territories (PICTs). Arthropod-borne virus (arbovirus) infections are major public health concern in the PICTs, with dengue virus (DENV), a Flavivirus of the Flaviviridae family, as the most common circulating and widespread arbovirus.
      • Singh N.
      • Kiedrzynski T.
      • Lepers C.
      • Benyon E.K.
      Dengue in the Pacific--an update of the current situation.
      In FP, DENV outbreaks have been recorded since the 40s.
      • Singh N.
      • Kiedrzynski T.
      • Lepers C.
      • Benyon E.K.
      Dengue in the Pacific--an update of the current situation.
      • Descloux E.
      • Cao-Lormeau V.M.
      • Roche C.
      • De Lamballerie X.
      Dengue 1 diversity and microevolution, French Polynesia 2001-2006: connection with epidemiology and clinics.
      • Cao-Lormeau V.M.
      • Roche C.
      • Aubry M.
      • Teissier A.
      • Lastere S.
      • Daudens E.
      • et al.
      Recent emergence of dengue virus serotype 4 in French Polynesia results from multiple introductions from other South Pacific Islands.
      • Cao-Lormeau V.M.
      • Roche C.
      • Musso D.
      • Mallet H.-P.
      • Dalipanda T.
      • Dofai A.
      • et al.
      Dengue virus type 3, South pacific islands, 2013.
      DENV used to be the only arbovirus isolated until October 2013 when FP experienced the largest outbreak of Zika virus (ZIKV) ever reported,
      • Cao-Lormeau V.M.
      • Roche C.
      • Teissier A.
      • Robin E.
      • Berry A.-L.
      • Mallet H.-P.
      • et al.
      Zika virus, French polynesia, South pacific, 2013.
      • Musso D.
      • Nilles E.J.
      • Cao-Lormeau V.M.
      Rapid spread of emerging Zika virus in the Pacific area.
      followed by an outbreak of chikungunya virus (CHIKV) that started in October 2014.
      • Aubry M.
      • Teissier A.
      • Roche C.
      • Richard V.
      • Shan Yan A.
      • Zisou K.
      • et al.
      Chikungunya outbreak, French Polynesia, October 2014.
      These two viruses, belonging respectively to Flavivirus and Alphavirus genera, have also been recently involved in outbreaks in other PICTs.
      • Musso D.
      • Nilles E.J.
      • Cao-Lormeau V.M.
      Rapid spread of emerging Zika virus in the Pacific area.
      • Cao-Lormeau V.M.
      • Musso D.
      Emerging arboviruses in the Pacific.
      • Duffy M.R.
      • Chen T.-H.
      • Hancock W.T.
      • Powers A.M.
      • Kool J.L.
      • Lanciotti R.S.
      • et al.
      Zika virus outbreak on Yap Island, Federated States of Micronesia.
      Other arboviruses have been reported in PICTs, but not in FP, such as Japanese encephalitis virus (JEV)
      • Baig S.
      • Fox K.
      • Jee Y.
      • O’Connor P.
      • Hombach J.
      • Wang S.
      • et al.
      Japanese Encephalitis Surveillance and Immunization — Asia and the Western Pacific, 2012.
      and Ross River virus (RRV).
      • Barber B.
      • Denholm J.T.
      • Spelman D.
      Ross River virus.
      RRV is an arbovirus belonging to the Alphavirus genus of the Togaviridae family.
      • Harley D.
      • Sleigh A.
      • Ritchie S.
      Ross River Virus Transmission, Infection, and Disease: a Cross-Disciplinary Review.
      It was first isolated from Aedes vigilax mosquitoes in Australia in 1959
      • Doherty R.L.
      • Whitehead R.H.
      • Gorman B.M.
      • O’Gower A.K.
      The isolation of a third group A arbovirus in Australia with preliminary observations on its relationship to epidemic polyarthritis.
      and further from more than 30 mosquito species.
      • Harley D.
      • Sleigh A.
      • Ritchie S.
      Ross River Virus Transmission, Infection, and Disease: a Cross-Disciplinary Review.
      The most common clinical manifestations of RRV infections are fever, arthralgia and rash. Asymptomatic infections range from 55 to 75%.
      • Harley D.
      • Sleigh A.
      • Ritchie S.
      Ross River Virus Transmission, Infection, and Disease: a Cross-Disciplinary Review.
      • Aaskov J.G.
      • Mataika J.U.
      • Lawrence G.W.
      • Rabukawaqa V.
      • Tucker M.M.
      • Miles J.A.
      • et al.
      An epidemic of Ross River virus infection in Fiji, 1979.
      • Russell R.C.
      • Cope S.E.
      • Yund A.J.
      • Hueston L.
      Combatting the enemy-mosquitoes and Ross River virus in a joint military exercise in tropical Australia.
      • Lau C.
      • Weinstein P.
      • Slaney D.
      Imported cases of Ross River virus disease in New Zealand - a travel medicine perspective.
      The first reports of unusual epidemics of “pain, skin eruption and general manifestations” thought to have been due to RRV occurred in 1928 in New South Wales, Australia.
      • Edwards A.
      An unusual epidemic.
      • Nimmo J.
      An unusual epidemic.
      A large RRV outbreak occurred in the Pacific in 1979-1980 with over 500.000 cases reported,
      • Lau C.
      • Weinstein P.
      • Slaney D.
      Imported cases of Ross River virus disease in New Zealand - a travel medicine perspective.
      and affected several PICTs including Fiji, Cook Islands, American Samoa, New Caledonia, Wallis & Futuna, and Vanuatu.
      • Aaskov J.G.
      • Mataika J.U.
      • Lawrence G.W.
      • Rabukawaqa V.
      • Tucker M.M.
      • Miles J.A.
      • et al.
      An epidemic of Ross River virus infection in Fiji, 1979.
      • Rosen L.
      • Gubler D.J.
      • Bennett P.H.
      Epidemic polyarthritis (Ross River) virus infection in the Cook Islands.
      • Tesh R.B.
      • McLean R.G.
      • Shroyer D.A.
      • Calisher C.H.
      • Rosen L.
      Ross River virus (Togaviridae: Alphavirus) infection (epidemic polyarthritis) in American Samoa.
      • Panon G.
      • Le Gonidec G.
      • Fauran P.
      Laboratory diagnosis of epidemic polyarthritis (Ross River virus) in New Caledonia.
      • Fauran P.
      • Le Gonidec G.
      • Rodhain F.
      Research on viral infections in South Pacific mosquitoes under natural conditions.
      • Fauran P.
      • Donaldson M.
      • Harper J.
      • Oseni R.A.
      • Aaskov J.G.
      Characterization of Ross River viruses isolated from patients with polyarthritis in New Caledonia and Wallis and Futuna Islands.
      In FP, due to the neighbouring RRV epidemics, patients presenting with a “dengue like syndrome” were routinely tested for RRV in addition to DENV between April and August 1980 then sporadically until February 1984. Although no RRV infection was detected during this period (unpublished data), circulation of RRV in FP cannot be excluded. After the 1979-1980 outbreak, RRV apparently disappeared from the PICTs. However, cases of RRV infections were subsequently reported from travelers returning from Fiji: 2 Canadian travelers, respectively in 2003 and 2004,
      • Klapsing P.
      • MacLean J.D.
