Search

We present the molecular identification of Apodemus agrarius (striped field mouse) as reservoir host of the Dobrava-Belgrade virus (DOBV) lineage DOBV-Aa in 3 federal states of Germany. Phylogenetic analyses provided evidence for multiple spillover of DOBV-Aa to A. flavicollis, a crucial prerequisite for host switch and genetic reassortment. [ABSTRACT FROM AUTHOR]

Crimean-Congo hemorrhagic fever virus (CCHFV) is an emerging tick-borne pathogen in different European countries. Since 2013, clinical and fatality cases associated with CCHFV infection have been reported in humans in Spain. During the last few years, endemic circulation of this virus has been detected in ticks and wild ungulates in this country, but the role of other sympatric wild species in the sylvatic cycle of this multi-host virus remains poorly understood. The aims of the present study were to assess exposure to CCHFV in wild lagomorphs in southern Spain, a CCHFV endemic area, and to determine the presence of the virus in ticks feeding on these species. Serum samples from 473 European wild rabbits (Oryctolagus cuniculus) and 162 Iberian hares (Lepus granatensis), and 120 ticks feeding on 85 of these wild lagomorphs were collected on 133 hunting grounds between 2018/2019 and 2021/2022 hunting seasons. The presence of antibodies against CCHFV was assessed in all serum samples using a commercial ELISA, whereas ticks were tested for the presence of CCHFV-RNA by a multiplex RT-qPCR that detects all known genotypes of this virus. None of the 635 (0.0 %; 95 %CI: 0.0-0.6) lagomorphs tested had anti-CCHFV antibodies and CCHFV-RNA was not found in any of the 120 (0.0 %; 95 %CI: 0.0-3.0) ticks analyzed. To the best of the authors knowledge, this is the first epidemiological study conducted on CCHFV in Iberian hare worldwide. Our findings indicate absence of exposure to CCHFV in European wild rabbit and Iberian hare populations, as well as in their ticks, which suggests that they do not seem to play a relevant role in the epidemiology of CCHFV in Mediterranean ecosystems of southern Spain., Competing Interests: Declaration of Competing Interest None of the authors of this study has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The structure of cellular prion proteins encoded by the prion protein gene (PRNP) impacts susceptibility to transmissible spongiform encephalopathies, including chronic wasting disease (CWD) in deer. The recent emergence of CWD in Northern European reindeer (Rangifer tarandus), moose (Alces alces alces) and red deer (Cervus elaphus), in parallel with the outbreak in North America, gives reason to investigate PRNP variation in European deer, to implement risk assessments and adjust CWD management for deer populations under threat. We here report PRNP-sequence data from 911 samples of German red, roe (Capreolus capreolus), sika (Cervus nippon) and fallow deer (Dama dama) as well as additional data from 26 Danish red deer close to the German border and four zoo species not native to Germany. No PRNP sequence variation was observed in roe and fallow deer, as previously described for populations across Europe. In contrast, a broad PRNP variation was detected in red deer, with non-synonymous polymorphisms at codons 98, 226 and 247 as well as synonymous mutations at codons 21, 78, 136 and 185. Moreover, a novel 24 bp deletion within the octapeptide repeat was detected. In summary, 14 genotypes were seen in red deer with significant differences in their geographical distribution and frequencies, including geographical clustering of certain genotypes, suggesting "PRNP-linages" in this species. Based on data from North American CWD and the genotyping results of the European CWD cases, we would predict that large proportions of wild cervids in Europe might be susceptible to CWD once introduced to naive populations., (© 2024. The Author(s).)

Background: Hepatitis E virus (HEV) is transmitted by the fecal route, usually through contaminated water in humans and/or infected animals, especially pigs. The objective of this study was to evaluate the level of anti-HEV antibodies in a panel of pig sera and to identify HEV in pig feces in farms. Methodology: The presence of HEV antibodies was tested by an in-house ELISA and a commercial ELISA IDvet. HEV genome was assessed by nested RT-PCR, and then, genotype was identified by sequencing (MinION Nanopore technology). Results: In 2017-2019, the 43% seroprevalence found in Forest Guinea was significantly higher than the 7% found in the Lower region ( p < 0.01). Presence of HEV genotype 3c was demonstrated during a secondary study in the Lower region (Conakry) in 2022. Conclusion: The presence of HEV-3c in pigs calls for an evaluation of seroprevalence in human populations and for a HEV genotype human circulation check. Contribution Heading: This study is the first report, to our knowledge, of seroprevalence and characterization of HEV infection in pigs in Guinea.

