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U1 snRNP (U1), in addition to its splicing role, protects pre-mRNAs from drastic premature termination by cleavage and polyadenylation (PCPA) at cryptic polyadenylation signals (PASs) in introns. Here, a high-throughput sequencing strategy of differentially expressed transcripts (HIDE-seq) mapped PCPA sites genome wide in divergent organisms. Surprisingly, whereas U1 depletion terminated most nascent gene transcripts within ~1 kb, moderate functional U1 level decreases, insufficient to inhibit splicing, dose-dependently shifted PCPA downstream and elicited mRNA 3' UTR shortening and proximal 3' exon switching characteristic of activated immune and neuronal cells, stem cells, and cancer. Activated neurons' signature mRNA shortening could be recapitulated by U1 decrease and antagonized by U1 overexpression. Importantly, we show that rapid and transient transcriptional upregulation inherent to neuronal activation physiology creates U1 shortage relative to pre-mRNAs. Additional experiments suggest cotranscriptional PCPA counteracted by U1 association with nascent transcripts, a process we term telescripting, ensuring transcriptome integrity and regulating mRNA length.
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Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Animais , Linhagem Celular , Drosophila melanogaster , Estudo de Associação Genômica Ampla , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Células NIH 3T3 , Neurônios/metabolismo , Processamento de Terminações 3' de RNA , Splicing de RNARESUMO
Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus recognized by the World Health Organization as an emerging infectious disease of growing concern. Utilizing phylodynamic and phylogeographic methods, we have reconstructed the origin and transmission patterns of SFTSV lineages and the roles demographic, ecological, and climatic factors have played in shaping its emergence and spread throughout Asia. Environmental changes and fluctuations in tick populations, exacerbated by the widespread use of pesticides, have contributed significantly to its geographic expansion. The increased adaptability of Lineage L2 strains to the Haemaphysalis longicornis vector has facilitated the dispersal of SFTSV through Southeast Asia. Increased surveillance and proactive measures are needed to prevent further spread to Australia, Indonesia, and North America.
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Phlebovirus , Filogeografia , Febre Grave com Síndrome de Trombocitopenia , Phlebovirus/genética , Animais , Sudeste Asiático , Febre Grave com Síndrome de Trombocitopenia/virologia , Febre Grave com Síndrome de Trombocitopenia/transmissão , Humanos , Filogenia , Vetores Aracnídeos/virologia , Carrapatos/virologia , Ixodidae/virologia , Espécies IntroduzidasRESUMO
We identified 3 clades of dengue virus serotype 3 belonging to genotype III isolated during 2019-2020 in Jamaica by using whole-genome sequencing and phylogenomic and phylogeographic analyses. The viruses likely originated from Asia in 2014. Newly expanded molecular surveillance efforts in Jamaica will guide appropriate public health responses.
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BACKGROUND: Diagnosis of people living with HIV (PLHIV) is the first step toward achieving the new Fast Track Strategy to end AIDS by 2030: 95-95-95. However, reaching PLHIV is especially difficult in resource-limited settings such as the Democratic Republic of Congo (DRC), where reliable prevalence data is lacking. This study evaluated the prevalence of HIV in patients in the urban Kinshasa area. METHODS: Individuals seeking healthcare were tested for HIV between February 2017 and July 2018 at existing Kinshasa urban clinics. The study was conducted in two phases. Case finding was optimized in a pilot study phase using a modified cell phone-based Open\Data Kit (ODK) collection system. HIV prevalence was then determined from data obtained between March-July of 2018 from 8320 individuals over the age of 18 years receiving care at one of 47 clinics in Kinshasa. RESULTS: The prevalence of HIV in our study was 11.0% (95% CI 10.3-11.6%) overall and 8.14% in the subset of N = 1240 participants who were healthy mothers seeking prenatal care. These results are in sharp contrast to President's Emergency Plan for AIDS Relief (PEPFAR) estimates of 2.86%, but are consistent with data from surrounding countries. CONCLUSION: While this data is sub-national and reflects an urban healthcare setting, given the large population of Kinshasa and rapidly changing age demographics, the results suggest that HIV prevalence in the DRC is substantially higher than previously reported.
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Infecções por HIV , Saúde da População Urbana , Adulto , República Democrática do Congo/epidemiologia , Feminino , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Humanos , Pessoa de Meia-Idade , Projetos Piloto , Gravidez , Cuidado Pré-NatalRESUMO
The prevalence of chronic hepatitis C virus (HCV) and the presence of human pegivirus 2 (HPgV-2) have not been examined in Cameroon, although HCV has been associated with HPgV-2 infections previously. Herein we aimed to characterize the burden and genetic diversity of HCV and the presence of HPgV-2 in Cameroon. Retrospective plasma specimens collected from N = 12 369 consenting subjects in South Cameroon from 2013 to 2016 were included in the study. The majority (97.1%) of participants were patients seeking health care. All specimens were screened for HCV using the Abbott RealTime HCV viral load assay and positive specimens with remaining volume were also screened for HPgV-2 antibodies on the Abbott ARCHITECT instrument, followed by molecular characterization. Overall, HCV RNA was detected in 305 (2.47%; 95% CI: 2.21%-2.75%) specimens. Notably, the prevalence of HCV RNA was 9.09% amongst participants over age 40 and 3.81% amongst males. Phylogenetic classification of N = 103 HCV sequences identified genotypes 1 (19.4%), 2 (15.5%) and 4 (65.1%) within the study cohort. Amongst HCV RNA-positive specimens, N = 28 (10.6%; 95% CI: 7.44%-14.90%) specimens also had detectable HPgV-2 antibodies. Of these, N = 2 viremic HPgV-2 infections were confirmed by sequencing and shared 93-94 median % identity with strains found on other continents. This is the first study to determine the prevalence of chronic HCV in Cameroon, and the discovery of HPgV-2 in this study cohort expands the geography of HPgV-2 to the African continent, indicating a widespread distribution exists.
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Anticorpos Antivirais/sangue , Monitoramento Epidemiológico , Infecções por Flaviviridae/epidemiologia , Flaviviridae/isolamento & purificação , Hepatite C Crônica/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Camarões/epidemiologia , Criança , Pré-Escolar , Coinfecção/epidemiologia , Coinfecção/virologia , Feminino , Flaviviridae/genética , Infecções por Flaviviridae/sangue , Hepacivirus/genética , Hepatite C Crônica/sangue , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Filogenia , Prevalência , RNA Viral/sangue , Estudos Retrospectivos , Adulto JovemRESUMO
Hepatitis C virus (HCV) and human pegivirus (HPgV), formerly GBV-C, are the only known human viruses in the Hepacivirus and Pegivirus genera, respectively, of the family Flaviviridae. We present the discovery of a second pegivirus, provisionally designated human pegivirus 2 (HPgV-2), by next-generation sequencing of plasma from an HCV-infected patient with multiple bloodborne exposures who died from sepsis of unknown etiology. HPgV-2 is highly divergent, situated on a deep phylogenetic branch in a clade that includes rodent and bat pegiviruses, with which it shares <32% amino acid identity. Molecular and serological tools were developed and validated for high-throughput screening of plasma samples, and a panel of 3 independent serological markers strongly correlated antibody responses with viral RNA positivity (99.9% negative predictive value). Discovery of 11 additional RNA-positive samples from a total of 2440 screened (0.45%) revealed 93-94% nucleotide identity between HPgV-2 strains. All 12 HPgV-2 RNA-positive cases were identified in individuals also testing positive for HCV RNA (12 of 983; 1.22%), including 2 samples co-infected with HIV, but HPgV-2 RNA was not detected in non-HCV-infected individuals (p<0.0001), including those singly infected by HIV (p = 0.0075) or HBV (p = 0.0077), nor in volunteer blood donors (p = 0.0082). Nine of the 12 (75%) HPgV-2 RNA positive samples were reactive for antibodies to viral serologic markers, whereas only 28 of 2,429 (1.15%) HPgV-2 RNA negative samples were seropositive. Longitudinal sampling in two individuals revealed that active HPgV-2 infection can persist in blood for at least 7 weeks, despite the presence of virus-specific antibodies. One individual harboring both HPgV-2 and HCV RNA was found to be seronegative for both viruses, suggesting a high likelihood of simultaneous acquisition of HCV and HPgV-2 infection from an acute co-transmission event. Taken together, our results indicate that HPgV-2 is a novel bloodborne infectious virus of humans and likely transmitted via the parenteral route.
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Infecções por Flaviviridae/virologia , Vírus GB C/genética , Hepacivirus/genética , Hepatite C/virologia , Hepatite Viral Humana/virologia , Sequência de Bases , Coinfecção/genética , Coinfecção/virologia , Feminino , Infecções por Flaviviridae/genética , Hepatite C/genética , Hepatite Viral Humana/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e.,switchingmechanismat 5' end ofRNAtranscript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median â¼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance.
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Genoma Viral , Infecções por HIV/virologia , HIV/classificação , HIV/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Plasma/virologia , Análise de Sequência de DNA/métodos , Camarões , Genótipo , HIV/genética , Humanos , FilogeniaRESUMO
A novel blood-borne human pegivirus (HPgV), HPgV-2, was recently identified in hepatitis C virus (HCV)-infected individuals and individuals who had received multiple transfusions. Robust serological assays capable of detecting antibodies in HPgV-2-infected individuals are needed to establish global seroprevalence rates and potential disease associations. The two objectives of this study were to determine the utility of mammalian cell-expressed HPgV-2 E2 glycoprotein or bacterium-expressed nonstructural protein 4AB (NS4AB) in detecting past or present infections and to compare the total prevalence (antibody and RNA positive) of HPgV-2 with that of the other human pegivirus, HPgV-1 (GB virus C [GBV-C]). HPgV-2 E2 antibodies were detected in 13 (92.86%) of 14 HPgV-2-viremic cases, and NS4AB antibodies were detected in 8 (57.14%) of 14 cases. The HPgV-2 seroprevalence was significantly higher (P < 0.0001) among HCV-infected individuals (3.31% [24 of 726 samples]) than among non-HCV-infected individuals (0.30% [4 of 1,348 samples]). Of 31 anti-E2-positive samples, 22 had supplemental supporting data; 12 samples were HPgV-2 RNA positive and 10 nonviremic samples were antibody positive for peptides or NS4AB. The total prevalence of HPgV-1 (35.00%) was significantly higher than that of HPgV-2 (1.33%) in all populations tested (P < 0.0001). For HPgV-1, codetection of antibodies to E2 and RNA was infrequent (5.88%). In contrast, antibodies to E2 were detected in most HPgV-2-viremic individuals (92.86%), as is observed among individuals chronically infected with HCV, most of whom are antibody positive for HCV E2. Our studies indicate that HPgV-2 circulates with HCV and displays a profile similar to the serological profile of HCV-infected persons, although the pathogenicity of this virus has yet to be established.
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Anticorpos Antivirais/sangue , Infecções por Flaviviridae/epidemiologia , Infecções por Flaviviridae/virologia , Flaviviridae/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Infecções por Flaviviridae/imunologia , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Estudos SoroepidemiológicosRESUMO
In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA-pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.
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Poliadenilação , Precursores de RNA/metabolismo , Splicing de RNA , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Pareamento de Bases , Sequência de Bases , Células HeLa , Humanos , Íntrons/genética , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Poliadenilação/efeitos dos fármacos , Poliadenilação/genética , Piranos/farmacologia , Precursores de RNA/genética , Splicing de RNA/efeitos dos fármacos , Splicing de RNA/genética , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/antagonistas & inibidores , Ribonucleoproteína Nuclear Pequena U1/genética , Compostos de Espiro/farmacologiaRESUMO
The motor neuron (MN) degenerative disease, spinal muscular atrophy (SMA) is caused by deficiency of SMN (survival motor neuron), a ubiquitous and indispensable protein essential for biogenesis of snRNPs, key components of pre-mRNA processing. However, SMA's hallmark MN pathology, including neuromuscular junction (NMJ) disruption and sensory-motor circuitry impairment, remains unexplained. Toward this end, we used deep RNA sequencing (RNA-seq) to determine if there are any transcriptome changes in MNs and surrounding spinal cord glial cells (white matter, WM) microdissected from SMN-deficient SMA mouse model at presymptomatic postnatal day 1 (P1), before detectable MN pathology (P4-P5). The RNA-seq results, previously unavailable for SMA at any stage, revealed cell-specific selective mRNA dysregulations (~300 of 11,000 expressed genes in each, MN and WM), many of which are known to impair neurons. Remarkably, these dysregulations include complete skipping of agrin's Z exons, critical for NMJ maintenance, strong up-regulation of synapse pruning-promoting complement factor C1q, and down-regulation of Etv1/ER81, a transcription factor required for establishing sensory-motor circuitry. We propose that dysregulation of such specific MN synaptogenesis genes, compounded by many additional transcriptome abnormalities in MNs and WM, link SMN deficiency to SMA's signature pathology.
