www.nature.com/scientificreports OPEN Reproducing the Rift Valley fever virus mosquito‑lamb‑mosquito transmission cycle Paul J. Wichgers Schreur1, Rianka P. M. Vloet1, Jet Kant1, Lucien van Keulen1, Jose L. Gonzales1, Tessa M. Visser2, Constantianus J. M. Koenraadt2, Chantal B. F. Vogels2,4 & Jeroen Kortekaas1,3* Rift Valley fever virus (RVFV) is a mosquito‑borne bunyavirus that is pathogenic to ruminants and humans. The virus is endemic to Africa and the Arabian Peninsula where outbreaks are characterized by abortion storms and mortality of newborns, particularly in sheep herds. Vector competence experiments in laboratory settings have suggested that over 50 mosquito species are capable of transmitting RVFV. Transmission of mosquito‑borne viruses in the feld is however infuenced by numerous factors, including population densities, blood feeding behavior, extrinsic incubation period, longevity of vectors, and viremia levels in vertebrate hosts. Animal models to study these important aspects of RVFV transmission are currently lacking. In the present work, RVFV was transmitted to European (Texel‑swifter cross‑breed) lambs by laboratory‑reared Aedes aegypti mosquitoes that were infected either by membrane feeding on a virus‑spiked blood meal or by feeding on lambs that developed viremia after intravenous inoculation of RVFV. Feeding of mosquitoes on viremic lambs resulted in strikingly higher infection rates as compared to membrane feeding. Subsequent transmission of RVFV from lamb to lamb by infected mosquitoes was highly efcient in both models. The animal models described here can be used to study mosquito‑mediated transmission of RVFV among the major natural target species and to evaluate the efcacy of vaccines against mosquito‑ mediated RVFV infection. Rif Valley fever virus (RVFV) is a mosquito-borne RNA virus that causes severe disease in ruminants and humans1. Te virus is transmitted via mosquitoes, predominantly of the genera Aedes and Culex2. Sheep are the most susceptible species, with disease being most severe in young lambs. RVFV has a tropism for the liver, with replication in hepatocytes resulting in focal to widespread hepatic necrosis 3,4. In severe cases, hepatic necrosis is associated with bleeding tendencies, shock and death. In pregnant ewes, RVFV also targets maternal epithe- lial cells of the placenta and fetal trophoblasts, resulting in severe placental pathology and abortion 5. Heavy mortalities among newborn lambs and abortion storms are key characteristics of RVF outbreaks. Infection of humans generally manifests as a self-resolving febrile illness, whereas a small percentage of patients develop encephalitis or hemorrhagic fever, the latter with high case fatality3. Although the risk of RVFV infection during human pregnancy is yet unclear, RVFV infections have been associated with miscarriage and recent studies have demonstrated that the virus replicates efciently in human placental explants5,6. Te threat that RVFV poses to human health is acknowledged by the World Health Organization that placed RVFV on the Blueprint list of priority diseases likely to cause future epidemics in the face of insufcient or absent countermeasures 7. Since its frst description in the 1930s, RVFV has caused severe outbreaks on the African continent and the Arabian Peninsula, where the disease has remained enzootic and continues to cause epizootics and epidemics1,4. Interepizootic maintenance of RVFV is possibly explained by vertical transmission of the virus to the eggs of foodwater Aedes mosquitoes8. Tese species oviposit near shallow wetlands, known as dambos, which dry out during periods of drought and food during heavy rains. Tese dambos provide ideal breeding grounds, as the eggs of foodwater mosquitoes depend on desiccation and rehydration for hatching. In desiccated form, the eggs can remain viable for long periods of time, possibly explaining how RVFV is maintained during periods of low or even apparent absent mosquito activity. Although this hypothesis is plausible, vertical transmission 1Wageningen Bioveterinary Research, Lelystad, The Netherlands. 2Laboratory of Entomology, Wageningen University & Research, Wageningen, The Netherlands. 3Laboratory of Virology, Wageningen University & Research, Wageningen, The Netherlands. 