Accumulation of mutations in antibody and CD8 T cell epitopes in a B cell depleted lymphoma patient with chronic SARS-CoV-2 infection.

Antibodies against the spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can drive adaptive evolution in immunocompromised patients with chronic infection. Here we longitudinally analyze SARS-CoV-2 sequences in a B cell-depleted, lymphoma patient with chronic, ultimately fatal infection, and identify three mutations in the spike protein that dampen convalescent plasma-mediated neutralization of SARS-CoV-2. Additionally, four mutations emerge in non-spike regions encoding three CD8 T cell epitopes, including one nucleoprotein epitope affected by two mutations. Recognition of each mutant peptide by CD8 T cells from convalescent donors is reduced compared to its ancestral peptide, with additive effects resulting from double mutations. Querying public SARS-CoV-2 sequences shows that these mutations have independently emerged as homoplasies in circulating lineages. Our data thus suggest that potential impacts of CD8 T cells on SARS-CoV-2 mutations, at least in those with humoral immunodeficiency, warrant further investigation to inform on vaccine design.

Genetic diversity and spatial distribution of Burkholderia mallei by core genome-based multilocus sequence typing analysis.

Burkholderia mallei is the etiological agent of glanders, a highly contagious and often fatal disease in equids. Due to the high genetic clonality of B. mallei, high-resolution typing assays are necessary to differentiate between individual strains. Here we report on the development and validation of a robust and reproducible core genome-based Multi Locus Sequence Typing Assay (cgMLST) for B. mallei, which is based on 3328 gene targets and enables high-resolution typing at the strain level. The assay was validated using a set of 120 B. mallei genomes from public databases and 23 newly sequenced outbreak strains from in-house strain collections. In this cgMLST analysis, strains from different geographic regions were clearly distinguished by at least 70 allele differences, allowing spatial clustering while closely related and epidemiologically related strains were separated by only zero to three alleles. Neither the different sequencing technologies nor the assembly strategies had an influence on the cgMLST results. The developed cgMLST is highly robust, reproducible and can be used for outbreak investigations, source tracking and molecular characterization of new B. mallei isolates.

Expanding the Burkholderia pseudomallei Complex with the Addition of Two Novel Species: Burkholderia mayonis sp. nov. and Burkholderia savannae sp. nov.

Distinct Burkholderia strains were isolated from soil samples collected in tropical northern Australia (Northern Territory and the Torres Strait Islands, Queensland). Phylogenetic analysis of 16S rRNA and whole genome sequences revealed these strains were distinct from previously described Burkholderia species and assigned them to two novel clades within the B. pseudomallei complex (Bpc). Because average nucleotide identity and digital DNA-DNA hybridization calculations are consistent with these clades representing distinct species, we propose the names Burkholderia mayonis sp. nov. and Burkholderia savannae sp. nov. Strains assigned to B. mayonis sp. nov. include type strain BDU6T (=TSD-80; LMG 29941; ASM152374v2) and BDU8. Strains assigned to B. savannae sp. nov. include type strain MSMB266T (=TSD-82; LMG 29940; ASM152444v2), MSMB852, BDU18, and BDU19. Comparative genomics revealed unique coding regions for both putative species, including clusters of orthologous genes associated with phage. Type strains of both B. mayonis sp. nov. and B. savannae sp. nov. yielded biochemical profiles distinct from each other and from other species in the Bpc, and profiles also varied among strains within B. mayonis sp. nov. and B. savannae sp. nov. Matrix-assisted laser desorption ionization time-of-flight (MLST) analysis revealed a B. savannae sp. nov. cluster separate from other species, whereas B. mayonis sp. nov. strains did not form a distinct cluster. Neither B. mayonis sp. nov. nor B. savannae sp. nov. caused mortality in mice when delivered via the subcutaneous route. The addition of B. mayonis sp. nov. and B. savannae sp. nov. results in a total of eight species currently within the Bpc. IMPORTANCEBurkholderia species can be important sources of novel natural products, and new species are of interest to diverse scientific disciplines. Although many Burkholderia species are saprophytic, Burkholderia pseudomallei is the causative agent of the disease melioidosis. Understanding the genomics and virulence of the closest relatives to B. pseudomallei, i.e., the other species within the B. pseudomallei complex (Bpc), is important for identifying robust diagnostic targets specific to B. pseudomallei and for understanding the evolution of virulence in B. pseudomallei. Two proposed novel species, B. mayonis sp. nov. and B. savannae sp. nov., were isolated from soil samples collected from multiple locations in northern Australia. The two proposed species belong to the Bpc but are phylogenetically distinct from all other members of this complex. The addition of B. mayonis sp. nov. and B. savannae sp. nov. results in a total of eight species within this significant complex of bacteria that are available for future studies.

