Laboratory strains of Aedes aegypti are competent to Brazilian Zika virus

Abstract

The Zika virus outbreaks are unprecedented human threat in relation to congenital malformations and neurological/autoimmune complications. Since this virus has high potential to spread in regions presenting the vectors, improvement in mosquito control is a top priority. Thus, Aedes aegypti laboratory strains will be fundamental to support studies in different research fields implicated on Zika-mosquito interactions which are the basis for the development of innovative control methods. In this sense, our aim was to determine the main infection aspects of a Brazilian Zika strain in reference Aedes aegypti laboratory mosquitoes. We orally exposed Rockefeller, Higgs and Rexville mosquitoes to the Brazilian ZIKV (ZIKVBR) and qRT-PCR was applied to determine the infection, dissemination and detection rates of ZIKV in the collected saliva as well as viral levels in mosquito tissues. The three strains sustain the virus development but Higgs showed significantly lower viral loads in bodies at 14 days post-infection (dpi) and the lowest prevalences in bodies and heads. The Rockefeller strain was the most susceptible at 7 dpi but similar dissemination rates were observed at 14 dpi. Although variations exist, the ZIKVBR RNA shows detectable levels in saliva of the three strains at 14 dpi but is only detected in Rockefeller at 7 dpi. Moreover, saliva samples from the three strains were confirmed to be infectious when intrathoracically injected into mosquitoes. The ZIKVBR kinetics was monitored in Rockefeller mosquitoes and virus could be identified in the heads at 4 dpi but was more consistently detected late in infection. Our study presents the first evaluation on how Brazilian Zika virus behaves in reference Aedes aegypti strains and shed light on how the infection evolves over time. Vector competence and hallmarks of the ZIKVBR development were revealed in laboratory mosquitoes, providing additional information to accelerate studies focused on ZIKV-mosquito interactions.

Fig 1. ZIKV^BR infection rates and viral levels in bodies and heads from Ae. aegypti laboratory strains.

The infection rate and viral levels per tissue were individually recorded in ROCK, HWE and RED females at 7 and 14 days following a ZIKV-infected blood meal (dpi). (A) Viral prevalence and infection levels in the bodies. Each body sample is represented by a solid circle. The infection prevalences between HWE x ROCK and HWE x RED at 7 (*p = 0.0197) and 14 dpi (*p = 0.0436) were significant different by Fisher’s exact test. The viral infection levels of the HWE were significantly lower in the bodies at 14 dpi compared to the ROCK (***p<0.001) or RED (**p<0.01) by two-way ANOVA with Bonferroni post-tests. (B) Viral prevalence and infection levels in the heads. Individual heads are indicated by open triangles. The infection prevalences between HWE x ROCK (**p = 0.0033) at 7 dpi and HWE x ROCK and HWE x RED (**p = 0.0033) at 14 dpi were significantly different by Fisher’s exact test. Black bars indicate the mean viral copy numbers and the dashed grey line demonstrates the detection limit.

Fig 2. ZIKV^BR infection, dissemination and detection rates of ZIKV in the collected saliva of Ae. aegypti laboratory strains.

The infection rate (number of infected bodies/total bodies analyzed), dissemination rate (number of infected heads/number of infected bodies) and the detection rate of ZIKV in the collected saliva (number of infected saliva sample/number of infected heads) were estimated in females from ROCK, HWE and RED strains at 7 and 14 days following a ZIKV-infected blood meal (dpi). The bars are representing the percentage values.

Fig 3. ZIKV^BR prevalence and viral levels in the collected saliva from Ae. aegypti laboratory strains.

The prevalence and viral levels per saliva sample were individually recorded in the ROCK, HWE and RED strains at 7 and 14 days following a ZIKV-infected blood meal (dpi). Each saliva sample is represented by a solid star. The prevalence of positive saliva between ROCK x RED was statistically different at 14 dpi (*p = 0.0436). Black bars indicate the mean viral copy numbers and the dashed grey line demonstrates the detection limit.

Fig 4. ZIKV^BR kinetics in bodies and heads of Ae. aegypti from the ROCK strain.

The infection rate and viral levels per tissue were individually recorded at 1, 4, 7, 11 and 14 days following a ZIKV-infected blood meal (dpi). Solid circles and open triangles represent each body and head sample, respectively, from the ROCK females. Solid (body) and dashed (head) blue lines indicate the viral infection progression (mean levels) during the time-course experiment. Dashed blue bars indicate early (1 to 4 dpi) and late (7 to 14 dpi) infection stages in heads in which a significant difference in infection prevalence was observed between the two stages using Fisher’s exact test (*, 1 dpi x 7 dpi—p = 0.0031; 1 dpi x 11 dpi—p = 0.0007; 1dpi x 14 dpi—p<0.0001; 4 dpi x 7 dpi—p = 0.0198; 4 dpi x 11 dpi—p = 0.0055; 4 dpi x 14 dpi—p = 0.0001). The dashed grey line demonstrates the detection limit.