RESUMO
Zika virus (ZIKV) disease continues to be a threat to public health, and it is estimated that millions of people have been infected and that there have been more cases of serious complications than those already reported. Despite many studies on the pathogenesis of ZIKV, several of the genes involved in the malformations associated with viral infection are still unknown. In this work, the morphological and molecular changes in the cortex and cerebellum of mice infected with ZIKV were evaluated. Neonatal BALB/c mice were inoculated with ZIKV intraperitoneally, and the respective controls were inoculated with a solution devoid of the virus. At day 10 postinoculation, the mice were euthanized to measure the expression of the markers involved in cortical and cerebellar neurodevelopment. The infected mice presented morphological changes accompanied by calcifications, as well as a decrease in most of the markers evaluated in the cortex and cerebellum. The modifications found could be predictive of astrocytosis, dendritic pathology, alterations in the regulation systems of neuronal excitation and inhibition, and premature maturation, conditions previously described in other models of ZIKV infection and microcephaly.
Assuntos
Infecção por Zika virus , Zika virus , Animais , Camundongos , Cerebelo , Gliose , Camundongos Endogâmicos BALB CRESUMO
The spread of Zika virus (ZIKV) from the African continent to the Americas promoted its molecular evolution, as reflected by mutations in its RNA genome. Most of the ZIKV genome sequences in the GenBank database have incomplete 5' and 3' UTR sequences, reflecting the deficiency of whole-genome sequencing technologies to resolve the sequences of the genome ends. We modified a protocol for rapid amplification of cDNA ends (RACE) to determine the complete sequences of the 5' and 3' UTRs of a previously reported ZIKV isolate (GenBank no. MH544701.1). This strategy is useful for determining 5' and 3' UTR sequences of ZIKV isolates and will be useful for comparative genomics applications.
Assuntos
Infecção por Zika virus , Zika virus , Humanos , Zika virus/genética , Regiões 3' não Traduzidas/genética , RNA Viral/genética , Evolução Molecular , Regiões 5' não Traduzidas/genética , Genoma Viral/genéticaRESUMO
Introduction: Monkeypox virus (MPXV) is an enveloped double-stranded DNA virus with a genome of approximately 197.209 bp. The current classification divides MPXV into three clades: Clade I (Central African or Congo Basin clade) and clades IIa and IIb (West African clades). Objective: To report the complete genome and phylogenetic analysis of a human monkeypox case detected in Colombia. Materials and methods: Exudate from vesicular lesions was obtained from a male patient with recent travel history to Spain. A direct genomic approach was implemented in which total DNA from the sample was purified through a column-based method, followed by sequencing on the Nanopore GridION. Reads were aligned against the MPXV reference genome using minimap2 v.2.24 and phylogenetic inference was performed using maximum likelihood estimation. Results: A total of 11.951 reads mapped directly to a reference genome with 96.8% of coverage (190.898 bp). Conclusion: Phylogenetic analysis of the MPXV circulating in Colombia demonstrated its close relationship to clade IIb responsible for the multi-country outbreak in 2022.
Introducción. El virus de la viruela del mono (MPXV) está compuesto por un genoma de ADN bicatenario, aproximadamente, de 197.209 pb. La clasificación actual agrupa el MPXV en tres clados: clado I (de la cuenca del Congo en África central), y clados IIa y IIb (de África occidental). Objetivo. Reportar el genoma completo y el análisis filogenético de un caso humano de viruela símica detectado en Colombia. Materiales y métodos. Se obtuvo exudado de lesiones vesiculares de un paciente varón con el antecedente de un viaje reciente a España. Se implementó un enfoque directo, en el cual se purificó el ADN total de la muestra mediante un método basado en columnas, seguido de la secuenciación directa en la plataforma Nanopore GridION. Las lecturas se alinearon con el genoma de referencia del MPXV, utilizando minimap2, v.2.24, y la inferencia filogenética fue realizada mediante la estimación por máxima verosimilitud. Resultados. Un total de 11.951 lecturas se alinearon directamente con el genoma de referencia con una cobertura del 96,8 % (190.898 pb). Conclusión. El análisis filogenético del MPXV circulante en Colombia demostró su estrecha relación con el clado de África occidental (clado IIb) responsable del brote en múltiples países en el 2022.
Assuntos
Monkeypox virus , Sequenciamento por Nanoporos , Filogenia , ColômbiaRESUMO
Dengue is a mosquito-borne disease that is of major importance in public health. Although it has been extensively studied at the molecular level, sequencing of the 5' and 3' ends of the untranslated regions (UTR) commonly requires specific approaches for completion and corroboration. The present study aimed to characterize the 5' and 3' ends of dengue virus types 1 to 4. The 5' and 3' ends of twenty-nine dengue virus isolates from acute infections were amplified through a modified protocol of the rapid amplification cDNA ends approach. For the 5' end cDNA synthesis, specific anti-sense primers for each serotype were used, followed by polyadenylation of the cDNA using a terminal transferase and subsequent PCR amplification with oligo(dT) and internal specific reverse primer. At the 3' end of the positive-sense viral RNA, an adenine tail was directly synthetized using an Escherichia coli poly(A) polymerase, allowing subsequent hybridization of the oligo(dT) during cDNA synthesis. The incorporation of the poly(A) tail at the 5' and 3' ends of the dengue virus cDNA and RNA, respectively, allowed for successful primer hybridization, PCR amplification and direct sequencing. This approach can be used for completing dengue virus genomes obtained through direct and next-generation sequencing methods.