Preliminary results on an autochthonous dengue outbreak in Lombardy Region, Italy, August 2023.

In August 2023, six locally acquired dengue virus 1 infections were detected in Lodi province, Lombardy Region, in northern Italy, where the vector Aedes albopictus is present. Four cases were hospitalised, none died. The viruses clustered with Peruvian and Brazilian strains collected between 2021 and 2023. This preliminary report highlights the importance of continued integrated surveillance of imported vector-borne virus infections and the potential for tropical disease outbreaks in highly populated regions of northern Italy where competent vectors are present.

External Quality Assessment of SARS-CoV-2 Sequencing: an ESGMD-SSM Pilot Trial across 15 European Laboratories.

This first pilot trial on external quality assessment (EQA) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) whole-genome sequencing, initiated by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for Genomic and Molecular Diagnostics (ESGMD) and the Swiss Society for Microbiology (SSM), aims to build a framework between laboratories in order to improve pathogen surveillance sequencing. Ten samples with various viral loads were sent out to 15 clinical laboratories that had free choice of sequencing methods and bioinformatic analyses. The key aspects on which the individual centers were compared were the identification of (i) single nucleotide polymorphisms (SNPs) and indels, (ii) Pango lineages, and (iii) clusters between samples. The participating laboratories used a wide array of methods and analysis pipelines. Most were able to generate whole genomes for all samples. Genomes were sequenced to various depths (up to a 100-fold difference across centers). There was a very good consensus regarding the majority of reporting criteria, but there were a few discrepancies in lineage and cluster assignments. Additionally, there were inconsistencies in variant calling. The main reasons for discrepancies were missing data, bioinformatic choices, and interpretation of data. The pilot EQA was overall a success. It was able to show the high quality of participating laboratories and provide valuable feedback in cases where problems occurred, thereby improving the sequencing setup of laboratories. A larger follow-up EQA should, however, improve on defining the variables and format of the report. Additionally, contamination and/or minority variants should be a further aspect of assessment.

Performance and Application of 16S rRNA Gene Cycle Sequencing for Routine Identification of Bacteria in the Clinical Microbiology Laboratory.

This review provides a state-of-the-art description of the performance of Sanger cycle sequencing of the 16S rRNA gene for routine identification of bacteria in the clinical microbiology laboratory. A detailed description of the technology and current methodology is outlined with a major focus on proper data analyses and interpretation of sequences. The remainder of the article is focused on a comprehensive evaluation of the application of this method for identification of bacterial pathogens based on analyses of 16S multialignment sequences. In particular, the existing limitations of similarity within 16S for genus- and species-level differentiation of clinically relevant pathogens and the lack of sequence data currently available in public databases is highlighted. A multiyear experience is described of a large regional clinical microbiology service with direct 16S broad-range PCR followed by cycle sequencing for direct detection of pathogens in appropriate clinical samples. The ability of proteomics (matrix-assisted desorption ionization-time of flight) versus 16S sequencing for bacterial identification and genotyping is compared. Finally, the potential for whole-genome analysis by next-generation sequencing (NGS) to replace 16S sequencing for routine diagnostic use is presented for several applications, including the barriers that must be overcome to fully implement newer genomic methods in clinical microbiology. A future challenge for large clinical, reference, and research laboratories, as well as for industry, will be the translation of vast amounts of accrued NGS microbial data into convenient algorithm testing schemes for various applications (i.e., microbial identification, genotyping, and metagenomics and microbiome analyses) so that clinically relevant information can be reported to physicians in a format that is understood and actionable. These challenges will not be faced by clinical microbiologists alone but by every scientist involved in a domain where natural diversity of genes and gene sequences plays a critical role in disease, health, pathogenicity, epidemiology, and other aspects of life-forms. Overcoming these challenges will require global multidisciplinary efforts across fields that do not normally interact with the clinical arena to make vast amounts of sequencing data clinically interpretable and actionable at the bedside.

Characterization update of HIV-1 M subtypes diversity and proposal for subtypes A and D sub-subtypes reclassification.