After the detection of bovine spongiform encephalopathy (BSE), and a zoonotic transmissible spongiform encephalopathy (TSE) caused by the pathological prion protein (PrP Sc ) in two goats, the investigation of goat prions became of greater interest. Therefore, a broad collection of European goat TSE isolates, including atypical scrapie, CH1641 and goat BSE as reference prion strains were biochemically characterised and subsequently inoculated into seven rodent models for further analysis (already published results of this comprehensive study are reviewed here for comparative reasons). We report here the histopathological and immunohistochemical data of this goat TSE panel, obtained after the first passage in Tgshp IX (tg-shARQ) mice, which overexpress the ovine prion protein. In addition to the clear-cut discrimination of all reference prion strains from the classical scrapie (CS) isolates, we were further able to determine three categories of CS strains. The investigation further indicates the occurrence of sub-strains that slightly resemble distant TSE strains, such as BSE or CH1641, reinforcing the theory that CS is not a single strain but a mixture of sub-strains, existing at varying extents in one isolate. This study further proved that Tgshp IX is a potent and reliable tool for the in-depth characterisation of prion strains.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the post-transcriptional expression of target genes. Virus-encoded miRNAs play an important role in the replication of viruses, modulate gene expression in both the virus and host, and affect their persistence and immune evasion in hosts. This renders viral miRNAs as potential targets for therapeutic applications, especially against pathogenic viruses that infect humans and animals. Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic RNA virus that causes severe disease in both humans and livestock. High mortality among newborn lambs and abortion storms are key characteristics of an RVF outbreak. To date, limited information is available on RVFV-derived miRNAs. In this study, computational methods were used to analyse the RVFV genome for putative pre-miRNA genes, which were then analysed for the presence of mature miRNAs. We detected 19 RVFV-encoded miRNAs and identified their potential mRNAs targets in sheep ( Ovis aries) , the most susceptible host. The identification of significantly enriched O. aries genes in association with RVFV miRNAs will help elucidate the molecular mechanisms underlying RVFV pathogenesis and potentially uncover novel drug targets for RVFV.

Crimean-Congo hemorrhagic fever (CCHF) is a zoonotic tick-borne disease, caused by an arbovirus of the genus Orthonairovirus and the family Nairoviridae. Crimean-Congo hemorrhagic fever virus (CCHFV) is widespread in several regions of the world. While the virus is not pathogenic to all susceptible livestock and wild mammals, it can lead to severe hemorrhagic fever in humans. In this review, we compiled published data on CCHFV infections in humans, animals, and ticks in Tunisia. Based on that, we discussed the epidemiology and the distribution patterns of CCHFV infections highlighting the risk factors for this virus in the country. CCHF infection prevalence in humans was estimated to 2.76% (5/181) and 5% (2/38) in Tunisian febrile patients and Tunisian slaughterhouse workers, respectively. Concurrently, seroprevalence in domestic ungulates (sheep, goats, cattle, and dromedaries) ranged from zero to 89.7%, and only one Hyalomma impeltatum tick specimen collected from dromedaries in southern Tunisian was positive for CCHFV by reverse transcriptase-polymerase chain reaction (0.6%; 1/165). As Tunisian studies on CCHFV are geographically scattered and limited due to very small sample sizes, further studies are needed to improve the knowledge on the epidemiology of CCHF in Tunisia.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic orthonairovirus of public health concern and widespread geographic distribution. Several animal species are known to seroconvert after infection with CCHFV without showing clinical symptoms. The commercial availability of a multi-species ELISA has led to an increase in recent serosurveillance studies as well as in the range of species reported to be exposed to CCHFV in the field, including wild boar (Sus scrofa). However, development and validation of confirmatory serological tests for swine based on different CCHFV antigens or test principles are hampered by the lack of defined control sera from infected and non-infected animals. For the detection of anti-CCHFV antibodies in swine, we established a swine-specific in-house ELISA using a panel of swine sera from CCHFV-free regions and regions with reported CCHFV circulation. We initially screened more than 700 serum samples from wild boar and domestic pigs and observed a correlation of ≃67% between the commercial and the in-house test. From these sera, we selected a panel of 60 samples that were further analyzed in a newly established indirect immunofluorescence assay (iIFA) and virus neutralization test. ELISA-non-reactive samples tested negative. Interestingly, only a subset of samples reactive in both ELISA and iIFA displayed CCHFV-neutralizing antibodies. The observed partial discrepancy between the tests may be explained by different test sensitivities, antibody cross-reactivities or suggests that the immune response to CCHFV in swine is not necessarily associated with eliciting neutralizing antibodies. Overall, this study highlights that meaningful CCHFV serology in swine, and possibly other species, should involve the performance of multiple tests and careful interpretation of the results., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

We conducted a cross-sectional study in wild boar and extensively managed Iberian pig populations in a hotspot area of Crimean-Congo hemorrhagic fever virus (CCHFV) in Spain. We tested for antibodies against CCHFV by using 2 ELISAs in parallel. We assessed the presence of CCHFV RNA by means of reverse transcription quantitative PCR protocol, which detects all genotypes. A total of 113 (21.8%) of 518 suids sampled showed antibodies against CCHFV by ELISA. By species, 106 (39.7%) of 267 wild boars and 7 (2.8%) of 251 Iberian pigs analyzed were seropositive. Of the 231 Iberian pigs and 231 wild boars analyzed, none tested positive for CCHFV RNA. These findings indicate high CCHFV exposure in wild boar populations in endemic areas and confirm the susceptibility of extensively reared pigs to CCHFV, even though they may only play a limited role in the enzootic cycle.

