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1.
Brief Bioinform ; 25(4)2024 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-38833323

RESUMEN

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.


Asunto(s)
COVID-19 , Secuenciación de Nucleótidos de Alto Rendimiento , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , COVID-19/virología , COVID-19/diagnóstico , COVID-19/epidemiología , Glicoproteína de la Espiga del Coronavirus/genética , Mutación , Genoma Viral , Eslovenia/epidemiología
2.
Emerg Infect Dis ; 29(3): 649-652, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36703245

RESUMEN

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.


Asunto(s)
Monkeypox virus , Mpox , Humanos , Finlandia , Mutación
3.
Emerg Infect Dis ; 27(9): 2450-2453, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34424180

RESUMEN

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.


Asunto(s)
COVID-19 , Hurones , Animales , Humanos , SARS-CoV-2 , Eslovenia
4.
Front Public Health ; 12: 1406777, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38813418

RESUMEN

Introduction: Residents of long-term care facilities (LTCFs) are at high risk of morbidity and mortality due to COVID-19, especially when new variants of concern (VOC) emerge. To provide intradisciplinary data in order to tailor public health interventions during future epidemics, available epidemiologic and genomic data from Slovenian LTCFs during the initial phases of the COVID-19 pandemic was analyzed. Methods: The first part of the study included SARS-CoV-2 reverse-transcription Real-Time PCR (rtRT-PCR) positive LTCF residents, from 21 facilities with COVID-19 outbreaks occurring in October 2020. The second part of the study included SARS-CoV-2 rtRT-PCR positive LTCF residents and staff between January and April 2021, when VOC Alpha emerged in Slovenia. Next-generation sequencing (NGS) was used to acquire SARS-CoV-2 genomes, and lineage determination. In-depth phylogenetic and mutational profile analysis were performed and coupled with available field epidemiological data to assess the dynamics of SARS-CoV-2 introduction and transmission. Results: 370/498 SARS-CoV-2 positive residents as well as 558/699 SARS-CoV-2 positive residents and 301/358 staff were successfully sequenced in the first and second part of the study, respectively. In October 2020, COVID-19 outbreaks in the 21 LTCFs were caused by intra-facility transmission as well as multiple independent SARS-CoV-2 introductions. The Alpha variant was confirmed in the first LTCF resident approximately 1.5 months after the first Alpha case was identified in Slovenia. The data also showed a slower replacement of existing variants by Alpha in residents compared to staff and the general population. Discussion: Multiple SARS CoV-2 introductions as well as intra-facility spreading impacted disease transmission in Slovenian LTCFs. Timely implementation of control measures aimed at limiting new introductions while controlling in-facility transmission are of paramount importance, especially as new VOCs emerge. Sequencing, in conjunction with epidemiological data, can facilitate the determination of the need for future improvements in control measures to protect LTCF residents from COVID-19 or other respiratory infections.


Asunto(s)
COVID-19 , Cuidados a Largo Plazo , SARS-CoV-2 , Humanos , COVID-19/epidemiología , COVID-19/transmisión , COVID-19/prevención & control , Eslovenia/epidemiología , SARS-CoV-2/genética , Cuidados a Largo Plazo/estadística & datos numéricos , Anciano , Femenino , Masculino , Brotes de Enfermedades , Anciano de 80 o más Años , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , Persona de Mediana Edad
5.
Artículo en Inglés | MEDLINE | ID: mdl-38179904

RESUMEN

Monkeypox virus (MPXV), originally endemic in West Africa (Clade II) and Central Africa (Clade I), has recently emerged worldwide and has reinforced the need for rapid and accurate MPXV diagnostics. This review presents and critically discusses the range of virological methods for laboratory diagnosis and characterization of MPXV as well as related lessons learned and practical experience gained from the 2022 Mpox global outbreak. Real-time PCR is currently considered the diagnostic gold standard and ensures accurate and timely confirmation of suspected Mpox cases based on suspicious skin lesions, and digital PCR improves the precision of MPXV DNA quantification. Whole genome sequencing reveals the diversity within the Clade IIb outbreak and highlights the role of microevolution in the adaptation of the virus to the human host. Continuous genomic surveillance is important for better understanding of human-to-human transmission and prevention of the emergence of variola virus-like strains. Traditional virological methods such as electron microscopy and virus isolation remain essential for comprehensive virus characterization, particularly in the context of vaccine and antiviral drug development. Despite the current challenges, serological tests detecting a range of anti-MPXV antibodies are important adjunct diagnostic and research tools for confirmation of late-presenting or asymptomatic MPXV cases, contact tracing, epidemiological studies, seroepidemiological surveys, and better understanding of the role of IgG and neutralizing antibodies in the immune response to infection and vaccination. A multidisciplinary approach combining advanced molecular techniques with traditional virological methods is important for rapid and reliable diagnosis, surveillance, and control of the outbreak.


Asunto(s)
Monkeypox virus , Mpox , Humanos , Técnicas de Laboratorio Clínico , Brotes de Enfermedades/prevención & control , Monkeypox virus/genética , Mpox/diagnóstico , Mpox/epidemiología
6.
J Clin Virol ; 171: 105620, 2024 04.
Artículo en Inglés | MEDLINE | ID: mdl-38237303

RESUMEN

Simultaneously characterising the genomic information of coronaviruses and the underlying nasal microbiome from a single clinical sample would help characterise infection and disease. Metatranscriptomic approaches can be used to sequence SARS-CoV-2 (and other coronaviruses) and identify mRNAs associated with active transcription in the nasal microbiome. However, given the large sequence background, unenriched metatranscriptomic approaches often do not sequence SARS-CoV-2 to sufficient read and coverage depth to obtain a consensus genome, especially with moderate and low viral loads from clinical samples. In this study, various enrichment methods were assessed to detect SARS-CoV-2, identify lineages and define the nasal microbiome. The methods were underpinned by Oxford Nanopore long-read sequencing and variations of sequence independent single primer amplification (SISPA). The utility of the method(s) was also validated on samples from patients infected seasonal coronaviruses. The feasibility of profiling the nasal microbiome using these enrichment methods was explored. The findings shed light on the performance of different enrichment strategies and their applicability in characterising the composition of the nasal microbiome.


