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Q fever (QF) and Rift Valley fever (RVF) are endemic zoonotic diseases in African countries, causing significant health and economic burdens. Accurate prevalence estimates, crucial for disease control, rely on robust diagnostic tests. While enzyme-linked immunosorbent assays (ELISA) are not the gold standard, they offer rapid, cost-effective, and practical alternatives. However, varying results from different tests and laboratories can complicate comparing epidemiological studies. This study aimed to assess the agreement of test results for QF and RVF in humans and livestock across different laboratory conditions and, for humans, different types of diagnostic tests. We measured inter-laboratory agreement using concordance, Cohen's kappa, and prevalence and bias-adjusted kappa (PABAK) on 91 human and 102 livestock samples collected from rural regions in Chad. The serum aliquots were tested using ELISA in Chad, and indirect immunofluorescence assay (IFA) (for human QF and RVF) and ELISA (for livestock QF and RVF) in Switzerland and Germany. Additionally, we examined demographic factors influencing test agreement, including district, setting (village vs. camp), sex, age, and livestock species of the sampled individuals. The inter-laboratory agreement ranged from fair to moderate. For humans, QF concordance was 62.5%, Cohen's kappa was 0.31, RVF concordance was 81.1%, and Cohen's kappa was 0.52. For livestock, QF concordance was 92.3%, Cohen's kappa was 0.59, RVF concordance was 94.0%, and Cohen's kappa was 0.59. Multivariable analysis revealed that QF test agreement is significantly higher in younger humans and people living in villages compared to camps and tends to be higher in livestock from Danamadji compared to Yao, and in small ruminants compared to cattle. Additionally, RVF agreement was found to be higher in younger humans. Our findings emphasize the need to consider sample conditions, test performance, and influencing factors when conducting and interpreting epidemiological seroprevalence studies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hungerbühler 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.)

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.

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.

The present study investigated the seropositivity rate of Hepatitis E virus (HEV) in domestic and working animals in Namibia, which included dogs, cats, horses, and donkeys. HEV poses a growing threat as a significant cause of human hepatitis globally and has several genotypes of varying zoonotic potential. As epidemiological data on the seroprevalence of HEV in Namibia is scarce, a serosurvey was conducted on archived serum samples of 374 dogs, 238 cats, 98 horses, and 60 donkeys collected between 2018 and 2022 from different regions, to assess the potential of these animals as sources of HEV infection. The findings revealed that 10.43% ( n  = 39/374) canine and 5.88% ( n  = 14/238) feline samples tested positive for HEV antibodies, whereas no seropositivity was detected in horses and donkeys. The study further examined the risk factors associated with HEV seropositivity, including animal sex, age, and geographical region, and noted a higher prevalence in dogs living in areas with intensive pig farming. Although there is no direct evidence indicating that these animals served as major reservoirs for HEV transmission to humans, the study underscores the importance of preventive measures to minimize contact exposure with pets considering the potential zoonotic risk, especially for susceptible risk groups. Further research is needed to explore the zoonotic potential of domestic animals and the epidemiological links between animal and human HEV transmissions in Namibia., 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 © 2024 Molini, Franzo, de Villiers, van Zyl, de Villiers, Khaiseb, Busch, Knauf, Dietze and Eiden.)

Throughout history, humans have relied on wood for constructions, tool production or as an energy source. How and to what extent these human activities have impacted plant abundance and composition over a long-term perspective is, however, not well known. To address this knowledge gap, we combined 44,239 precisely dated tree-ring samples from economically and ecologically important tree species (spruce, fir, pine, oak) from historical buildings, and pollen-based plant cover estimates using the REVEALS model from 169 records for a total of 34 1° × 1° grid cells for Central Europe. Building activity and REVEALS estimates were compared for the entire study region (4-15°E, 46-51°N), and for low (<500 m asl) and mid/high elevations (≥500 m asl) in 100-year time windows over the period 1150-1850. Spruce and oak were more widely used in wooden constructions, amounting to 35 % and 32 %, respectively, compared to pine and fir. Besides wood properties and species abundance, tree diameters of harvested individuals, being similar for all four species, were found to be the most crucial criterion for timber selection throughout the last millennium. Regarding land use changes, from the 1150-1250's onwards, forest cover generally decreased due to deforestation until 1850, especially at lower elevations, resulting in a more heterogeneous landscape. The period 1650-1750 marks a distinct change in the environmental history of Central Europe; increasing agriculture and intense forest management practices were introduced to meet the high demands of an increasing population and intensifying industrialization, causing a decrease in palynological diversity, especially at low elevations. Likely the characteristic vegetation structure and composition of contemporary landscapes originated from that period. We further show that land use has impacted vegetation composition and diversity at an increasing speed leading to a general homogenization of landscapes through time, highlighting the limited environmental benefits of even-aged plantation forestry., 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.)

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.

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.)

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 synthesis and characterization of sol-gel-derived cornhusk support for low-temperature catalytic methane combustion (LTCMC) were investigated in this study. The prepared cornhusk support was impregnated with palladium and cerium oxide (Pd/CeO 2 ) via the classical incipient wetness method. The resulting catalyst was characterized using various techniques, including X-ray diffraction (XRD), N 2 physisorption (BET), transmission electron microscopy (TEM), and hydrogen temperature-programmed reduction (H 2 -TPR). The catalytic performance of the Pd/CeO 2 /CHSiO 2 catalyst was evaluated for methane combustion in the temperature range of 150-600 °C using a temperature-controlled catalytic flow reactor, and its performance was compared with a commercial catalyst. The results showed that the Pd/CeO 2 dispersed on SiO 2 from the cornhusk ash support (Pd/CeO 2 /CHSiO 2 ) catalyst exhibited excellent catalytic activity for methane combustion, with a conversion of 50% at 394 °C compared with 593 °C for the commercial silica catalyst (Pd/CeO 2 /commercial). Moreover, the Pd/CeO 2 /CHSiO 2 catalyst displayed better catalytic stability after 10 h on stream, with a 7% marginal loss in catalytic activity compared with 11% recorded for the Pd/CeO 2 /commercial catalyst. The N 2 physisorption and H 2 -TPR results indicated that the cornhusk SiO 2 support possessed a higher surface area and strong reducibility than the synthesized commercial catalyst, contributing to the enhanced catalytic activity of the Pd/CeO 2 /SiO 2 catalyst. Overall, the SiO 2 generated from cornhusk ash exhibited promising potential as a low-cost and environmentally friendly support for LTCMC catalysts.

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.

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.

The hepatitis E virus (HEV) is the main cause of viral acute hepatitis in the world, affecting more than 20 million people annually. During the acute phase of infection, HEV can be detected in various body fluids, which has a significant impact in terms of transmission, diagnosis or extrahepatic manifestations. Several studies have isolated HEV in the genitourinary tract of humans and animals, which could have important clinical and epidemiological implications. So, our main objective was to evaluate the presence of HEV in testis of naturally infected wild boars (Sus scrofa). For it, blood, liver, hepatic lymph node and testicle samples were collected from 191 male wild boars. The presence of HEV was evaluated in serum by PCR, as well as in tissues by PCR and immunohistochemistry. Four animals (2.09%; 95%CI: 0.82-5.26) showed detectable HEV RNA in serum, being confirmed the presence of HEV-3f genotype in three of them by phylogenetic analysis. HEV was also detected in liver and/or hepatic lymph nodes of the four animals by RT-PCR, as well as by immunohistochemistry analysis. Only one of these wild boars also showed detectable viral load in testis, observing HEV-specific labelling in a small number of fibroblasts and some Sertoli cells. Our results confirm the presence of HEV genotype 3 in naturally infected wild boar testis, although no associated tissue damage was evidenced. This study does not allow us to discard semen as a possible source of HEV transmission in suids. Future experimental studies are necessary to evaluate the impact of HEV genotype 3 on fertility and the possibility of transmission through sexual contact in this specie., (© 2022 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Hepatitis E is a major cause of acute liver disease in humans worldwide. The infection is caused by hepatitis E virus (HEV) which is transmitted in Europe to humans primarily through zoonotic foodborne transmission from domestic pigs, wild boar, rabbits, and deer. HEV belongs to the family Hepeviridae , and possesses a positive-sense, single stranded RNA genome. This agent usually causes an acute self-limited infection in humans, but in people with low immunity, e.g., immunosuppressive therapy or underlying liver diseases, the infection can evolve to chronicity and is able to induce a variety of extrahepatic manifestations. Pig and wild boar have been identified as the primary animal reservoir in Europe, and consumption of raw and undercooked pork is known to pose a potential risk of foodborne HEV infection. In this study, we analysed pig and wild boar liver, faeces, and muscle samples collected in 2019 in Mecklenburg-Western Pomerania, north-east Germany. A total of 393 animals of both species were investigated using quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR), conventional nested RT-PCR and sequence analysis of amplification products. In 33 animals, HEV RNA was detected in liver and/or faeces. In one individual, viral RNA was detected in muscle tissue. Sequence analysis of a partial open reading frame 1 region demonstrated a broad variety of genotype 3 (HEV-3) subtypes. In conclusion, the study demonstrates a high, but varying prevalence of HEV RNA in swine populations in Mecklenburg-Western Pomerania. The associated risk of foodborne HEV infection needs the establishment of sustainable surveillance and treatment strategies at the interface between humans, animals, and the environment within a One Health framework.

Livestock industry supports the livelihood of around 1.3 billion people in the world, with swine industry contributing with 30% of total livestock production worldwide. To maintain and guarantee this production, a pivotal point according to the OIE is addressing potential biohazards. To control them, permanent sero-surveillance is crucial to achieve more focused veterinary public health intervention and prevention strategies, to break the chains of transmission, and to enable fast responses against outbreaks. Within this context, multiplex assays are powerful tools with the potential to simplify surveillance programs, since they reduce time, labour, and variability within analysis. In the present work, we developed a multiplex bead-based assay for the detection of specific antibodies to six relevant pathogens affecting swine: ASFV, CSFV, PRRSV, SIV, TB and HEV. The most immunogenic target antigen of each pathogen was selected as the target protein to coat different microsphere regions in order to develop this multiplex assay. A total of 1544 serum samples from experimental infections as well as field samples were included in the analysis. The 6-plex assay exhibited credible diagnostic parameters with sensitivities ranging from 87.0% to 97.5% and specificities ranging from 87.9% to 100.0%, demonstrating it to be a potential high throughput tool for surveillance of infectious diseases in swine., (© 2021 Wiley-VCH GmbH.)

West Nile virus (WNV) is an emerging infectious pathogen circulating between mosquitoes and birds but also infecting mammals. WNV has become autochthonous in Germany, causing striking mortality rates in avifauna and occasional diseases in humans and horses. We therefore wanted to assess the possible role of free-ranging poultry in the WNV transmission cycle and infected 15 goslings with WNV lineage 2 (German isolate). The geese were monitored daily and sampled regularly to determine viremia, viral shedding, and antibody development by molecular and serological methods. Geese were euthanized at various time points post-infection (pi). All infected geese developed variable degrees of viremia from day 1 to day 10 (maximum) and actively shed virus from days 2 to 7 post-infection. Depending on the time of death, the WN viral genome was detected in all examined tissue samples in at least one individual by RT-qPCR and viable virus was even re-isolated, except for in the liver. Pathomorphological lesions as well as immunohistochemically detectable viral antigens were found mainly in the brain. Furthermore, all of the geese seroconverted 6 days pi at the latest. In conclusion, geese are presumably not functioning as important amplifying hosts but are suitable sentinel animals for WNV surveillance.

Rift Valley fever phlebovirus (RVFV) is an arthropod-borne virus that can cause severe disease in ruminants and humans. Epidemics occur mainly after heavy rainfall, which leads to a significant increase in the occurrence of RVFV-transmitting mosquitoes. During inter-epidemic periods, the virus is assumed to be maintained between mosquitoes, susceptible livestock and yet unknown wildlife. The widespread rodent Rattus rattus (black rat) has been suspected to be involved in RVFV maintenance. In order to elucidate its susceptibility and thus its possible role in the transmission cycle of the virus, an experimental infection study was performed. Black rats were subcutaneously infected with highly virulent RVFV strain 35/74 and euthanized on days 3, 14 and 28 post-infection. Additional black rats served as non-infected contact animals. The infected black rats showed high susceptibility to RVFV infection. Generation of RVFV-neutralizing antibodies was found, and the rats developed viraemias lasting up to 17 days. Viral RNA was found in tissues until the last day of the experiment. However, neither a clinical manifestation nor virus-induced histopathological lesions were observed in any rat. These findings indicate the persistence of RVFV in black rats without affecting the animals. In contact animals, no evidence of horizontal RVFV transmission was found, although the co-housed infected rats showed oral, rectal and conjunctival RVFV shedding. Results of this study point to an involvement of black rats in the RVFV transmission cycle, and further studies are needed to investigate their potential role in the maintenance of the virus., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

In Mauritania, several mosquito-borne viruses have been reported that can cause devastating diseases in animals and humans. However, monitoring data on their occurrence and local distribution are limited. Rift Valley fever virus (RVFV) is an arthropod-borne virus that causes major outbreaks throughout the African continent and the Arabian Peninsula. The first Rift Valley fever (RVF) epidemic in Mauritania occurred in 1987 and since then the country has been affected by recurrent outbreaks of the disease. To gain information on the occurrence of RVFV as well as other mosquito-borne viruses and their vectors in Mauritania, we collected and examined 4,950 mosquitoes, belonging to four genera and 14 species. The mosquitoes were captured during 2018 in the capital Nouakchott and in southern parts of Mauritania. Evidence of RVFV was found in a mosquito pool of female Anopheles pharoensis mosquitoes collected in December on a farm near the Senegal River. At that time, 37.5% of 16 tested Montbéliarde cattle on the farm showed RVFV-specific IgM antibodies. Additionally, we detected IgM antibodies in 10.7% of 28 indigenous cattle that had been sampled on the same farm one month earlier. To obtain information on potential RVFV reservoir hosts, blood meals of captured engorged mosquitoes were analyzed. The mosquitoes mainly fed on humans (urban areas) and cattle (rural areas), but also on small ruminants, donkeys, cats, dogs and straw-colored fruit bats. Results of this study demonstrate the circulation of RVFV in Mauritania and thus the need for further research to investigate the distribution of the virus and its vectors. Furthermore, factors that may contribute to its maintenance should be analyzed more closely. In addition, two mosquito pools containing Aedes aegypti and Culex quinquefasciatus mosquitoes showed evidence of dengue virus (DENV) 2 circulation in the city of Rosso. Further studies are therefore needed to also examine DENV circulation in Mauritania., Competing Interests: The authors have declared that no competing interests exist.

West Nile virus (WNV) and Usutu virus (USUV) are important flaviviruses circulating in Germany. While USUV was first reported more than 10 years ago, WNV has only reached the country in 2018. Wild birds are important amplifying hosts for both viruses. Therefore, we have been monitoring the bird population in different regions of Germany by a previously established network for many years. This report summarizes the results of molecular and/or serological methods of 2345 blood samples from birds of 22 different orders and over 2900 bird carcasses from 2019 and 2020. USUV RNA circulation was found in different regions of Germany, with emphasis on USUV lineages Europe 3 and Africa 3. Increased evidence of USUV lineage Europe 2 was detected in eastern Germany. WNV RNA was found only in birds from the eastern part of the country. The seroprevalence for USUV was between 3.11% and 7.20% in all three regions investigated, whereas the WNV seroprevalence spanned from 14.77% to 16.15% in eastern Germany, with a noticeable tendency for a westward and southward expansion in both years. Thus, wild bird monitoring for WNV and USUV can serve as an early warning system for a human exposure risk.

Zoonotic hepatitis E virus (HEV) infection is an emerging cause of acute viral hepatitis in developed countries. Known reservoirs of zoonotic genotype 3 (HEV-3) are mainly pigs and wild boar, and to a lesser extent rabbits and deer. Rabbit hepatitis E virus (HEV-3ra) is prevalent in rabbits worldwide and represents a particular risk for zoonotic infection. Current understanding of the molecular mechanisms of HEV pathogenesis is incomplete, particularly due to the limited availability of efficient and reliable cell culture systems. In order to identify genomic regions responsible for HEV propagation in cell culture, we developed a modular chimeric reporter replicon system based on cell culture-adapted (Kernow-C1/p6 and 47832mc) and rabbit-derived HEV strains. Replication in HepG2 cells was monitored on the basis of a Gaussia luciferase reporter gene that was inserted in place of the open reading frame (ORF) 2 of the HEV genome. Luciferase activity of rabbit HEV-derived replicons was significantly lower than that of Kernow-C1/p6 and 47832mc replicons. Serial exchanges of defined ORF1 segments within the Kernow-C1/p6 replicon backbone indicated that HEV replication in HepG2 cells is not determined by a single domain but rather by an interplay of longer segments of the ORF1-derived nonstructural polyprotein. This implies that a specific combination of viral factors is required for efficient HEV propagation in cell culture.

The emergence of West Nile virus (WNV) and Usutu virus (USUV) in Europe resulted in significant outbreaks leading to avifauna mortality and human infections. Both viruses have overlapping geographical, host and vector ranges, and are often co-circulating in Europe. In Germany, a nationwide bird surveillance network was established to monitor these zoonotic arthropod-borne viruses in migratory and resident birds. In this framework, co-infections with WNV and USUV were detected in six dead birds collected in 2018 and 2019. Genomic sequencing and phylogenetic analyses classified the detected WNV strains as lineage 2 and the USUV strains as lineages Africa 2 (n = 2), Africa 3 (n = 3) and Europe 2 (n = 1). Preliminary attempts to co-propagate both viruses in vitro failed. However, we successfully cultivated WNV from two animals. Further evidence for WNV-USUV co-infection was obtained by sampling live birds in four zoological gardens with confirmed WNV cases. Three snowy owls had high neutralizing antibody titres against both WNV and USUV, of which two were also positive for USUV-RNA. In conclusion, further reports of co-infections in animals as well as in humans are expected in the future, particularly in areas where both viruses are present in the vector population., (© 2021 The Authors. Transboundary and Emerging Diseases published by Wiley-VCH GmbH.)

Rift Valley fever (RVF) is a zoonotic disease caused by RVF Phlebovirus (RVFV). The RVFV MP-12 vaccine strain is known to exhibit residual virulence in the case of a deficient interferon type 1 response. The hypothesis of this study is that virus replication and severity of lesions induced by the MP-12 strain in immunocompromised mice depend on the specific function of the disturbed pathway. Therefore, 10 strains of mice with deficient innate immunity (B6-IFNAR tmAgt , C.129S7(B6)-Ifng tm1Ts /J, B6-TLR3 tm1Flv , B6-TLR7 tm1Aki , NOD/ShiLtJ), helper T-cell- (CD4 tm1Mak ), cytotoxic T-cell- (CD8 atm1Mak ), B-cell- (Igh-J tm1Dhu N?+N2), combined T- and B-cell- (NU/J) and combined T-, B-, natural killer (NK) cell- and macrophage-mediated immunity (NOD.Cg-Prkdc scid Il2rg tm1WjI /SzJ (NSG) mice) were subcutaneously infected with RVFV MP-12. B6-IFNAR tmAgt mice were the only strain to develop fatal disease due to RVFV-induced severe hepatocellular necrosis and apoptosis. Notably, no clinical disease and only mild multifocal hepatocellular necrosis and apoptosis were observed in NSG mice, while immunohistochemistry detected the RVFV antigen in the liver and the brain. No or low virus expression and no lesions were observed in the other mouse strains. Conclusively, the interferon type 1 response is essential for early control of RVFV replication and disease, whereas functional NK cells, macrophages and lymphocytes are essential for virus clearance.

Ngari virus (NRIV) has been mostly detected during concurrent outbreaks of Rift Valley fever virus (RVFV). NRIV is grouped in the genus Orthobunyavirus within the Bunyaviridae family and RVFV in the genus Phlebovirus in the family Phenuiviridae . Both are zoonotic arboviruses and can induce hemorrhagic fever displaying the same clinical picture in humans and small ruminants. To investigate if NRIV and its parental viruses, Bunyamwera virus (BUNV) and Batai virus (BATV), played a role during the Mauritanian RVF outbreak in 2015/16, we analyzed serum samples of sheep and goats from central and southern regions in Mauritania by quantitative real-time RT-PCR, serum neutralization test (SNT) and ELISA. 41 of 458 samples exhibited neutralizing reactivity against NRIV, nine against BATV and three against BUNV. Moreover, complete virus genomes from BUNV could be recovered from two sheep as well as two NRIV isolates from a goat and a sheep. No RVFV-derived viral RNA was detected, but 81 seropositive animals including 22 IgM-positive individuals were found. Of these specimens, 61 samples revealed antibodies against RVFV and at least against one of the three orthobunyaviruses. An indirect ELISA based on NRIV/BATV and BUNV derived Gc protein was established as complement to SNT, which showed high performance regarding NRIV, but decreased sensitivity and specificity regarding BATV and BUNV. Moreover, we observed high cross-reactivity among NRIV and BATV serological assays. Taken together, the data indicate the co-circulation of at least BUNV and NRIV in the Mauritanian sheep and goat populations., 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 © 2021 Cichon, Barry, Stoek, Diambar, Ba, Ziegler, Rissmann, Schulz, Haki, Höper, Doumbia, Bah, Groschup and Eiden.)

Background & Aims: Hepatitis E virus (HEV) infections are prevalent worldwide. Various viruses have been detected in the ejaculate and can outlast the duration of viremia, indicating replication beyond the blood-testis barrier. HEV replication in diverse organs, however, is still widely misunderstood. We aimed to determine the occurrence, features and morphology of HEV in the ejaculate., Methods: The presence of HEV in testis was assessed in 12 experimentally HEV-genotype 3-infected pigs. We further tested ejaculate, urine, stool and blood from 3 chronically HEV genotype 3-infected patients and 6 immunocompetent patients with acute HEV infection by HEV-PCR. Morphology and genomic characterization of HEV particles from various human compartments were determined by HEV-PCR, density gradient measurement, immune-electron microscopy and genomic sequencing., Results: In 2 of the 3 chronically HEV-infected patients, we observed HEV-RNA (genotype 3c) in seminal plasma and semen with viral loads >2 logs higher than in the serum. Genomic sequencing showed significant differences between viral strains in the ejaculate compared to stool. Under ribavirin-treatment, HEV shedding in the ejaculate continued for >9 months following the end of viremia. Density gradient measurement and immune-electron microscopy characterized (enveloped) HEV particles in the ejaculate as intact., Conclusions: The male reproductive system was shown to be a niche of HEV persistence in chronic HEV infection. Surprisingly, sequence analysis revealed distinct genetic HEV variants in the stool and serum, originating from the liver, compared to variants in the ejaculate originating from the male reproductive system. Enveloped HEV particles in the ejaculate did not morphologically differ from serum-derived HEV particles., Lay Summary: Enveloped hepatitis E virus particles could be identified by PCR and electron microscopy in the ejaculate of immunosuppressed chronically infected patients, but not in immunocompetent experimentally infected pigs or in patients with acute self-limiting hepatitis E., Competing Interests: Conflict of interest Authors declare that they have no conflict of interests regarding this manuscript. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to the genus Orthonairovirus (Nairovididae) and is a (re)emerging tick-borne pathogen. It is endemic in most parts of Africa, Asia and southern Europe, and can cause severe hemorrhagic symptoms in humans, with high fatality rates (5-30%)., Methods: Hyalomma ticks were collected from four different livestock herds (cattle and camels) in Mauritania in 2018. The tick species were determined morphologically and confirmed molecularly by using the cytochrome oxidase 1 gene marker. For the detection of CCHFV, ticks were tested individually by one-step multiplex real-time reverse-transcriptase quantitative polymerase chain reaction. The small segment of all positive samples was sequenced to determine the CCHFV genotype., Results: In total, 39 of the 1523 ticks (2.56%) collected from 63 cattles and 28 camels tested positive for CCHFV. Three Hyalomma species were identified. Hyalomma rufipes had the largest proportion of positivity (5.67%; 16/282), followed by Hyalomma dromedarii (1.89%; 23/1214). No Hyalomma impeltatum tested positive (0%; 0/21). Positive ticks were found in only six out of 91 host animals. Viral sequence analysis revealed the presence of two different CCHFV lineages (Africa I and Africa III)., Conclusions: In this study, 2.56% of Hyalomma ticks collected from camels and cattle in Mauritania tested positive for CCHFV. However, the true prevalence of CCHFV in unfed ticks may be lower, as a considerable number of ticks may have been passively infected during blood-feeding by co-feeding ticks or due to viremia of the host. The results indicate the need to track the actual area of circulation of this virus.

Hepatitis E is an emerging viral disease that is the leading cause of viral hepatitis in the world. The vast majority of hepatitis E cases in developed countries are caused by zoonotic genotypes 3 and 4 of hepatitis E virus (HEV) for which pig and wild boar and to lesser extent rabbits are the main reservoir. According to recent reports rabbits are a source of human HEV infection and highlight the risk of zoonotic foodborne transmission. Here we report the molecular analysis of a novel HEV strain identified in a rabbit during a countrywide surveillance of rabbits and hares in Germany, 2016. The analysis of the complete genome reveals characteristics of a putative novel recombinant subtype of the species Orthohepevirus A within the clade of genotype 3 but not closely related to any known subtypes. Importantly, the genome of this strain possesses a nucleotide exchange in the overlapping region of open reading frames ORF2/ORF3 interfering with a broadly applied diagnostic real-time RT-PCR. In conclusion, a new type of HEV strain was identified in a German rabbit with atypical and novel sequence characteristics.

Rift Valley fever phlebovirus (RVFV) is a zoonotic arthropod-borne virus, which has led to devastating epidemics in African countries and on the Arabian Peninsula. Results of in-vivo, in-vitro and field studies suggested that amphibians and reptiles may play a role as reservoir hosts of RVFV, promoting its maintenance during inter-epidemic periods. To elucidate this hypothesis, we examined two newly established reptile-derived cell lines (Egyptian cobra and Chinese pond turtle) and five previously generated reptile- and amphibian-derived cell lines for their replicative capacity for three low- and high-pathogenic RVFV strains. At different time points after infection, viral loads (TCID 50 ), genome loads and the presence of intracellular viral antigen (immunofluorescence) were assessed. Additionally, the influence of temperatures on the replication was examined. Except for one cell line (read-eared slider), all seven cell lines were infected by all three RVFV strains. Two different terrapin-derived cell lines (Common box turtle, Chinese pond turtle) were highly susceptible. A temperature-dependent replication of RVFV was detected for both amphibian and reptile cells. In conclusion, the results of this study indicate the general permissiveness of amphibian and reptile cell lines to RVFV and propose a potential involvement of terrapins in the virus ecology.

Rift Valley fever phlebovirus (RVFV, Phenuiviridae) is an emerging arbovirus that can cause potentially fatal disease in many host species including ruminants and humans. Thus, tools to detect this pathogen within tissue samples from routine diagnostic investigations or for research purposes are of major interest. This study compares the immunohistological usefulness of several mono- and polyclonal antibodies against RVFV epitopes in tissue samples derived from natural hosts of epidemiologic importance (sheep), potentially virus transmitting insect species (Culex quinquefasciatus, Aedes aegypti) as well as scientific infection models (mouse, Drosophila melanogaster, C6/36 cell pellet). While the nucleoprotein was the epitope most prominently detected in mammal and mosquito tissue samples, fruit fly tissues showed expression of glycoproteins only. Antibodies against non-structural proteins exhibited single cell reactions in salivary glands of mosquitoes and the C6/36 cell pellet. However, as single antibodies exhibited a cross reactivity of varying degree in non-infected specimens, a careful interpretation of positive reactions and consideration of adequate controls remains of critical importance. The results suggest that primary antibodies directed against viral nucleoproteins and glycoproteins can facilitate RVFV detection in mammals and insects, respectively, and therefore will allow RVFV detection for diagnostic and research purposes.

Prion diseases like scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jakob disease (CJD) in humans are fatal neurodegenerative diseases characterized by the conformational conversion of the normal, mainly α-helical cellular prion protein (PrP C ) into the abnormal β-sheet rich infectious isoform PrP Sc . Various therapeutic or prophylactic approaches have been conducted, but no approved therapeutic treatment is available so far. Immunisation against prions is hampered by the self-tolerance to PrP C in mammalian species. One strategy to avoid this tolerance is presenting PrP variants in virus-like particles (VLPs). Therefore, we vaccinated C57/BL6 mice with nine prion peptide variants presented by hamster polyomavirus capsid protein VP1/VP2-derived VLPs. Mice were subsequently challenged intraperitoneally with the murine RML prion strain. Importantly, one group exhibited significantly increased mean survival time of 240 days post-inoculation compared with 202 days of the control group. These data show that immunisation with VLPs presenting PrP peptides may represent a promising strategy for an effective vaccination against transmissible spongiform encephalitis agents.

Crimean-Congo hemorrhagic fever virus (CCHFV) is one of the most widespread zoonotic arthropod-borne viruses in many parts of Africa, Europe and Asia. It belongs to the family of Nairoviridae in the genus of Orthonairovirus. The main reservoir and vector are ticks of the genus Hyalomma. Livestock animals (such as cattle, small ruminants and camels) develop a viremias lasting up to two weeks with absence of clinical symptoms, followed by seroconversion. This study was carried out to assess risk factors that affect seroprevalence rates in different species. In total, 928 livestock animal samples (cattle = 201; sheep = 247; goats = 233; camels = 247) from 11 out of 13 regions in Mauritania were assayed for CCHFV-specific immunoglobulin G (IgG) antibodies using enzyme-linked immunosorbent assays (ELISA) (including a novel indirect camel-IgG-specific CCHFV ELISA). Inconclusive results were resolved by an immunofluorescence assay (IFA). A generalized linear mixed-effects model (GLMM) was used to draw conclusions about the impact of certain factors (age, species, sex and region) which might have influenced the CCHFV antibody status of surveyed animals. In goats and sheep, about 15% of the animals were seropositive, whereas in cattle (69%) and camels (81%), the prevalence rate was significantly higher. On average, cattle and camels were up to twice to four times older than small ruminants. Interestingly, the seroprevalence in all species was directly linked to the age of the animals, i.e. older animals had significantly higher seroprevalence rates than younger animals. The highest CCHFV seroprevalence in Mauritania was found in camels and cattle, followed by small ruminants. The large proportion of positive animals in cattle and camels might be explained by the high ages of the animals. Future CCHFV prevalence studies should at least consider the age of surveyed animals in order to avoid misinterpretations., Competing Interests: The authors have declared that no competing interests exist.

Background: Hepatitis E virus (HEV) is the leading cause of acute hepatitis throughout the world. Increasing blood component transfusion-associated HEV infections highlight the need for reliable virus inactivation procedures for plasma derivatives from pooled plasma donations., Study Design and Methods: An animal infection study was conducted to evaluate the efficiency of HEV inactivation by pasteurization during the manufacturing process of the von Willebrand Factor/Factor VIII (VWF/FVIII) concentrate Haemate P/Humate-P (CSL Behring, Marburg, Germany). For this purpose, groups of pigs were inoculated with stabilized VWF/FVIII intermediate spiked with HEV-positive liver homogenate and exposed to increasing incubation times of 0, 3, 6, and 10 h at 60°C. Animals were evaluated for virus replication over 27 days and in a subsequent trial over 92 days., Results: Virus replication was detected in animals up to the 6-h pasteurization group. In contrast, pasteurization for 10 h did not reveal virus detection when the observation period was 27 days. In an additional experiment using the 10-h pasteurized material, two individuals started virus excretion and seroconverted when the observation period was extended to 92 days. Based on the total infection rate (2 of 12) of the animals inoculated with the sample pasteurized for 10 h, a virus reduction factor of at least 4.7 log 10 is calculated., Conclusion: This study demonstrates that pasteurization at 60°C for 10 h of an HEV-positive plasma derivative leads to the effective reduction of infectivity, resulting in a VWF/FVIII product with an appropriate margin of safety for HEV., (© 2021 The Authors. Transfusion published by Wiley Periodicals LLC. on behalf of AABB.)

Compared to free antigens, antigens immobilized on scaffolds, such as nanoparticles, generally show improved immunogenicity. Conventionally, antigens are conjugated to scaffolds through genetic fusion or chemical conjugation, which may result in impaired assembly or heterogeneous binding and orientation of the antigens. By combining two emerging technologies-i.e., self-assembling multimeric protein scaffold particles (MPSPs) and bacterial superglue-these shortcomings can be overcome and antigens can be bound on particles in their native conformation. In the present work, we assessed whether this technology could improve the immunogenicity of a candidate subunit vaccine against the zoonotic Rift Valley fever virus (RVFV). For this, the head domain of glycoprotein Gn, a known target of neutralizing antibodies, was coupled on various MPSPs to further assess immunogenicity and efficacy in vivo. The results showed that the Gn head domain, when bound to the lumazine synthase-based MPSP, reduced mortality in a lethal mouse model and protected lambs, the most susceptible RVFV target animals, from viremia and clinical signs after immunization. Furthermore, the same subunit coupled to two other MPSPs ( Geobacillus stearothermophilus E2 or a modified KDPG Aldolase) provided full protection in lambs as well.

Tick-borne encephalitis virus (TBEV), a member of the family Flaviviridae, is the most important tick-transmitted arbovirus in Europe. It can cause severe illnesses in humans and in various animal species. The main mechanism for the spread of TBEV into new areas is considered to be the translocation of infected ticks. To find out whether ducks can function as a natural virus reservoir in addition to serving as passive transport vectors, we carried out an experimental TBEV challenge study to reveal their susceptibility and resulting pathogenesis. Nineteen ducks were inoculated subcutaneously with TBEV strain 'Neudoerfl' and monitored for 21 days. Blood, oropharyngeal and cloacal swabs were collected throughout the experiment and organ samples upon necropsy at the end of the study. All samples were tested for TBEV-RNA by real-time polymerase chain reaction. TBEV-specific antibodies were determined by virus neutralization test and ELISA. Organ samples were examined histopathologically and by immunohistochemistry. The inoculated ducks did not show any clinical symptoms. TBEV-specific RNA was detected in all brain samples as well as in a few blood and swab samples. Moreover, all challenged birds produced TBEV antibodies and showed a mild to severe acute to subacute necrotizing encephalitis. TBEV-specific antigen was detected in the brain of 14 ducks by immunohistochemistry. The short and low viremic phases, as well as the low virus load in tissues, suggest that ducks should not be considered as reservoir hosts. However, due to the high antibody levels, ducks can serve as sentinel species for the detection of natural TBEV foci., (© 2020 The Authors. Transboundary and Emerging Diseases published by Blackwell Verlag GmbH.)

Arthropod-borne Batai virus (BATV) is an Orthobunyavirus widely distributed throughout European livestock and has, in the past, been linked to febrile diseases in humans. In Germany, BATV was found in mosquitoes and in one captive harbor seal, and antibodies were recently detected in various ruminant species. We have, therefore, conducted a follow-up study in ruminants from Saxony-Anhalt, the most affected region in Eastern Germany. A total of 325 blood samples from apparently healthy sheep, goats, and cattle were tested using a BATV-specific qRT-PCR and SNT. Even though viral RNA was not detected, the presence of antibodies was confirmed in the sera of all three species: sheep (16.5%), goats (18.3%), and cattle (41.4%). Sera were further analyzed by a glycoprotein Gc-based indirect ELISA to evaluate Gc-derived antibodies as a basis for a new serological test for BATV infections. Interestingly, the presence of neutralizing antibodies was not directly linked to the presence of BATV Gc antibodies. Overall, our results illustrate the high frequency of BATV infections in ruminants in Eastern Germany.

Rift Valley fever phlebovirus (RVFV) is an arthropod-borne zoonotic pathogen, which is endemic in Africa, causing large epidemics, characterized by severe diseases in ruminants but also in humans. As in vitro and field investigations proposed amphibians and reptiles to potentially play a role in the enzootic amplification of the virus, we experimentally infected African common toads and common agamas with two RVFV strains. Lymph or sera, as well as oral, cutaneous and anal swabs were collected from the challenged animals to investigate seroconversion, viremia and virus shedding. Furthermore, groups of animals were euthanized 3, 10 and 21 days post-infection (dpi) to examine viral loads in different tissues during the infection. Our data show for the first time that toads are refractory to RVFV infection, showing neither seroconversion, viremia, shedding nor tissue manifestation. In contrast, all agamas challenged with the RVFV strain ZH501 carried virus genomes in the spleens at 3 dpi, but the animals displayed neither viremia nor virus shedding. In conclusion, the results of this study indicate that amphibians are not susceptible and reptiles are only susceptible to a low extent to RVFV, indicating that both species play, if at all, rather a subordinate role in the RVF virus ecology.

West Nile virus (WNV) is a mosquito-borne virus that originates from Africa and at present causes neurological disease in birds, horses, and humans all around the globe. As West Nile fever is an important zoonosis, the role of free-ranging domestic poultry as a source of infection for humans should be evaluated. This study examined the pathogenicity of an Italian WNV lineage 1 strain for domestic poultry (chickens, ducks, and geese) held in Germany. All three species were subcutaneously injected with WNV, and the most susceptible species was also inoculated via mosquito bite. All species developed various degrees of viremia, viral shedding (oropharyngeal and cloacal), virus accumulation, and pathomorphological lesions. Geese were most susceptible, displaying the highest viremia levels. The tested waterfowl, geese, and especially ducks proved to be ideal sentinel species for WNV due to their high antibody levels and relatively low blood viral loads. None of the three poultry species can function as a reservoir/amplifying host for WNV, as their viremia levels most likely do not suffice to infect feeding mosquitoes. Due to the recent appearance of WNV in Germany, future pathogenicity studies should also include local virus strains.

Background: Micronutrient supplementation has been extensively explored as a strategy to improve health and reduce risk of chronic diseases. Fat-soluble vitamins like A and E with their antioxidant properties and mechanistic interactions with lipoproteins, have potentially a key impact on lipid metabolism and lipidemia., Objective: The impact of micronutrients on lipid metabolism requires further investigation including characterization of plasma lipidome following supplementation and any cause-effect on circulating lipids., Design: In this study, we elucidate the effect and associations of a multi-micronutrient intervention in Brazilian children and teens with lipoprotein alterations and lipid metabolism., Results: Our analysis suggests a combination of short and long-term impact of supplementation on lipid metabolism, potentially mediated primarily by α-tocopherol (vitamin E) and retinol (vitamin A). Among the lipid classes, levels of phospholipids, lysophospholipids, and cholesterol esters were impacted the most along with differential incorporation of stearic, palmitic, oleic and arachidonic acids. Integrated analysis with proteomic data suggested potential links to supplementation-mediated alterations in protein levels of phospholipases and pyruvate dehydrogenase kinase 1 (PDK1)., Conclusions: Associations between the observed differences in lipidemia, total triglyceride, and VLDL-cholesterol levels suggest that micronutrients may play a role in reducing these risk factors for cardiovascular disease in children. This would require further investigation., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

Rift Valley fever virus (RVFV) is a zoonotic arbovirus that causes severe disease in humans and ruminants. The infection is characterized by abortions in pregnant animals, high mortality in neonates as well as febrile illness in humans that develop in 1% of cases encephalitis or hemorrhagic fever. There is presently no specific antiviral treatment for RVFV infection available. In this study, two monoclonal antibodies (mAbs), raised against glycoprotein Gn, were applied in a therapeutic study. Treatment of RVFV infected mice with neutralizing mAb Gn3 alone at two different time points (30 minutes before or 30 minutes after virus challenge) showed only moderate efficacy of about 58.3% survival in both applications. However, a combination therapy together with non-neutralizing mAb Gn32 demonstrated complete protection (100% survival) when applied 30 minutes after the lethal challenge dose. The increase of mAb efficacy is probably based on cooperative neutralization effects. These data suggest that a combination therapy with mAbs Gn3 and Gn32 could be an effective treatment option against RVFV infection., Competing Interests: The authors have declared that no competing interests exist.

Hepatitis E virus (HEV) is the main course for acute hepatitis in humans throughout the world. Human associated genotypes 1 and 2 as well as zoonotic genotypes 3 and 4 are grouped in the species Orthohepevirus A . In addition, a large variety of HEV-related viruses has been found in vertebrates including carnivores, rats, bats, and chickens, which were classified in species Orthohepevirus B-D . In 2015, partial genome sequences of a novel hepevirus were detected in feces of red foxes ( Vulpes vulpes ). However, no further information about virus circulation and the prevalence in foxes was available. We therefore assayed a unique panel of 880 transudates, which was collected from red foxes over 19 years (1993-2012) in Brandenburg, Germany, for HEV-related viral RNA and antibodies. Our results demonstrate a high antibody prevalence of HEV in red foxes, which oscillated annually between 40 and 100%. Molecular screening of the transudates revealed only a single RNA-positive sample, which was assigned to the carnivore species Orthohepevirus C based on the amplified partial sequence. These data indicate that the virus is circulating widely in the fox population and that foxes are carriers of this virus., (Copyright © 2020 Eiden, Dähnert, Spoerel, Vina-Rodriguez, Schröder, Conraths and Groschup.)

Sindbis virus (SINV) is an arbovirus causing clinical symptoms such as arthritis, rash and fever following human infections in Fennoscandia. Its transmission cycle involves mosquito species as vectors as well as wild birds that act as natural reservoir hosts. In Germany, SINV was first time observed in 2009 in different mosquito species in the Upper Rhine valley and one year later in a hooded crow in Berlin. Recently, SINV was also detected repeatedly at various locations in Germany in the context of a mosquitoes monitoring program for arboviruses. In this study, we detected for just the second time a SINV infection in a diseased wild bird (common wood pigeon) from Central Europe. SINV was isolated by cell culture and the complete SINV genome sequence was determined. Phylogenetic analyses revealed a close affiliation to SINV genotype I with a high similarity to human isolate sequences from Finland, Sweden and Russia. The isolate was genetically distinct from the first avian isolate suggesting the circulation of at least two different SINV strains in Germany. In order to reveal the infection frequency in SINV positive mosquito regions 749 bird blood samples were assayed serologically and SINV antibodies found primarily in resident birds. SINV is therefore endemically circulating in mosquitoes in Germany, which results in occasional bird infections. No data are yet available on zoonotic transmission to humans., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Rift Valley fever (RVF) is a zoonotic vector borne infectious disease of major medical and veterinary importance particularly in sub-Saharan Africa. However, there is dearth of epidemiological knowledge of the disease in Cameroon. We conducted a cross-sectional study (January 2016-January 2017) to investigate the seroprevalence and potential risk factors of Rift Valley fever virus (RVFV) in sheep and goats in the North region of Cameroon. Stratified sampling approach was used to select herds where sera were collected from 680 randomly selected small ruminants (355 goats and 325 sheep) in eight localities (Kismatari, Lagdo, Pitoa, Garoua, Bocklé, Dembo, Poli and Touboro) within three administrative divisions (Bénoué, Mayo-Rey and Faro) in the North region. Anti-RVFV antibodies were detected using a competitive Enzyme-Linked Immunosorbent Assay (ELISA), while a capture ELISA was used for the detection of specific RVFV-Immunoglobulin M (Ig-M) antibodies. We evaluated the associated potential risk factors of RVF in small ruminants based on observations of animal-related intrinsic and extrinsic factors in combination with serological results. The results revealed that 3.4% (95% confidence interval (CI): 2.2-5.1%) of sampled animals and 24.6% (95% CI: 15.1-37.1%) of 65 sampled herds were seropositive for anti-RVFV antibodies and no difference in seropositivity between sheep and goats at individual animal as well as at herd levels was observed. Localities along hydrographic or large water banks such as Kismatari (OR: 14.333, (95% CI: 1.436-145.088)) and Pitoa (OR = 11.467 (95% CI: 1.249-50.306)) were significantly associated to RVFV antibody seroprevalence in a simple logistic regression. In addition, the multiple regression analysis showed that age and access to water points significantly influenced RVFV antibody seroprevalence in small ruminants. This study revealed that anti-RVFV antibodies are present in sheep and goats in the North region of Cameroon. It highlights the likely endemic circulation of RVFV in the considered localities despite the absence of clinical cases reported in animals or humans. Under these conditions, it is necessary to set up an early warning, surveillance and control strategy based on epizootic risk., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 R. Poueme et al.)