Hepatitis E virus (HEV) is the main cause of acute hepatitis in humans worldwide and is responsible for a large number of outbreaks especially in Africa. Human infections are mainly caused by genotypes 1 and 2 of the genus Paslahepevirus , which are exclusively associated with humans. In contrast, viruses of genotypes 3 and 4 are zoonotic and have their main reservoir in domestic and wild pigs, from which they can be transmitted to humans primarily through the consumption of meat products. Both genotypes 3 and 4 are widespread in Europe, Asia, and North America and lead to sporadic cases of hepatitis E. However, there is little information available on the prevalence of these genotypes and possible transmission routes from animal reservoirs to humans in African countries. We therefore analysed 1086 pig sera collected in 2016/2017 in four districts in Sierra Leone for antibodies against HEV using a newly designed in-house ELISA. In addition, the samples were also analysed for HEV RNA by quantitative real-time RT-PCR. The overall seroprevalence in Sierra Leone was low with only 44 positive sera and a prevalence of 4.0%. Two serum pools were RT-PCR-positive and recovered partial sequences clustered into the genotype 3 (HEV-3) of the order Paslahepevirus , species Paslahepevirus balayani . The results are the first evidence of HEV-3 infection in pigs from Sierra Leone and demonstrate a low circulation of the virus in these animals to date. Further studies should include an examination of humans, especially those with close contact with pigs and porcine products, as well as environmental sampling to evaluate public health effects within the framework of a One Health approach.

Although pigs are naturally susceptible to Reston virus and experimentally to Ebola virus (EBOV), their role in Orthoebolavirus ecology remains unknown. We tested 888 serum samples collected from pigs in Guinea during 2017-2019 (between the 2013-16 epidemic and its resurgence in 2021) by indirect ELISA against the EBOV nucleoprotein. We identified 2 hotspots of possible pig exposure by IgG titer levels: the northern coast had 48.7% of positive serum samples (37/76), and Forest Guinea, bordering Sierra Leone and Liberia, where the virus emerged and reemerged, had 50% of positive serum samples (98/196). The multitarget Luminex approach confirms ELISA results against Ebola nucleoprotein and highlights cross-reactivities to glycoprotein of EBOV, Reston virus, and Bundibugyo virus. Those results are consistent with previous observations of the circulation of Orthoebolavirus species in pig farming regions in Sierra Leone and Ghana, suggesting potential risk for Ebola virus disease in humans, especially in Forest Guinea.

We tested for Rift Valley fever virus (RVFV) from at least 15 species of non-human primates. RVFV IgG/IgM antibodies were detected in 3.7% (2 out of 53) of chimpanzees (Pan troglodytes) and in 1.4% (1 out of 72) of unidentified non-human primate species. This study was the first investigation of RVFV in monkeys in Cameroon., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The synthesis of a library of halogenated rocaglate derivatives belonging to the flavagline class of natural products, of which silvestrol is the most prominent example, is reported. Their antiviral activity and cytotoxicity profile against a wide range of pathogenic viruses, including hepatitis E, Chikungunya, Rift Valley Fever virus and SARS-CoV-2, were determined. The incorporation of halogen substituents at positions 4', 6 and 8 was shown to have a significant effect on the antiviral activity of rocaglates, some of which even showed enhanced activity compared to CR-31-B and silvestrol.

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide. Up to now, no approved treatment nor a globally licensed vaccine is available. Several recombinant HEV vaccines have been developed to protect against HEV infection in humans, including the commercially available Hecolin vaccine, which are mainly based on HEV genotype 1. However, the efficacy of these vaccines against other HEV genotypes, especially genotype 3 is unknown. In this study, we evaluated the protective efficacy of Hecolin® and a novel genotype 3-based vaccine p239(gt3) against HEV-3 in a pig infection model. Pigs were divided into three groups: one group was vaccinated with Hecolin®, the second group was vaccinated with p239(gt3), and the control group received no vaccine. All pigs were subsequently challenged with HEV genotype 3 to assess the effectiveness of the vaccines. Although all immunised animals developed a high titer of neutralizing antibodies, the results showed that both vaccine applications could not provide complete protection against HEV (gt3) infection: Two out of four animals of the Hecolin® group displayed even virus shedding, and viral RNA could be detected in bile and/or liver of three out of four animals in both vaccination groups. Only one out of four animals in each group was fully protected. Neither Hecolin® nor the novel p239(gt3) vaccine provided sufficient protection against genotype 3 infection. While Hecolin® only partial protected pigs from HEV shedding, the novel p239(gt3) vaccine was at least able to prevent infected pigs from virus shedding. The results highlight the need for further development of HEV vaccines that exhibit broad protection against multiple HEV genotypes and the use of appropriate animal infection models., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Usutu virus (USUV) is a flavivirus transmitted to avian species through mosquito bites that causes mass mortalities in wild and captive bird populations. However, several cases of positive dead birds have been recorded during the winter, a vector-free period. To explain how USUV "overwinters", the main hypothesis is bird-to-bird transmission, as shown for the closely related West Nile virus. To address this question, we experimentally challenged canaries with intranasal inoculation of USUV, which led to systemic dissemination of the virus, provided the inoculated dose was sufficient (>10 2 TCID 50 ). We also highlighted the oronasal excretion of infectious viral particles in infected birds. Next, we co-housed infected birds with naive sentinels, to determine whether onward transmission could be reproduced experimentally. We failed to detect such transmission but demonstrated horizontal transmission by transferring sputum from an infected to a naive canary. In addition, we evaluated the cellular tropism of respiratory mucosa to USUV in vitro using a canary tracheal explant and observed only limited evidence of viral replication. Further research is then needed to assess if and how comparable bird-to-bird transmission occurs in the wild.

