Excretion Dynamics of Arboviruses in Mosquitoes and the Potential Use in Vector Competence Studies and Arbovirus Surveillance

The increasing threat of arboviruses such as West Nile virus (WNV) and Usutu virus (USUV) requires the fast and efficient surveillance of these viruses. The examination of mosquitoes takes up an important part; however, these investigations are usually very time-consuming. An alternative sample type for arbovirus surveillance might be mosquito excreta. In order to determine the excretion dynamics under laboratory conditions, laboratory colonies of Aedes vexans and Culex pipiens biotype molestus were infected with WNV, USUV or tick-borne encephalitis virus (TBEV). After infection, the excreta were sampled and investigated for viral RNA. Excretion of viral RNA together with infectious blood meal could be detected up to five days after infection. Further excretion seemed to correlate with a disseminated infection in mosquitoes, at least after USUV infection. In addition, it could be determined that the amount of viral RNA in the excretions correlated positively with the viral load in the mosquito bodies. Overall, this study shows that the usage of mosquito excreta as a sample type for surveillance enables the detection of endemic viruses (WNV, USUV) as well as non-mosquito-borne viruses (TBEV). In addition, examination of viral shedding during vector competence studies can provide insights into the course of infection without sacrificing animals

Vector Competence of German Aedes punctor (Kirby, 1837) for West Nile Virus Lineages 1 and 2

West Nile virus (WNV) is a zoonotic flavivirus transmitted by mosquitoes as a biological vector. Because of its biting behavior, the widespread snow-melt mosquito Aedes punctor could be a potential bridge vector for WNV to humans and nonhuman mammals. However, little is known on its role in transmission of WNV. The aim of this study was to determine the vector competence of German Ae. punctor for WNV lineages 1 and 2. Field-collected larvae and pupae were reared to adults and offered infectious blood containing either an Italian WNV lineage 1 or a German WNV lineage 2 strain via cotton stick feeding. Engorged females were incubated for 14/15 or 21 days at 18 °C. After incubation; surviving mosquitoes were dissected and forced to salivate. Mosquito bodies with abdomens, thoraces and heads, legs plus wings and saliva samples were investigated for WNV RNA by RT-qPCR. Altogether, 2/70 (2.86%) and 5/85 (5.88%) mosquito bodies were found infected with WNV lineage 1 or 2, respectively. In two mosquitoes, viral RNA was also detected in legs and wings. No saliva sample contained viral RNA. Based on these results, we conclude that Ae. punctor does not play an important role in WNV transmission in Germany

Cellular co-infections of West Nile virus and Usutu virus influence virus growth kinetics

Abstract The mosquito-borne flaviviruses West Nile virus (WNV) and Usutu virus (USUV) pose a significant threat to the health of humans and animals. Both viruses co-circulate in numerous European countries including Germany. Due to their overlapping host and vector ranges, there is a high risk of co-infections. However, it is largely unknown if WNV and USUV interact and how this might influence their epidemiology. Therefore, in-vitro infection experiments in mammalian (Vero B4), goose (GN-R) and mosquito cell lines (C6/36, CT) were performed to investigate potential effects of co-infections in vectors and vertebrate hosts. The growth kinetics of German and other European WNV and USUV strains were determined and compared. Subsequently, simultaneous co-infections were performed with selected WNV and USUV strains. The results show that the growth of USUV was suppressed by WNV in all cell lines. This effect was independent of the virus lineage but depended on the set WNV titre. The replication of WNV also decreased in co-infection scenarios on vertebrate cells. Overall, co-infections might lead to a decreased growth of USUV in mosquitoes and of both viruses in vertebrate hosts. These interactions can strongly affect the epidemiology of USUV and WNV in areas where they co-circulate

In action—an early warning system for the detection of unexpected or novel pathogens