Koala retrovirus (KoRV) is in the process of endogenization into the koala (Phascolarctos cinereus) genome and is currently spreading through the Australian koala population. Understanding how the koala's immune system responds to KoRV infection is critical for developing an efficacious vaccine to protect koalas. To this end, we analyzed the antibody response of 235 wild koalas, sampled longitudinally over a four-year period, that harbored KoRV-A, and with or without KoRV-B. We found that the majority of the sampled koalas were able to make anti-KoRV antibodies, and that there was a linear increase in anti-KoRV IgG levels in koalas up to approximately seven years of age and then a gradual decrease thereafter. Koalas infected with both KoRV-A and KoRV-B were found to have slightly higher anti-KoRV IgG titers than koalas with KoRV-A alone and there was an inverse relationship between anti-KoRV IgG levels and circulating KoRV viral load. Finally, we identified distinct epitopes on the KoRV envelope protein that were recognized by antibodies. Together, these findings provide insight into the koala's immune response to KoRV and may be useful in the development of a therapeutic KoRV vaccine.

Wild birds play an important role as reservoir hosts and vectors for zoonotic arboviruses and foster their spread. Usutu virus (USUV) has been circulating endemically in Germany since 2011, while West Nile virus (WNV) was first diagnosed in several bird species and horses in 2018. In 2017 and 2018, we screened 1709 live wild and zoo birds with real-time polymerase chain reaction and serological assays. Moreover, organ samples from bird carcasses submitted in 2017 were investigated. Overall, 57 blood samples of the live birds (2017 and 2018), and 100 organ samples of dead birds (2017) were positive for USUV-RNA, while no WNV-RNA-positive sample was found. Phylogenetic analysis revealed the first detection of USUV lineage Europe 2 in Germany and the spread of USUV lineages Europe 3 and Africa 3 towards Northern Germany. USUV antibody prevalence rates were high in Eastern Germany in both years. On the contrary, in Northern Germany, high seroprevalence rates were first detected in 2018, with the first emergence of USUV in this region. Interestingly, high WNV-specific neutralizing antibody titers were observed in resident and short-distance migratory birds in Eastern Germany in 2018, indicating the first signs of a local WNV circulation.

Background: Traumatic brain injury (TBI) is recognized as a metabolic disease, characterized by acute cerebral glucose hypo-metabolism. Adaptive metabolic responses to TBI involve the utilization of alternative energy substrates, such as ketone bodies. Cerebral microdialysis (CMD) has evolved as an accurate technique allowing continuous sampling of brain extracellular fluid and assessment of regional cerebral metabolism. We present the successful application of a combined hypothesis- and data-driven metabolomics approach using repeated CMD sampling obtained routinely at patient bedside. Investigating two patient cohorts (n = 26 and n = 12), we identified clinically relevant metabolic patterns at the acute post-TBI critical care phase., Methods: Clinical and CMD metabolomics data were integrated and analysed using in silico and data modelling approaches. We used both unsupervised and supervised multivariate analysis techniques to investigate structures within the time series and associations with patient outcome., Findings: The multivariate metabolite time series exhibited two characteristic brain metabolic states that were attributed to changes in key metabolites: valine, 4-methyl-2-oxovaleric acid (4-MOV), isobeta-hydroxybutyrate (iso-bHB), tyrosyine, and 2-ketoisovaleric acid (2-KIV). These identified cerebral metabolic states differed significantly with respect to standard clinical values. We validated our findings in a second cohort using a classification model trained on the cerebral metabolic states. We demonstrated that short-term (therapeutic intensity level (TIL)) and mid-term patient outcome (6-month Glasgow Outcome Score (GOS)) can be predicted from the time series characteristics., Interpretation: We identified two specific cerebral metabolic patterns that are closely linked to ketometabolism and were associated with both TIL and GOS. Our findings support the view that advanced metabolomics approaches combined with CMD may be applied in real-time to predict short-term treatment intensity and long-term patient outcome., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Hepatitis E virus (HEV) is a recognized zoonotic disease; autochthonous infections in Europe are caused to a great extent by HEV genotype 3. Pigs and wild boar are the main reservoirs for this genotype and normally they develop no or only subclinical symptoms with mild histopathological lesions. However, co-infections with other pig pathogens can lead to severe cases in pigs, including liver hemorrhage and necrosis. During a monitoring program 2016 in Saxony, Germany, farmed pigs with various clinical outcomes including fatalities were analysed for HEV and concurrent infections. We could detect eight HEV infected pigs from which six were co-infected with porcine circovirus 2 (PCV2). Phylogenetic analysis revealed HEV sub-genotypes 3e and 3f as well as PCV2 genotypes 2b and 2d. A direct correlation of the co-infection to the course of disease could not be determined, but the results provide hints that the immune modulatory effects of PCV2 combined with HEV influence the disease pattern in pigs., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)