      • Glaze S.
      • McClean K.L.
      • Drebot M.A.
      • Lanciotti R.S.
      • et al.
      Ross River virus disease reemergence, Fiji, 2003–2004.
      and 5 New Zealand travelers, respectively in 1997, 2000, 2001, 2003 and 2009.
      • Lau C.
      • Weinstein P.
      • Slaney D.
      Imported cases of Ross River virus disease in New Zealand - a travel medicine perspective.
      Evidence of RRV infections were also found among residents of Papua New Guinea.
      • Hii J.
      • Dyke T.
      • Dagoro H.
      • Sanders R.C.
      Health impact assessments of malaria and Ross River virus infection in the Southern Highlands Province of Papua New Guinea.
      To date, few data are available about the circulation, incidence and prevalence of RRV in the Pacific. To our knowledge, 13 serosurveys have been conducted for RRV in the region.
      • Harley D.
      • Sleigh A.
      • Ritchie S.
      Ross River Virus Transmission, Infection, and Disease: a Cross-Disciplinary Review.
      • Aaskov J.G.
      • Mataika J.U.
      • Lawrence G.W.
      • Rabukawaqa V.
      • Tucker M.M.
      • Miles J.A.
      • et al.
      An epidemic of Ross River virus infection in Fiji, 1979.
      • Tesh R.B.
      • McLean R.G.
      • Shroyer D.A.
      • Calisher C.H.
      • Rosen L.
      Ross River virus (Togaviridae: Alphavirus) infection (epidemic polyarthritis) in American Samoa.
      Among the studies performed in Australia,
      • Harley D.
      • Sleigh A.
      • Ritchie S.
      Ross River Virus Transmission, Infection, and Disease: a Cross-Disciplinary Review.
      seropositivity ranged from 8% for sera collected in North and Southeast Queensland, Darwin and Northern territory area in 1974-1975
      • Stallman N.D.
      • Wiemers M.A.
      • Bourke A.T.
      Serology in diagnosis and surveys of man.
      to up to 55% for sera collected from aboriginal communities in North Queensland and Northern territory in 1966-1971.
      • Doherty R.L.
      • Gorman B.M.
      • Whitehead R.H.
      • Carley J.G.
      Studies of epidemic polyarthritis: the significance of three group A arboviruses isolated from mosquitoes in Queensland.
      In a serosurvey conducted by Tesh et al. in several PICTs,
      • Tesh R.B.
      • Gajdusek D.C.
      • Garruto R.M.
      • Cross J.H.
      • Rosen L.
      The distribution and prevalence of group A arbovirus neutralizing antibodies among human populations in Southeast Asia and the Pacific islands.
      evidence of RRV infections was found among populations sampled in Papua New Guinea between 1960 and1969 and in Solomon Islands between 1960 and 1966, with rates of RRV seropositivity among localities ranging from 10% to 64% and 1% to 17%, respectively. In contrast, no RRV antibodies were found in sera collected in Vanuatu in 1963 and 1972, New Caledonia in 1963, Palau in 1961 and 1974, and American Samoa in 1962. In a study further conducted in American Samoa, 43.8% of the people sampled at the end of the 1979-1980 RRV outbreak had evidence of RRV infection, whereas those previously sampled in 1972 had no RRV antibodies.
      • Tesh R.B.
      • McLean R.G.
      • Shroyer D.A.
      • Calisher C.H.
      • Rosen L.
      Ross River virus (Togaviridae: Alphavirus) infection (epidemic polyarthritis) in American Samoa.
      In Fiji, seroprevalence was over 90% in some communities after the RRV outbreak in 1979-1980.
      • Aaskov J.G.
      • Mataika J.U.
      • Lawrence G.W.
      • Rabukawaqa V.
      • Tucker M.M.
      • Miles J.A.
      • et al.
      An epidemic of Ross River virus infection in Fiji, 1979.
      In the only study conducted among blood donors, seroprevalence for RRV antibodies was 8.4% from the 2,952 sera collected in South Australia in 1992.
      • Weinstein P.
      • Worswick D.
      • Macintyre A.
      • Cameron S.
      Human sentinels for arbovirus surveillance and regional risk classification in South Australia.
      To our knowledge, except for Australia where RRV is the most common and widespread arboviral disease with about 5,000 cases notified annually,
      • Barber B.
      • Denholm J.T.
      • Spelman D.
      Ross River virus.
      • Yu W.
      • Mengersen K.
      • Dale P.
      • Mackenzie J.S.
      • Toloo G.S.
      • Wang X.
      • et al.
      Epidemiologic patterns of Ross River virus disease in Queensland, Australia, 2001–2011.
      and to a lesser extent for Fiji,
      • Lau C.
      • Weinstein P.
      • Slaney D.
      Imported cases of Ross River virus disease in New Zealand - a travel medicine perspective.
      • Klapsing P.
      • MacLean J.D.
      • Glaze S.
      • McClean K.L.
      • Drebot M.A.
      • Lanciotti R.S.
      • et al.
      Ross River virus disease reemergence, Fiji, 2003–2004.
      there are no recent data about circulation of RRV in the Pacific area.
      Due to the incidence of RRV infections in Australia and frequent population movements across the Pacific region, the risk of emergence of this virus in PICTs, as happened in the early 80s,
      • Aaskov J.G.
      • Mataika J.U.
      • Lawrence G.W.
      • Rabukawaqa V.
      • Tucker M.M.
      • Miles J.A.
      • et al.
      An epidemic of Ross River virus infection in Fiji, 1979.
      • Rosen L.
      • Gubler D.J.
      • Bennett P.H.
      Epidemic polyarthritis (Ross River) virus infection in the Cook Islands.
      • Tesh R.B.
      • McLean R.G.
      • Shroyer D.A.
      • Calisher C.H.
      • Rosen L.
      Ross River virus (Togaviridae: Alphavirus) infection (epidemic polyarthritis) in American Samoa.
      • Panon G.
      • Le Gonidec G.
      • Fauran P.
      Laboratory diagnosis of epidemic polyarthritis (Ross River virus) in New Caledonia.
      • Fauran P.
      • Le Gonidec G.
      • Rodhain F.
      Research on viral infections in South Pacific mosquitoes under natural conditions.
      • Fauran P.
      • Donaldson M.
      • Harper J.
      • Oseni R.A.
      • Aaskov J.G.
      Characterization of Ross River viruses isolated from patients with polyarthritis in New Caledonia and Wallis and Futuna Islands.
      is high. More recently, the detection of sporadic cases of RRV infections from Fiji
      • Lau C.
      • Weinstein P.
      • Slaney D.
      Imported cases of Ross River virus disease in New Zealand - a travel medicine perspective.
      • Klapsing P.
      • MacLean J.D.
      • Glaze S.
      • McClean K.L.
      • Drebot M.A.
      • Lanciotti R.S.
      • et al.
      Ross River virus disease reemergence, Fiji, 2003–2004.
      shows that this pathogen remains a serious threat to the PICTs. In order to determine if RRV has already been introduced and has circulated in FP, we conducted a serological survey on 593 blood donors sera collected from July 2011 to October 2013.