Genetic Characterization of Brucella spp.: Whole Genome Sequencing-Based Approach for the Determination of Multiple Locus Variable Number Tandem Repeat Profiles.

Brucellosis is an important zoonosis that is emerging in some regions of the world, gaining increased relevance with the inclusion of the causing agent Brucella spp. in the class B bioterrorism group. Until now, multi-locus VNTR Analysis (MLVA) based on 16 loci has been considered as the gold standard for Brucella typing. However, this methodology is laborious, and, with the rampant release of Brucella genomes, the transition from the traditional MLVA to whole genome sequencing (WGS)-based typing is on course. Nevertheless, in order to avoid a disruptive transition with the loss of massive genetic data obtained throughout the last decade and considering that the transition timings will vary considerably among different countries, it is important to determine WGS-based MLVA alleles of the nowadays sequenced genomes. On this regard, we aimed to evaluate the performance of a Python script that had been previously developed for the rapid in silico extraction of the MLVA alleles, by comparing it to the PCR-based MLVA procedure over 83 strains from different Brucella species. The WGS-based MLVA approach detected 95.3% of all possible 1,328 hits (83 strains×16 loci) and showed an agreement rate with the PCR-based MLVA procedure of 96.4% for MLVA-16. According to our dataset, we suggest the use of a minimal depth of coverage of ~50x and a maximum number of ~200 contigs as guiding "boundaries" for the future application of the script. In conclusion, the evaluated script seems to be a very useful and robust tool for the in silico determination of MLVA profiles of Brucella strains, allowing retrospective and prospective molecular epidemiological studies, which are important for maintaining an active epidemiological surveillance of brucellosis.

COVID-19 in Patients Receiving CD20-depleting Immunochemotherapy for B-cell Lymphoma.

The clinical and immunological impact of B-cell depletion in the context of coronavirus disease 2019 (COVID-19) is unclear. We conducted a prospectively planned analysis of COVID-19 in patients who received B-cell depleting anti-CD20 antibodies and chemotherapy for B-cell lymphomas. The control cohort consisted of age- and sex-matched patients without lymphoma who were hospitalized because of COVID-19. We performed detailed clinical analyses, in-depth cellular and molecular immune profiling, and comprehensive virological studies in 12 patients with available biospecimens. B-cell depleted lymphoma patients had more severe and protracted clinical course (median hospitalization 88 versus 17 d). All patients actively receiving immunochemotherapy (n = 5) required ICU support including long-term mechanical ventilation. Neutrophil recovery following granulocyte colony stimulating factor stimulation coincided with hyperinflammation and clinical deterioration in 4 of the 5 patients. Immune cell profiling and gene expression analysis of peripheral blood mononuclear cells revealed early activation of monocytes/macrophages, neutrophils, and the complement system in B-cell depleted lymphoma patients, with subsequent exacerbation of the inflammatory response and dysfunctional interferon signaling at the time of clinical deterioration of COVID-19. Longitudinal immune cell profiling and functional in vitro assays showed SARS-CoV-2-specific CD8+ and CD4+ T-effector cell responses. Finally, we observed long-term detection of SARS-CoV-2 in respiratory specimens (median 84 versus 12 d) and an inability to mount lasting SARS-CoV-2 antibody responses in B-cell depleted lymphoma patients. In summary, we identified clinically relevant particularities of COVID-19 in lymphoma patients receiving B-cell depleting immunochemotherapies.

Liver transplantation in a patient after COVID-19 - Rapid loss of antibodies and prolonged viral RNA shedding.

