Comparison of Chikungunya Virus and Zika Virus Replication and Transmission Dynamics in Aedes aegypti Mosquitoes

Abstract

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are arthropod-borne viruses transmitted mainly by Aedes aegypti mosquitoes. These viruses have become endemic in large parts of North, Central, and South America. Arboviruses persistently infect mosquitoes throughout their life span and become infectious (i.e., expectorate infectious virus in saliva) after a period of time called the extrinsic incubation period (EIP). The duration of this infectiousness, however, is not well characterized. This is an important shortcoming because many epidemiological models assume that mosquitoes continue to be infectious for the duration of their life span. To define the duration of infectiousness for CHIKV and ZIKV, mosquitoes were infected orally with these viruses. Every 2 days, legs/wings, midguts, salivary glands, and saliva were collected from 30 to 60 mosquitoes and viral load measured. In CHIKV-infected mosquitoes, infectious virus in saliva peaked early (2-4 dpi), and then decreased rapidly and was rarely observed after 10 dpi. Viral RNA in infected tissues also decreased after the initial peak (4-8 dpi) but did so much less drastically. In ZIKV-infected mosquitoes, the infectious virus in saliva peaked at 12-14 dpi and dropped off only slightly after 14 dpi. In infected tissues, viral RNA increased early during infection, and then plateaued after 6-10 days. Our findings suggest that significant variation exists in the duration of the infectious period for arboviruses that is in part influenced by virus clearance from expectorated saliva.

Figure 1.

Chikungunya virus (CHIKV) RNA copy numbers and infectious virus in Aedes aegypti mosquito tissues. Viral RNA copy numbers in CHIKV-infected midguts (A), legs/wings (B), salivary glands (C), and saliva (D) from 2 to 20 dpi are shown. All viral copy numbers were quantified using quantitative real-time reverse transcriptase–PCR. Infectious virus titers in saliva samples (E) from 2 to 20 dpi were titrated by plaque assay on Vero cells. The geometric mean of 50–60 mosquito samples from two replicate experiments is shown. Error bars indicate the 95% CI. The limit of detection was 335 copy numbers (correlates to 36.5 CT value) for viral RNA and 3.3 PFU for infectious virus. This figure appears in color at www.ajtmh.org.

Figure 2.

Chikungunya virus (CHIKV) infectious virus in saliva of Aedes aegypti mosquitoes fed on honey or sugar. Chikungunya virus was titrated in saliva samples of Ae. aegypti mosquitoes fed on either a filter card soaked in honey/sugar solution (A) or a sugar cube (B) following infection. Mosquito infection status was confirmed in mosquito leg and body tissues by plaque assay. The geometric mean and individual data points from 30 mosquito samples are shown. Error bars indicate the 95% CI. The limit of detection was 3.3 PFU per saliva sample. This figure appears in color at www.ajtmh.org.

Figure 3.

Zika virus (ZIKV) RNA copy numbers and infectious virus in Aedes aegypti mosquito tissues. Viral RNA copy numbers in ZIKV-infected midguts (A), legs/wings (B), salivary glands (C), and saliva (D) from 2 to 20 dpi are shown. All viral copy numbers were quantified using quantitative real-time reverse transcriptase–PCR. Infectious virus titers in saliva samples (E) from 2 to 20 dpi were titrated by plaque assay on Vero cells. The geometric mean of 30 mosquito samples from one experiment is shown. Error bars indicate the 95% CI. The limit of detection was 335 copy numbers (correlates to 36.5 CT value) for viral RNA and 3.3 PFU for infectious virus. This figure appears in color at www.ajtmh.org.

Figure 4.

General trends of Chikungunya virus (CHIKV) and Zika virus (ZIKV) RNA copy numbers and infectious virus in Aedes aegypti mosquito saliva. Geometric mean of viral RNA copy numbers (A) and infectious virus (B) from CHIKV and ZIKV saliva samples from 2 to 20 dpi are shown. Percent of positive samples of viral RNA copy numbers (C) and infectious virus (D) from CHIKV and ZIKV saliva samples from 2 to 20 dpi are shown. This figure appears in color at www.ajtmh.org.