Three avian viral pathogens circulate in Germany with particular importance for animal disease surveillance due to their zoonotic potential, their impact on wild bird populations and/or poultry farms: Highly pathogenic (HP) avian influenza virus (AIV) of subtype H5 (HPAIV H5), Usutu virus (USUV), and West Nile virus (WNV). Whereas HPAIV H5 has been mainly related to epizootic outbreaks in winter, the arthropod-borne viruses USUV and WNV have been detected more frequently during summer months corresponding to peak mosquito activity. Since 2021, tendencies of a potentially year-round, i.e. enzootic, status of HPAIV in Germany have raised concerns that Orthomyxoviruses (AIV) and Flaviviruses (USUV, WNV) may not only circulate in the same region, but also at the same time and in the same avian host range. In search of a host species group suitable for a combined surveillance approach for all mentioned pathogens, we retrospectively screened and summarized case reports, mainly provided by the respective German National Reference Laboratories (NRLs) from 2006 to 2021. Our dataset revealed an overlap of reported infections among nine avian genera. We identified raptors as a particularly affected host group, as the genera Accipiter , Bubo , Buteo , Falco , and Strix represented five of the nine genera, and highlighted their role in passive surveillance. This study may provide a basis for broader, pan-European studies that could deepen our understanding of reservoir and vector species, as HPAIV, USUV, and WNV are expected to further become established and/or spread in Europe in the future and thus improved surveillance measures are of high importance.

To assess pastoralists' and agropastoralists' knowledge on Rift Valley fever (RVF), participatory epidemiological studies were conducted with 215 livestock keepers and 27 key informants in Napak, Butebo, Isingiro and Lyantonde districts, Uganda, between January and February 2022. Livestock keepers in all four districts had knowledge of RVF and even had local names or descriptions for it. Pastoralists and agropastoralists possessed valuable knowledge of RVF clinical descriptions and epidemiological risk factors such as the presence of infected mosquitoes, living in flood-prone areas, and excessive rainfall. RVF was ranked among the top ten most important cattle diseases. Pastoralists called RVF Lonyang, symbolizing a disease associated with jaundice, high fever, abortions in pregnant cows, and sudden death in calves. Key informants identified infected domestic animals, the presence of infected mosquitoes, livestock movement and trade, and infected wild animals as risk pathways for the introduction of RVF into an area. Drinking raw blood and milk was perceived as the most likely pathway for human exposure to RVF virus; while the highest consequence was high treatment costs. The results indicate that pastoralists provided key epidemiological information that could be essential for designing an effective national RVF surveillance and early warning system., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicting interests that may have inappropriately influenced them in writing this original research article., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The consumption of raw or undercooked meat products poses a serious risk for human hepatitis E virus (HEV) infections. In many high-income countries, domestic pigs and wild boars represent the main animal reservoirs for HEV and are usually identified by reverse transcription-PCR and antibody enzyme-linked immunosorbent assay (ELISA). In order to characterize the humoral immune response in more detail, a cell culture-based serum neutralization assay using a culture-adapted HEV strain was established here. Measurement of neutralizing antibodies was only possible after removing the viral quasi-envelope by detergent treatment. Serum samples of 343 wild boars from Northern Germany were first analyzed for anti-HEV IgG using an in-house ELISA, resulting in 19% positive samples. Subsequently, a subset of 41 representative samples was tested with the neutralization assay, and the results correlated well with those obtained by ELISA. Not only the human HEV strain 47832c but also two porcine HEV strains were shown to be neutralized by porcine serum antibodies. Neutralizing activity was also found in samples containing both HEV-specific antibodies and HEV RNA. Testing of serum samples derived from two experimentally infected domestic pigs showed a steep increase in neutralizing activity at 24 or 51 days post infection, dependent on the used infectious dose. The developed assay can be useful for characterization of the humoral immune response after HEV infection and for assessing the efficiency of HEV vaccine candidates., Competing Interests: The authors declare no conflict of interest.

