RESUMO
Dengue has become one of the vector-borne diseases that affect humans worldwide. In Latin American countries, Colombia is historically one of the most affected by epidemics of this flavivirus. The underreporting of signs and symptoms of probable cases of dengue, the lack of characterization of the serotypes of the infection, and the few detailed studies of postmortem necropsies of patients are among other conditions that have delayed progress in the knowledge of the pathogenesis of the disease. This study presents the results of fragment sequencing assays on paraffin-embedded tissue samples from fatal DENV cases during the 2010 epidemic in Colombia. We found that the predominant serotype was DENV-2, with the Asian/American genotype of lineages 1 and 2. This work is one of the few reports of the circulating genotypes of dengue during the 2010 epidemic in Colombia, one of the most lethal dates in the country's history.
Assuntos
Vírus da Dengue , Dengue , Humanos , Vírus da Dengue/genética , Dengue/epidemiologia , Parafina , Genótipo , Filogenia , SorogrupoRESUMO
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
Coronavirus disease (COVID-19) in Colombia was first diagnosed in a traveler arriving from Italy on February 26, 2020. However, limited data are available on the origins and number of introductions of COVID-19 into the country. We sequenced the causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), from 43 clinical samples we collected, along with another 79 genome sequences available from Colombia. We investigated the emergence and importation routes for SARS-CoV-2 into Colombia by using epidemiologic, historical air travel, and phylogenetic observations. Our study provides evidence of multiple introductions, mostly from Europe, and documents >12 lineages. Phylogenetic findings validate the lineage diversity, support multiple importation events, and demonstrate the evolutionary relationship of epidemiologically linked transmission chains. Our results reconstruct the early evolutionary history of SARS-CoV-2 in Colombia and highlight the advantages of genome sequencing to complement COVID-19 outbreak investigations.
Assuntos
COVID-19/epidemiologia , COVID-19/virologia , Genoma Viral , Genômica/métodos , Filogenia , SARS-CoV-2/genética , Colômbia/epidemiologia , Humanos , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Knockdown resistance (kdr) is a well-characterized target-site insecticide resistance mechanism that is associated with DDT and pyrethroid resistance. Even though insecticide resistance to pyrethroids and DDT have been reported in Anopheles albimanus, Anopheles benarrochi sensu lato (s.l.), Anopheles darlingi, Anopheles nuneztovari s.l., and Anopheles pseudopunctipennis s.l. malaria vectors in Latin America, there is a knowledge gap on the role that kdr resistance mechanisms play in this resistance. The aim of this study was to establish the role that kdr mechanisms play in pyrethroid and DDT resistance in the main malaria vectors in Colombia, in addition to previously reported metabolic resistance mechanisms, such as mixed function oxidases (MFO) and nonspecific esterases (NSE) enzyme families. METHODS: Surviving (n = 62) and dead (n = 67) An. nuneztovari s.l., An. darlingi and An. albimanus mosquitoes exposed to diagnostic concentrations of DDT and pyrethroid insecticides were used to amplify and sequence a ~ 225 bp fragment of the voltage-gated sodium channels (VGSC) gene. This fragment spanning codons 1010, 1013 and 1014 at the S6 segment of domain II to identify point mutations, which have been associated with insecticide resistance in different species of Anopheles malaria vectors. RESULTS: No kdr mutations were detected in the coding sequence of this fragment in 129 samples, 62 surviving mosquitoes and 67 dead mosquitoes, of An. darlingi, An. nuneztovari s.l. and An. albimanus. CONCLUSION: Mutations in the VGSC gene, most frequently reported in other species of the genus Anopheles resistant to pyrethroid and DDT, are not associated with the low-intensity resistance detected to these insecticides in some populations of the main malaria vectors in Colombia. These results suggest that metabolic resistance mechanisms previously reported in these populations might be responsible for the resistance observed.
Assuntos
Anopheles/genética , DDT/farmacologia , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Mosquitos Vetores/genética , Piretrinas/farmacologia , Animais , Anopheles/efeitos dos fármacos , Colômbia , Malária , Especificidade da EspécieRESUMO
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
Dengue is the most important arthropod-borne viral infection of humans. However, its viral pathogenesis is still unknown. The information collected from dengue fatal cases is crucial for understanding the complex interactions between virulence and host factors. This study aimed to establish possible associations between the clinical characteristics, histopathological changes, replication, and tissue location of viral serotypes in dengue fatal cases. Clinical and histopathological characterizations, antigen localization in tissue, and detection of the infecting serotype and replication using real-time polymerase chain reaction were all performed on the dengue fatal cases. The majority of the cases involved people under the age of 20. Bleeding (48.3%), abdominal pain (44.8%), myalgia (52.9%), and headache (48.3%) were the most common clinical manifestations in the cases. There was multiorgan pathology, with histopathological changes primarily in the liver, spleen, and lung. Similarly, the viral antigen was found primarily in these organs; however, there were no associations between tissue changes, viral location, infecting serotypes, and replication processes. Dengue infection should be considered a multiorgan disease, the outcome of which is possibly not associated with the infecting viral serotype.
