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 in vitro and in vivo 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.

Differentiation of Antibodies against Selected Simbu Serogroup Viruses by a Glycoprotein Gc-Based Triplex ELISA.

The Simbu serogroup of orthobunyaviruses includes several pathogens of veterinary importance, among them Schmallenberg virus (SBV), Akabane virus (AKAV) and Shuni virus (SHUV). They infect predominantly ruminants and induce severe congenital malformation. In adult animals, the intra vitam diagnostics by direct virus detection is limited to only a few days due to a short-lived viremia. For surveillance purposes the testing for specific antibodies is a superior approach. However, the serological differentiation is hampered by a considerable extent of cross-reactivity, as viruses were assigned into this serogroup based on antigenic relatedness. Here, we established a glycoprotein Gc-based triplex enzyme-linked immunosorbent assay (ELISA) for the detection and differentiation of antibodies against SBV, AKAV, and SHUV. A total of 477 negative samples of various ruminant species, 238 samples positive for SBV-antibodies, 36 positive for AKAV-antibodies and 53 SHUV antibody-positive samples were tested in comparison to neutralization tests. For the newly developed ELISA, overall diagnostic specificities of 84.56%, 94.68% and 89.39% and sensitivities of 89.08%, 69.44% and 84.91% were calculated for SBV, AKAV and SHUV, respectively, with only slight effects of serological cross-reactivity on the diagnostic specificity. Thus, this test system could be used for serological screening in suspected populations or as additional tool during outbreak investigations.

Full-genome sequencing of German rabbit haemorrhagic disease virus uncovers recombination between RHDV (GI.2) and EBHSV (GII.1).

Rabbit haemorrhagic disease virus (RHDV; genotypes GI.1 and GI.2) and European brown hare syndrome virus (EBHSV; genotype GII.1) are caliciviruses belonging to the genus Lagovirus. These viruses pose a serious threat to wild and domestic rabbit and hare populations around the world. In recent years, an expanding genetic diversity has been described within the genus, with recombination events occurring between the different genotypes. Here, we generated and analysed 56 full-genome sequences of RHDV and EBHSV from rabbit and hare livers, collected in Germany between the years 2013 and 2020. We could show that genotype Gl.2 (RHDV-2) almost entirely replaced Gl.1 (classical RHDV) in the German rabbit population. However, GI.1 is still present in Germany and has to be included into disease control and vaccination strategies. Three recombinant strains were identified from rabbit samples that contain the structural genes of genotype Gl.2 and the non-structural genes of genotype Gl.1b. Of special interest is the finding that sequences from two hare samples showed recombination events between structural genes of RHDV Gl.2 and non-structural genes of EBHSV GII.1, a recombination between different genogroups that has not been described before. These findings lead to the assumption that also a recombination of the non-structural genes of RHDV Gl.2 with the structural genes of EBHSV Gll.1 might be possible and therefore increase the potential genetic variability of lagoviruses immensely. Our findings underline the importance of whole genome analysis with next-generation sequencing technology as one of new tools now available for in-depth studies that allow in depth molecular epidemiology with continuous monitoring of the genetic variability of viruses that would otherwise likely stay undetected if only routine diagnostic assays are used.

Preparedness needs research: How fundamental science and international collaboration accelerated the response to COVID-19.

The first cluster of patients suffering from coronavirus disease 2019 (COVID-19) was identified on December 21, 2019, and as of July 29, 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been linked with 664,333 deaths and number at least 16,932,996 worldwide. Unprecedented in global societal impact, the COVID-19 pandemic has tested local, national, and international preparedness for viral outbreaks to the limits. Just as it will be vital to identify missed opportunities and improve contingency planning for future outbreaks, we must also highlight key successes and build on them. Concomitant to the emergence of a novel viral disease, there is a 'research and development gap' that poses a threat to the overall pace and quality of outbreak response during its most crucial early phase. Here, we outline key components of an adequate research response to novel viral outbreaks using the example of SARS-CoV-2. We highlight the exceptional recent progress made in fundamental science, resulting in the fastest scientific response to a major infectious disease outbreak or pandemic. We underline the vital role of the international research community, from the implementation of diagnostics and contact tracing procedures to the collective search for vaccines and antiviral therapies, sustained by unique information sharing efforts.