Hepatitis E virus (HEV) genotype 3 infections in Germany are mainly transmitted zoonotically through the consumption of swine meat. Furthermore, there is evidence that pets might come into contact with HEV, but the relevance of companion animals as possible sources of HEV transmission in Germany still needs to be defined. A monitoring study was therefore carried out on dogs, cats, and horses from Germany. In total 365 serum samples from pets (124 dogs, 119 cats, and 122 horses) were tested for HEV by PCR and for anti-HEV antibodies by a commercial ELISA. The HEV seroprevalence determined by the sero-assay varied significantly between dogs (10%), cats (6%), and horses (2%). Liver injury-related enzymes, alanine transaminase (ALT), and aspartate transaminase (AST) showed no differences between HEV-positive or negative animals. None of the pet serum samples tested positive for PCR. This serological study suggests that dogs and cats are significantly exposed to HEV in Germany, while horses are of minor relevance., (© 2023. The Author(s).)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Usutu virus (USUV) is a mosquito-borne flavivirus that is widely distributed in southern and central Europe. The zoonotic virus circulates primarily between birds and mosquitoes, can, however, in rare cases infect other mammals including humans. In the past, USUV has been repeatedly associated with mass mortalities in birds, primarily blackbirds and owls. Birds commonly succumb either due to the peracute nature of the infection or due to severe encephalitis. In Germany, USUV has spread rapidly since its first detection in 2010 in mosquitoes under the presence of susceptible host and vector species. Nonetheless, there is to date limited access to whole genome sequences resulting in the absence of in-depth phylogenetic and phylodynamic analyses. In this study, 118 wild and captive birds were sequenced using a nanopore sequencing platform with prior target enrichment via amplicons. Due to the high abundancy of Europe 3 and Africa 3 in Germany an ample quantity of associated whole genome sequences was generated and the most recent common ancestor could be determined for each lineage. The corresponding clock phylogeny revealed an introduction of USUV Europe 3 and Africa 3 into Germany three years prior to their first isolation in the avifauna in 2011 and 2014, respectively. Based on the clustering and temporal history of the lineages, evidence exists for the genetic evolution of USUV within Germany as well as new introductions thereof into the country., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Bergmann et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The family Phenuiviridae comprises viruses with 2-8 segments of negative-sense or ambisense RNA, comprising 8.1-25.1 kb in total. Virions are typically enveloped with spherical or pleomorphic morphology but can also be non-enveloped filaments. Phenuivirids infect animals including livestock and humans, birds, plants or fungi, as well as arthropods that serve as single hosts or act as biological vectors for transmission to animals or plants. Phenuivirids include important pathogens of humans, livestock, seafood and agricultural crops. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Phenuiviridae , which is available at ictv.global/report/phenuiviridae.

Usutu virus (USUV) and West Nile virus (WNV) are closely related pathogens circulating between mosquitoes and birds, but also infecting mammals as dead-end hosts. Both viruses share the same susceptible hosts, vectors, and even distribution areas in Central Europe. The aim of the study was, therefore, to understand their amplification potential and interference upon a successive infection. Two-week old geese were initially infected with an USUV isolate from Germany and with a German WNV isolate17 days later. The geese were susceptible to the USUV and the WNV infections, as evidenced by specific flavivirus antibodies in all of the birds. Furthermore, in half of the USUV-inoculated geese, USUV genomes were detected in the blood and swab samples 2-4 days post-infection. Additionally, most of the examined organs contained USUV genomes and showed signs of encephalitis and ganglioneuritis. Interestingly, upon a sequential infection with WNV, the genome copy numbers in all of the examined samples were significantly lower and less frequent than after a WNV mono-infection. Similarly, the histopathological lesions were less severe. Therefore, it can be concluded that a previous USUV infection can protect birds from clinical disease in a subsequent WNV infection.

The use of wild animals in research is complicated due to the capture and housing conditions, as well as to legal aspects, making it difficult to develop in vivo and in vitro models for the study of pathologies that affect these species. Here we validate an in vitro model of tendon-derived mesenchymal cells (TDSC) from Eurasian blackbird ( Turdus merula ) cadaveric samples. Through the expression of surface markers and the ability to differentiate into multiple lineages, the nature of the cells was confirmed. We then evaluated Mesenchymal Stem Cells (MSCs) as an infection model for the Usutu Flavivirus. To this aim, blackbird TDSCs were compared to Vero E6 cells, commonly used in Flavivirus studies. Both cells showed permissiveness to USUV infection as confirmed by immunocytochemistry. Moreover, TDSCs exhibited replication kinetics similar to, although slightly lower than, Vero E6, confirming these cells as a pertinent study model for the study of the pathogenesis of USUV. In this work, we isolated and characterized tendon-derived mesenchymal stem cells, which represent an interesting and convenient in vitro model for the study of wildlife species in laboratories., Competing Interests: The authors declare no conflict of interest.