RESUMO
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 , Mpox , Colômbia , Humanos , Masculino , Mpox/diagnóstico , Mpox/epidemiologia , Mpox/patologia , Monkeypox virus/genética , Filogenia , Análise de Sequência de DNARESUMO
The E484K mutation at the SARS-CoV-2 Spike protein emerged independently in different variants around the world and has been widely associated with immune escape from neutralizing antibodies generated during previous infection or vaccination. In this work, the B.1â¯+â¯L249S+E484K lineage was isolated along with A.1, B.1.420, and B.1.111 SARS-CoV-2 lineages without the E484K mutation and the neutralizing titer of convalescent sera was compared using microneutralization assays. While no significant differences in the neutralizing antibody titers were found between A.1 and B.lineages without the E484K mutation, the neutralizing titers against B.1â¯+â¯L249S+E484K were 1.5, 1.9, 2.1, and 1.3-fold lower than against A.1, B.1.420, B.1.111-I, and B.1.111-II, respectively. However, molecular epidemiological data indicate that there is no increase in the transmissibility rate associated with this new lineage. This study supports the capability of new variants with the E484K mutation to be resistant to neutralization by humoral immunity, and therefore the need to intensify surveillance programs to determine if these lineages represent a risk for public health.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Glicoproteína da Espícula de Coronavírus , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/imunologia , Humanos , Imunidade Humoral , Mutação , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Several SARS-CoV-2 variants of concern (VOC) and interest (VOI) co-circulate in Colombia, and determining the neutralizing antibody (nAb) responses is useful to improve the efficacy of COVID-19 vaccination programs. Thus, nAb responses against SARS-CoV-2 isolates from the lineages B.1.111, P.1 (Gamma), B.1.621 (Mu), AY.25.1 (Delta), and BA.1 (Omicron), were evaluated in serum samples from immunologically naïve individuals between 9 and 13 weeks after receiving complete regimens of CoronaVac, BNT162b2, ChAdOx1, or Ad26.COV2.S, using microneutralization assays. An overall reduction of the nAb responses against Mu, Delta, and Omicron, relative to B.1.111 and Gamma was observed in sera from vaccinated individuals with BNT162b2, ChAdOx1, and Ad26.COV2.S. The seropositivity rate elicited by all the vaccines against B.1.111 and Gamma was 100%, while for Mu, Delta, and Omicron ranged between 32 to 87%, 65 to 96%, and 41 to 96%, respectively, depending on the vaccine tested. The significant reductions in the nAb responses against the last three dominant SARS-CoV-2 lineages in Colombia indicate that booster doses should be administered following complete vaccination schemes to increase the nAb titers against emerging SARS-CoV-2 lineages.
RESUMO
BACKGROUND: The higher number of cases and deaths caused by COVID-19 in Colombia occurred during the third epidemic peak, where the Mu variant was associated with 50% of the cases. OBJECTIVE: To evaluate the association between the clinical outcome of COVID-19 with health conditions and SARS-CoV-2 lineages. METHODS: In this study, clinical metadata and SARS-CoV-2 lineages from 535 patients with different degrees of COVID-19 severity were obtained after the SARS-CoV-2 genomic surveillance in Colombia. Then, the associations between these variables were determined using a multidimensional unfolding analysis. RESULTS: Asymptomatic, symptomatic, severe, and deceased outcomes represented 15.2%, 29.7%, 7.3%, and 47.8% of the cases, respectively. Males tend to develop more serious COVID-19, and severe or fatal outcomes were typically observed in patients aged >60 years with comorbidities, including chronic obstructive pulmonary disease, heart disease, kidney disease, obesity, asthma, and smoking history. The SARS-CoV-2 Mu and Gamma variants dominated the third epidemic peak and accounted for most fatal cases with odd ratio values of 128.2 (CI 53.0-310.1) and 18.6 (CI 8.294-41.917). CONCLUSION: This study shows the high impact of SARS-CoV-2 lineages with higher prevalence on public health and the importance of monitoring COVID-19 risk factors to control the associated mortality.
