Infection, dissemination, and transmission efficiencies of Zika virus in Aedes aegypti after serial passage in mosquito or mammalian cell lines or alternating passage in both cell types

Abstract

Background: Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) with an urban transmission cycle that primarily involves humans and Aedes aegypti. Evidence suggests that the evolution of some arboviruses is constrained by their dependency on alternating between disparate (vertebrate and invertebrate) hosts. The goals of this study are to compare the genetic changes that occur in ZIKV after serial passaging in mosquito or vertebrate cell lines or alternate passaging in both cell types and to compare the replication, dissemination, and transmission efficiencies of the cell culture-derived viruses in Ae. aegypti.

Methods: An isolate of ZIKV originally acquired from a febrile patient in Yucatan, Mexico, was serially passaged six times in African green monkey kidney (Vero) cells or Aedes albopictus (C6/36) cells or both cell types by alternating passage. A colony of Ae. aegypti from Yucatan was established, and mosquitoes were challenged with the cell-adapted viruses. Midguts, Malpighian tubules, ovaries, salivary glands, wings/legs and saliva were collected at various times after challenge and tested for evidence of virus infection.

Results: Genome sequencing revealed the presence of two non-synonymous substitutions in the premembrane and NS1 regions of the mosquito cell-adapted virus and two non-synonymous substitutions in the capsid and NS2A regions of both the vertebrate cell-adapted and alternate-passaged viruses. Additional genetic changes were identified by intrahost variant frequency analysis. Virus maintained by continuous C6/36 cell passage was significantly more infectious in Ae. aegypti than viruses maintained by alternating passage and consecutive Vero cell passage.

Conclusions: Mosquito cell-adapted ZIKV displayed greater in vivo fitness in Ae. aegypti compared to the other viruses, indicating that obligate cycling between disparate hosts carries a fitness cost. These data increase our understanding of the factors that drive ZIKV adaptation and evolution and underscore the important need to consider the in vivo passage histories of flaviviruses to be evaluated in vector competence studies.

Keywords: Adaptive mutations; Aedes aegypti; Flavivirus; Vectorial competence; Zika virus.

Fig. 1

Intrahost variant frequency analysis of strains tested. Intrahost variant (iSNVs) frequency analysis of four strain tested. distributed across the ZIKV CDS (a). iSNV frequency analysis of passage-specific mutations (b–h). The sum of novel variants not found in ZIKV-6C, ZIKV-6V and ZIKV-6A, which are unique to those observed in ZIKV-I (i). Genetic divergence (F_ST) of ZIKV-6C, ZIKV-6V and ZIKV-6A was compared with ZIKV-I (j)

Fig. 2

Midgut infection and dissemination rates of Ae. Aegypti infected with host-cell adapted Zika virus. Midgut infection rates of Ae. aegytpi infected with host cell-adapted Zika virus at (a) 14 days and (b) 21 days after infectious blood meal (Kruskal-Wallis test, X² = 70.288, df = 3, P ≤ 0.05 and X² = 58.807, df = 3, P ≤ 0.05, respectively). Dissemination rates to organs other than midgut of Ae. aegytpi challenged with host cell-adapted Zika virus at c 14 days (Kruskal-Wallis test, X² = 3.280, df = 2, P ≥ 0.05) and d 21 days after infectious blood meal (not statistically proven because all three strains have a ratio of 1). Bars with a different letter are statistically significant

Fig. 3

Salivary gland infection and transmission rates of Ae. aegytpi infected with host cell-adapted Zika virus. Salivary glands infection rates of Ae. aegytpi challenged with host cell-adapted Zika virus at a 14 days and b 21 days after infectious blood meal (Kruskal-Wallis test, X² = 1.129, df = 3, P ≥ 0.05 and X² = 1.858, df = 2, P ≥ 0.05, respectively). Transmission rates of Ae. aegypti challenged with host cell-adapted Zika virus at c 14 days and d 21 days after infectious blood meal (Mann-Whitney U-test, Z = 1.449, P ≥ 0.05 and Z = − 0.062, P ≥ 0.05, respectively). Bars with the same letter are not statistically significant

Fig. 4

RNA copy numbers in organs and body parts from mosquitoes challenged with ZIKV-I and ZIKV-6C. The study was performed using: a Malpighian tubules, b midguts, c ovaries, d salivary glands and e wings/legs. The amount of viral RNA in each organ over time was determined by Friedman test. The amount of viral RNA in different organs was compared at the same time point using Mann-Whitney U and Kruskal-Wallis tests. P ≤ 0.05 was considered significant. ZIKV-6V and ZIKV-6A were not included in the analysis because a small number of mosquitoes tested positive for these viruses, preventing a robust statistical analysis