BACKGROUND: The large and constantly evolving HIV-1 pandemic has led to an increasingly complex diversity. Because of some taxonomic difficulties among the most diverse HIV-1 subtypes, and taking advantage of the large amount of sequence data generated in the recent years, we investigated novel lineage patterns among the main HIV-1 subtypes. RESULTS: All HIV full-length genomes available in public databases were analysed (n = 2017). Maximum likelihood phylogenies and pairwise genetic distance were obtained. Clustering patterns and mean distributions of genetic distances were compared within and across the current groups, subtypes and sub-subtypes of HIV-1 to detect and analyse any divergent lineages within previously defined HIV lineages. The level of genetic similarity observed between most HIV clades was deeply consistent with the current classification. However, both subtypes A and D showed evidence of further intra-subtype diversification not fully described by the nomenclature system at the time and could be divided into several distinct sub-subtypes. CONCLUSIONS: With this work, we propose an updated nomenclature of sub-types A and D better reflecting their current genetic diversity and evolutionary patterns. Allowing a more accurate nomenclature and classification system is a necessary step for easier subtyping of HIV strains and a better detection or follow-up of viral epidemiology shifts.

New and continuing developments at PROSITE.

PROSITE (http://prosite.expasy.org/) consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule a collection of rules, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE signatures, together with ProRule, are used for the annotation of domains and features of UniProtKB/Swiss-Prot entries. Here, we describe recent developments that allow users to perform whole-proteome annotation as well as a number of filtering options that can be combined to perform powerful targeted searches for biological discovery. The latest version of PROSITE (release 20.85, of 30 August 2012) contains 1308 patterns, 1039 profiles and 1041 ProRules.

pfsearchV3: a code acceleration and heuristic to search PROSITE profiles.

SUMMARY: The PROSITE resource provides a rich and well annotated source of signatures in the form of generalized profiles that allow protein domain detection and functional annotation. One of the major limiting factors in the application of PROSITE in genome and metagenome annotation pipelines is the time required to search protein sequence databases for putative matches. We describe an improved and optimized implementation of the PROSITE search tool pfsearch that, combined with a newly developed heuristic, addresses this limitation. On a modern x86_64 hyper-threaded quad-core desktop computer, the new pfsearchV3 is two orders of magnitude faster than the original algorithm. AVAILABILITY AND IMPLEMENTATION: Source code and binaries of pfsearchV3 are freely available for download at http://web.expasy.org/pftools/#pfsearchV3, implemented in C and supported on Linux. PROSITE generalized profiles including the heuristic cut-off scores are available at the same address.

The genome of the fire ant Solenopsis invicta.

Ants have evolved very complex societies and are key ecosystem members. Some ants, such as the fire ant Solenopsis invicta, are also major pests. Here, we present a draft genome of S. invicta, assembled from Roche 454 and Illumina sequencing reads obtained from a focal haploid male and his brothers. We used comparative genomic methods to obtain insight into the unique features of the S. invicta genome. For example, we found that this genome harbors four adjacent copies of vitellogenin. A phylogenetic analysis revealed that an ancestral vitellogenin gene first underwent a duplication that was followed by possibly independent duplications of each of the daughter vitellogenins. The vitellogenin genes have undergone subfunctionalization with queen- and worker-specific expression, possibly reflecting differential selection acting on the queen and worker castes. Additionally, we identified more than 400 putative olfactory receptors of which at least 297 are intact. This represents the largest repertoire reported so far in insects. S. invicta also harbors an expansion of a specific family of lipid-processing genes, two putative orthologs to the transformer/feminizer sex differentiation gene, a functional DNA methylation system, and a single putative telomerase ortholog. EST data indicate that this S. invicta telomerase ortholog has at least four spliceforms that differ in their use of two sets of mutually exclusive exons. Some of these and other unique aspects of the fire ant genome are likely linked to the complex social behavior of this species.

How much should we sequence? An analysis of the Swiss SARS-CoV-2 surveillance effort.