Usutu virus (USUV) and West Nile virus (WNV) are known to cause diseases and mortalities in bird populations. Since 2010/2011, USUV has circulated in Germany and spread nationwide, while WNV was only introduced into East Germany in 2018. The zoological garden investigated is located in Northern Germany, where USUV infections in wild birds have been detected for several years. In this longitudinal study conducted over a four-year period, zoo birds were sampled biannually and screened for molecular and serological evidence of USUV and WNV. USUV genomes were detected in eight of the sampled birds and whole-genome sequences revealed the circulation of USUV lineages Europe 3 and Africa 3. Of the eight birds infected with USUV during the study period, four died after the infection, while four survived without displaying clinical signs. Furthermore, in a few of the birds, a USUV (re-)infection was confirmed on a serological level with three birds producing USUV-neutralizing antibodies (nAbs) over a period of four years. Nonetheless, in two birds sampled throughout this longitudinal study, neither a USUV nor a WNV infection was evident. In 2022, WNV nAbs were detected for the first time in a juvenile zoo bird, indicating the introduction of the virus into this region.

Nairobi sheep disease virus (NSDV) belongs to the Orthonairovirus genus in the Bunyavirales order and is genetically related to human-pathogenic Crimean-Congo hemorrhagic fever virus (CCHFV). NSDV is a zoonotic pathogen transmitted by ticks and primarily affects naïve small ruminants in which infection leads to severe and often fatal hemorrhagic gastroenteritis. Despite its veterinary importance and the striking similarities in the clinical picture between NSDV-infected ruminants and CCHFV patients, the molecular pathogenesis of NSDV and its interactions with the host cell are largely unknown. Here, we identify the membrane-bound proprotein convertase site-1 protease (S1P), also known as subtilisin/kexin-isozyme-1 (SKI-1), as a host factor affecting NSDV infectivity. Absence of S1P in SRD-12B cells, a clonal CHO-K1 cell variant with a genetic defect in the S1P gene (MBTPS1), results in significantly decreased NSDV infectivity while transient complementation of SKI-1/S1P rescues NSDV infection. SKI-1/S1P is dispensable for virus uptake but critically required for production of infectious virus progeny. Moreover, we provide evidence that SKI-1/S1P is involved in the posttranslational processing of the NSDV glycoprotein precursor. Our results demonstrate the role of SKI-1/S1P in the virus life cycle of NSDV and suggest that this protease is a common host factor for orthonairoviruses and may thus represent a promising broadly-effective, indirect antiviral target., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Since 2018, autochthonous West Nile virus (WNV) infections have been regularly reported in eastern-central Germany. While clinically apparent infections in humans and horses are not frequent, seroprevalence studies in horses may allow the tracing of WNV and related flaviviruses transmission, such as tick-borne encephalitis virus (TBEV) and Usutu virus (USUV), and consequently help to estimate the risk of human infections. Hence, the aim of our study was to follow the seropositive ratio against these three viruses in horses in Saxony, Saxony Anhalt, and Brandenburg and to describe their geographic distribution for the year 2021. In early 2022, i.e., before the virus transmission season, sera from 1232 unvaccinated horses were tested using a competitive pan-flavivirus ELISA (cELISA). In order to estimate the true seropositive ratio of infection with WNV, TBEV, and USUV for 2021, positive and equivocal results were confirmed by a virus neutralization test (VNT). In addition, possible risk factors for seropositivity using questionnaires were analyzed using logistic regression based on questionnaires similar to our previous study from 2020. In total, 125 horse sera reacted positive in the cELISA. Based on the VNT, 40 sera showed neutralizing antibodies against WNV, 69 against TBEV, and 5 against USUV. Three sera showed antibodies against more than one virus, and eight were negative based on the VNT. The overall seropositive ratio was 3.3% (95% CI: 2.38-4.40) for WNV, 5.6% (95% CI: 4.44-7.04) for TBEV, and 0.4% (95% CI: 0.14-0.98) for USUV infections. While age and number of horses on the holding were factors predicting TBEV seropositivity, no risk factors were discovered for WNV seropositivity. We conclude that horses are useful sentinels to determine the flavivirus circulation in eastern-central Germany, as long as they are not vaccinated against WNV.

