High level of vector competence of Aedes aegypti and Aedes albopictus from ten American countries as a crucial factor in the spread of Chikungunya virus

Abstract

Chikungunya virus (CHIKV) causes a major public health problem. In 2004, CHIKV began an unprecedented global expansion and has been responsible for epidemics in Africa, Asia, islands in the Indian Ocean region, and surprisingly, in temperate regions, such as Europe. Intriguingly, no local transmission of chikungunya virus (CHIKV) had been reported in the Americas until recently, despite the presence of vectors and annually reported imported cases. Here, we assessed the vector competence of 35 American Aedes aegypti and Aedes albopictus mosquito populations for three CHIKV genotypes. We also compared the number of viral particles of different CHIKV strains in mosquito saliva at two different times postinfection. Primarily, viral dissemination rates were high for all mosquito populations irrespective of the tested CHIKV isolate. In contrast, differences in transmission efficiency (TE) were underlined in populations of both species through the Americas, suggesting the role of salivary glands in selecting CHIKV for highly efficient transmission. Nonetheless, both mosquito species were capable of transmitting all three CHIKV genotypes, and TE reached alarming rates as high as 83.3% and 96.7% in A. aegypti and A. albopictus populations, respectively. A. albopictus better transmitted the epidemic mutant strain CHIKV_0621 of the East-Central-South African (ECSA) genotype than did A. aegypti, whereas the latter species was more capable of transmitting the original ECSA CHIKV_115 strain and also the Asian genotype CHIKV_NC. Therefore, a high risk of establishment and spread of CHIKV throughout the tropical, subtropical, and even temperate regions of the Americas is more real than ever.

Importance: Until recently, the Americas had never reported chikungunya (CHIK) autochthonous transmission despite its global expansion beginning in 2004. Large regions of the continent are highly infested with Aedes aegypti and Aedes albopictus mosquitoes, and millions of dengue (DEN) cases are annually recorded. Indeed, DEN virus and CHIK virus (CHIKV) share the same vectors. Due to a recent CHIK outbreak affecting Caribbean islands, the need for a Pan-American evaluation of vector competence was compelling as a key parameter in assessing the epidemic risk. We demonstrated for the first time that A. aegypti and A. albopictus populations throughout the continent are highly competent to transmit CHIK irrespective of the viral genotypes tested. The risk of CHIK spreading throughout the tropical, subtropical, and even temperate regions of the Americas is more than ever a reality. In light of our results, local authorities should immediately pursue and reinforce epidemiological and entomological surveillance to avoid a severe epidemic.

FIG 1

Mosquito populations tested. The color code indicates localities where only A. aegypti (red), only A. albopictus (blue), and both A. aegypti and A. albopictus (green) mosquitoes were collected. TYS, Tyson, MO; VRB, Vero Beach, FL; MXC, Chiapas, Mexico; PAN, Panamá, Panama; DEL, Delta Amacuro, Venezuela; TUM, Tumbes, Peru; PUM, Punchana, Peru; MAN, Manaus, Brazil; STR, Santarém, Brazil; PNM, Parnamirim, Brazil; CAB, Campos Belos, Brazil; CPG, Campo Grande, Brazil; JRB, Jurujuba, Brazil; PAQ, Paquetá, Brazil; VAZ, Vaz Lobo, Brazil; BEL, Belford Roxo, Brazil; SAN, Santos, Brazil; BMA, Monteagudo, Bolivia; SDG, Salto del Guairá, Paraguay; ASU, Asunción, Paraguay; SAL, Salto, Uruguay; MIA, Misiones, Argentina; ACO, Corrientes, Argentina; BUE, Buenos Aires, Argentina.

FIG 2

Dissemination (A) and transmission (B) efficiencies of two CHIKV isolates and two clones of the respective viral isolates in A. albopictus mosquitoes from Paquetá, Rio de Janeiro, Brazil. At days 1, 2, 3, 7, and 10 after an infectious blood meal, mosquitoes were sacrificed, and heads and saliva were collected for determination of their infectious status. Mosquito heads were individually ground in 250 μl Leibovitz L15 medium supplemented with 4% FBS, following inoculation onto an A. albopictus C6/36 cell monolayer in 96-well plates and incubation at 28°C for 3 days. Plates were fixed with 3.6% formaldehyde, washed three times with PBS, and analyzed by indirect immunofluorescence assay (IFA). For saliva collection, each mosquito had the wings and legs removed, and the proboscis was inserted into a 20-μl tip containing 5 μl of FBS. After 45 min of salivation, FBS containing saliva was expelled into 45 μl of Leibovitz L15 medium and inoculated onto an A. albopictus C6/36 cell monolayer in 96-well plates. Plates were incubated and stained (IFA) as described in Materials and Methods. Dissemination efficiency corresponds to the proportion of mosquito females with disseminated virus in the head among the tested mosquitoes. Transmission efficiency corresponds to the proportion of mosquitoes with infectious saliva among the tested mosquitoes. CHIKV_0621 is a strain isolated from La Réunion (E1-226V substitution), CHIKV_115 is a strain isolated from La Réunion (E1-226A), CHIKV_0621 (V) is a clone corresponding to a single virus isolated from CHIKV_0621, and CHIKV_115 (A) is a clone corresponding to a single virus isolated from CHIKV_115. Clones were provided by C. Arias-Goeta, Institut Pasteur, Paris, France.