      2. Materials and Methods

      2.1 Participants

      Blood donors were recruited by the French Polynesian blood bank centre (Tahiti, FP). All blood donors lived in Tahiti, the most inhabited island of FP, and were over 18 years old. Written consent for the use of a serum sample for serological testing was obtained before blood collection for all the blood donors included in this study. Volunteers to blood donation reporting a “dengue like syndrome” in the past 3 months, or any symptoms of acute or recent infection, were excluded from blood donation. None of the blood donors has been sampled specifically for the purpose of this study. A medical questionnaire was completed by the physician of the blood bank center before blood collection, in accordance with the recommendations for blood donation. The number of years of residence in FP and the past history of travel outside FP were recorded for all blood donors.
      A total of 593 blood donors recruited from July 2011 to October 2013 were included in this study. Based on their travel history, blood donors were divided into 3 groups: group 1 is composed of residents who were born in FP and have never travelled abroad (132 blood donors); group 2 of residents who were born in FP and have travelled at least once abroad (290 blood donors); and group 3 of immigrants (171 blood donors). Within groups 2 and 3, blood donors reporting past travel in countries with or without reported RRV autochthonous infections (Figure 1) were separated in 2 subgroups, a and b respectively (Table 1).
      Figure thumbnail gr1
      Figure 1Circulation of Ross river virus in the Pacific. Countries with reported Ross River virus autochthonous infections are circled in red. French Polynesia is circled in blue.
      Table 1Characteristics of the blood donors included in the study
      Blood donorsBorn in FPImmigrantsTotal
      Group 1Group 2aGroup 2bTotal group 2Group 3aGroup 3bTotal group 3
      Number13214514529010170171593
      %22.2624.4524.4548.9017.0311.8028.84100
      Age
      Range18-5918-6918-6318-6919-7519-7219-7518-75
      Mean32.8038.1331.9335.0344.7841.1143.2836.91
      Median32.0037.0029.0033.0046.0041.0042.0036.00
      Years of residence in FP
      Range18-5912-6915-6312-690-490-500-500-69
      Mean32.8037.4731.6534.5617.1611.4114.8128.47
      Median32.0036.0029.0032.0016.007.5012.0027.00
      Group 1: residents who were born in FP and have never travelled abroad; group 2a: residents who were born in FP and have travelled at least once in a country with reported RRV infections; group 2b: residents who were born in FP and have travelled at least once but in countries without reported RRV infections; group 3a: immigrants who have travelled at least once in a country with reported RRV infections; group 3b: immigrants who have travelled at least once but in countries without reported RRV infections.

      2.2 Sample Collection

      Blood samples for serological testing were collected in BD Vacutainer® Blood Collection tubes and stored at + 4 °C for maximum 1 day at the blood bank centre before shipment to the “Institut Louis Malardé” (ILM), Tahiti, FP. At ILM, blood samples were centrifuged for 15 minutes at 1,300 g then sera were retrieved and frozen at – 20 °C until processing.