Proactive approaches in preventing future epidemics include pathogen discovery prior to their emergence in human and/or animal populations. Playing an important role in pathogen discovery, high-throughput sequencing (HTS) enables the characterization of microbial and viral genetic diversity within a given sample. In particular, metagenomic HTS allows the unbiased taxonomic profiling of sequences; hence, it can identify novel and highly divergent pathogens such as viruses. Newly discovered viral sequences must be further investigated using genomic characterization, molecular and serological screening, and/or invitro and invivo characterization. Several outbreak and surveillance studies apply unbiased generic HTS to characterize the whole genome sequences of suspected pathogens. In contrast, this study aimed to screen for novel and unexpected pathogens in previously generated HTS datasets and use this information as a starting point for the establishment of an early warning system (EWS). As a proof of concept, the EWS was applied to HTS datasets and archived samples from the 2018–9 West Nile virus (WNV) epidemic in Germany. A metagenomics read classifier detected sequences related to genome sequences of various members of Riboviria. We focused the further EWS investigation on viruses belonging to the families Peribunyaviridae and Reoviridae, under suspicion of causing co-infections in WNV-infected birds. Phylogenetic analyses revealed that the reovirus genome sequences clustered with sequences assigned to the species Umatilla virus (UMAV), whereas a new peribunyavirid, tentatively named ‘Hedwig virus’ (HEDV), belonged to a putative novel genus of the family Peribunyaviridae. In follow-up studies, newly developed molecular diagnostic assays detected fourteen UMAV-positive wild birds from different German cities and eight HEDV-positive captive birds from two zoological gardens. UMAV was successfully cultivated in mosquito C6/36 cells inoculated with a blackbird liver. In conclusion, this study demonstrates the power of the applied EWS for the discovery and characterization of unexpected viruses in repurposed sequence datasets, followed by virus screening and cultivation using archived sample material. The EWS enhances the strategies for pathogen recognition before causing sporadic cases and massive outbreaks and proves to be a reliable tool for modern outbreak preparedness

West Nile Virus Epidemic in Germany Triggered by Epizootic Emergence, 2019

One year after the first autochthonous transmission of West Nile virus (WNV) to birds and horses in Germany, an epizootic emergence of WNV was again observed in 2019. The number of infected birds and horses was considerably higher compared to 2018 (12 birds, two horses), resulting in the observation of the first WNV epidemy in Germany: 76 cases in birds, 36 in horses and five confirmed mosquito-borne, autochthonous human cases. We demonstrated that Germany experienced several WNV introduction events and that strains of a distinct group (Eastern German WNV clade), which was introduced to Germany as a single introduction event, dominated mosquito, birds, horse and human-related virus variants in 2018 and 2019. Virus strains in this clade are characterized by a specific-Lys2114Arg mutation, which might lead to an increase in viral fitness. Extraordinary high temperatures in 2018/2019 allowed a low extrinsic incubation period (EIP), which drove the epizootic emergence and, in the end, most likely triggered the 2019 epidemic. Spatiotemporal EIP values correlated with the geographical WNV incidence. This study highlights the risk of a further spread in Germany in the next years with additional human WNV infections. Thus, surveillance of birds is essential to provide an early epidemic warning and thus, initiate targeted control measures.Peer Reviewe

Interacting Regions of CD81 and Two of Its Partners, EWI-2 and EWI-2wint, and Their Effect on Hepatitis C Virus Infection*

CD81 is a tetraspanin protein that is involved in several essential cellular functions, as well as in the hepatitis C virus (HCV) infection. CD81 interacts with a high stoichiometry with its partner proteins EWI-2, EWI-2wint, and EWI-F. These latter proteins modify the functions of CD81 and can thereby potentially inhibit infection or modulate cell migration. Here, we characterized the cleavage of EWI-2 leading to the production of EWI-2wint, which has been shown to inhibit HCV infection. We determined the regions of EWI-2/EWI-2wint and CD81 that are important for their interaction and their functionality. More precisely, we identified a glycine zipper motif in the transmembrane domain of EWI-2/EWI-2wint that is essential for the interaction with CD81. In addition, we found that palmitoylation on two juxtamembranous cysteines in the cytosolic tail of EWI-2/EWI-2wint is required for their interaction with CD81 as well as with CD9, another tetraspanin. Thus, we have shown that palmitoylation of a tetraspanin partner protein can influence the interaction with a tetraspanin. We therefore propose that palmitoylation not only of tetraspanins, but also of their partner proteins is important in regulating the composition of complexes in tetraspanin networks. Finally, we identified the regions in CD81 that are necessary for its functionality in HCV entry and we demonstrated that EWI-2wint needs to interact with CD81 to exert its inhibitory effect on HCV infection