Experimental Adaptation of the Yellow Fever Virus to the Mosquito Aedes albopictus and Potential risk of urban epidemics in Brazil, South America

Abstract

Despite the availability of an efficient vaccine, Yellow fever (YF), a viral disease transmitted by mosquitoes, is still a threat. In Brazil, the yellow fever virus (YFV) has been restricted to a jungle cycle for more than 70 years. However, YFV has recently invaded populated cities in the Southeast such as Rio de Janeiro where the opportunistic mosquito Aedes albopictus is well established. Using in vivo passages of YFV in Ae. albopictus, we have selected viral strains presenting substitutions in NS1 gene. We did 10 passages of YFV-74018 on two distinct Ae. albopictus populations: (i) Manaus collected from a YFV-endemic area in Amazonia and (ii) PNMNI from a YFV-free area in the state of Rio de Janeiro. Full viral genomes were deep sequenced at each passage. We obtained two YFV strains presenting a non-synonymous substitution in the NS1 gene. Interestingly, they intervened at two different positions in NS1 gene according to the mosquito population: I2772T in Ae. albopictus Manaus and S3303N in Ae. albopictus PNMNI. Both substitutions reached fixation at the passage 10. Our data suggest that YFV has the potential for adaption to Ae. albopictus thereby posing a threat to most cities in South America where this mosquito is present.

Figure 1

Viral titers obtained at each passage of YFV on Ae. albopictus mosquitoes (A) using virus collected from head and (B) from saliva. Mosquitoes were orally infected with YFV-74018. After 21 days, mosquitoes were processed as follows: (i) heads were collected and ground, and (ii) mosquito saliva were pooled. Head and saliva homogenates were incubated on Ae. albopictus C6/36 cells for 8 days. Collected supernatants were used to initiate the next passage. 30 mosquitoes were used at each passage.

Figure 2

Experimental design to enhance YFV transmission by Ae. albopictus. The YFV-74018 was passaged 10 times on two populations of Ae. albopictus: (i) Manaus collected in a YFV-endemic area in Amazonia, and (ii) PNMNI collected from Rio de Janeiro, a YFV-free area. Each passage includes: the oral infection of mosquitoes with YFV, collection of mosquito saliva at day 21 post-infection, viral amplification on Ae. albopictus derived C6/36 cell culture, and initiation of the next passage using the viral suspension obtained. Control isolates were serially passaged 10 times on C6/36 cells.

Figure 3

Heatmap indicating variant frequency differences between the reference genome YFV-74018 and isolates selected after passages on Ae. albopictus Manaus (A) and PNMNI (B). All 10 passages were deep sequenced: P1 to P5 were performed using homogenates of mosquito heads, and P6-S to P10-S with mosquito saliva. The intensity of black corresponds to the variant frequency. Only variants for which the frequency is above 20% are shown.

Figure 4

Frequency of the T2772C variant detected after the passage P7-S in Ae. albopictus Manaus. This substitution corresponds to a non-synonymous change from isoleucine to threonine in NS1 gene.

Figure 5

Frequencies of the K1702K and S3303N variants detected after the passages P6-S and P7-S respectively in Ae. albopictus PNMNI. Only the substitution S3303N corresponds to a non-synonymous change from serine to asparagine in NS1 gene.