      2.3 Production of recombinant antigens from RRV and CHIKV

      Serum samples were tested for the presence of immunoglobulin G class antibodies (IgGs) against RRV by indirect ELISA using recombinant antigens from RRV. Given that CHIKV has circulated in the PICTs since 2011 and is antigenically related to RRV, recombinant antigens from CHIKV were also used to test serum samples.
      Recombinant antigens were produced in Drosophila S2 cells using the Drosophila S2 expression system (Life Technologies, USA). The synthetic genes (GeneCust, Luxembourg) encoding the soluble ectodomain of the envelope E2 glycoprotein (sE2) from RRV strain QML 1 (GenBank accession no. GQ433354) or CHIKV La Reunion strain 06-49 (GenBank accession no. AM258994) were cloned into the shuttle vector pMT/BiP/His A in which the SNAP-tag sequence (Covalys Biosciences AG, Switzerland) had been initially inserted as a stabilizing protein. The resulting plasmids encoding either the chimeric protein RR.sE2-SNAP or CHIK.sE2-SNAP were transfected into S2 cells to establish stable cell lines S2/RR.sE2-SNAP, and S2/CHIK.sE2,
      • Warter L.
      • Lee C.Y.
      • Thiagarajan R.
      • Grandadam M.
      • Lebecque S.
      • Lin R.T.P.
      • et al.
      Chikungunya virus envelope-specific human monoclonal antibodies with broad neutralization potency.
      according to the manufacturer's recommendations (Life Technologies, USA). After a 10-days cadmium induction of the stable S2 cell lines grown in 1-Liter spinner, cell supernatants were recovered and secreted soluble His-tagged recombinant viral antigens were purified on chelating and size-exclusion chromatography columns. The protein amounts of highly purified RR.sE2-SNAP and CHIK.sE2-SNAP were determined using a BCA protein assay kit (Thermo Scientific, USA), according to manufacturer's instructions. At least 50 mg of each recombinant viral antigen (concentration: 0.5 to 1 mg/ml) were obtained from a single batch of 1-L of S2 cell culture. The samples of purified RR.sE2-SNAP and CHIK.sE2-SNAP proteins were kept at -80 °C.

      2.4 Protocol of indirect ELISA

      For the capture of anti-RRV and anti-CHIKV IgGs, RR.sE2-SNAP, CHIK.sE2-SNAP, and antigen control (SNAP) were diluted at the final concentration of 2 μg/mL in phosphate buffer saline (PBS, Biomérieux, France), and 100 μL of protein sample were coated in 96-well plates overnight at +4 °C. After incubation, antigen sample was removed and wells were blocked with PBS containing 3% of skimmed milk (Régilait, France) and 1% of sodium azide (Merck, Germany). Sera of participants, as well as negative and positive control sera, were diluted 1:400 in PBS with 3% skimmed milk, 0.05% Tween 20 and 1% sodium azide. Then, each diluted serum was added in 2 wells containing either RR.sE2 or CHIK.sE2 recombinant antigens, and in 2 wells containing only SNAP proteins. Plates were incubated for 1 hour at 41 °C and subsequently washed using PBS with 0.05% Tween 20 (Merck, Germany). Peroxidase-conjugated goat anti-human IgG antibodies (Jackson ImmunoResearch, USA) were added in all wells at a dilution of 1:10 000 in PBS with 3% skimmed milk and 0.05% Tween 20. Plates were incubated for 1 hour at 41 °C and washed before addition of peroxidase substrate tetramethylbenzidine (TMB, KPL, USA). Plates were stored at room temperature in the dark for 5 minutes and the enzymatic reaction between the peroxidase and its substrate was stopped by the addition of TMB Stop Solution (KPL, USA). The absorbance was read at a wavelength of 450 nm (OD450) using a microplate photometer (Thermo Scientific, USA).
      Sera found positive for RRV IgGs using recombinants antigens were also tested using the kit Panbio Ross River Virus IgG ELISA (Panbio diagnostics, USA), following manufacturer's recommendations.

      2.5 Serological analysis

      For each serum, average absorbance was calculated using the OD450 values measured in the 2 wells containing the same recombinant antigen or SNAP proteins. Specific absorbance was then determined by deducting the mean absorbance value obtained with SNAP to the mean absorbance value found for each recombinant antigen. Sera with a specific absorbance value ≥ 0.2 were considered positive for the presence of IgGs against the virus tested.

      2.6 Statistical analysis

      To assess the impact of age and travel history on RRV seroprevalence among participants, Fisher's test was performed using the GraphPad Prism 6 version 6.03 software. P values less than 0.05 were considered as statistically significant.

      2.7 Ethics Statement

      The recruitment of participants and processing of blood samples were conducted in accordance with the Ethics Committee of French Polynesia under reference n°60/CEPF (06/27/2013). Blood sample and personal data of each participant were anonymized at the French Polynesian blood bank centre before sending to ILM.