West Nile virus (WNV) is the most widespread arthropod-borne (arbo) virus and the primary cause of arboviral encephalitis globally. Members of WNV species genetically diverged and are classified into different hierarchical groups below species rank. However, the demarcation criteria for allocating WNV sequences into these groups remain individual and inconsistent, and the use of names for different levels of the hierarchical levels is unstructured. In order to have an objective and comprehensible grouping of WNV sequences, we developed an advanced grouping workflow using the 'affinity propagation clustering' algorithm and newly included the 'agglomerative hierarchical clustering' algorithm for the allocation of WNV sequences into different groups below species rank. In addition, we propose to use a fixed set of terms for the hierarchical naming of WNV below species level and a clear decimal numbering system to label the determined groups. For validation, we applied the refined workflow to WNV sequences that have been previously grouped into various lineages, clades, and clusters in other studies. Although our workflow regrouped some WNV sequences, overall, it generally corresponds with previous groupings. We employed our novel approach to the sequences from the WNV circulation in Germany 2020, primarily from WNV-infected birds and horses. Besides two newly defined minor (sub)clusters comprising only three sequences each, Subcluster 2.5.3.4.3c was the predominant WNV sequence group detected in Germany from 2018 to 2020. This predominant subcluster was also associated with at least five human WNV infections in 2019-20. In summary, our analyses imply that the genetic diversity of the WNV population in Germany is shaped by enzootic maintenance of the dominant WNV subcluster accompanied by sporadic incursions of other rare clusters and subclusters. Moreover, we show that our refined approach for sequence grouping yields meaningful results. Although we primarily aimed at a more detailed WNV classification, the presented workflow can also be applied to the objective genotyping of other virus species., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press.)

West Nile virus (WNV) is known to cause disease and death in humans and various animals worldwide. WNV has circulated in Germany since 2018. In 2020, four birds tested positive for the WNV genome at Zoopark Erfurt (Thuringia). Moreover, virus neutralization assays detected neutralizing antibodies (nAb) against WNV in 28 birds. In addition, nAb against WNV and Usutu virus (USUV) were found in 14 birds. To protect valuable animals and to reduce the risk of viral transmission from birds to humans, we performed a field study on WNV vaccination at the zoo. To conduct the study, 61 birds from the zoo were categorized into three groups and subjected to a vaccination regimen, where each bird received either 1.0 mL, 0.5 mL, or 0.3 mL of a commercial inactivated WNV vaccine three times. The vaccinations were administered at three-week intervals, or as per modified vaccination schedules. Furthermore, 52 birds served as non-vaccinated controls. Adverse vaccination reactions were absent. The greatest increase in nAb titres was observed in birds that received 1.0 mL of vaccine. However, pre-existing antibodies to WNV and USUV appeared to have a major effect on antibody development in all groups and in all bird species, whereas sex and age had no effect. After vaccination, no death was detected in vaccinated birds for more than 1 year.

Bovine spongiform encephalopathy (BSE) belongs to the group of transmissible spongiform encephalopathies and is associated with the accumulation of a pathological isoform of the host-encoded glycoprotein, designated prion protein (PrP Sc ). Classical BSE (C-type) and two atypical BSE forms (L- and H-type) are known, and can be discriminated by biochemical characteristics. The goal of our study was to identify type-specific PrP Sc profiles by using Immunohistochemistry. In our study, brain samples from 21 cattle, intracerebrally inoculated with C-, H-, and L-type BSE, were used. In addition, the corresponding samples from three orally C-type BSE infected animals were also included. From all animals, a lesion and PrP Sc -profiles of six brain regions were determined. The lesion profile and the neuroanatomical distribution of PrP Sc was highly consistent between the groups, but the immunohistochemical analysis revealed a distinct PrP Sc profile for the different BSE-types, which included both the topographic and cellular pattern of PrP Sc . This qualitative and quantitative analysis of PrP Sc affected structures sheds new light into the pathogenesis of the different BSE types. Furthermore, immunohistochemical characterization is supported as an additional diagnostic tool in BSE surveillance programs, especially when only formalin-fixed tissue samples are available.

Background: Ticks are obligate hematophagous arthropods capable of transmitting a great variety of endemic and emerging pathogens causing diseases in animals and humans., Objectives: The aim of this study was to investigate the presence of Bartonella spp., Rickettsia spp., Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum in ticks collected from cattle in Benin and Togo., Methods: Overall, 396 (148 males, 205 females and 43 nymphs) ticks were collected from cattle in 17 districts (Benin and Togo) between 2019 and 2020. Ticks were pooled into groups of 2-6 ticks per pool according to individual host, location, species and developmental stage. The DNA of each pool was extracted for molecular screening., Results: PCR results revealed that 20 tick pools were positive for Bartonella spp. (Benin and Togo) and 23 tick pools positive for Rickettsia spp. (Benin), while all pools were negative for A. phagocytophilum and B. burgdorferi s.l. Sequence analysis of positive Rickettsia samples revealed the presence of Rickettsia aeschlimannii., Conclusions: The present study highlights the presence of zoonotic agents in ticks collected from cattle in Benin and Togo. This information will raise awareness of tick-borne diseases among physicians and veterinarians, stimulate further studies to monitor these pathogens, and advise on necessary measures to control the spread of these zoonoses., (© 2022 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