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Regulação da Expressão Gênica/fisiologia , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Proteínas do Complexo SMN/deficiência , Sinapses/fisiologia , Transcriptoma/genética , Animais , Sequência de Bases , Complemento C1q/genética , Proteínas de Ligação a DNA/genética , Imunofluorescência , Humanos , Camundongos , Dados de Sequência Molecular , Neuroglia/metabolismo , RNA Mensageiro/metabolismo , Proteínas do Complexo SMN/metabolismo , Análise de Sequência de RNA , Sinapses/genética , Fatores de Transcrição/genéticaRESUMO
Introduction: Mayaro Fever (MF) is a tropical disease caused by the Mayaro virus (MAYV), with outbreaks documented in Latin America. Methods: A hospital-based fever surveillance in Leticia, Colombian Amazon, collected sera from 1,460 patients aged 5-89 between December 2020 and April 2023. Results: Dengue and malaria were the main diagnoses (19.4 and 5.8%, respectively), leaving 71.4% of cases unidentified after testing. Metagenomic sequencing and real-time RT-qPCR testing identified MAYV in two patients (25-year-old male and an 80-year-old female) exhibiting typical symptoms, of MF including rash, joint pain, and fever. Phylogenetics analysis of these two viruses revealed a close relationship to Peruvian strains within the MAYV D genotype. Discussion: The study of AFI in Leticia, Colombia, identified dengue as prevalent, with malaria, COVID-19, Influenza, and Zika viruses also detected. Despite extensive testing, most cases remained unexplained until metagenomic sequencing revealed MAYV, previously unseen in Colombia but known in neighboring countries. Conclusion: This study presents the first near full-length genomes of MAYV in Colombia, highlighting the need for further seroprevalence studies and enhanced surveillance to understand and control the spread of the virus in the region.
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Introduction: Acute febrile illnesses (AFI) in developing tropical and sub-tropical nations are challenging to diagnose due to the numerous causes and non-specific symptoms. The proliferation of rapid diagnostic testing and successful control campaigns against malaria have revealed that non-Plasmodium pathogens still contribute significantly to AFI burden. Thus, a more complete understanding of local trends and potential causes is important for selecting the correct treatment course, which in turn will reduce morbidity and mortality. Next-generation sequencing (NGS) in a laboratory setting can be used to identify known and novel pathogens in individuals with AFI. Methods: In this study, plasma was collected from 228 febrile patients tested negative for malaria at clinics across Senegal from 2020-2022. Total nucleic acids were extracted and converted to metagenomic NGS libraries. To identify viral pathogens, especially those present at low concentration, an aliquot of each library was processed with a viral enrichment panel and sequenced. Corresponding metagenomic libraries were also sequenced to identify non-viral pathogens. Results and Discussion: Sequencing reads for pathogens with a possible link to febrile illness were identified in 51/228 specimens, including (but not limited to): Borrelia crocidurae (N = 7), West Nile virus (N = 3), Rickettsia felis (N = 2), Bartonella quintana (N = 1), human herpesvirus 8 (N = 1), and Saffold virus (N = 1). Reads corresponding to Plasmodium falciparum were detected in 19 specimens, though their presence in the cohort was likely due to user error of rapid diagnostic testing or incorrect specimen segregation at the clinics. Mosquito-borne pathogens were typically detected just after the conclusion of the rainy season, while tick-borne pathogens were mostly detected before the rainy season. The three West Nile virus strains were phylogenetically characterized and shown to be related to both European and North American clades. Surveys such as this will increase the understanding of the potential causes of non-malarial AFI, which may help inform diagnostic and treatment options for clinicians who provide care to patients in Senegal.
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Mining the genome sequence of Burkholderia thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster resembling that of FR901464 (4), a prototype spliceosome inhibitor produced by Pseudomonas sp. No. 2663. Transcriptional analysis revealed a cultivation condition in which a regulatory gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, named thailanstatins A (1), B (2), and C (3), were isolated from the fermentation broth of B. thailandensis MSMB43. Thailanstatins are proposed to be biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase pathway. They differ from 4 by lacking an unstable hydroxyl group and by having an extra carboxyl moiety; those differences endow thailanstatins with a significantly greater stability than 4 as tested in phosphate buffer at pH 7.4. In vitro assays showed that thailanstatins inhibit pre-mRNA splicing as potently as 4, with half-maximal inhibitory concentrations in the single to sub-µM range. Cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range. This work provides new chemical entities for research and development and new structure-activity information for chemical optimization of related spliceosome inhibitors.
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Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Burkholderia/química , Piranos/isolamento & purificação , Piranos/farmacologia , Antineoplásicos/química , Ensaios de Seleção de Medicamentos Antitumorais , Genômica , Humanos , Família Multigênica , Pseudomonas/química , Piranos/química , Precursores de RNA/efeitos dos fármacos , Compostos de Espiro/química , Compostos de Espiro/isolamento & purificação , Relação Estrutura-AtividadeRESUMO
IMPORTANCE: Picobirnaviruses (PBVs) are highly heterogeneous viruses encoding a capsid and RdRp. Detected in a wide variety of animals with and without disease, their association with gastrointestinal and respiratory infections, and consequently their public health importance, has rightly been questioned. Determining the "true" host of Picobirnavirus lies at the center of this debate, as evidence exists for them having both vertebrate and prokaryotic origins. Using integrated and time-stamped phylogenetic approaches, we show they are contemporaneous viruses descending from two different ancestors: avian Reovirus and fungal Partitivirus. The fungal PBV-R2 species emerged with a single segment (RdRp) until it acquired a capsid from vertebrate PBV-R1 and PBV-R3 species. Protein and RNA folding analyses revealed how the former came to resemble the latter over time. Thus, parallel evolution from disparate hosts has driven the adaptation and genetic diversification of the Picobirnaviridae family.
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Picobirnavirus , Infecções por Vírus de RNA , Animais , Filogenia , Picobirnavirus/genética , Fezes , Infecções por Vírus de RNA/veterinária , Proteínas do Capsídeo/genética , RNA Polimerase Dependente de RNA/genéticaRESUMO
Characterized by high genetic diversity, broad host range, and resistance to adverse conditions, coupled with recent reports of neurotropic astroviruses circulating in humans, mamastroviruses pose a threat to public health. The current astrovirus classification system based on host source prevents determining whether strains with distinct tropism or virulence are emerging. By using integrated phylogeny, we propose a standardized demarcation of species and genotypes, with reproducible cut-off values that reconcile the pairwise sequence distribution, genetic distances between lineages, and the topological reconstruction of the Mamastrovirus genus. We further define the various links established by co-evolution and resolve the dynamics of transmission chains to identify host-jump events and the sources from which different mamastrovirus species circulating in humans have emerged. We observed that recombination is relatively infrequent and restricted to within genotypes. The well-known "human" astrovirus, defined here as mamastrovirus species 7, has co-speciated with humans, while there have been two additional host-jumps into humans from distinct hosts. Newly defined species 6 genotype 2, linked to severe gastroenteritis in children, resulted from a marmot to human jump taking place â¼200 years ago while species 6 genotype 7 (MastV-Sp6Gt7), linked to neurological disease in immunocompromised patients, jumped from bovines only â¼50 years ago. Through demographic reconstruction, we determined that the latter reached coalescent viral population growth only 20 years ago and is evolving at a much higher evolutionary rate than other genotypes infecting humans. This study constitutes mounting evidence of MastV-Sp6Gt7 active circulation and highlights the need for diagnostics capable of detecting it.
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Infecções por Astroviridae , Astroviridae , Gastroenterite , Mamastrovirus , Criança , Humanos , Animais , Bovinos , Mamastrovirus/genética , Infecções por Astroviridae/epidemiologia , Filogenia , FezesRESUMO
Metagenomic next-generation sequencing (mNGS) has enabled the high-throughput multiplexed identification of sequences from microbes of potential medical relevance. This approach has become indispensable for viral pathogen discovery and broad-based surveillance of emerging or re-emerging pathogens. From 2015 to 2019, plasma was collected from 9586 individuals in Cameroon and the Democratic Republic of the Congo enrolled in a combined hepatitis virus and retrovirus surveillance program. A subset (n = 726) of the patient specimens was analyzed by mNGS to identify viral co-infections. While co-infections from known blood-borne viruses were detected, divergent sequences from nine poorly characterized or previously uncharacterized viruses were also identified in two individuals. These were assigned to the following groups by genomic and phylogenetic analyses: densovirus, nodavirus, jingmenvirus, bastrovirus, dicistrovirus, picornavirus, and cyclovirus. Although of unclear pathogenicity, these viruses were found circulating at high enough concentrations in plasma for genomes to be assembled and were most closely related to those previously associated with bird or bat excrement. Phylogenetic analyses and in silico host predictions suggested that these are invertebrate viruses likely transmitted through feces containing consumed insects or through contaminated shellfish. This study highlights the power of metagenomics and in silico host prediction in characterizing novel viral infections in susceptible individuals, including those who are immunocompromised from hepatitis viruses and retroviruses, or potentially exposed to zoonotic viruses from animal reservoir species.
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Quirópteros , Coinfecção , Viroses , Vírus , Animais , Vírus Satélites/genética , Metagenômica , Filogenia , Vírus/genética , Retroviridae/genética , Vírus de Hepatite/genética , Insetos/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Dengue virus (DENV) is the etiological agent of dengue fever (DF), which is among the most prevalent vector-borne diseases in the tropics. In 2022, the Colombian health surveillance system reported more than 69,000 cases of DF. As part of a hospital-based fever surveillance study, acute-phase sera were collected from 4,545 patients with suspected dengue between 2020 and 2023 in three municipalities of Colombia. Combined reverse transcription-polymerase chain reaction and antigen rapid testing confirmed that 376 patients (8.3%) had DF. The virus was isolated in cell culture from 166 of these patients (44.1%), and genome sequencing was performed successfully on 122 (73.5%). Three DENV serotypes (1, 2, and 3) were identified. Phylogenetic analyses of the DENV-2 sequences revealed that 42 of 50 of the isolates (84%) belonged to the DENV-2 cosmopolitan genotype lineage, clustering with sequences from Asia, Peru, and Brazil. We report the detection, isolation, and whole-genome sequencing (11 Kb) of the DENV-2 cosmopolitan genotype and its recent introduction to Colombia.
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Vírus da Dengue , Dengue , Humanos , Sorogrupo , Filogenia , Colômbia/epidemiologia , GenótipoRESUMO
The first 18 months of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections in Colombia were characterized by three epidemic waves. During the third wave, from March through August 2021, intervariant competition resulted in Mu replacing Alpha and Gamma. We employed Bayesian phylodynamic inference and epidemiological modeling to characterize the variants in the country during this period of competition. Phylogeographic analysis indicated that Mu did not emerge in Colombia but acquired increased fitness there through local transmission and diversification, contributing to its export to North America and Europe. Despite not having the highest transmissibility, Mu's genetic composition and ability to evade preexisting immunity facilitated its domination of the Colombian epidemic landscape. Our results support previous modeling studies demonstrating that both intrinsic factors (transmissibility and genetic diversity) and extrinsic factors (time of introduction and acquired immunity) influence the outcome of intervariant competition. This analysis will help set practical expectations about the inevitable emergences of new variants and their trajectories. IMPORTANCE Before the appearance of the Omicron variant in late 2021, numerous SARS-CoV-2 variants emerged, were established, and declined, often with different outcomes in different geographic areas. In this study, we considered the trajectory of the Mu variant, which only successfully dominated the epidemic landscape of a single country: Colombia. We demonstrate that Mu competed successfully there due to its early and opportune introduction time in late 2020, combined with its ability to evade immunity granted by prior infection or the first generation of vaccines. Mu likely did not effectively spread outside of Colombia because other immune-evading variants, such as Delta, had arrived in those locales and established themselves first. On the other hand, Mu's early spread within Colombia may have prevented the successful establishment of Delta there. Our analysis highlights the geographic heterogeneity of early SARS-CoV-2 variant spread and helps to reframe the expectations for the competition behaviors of future variants.
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COVID-19 , Humanos , Teorema de Bayes , COVID-19/epidemiologia , Colômbia/epidemiologia , SARS-CoV-2/genéticaRESUMO
Pathogens carried by insects, such as bunyaviruses, are frequently transmitted into human populations and cause diseases. Knowing which spillover events represent a public health threat remains a challenge. Metagenomic next-generation sequencing (mNGS) can support infectious disease diagnostics by enabling the detection of any pathogen from clinical specimens. mNGS was performed on blood samples to identify potential viral coinfections in human immunodeficiency virus (HIV)-positive individuals from Kinshasa, the Democratic Republic of the Congo (DRC), participating in an HIV diversity cohort study. Time-resolved phylogenetics and molecular assay development assisted in viral characterization. The nearly complete genome of a novel orthobunyavirus related to Nyangole virus, a virus previously identified in neighboring Uganda, was assembled from a hepatitis B virus-positive patient. A quantitative polymerase chain reaction assay was designed and used to screen >2,500 plasma samples from Cameroon, the DRC, and Uganda, failing to identify any additional cases. The recent sequencing of a US Center for Disease Control Arbovirus Reference Collection revealed that this same virus, now named Bangui virus, was first isolated in 1970 from an individual in the Central African Republic. Time-scaled phylogenetic analyses of Bangui with the related Anopheles and Tanga serogroup complexes indicate that this virus emerged nearly 10,000 years ago. Pervasive and episodic models further suggest that this virus is under purifying selection and that only distant common ancestors were subject to positive selection events. This study represents only the second identification of a Bangui virus infection in over 50 years. The presumed rarity of Bangui virus infections in humans can be explained by its constraint to an avian host and insect vector, precluding efficient transmission into the human population. Our results demonstrate that molecular phylogenetic analyses can provide insights into the threat posed by novel or re-emergent viruses identified by mNGS.
RESUMO
Arbovirus infections are frequent causes of acute febrile illness (AFI) in tropical countries. We conducted health facility-based AFI surveillance at four sites in Colombia (Cucuta, Cali, Villavicencio, Leticia) during 2019-2022. Demographic, clinical and risk factor data were collected from persons with AFI that consented to participate in the study (n = 2,967). Serologic specimens were obtained and tested for multiple pathogens by RT-PCR and rapid test (Antigen/IgM), with 20.7% identified as dengue positive from combined testing. Oropouche virus (OROV) was initially detected in serum by metagenomic next-generation sequencing (mNGS) and virus target capture in a patient from Cúcuta. Three additional infections from Leticia were confirmed by conventional PCR, sequenced, and isolated in tissue culture. Phylogenetic analysis determined there have been at least two independent OROV introductions into Colombia. To assess OROV spread, a RT-qPCR dual-target assay was developed which identified 87/791 (10.9%) viremic cases in AFI specimens from Cali (3/53), Cucuta (3/19), Villavicencio (38/566), and Leticia (43/153). In parallel, an automated anti-nucleocapsid antibody assay detected IgM in 27/503 (5.4%) and IgG in 92/568 (16.2%) patients screened, for which 24/68 (35.3%) of PCR positives had antibodies. Dengue was found primarily in people aged <18 years and linked to several clinical manifestations (weakness, skin rash and petechiae), whereas Oropouche cases were associated with the location, climate phase, and odynophagia symptom. Our results confirm OROV as an emerging pathogen and recommend increased surveillance to determine its burden as a cause of AFI in Colombia.