      3. Results

      Among the 593 blood donors, 204 (34.40%), 18 (3.03%) and 10 (1.69%) were found seropositive for RRV, CHIKV or both viruses IgGs, respectively (Table 2). Seroprevalence in different groups and subgroups ranged from 21.43% (group 3b) to 42.42% (group 1) for RRV and it ranged from 0% (group 3b) to 5.94% (group 3a) for CHIKV. Percentages of RRV seropositivity were not significantly different between groups except for group 3b against groups 1 and 2b (P values were 0.0032 and 0.0408, respectively). Among the 204 blood donors seropositive for RRV IgGs, 14 were also found positive using the kit Panbio Ross River Virus IgG ELISA.
      Table 2Seropositivity for RRV and CHIKV in each group of blood donors
      Blood donorsBorn in FPImmigrantsTotal
      Group 1Group 2aGroup 2bTotal group 2Group 3aGroup 3bTotal group 3
      Seropositive for RRV
      Total56 (2)
      Number of blood donors tested positive for RRV with the kit Panbio Ross River Virus IgG Elisa.
      49 (6)52 (5)101 (11)32 (1)15 (0)47 (1)204 (14)
      %42.4233.7935.8634.8331.6821.4327.4834.40
      Seropositive for CHIKV
      Total2641060618
      %1.514.142.763.455.9403.513.03
      Seropositive for both RRV and CHIKV
      Total143720210
      %0.762.762.072.411.980.001.171.69
      a Number of blood donors tested positive for RRV with the kit Panbio Ross River Virus IgG Elisa.
      Seroprevalence for RRV according to the year of birth or the length of stay in FP of blood donors that have never travelled in areas with known circulation of RRV is reported in Table 3. Among blood donors born after 1981 belonging to groups 1 and 2b, 37.09% and 31.16% were seropositive for RRV, respectively. In the two groups, the rate of positivity for RRV IgGs increased with age. Among blood donors of group 3b who arrived in FP during the years 1982-2000, 2001-2005 and 2006-2013, 40%, 25% and 5.88% were seropositive for RRV, respectively.
      Table 3Seropositivity for RRV according to the year of birth or the number of years of residence for blood donors that have never travelled in countries with reported RRV infections
      Blood donors seropositive for RRVGroup 1Group 2bGroup 3b
      Born in FP
      Born before 198133/70 (47.14%)28/68 (41.17%)
      Born from 1982 to 19878/20 (40.00%)12/37 (32.43%)
      Born from 1988 to 199210/27 (37.03%)10/31 (32.25%)
      Born after 19935/15 (33.33%)2/9 (22.22%)
      Total born after 198123/62 (37.09%)24/77 (31.16%)
      Total born before and after 198156/132 (42.42%)52/145 (35.86%)
      Immigrants
      Arrived in FP before 19813/8 (37.5%)
      Arrived in FP between 1982-20008/20 (40.00%)
      Arrived in FP between 2001-20052/8 (25.00%)
      Arrived in FP between 2006-20132/34 (5.88%)
      Total arrived in FP after 198112/62 (19.35%)
      Total arrived in FP before and after 198115/70 (21.43%)

