Efficient SARS-CoV-2 variant detection and monitoring with Spike Screen next-generation sequencing.

The emergence and rapid spread of SARS-CoV-2 prompted the global community to identify innovative approaches to diagnose infection and sequence the viral genome because at several points in the pandemic positive case numbers exceeded the laboratory capacity to characterize sufficient samples to adequately respond to the spread of emerging variants. From week 10, 2020, to week 13, 2023, Slovenian routine complete genome sequencing (CGS) surveillance network yielded 41 537 complete genomes and revealed a typical molecular epidemiology with early lineages gradually being replaced by Alpha, Delta, and finally Omicron. We developed a targeted next-generation sequencing based variant surveillance strategy dubbed Spike Screen through sample pooling and selective SARS-CoV-2 spike gene amplification in conjunction with CGS of individual cases to increase throughput and cost-effectiveness. Spike Screen identifies variant of concern (VOC) and variant of interest (VOI) signature mutations, analyses their frequencies in sample pools, and calculates the number of VOCs/VOIs at the population level. The strategy was successfully applied for detection of specific VOC/VOI mutations prior to their confirmation by CGS. Spike Screen complemented CGS efforts with an additional 22 897 samples sequenced in two time periods: between week 42, 2020, and week 24, 2021, and between week 37, 2021, and week 2, 2022. The results showed that Spike Screen can be applied to monitor VOC/VOI mutations among large volumes of samples in settings with limited sequencing capacity through reliable and rapid detection of novel variants at the population level and can serve as a basis for public health policy planning.

Intrahost Monkeypox Virus Genome Variation in Patient with Early Infection, Finland, 2022.

Monkeypox virus was imported into Finland during late May-early June 2022. Intrahost viral genome variation in a sample from 1 patient comprised a major variant with 3 lineage B.1.3-specific mutations and a minor variant with ancestral B.1 nucleotides. Results suggest either ongoing APOBEC3 enzyme-mediated evolution or co-infection.

Transmission of SARS-CoV-2 from Human to Domestic Ferret.

We report a case of natural infection with severe acute respiratory syndrome coronavirus 2 transmitted from an owner to a pet ferret in the same household in Slovenia. The ferret had onset of gastroenteritis with severe dehydration. Whole-genome sequencing of the viruses isolated from the owner and ferret revealed a 2-nt difference.

Prospective Longitudinal Study of Dynamics of Human Papillomavirus 6 and 11 Infection in Anogenital Hairs and Eyebrows of Male Patients with Anogenital Warts and Age-Matched Controls.

To better understand the natural history of anogenital warts (AGWs) and the dynamics of HPV6/11 infection in regional hairs, 32 newly diagnosed male patients with AGWs and 32 age-matched healthy controls were closely followed. During enrollment and six follow-up visits (every 2.6 months), 43 AGW tissues and 1232 anogenital and eyebrow hair samples were collected. This is the closest longitudinal monitoring of AGW patients to date. Patients were treated according to standards of care. The HPV6/11 prevalence was 19.9% in the patients' hair samples (HPV6 B1 in 53.1%) and 0% in the controls. The highest HPV6/11 prevalence was found in pubic hairs (29.0%) and the lowest in eyebrows (7.1%). The odds of having HPV6/11-positive hairs increased with smoking, shaving the anogenital region, and age. A close association between HPV6/11 presence in hairs and clinically visible AGWs was observed. The proportion of patients with visible AGWs and HPV6/11-positive hairs declined during follow-up with similar trends. No particular HPV6/11 variant was linked with an increased AGW recurrence, but the sublineage HPV6 B1 showed significantly higher clearance from hairs. Despite treatment, 78.1% and 62.5% of the AGW patients experienced one and two or more post-initial AGW episodes, respectively. The patients with HPV6/11-positive hairs or visible AGWs at a preceding visit demonstrated substantially higher odds of presenting with visible AGWs at a subsequent visit.

Comprehensive analysis of 66 complete molluscum contagiosum virus (MOCV) genomes: characterization and functional annotation of 47 novel complete MOCV genomes, including the first genome of MOCV genotype 3, and a proposal for harmonized MOCV genotyping indexing.

Four molluscum contagiosum virus (MOCV) genotypes (MOCV1-4) and four subtype variants (MOCV1p, MOCV1va, MOCV1vb, and MOCV1vc) were partially characterized using restriction enzyme profiling in the early 1980s/1990s. However, complete genome sequences of only MOCV1 and MOCV2 are available. The evolutionary pathways of MOCV genotypes and subtype variants with unavailable sequences remain unclear, and also whether all MOCV genotypes/subtype variants can be reliably detected and appropriately categorized using available PCR-based protocols. We de novo fully characterized and functionally annotated 47 complete MOCV genomes, including two putative non-MOCV1/2 isolates, expanding the number of fully characterized MOCV genomes to 66. To ascertain the placement of any putative novel MOCV sequence into the restriction profiling typing scheme, we developed an original framework for extracting complete MOCV genome sequence-based restriction profiles and matching them with reference restriction profiles. We confirmed that two putative non-MOCV1/2 isolates represent the first complete genomes of MOCV3. Comprehensive phylogenomic, recombination, and restriction enzyme recognition site analysis of all 66 currently available MOCV genomes showed that they can be agglomerated into six phylogenetic subgroups (PG1-6), corresponding to the subtype variants from the pioneering studies. PG5 was a novel subtype variant of MOCV2, but no PGs corresponded to the subtype variants MOCV1vb or MOCV4. We showed that the phylogenetic subgroups may have diverged from the prototype MOCV genotype lineages following large-scale recombination events and hinted at partial sequence content of MOCV4 and direction of recombinant transfer in the events that spawned PG5 and the yet undetected subtype variant MOCV1vb.IMPORTANCEFour molluscum contagiosum virus (MOCV) genotypes (MOCV1-4) and four subtype variants were partially characterized using restriction enzyme profiling in the 1980s/1990s, but complete genome sequences of only MOCV1 and MOCV2 are available. The evolutionary pathways whereby genotypes/subtype variants with unavailable sequences emerged and whether all MOCVs can be detected using current diagnostic approaches remain unclear. We fully characterized 47 novel complete MOCV genomes, including the first complete MOCV3 genome, expanding the number of fully characterized genomes to 66. For reliably classifying the novel non-MOCV1/2 genomes, we developed and validated a framework for matching sequence-derived restriction maps with those defining MOCV subtypes in pioneering studies. Six phylogenetic subgroups (PG1-6) were identified, PG5 representing a novel MOCV2 subtype. The phylogenetic subgroups diverged from the prototype lineages following large-scale recombination events and hinted at partial sequence content of MOCV4 and direction of recombinant transfer in the events spawning PG5 and yet undetected MOCV1vb variant.

Molecular epidemiology of the 2022 monkeypox virus outbreak in Slovenia.

INTRODUCTION: Monkeypox virus (MPXV), typically endemic in West and Central Africa, has raised global concern due to the recent outbreak in several non-endemic countries with human-to-human transmission. Here we present a comprehensive analysis of MPXV genomes from Slovenia. METHODS: Two real-time polymerase chain reaction (RT-PCR) assays for Orthopoxvirus (OPV) and MPXV genes were used for laboratory confirmation of mpox. Complete MPXV genomic sequences were obtained using nanopore long reads and Illumina technology. Phylogenetic analyses compared the Slovenian MPXV sequences with the global sequences. RESULTS: A total of 49 laboratory-confirmed mpox cases were diagnosed in Slovenia in 2022, mainly affecting males under 40. In 48 cases, a complete genome sequence was obtained and phylogenetic analysis revealed five distinct lineages (B.1, B.1.14, B.1.2, B.1.3, and A.2.1), with B.1 and B.1.3 dominating, suggesting multiple introductions into Slovenia. Genome analysis revealed significant divergence from the reference MPXV-M5312_HM12_Rivers. CONCLUSIONS: The genetic diversity observed in the Slovenian MPXV sequences sheds light on the complex dynamics of the 2022 mpox outbreak and highlights the need for further research to understand the impact of mutations on MPXV functional characteristics and their role in the evolution and diversification of current lineages.

Complete Genome Sequencing of Tick-Borne Encephalitis Virus Directly from Clinical Samples: Comparison of Shotgun Metagenomic and Targeted Amplicon-Based Sequencing.

The clinical presentation of tick-borne encephalitis virus (TBEV) infection varies from asymptomatic to severe meningoencephalitis or meningoencephalomyelitis. The TBEV subtype has been suggested as one of the most important risk factors for disease severity, but TBEV genetic characterization is difficult. Infection is usually diagnosed in the post-viremic phase, and so relevant clinical samples of TBEV are extremely rare and, when present, are associated with low viral loads. To date, only two complete TBEV genomes sequenced directly from patient clinical samples are publicly available. The aim of this study was to develop novel protocols for the direct sequencing of the TBEV genome, enabling studies of viral genetic determinants that influence disease severity. We developed a novel oligonucleotide primer scheme for amplification of the complete TBEV genome. The primer set was tested on 21 clinical samples with various viral loads and collected over a 15-year period using the two most common sequencing platforms. The amplicon-based strategy was compared to direct shotgun sequencing. Using the novel primer set, we successfully obtained nearly complete TBEV genomes (>90% of genome) from all clinical samples, including those with extremely low viral loads. Comparison of consensus sequences of the TBEV genome generated using the novel amplicon-based strategy and shotgun sequencing showed no difference. We conclude that the novel primer set is a powerful tool for future studies on genetic determinants of TBEV that influence disease severity and will lead to a better understanding of TBE pathogenesis.

First Report of Phodopus sungorus Papillomavirus Type 1 Infection in Roborovski Hamsters (Phodopus roborovskii).

Papillomaviruses (PVs) are considered highly species-specific with cospeciation as the main driving force in their evolution. However, a recent increase in the available PV genome sequences has revealed inconsistencies in virus-host phylogenies, which could be explained by adaptive radiation, recombination, host-switching events and a broad PV host range. Unfortunately, with a relatively low number of animal PVs characterized, understanding these incongruities remains elusive. To improve knowledge of biology and the spread of animal PV, we collected 60 swabs of the anogenital and head and neck regions from a healthy colony of 30 Roborovski hamsters (Phodopus roborovskii) and detected PVs in 44/60 (73.3%) hamster samples. This is the first report of PV infection in Roborovski hamsters. Moreover, Phodopus sungorus papillomavirus type 1 (PsuPV1), previously characterized in Siberian hamsters (Phodopus sungorus), was the only PV detected in Roborovski hamsters. In addition, after a detailed literature search, review and summary of published evidence and construction of a tanglegram linking the cladograms of PVs and their hosts, our findings were discussed in the context of available knowledge on PVs described in at least two different host species.

HIV-1 Unique Recombinant Forms Identified in Slovenia and Their Characterization by Near Full-Length Genome Sequencing.

Surveillance of HIV circulating recombinant forms (CRFs) is important because HIV diversity can affect various aspects of HIV infection from prevention to diagnosis and patient management. A comprehensive collection of pol sequences obtained from individuals diagnosed with HIV-1 from 2000 to 2016 in Slovenia was subtyped to identify possible unique recombinant forms (URFs). Selected samples were subjected to near full-length genome (NFLG) sequencing and detailed recombination analyses. Discordant subtyping results were observed for 68/387 (17.6%) sequences and 20 sequences were identified as the most probable URFs and selected for NFLG characterization. Further, 11 NFLGs and two sequences of >7000 base pairs were obtained. Seven sequences were identified as "pure" subtypes or already characterized CRFs: subtype B (n = 5), sub-subtype A6 (n = 1), and CRF01_AE (n = 1). The remaining six sequences were determined to be URFs; four displayed a single recombination event and two exhibited a complex recombination pattern involving several subtypes or CRFs. Finally, three HIV strains were recognized as having epidemic potential and could be further characterized as new CRFs. Our study shows that the identification of new CRFs is possible, even in countries where HIV diversity is considered limited, emphasizing the importance of the surveillance of HIV recombinant forms.

Viral Metagenomic Profiling of Croatian Bat Population Reveals Sample and Habitat Dependent Diversity.

To date, the microbiome, as well as the virome of the Croatian populations of bats, was unknown. Here, we present the results of the first viral metagenomic analysis of guano, feces and saliva (oral swabs) of seven bat species (Myotis myotis, Miniopterus schreibersii, Rhinolophus ferrumequinum, Eptesicus serotinus, Myotis blythii, Myotis nattereri and Myotis emarginatus) conducted in Mediterranean and continental Croatia. Viral nucleic acids were extracted from sample pools, and analyzed using Illumina sequencing. The presence of 63 different viral families representing all seven Baltimore groups were confirmed, most commonly insect viruses likely reflecting the diet of insectivorous bats. Virome compositions of our samples were largely impacted by the sample type: invertebrate-infecting viruses were most frequently found in feces, bacterial viruses in guano, whereas vertebrate-infecting viruses were most common in swabs. Most vertebrate-infecting virus sequences were assigned to retroviruses, parvoviruses, iridoviruses, and poxviruses. We further report the complete genome sequence of a novel adeno-associated virus, densovirus and a near complete length genome sequence of a novel iflavirus. Additionally, one of the most interesting findings in this study was the difference in viromes between two contrasting habitats, the continental and Mediterranean Croatia.

Whole genome sequencing characterization of Slovenian carbapenem-resistant Klebsiella pneumoniae, including OXA-48 and NDM-1 producing outbreak isolates.

