The summer of 2018 in Germany was the second hottest and driest on record. These generally extremely favorable climatic conditions most likely triggered the further expansion and the efficient propagation of the zoonotic arthropod-borne West Nile virus in many Southern/Southeastern and even Central European countries. WNV infections were detected for the first time in resident wild and aviary birds, such as common blackbirds, northern goshawks and great grey owls in Eastern and Southeastern Germany. The causative WNV strain belonged to the central European subclade II. Phylogeographic analysis indicated a single introduction event of WNV into Germany, most likely in 2016 from Czech Republic, and also a unique non-synonymous mutation in the NS3 gene. Extraordinary high temperatures in 2018 presumably led to decreased averaged extrinsic incubation period values for WNV in mosquitoes, leading to rapid virus amplification and greater transmission risk for vertebrates in Germany. Blood transfusion services and clinicians in Germany should be aware of these possible WNV infection risks in humans especially during late summer.

Two novel 1-step real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays for the simultaneous detection of West Nile virus (WNV) lineage 1 and 2 strains were developed. Primers and the probe of assay 1 target the 5′-untranslated region (UTR), whereas the amplicon of assay 2 is located in the nonstructural region NS2A, which enables an unambiguous and independent WNV diagnosis based on 2 different amplicons. Both assays allow the detection of as few as 2–4 genome copies of WNV strains NY99, Uganda B956, Kunjin, and Sarafend (all cultured on Vero cells). A new synthetic RNA mutant of the 5′-UTR amplicon, which contains 6 twist inverted base-pair changes at the probe attachment site, was used as external calibrator control.

Hepatitis E virus (HEV) is the causative agent of acute hepatitis E in humans in developing countries, but sporadic and autochthonous cases do also occur in industrialised countries. In Europe, food-borne zoonotic transmission of genotype 3 (gt3) has been associated with domestic pig and wild boar. However, little is known about the course of HEV infection in European wild boar and their role in HEV transmission to domestic pigs. To investigate the transmissibility and pathogenesis of wild boar-derived HEVgt3, we inoculated four wild boar and four miniature pigs intravenously. Using quantitative real-time RT-PCR viral RNA was detected in serum, faeces and in liver, spleen and lymph nodes. The antibody response evolved after fourteen days post inoculation. Histopathological findings included mild to moderate lymphoplasmacytic hepatitis which was more prominent in wild boar than in miniature pigs. By immunohistochemical methods, viral antigens were detected mainly in Kupffer cells and liver sinusoidal endothelial cells, partially associated with hepatic lesions, but also in spleen and lymph nodes. While clinical symptoms were subtle and gross pathology was inconspicuous, increased liver enzyme levels in serum indicated hepatocellular injury. As the faecal-oral route is supposed to be the most likely transmission route, we included four contact animals to prove horizontal transmission. Interestingly, HEVgt3-infection was also detected in wild boar and miniature pigs kept in contact to intravenously inoculated wild boar. Given the high virus loads and long duration of viral shedding, wild boar has to be considered as an important HEV reservoir and transmission host in Europe.

An increasing number of indigenous cases of hepatitis E caused by genotype 3 viruses (HEV-3) have been diagnosed all around the word, particularly in industrialized countries. Hepatitis E is a zoonotic disease and accumulating evidence indicates that domestic pigs and wild boars are the main reservoirs of HEV-3. A detailed analysis of HEV-3 subtypes could help to determine the interplay of human activity, the role of animals as reservoirs and cross species transmission. Although complete genome sequences are most appropriate for HEV subtype determination, in most cases only partial genomic sequences are available. We therefore carried out a subtype classification analysis, which uses regions from all three open reading frames of the genome. Using this approach, more than 1000 published HEV-3 isolates were subtyped. Newly recovered HEV partial sequences from hunted German wild boars were also included in this study. These sequences were assigned to genotype 3 and clustered within subtype 3a, 3i and, unexpectedly, one of them within the subtype 3b, a first non-human report of this subtype in Europe.

The genusFlavivirusincludes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of mostFlavivirusspecies are available, there has been also a demand for a broad-rangeFlavivirusassay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all knownFlavivirusspecies. This assay has been used as a screening and confirmation tool forFlaviviruspresence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-sixFlavivirusstrains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented.