Rapid Amplicon Nanopore Sequencing (RANS) for the Differential Diagnosis of Monkeypox Virus and Other Vesicle-Forming Pathogens.

As of July 2022, more than 16,000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently, MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (the causative agent of smallpox). Following the eradication of smallpox, there was a significant decrease in smallpox-related morbidity and the population's immunity to other OPV-related diseases such as MPX. In parallel, there was a need for differential diagnosis between the different OPVs' clinical manifestations and diseases with similar symptoms (i.e., chickenpox, herpes simplex). The current study aimed to provide a rapid genetic-based diagnostic tool for accurate and specific identification of MPXV and additional related vesicle-forming pathogens. We initially assembled a list of 14 relevant viral pathogens, causing infectious diseases associated with vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we termed rapid amplicon nanopore sequencing (RANS). The RANS approach uses diagnostic regions that harbor high homology in their boundaries and internal diagnostic SNPs that, when sequenced, aid the discrimination of each pathogen within a group. During a multiplex PCR amplification, a dA tail and a 5'-phosphonate were simultaneously added, thus making the PCR product ligation ready for nanopore sequencing. Following rapid sequencing (a few minutes), the reads were compared to a reference database and the nearest strain was identified. We first tested our approach using samples of known viruses cultured in cell lines. All the samples were identified correctly and swiftly. Next, we examined a variety of clinical samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the PCR-positive MPXV samples and mapped them to strains that were sequenced during the 2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we obtained definite results, identifying other vesicle-forming viruses: Human herpesvirus 3, Human herpesvirus 2, and Molluscum contagiosum virus. This work was a proof-of-concept study, demonstrating the potential of the RANS approach for rapid and discriminatory identification of a panel of closely related pathogens. The simplicity and affordability of our approach makes it straightforward to implement in any genetics lab. Moreover, other differential diagnostics panels might benefit from the implementation of the RANS approach into their diagnostics pipelines.

Whole genome sequencing and taxonomic profiling of two Pantoea sp. isolated from environmental samples in Israel.

OBJECTIVE: As part of a research aiming at the isolation of bacteria secreting growth inhibiting compounds, cultures of Francisella tularensis were implanted in environmental samples and monitored for inhibition zones on agar. Two antibiotic-like secreting bacteria were isolated, their genomic sequence was deciphered and taxonomic profiling analysis classified them as belonging to the Pantoea genus. DATA DESCRIPTION: Two bacterial isolates exhibiting growth inhibition zones to F. tularensis (LVS) were analyzed using the Oxford Nanopore Technology (ONT). Preliminary de novo assembly of the reads was performed, followed by taxonomic profiling based on Multi Locus Sequence Analysis (MLSA) and implementation of the Average Nucleotide Identity (ANI) measure. The genomic sequences resulted in the identification of two different Pantoea species, denoted EnvD and EnvH. Subsequent de novo genome assembly generated 5 and 10 contigs for EnvD and EnvH, respectively. The largest contig (4,008,183 bps and 3,740,753 bps for EnvD and EnvH, respectively), overlaps to a major extent to the chromosome of closely related Pantoea species. ANI values calculated for both isolates revealed two apparently new species of the Pantoea genus. Our study deciphered the identity of two bacteria producing antibiotic-like compounds, and the genomic sequence revealed they represent distinct Pantoea species.