Mosquito-borne Flaviviruses : Vector and Avian Host Susceptibility for West Nile Virus and Usutu Virus in Germany

Flaviviruses are known to cause a variety of diseases in humans as well as animals, ranging from mild febrile illnesses to severe hemorrhagic or neuroinvasive manifestations. West Nile virus (WNV) and Usutu virus (USUV) are closely related flaviviruses circulating in an enzootic sylvatic cycle, consisting of mosquitoes (particularly Culex species) as vectors and birds as amplifying and/or reservoir hosts. Humans and equines, however, are considered dead-end hosts as they can develop severe clinical diseases but cannot in turn infect naïve feeding mosquitoes. Globalization, with increased international trade, in combination with climate change is facilitating the worldwide spread of vectors and their pathogens. To date WNV is the most dispersed zoonotic arbovirus, with detections in every continent aside Antarctica. Outbreaks are recorded yearly and the virus is becoming an increasing veterinary and public health concern. At the same time, USUV is also expanding its geographical range and colonizing new ecological niches. Since the first isolation of USUV (2010) and WNV (2018) in Germany, both viruses have become endemic. They are overwintering either in infected mosquito females or in natural reservoir hosts with a yearly reemergence predominantly towards the end of the mosquito transmission seasons (from August to October). This has not only emphasized the necessity for evidence-based passive and active surveillance programs, but also for the completion of infection studies focusing on the pathogenesis of these viruses and the immunocompetence of their hosts. The experiments in the frame of this thesis, therefore, began to uncover vector species involved in the WNV and USUV transmission cycles in Germany and to tackle the question of whether domestic poultry could play a role as an amplifying and/or reservoir host for WNV. In the first study (manuscript I) German Cx. pipiens biotype molestus and Cx. torrentium species proved to be vector-competent for an USUV Africa lineage 2 strain, isolated in Germany from a great grey owl (2015). Results were comparable to that of an established Serbian Cx. pipiens biotype molestus colony, while a Malaysian Ae. aegpyti colony could not transmit USUV in its saliva. To our knowledge, this is the first confirmation of Cx. torrentium transmitting USUV under laboratory settings. The transmission rate of the German Cx. pipiens biotype molestus colony was dependent on the viral titer in the blood meal. When the blood meal titer declined from 107.4 to 105.1 TCID50/mL the infection rate dropped and the mosquitoes no longer developed a disseminated infection nor transmitted USUV in their saliva, neither after 14 nor 21 dpi. With the first isolation of WNV in Germany in 2018, the question of possible vector species was no longer confined to USUV. The second study (manuscript II), therefore, comprised vector competence studies with indigenous mosquito species and the first recorded WNV lineage 2 strain isolated from a great grey owl in eastern Germany. Two populations of fieldderived Cx. pipiens biotype pipiens from the northeast of Germany (Brandenburg) could readily transmit WNV in their saliva two and three weeks after feeding on an infectious blood meal. The same was true for a German Cx. pipiens biotype molestus colony from northern Germany (Lower Saxony). The transmission efficiency was, however, lower for biotype molestus than for biotype pipiens. The study also confirmed that the vector competence of Cx. pipiens biotype pipiens was dependent on the incubation temperature. The infection rates were fairly similar across all temperature gradients, yet the dissemination rates and transmission efficiencies were significantly greater at 25 °C and 28 °C compared to only 18 °C. Surprisingly, however, individual mosquitoes were still able to transmit WNV after incubation at 18 °C, an indication for the adaptation of the vector and the virus to temperate climates. An invasive Ae. albopictus population, established in central Germany (Thuringia), was also susceptible to WNV, but did not transmit infectious saliva to the same extent as the Culex species. The third study (manuscript III) aimed to establish a method by which to successfully infect Culex species with WNV via intrathoracic injections. This procedure was not only an essential requirement for the mosquito-to-bird infection models, but also enabled an assessment of the functionality and importance of the midgut barrier for WNV in Culex species. The intrathoracic injection of Cx. pipiens biotype pipiens and Cx. torrentium with a European WNV lineage 1 strain guaranteed in both species a disseminated infection with approximately every second mosquito secreting infectious saliva 10/11 dpi. The results verified the significance of the midgut barrier, as it appears to limit initial virus replication in the midgut and subsequently also virus dissemination to secondary tissues. Finally, the fourth study (manuscript IV) examined the interactions between susceptible vectors and hosts i.e., domestic poultry. It was designed to mimic the natural mosquito-to-bird transmission pathway of WNV and to compare subsequent disease pathogenesis to that of subcutaneously injected birds. In the first experiments, juvenile chickens, ducks, and geese were injected subcutaneously with a European WNV lineage 1 strain and monitored and sampled for three weeks post infection. The majority of the birds did not develop any clinical signs aside from one duck that succumbed 2 dpi (associated with technopathies) and two geese that possibly developed a persistent infection. They showed clinical manifestations only in the third week yet the pathological alterations (severe splenitis) were typical for an acute infection. Chickens were least susceptible, while ducks displayed the highest antibody levels and geese the highest viremia. Viral shedding was observed in all three species from 2 to 5 dpi, and was mainly via the oropharynx in chickens and geese and via the cloaca in ducks. Macroscopic lesions were observed in the ducks and geese and included in particular pale and mottled appearance of the myocardium. All three species developed histological lesions, which are characteristic for a viral infection and were primarily detected in the hearts (myocarditis) and brains (encephalitis) of the ducks and the spleens (hyperplasia/splenitis) and brains (encephalitis) of the geese. As geese were most susceptible to WNV after its subcutaneous injection, this species was also used for the mosquito-to-bird infection model. For this purpose, field-derived Cx. pipiens biotype pipiens mosquitoes from northeast Germany (Brandenburg) were intrathoracically injected with the WNV lineage 1 strain and allowed to feed on juvenile geese 10 dpi. Four of the eight exposed geese developed viremia, shed virus, seroconverted, and exhibited pathological lesions. The geese infected via mosquito bite displayed higher viremia levels and more diverse pathological lesions than those injected subcutaneously, providing evidence for a mosquito saliva-induced modulation of WNV pathogenesis in geese. In conclusion, these studies proved various Culex species to be vector-competent for USUV and/or WNV in Germany and verified that domestic poultry is unlikely to act as an amplifying or reservoir host for WNV lineage 1 in nature. Ducks and geese appear to be suitable sentinel species. They can monitor the spread of WNV and ideally detect WNV epidemics prior to the onset of human cases.