      4. Discussion

      Arboviruses are emerging pathogens in Oceania
      • Cao-Lormeau V.M.
      • Musso D.
      Emerging arboviruses in the Pacific.
      • Kline K.
      • McCarthy J.S.
      • Pearson M.
      • Loukas A.
      • Hotez P.J.
      Neglected tropical diseases of Oceania: review of their prevalence, distribution, and opportunities for control.
      and, except for DENV, few data are available about their circulation, incidence and prevalence in the region. In particular, the spread of RRV in the Pacific region is unknown. In the present study, sample collection was conducted in FP from July 2011 to early October 2013. Even if the first CHIKV outbreak in FP was recorded in October 2014, CHIKV had begun circulating in other PICTs from 2011.
      • Cao-Lormeau V.M.
      • Musso D.
      Emerging arboviruses in the Pacific.
      Consequently, a previous silent circulation of CHIKV in FP cannot be excluded. To insure that the detection of RRV IgGs was not due to cross-reaction of RRV recombinant antigens with CHIKV antibodies resulting from a non-detected previous infection, all sera were tested for the presence of CHIKV IgGs, in addition to RRV IgGs. The low rate of immunization against CHIKV measured among blood donors (3.03%) suggests that the virus did not actively circulate in FP before 2014. This hypothesis is supported by the large magnitude of the CHIKV outbreak that started in October 2014, with at least 25% of the population estimated to have been infected.
      • Nhan T.X.
      • Musso D.
      The burden of chikungunya in the Pacific.
      Moreover, the small number of sera found positive for both RRV and CHIKV IgGs (10/593) suggests the absence or limited cross-reactions between the two Alphaviruses with our ELISA assay.
      The 204 sera found positive for RRV IgGs using recombinant antigens were also tested with a commercial serological assay (kit Panbio Ross River Virus IgG ELISA), and 14 (6.86%) of them were found positive with both assays, confirming the circulation of RRV in FP. Discordant results between in-house and commercial serological tests have already been reported for other arboviruses. For instance, two commercial ELISA kits evaluated for the detection of CHIKV IgGs in serum samples previously tested positive with in-house ELISAs from 2 National Reference Centers for Arboviruses showed sensitivities of 52% and 88%.
      • Prat C.M.
      • Flusin O.
      • Panella A.
      • Tenebray B.
      • Lanciotti R.
      • Leparc-Goffart I.
      Evaluation of commercially available serologic diagnostic tests for chikungunya virus.
      Moreover, when comparing the performance of several serological tests for detecting JEV-specific IgGs in 29 vaccinated children, the in-house assay detected neutralizing antibodies in all children whereas the commercial ELISA assay showed no positive reaction.
      • Cha G.W.
      • Cho J.E.
      • Ju Y.R.
      • Hong Y.-J.
      • Han M.G.
      • Lee W.-J.
      • et al.
      Comparison of four serological tests for detecting antibodies to Japanese encephalitis virus after vaccination in children.
      In another study conducted on serum samples from 100 persons vaccinated against JEV, only 6.6% of the sera found positive with the in-house assay were also detected positive using a commercial IgG ELISA test.
      • Litzba N.
      • Klade C.S.
      • Lederer S.
      • Niedrig M.
      Evaluation of serological diagnostic test systems assessing the immune response to Japanese encephalitis vaccination.
      The discrepancy between the results obtained with laboratory and commercial assays may be related to the cut-off used. For a diagnosis purpose (commercial kit), the cut-off is high because specificity is preferred to sensitivity. Moreover, diagnostic kits are dedicated to the detection of recent rather than past infections and should not be used to screen general population, which is the purpose of seroprevalence studies. According to the manufacturer's protocol, the cut-off of the commercial kit used in our study was determined on an Australian population and may require adjustment depending on the population analyzed. Consequently, the cut-off of the commercial assay should be adapted for the analysis of the serological results obtained from the French Polynesian population.
      The overall rate of RRV seropositive blood donors found in our study was surprisingly high (34.40%) given that RRV infection cases had never been reported in FP. The absence of significant difference in prevalence between blood donors who were born in FP and have travelled or not in areas with or without RRV reported infections (groups 1 and 2), suggests that they have been infected in FP. The high rates of RRV seropositivity found among blood donors who were born in FP after 1981, whatever they had never travelled abroad (group 1, 37.09%) or had travelled only in areas without RRV reported cases (group 2b, 31.16%), support the idea that RRV has circulated in FP after the large outbreak that occurred in 1979-1980 in the PICTs. The lowest (21.43%) and highest (42.42%) RRV seroprevalence rates are found for the immigrant group that is supposed not to have been exposed elsewhere than in FP (group 3b) and for the group of residents who were born in FP and have never travelled abroad (group 1), respectively, and are significantly different between the two groups (P=0.0032). Together with the median time of residence in FP for the immigrants (7.50 years) which is 4 times lower than the one found for the blood donors born in FP (32.00 years), these data suggest that the increase in RRV seroprevalence is linked to the time of residence in FP. The increase with age of the acquisition of RRV IgGs among the residents born in FP and that could have been infected only locally (goups 1 and 2b) is consistent with a silent continuous circulation of RRV during the last decades. This is confirmed by the detection of 12 RRV seropositive blood donors among immigrants who arrived in FP between 1982-2013 and who have never travelled in countries with reported RRV infections (group 3b). The finding that RRV has circulated in FP is concordant with the presence of several mosquito species that can potentially transmit the virus, including Aedes aegypti, Aedes polynesiensis, Culex annulirostris, Culex quinquefasciatus and Culex sitiens.
      • Harley D.
      • Sleigh A.
      • Ritchie S.
      Ross River Virus Transmission, Infection, and Disease: a Cross-Disciplinary Review.
      • Rosen L.
      • Gubler D.J.
      • Bennett P.H.
      Epidemic polyarthritis (Ross River) virus infection in the Cook Islands.
      • Gubler D.J.
      Transmission of Ross River virus by Aedes polynesiensis and Aedes aegypti.
      • Guillaumot L.
      Arboviruses and their vectors in the Pacific - Status report.
      • Lardeux F.J.
      Biological control of Culicidae with the copepod Mesocyclops aspericornis and larvivorous fish (Poeciliidae) in a village of French Polynesia.
      • Mercer D.R.
      • Bossin H.
      • Sang M.C.
      • O’Connor L.
      • Dobson S.L.
      Monitoring temporal abundance and spatial distribution of Aedes polynesiensis using BG-Sentinel traps in neighboring habitats on Raiatea, Society Archipelago, French Polynesia.
      • Rageau J.
      Inventaire des arthropodes d’intérêt médical et vétérinaire dans les territoires français du Pacifique sud.
      RRV may have circulated in FP without being detected for several reasons. The main one is that 55% to 75% of RRV infections are asymptomatic,
      • Harley D.
      • Sleigh A.
      • Ritchie S.
      Ross River Virus Transmission, Infection, and Disease: a Cross-Disciplinary Review.
      • Aaskov J.G.
      • Mataika J.U.
      • Lawrence G.W.
      • Rabukawaqa V.
      • Tucker M.M.
      • Miles J.A.
      • et al.
      An epidemic of Ross River virus infection in Fiji, 1979.
      • Russell R.C.
      • Cope S.E.
      • Yund A.J.
      • Hueston L.
      Combatting the enemy-mosquitoes and Ross River virus in a joint military exercise in tropical Australia.
      • Lau C.
      • Weinstein P.
      • Slaney D.
      Imported cases of Ross River virus disease in New Zealand - a travel medicine perspective.
      and many symptomatic patients have mild symptoms that may not require to seek medical attention. In addition, RRV infections can have clinical presentations similar to DENV infections. However, with laboratory capacities for arboviruses detection mainly focused on DENV, patients presenting with a “dengue like syndrome” but tested negative for DENV are not systematically investigated for other arboviruses. At last, virus isolation is rarely achieved, probably because RRV does not persist beyond the early stages of disease,
      • Harley D.
      • Sleigh A.
      • Ritchie S.
      Ross River Virus Transmission, Infection, and Disease: a Cross-Disciplinary Review.
      which may explain why despite laboratory diagnosis, no RRV infectious cases were detected in FP between 1980-1984.
      Only adults were tested in this study but since most infections occur between the ages of 20 and 60, peaking between 30 and 40,
      • Lau C.
      • Weinstein P.
      • Slaney D.
      Imported cases of Ross River virus disease in New Zealand - a travel medicine perspective.
      • Russell R.C.
      • Dwyer D.E.
      Arboviruses associated with human disease in Australia.
      it should be relevant. Thus, despite the exclusion of children and discrepant results in the percentage of RRV positive blood donors depending on serological methodology used, we can conclude to a silent circulation of RRV in FP. Irrefutable evidence of autochthonous RRV transmission in FP would require the isolation of the virus or of its RNA from a resident with no recent travel history. Attempts to isolate RRV will be performed in a second study on sera collected from patients presenting with symptoms suggestive of an arbovirus infection and tested negative for DENV, ZIKV and CHIKV.
      Like RRV, it is possible that other arboviruses may also have circulated in FP and in other PICTs without being detected. Indeed, the occurrence of West Nile virus or JEV introductions respectively from the USA and Japan are possible since many PICTs have direct and frequent flights from these countries, and several mosquito species established in the PICTs may transmit these viruses.
      • Guillaumot L.
      Arboviruses and their vectors in the Pacific - Status report.
      Due to the growing risk of emergence of new arboviruses in the Pacific, joint efforts should be done to improve local and regional surveillance,
      • Derraik J.G.B.
      • Slaney D.
      • Nye E.R.
      • Weinstein P.
      Vector-borne disease prevention: the need for a joint South Pacific approach.
      as well as vector control.