During the SARS-CoV-2 pandemic, many countries directed substantial resources toward genomic surveillance to detect and track viral variants. There is a debate over how much sequencing effort is necessary in national surveillance programs for SARS-CoV-2 and future pandemic threats. We aimed to investigate the effect of reduced sequencing on surveillance outcomes in a large genomic data set from Switzerland, comprising more than 143k sequences. We employed a uniform downsampling strategy using 100 iterations each to investigate the effects of fewer available sequences on the surveillance outcomes: (i) first detection of variants of concern (VOCs), (ii) speed of introduction of VOCs, (iii) diversity of lineages, (iv) first cluster detection of VOCs, (v) density of active clusters, and (vi) geographic spread of clusters. The impact of downsampling on VOC detection is disparate for the three VOC lineages, but many outcomes including introduction and cluster detection could be recapitulated even with only 35% of the original sequencing effort. The effect on the observed speed of introduction and first detection of clusters was more sensitive to reduced sequencing effort for some VOCs, in particular Omicron and Delta, respectively. A genomic surveillance program needs a balance between societal benefits and costs. While the overall national dynamics of the pandemic could be recapitulated by a reduced sequencing effort, the effect is strongly lineage-dependent-something that is unknown at the time of sequencing-and comes at the cost of accuracy, in particular for tracking the emergence of potential VOCs.IMPORTANCESwitzerland had one of the most comprehensive genomic surveillance systems during the COVID-19 pandemic. Such programs need to strike a balance between societal benefits and program costs. Our study aims to answer the question: How would surveillance outcomes have changed had we sequenced less? We find that some outcomes but also certain viral lineages are more affected than others by sequencing less. However, sequencing to around a third of the original effort still captured many important outcomes for the variants of concern such as their first detection but affected more strongly other measures like the detection of first transmission clusters for some lineages. Our work highlights the importance of setting predefined targets for a national genomic surveillance program based on which sequencing effort should be determined. Additionally, the use of a centralized surveillance platform facilitates aggregating data on a national level for rapid public health responses as well as post-analyses.

Identification of an OPR protein involved in the translation initiation of the PsaB subunit of photosystem I.

Genetic analysis of mutants deficient in the biosynthesis of the photosystem I complex has revealed several nucleus-encoded factors that act at different post-transcriptional steps of chloroplast gene expression. Here we have identified and characterized the gene affected in the tab 1-F15 mutant, which is specifically deficient in the translation of the photosystem I reaction center protein PsaB as the result of a single nucleotide deletion. This gene encodes Tab 1, a 1287 amino acid protein that contains 10 tandem 38-40 amino acid degenerate repeats of the PPPEW/OPR (octatricopeptide repeat) family, first described for the chloroplast translation factor Tbc2. These repeats are involved in the binding of Tab 1 to the 5'-untranslated region of the psaB mRNA based on gel mobility shift assays. Tab 1 is part of a large family of proteins in Chlamydomonas that are also found in several bacteria and protozoans, but are rare in land plants.

SAS-6 defines a protein family required for centrosome duplication in C. elegans and in human cells.

The mechanisms that ensure centrosome duplication are poorly understood. In Caenorhabditis elegans, ZYG-1, SAS-4, SAS-5 and SPD-2 are required for centriole formation. However, it is unclear whether these proteins have functional homologues in other organisms. Here, we identify SAS-6 as a component that is required for daughter centriole formation in C. elegans. SAS-6 is a coiled-coil protein that is recruited to centrioles at the onset of the centrosome duplication cycle. Our analysis indicates that SAS-6 and SAS-5 associate and that this interaction, as well as ZYG-1 function, is required for SAS-6 centriolar recruitment. SAS-6 is the founding member of an evolutionarily conserved protein family that contains the novel PISA motif. We investigated the function of the human homologue of SAS-6. GFP-HsSAS-6 localizes to centrosomes and its overexpression results in excess foci-bearing centriolar markers. Furthermore, siRNA-mediated inactivation of HsSAS-6 in U2OS cells abrogates centrosome overduplication following aphidicolin treatment and interferes with the normal centrosome duplication cycle. Therefore, HsSAS-6 is also required for centrosome duplication, indicating that the function of SAS-6-related proteins has been widely conserved during evolution.

PROSITE, a protein domain database for functional characterization and annotation.

PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of these profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. PROSITE is largely used for the annotation of domain features of UniProtKB/Swiss-Prot entries. Among the 983 (DNA-binding) domains, repeats and zinc fingers present in Swiss-Prot (release 57.8 of 22 September 2009), 696 ( approximately 70%) are annotated with PROSITE descriptors using information from ProRule. In order to allow better functional characterization of domains, PROSITE developments focus on subfamily specific profiles and a new profile building method giving more weight to functionally important residues. Here, we describe AMSA, an annotated multiple sequence alignment format used to build a new generation of generalized profiles, the migration of ScanProsite to Vital-IT, a cluster of 633 CPUs, and the adoption of the Distributed Annotation System (DAS) to facilitate PROSITE data integration and interchange with other sources. The latest version of PROSITE (release 20.54, of 22 September 2009) contains 1308 patterns, 863 profiles and 869 ProRules. PROSITE is accessible at: http://www.expasy.org/prosite/.

PeroxiBase: a database with new tools for peroxidase family classification.