A CRISPR/Cas9 Generated Bovine CD46-knockout Cell Line-A Tool to Elucidate the Adaptability of Bovine Viral Diarrhea Viruses (BVDV).

Bovine viral diarrhea virus (BVDV) entry into a host cell is mediated by the interaction of the viral glycoprotein E2 with the cellular transmembrane CD46 receptor. In this study, we generated a stable Madin-Darby Bovine Kidney (MDBK) CD46-knockout cell line to study the ability of different pestivirus A and B species (BVDV-1 and -2) to escape CD46-dependent cell entry. Four different BVDV-1/2 isolates showed a clearly reduced infection rate after inoculation of the knockout cells. However, after further passaging starting from the remaining virus foci on the knockout cell line, all tested virus isolates were able to escape CD46-dependency and grew despite the lack of the entry receptor. Whole-genome sequencing of the escape-isolates suggests that the genetic basis for the observed shift in infectivity is an amino acid substitution of an uncharged (glycine/asparagine) for a charged amino acid (arginine/lysine) at position 479 in the ERNS in three of the four isolates tested. In the fourth isolate, the exchange of a cysteine at position 441 in the ERNS resulted in a loss of ERNS dimerization that is likely to influence viral cell-to-cell spread. In general, the CD46-knockout cell line is a useful tool to analyze the role of CD46 for pestivirus replication and the virus-receptor interaction.

Development of within-herd immunity and long-term persistence of antibodies against Schmallenberg virus in naturally infected cattle.

BACKGROUND: In 2011, the teratogenic, insect-transmitted Schmallenberg virus (SBV) emerged at the German/Dutch border region and subsequently spread rapidly throughout the European continent. In cattle, one of the major target species of SBV, first antibodies are detectable between one and three weeks after infection, but the duration of humoral immunity is unknown. To assess the course of immunity in individual animals and the development of the within-herd seroprevalence, cattle kept in a German farm with a herd size of about 300 lactating animals were annually blood sampled between December 2011 and December 2017 and tested for the presence of SBV-specific antibodies. RESULTS: During the monitored period, the within-herd seroprevalence declined from 74.92% in 2011 to 39.93% in 2015 and, thereafter, slightly increased to 49.53% in 2016 and 48.44% in 2017. From the animals that were tested in 2014 and 2015 for the first time (between 24 and 35 months of age) only 14.77% and 7.45%, respectively, scored positive. Thereafter, the seropositivity rate of this age group rose markedly to 58.04% in 2016 and 48.10% in 2017 indicating a circulation of SBV. Twenty-three individual animals were consistently sampled once per year between 2011 and 2017 after the respective insect vector season, 17 of them tested positive at the first sampling. Fourteen animals were still seropositive in December 2017, while three cattle (17.65%) became seronegative. CONCLUSIONS: The regular re-emergence of SBV in Central Europe is a result of decreasing herd immunity caused by the replacement of animals by seronegative youngstock rather than of a drop of antibody levels in previously infected individual animals. The consequences of the overall decline in herd seroprevalence may be increasing virus circulation and more cases of fetal malformation caused by infection of naïve dams during gestation.

Budget impact model for oncopharmacogenetics from the perspective of mandatory basic health insurance in Switzerland using the example of breast cancer.

Single-nucleotide polymorphisms (SNPs) can severely impact individual drug response and health outcomes in cancer patients. Genetic tests to screen for marker SNPs are available to adjust the drug dose of oncologicals to the patient's needs. However, it is unclear whether the positive effects outbalance the increased costs or even lead to an overall cost reduction. This very pragmatic analysis used three frequently used oncologicals for the treatment of breast cancer to evaluate whether preemptive pharmacogenetic testing may have a cost-reducing impact on health care spending in the Swiss health care system. Our results indicate that oncopharmacogenetics might help to reduce health care costs (ie, by avoiding adverse drug effects) and to increase efficiency of drugs in oncologic patients.