FIG 3

Transmission efficiency of three CHIKV isolates in 35 A. albopictus and A. aegypti populations from 10 American countries at day 7 postinfection. After an infectious blood meal, mosquitoes were sacrificed, and saliva was collected from individual mosquitoes and titrated by focus fluorescent assay on A. albopictus C6/36 cells to determine infectious status. Transmission efficiency corresponds to the proportion of mosquitoes with infectious saliva among those tested. Viral strains are as follows: CHIKV_0621 was isolated from La Réunion (ECSA genotype, E1-226V and E1-98A substitutions), CHIKV_115 was isolated from La Réunion (ECSA genotype, E1-226A and E1-98A substitutions), and CHIKV_NC was isolated from New Caledonia (Asian genotype, E1-226A and E1-98T substitutions). Mosquito populations are as follows (from north to south): TYS, Tyson, MO; VRB, Vero Beach, FL; MXC, Chiapas, Mexico; PAN, Panamá, Panama; DEL, Delta Amacuro, Venezuela; TUM, Tumbes, Peru; PUM, Punchana, Peru; MAN, Manaus, Brazil; STR, Santarém, Brazil; PNM, Parnamirim, Brazil; CAB, Campos Belos, Brazil; CPG, Campo Grande, Brazil; JRB, Jurujuba, Brazil; PAQ, Paquetá, Brazil; VAZ, Vaz Lobo, Brazil; BEL, Belford Roxo, Brazil; SAN, Santos, Brazil; BMA, Monteagudo, Bolivia; SDG, Salto del Guairá, Paraguay; ASU, Asunción, Paraguay; SAL, Salto, Uruguay; MIA, Misiones, Argentina; ACO, Corrientes, Argentina; and BUE, Buenos Aires, Argentina. Error bars show 95% confidence intervals.

FIG 4

Transmission efficiency of CHIKV_0621 and CHIKV_115 isolates in 35 A. aegypti and A. albopictus populations from 10 American countries at day 7 postinfection. Transmission efficiency corresponds to the proportion of mosquitoes with infectious saliva among those tested. The color code indicates different degrees of transmission efficiency (TE): yellow, mosquito strains with TE ≤ 30% (low TE); pale orange, strains with 30% < TE < 70% (moderate TE); red, strains with TE ≥ 70% (high TE). The viral strains are as follows: CHIKV_0621 was isolated from La Réunion (ECSA genotype, E1-226V substitution) and CHIKV_115 isolated from La Réunion (ECSA genotype, E1-226A substitution). The mosquito populations are as follows (from north to south): TYS, Tyson, MO; VRB, Vero Beach, FL; MXC, Chiapas, Mexico; PAN, Panamá, Panama; DEL, Delta Amacuro, Venezuela; TUM, Tumbes, Peru; PUM, Punchana, Peru; MAN, Manaus, Brazil; STR, Santarém, Brazil; PNM, Parnamirim, Brazil; CAB, Campos Belos, Brazil; CPG, Campo Grande, Brazil; JRB, Jurujuba, Brazil; PAQ, Paquetá, Brazil; VAZ, Vaz Lobo, Brazil; BEL, Belford Roxo, Brazil; SAN, Santos, Brazil; BMA, Monteagudo, Bolivia; SDG, Salto del Guairá, Paraguay; ASU, Asunción, Paraguay; SAL, Salto, Uruguay; MIA, Misiones, Argentina; ACO, Corrientes, Argentina; BUE, Buenos Aires, Argentina.

FIG 5

Viral loads of three CHIKV isolates in saliva of A. albopictus and A. aegypti mosquitoes from 35 populations from the Americas at day 7 postinfection. At day 7 after an infectious blood meal, mosquitoes were sacrificed, and saliva was collected from individual mosquitoes and titrated by focus fluorescent assay on A. albopictus C6/36 cells. The viral strains are as follows: CHIKV_0621 was isolated from La Réunion (ECSA genotype, E1-226V and E1-98A substitutions), CHIKV_115 was isolated from La Réunion (ECSA genotype, E1-226A and E1-98A substitutions), and CHIKV_NC was isolated from New Caledonia (Asian genotype, E1-226A and E1-98T substitutions). The mosquito populations are as follows (from north to south): TYS, Tyson, MO; VRB, Vero Beach, FL; MXC, Chiapas, Mexico; PAN, Panamá, Panama; DEL, Delta Amacuro, Venezuela; TUM, Tumbes, Peru; PUM, Punchana, Peru; MAN, Manaus, Brazil; STR, Santarém, Brazil; PNM, Parnamirim, Brazil; CAB, Campos Belos, Brazil; CPG, Campo Grande, Brazil; JRB, Jurujuba, Brazil; PAQ, Paquetá, Brazil; VAZ, Vaz Lobo, Brazil; BEL, Belford Roxo, Brazil; SAN, Santos, Brazil; BMA, Monteagudo, Bolivia; SDG, Salto del Guairá, Paraguay; ASU, Asunción, Paraguay; SAL, Salto, Uruguay; MIA, Misiones, Argentina; ACO, Corrientes, Argentina; BUE, Buenos Aires, Argentina. Error bars refer to the standard error of the mean titer for each pairing of mosquito population and virus strain.