On the African continent, a large number of arthropod-borne viruses (arboviruses) with zoonotic potential have been described, and yet little is known of most of these pathogens, including their actual distribution or genetic diversity. In this study, we evaluated as a proof-of-concept the effectiveness of the nonspecific sequencing technique sequence-independent single primer amplification (SISPA) on third-generation sequencing techniques (MinION sequencing, Oxford Nanopore Technologies, Oxford, UK) by comparing the sequencing results from six different samples of arboviruses known to be circulating in Africa (Crimean-Congo hemorrhagic fever virus (CCHFV), Rift Valley fever virus (RVFV), Dugbe virus (DUGV), Nairobi sheep disease virus (NSDV), Middleburg virus (MIDV) and Wesselsbron virus (WSLV)). All sequenced samples were derived either from previous field studies or animal infection trials. Using this approach, we were able to generate complete genomes for all six viruses without the need for virus-specific whole-genome PCRs. Higher Cq values in diagnostic RT-qPCRs and the origin of the samples (from cell culture or animal origin) along with their quality were found to be factors affecting the success of the sequencing run. The results of this study may stimulate the use of metagenomic sequencing approaches, contributing to a better understanding of the genetic diversity of neglected arboviruses.

In March 2022, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was amended and emended. The phylum was expanded by two new families (bunyaviral Discoviridae and Tulasviridae), 41 new genera, and 98 new species. Three hundred forty-nine species were renamed and/or moved. The accidentally misspelled names of seven species were corrected. This article presents the updated taxonomy of Negarnaviricota as now accepted by the ICTV., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Unlike farm animals, wild animals are not subject to continuous health surveillance. Individual projects designed to screen wildlife populations for specific pathogens are, therefore, also of great importance for human health. In this context, the possible formation of a reservoir for highly pathogenic zoonotic pathogens is a focus of research. Two of these pathogens that have received particular attention during the last years are the novel severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), due to its fast global spread and high impact to the human health, and, since its introduction into Germany, the flavivirus West Nile virus (WNV). Especially in combination with invasive vertebrate species (e.g., raccoons ( Procyon lotor ) and raccoon dogs ( Nyctereutes procyonoides ) in Germany), risk analysis must be done to enable health authorities to assess the potential for the establishment of new wild life reservoirs for pathogens. Therefore, samples were collected from raccoons and raccoon dogs and analyzed for the presence of SARS-CoV-2 and WNV infections in these populations. Molecular biological and serological data obtained imply that no SARS-CoV-2 nor WNV reservoir has been established in these two wild life species yet. Future investigations need to keep an eye on these invasive carnivore populations, especially since the close contact of these animals to humans, mainly in urban areas, would make animal-human transmission a challenge for human health.

Rift Valley fever phlebovirus (RVFV) causes Rift Valley fever (RVF), an emerging zoonotic disease that causes abortion storms and high mortality rates in young ruminants as well as severe or even lethal complications in a subset of human patients. This study investigates the pathomechanism of intranuclear inclusion body formation in severe RVF in a mouse model. Liver samples from immunocompetent mice infected with virulent RVFV 35/74, and immunodeficient knockout mice that lack interferon type I receptor expression and were infected with attenuated RVFV MP12 were compared to livers from uninfected controls using histopathology and immunohistochemistry for RVFV nucleoprotein, non-structural protein S (NSs) and pro-apoptotic active caspase-3. Histopathology of the livers showed virus-induced, severe hepatic necrosis in both mouse strains. However, immunohistochemistry and immunofluorescence revealed eosinophilic, comma-shaped, intranuclear inclusions and an intranuclear (co-)localization of RVFV NSs and active caspase-3 only in 35/74-infected immunocompetent mice, but not in MP12-infected immunodeficient mice. These results suggest that intranuclear accumulation of RVFV 35/74 NSs is involved in nuclear translocation of active caspase-3, and that nuclear NSs and active caspase-3 are involved in the formation of the light microscopically visible inclusion bodies.

Usutu virus (USUV) is a mosquito-borne zoonotic virus and one of the causes of flavivirus encephalitis in birds and occasionally in humans. USUV rapidly disperses in a susceptible host and vector environment, as is the case in South and Central Europe. However, compared to other flaviviruses, USUV has received less research attention and there is therefore limited access to whole-genome sequences and also to in-depth phylogenetic and phylodynamic analyses. To ease future molecular studies, this study compares first- (partial sequencing via Sanger), second- (Illumina), and third-generation (MinION Nanopore) sequencing platforms for USUV. With emphasis on MinION Nanopore sequencing, cDNA-direct and target-enrichment (amplicon-based) sequencing approaches were validated in parallel. The study was based on four samples from succumbed birds commonly collected throughout Germany. The samples were isolated from various sample matrices, organs as well as blood cruor, and included three different USUV lineages. We concluded that depending on the focus of a research project, amplicon-based MinION Nanopore sequencing can be an ideal cost- and time-effective alternative to Illumina in producing optimal genome coverage. It can be implemented for an array of lab- or field-based objectives, including among others: phylodynamic studies and the analysis of viral quasispecies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Holicki, Bergmann, Stoek, Schulz, Groschup, Ziegler and Sadeghi.)