Peroxidases (EC 1.11.1.x), which are encoded by small or large multigenic families, are involved in several important physiological and developmental processes. They use various peroxides as electron acceptors to catalyse a number of oxidative reactions and are present in almost all living organisms. We have created a peroxidase database (http://peroxibase.isb-sib.ch) that contains all identified peroxidase-encoding sequences (about 6000 sequences in 940 organisms). They are distributed between 11 superfamilies and about 60 subfamilies. All the sequences have been individually annotated and checked. PeroxiBase can be consulted using six major interlink sections 'Classes', 'Organisms', 'Cellular localisations', 'Inducers', 'Repressors' and 'Tissue types'. General documentation on peroxidases and PeroxiBase is accessible in the 'Documents' section containing 'Introduction', 'Class description', 'Publications' and 'Links'. In addition to the database, we have developed a tool to classify peroxidases based on the PROSITE profile methodology. To improve their specificity and to prevent overlaps between closely related subfamilies the profiles were built using a new strategy based on the silencing of residues. This new profile construction method and its discriminatory capacity have been tested and validated using the different peroxidase families and subfamilies present in the database. The peroxidase classification tool called PeroxiScan is accessible at the following address: http://peroxibase.isb-sib.ch/peroxiscan.php.

Quantification of the spread of SARS-CoV-2 variant B.1.1.7 in Switzerland.

BACKGROUND: In December 2020, the United Kingdom (UK) reported a SARS-CoV-2 Variant of Concern (VoC) which is now named B.1.1.7. Based on initial data from the UK and later data from other countries, this variant was estimated to have a transmission fitness advantage of around 40-80 % (Volz et al., 2021; Leung et al., 2021; Davies et al., 2021). AIM: This study aims to estimate the transmission fitness advantage and the effective reproductive number of B.1.1.7 through time based on data from Switzerland. METHODS: We generated whole genome sequences from 11.8 % of all confirmed SARS-CoV-2 cases in Switzerland between 14 December 2020 and 11 March 2021. Based on these data, we determine the daily frequency of the B.1.1.7 variant and quantify the variant's transmission fitness advantage on a national and a regional scale. RESULTS: We estimate B.1.1.7 had a transmission fitness advantage of 43-52 % compared to the other variants circulating in Switzerland during the study period. Further, we estimate B.1.1.7 had a reproductive number above 1 from 01 January 2021 until the end of the study period, compared to below 1 for the other variants. Specifically, we estimate the reproductive number for B.1.1.7 was 1.24 [1.07-1.41] from 01 January until 17 January 2021 and 1.18 [1.06-1.30] from 18 January until 01 March 2021 based on the whole genome sequencing data. From 10 March to 16 March 2021, once B.1.1.7 was dominant, we estimate the reproductive number was 1.14 [1.00-1.26] based on all confirmed cases. For reference, Switzerland applied more non-pharmaceutical interventions to combat SARS-CoV-2 on 18 January 2021 and lifted some measures again on 01 March 2021. CONCLUSION: The observed increase in B.1.1.7 frequency in Switzerland during the study period is as expected based on observations in the UK. In absolute numbers, B.1.1.7 increased exponentially with an estimated doubling time of around 2-3.5 weeks. To monitor the ongoing spread of B.1.1.7, our plots are available online.

Functional differentiation of tbf1 orthologues in fission and budding yeasts.

In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function but also has other roles in the global regulation of transcription. We have identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by transspecies complementation that its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues. Our findings indicate that an essential function of this family of proteins has diverged in the budding and fission yeasts and suggest that effects on telomere length or structure are not the primary cause of inviability in S. pombe tbf1 null strains.

The 20 years of PROSITE.

PROSITE consists of documentation entries describing protein domains, families and functional sites, as well as associated patterns and profiles to identify them. It is complemented by ProRule, a collection of rules based on profiles and patterns, which increases the discriminatory power of profiles and patterns by providing additional information about functionally and/or structurally critical amino acids. In this article, we describe the implementation of a new method to assign a status to pattern matches, the new PROSITE web page and a new approach to improve the specificity and sensitivity of PROSITE methods. The latest version of PROSITE (release 20.19 of 11 September 2007) contains 1319 patterns, 745 profiles and 764 ProRules. Over the past 2 years, about 200 domains have been added, and now 53% of UniProtKB/Swiss-Prot entries (release 54.2 of 11 September 2007) have a PROSITE match. PROSITE is available on the web at: http://www.expasy.org/prosite/.

MyHits: improvements to an interactive resource for analyzing protein sequences.

