Enhanced fitness of SARS-CoV-2 variant of concern Alpha but not Beta.

Emerging variants of concern (VOCs) are driving the COVID-19 pandemic1,2. Experimental assessments of replication and transmission of major VOCs and progenitors are needed to understand the mechanisms of replication and transmission of VOCs3. Here we show that the spike protein (S) from Alpha (also known as B.1.1.7) and Beta (B.1.351) VOCs had a greater affinity towards the human angiotensin-converting enzyme 2 (ACE2) receptor than that of the progenitor variant S(D614G) in vitro. Progenitor variant virus expressing S(D614G) (wt-S614G) and the Alpha variant showed similar replication kinetics in human nasal airway epithelial cultures, whereas the Beta variant was outcompeted by both. In vivo, competition experiments showed a clear fitness advantage of Alpha over wt-S614G in ferrets and two mouse models-the substitutions in S were major drivers of the fitness advantage. In hamsters, which support high viral replication levels, Alpha and wt-S614G showed similar fitness. By contrast, Beta was outcompeted by Alpha and wt-S614G in hamsters and in mice expressing human ACE2. Our study highlights the importance of using multiple models to characterize fitness of VOCs and demonstrates that Alpha is adapted for replication in the upper respiratory tract and shows enhanced transmission in vivo in restrictive models, whereas Beta does not overcome Alpha or wt-S614G in naive animals.

Lymphocytic Choriomeningitis Virus Lineage V in Wood Mice, Germany.

We identified a novel lineage of lymphocytic choriomeningitis virus, tentatively named lineage V, in wood mice (Apodemus sylvaticus) from Germany. Wood mouse-derived lymphocytic choriomeningitis virus can be found across a substantially greater range than previously thought. Increased surveillance is needed to determine its geographic range and zoonotic potential.

Reemergence of Lymphocytic Choriomeningitis Mammarenavirus, Germany.

Lymphocytic choriomeningitis mammarenavirus (LCMV) is a globally distributed zoonotic pathogen transmitted by house mice (Mus musculus). We report the reemergence of LCMV (lineages I and II) in wild house mice (Mus musculus domesticus) and LCMV lineage I in a diseased golden lion tamarin (Leontopithecus rosalia) from a zoo in Germany.

Field survey and molecular characterization of apicomplexan parasites in small mammals from military camps in Afghanistan.

Small mammals are an important reservoir for causative agents of numerous infectious diseases, including zoonotic and vector-borne diseases. The occurrence of these pathogens represents a regional but permanent threat for humans and animals in general and might especially weaken military personnel and companion animals in abroad missions. In our study, small mammals collected in military camps in Afghanistan (Feyzabad, Mazar-e Sharif, and Kunduz) were investigated for the presence of apicomplexans using histopathology and molecular methods. For this purpose, well-established and newly developed real-time PCR assays were applied. A high prevalence was detected not only in house mice (Mus musculus), but also in shrews (Crocidura cf. suaveolens) and grey dwarf hamsters (Cricetulus migratorius). The molecular characterization based on the 18S rRNA gene revealed a close relationship to a cluster of Hepatozoon sp. detected in voles of the genus Microtus. Hepatozoon canis DNA was detected in one house mouse as well as in two Rhipicephalus ticks from a dog puppy. In addition, around 5% of the house mice were found to be infected with far related adeleorinids showing the highest sequence identity of 91.5% to Klossiella equi, the only published Klossiella sequence at present. For their better phylogenetic characterization, we conducted metagenomics by sequencing of two selected samples. The resulting 18S rRNA gene sequences have a length of about 2400 base pairs including an insertion of about 500 base pairs and are 100% identical to each other. Histopathology together with organ tropism and detection rates verified this sequence as of Klossiella muris. In conclusion, we documented naturally occurring protozoan stages and the additional taxonomic characterization of a well-known commensal in mice by applying a combination of different approaches. The study is of medical, social, and biological importance for ensuring human and animal health in military camps and also stresses the required awareness for the potential risk of zoonoses.

High viral loads: what drives fatal cases of COVID-19 in vaccinees? - an autopsy study.