      Acknowledgments

      We are grateful to Jérôme Viallon from the Institut Louis Malardé, French Polynesia, for technical support, and to Philippe Buchy from the Institut Pasteur in Cambodia, for providing CHIKV positive control serum.
      This study was supported by the “Contrat de projets 2012-2014” (Convention No. 7331/MSS/DSP, 08/31/2012).
      Conflict of interest statement: None of the authors have any conflict of interest (financial or personal) in this study.
      Ethical approval statement: This study was approved by the Ethics Committee of French Polynesia under reference n°60/CEPF (06/27/2013).

      References

        • Singh N.
        • Kiedrzynski T.
        • Lepers C.
        • Benyon E.K.
        Dengue in the Pacific--an update of the current situation.
        Pac Health Dialog. 2005; 12: 111-119
        • Descloux E.
        • Cao-Lormeau V.M.
        • Roche C.
        • De Lamballerie X.
        Dengue 1 diversity and microevolution, French Polynesia 2001-2006: connection with epidemiology and clinics.
        PLoS Negl Trop Dis. 2009; 3: e493
        • Cao-Lormeau V.M.
        • Roche C.
        • Aubry M.
        • Teissier A.
        • Lastere S.
        • Daudens E.
        • et al.
        Recent emergence of dengue virus serotype 4 in French Polynesia results from multiple introductions from other South Pacific Islands.
        PLoS One. 2011; 6: e29555
        • Cao-Lormeau V.M.
        • Roche C.
        • Musso D.
        • Mallet H.-P.
        • Dalipanda T.
        • Dofai A.
        • et al.
        Dengue virus type 3, South pacific islands, 2013.
        Emerg Infect Dis. 2014; 20: 1034-1036
        • Cao-Lormeau V.M.
        • Roche C.
        • Teissier A.
        • Robin E.
        • Berry A.-L.
        • Mallet H.-P.
        • et al.
        Zika virus, French polynesia, South pacific, 2013.
        Emerg Infect Dis. 2014; 20: 1085-1086
        • Musso D.
        • Nilles E.J.
        • Cao-Lormeau V.M.
        Rapid spread of emerging Zika virus in the Pacific area.
        Clin Microbiol Infect. 2014; 20 (O595-6)
        • Aubry M.
        • Teissier A.
        • Roche C.
        • Richard V.
        • Shan Yan A.
        • Zisou K.
        • et al.
        Chikungunya outbreak, French Polynesia, October 2014.
        Emerg Infect Dis. 2015; 21: 724-726
        • Cao-Lormeau V.M.
        • Musso D.
        Emerging arboviruses in the Pacific.
        Lancet. 2014; 384: 1571-1572
        • Duffy M.R.
        • Chen T.-H.
        • Hancock W.T.
        • Powers A.M.
        • Kool J.L.
        • Lanciotti R.S.
        • et al.
        Zika virus outbreak on Yap Island, Federated States of Micronesia.
        N Engl J Med. 2009; 360: 2536-2543
        • Baig S.
        • Fox K.
        • Jee Y.
        • O’Connor P.
        • Hombach J.
        • Wang S.
        • et al.
        Japanese Encephalitis Surveillance and Immunization — Asia and the Western Pacific, 2012.
        Morb Mortal Wkly Rep. 2013; 62: 658-662
        • Barber B.
        • Denholm J.T.
        • Spelman D.
        Ross River virus.
        Aust Fam Physician. 2009; 38: 586-589
        • Harley D.
        • Sleigh A.
        • Ritchie S.
        Ross River Virus Transmission, Infection, and Disease: a Cross-Disciplinary Review.
        Clin Microbiol Rev. 2001; 14: 909-932
        • Doherty R.L.
        • Whitehead R.H.
        • Gorman B.M.
        • O’Gower A.K.
        The isolation of a third group A arbovirus in Australia with preliminary observations on its relationship to epidemic polyarthritis.
        Aust J Sci. 1963; 26: 183-184
        • Aaskov J.G.
        • Mataika J.U.
        • Lawrence G.W.
        • Rabukawaqa V.
        • Tucker M.M.
        • Miles J.A.
        • et al.
        An epidemic of Ross River virus infection in Fiji, 1979.
        Am J Trop Med Hyg. 1981; 30: 1053-1059
        • Russell R.C.
        • Cope S.E.
        • Yund A.J.
        • Hueston L.
        Combatting the enemy-mosquitoes and Ross River virus in a joint military exercise in tropical Australia.
        Am J Trop Med Hyg. 1998; 59: S307
        • Lau C.
        • Weinstein P.
        • Slaney D.
        Imported cases of Ross River virus disease in New Zealand - a travel medicine perspective.
        Travel Med Infect Dis. 2012; 10: 129-134
        • Edwards A.
        An unusual epidemic.
        Med J Aust. 1928; 1: 664-665
        • Nimmo J.
        An unusual epidemic.
        Med J Aust. 1928; 1: 549-550
        • Rosen L.
        • Gubler D.J.
        • Bennett P.H.
        Epidemic polyarthritis (Ross River) virus infection in the Cook Islands.
        Am J Trop Med Hyg. 1981; 30: 1294-1302
        • Tesh R.B.
        • McLean R.G.
        • Shroyer D.A.
        • Calisher C.H.
        • Rosen L.
        Ross River virus (Togaviridae: Alphavirus) infection (epidemic polyarthritis) in American Samoa.
        Trans R Soc Trop Med Hyg. 1981; 75: 426-431
        • Panon G.
        • Le Gonidec G.
        • Fauran P.
        Laboratory diagnosis of epidemic polyarthritis (Ross River virus) in New Caledonia.
        Bull Soc Pathol Exot Filiales. 1983; 76: 755-760