4Present address: Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, CT, USA. *email: [email protected] Scientifc Reports | (2021) 11:1477 | https://doi.org/10.1038/s41598-020-79267-1 1 Vol.:(0123456789) www.nature.com/scientificreports/ of RVFV to mosquito eggs has been demonstrated only once, and in only one mosquito species, namely Aedes (Neomelaniconion) mcintoshi Huang, a species cited before 1985 as Ae. lineatopennis8. Additionally, evidence is accumulating that RVFV is maintained in mosquito-ruminant-mosquito cycles in the absence of outbreaks 9,10. Apart from re-emergence of RVFV in enzootic areas, climate change, globalization, increasing populations of susceptible animals, and changing ecologies of mosquito vectors may facilitate future incursions into cur- rently unafected areas. Tis concern is fueled by data that suggest that over 50 mosquito species, many of which with a global distribution, can potentially act as vectors of RVFV 2. Tese data are based on isolation of RVFV from feld-collected mosquitoes and from vector competence experiments in laboratory settings, in which the virus is taken up by mosquitoes by membrane feeding on a RVFV-spiked blood meal 2,11,12. However, to more frmly establish the ability of a mosquito species to act as a vector, experiments are required in which the virus is transmitted among susceptible animals via infected mosquitoes. In such experiments, factors such as mosquito population density, blood feeding behavior, and longevity can be taken into account to determine the vectorial capacity of a mosquito vector13. Furthermore, such experiments could address the infuence of viremia levels in diferent vertebrate hosts on mosquito-mediated transmission. In the present work, Aedes aegypti mosquitoes were infected with RVFV afer membrane feeding on a virus- spiked blood meal or afer feeding on lambs that developed viremia afer intravenous inoculation of RVFV. Te virus was subsequently transmitted by these mosquitoes to naive lambs. Te presented animal models can be used to study environmental, vector and host factors that contribute to RVFV transmission and to determine the efcacy of vaccines against natural, mosquito-mediated infection. Results Infection of lambs via intravenous inoculation of RVFV followed by transmission of the virus between lambs and mosquitoes. A group of 6 lambs (Group 1, #157–162) was intravenously (IV) inoc- ulated with RVFV on day 0 (Fig. 1a, Experiment 1). Upon onset of pyrexia, previously shown to correspond with peak viremia14, mosquitoes were allowed to take a blood meal. To this end, mosquitoes were placed on sedated lambs in cardboard containers, bound to each of the hind legs with elastic tape (Fig. 1b,c). Te average mosquito feeding rate was 85% (± 7%) (Table 1). Fully engorged mosquitoes were collected and maintained in an insect incubator as separate groups per lamb. All IV inoculated lambs developed pyrexia (Fig. 2a), became listless and were disinclined to feed from day 2 onwards. Analysis of daily collected plasma samples with reverse-tran- scriptase quantitative PCR (RT-qPCR) and virus isolation demonstrated that all lambs developed high viremia, which peaked on day 2 post inoculation (Fig. 2b), confrming that mosquito feeding occurred at peak viremia. One lamb (#159) acutely succumbed to the infection on day 4, one lamb acutely succumbed on day 5 (#157), one lamb had to be euthanized afer reaching a humane end-point (HEP) on day 6 (#161) and one on day 7 (#162), resulting in a case fatality rate (CFR) of 67% (Fig. 2c). Necropsies of fatal cases revealed typical pathological manifestations of RVFV infection, with focal to widespread hepatic necrosis (Fig. 3a–c). Te average survival rate of blood-fed mosquitoes afer 13 days of incubation was 88% (± 8%) (Table 1). Fol- lowing this incubation period, the 6 groups of surviving mosquitoes were each placed on naive lambs (Group 2, #169–174, Fig. 1a) (on average 72 mosquitoes per lamb) and allowed to take a second blood meal as described above. Te average feeding rate of these mosquitoes was 76% (± 14%) (Table 1). Two days post feeding (dpf) on the naive lambs (day 17), all lambs developed high fever (Fig. 2d). One lamb (#173) reached a HEP and was euthanized on day 18 (dpf 3), two lambs (#169 and #174) succumbed acutely on day 19 (dpf 4) and one (#170) on day 20 (dpf 5), resulting in a CFR of 67% (Fig. 2f). Similar as observed in Group 1, all lambs that succumbed to the infection revealed typical signs of RVFV infection at necropsy. Analysis of plasma samples by RT-qPCR and virus isolation confrmed that peak viremia occurred on 2 dpf (day 17) (Fig. 2e). As all mosquito-exposed lambs developed viremia, transmission of RVFV by Ae. aegypti mosquitoes from viremic lambs to naive lambs was deemed very efcient. To retrospectively assess the number of RVFV-positive mosquitoes that had fed on each naive lamb, mos- quito saliva samples were collected by forced salivation and assayed for the presence of virus. Tese analyses demonstrated that 38% (± 9%) of the mosquitoes contained RVFV in saliva and suggested that each of the lambs from Group 2 were exposed to a minimum of 14 and a maximum of 26 saliva-positive mosquitoes (Table 1).