Clinically inapparent mpox virus (MPXV) infections among clients of three anonymous Community Based Voluntary Counselling and Testing centres in Berlin, Germany, 2022-2023.

INTRODUCTION: Since the mpox outbreak in 2022, it was unclear if and how often infections with mpox virus (MPXV) were clinically inapparent, i.e. not presenting to clinical care with mpox symptoms. Moreover, it was hypothesized that MPXV circulated in the affected communities before the outbreak was officially detected. METHODS: We retrospectively tested rectal and urethral swabs, and pooled samples for presence of MPXV. Samples were obtained from routine STI testing of three anonymous Community Based Voluntary Counselling and Testing (CBVCT) centres in Berlin, in 2022 and 2023. Testing results were linked to anonymously provided behavioural data. RESULTS: Overall, 9,053 samples from 6,600 client visits were included. Clinically inapparent MPXV infections were detectable in 1.1% of the samples. We did not find MPXV infections in the month before the first cases appeared in Berlin or between October 2022 and January 2023 when case numbers were low in Germany. However, during the outbreak period in 2022, we found clinically inapparent MPXV infections among 2.2% of the clients and during summer/autumn 2023 among 0.3%. The number of condomless anal/vaginal intercourse partners within the previous 6 months and PrEP use were identified as predictors of clinically inapparent MPXV infection. CONCLUSION: Clinically inapparent MPXV infections occurred during the mpox outbreak in Berlin in 2022 and post-outbreak in summer/autumn 2023. Unrecognized MPXV circulation in Berlin before the recognition of the outbreak in May 2022 appears unlikely. However, low-level sustained circulation of clinically inapparent MPXV infections need to be acknowledged in mpox prevention strategies.

Sequence-based identification of inositol monophosphatase-like histidinol-phosphate phosphatases (HisN) in Corynebacterium glutamicum, Actinobacteria, and beyond.

BACKGROUND: The eighth step of L-histidine biosynthesis is carried out by an enzyme called histidinol-phosphate phosphatase (HolPase). Three unrelated HolPase families are known so far. Two of them are well studied: HAD-type HolPases known from Gammaproteobacteria like Escherichia coli or Salmonella enterica and PHP-type HolPases known from yeast and Firmicutes like Bacillus subtilis. However, the third family of HolPases, the inositol monophosphatase (IMPase)-like HolPases, present in Actinobacteria like Corynebacterium glutamicum (HisN) and plants, are poorly characterized. Moreover, there exist several IMPase-like proteins in bacteria (e.g. CysQ, ImpA, and SuhB) which are very similar to HisN but most likely do not participate in L-histidine biosynthesis. RESULTS: Deletion of hisN, the gene encoding the IMPase-like HolPase in C. glutamicum, does not result in complete L-histidine auxotrophy. Out of four hisN homologs present in the genome of C. glutamicum (impA, suhB, cysQ, and cg0911), only cg0911 encodes an enzyme with HolPase activity. The enzymatic properties of HisN and Cg0911 were determined, delivering the first available kinetic data for IMPase-like HolPases. Additionally, we analyzed the amino acid sequences of potential HisN, ImpA, SuhB, CysQ and Cg0911 orthologs from bacteria and identified six conserved sequence motifs for each group of orthologs. Mutational studies confirmed the importance of a highly conserved aspartate residue accompanied by several aromatic amino acid residues present in motif 5 for HolPase activity. Several bacterial proteins containing all identified HolPase motifs, but showing only moderate sequence similarity to HisN from C. glutamicum, were experimentally confirmed as IMPase-like HolPases, demonstrating the value of the identified motifs. Based on the confirmed IMPase-like HolPases two profile Hidden Markov Models (HMMs) were build using an iterative approach. These HMMs allow the fast, reliable detection and differentiation of the two paralog groups from each other and other IMPases. CONCLUSION: The kinetic data obtained for HisN from C. glutamicum, as an example for an IMPase-like HolPases, shows remarkable differences in enzyme properties as compared to HAD- or PHP-type HolPases. The six sequence motifs and the HMMs presented in this study can be used to reliably differentiate between IMPase-like HolPases and IMPase-like proteins with no such activity, with the potential to enhance current and future genome annotations. A phylogenetic analysis reveals that IMPase-like HolPases are not only present in Actinobacteria and plant but can be found in further bacterial phyla, including, among others, Proteobacteria, Chlorobi and Planctomycetes.