North American House Sparrows Are Competent for Usutu Virus Transmission

Abstract

Usutu virus (USUV, Flaviviridae) is an emerging mosquito-borne virus that has been implicated in neuroinvasive disease in humans and epizootic deaths in wild birds. USUV is maintained in an enzootic cycle between ornithophilic mosquitoes, primarily Culex spp., and wild birds, predominantly passerine species. However, limited experimental data exist on the species competent for USUV transmission. Here, we demonstrate that house sparrows are susceptible to multiple USUV strains. Our study also revealed that Culex quinquefasciatus mosquitoes are susceptible to USUV, with a significantly higher infection rate for the Netherlands 2016 USUV strain compared to the Uganda 2012 USUV strain at 50% and 19%, respectively. To assess transmission between avian host and mosquito vector, we allowed mosquitoes to feed on either juvenile chickens or house sparrows inoculated with USUV. Both bird models transmitted USUV to C. quinquefasciatus mosquitoes. Linear regression analyses indicated that C. quinquefasciatus infection rates were positively correlated with avian viremia levels, with 3 to 4 log₁₀ PFU/mL representing the minimum avian viremia threshold for transmission to mosquitoes. Based on the viremia required for transmission, house sparrows were estimated to more readily transmit the Netherlands 2016 strain compared to the Uganda 2012 strain. These studies provide insights on a competent reservoir host of USUV. IMPORTANCE Usutu virus (USUV) is a zoonotic mosquito-borne virus that can cause neuroinvasive disease, including meningitis and encephalitis, in humans and has resulted in hundreds of thousands of deaths in wild birds. The perpetuation of USUV in nature is dependent on transmission between Culex spp. mosquitoes and various avian species. To date, few experimental data exist for determining which bird species are important for the maintenance of USUV. Our studies showed that house sparrows can transmit infectious Usutu virus, indicating their role as a competent host species. By identifying reservoir species of USUV, we can predict areas of USUV emergence and mitigate its impacts on global human and wildlife health.

Keywords: arbovirus; house sparrow; mosquito; transmission.

FIG 1

Culex quinquefasciatus mosquitoes are susceptible to Usutu virus (USUV) infection. (A) Proportion of mosquitoes found positive for infectious USUV out of the total number of mosquitoes that were fed on a bloodmeal containing the Netherlands 2016 or Uganda 2012 USUV strain. Mosquito bodies represent infection rates; legs and wings represent dissemination rates; saliva represents transmission rates. Fisher’s exact test was used to compare proportions. ***, P < 0.001. (B) Viral titers in mosquito bodies. (C) Viral titers in mosquito legs and wings. (D) Viral titers in mosquito saliva. Circles represent individual samples; horizontal lines represent the mean; error bars represent standard deviation. Limit of detection (LOD) is represented by the dashed line. Mann-Whitney test was used to compare viruses. **, P < 0.01.

FIG 2

Low antibody response (LAS) chicken model transmits USUV to C. quinquefasciatus mosquitoes. (A) Viremia in LAS chicks inoculated with Netherlands 2016 or Uganda 2012 USUV strain. Circles represent individual samples; horizontal lines represent the mean; error bars represent standard deviation. Limit of detection is represented by the dashed line. Mann-Whitney test was used to compare viruses. *, P < 0.05. (B) Proportion of mosquito bodies positive for infectious USUV out of the total number of mosquitoes fed on either Netherlands 2016-inoculated or Uganda 2012-inoculated LAS chicks. Fisher’s exact test was used to compare proportions; no significance was observed. (C) USUV titers in mosquito bodies fed on USUV-inoculated LAS chicks. Circles represent individual samples; horizontal lines represent the mean; error bars represent standard deviation. Limit of detection is represented by the dashed line. Mann-Whitney test was used to compare viruses; no significance was observed.

FIG 3

House sparrows are susceptible to African and European USUV strains. (A) Viremia in house sparrows inoculated with Netherlands 2016 or Uganda 2012 USUV strain. Circles represent individual samples; horizontal lines represent the mean; error bars represent standard deviation. Limit of detection is represented by the dashed line. Multiple Mann-Whitney tests using the Holm-Sidak method were used to compare viruses; no differences were observed. (B) Neutralizing antibody response was measured by plaque reduction neutralization test (PRNT); samples were considered positive if they reached the 90% reduction threshold (PRNT₉₀). Highest reciprocal serum titer where 90% reduction was reached is shown; circles represent individual serum samples. The geometric mean titer (GMT) of each group is represented by a solid line. Samples that did not reach 90% reduction threshold were considered negative and graphed at half-LOD, 10. A Mann-Whitney test was used to compare GMTs; no significance was observed. (C) Brain tissue collected on 14 dpi from house sparrow inoculated with Netherlands 2016 USUV strain, with perivascular cuffing (arrow) (hematoxylin and eosin [H&E] stain). (D) Heart tissues collected on 14 dpi from house sparrow inoculated with Uganda 2012 USUV strain, with foci of inflammation and myocardial degeneration (arrow) (H&E stain). (E) Liver tissue from house sparrow inoculated with Uganda 2012 USUV strain, with lesions of lymphocytic inflammation (arrow) and hepatic degeneration (arrowhead) (H&E stain). Scale bars = 50 μm.

FIG 4

House sparrows transmit USUV to C. quinquefasciatus mosquitoes. (A) Proportion of mosquito bodies positive for USUV out of the total number of mosquitoes fed on either Netherlands 2016-inoculated or Uganda 2012-inoculated house sparrows. Fisher’s exact test was used to compare proportions; no significance was observed. (B) USUV titers in mosquito bodies fed on USUV-inoculated house sparrows. Circles represent individual samples; horizontal lines represent the mean; error bars represent standard deviation. Limit of detection is represented by the dashed line. Mann-Whitney test was used to compare viruses; no significant difference was observed.

FIG 5

Estimated USUV infectiousness relative to avian viremia level. (A) Linear regression analysis of Netherlands 2016 avian viremia titer (log₁₀ PFU/mL) compared to C. quinquefasciatus mosquito USUV infection rates. (B) Linear regression analysis of Uganda 2012 avian viremia titer (log₁₀ PFU/mL) compared to C. quinquefasciatus mosquito USUV infection rates. Open circles represent mosquitoes that fed on LAS chicks; closed circles represent mosquitoes that fed on house sparrows. Dashed lines represent 95% confidence intervals. (C) Estimated daily infectiousness of house sparrows inoculated with either Netherlands 2016 or Uganda 2012 over the course of 7 days. Circles represent individual samples; horizontal lines represent the mean; error bars represent standard deviation. Multiple Mann-Whitney tests using the Holm-Sidak method were used to compare viruses. **, P < 0.01.