The rate of SARS-CoV-2 infections in vaccinees has become a relevant serious issue. This study aimed to determine the causes of death, histological organ alteration, and viral spread in relation to demographic, clinical-pathological, viral variants, and vaccine types for deceased individuals with proven SARS-CoV-2 infection after vaccination who died between January and November 2021. Twenty-nine consecutively collected cases were analyzed and compared to 141 nonvaccinated control cases. Autopsies were performed on 16 partially and 13 fully vaccinated individuals. Most patients were elderly and suffered from several relevant comorbidities. Real-time RT-PCR (RT-qPCR) identified a significantly increased rate of generalized viral dissemination within organ systems in vaccinated cases versus nonvaccinated cases (45% vs. 16%, respectively; P = 0.008) mainly with Ct-values of higher than 25 in non-respiratory samples. However, vaccinated cases also showed high viral loads, reaching Ct-values below 10, especially in the upper airways and lungs. This was accompanied by high rates of pulmonal bacterial or mycotic superinfections and the occurrence of immunocompromising factors, such as malignancies, immunosuppressive drug intake, or decreased immunoglobulin levels. All these findings were particularly accentuated in partially vaccinated patients compared to fully vaccinated individuals. The virus dissemination observed in our case study may indicate that patients with an impaired immune system have a decreased ability to eliminate the virus. However, the potential role of antibody-dependent enhancement must also be ruled out in future studies. Fatal cases of COVID-19 in vaccinees were rare and often associated with severe comorbidities or other immunosuppressive conditions.

Prolonged SARS-CoV-2 RNA Shedding from Therapy Cat after Cluster Outbreak in Retirement Home.

We report a therapy cat in a nursing home in Germany infected with severe acute respiratory syndrome coronavirus 2 during a cluster outbreak in the home residents. Although we confirmed prolonged presence of virus RNA in the asymptomatic cat, genome sequencing showed no further role of the cat in human infections on site.

Economic evaluation of whole genome sequencing for pathogen identification and surveillance - results of case studies in Europe and the Americas 2016 to 2019.

BackgroundWhole genome sequencing (WGS) is increasingly used for pathogen identification and surveillance.AimWe evaluated costs and benefits of routine WGS through case studies at eight reference laboratories in Europe and the Americas which conduct pathogen surveillance for avian influenza (two laboratories), human influenza (one laboratory) and food-borne pathogens (five laboratories).MethodsThe evaluation focused on the institutional perspective, i.e. the 'investment case' for implementing WGS compared with conventional methods, based on costs and benefits during a defined reference period, mostly covering at least part of 2017. A break-even analysis estimated the number of cases of illness (for the example of Salmonella surveillance) that would need to be avoided through WGS in order to 'break even' on costs.ResultsOn a per-sample basis, WGS was between 1.2 and 4.3 times more expensive than routine conventional methods. However, WGS brought major benefits for pathogen identification and surveillance, substantially changing laboratory workflows, analytical processes and outbreaks detection and control. Between 0.2% and 1.1% (on average 0.7%) of reported salmonellosis cases would need to be prevented to break even with respect to the additional costs of WGS.ConclusionsEven at cost levels documented here, WGS provides a level of additional information that more than balances the additional costs if used effectively. The substantial cost differences for WGS between reference laboratories were due to economies of scale, degree of automation, sequencing technology used and institutional discounts for equipment and consumables, as well as the extent to which sequencers are used at full capacity.

Susceptibility of Raccoon Dogs for Experimental SARS-CoV-2 Infection.

Raccoon dogs might have been intermediate hosts for severe acute respiratory syndrome-associated coronavirus in 2002-2004. We demonstrated susceptibility of raccoon dogs to severe acute respiratory syndrome coronavirus 2 infection and transmission to in-contact animals. Infected animals had no signs of illness. Virus replication and tissue lesions occurred in the nasal conchae.

Untargeted metagenomics shows a reliable performance for synchronous detection of parasites.

Shotgun metagenomics with high-throughput sequencing (HTS) techniques is increasingly used for pathogen identification and characterization. While many studies apply targeted amplicon sequencing, here we used untargeted metagenomics to simultaneously identify protists and helminths in pre-diagnosed faecal and tissue samples. The approach starts from RNA and operates without an amplification step, therefore allowing the detection of all eukaryotes, including pathogens, since it circumvents the bias typically observed in amplicon-based HTS approaches. The generated metagenomics datasets were analysed using the RIEMS tool for initial taxonomic read assignment. Mapping analyses against ribosomal reference sequences were subsequently applied to extract 18S rRNA sequences abundantly present in the sequence datasets. The original diagnosis, which was based on microscopy and/or PCR, could be confirmed in nearly all cases using ribosomal RNA metagenomics. In addition to the pre-diagnosed taxa, we detected other intestinal eukaryotic parasites of uncertain pathogenicity (of the genera Dientamoeba, Entamoeba, Endolimax, Hymenolepis) that are often excluded from routine diagnostic protocols. The study clearly demonstrates the applicability of untargeted RNA metagenomics for the parallel detection of parasites.

Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated In Silico High-Throughput Sequencing Data Sets.

Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants' analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.

Filling gaps in the microsporidian tree: rDNA phylogeny of Chytridiopsis typographi (Microsporidia: Chytridiopsida).

Microsporidia are intracellular eukaryotic parasites of animals, characterized by unusual morphological and genetic features. They can be divided in three main groups, the classical microsporidians presenting all the features of the phylum and two putative primitive groups, the chytridiopsids and metchnikovellids. Microsporidia originated from microsporidia-like organisms belonging to a lineage of chytrid-like endoparasites basal or sister to the Fungi. Genetic and genomic data are available for all members, except chytridiopsids. Herein, we filled this gap by obtaining the rDNA sequence (SSU-ITS-partial LSU) of Chytridiopsis typographi (Chytridiopsida), a parasite of bark beetles. Our rDNA molecular phylogenies indicate that Chytridiopsis branches earlier than metchnikovellids, commonly thought ancestral, forming the more basal lineage of the Microsporidia. Furthermore, our structural analyses showed that only classical microsporidians present 16S-like SSU rRNA and 5.8S/LSU rRNA gene fusion, whereas the standard eukaryote rRNA gene structure, although slightly reduced, is still preserved in the primitive microsporidians, including 18S-like SSU rRNA with conserved core helices, and ITS2-like separating 5.8S from LSU. Overall, our results are consistent with the scenario of an evolution from microsporidia-like rozellids to microsporidians, however suggesting for metchnikovellids a derived position, probably related to marine transition and adaptation to hyperparasitism. The genetic and genomic data of additional members of Chytridiopsida and Rozellomycota will be of great value, not only to resolve phylogenetic relationships but also to improve our understanding of the evolution of these fascinating organisms.

New insights from molecular phylogenetics of amoebophagous fungi (Zoopagomycota, Zoopagales).

Amoebophagous fungi are represented in all fungal groups: Basidiomycota, Ascomycota, Zygomycota, and Chytridiomycota. The amoebophagous fungi, within the zygomycota (Zoopagales, Zoopagomycota), mainly affect naked amoebae as ectoparasites or endoparasites. It is rather difficult to isolate members of the Zoopagales, because of their parasitic lifestyle, and to bring them into culture. Consequently, gene sequences of this group are undersampled, and its species composition and phylogeny are relatively unknown. In the present study, we were able to isolate amoebophagous fungi together with their amoeba hosts from various habitats (moss, pond, bark, and soil). Altogether, four fungal strains belonging to the genera Acaulopage and Stylopage plus one unidentified isolate were detected. Sequences of the 18S rDNA and the complete ITS region and partial 28S (LSU) rDNA were generated. Subsequent phylogenetic analyses showed that all new isolates diverge at one branch together with two environmental clonal sequences within the Zoopagomycota. Here, we provide the first molecular characterization of the genus Stylopage. Stylopage is closely related to the genus Acaulopage. In addition, taxonomy and phylogeny of amoebophagous fungi and their ecological importance are reviewed based on new sequence data, which includes environmental clonal sequences.

Culturing Heterotrophic Protists from the Baltic Sea: Mostly the "Usual Suspects" but a Few Novelties as Well.

The study of cultured strains has a long tradition in protistological research and has greatly contributed to establishing the morphology, taxonomy, and ecology of many protist species. However, cultivation-independent techniques, based on 18S rRNA gene sequences, have demonstrated that natural protistan assemblages mainly consist of hitherto uncultured protist lineages. This mismatch impedes the linkage of environmental diversity data with the biological features of cultured strains. Thus, novel taxa need to be obtained in culture to close this knowledge gap. In this study, traditional cultivation techniques were applied to samples from coastal surface waters and from deep oxygen-depleted waters of the Baltic Sea. Based on 18S rRNA gene sequencing, 126 monoclonal cultures of heterotrophic protists were identified. The majority of the isolated strains were affiliated with already cultured and described taxa, mainly chrysophytes and bodonids. This was likely due to "culturing bias" but also to the eutrophic nature of the Baltic Sea. Nonetheless, ~ 12% of the isolates in our culture collection showed highly divergent 18S rRNA gene sequences compared to those of known organisms and thus may represent novel taxa, either at the species level or at the genus level. Moreover, we also obtained evidence that some of the isolated taxa are ecologically relevant, under certain conditions, in the Baltic Sea.

Molecular identification of bacterial endosymbionts of Sappinia strains.

The genus Sappinia comprises free-living amoebae occurring worldwide in a variety of habitats such as soils, plant matter and freshwater ponds, but also animal faeces, and includes at present three species, S. pedata, S. diploidea and S. platani. The genus is potentially pathogenic, as indicated by the identification of S. pedata in a case of human amoebic encephalitis. Electron microscopy studies on some strains already revealed intracellular bacteria in Sappinia. In the current study, we performed 16S ribosomal RNA gene (rDNA) analysis of these bacterial endosymbionts. We first inferred relationships among Sappinia strains on the basis of 18S rDNA, demonstrating that S. pedata emerged as sister to a larger clade including S. diploidea, S. platani and a few 'S. diploidea-like' strains. Thus, bacterial 16S rDNA was searched for in representative strains of each Sappinia species/subgroup. We found that Sappinia strains were associated to distinct species of Flavobacterium or Pedobacter (phylum Bacteroidetes). These appear to be distributed following the amoebal host subgroups, and are not directly related to other Bacteroidetes species known as interacting with free-living amoebae. While all the endosymbionts' close relatives are known to grow on agar, bacteriological media inoculated with amoebal extracts remained negative. Overall, results indicate that the recovered bacteria are likely specific obligate endosymbionts of Sappinia species. Further studies, including additional amoebal strains and deep morphological and molecular analyses, will be necessary to confirm this hypothesis.

Molecular identification of Nucleophaga terricolae sp. nov. (Rozellomycota), and new insights on the origin of the Microsporidia.

Microsporidia are widespread endoparasites of animals, including humans. They are characterized by highly modified morphological and genetic features that cause difficulties in elucidating their enigmatic origin and evolution. Recent advances, however, indicate that the Microsporidia have emerged from the Rozellomycota, forming together either the most basal lineage of the Fungi or its closer relative. The Rozellomycota comprise a huge diversity of uncultured environmental clones, with a very few known species endoparasitic of algae and water moulds, like the chytrid-like Rozella, and of free-living amoebae, like Nucleophaga and the microsporidia-like Paramicrosporidium. A possible ancestral microsporidium, Mitosporidium, has recently been described from the water flea Daphnia, since the phylogenomic reconstruction showed that it branches to the root of the microsporidian tree, while the genome analysis revealed a fungal-like nuclear genome and the persistence of a mitochondrial genome. Here we report the 18S rDNA molecular phylogeny of an additional microsporidium-like endoparasite of amoebae, which has a developmental cycle almost identical to that of Nucleophaga amoebae. Our results show that the endoparasite is closely related to N. amoebae, forming a distinct species, for which we propose the name Nucleophaga terricolae. Furthermore, the Nucleophaga lineage is recovered as sister to the Microsporidia while Mitosporidium turns out to be member of a well-supported group of environmental clones. These results raise the question about the actual ancestry of the Microsporidia within the Rozellomycota. A precise and robust phylogeny will require further comparative genomic studies of these various strains, and should also consider the primitive microsporidia, for which genetic data are still lacking, because all these organisms are essentially morphologically similar.

Pathogen dynamics and discovery of novel viruses and enzymes by deep nucleic acid sequencing of wastewater.

Wastewater contains an extensive reservoir of genetic information, yet largely unexplored. Here, we analyzed by high-throughput sequencing total nucleic acids extracted from wastewater samples collected during a 17 month-period in Berlin, Germany. By integrating global wastewater datasets and applying a novel computational approach to accurately identify viral strains within sewage RNA-sequencing data, we demonstrated the emergence and global dissemination of a specific astrovirus strain. Astrovirus abundance and sequence variation mirrored temporal and spatial patterns of infection, potentially serving as footprints of specific timeframes and geographical locations. Additionally, we revealed more than 100,000 sequence contigs likely originating from novel viral species, exhibiting distinct profiles in total RNA and DNA datasets and including undescribed bunyaviruses and parvoviruses. Finally, we identified thousands of new CRISPR-associated protein sequences, including Transposase B (TnpB), a class of compact, RNA-guided DNA editing enzymes. Collectively, our findings underscore the potential of high-throughput sequencing of total nucleic acids derived from wastewater for a broad range of applications.