Asunto(s)
COVID-19 , Microbiota , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Genoma Viral , Microbiota/genética , Nasofaringe
7.
Acta Dermatovenerol Alp Pannonica Adriat ; 32(3): 111-117, 2023 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-37749971

RESUMEN

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.


Asunto(s)
Monkeypox virus , Mpox , Masculino , Humanos , Monkeypox virus/genética , Epidemiología Molecular , Eslovenia/epidemiología , Mpox/diagnóstico , Mpox/epidemiología , Filogenia , Brotes de Enfermedades
8.
Microorganisms ; 11(12)2023 Dec 12.
Artículo en Inglés | MEDLINE | ID: mdl-38138107

RESUMEN

In this article, we report on a rare case of acute respiratory distress syndrome (ARDS) caused by the Puumala orthohantavirus (PUUV), which is typically associated with hemorrhagic fever with renal syndrome (HFRS). This is the first documented case of PUUV-associated ARDS in Southeast Europe. The diagnosis was confirmed by serum RT-PCR and serology and corroborated by phylogenetic analysis and chemokine profiling. The patient was a 23-year-old male from Zagreb, Croatia, who had recently traveled throughout Europe. He presented with fever, headache, abdominal pain, and sudden onset of ARDS. Treatment involved high-flow nasal cannula oxygen therapy and glucocorticoids, which resulted in a full recovery. A systematic literature review identified 10 cases of hantavirus pulmonary syndrome (HPS) caused by PUUV in various European countries and Turkey between 2002 and 2023. The median age of patients was 53 years (range 24-73), and six of the patients were male. Most patients were treated in intensive care units, but none received antiviral therapy targeting PUUV. Eight patients survived hospitalization. The presented case highlights the importance of considering HPS in the differential diagnosis of ARDS, even in areas where HFRS is the dominant form of hantavirus infection.

9.
Viruses ; 14(6)2022 06 10.
Artículo en Inglés | MEDLINE | ID: mdl-35746738

RESUMEN

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.


Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas , Encefalitis Transmitida por Garrapatas , Secuencia de Bases , Virus de la Encefalitis Transmitidos por Garrapatas/genética , Encefalitis Transmitida por Garrapatas/diagnóstico , Genes Virales , Humanos , Secuenciación Completa del Genoma
10.
Viruses ; 12(7)2020 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-32635155

RESUMEN

West Nile virus (WNV) is a flavivirus transmitted by mosquitoes. Birds are the reservoir for the virus; humans, horses and other mammals are dead-end hosts. Infections caused by WNV in humans can vary from asymptomatic infections to West Nile fever (WNF) or West Nile neuroinvasive disease (WNND). In 1995, a serosurvey was performed in Slovenia on forest workers, and WNV specific IgG antibodies were confirmed in 6.8% of the screened samples, indicating that WNV is circulating in Slovenia. No human disease cases were detected in Slovenia until 2013, when the first case of WNV infection was confirmed in a retrospective study in a 79-year old man with meningitis. In 2018, three patients with WNND were confirmed by laboratory tests, with detection of IgM antibodies in the cerebrospinal fluid of the patients. In one of the patients, WNV RNA was detected in the urine sample. In 2017, 2018 and 2019, a mosquito study was performed in Slovenia. Mosquitoes were sampled on 14 control locations and 35 additional locations in 2019. No WNV was detected in mosquitoes in 2017 and 2019, but we confirmed the virus in a pool of Culex sp. mosquitoes in 2018. The virus was successfully isolated, and complete genome sequence was acquired. The whole genome of the WNV was also sequenced from the patient's urine sample. The whole genome sequences of the WNV virus detected in Slovenian patient and mosquito indicate the virus most likely spread from the north, because of the geographic proximity and because the sequences cluster with the Austrian and Hungarian sequences. A sentinel study was performed on dog sera samples, and we were able to confirm IgG antibodies in 1.8% and 4.3% of the samples in 2017 and 2018, respectively. Though Slovenia is not a highly endemic country for WNV, we have established that the virus circulates in Slovenia.


Asunto(s)
Enfermedades de los Perros/virología , Fiebre del Nilo Occidental/veterinaria , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/aislamiento & purificación , Anciano , Animales , Anticuerpos Antivirales/sangre , Culex , Culicidae/clasificación , Culicidae/fisiología , Culicidae/virología , Enfermedades de los Perros/sangre , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/epidemiología , Perros , Femenino , Genoma Viral , Humanos , Masculino , Persona de Mediana Edad , Filogenia , Estudios Retrospectivos , Eslovenia/epidemiología , Fiebre del Nilo Occidental/sangre , Fiebre del Nilo Occidental/epidemiología , Virus del Nilo Occidental/clasificación , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/inmunología
11.
Microbiol Resour Announc ; 8(37)2019 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-31515334

RESUMEN

A case of chikungunya virus infection was imported from Thailand into Slovenia in late 2018. The infection was diagnosed using real-time reverse transcription-PCR, the virus was isolated in cell culture, and the whole genome was sequenced. Phylogenetic analysis of the nearly complete viral genome indicated that the virus belongs to the Indian Ocean lineage but does not possess the A226V mutation in the envelope protein E1.

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