        • Fauran P.
        • Le Gonidec G.
        • Rodhain F.
        Research on viral infections in South Pacific mosquitoes under natural conditions.
        Bull Soc Pathol Exot Filiales. 1984; 77: 628-636
        • Fauran P.
        • Donaldson M.
        • Harper J.
        • Oseni R.A.
        • Aaskov J.G.
        Characterization of Ross River viruses isolated from patients with polyarthritis in New Caledonia and Wallis and Futuna Islands.
        Am J Trop Med Hyg. 1984; 33: 1228-1231
        • Klapsing P.
        • MacLean J.D.
        • Glaze S.
        • McClean K.L.
        • Drebot M.A.
        • Lanciotti R.S.
        • et al.
        Ross River virus disease reemergence, Fiji, 2003–2004.
        Emerg Infect Dis. 2005; 11: 613-615
        • Hii J.
        • Dyke T.
        • Dagoro H.
        • Sanders R.C.
        Health impact assessments of malaria and Ross River virus infection in the Southern Highlands Province of Papua New Guinea.
        P N G Med J. 1997; 40: 14-25
        • Stallman N.D.
        • Wiemers M.A.
        • Bourke A.T.
        Serology in diagnosis and surveys of man.
        Arbovirus Res Aust. 1976; 1: 46-64
        • Doherty R.L.
        • Gorman B.M.
        • Whitehead R.H.
        • Carley J.G.
        Studies of epidemic polyarthritis: the significance of three group A arboviruses isolated from mosquitoes in Queensland.
        Australas Ann Med. 1964; 13: 322-327
        • Tesh R.B.
        • Gajdusek D.C.
        • Garruto R.M.
        • Cross J.H.
        • Rosen L.
        The distribution and prevalence of group A arbovirus neutralizing antibodies among human populations in Southeast Asia and the Pacific islands.
        Am J Trop Med Hyg. 1975; 24: 664-675
        • Weinstein P.
        • Worswick D.
        • Macintyre A.
        • Cameron S.
        Human sentinels for arbovirus surveillance and regional risk classification in South Australia.
        Med J Aust. 1994; 160: 494-499
        • Yu W.
        • Mengersen K.
        • Dale P.
        • Mackenzie J.S.
        • Toloo G.S.
        • Wang X.
        • et al.
        Epidemiologic patterns of Ross River virus disease in Queensland, Australia, 2001–2011.
        Am J Trop Med Hyg. 2014; 91: 109-118
        • Warter L.
        • Lee C.Y.
        • Thiagarajan R.
        • Grandadam M.
        • Lebecque S.
        • Lin R.T.P.
        • et al.
        Chikungunya virus envelope-specific human monoclonal antibodies with broad neutralization potency.
        J Immunol. 2011; 186: 3258-3264
        • Kline K.
        • McCarthy J.S.
        • Pearson M.
        • Loukas A.
        • Hotez P.J.
        Neglected tropical diseases of Oceania: review of their prevalence, distribution, and opportunities for control.
        PLoS Negl Trop Dis. 2013; 7: e1755
        • Nhan T.X.
        • Musso D.
        The burden of chikungunya in the Pacific.
        Clin Microbiol Infect. 2015;
        • Prat C.M.
        • Flusin O.
        • Panella A.
        • Tenebray B.
        • Lanciotti R.
        • Leparc-Goffart I.
        Evaluation of commercially available serologic diagnostic tests for chikungunya virus.
        Emerg Infect Dis. 2014; 20: 2129-2132
        • Cha G.W.
        • Cho J.E.
        • Ju Y.R.
        • Hong Y.-J.
        • Han M.G.
        • Lee W.-J.
        • et al.
        Comparison of four serological tests for detecting antibodies to Japanese encephalitis virus after vaccination in children.
        Osong public Heal Res Perspect. 2014; 5: 286-291
        • Litzba N.
        • Klade C.S.
        • Lederer S.
        • Niedrig M.
        Evaluation of serological diagnostic test systems assessing the immune response to Japanese encephalitis vaccination.
        PLoS Negl Trop Dis. 2010; 4: e883
        • Gubler D.J.
        Transmission of Ross River virus by Aedes polynesiensis and Aedes aegypti.
        Am J Trop Med Hyg. 1981; 30: 1303-1306
        • Guillaumot L.
        Arboviruses and their vectors in the Pacific - Status report.
        Pac Heal Dialog. 2005; 12: 45-52
        • Lardeux F.J.
        Biological control of Culicidae with the copepod Mesocyclops aspericornis and larvivorous fish (Poeciliidae) in a village of French Polynesia.
        Med Vet Entomol. 1992; 6: 9-15
        • Mercer D.R.
        • Bossin H.
        • Sang M.C.
        • O’Connor L.
        • Dobson S.L.
        Monitoring temporal abundance and spatial distribution of Aedes polynesiensis using BG-Sentinel traps in neighboring habitats on Raiatea, Society Archipelago, French Polynesia.
        J Med Entomol. 2012; 49: 51-60
        • Rageau J.
        Inventaire des arthropodes d’intérêt médical et vétérinaire dans les territoires français du Pacifique sud.
        Med Vet Entomol. 1958; 3: 873-882
        • Russell R.C.
        • Dwyer D.E.
        Arboviruses associated with human disease in Australia.
        Microbes Infect. 2000; 2: 1693-1704
        • Derraik J.G.B.
        • Slaney D.
        • Nye E.R.
        • Weinstein P.
        Vector-borne disease prevention: the need for a joint South Pacific approach.
        N Z Med J. 2009; 122: 7-12