Detection of novel orthoparamyxoviruses, orthonairoviruses and an orthohepevirus in European white-toothed shrews.

While the viromes and immune systems of bats and rodents have been extensively studied, comprehensive data are lacking for insectivores (order Eulipotyphla) despite their wide geographic distribution. Anthropogenic land use and outdoor recreational activities, as well as changes in the range of shrews, may lead to an expansion of the human-shrew interface with the risk of spillover infections, as reported for Borna disease virus 1. We investigated the virome of 45 individuals of 4 white-toothed shrew species present in Europe, using metagenomic RNA sequencing of tissue and intestine pools. Moderate to high abundances of sequences related to the families Paramyxoviridae, Nairoviridae, Hepeviridae and Bornaviridae were detected. Whole genomes were determined for novel orthoparamyxoviruses (n=3), orthonairoviruses (n=2) and an orthohepevirus. The novel paramyxovirus, tentatively named Hasua virus, was phylogenetically related to the zoonotic Langya virus and Mòjiang virus. The novel orthonairoviruses, along with the potentially zoonotic Erve virus, fall within the shrew-borne Thiafora virus genogroup. The highest viral RNA loads of orthoparamyxoviruses were detected in the kidneys, in well-perfused organs for orthonairoviruses and in the liver and intestine for orthohepevirus, indicating potential transmission routes. Notably, several shrews were found to be coinfected with viruses from different families. Our study highlights the virus diversity present in shrews, not only in biodiversity-rich regions but also in areas influenced by human activity. This study warrants further research to characterize and assess the clinical implications and risk of these viruses and the importance of shrews as reservoirs in European ecosystems.

A safe, effective and adaptable live-attenuated SARS-CoV-2 vaccine to reduce disease and transmission using one-to-stop genome modifications.

Approved vaccines are effective against severe COVID-19, but broader immunity is needed against new variants and transmission. Therefore, we developed genome-modified live-attenuated vaccines (LAV) by recoding the SARS-CoV-2 genome, including 'one-to-stop' (OTS) codons, disabling Nsp1 translational repression and removing ORF6, 7ab and 8 to boost host immune responses, as well as the spike polybasic cleavage site to optimize the safety profile. The resulting OTS-modified SARS-CoV-2 LAVs, designated as OTS-206 and OTS-228, are genetically stable and can be intranasally administered, while being adjustable and sustainable regarding the level of attenuation. OTS-228 exhibits an optimal safety profile in preclinical animal models, with no side effects or detectable transmission. A single-dose vaccination induces a sterilizing immunity in vivo against homologous WT SARS-CoV-2 challenge infection and a broad protection against Omicron BA.2, BA.5 and XBB.1.5, with reduced transmission. Finally, this promising LAV approach could be applicable to other emerging viruses.

Impact of protist grazing on a key bacterial group for biogeochemical cycling in Baltic Sea pelagic oxic/anoxic interfaces.

Barrier zones between oxic and anoxic water masses (redoxclines) host highly active prokaryotic communities with important roles in biogeochemical cycling. In Baltic Sea pelagic redoxclines, Epsilonproteobacteria of the genus Sulfurimonas (subgroup GD17) have been shown to dominate chemoautotrophic denitrification. However, little is known on the loss processes affecting this prokaryotic group. In the present study, the protist grazing impact on the Sulfurimonas subgroup GD17 was determined for suboxic and oxygen/hydrogen sulphide interface depths of Baltic Sea redoxclines, using predator exclusion assays and bacterial amendment with the cultured representative 'Sulfurimonas gotlandica' strain GD1. Additionally, the principal bacterivores were identified by RNA-Stable Isotope Probing (RNA-SIP). The natural Sulfurimonas subgroup GD17 population grew strongly under oxygen/hydrogen sulphide interface conditions (doubling time: 1-1.5 days), but protist grazing could consume the complete new cell production per day. In suboxic samples, little or no growth of Sulfurimonas subgroup GD17 was observed. RNA-SIP identified five active grazers, belonging to typical redoxcline ciliates (Oligohymenophorea, Prostomatea) and globally widespread marine flagellate groups (MAST-4, Chrysophyta, Cercozoa). Overall, we demonstrate for the first time that protist grazing can control the growth, and potentially the vertical distribution, of a chemolithoautotrophic key-player of oxic/anoxic interfaces.