Reinforced surveillance systems aimed at monitoring the introduction of CHIKV have been implemented in 6 departments in southeastern France, including the Var department, where Ae. albopictus
has spread since its introduction in 2004, presumably from northern Italy (4
). On August 29, 2010, a 7-year-old girl (patient 1) with acute febrile syndrome, headache, and abdominal pain sought treatment in the city of Fréjus (Var) 1 day after she had returned from Rajasthan, India. Continuous CHIKV circulation in northern India districts has been reported during 2009–2010 (www.promedmail.org
). The patient’s serum sample was found positive for CHIKV infection by reverse transcription–PCR (RT-PCR) (5,6
). Three weeks after the notification of patient 1, another young girl (patient 2) experienced clinical symptoms that began on September 18 with fever, arthralgia, backache, headache, and retro-orbital pain. Patient 2 had no history of travel in areas endemic for CHIKV. She resided 2.5 km from patient 1. The serum specimen was positive for CHIKV diagnosis. Patient 2’s physician reported that a young girl (patient 3), a close friend of her patient, showed clinical symptoms compatible with CHIKV infection at the same time. Patient 3, who lives near patient 1, had invited patient 2 to spend the night of September 15 at her home. The 2 children reported numerous mosquito bites. A serum sample from patient 3 was collected 1 week after onset of fever and monoclonal antibody capture ELISA detected high titers of specific anti-CHIKV immunoglobulin M. The serum sample also showed a weak RT-PCR signal for CHIKV. Given that patients 2 and 3 did not report any recent travel to areas endemic for CHIKV, their illnesses were classified as autochthonous cases of CHIKV infection. No complications were recorded, but all 3 patients had persistent weakness and joint pain 3 months after the acute phase.
High densities of Ae. albopictus mosquitoes have been found in the Var department since 2008. Intensive mosquito control measures, including spraying for adult mosquitoes and destroying breeding sites, were undertaken around the patients’ residences and areas visited by confirmed case-patients. No further cases were found by the active case finding system (a local physician and laboratories network) implemented for 45 days after the declaration of the last autochthonous case.
A molecular study of France/2010 CHIKV strains isolated in Fréjus obtained from patients 1 (imported case) and 2 (autochthonous case) was performed. Viral genomic RNA was extracted from CHIKV grown once in mosquito C6/36 cells and then subjected to RT-PCR amplification by using a set of primers targeting the structural genes of CHIKV (7
). Paired sequence analysis of the E2–6K–E1 junction showed that the 2 France/2010 CHIKV strains display a divergence rate <0.05% at the nucleotide level, whereas 100% identity was observed at the amino acid level. Phylogenetic analysis demonstrated that these viral strains belong to a cluster that is closely related to strains from India within the ECSA lineage (). The France/2010 CHIKV isolate from patient 2 might be derived from an Indian strain introduced by patient 1 (index case). Genotypes E2-211T, E2-312M, E2-386A, 6K-8I, and E1-284E that are found in the currently circulating strains belonging to the ECSA lineage were identified in France/2010 CHIKV isolates (2,3,7
). These isolates also display the genotype E1-211E specifically shared by viral strains belonging to the Asian phylogenetic group (). The residue Ala at position E2-264 has not been previously described in any CHIKV strains.
Figure Phylogenetic relationships among chikungunya virus isolates from cases of chikungunya fever in France, based on complete E2-6K-E1 nucleotide sequence (2,771 nt) analysis. Gray shading indicates imported and autochthonous strains. Sequence alignments were (more ...)
Relevant amino acid substitutions identified between France/2010 CHIKV isolates (autochthonous and imported cases) versus a selection of CHIKV strains*
Recent attention has focused on the predominant role of E1 and E2 proteins in successful CHIKV infection of the anthropophilic Ae. albopictus
). Vector competence experiments with La Réunion/2006 CHIKV isolates demonstrated the importance of the newly acquired E1-Ala226Val substitution for efficient transmission by Ae. albopictus
mosquitoes during the 2006 outbreak in Réunion Island (7–10
). Italy/2007 CHIKV strains also exhibited the signature E1-226V genotype (11
from northern Italy and from southeastern France showed disseminated infection rates ranging from 75%–90% for CHIKV strains with E1-226V (10
). The 2 France/2010 CHIKV strains isolated in Fréjus have Ala at position E1-226 (). The presence of an Asp residue at position E2-60, found in most of the ECSA CHIKV strains, may in part counterbalance the less favorable transmission of E1-226A strain in Ae. albopictus
(). The Thr residue at position E2-211 potentiates the infectivity of CHIKV in Ae. albopictus
mosquitoes only in synergy with E1-226V. The presence of E2-211T in CHIKV isolates from France underlines the risk for emergence of a fully adapted viral variant if the E1-226V genotype was selected during continuous transmission within Ae. albopictus
populations in France (7,8,9