OBJECTIVES: The first hospital outbreak of carbapenemase-producing Enterobacteriaceae in Slovenia occurred in 2014-2016. Whole genome sequencing was used to analyse the population of carbapenem-resistant Klebsiella pneumoniae collected in Slovenia in 2014-2017, including OXA-48 and/or NDM-1 producing strains from the outbreak. METHODS: A total of 32 K. pneumoniae isolates were analysed using short-read sequencing. Multi-locus sequence typing and core genome multi-locus sequence typing were used to infer genetic relatedness. Antimicrobial resistance markers, virulence factors, plasmid content and wzi types were determined. Long-read sequencing was used for six isolates for detailed analysis of plasmids and their possible transmission. RESULTS: Overall, we detected 10 different sequence types (STs), the most common being ST437 (40.6%). Isolates from the initial outbreak belonged to ST437 (12/16) and ST147 (4/16). A second outbreak of four ST15 isolates was discovered. A new ST (ST3390) and two new wzi types (wzi-556, wzi-559) were identified. blaOXA-48 was found in 17 (53.1%) isolates, blaNDM-1 in five (15.6%), and a combination of blaOXA-48/NDM-1 in seven (21.9%) isolates. Identical plasmids carrying blaOXA-48 were found in outbreak isolates sequenced with long-read sequencing technology. CONCLUSIONS: Whole genome sequencing of Slovenian carbapenem-resistant K. pneumoniae isolates revealed multiple clusters of STs, two of which were involved in the first hospital outbreak of carbapenem producing K. pneumoniae in Slovenia. Transmission of the plasmid carrying blaOXA-48 between two STs was likely to have occurred. A previously unidentified second outbreak was also discovered, highlighting the importance of whole genome sequencing in detection and/or characterization of hospital outbreaks and surveillance of drug-resistant bacterial clones.

A novel reassortant mammalian orthoreovirus with a divergent S1 genome segment identified in a traveler with diarrhea.

Mammalian orthoreoviruses with reassortant genomes have recently been detected in various mammals and humans with respiratory, central nervous system, and gastrointestinal symptoms. This study describes the detection of the novel reassortant mammalian orthoreovirus SI-MRV07 in a traveler with gastroenteritis that returned from southeast Asia. The virus was initially detected with electron microscopy in stool, followed by propagation in the epithelial-like monkey kidney Marc145 cell line. Whole-genome sequencing revealed the reassortant nature of the genome segments, whereby the S1 genome segment was the most variable according to known sequences deposited in GenBank. Based on the nucleotide sequence of the S1 genome segment, the isolate clusters to serotype 2, close to the reference strain Jones T2J. The patient's serum showed the highest virus neutralization capacity toward SI-MRV07 and T2J isolates. This study provides additional insight into emerging mammalian orthoreoviruses with reassortant genomes and possible zoonotic potential, which should be carefully monitored in the future.

Novel Circo-Like Virus Detected in a Croatian Bat Population.

Viral metagenomics analysis of samples from bats has been carried out as part of bat rabies surveillance in Croatia. Here, we report the complete genome sequence of a novel circo-like virus isolated from a sample of Miniopterus schreibersii bat guano determined by Illumina next-generation sequencing.

Analysis of HIV-1 diversity, primary drug resistance and transmission networks in Croatia.

Molecular epidemiology of HIV-1 infection in treatment-naive HIV-1 infected persons from Croatia was investigated. We included 403 persons, representing 92.4% of all HIV-positive individuals entering clinical care in Croatia in 2014-2017. Overall prevalence of transmitted drug resistance (TDR) was estimated at 16.4%. Resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside RTI (NNRTIs) and protease inhibitors (PIs) was found in 11.4%, 6.7% and 2.5% of persons, respectively. Triple-class resistance was determined in 2.2% of individuals. In addition, a single case (1.0%) of resistance to integrase strand-transfer inhibitors (InSTIs) was found. Deep sequencing was performed on 48 randomly selected samples and detected additional TDR mutations in 6 cases. Phylogenetic inference showed that 347/403 sequences (86.1%) were part of transmission clusters and identified forward transmission of resistance in Croatia, even that of triple-class resistance. The largest TDR cluster of 53 persons with T215S was estimated to originate in the year 1992. Our data show a continuing need for pre-treatment HIV resistance testing in Croatia. Even though a low prevalence of resistance to InSTI was observed, surveillance of TDR to InSTI should be continued.

Clinical, Histopathological, and Virological Evaluation of 203 Patients With a Clinical Diagnosis of Molluscum Contagiosum.

Molluscum contagiosum (MC) manifests as small, umbilicated papules caused by the molluscum contagiosum virus (MCV). The extent of clinical misdiagnosis of MC is unknown. Combined clinical, histopathological, and virological evaluation of 203 consecutive patients with clinical diagnosis of MC treated at a university hospital during a 5-year period showed the correct clinical diagnosis in 188 of 203 (92.6%) patients. All 15 clinically misdiagnosed MC lesions were histopathologically and virologically confirmed as either common or anogenital warts caused by different human papillomaviruses. The MCV1/MCV2 subtypes ratio was 1.54:1, and the distribution of MCV subtypes differed across patients' age and anatomical location of lesions.