The MyHits web site (http://myhits.isb-sib.ch) is an integrated service dedicated to the analysis of protein sequences. Since its first description in 2004, both the user interface and the back end of the server were improved. A number of tools (e.g. MAFFT, Jacop, Dotlet, Jalview, ESTScan) were added or updated to improve the usability of the service. The MySQL schema and its associated API were revamped and the database engine (HitKeeper) was separated from the web interface. This paper summarizes the current status of the server, with an emphasis on the new services.

New developments in the InterPro database.

InterPro is an integrated resource for protein families, domains and functional sites, which integrates the following protein signature databases: PROSITE, PRINTS, ProDom, Pfam, SMART, TIGRFAMs, PIRSF, SUPERFAMILY, Gene3D and PANTHER. The latter two new member databases have been integrated since the last publication in this journal. There have been several new developments in InterPro, including an additional reading field, new database links, extensions to the web interface and additional match XML files. InterPro has always provided matches to UniProtKB proteins on the website and in the match XML file on the FTP site. Additional matches to proteins in UniParc (UniProt archive) are now available for download in the new match XML files only. The latest InterPro release (13.0) contains more than 13 000 entries, covering over 78% of all proteins in UniProtKB. The database is available for text- and sequence-based searches via a webserver (http://www.ebi.ac.uk/interpro), and for download by anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro). The InterProScan search tool is now also available via a web service at http://www.ebi.ac.uk/Tools/webservices/WSInterProScan.html.

Machine Learning Approach for Candida albicans Fluconazole Resistance Detection Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Candida albicans causes life-threatening systemic infections in immunosuppressed patients. These infections are commonly treated with fluconazole, an antifungal agent targeting the ergosterol biosynthesis pathway. Current Antifungal Susceptibility Testing (AFST) methods are time-consuming and are often subjective. Moreover, they cannot reliably detect the tolerance phenomenon, a breeding ground for the resistance. An alternative to the classical AFST methods could use Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass spectrometry (MS). This tool, already used in clinical microbiology for microbial species identification, has already offered promising results to detect antifungal resistance on non-azole tolerant yeasts. Here, we propose a machine-learning approach, adapted to MALDI-TOF MS data, to qualitatively detect fluconazole resistance in the azole tolerant species C. albicans. MALDI-TOF MS spectra were acquired from 33 C. albicans clinical strains isolated from 15 patients. Those strains were exposed for 3 h to 3 fluconazole concentrations (256, 16, 0 µg/mL) and with (5 µg/mL) or without cyclosporin A, an azole tolerance inhibitor, leading to six different experimental conditions. We then optimized a protein extraction protocol allowing the acquisition of high-quality spectra, which were further filtered through two quality controls. The first one consisted of discarding not identified spectra and the second one selected only the most similar spectra among replicates. Quality-controlled spectra were divided into six sets, following the sample preparation's protocols. Each set was then processed through an R based script using pre-defined housekeeping peaks allowing peak spectra positioning. Finally, 32 machine-learning algorithms applied on the six sets of spectra were compared, leading to 192 different pipelines of analysis. We selected the most robust pipeline with the best accuracy. This LDA model applied to the samples prepared in presence of tolerance inhibitor but in absence of fluconazole reached a specificity of 88.89% and a sensitivity of 83.33%, leading to an overall accuracy of 85.71%. Overall, this work demonstrated that combining MALDI-TOF MS and machine-learning could represent an innovative mycology diagnostic tool.

Functional characterization of Pneumocystis carinii brl1 by transspecies complementation analysis.

Pneumocystis jirovecii is a fungus which causes severe opportunistic infections in immunocompromised humans. The brl1 gene of P. carinii infecting rats was identified and characterized by using bioinformatics in conjunction with functional complementation in Saccharomyces cerevisiae and Schizosaccharomyces pombe. The ectopic expression of this gene rescues null alleles of essential nuclear membrane proteins of the Brr6/Brl1 family in both yeasts.

The PROSITE database.

The PROSITE database consists of a large collection of biologically meaningful signatures that are described as patterns or profiles. Each signature is linked to a documentation that provides useful biological information on the protein family, domain or functional site identified by the signature. The PROSITE database is now complemented by a series of rules that can give more precise information about specific residues. During the last 2 years, the documentation and the ScanProsite web pages were redesigned to add more functionalities. The latest version of PROSITE (release 19.11 of September 27, 2005) contains 1329 patterns and 552 profile entries. Over the past 2 years more than 200 domains have been added, and now 52% of UniProtKB/Swiss-Prot entries (release 48.1 of September 27, 2005) have a cross-reference to a PROSITE entry. The database is accessible at http://www.expasy.org/prosite/.