Saliva collection via capillary method may underestimate arboviral transmission by mosquitoes

Abstract

Background: Arthropod-borne viruses (arboviruses) impose a major health and economic burden on human populations globally, with mosquitoes serving as important vectors. Measuring the ability of a mosquito population to transmit an arbovirus is important in terms of evaluating its public health risk. In the laboratory, a variety of methods are used to estimate arboviral transmission by mosquitoes, including indirect methods involving viral detection from mosquito saliva collected by forced salivation. The accuracy of indirect methods to estimate arbovirus transmission to live animal hosts has not been fully evaluated.

Methods: We compared three commonly used proxies of arboviral transmission, namely, the presence of virus in mosquito legs, in salivary glands (SG) and in saliva collected in capillary tubes using forced salivation, with direct transmission estimates from mosquitoes to suckling mice. We analyzed five vector-virus combinations, including Aedes aegypti infected with chikungunya virus, West Nile virus and Zika virus; Culex quinquefasciatus infected with West Nile virus; and Aedes triseriatus infected with La Crosse virus.

Results: Comparatively, the methods of detecting virus infection in mosquito legs and in SG were equally accurate in predicting transmission. Overall, the presence of virus in mosquito legs was a more accurate predictor of transmission than the commonly implemented viral detection method using forced salivation into a capillary tube, and was subject to less technical variation.

Conclusions: These results suggest that, in general, forced salivation methods tend to underestimate virus transmission, and they provide confidence in the use of mosquito leg screens to evaluate the transmission potential of a mosquito population.

Keywords: Arbovirus; Proxy; Saliva; Transmission; Vector.

Fig. 1

Transmission rate estimated for Aedes aegypti mosquitoes injected with three different arboviruses, based on sample type. a CHIKV (N = 17), b WNV (N = 18/^#17), c ZIKV (N = 17). Error bars represent the 95% binomial confidence intervals. Asterisks (*) indicate significant comparisons based on a Fisher exact [23] test after Holm correction for multiple testing [24]. Abbreviations: CHIKV, Chikungunya virus; SG, salivary glands; WNV, West Nile Virus; ZIKV, Zika virus

Fig. 2

Transmission rate estimated for Culex quinquefasciatus mosquitoes injected with WNV, based on sample type at 7 dpi (dark-gray bars; N = 38) and 10 dpi (light-gray bars; N = 33^†). ^†SG were only dissected from 15 out of the 33 mosquitoes at 10 dpi and that only 6 mice were successfully exposed to WNV. Error bars represent the 95% binomial confidence intervals. Asterisks (*) indicate significant comparisons at 10 dpi based on a Fisher exact [23] test after Holm correction for multiple testing [24]. Abbreviations: dpi, Days post-infection

Fig. 3

Transmission rate estimated for Aedes triseriatus mosquitoes injected with LACV based on sample type (N = 17). Error bars represent the 95% binomial confidence intervals. Asterisks (*) indicate significant comparisons based on a Fisher exact [23] test after Holm correction for multiple testing [24]. Abbreviations: LACV, La Crosse virus

Fig. 4

Transmission rate estimated for orally infected Ae. aegypti mosquitoes with CHIKV based on sample type after 12 dpi (N = 8) (a) and 15 dpi (N = 15) (b). Error bars represent the 95% binomial confidence intervals. Asterisks (*) indicate significant comparisons based on a Fisher exact [23] test after Holm correction for multiple testing [24]

Fig. 5

Accuracy of proxies relative to direct virus transmission estimates to suckling mice. Vertical axis shows the difference in the percentage of transmission estimated between the proxy and the mice infections. Different shades of gray represent the different viruses. Experiments from intrathoracic injection are indicated by the larger circles and oral infections by triangles. Mean and standard deviation of the proxy are indicated by the small diamond and attached vertical line, respectively