To date, little is known about the duration and effectiveness of immunity as well as possible adverse late effects after an infection with SARS-CoV-2. Thus it is unclear, when and if liver transplantation can be safely offered to patients who suffered from COVID-19. Here, we report on a successful liver transplantation shortly after convalescence from COVID-19 with subsequent partial seroreversion as well as recurrence and prolonged shedding of viral RNA.

A headache with surprising outcome: first case of brucellosis caused by Brucella suis biovar 1 in Germany.

In July 2018, brucellosis was diagnosed in a German patient without a travel history to regions endemic for Brucella. Microbiological analysis, including whole-genome sequencing, revealed Brucella suis biovar 1 as the etiologic agent. Core-genome-based multilocus sequence-typing analysis placed the isolate in close proximity to strains originating from Argentina. Notably, despite a strong IgM response, the patient did not develop Brucella-specific IgG antibodies during infection. Here, we describe the clinical course of infection, the extensive epidemiological investigations, and discuss possible routes of transmission.

Use of Next Generation Sequencing to study two cowpox virus outbreaks.

BACKGROUND: Between 2008 and 2011 about 40 cases of human cowpox were reported from Germany and France. Infections had been acquired via close contact to infected, young pet rats. An identical and unique sequence of the hemagglutinin gene was found in various cowpox virus (CPXV) isolates pointing to a common source of infection. In a second CPXV outbreak in cats in a small animal clinic in Germany in 2015, four out of five hospitalized cats showed identical hemagglutinin sequences and thus, a hospital-acquired transmission had been assumed. Next-Generation Sequencing was performed in order to re-investigate the outbreaks, as epidemiological data could not confirm all cases. METHODS: Homogenates of lesion material from rats, cats and humans were cultivated in cell culture. The genomes of four virus isolates, nine CPXVs from our strain collections and from DNA of three paraffin-embedded lesion materials were determined by Next Generation Sequencing (NGS). For phylogenetic analyses a MAFFT-alignment was generated. A distance matrix based on concatenated SNPs was calculated and plotted as dendrogram using Unweighted Pair Group Method with Arithmetic mean (UPGMA) for visualization. RESULTS: Aligning of about 200.000 nucleotides of 8 virus isolates associated with the pet rat outbreak revealed complete identity of six genomes, the remainder two genomes differed in as little as 3 SNPs. When comparing this dataset with four already published CPXV genomes also associated with the pet rat outbreak, again a maximum difference of 3 SNPs was found. The outbreak which lasted from 2008 till 2011 was indeed caused by a single strain which has maintained an extremely high level of clonality over 4 years. Aligning genomic sequences from four cases of feline cowpox revealed 3 identical sequences and one sequence which differed in 65 nucleotides. Although identical hemagglutinin sequences had been obtained from four hospitalized cats, genomic sequencing proved that a hospital-acquired transmission had occurred in only three cats. CONCLUSION: Analyzing the rather short sequence of the hemagglutinin gene is not sufficient to conduct molecular trace back analyses. Instead, whole genome sequencing is the method of choice which can even be applied to paraffin-embedded specimens.

Newly emerging ulceroglandular tularaemia in Western Austria.

Tularaemia is a rare zoonotic disease caused by Francisella tularensis in humans. In Europe infections of humans and animals are mainly caused by F. tularensis subspecies holarctica. We report the first three documented cases of tularaemia in humans in Western Austria.

Five cases of vector-borne Francisella tularensis holarctica infections in south-western Germany and genetic diversity.

Tularemia is a rare zoonotic disease in Germany. Francisella tularensis has been isolated previously from ticks in southern Germany underscoring the importance of ticks (Ixodes ricinus) in tularemia transmission, but there have been only few reports from this region with single cases or small case series of tick-borne transmissions of tularemia. We report five cases of non-game animal associated tularemia diagnosed from 2010 to 2016 in southwestern Germany - Baden-Wuerttemberg. Our case series and molecular typing (MLVA) results add published clinical experience to this underdiagnosed disease and consolidate previous findings regarding tick-borne transmission of tularemia and phylogenetic diversity in Germany.

Whole genome sequencing of Brucella melitensis isolated from 57 patients in Germany reveals high diversity in strains from Middle East.

Brucellosis, a worldwide common bacterial zoonotic disease, has become quite rare in Northern and Western Europe. However, since 2014 a significant increase of imported infections caused by Brucella (B.) melitensis has been noticed in Germany. Patients predominantly originated from Middle East including Turkey and Syria. These circumstances afforded an opportunity to gain insights into the population structure of Brucella strains. Brucella-isolates from 57 patients were recovered between January 2014 and June 2016 with culture confirmed brucellosis by the National Consultant Laboratory for Brucella. Their whole genome sequences were generated using the Illumina MiSeq platform. A whole genome-based SNP typing assay was developed in order to resolve geographically attributed genetic clusters. Results were compared to MLVA typing results, the current gold-standard of Brucella typing. In addition, sequences were examined for possible genetic variation within target regions of molecular diagnostic assays. Phylogenetic analyses revealed spatial clustering and distinguished strains from different patients in either case, whereas multiple isolates from a single patient or technical replicates showed identical SNP and MLVA profiles. By including WGS data from the NCBI database, five major genotypes were identified. Notably, strains originating from Turkey showed a high diversity and grouped into seven subclusters of genotype II. MLVA analysis congruently clustered all isolates and predominantly matched the East Mediterranean genetic clade. This study confirms whole-genome based SNP-analysis as a powerful tool for accurate typing of B. melitensis. Furthermore it allows special allocation and therefore provides useful information on the geographic origin for trace-back analysis. However, the lack of reliable metadata in public databases often prevents a resolution below geographic regions or country levels and corresponding precise trace-back analysis. Once this obstacle is resolved, WGS-derived bacterial typing adds an important method to complement epidemiological surveys during outbreak investigations. This is the first report of a detailed genetic investigation of an extensive collection of B. melitensis strains isolated from human cases in Germany.

Burkholderia humptydooensis sp. nov., a New Species Related to Burkholderia thailandensis and the Fifth Member of the Burkholderia pseudomallei Complex.

During routine screening for Burkholderia pseudomallei from water wells in northern Australia in areas where it is endemic, Gram-negative bacteria (strains MSMB43T, MSMB121, and MSMB122) with a similar morphology and biochemical pattern to B. pseudomallei and B. thailandensis were coisolated with B. pseudomallei on Ashdown's selective agar. To determine the exact taxonomic position of these strains and to distinguish them from B. pseudomallei and B. thailandensis, they were subjected to a series of phenotypic and molecular analyses. Biochemical and fatty acid methyl ester analysis was unable to distinguish B. humptydooensis sp. nov. from closely related species. With matrix-assisted laser desorption ionization-time of flight analysis, all isolates grouped together in a cluster separate from other Burkholderia spp. 16S rRNA and recA sequence analyses demonstrated phylogenetic placement for B. humptydooensis sp. nov. in a novel clade within the B. pseudomallei group. Multilocus sequence typing (MLST) analysis of the three isolates in comparison with MLST data from 3,340 B. pseudomallei strains and related taxa revealed a new sequence type (ST318). Genome-to-genome distance calculations and the average nucleotide identity of all isolates to both B. thailandensis and B. pseudomallei, based on whole-genome sequences, also confirmed B. humptydooensis sp. nov. as a novel Burkholderia species within the B. pseudomallei complex. Molecular analyses clearly demonstrated that strains MSMB43T, MSMB121, and MSMB122 belong to a novel Burkholderia species for which the name Burkholderia humptydooensis sp. nov. is proposed, with the type strain MSMB43T (American Type Culture Collection BAA-2767; Belgian Co-ordinated Collections of Microorganisms LMG 29471; DDBJ accession numbers CP013380 to CP013382).IMPORTANCEBurkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The genus Burkholderia consists of a diverse group of species, with the closest relatives of B. pseudomallei referred to as the B. pseudomallei complex. A proposed novel species, B. humptydooensis sp. nov., was isolated from a bore water sample from the Northern Territory in Australia. B. humptydooensis sp. nov. is phylogenetically distinct from B. pseudomallei and other members of the B. pseudomallei complex, making it the fifth member of this important group of bacteria.

Extensive Recruitment of Plasma Blasts to the Cerebrospinal Fluid in Toscana Virus Encephalitis.

An unexpectedly extensive recruitment of B cells and plasma blasts to the cerebrospinal fluid (CSF) in a patient with Toscana virus (TOSV) encephalitis is described. Acute infection by TOSV was demonstrated by serological methods and by detection of TOSV-specific nucleic acid in the CSF by real-time polymerase chain reaction and sequencing.

Microevolution of Anthrax from a Young Ancestor (M.A.Y.A.) Suggests a Soil-Borne Life Cycle of Bacillus anthracis.

During an anthrax outbreak at the Pollino National Park (Basilicata, Italy) in 2004, diseased cattle were buried and from these anthrax-foci Bacillus anthracis endospores still diffuse to the surface resulting in local accumulations. Recent data suggest that B. anthracis multiplies in soil outside the animal-host body. This notion is supported by the frequent isolation of B. anthracis from soil lacking one or both virulence plasmids. Such strains represent an evolutionary dead end, as they are likely no longer able to successfully infect new hosts. This loss of virulence plasmids is explained most simply by postulating a soil-borne life cycle of the pathogen. To test this hypothesis we investigated possible microevolution at two natural anthrax foci from the 2004 outbreak. If valid, then genotypes of strains isolated from near the surface at these foci should be on a different evolutionary trajectory from those below residing in deeper-laying horizons close to the carcass. Thus, the genetic diversity of B. anthracis isolates was compared conducting Progressive Hierarchical Resolving Assays using Nucleic Acids (PHRANA) and next generation Whole Genome Sequencing (WGS). PHRANA was not discriminatory enough to resolve the fine genetic relationships between the isolates. Conversely, WGS of nine isolates from near-surface and nine from near-carcass revealed five isolate specific SNPs, four of which were found only in different near-surface isolates. In support of our hypothesis, one surface-isolate lacked plasmid pXO1 and also harbored one of the unique SNPs. Taken together, our results suggest a limited soil-borne life cycle of B. anthracis.

Genome Sequence of Bacillus anthracis Isolated from an Anthrax Burial Site in Pollino National Park, Basilicata Region (Southern Italy).

A Bacillus anthracis strain was isolated from a burial-site in Pollino National Park where a bovine died of anthrax and was buried in 2004. We report the first genome sequence of B. anthracis isolated in the Basilicata region (southern Italy), which is the highest risk area of anthrax infection in Italy.

Complete Genome Sequence of the Embu Virus Strain SPAn880.

We report the complete genome sequence of the Embu virus. The genome consists of 185,139 bp and is nearly identical to that of the Cotia virus. This is the first report on the Embu virus genome sequence, which has been considered an unclassified poxvirus until now.

Identification of factors influencing the Puumala virus seroprevalence within its reservoir in aMontane Forest Environment.

Puumala virus (PUUV) is a major cause of mild to moderate haemorrhagic fever with renal syndrome and is transmitted by the bank vole (Myodes glareolus). There has been a high cumulative incidence of recorded human cases in South-eastern Germany since 2004 when the region was first recognized as being endemic for PUUV. As the area is well known for outdoor recreation and the Bavarian Forest National Park (BFNP) is located in the region, the increasing numbers of recorded cases are of concern. To understand the population and environmental effects on the seroprevalence of PUUV in bank voles we trapped small mammals at 23 sites along an elevation gradient from 317 to 1420m above sea level. Generalized linear mixed effects models(GLMEM) were used to explore associations between the seroprevalence of PUUV in bank voles and climate and biotic factors. We found that the seroprevalence of PUUV was low (6%-7%) in 2008 and 2009, and reached 29% in 2010. PUUV seroprevalence was positively associated with the local species diversity and deadwood layer, and negatively associated with mean annual temperature, mean annual solar radiation, and herb layer. Based on these findings, an illustrative risk map for PUUV seroprevalence prediction in bank voles was created for an area of the national park. The map will help when planning infrastructure in the national park (e.g., huts, shelters, and trails).

Draft Genome Sequence of Strain BF-4, a Lysinibacillus-Like Bacillus Isolated during an Anthrax Outbreak in Bavaria.

We report the draft genome sequence of Lysinibacillus sp. strain BF-4. Strain BF-4 has a notably small genome for a free-living bacillus, with a size of 2.63 Mbp. In agreement with phenotypic observations, the genome lacks genes essential for endospore formation.