Assuntos
COVID-19 , Epidemias , Masculino , Humanos , SARS-CoV-2/genética , COVID-19/epidemiologia , Colômbia/epidemiologiaRESUMO
Global surveillance programs for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are showing the emergence of variants with mutations in the spike protein. Genomic and laboratory surveillance are important to determine if these variants may be more infectious or less susceptible to antiviral treatments and vaccine-induced antibodies. Three of the most predominant SARS-CoV-2 variants in Colombia during the epidemiological peaks of 2021 were isolated: Mu, a variant of interest; Gamma, a variant of concern; B.1.111, which lacks genetic markers associated with greater virulence. Microneutralization assays were performed by incubating 120 mean tissue culture infectious doses (TCID50) of each SARS-CoV-2 isolate with five two-fold serial dilutions of sera from 31 BNT162b2-vaccinated volunteers. The mean neutralization titer (MN50) was calculated by the Reed-Muench method. At the end of August, Mu represented 49% of coronavirus disease 2019 (COVID-19) cases in Colombia, followed by 25% of Gamma. In contrast, B.1.111 became almost undetectable. The evaluation of neutralizing antibodies suggests that patients vaccinated with BNT162b2 generate neutralizing antibody titers against the Mu variant at significantly lower concentrations relative to B.1.111 and Gamma. This study shows the importance of continuing surveillance programs of emerging variants, as well as the need to evaluate the neutralizing antibody response induced by other vaccines.
RESUMO
Coronavirus disease 2019 (COVID-19) is transmitted person-to-person mainly by close contact or droplets from respiratory tract. However, the actual time of viral shedding is still uncertain as well as the different routes of transmission. We aimed to characterize RNA shedding from nasopharyngeal and rectal samples in prolonged cases of mild COVID-19 in young male soldiers. Seventy patients from three different military locations were monitored after recommending to follow more strict isolation measures to prevent the spread of the virus. Then, nasopharyngeal, rectal, and blood samples were taken. SARS-CoV-2 RNA was detected by RT-PCR and specific antibodies by chemiluminescent immunoassays. The median nucleic acid conversion time (NACT) was 60 days (IQR: 7-85 days). Rectal swabs were taken in 60â% of patients. Seven patients (10â%) were positive in nasopharyngeal and rectal swabs, and five (7.14â%) remained positive in rectal swabs, but negative in nasopharyngeal samples. Four patients (5.71â%) that had been discharged, were positive again after 15 days. No significant difference was found in nucleic acid conversion time between age groups nor clinical classification. Maintaining distancing among different positive patients is essential as a possible re-exposure to the virus could cause a longer nucleic acid conversion time in SARS-COV-2 infections.
Assuntos
Anticorpos Antivirais/sangue , COVID-19 , Imunoglobulina G/sangue , RNA Viral/análise , COVID-19/diagnóstico , COVID-19/prevenção & controle , Surtos de Doenças , Humanos , Masculino , Militares , SARS-CoV-2 , Eliminação de Partículas ViraisRESUMO
The real-time reverse transcription-polymerase chain reaction (real-time RT-qPCR) has become a leading technique for the detection and quantification of arboviruses, including Chikungunya, Dengue, and Zika viruses. In this study, an updated real-time RT-qPCR assay was designed and evaluated together with a synthetic positive-control chimeric RNA for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses. Amplification assays were performed to verify the construct integrity and optimal reaction/thermal cycling conditions. The analytical sensitivity of the assay was determined for each virus in single and multiplex reactions, as well as the performance in the detection and viral load quantification of experimental samples. The real-time RT-qPCR assay presented here allowed for the simultaneous detection and quantification of Chikungunya, Dengue, and Zika viruses and could be applied in several studies where the accurate quantification of viral genomes is required.
Assuntos
Vírus Chikungunya/isolamento & purificação , Vírus da Dengue/isolamento & purificação , Testes Diagnósticos de Rotina/instrumentação , Reação em Cadeia da Polimerase/instrumentação , Zika virus/isolamento & purificação , Febre de Chikungunya/diagnóstico , Dengue/diagnóstico , Humanos , Infecção por Zika virus/diagnósticoRESUMO
COVID-19 pandemics has led to genetic diversification of SARS-CoV-2 and the appearance of variants with potential impact in transmissibility and viral escape from acquired immunity. We report a new and highly divergent lineage containing 21 distinctive mutations (10 non-synonymous, eight synonymous, and three substitutions in non-coding regions). The amino acid changes L249S and E484K located at the CTD and RBD of the Spike protein could be of special interest due to their potential biological role in the virus-host relationship. Further studies are required for monitoring the epidemiologic impact of this new lineage.
RESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic diversity has the potential to impact the virus transmissibility and the escape from natural infection- or vaccine-elicited neutralizing antibodies. Here, representative samples from circulating SARS-CoV-2 in Colombia between January and April 2021, were processed for genome sequencing and lineage determination following the nanopore amplicon ARTIC network protocol and PANGOLIN pipeline. This strategy allowed us to identify the emergence of the B.1.621 lineage, considered a variant of interest (VOI) with the accumulation of several substitutions affecting the Spike protein, including the amino acid changes I95I, Y144T, Y145S and the insertion 146 N in the N-terminal domain, R346K, E484K and N501Y in the Receptor binding Domain (RBD) and P681H in the S1/S2 cleavage site of the Spike protein. The rapid increase in frequency and fixation in a relatively short time in Magdalena, Atlantico, Bolivar, Bogotá D.C, and Santander that were near the theoretical herd immunity suggests an epidemiologic impact. Further studies will be required to assess the biological and epidemiologic roles of the substitution pattern found in the B.1.621 lineage.
Assuntos
Substituição de Aminoácidos , COVID-19/epidemiologia , Genoma Viral , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/transmissão , COVID-19/virologia , Colômbia/epidemiologia , Monitoramento Epidemiológico , Evolução Molecular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Filogenia , Filogeografia , Domínios Proteicos , SARS-CoV-2/classificação , SARS-CoV-2/patogenicidade , Índice de Gravidade de DoençaRESUMO
The COVID-19 pandemic caused by SARS-CoV-2 is a public health problem on a scale unprecedented in the last 100 years, as has been the response focused on the rapid genomic characterization of SARS-CoV-2 in virtually all regions of the planet. This pandemic emerged during the era of genomic epidemiology, a science fueled by continued advances in next-generation sequencing. Since its recent appearance, genomic epidemiology included the precise identification of new lineages or species of pathogens and the reconstruction of their genetic variability in real time, evidenced in past outbreaks of influenza H1N1, MERS, and SARS. However, the global and uncontrolled scale of this pandemic created a scenario where genomic epidemiology was put into practice en masse, from the rapid identification of SARS-CoV-2 to the registration of new lineages and their active surveillance throughout the world. Prior to the COVID-19 pandemic, the availability of genomic data on circulating pathogens in several Latin America and the Caribbean countries was scarce or nil. With the arrival of SARS-CoV-2, this scenario changed significantly, although the amount of available information remains scarce and, in countries such as Colombia, Brazil, Argentina, and Chile, the genomic information of SARS-CoV-2 was obtained mainly by research groups in genomic epidemiology rather than the product of a public health surveillance policy or program. This indicates the need to establish public health policies aimed at implementing genomic epidemiology as a tool to strengthen surveillance and early warning systems against threats to public health in the region.
La pandemia de COVID-19 causada por el SARS-CoV-2 es un problema de salud pública sin precedentes en los últimos 100 años, así como la respuesta centrada en la caracterización genómica del SARS-CoV-2 prácticamente en todas las regiones del planeta. Esta pandemia surgió durante la era de la epidemiología genómica impulsada por los continuos avances en la secuenciación de próxima generación. Desde su reciente aparición, la epidemiología genómica permitió la identificación precisa de nuevos linajes o especies de agentes patógenos y la reconstrucción de su variabilidad genética en tiempo real, lo que se hizo evidente en los brotes de influenza H1N1, MERS y SARS. Sin embargo, la escala global y descontrolada de esta pandemia ha generado una situación que obligó a utilizar de forma masiva herramientas de la epidemiología genómica como la rápida identificación del SARS-CoV-2 y el registro de nuevos linajes y su vigilancia activa en todo el mundo. Antes de la pandemia de COVID-19 la disponibilidad e datos genómicos de agentes patógenos circulantes en varios países de Latinoamérica y el Caribe era escasa o nula. Con la llegada del SARS-CoV-2 dicha situación cambió significativamente, aunque la cantidad de información disponible sigue siendo escasa y, en países como Colombia, Brasil, Argentina y Chile, la información genómica del SARS-CoV-2 provino principalmente de grupos de investigación en epidemiología genómica más que como producto de una política o programa de vigilancia en salud pública.
Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/epidemiologia , Genoma Viral , Disseminação de Informação , Epidemiologia Molecular/tendências , Pandemias , Pneumonia Viral/epidemiologia , Vigilância da População , RNA Viral/genética , Análise de Sequência de RNA , Sequência de Bases , COVID-19 , Região do Caribe , Doenças Transmissíveis Emergentes , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Planejamento em Desastres , Surtos de Doenças , Humanos , América Latina/epidemiologia , Epidemiologia Molecular/métodos , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Utilização de Procedimentos e Técnicas , Saúde Pública , RNA-Seq , SARS-CoV-2 , Desenvolvimento Sustentável , Viroses/epidemiologiaRESUMO
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.
Assuntos
Vírus da Dengue/genética , Dengue/virologia , RNA Viral/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Vírus da Dengue/metabolismo , Genoma Viral , Humanos , Poliadenilação , RNA Viral/metabolismoRESUMO
Introduction: SARS-CoV-2 has been identified as the new coronavirus causing an outbreak of acute respiratory disease in China in December, 2019. This disease, currently named COVID-19, has been declared as a pandemic by the World Health Organization (WHO). The first case of COVID-19 in Colombia was reported on March 6, 2020. Here we characterize an early SARS-CoV-2 isolate from the pandemic recovered in April, 2020. Objective: To describe the isolation and characterization of an early SARS-CoV-2 isolate from the epidemic in Colombia. Materials and methods: A nasopharyngeal specimen from a COVID-19 positive patient was inoculated on different cell lines. To confirm the presence of SARS-CoV-2 on cultures we used qRT-PCR, indirect immunofluorescence assay, transmission and scanning electron microscopy, and next-generation sequencing. Results: We determined the isolation of SARS-CoV-2 in Vero-E6 cells by the appearance of the cytopathic effect three days post-infection and confirmed it by the positive results in the qRT-PCR and the immunofluorescence with convalescent serum. Transmission and scanning electron microscopy images obtained from infected cells showed the presence of structures compatible with SARS-CoV-2. Finally, a complete genome sequence obtained by next-generation sequencing allowed classifying the isolate as B.1.5 lineage. Conclusion: The evidence presented in this article confirms the first isolation of SARSCoV-2 in Colombia. In addition, it shows that this strain behaves in cell culture in a similar way to that reported in the literature for other isolates and that its genetic composition is consistent with the predominant variant in the world. Finally, points out the importance of viral isolation for the detection of neutralizing antibodies, for the genotypic and phenotypic characterization of the strain and for testing compounds with antiviral potential.
Introducción. El nuevo coronavirus causante de un brote de enfermedad respiratoria aguda en China en diciembre de 2019 se identificó como SARS-CoV-2. La enfermedad, denominada COVID-19, fue declarada pandemia por la Organización Mundial de la Salud (OMS). El primer caso de COVID-19 en Colombia se reportó el 6 de marzo de 2020; en este estudio se caracterizó un aislamiento temprano del virus SARS-CoV-2 de una muestra ecolectada en abril de 2020. Objetivos. Describir y caracterizar una cepa temprana a partir de un aislamiento de SARSCoV-2 durante la pandemia en Colombia. Materiales y métodos. Se obtuvo una muestra de un paciente con COVID-19 confirmada por qRT-PCR; la muestra fue inoculada en diferentes líneas celulares hasta la aparición del efecto citopático. Para confirmar la presencia de SARS-CoV-2 en el cultivo, se utilizó la qRT-PCR a partir de los sobrenadantes, la inmunofluorescencia indirecta (IFI) en células Vero-E6, así como microscopía electrónica y secuenciación de nueva generación (nextgeneration sequencing). Resultados. Se confirmó el aislamiento de SARS-CoV-2 en células Vero-E6 por la aparición del efecto citopático tres días después de la infección, así como mediante la qRT-PCR y la IFI positiva con suero de paciente convaleciente positivo para SARS-CoV-2. Además, en las imágenes de microscopía electrónica de trasmisión y de barrido de células infectadas se observaron estructuras compatibles con viriones de SARS-CoV-2. Por último, se obtuvo la secuencia completa del genoma, lo que permitió clasificar el aislamiento como linaje B.1.5. Conclusiones. La evidencia presentada en este artículo permite confirmar el primer aislamiento de SARS-CoV-2 en Colombia. Además, muestra que esta cepa se comporta en cultivo celular de manera similar a lo reportado en la literatura para otros aislamientos y que su composición genética está acorde con la variante predominante en el mundo. Finalmente, se resalta la importancia que tiene el aislamiento viral para la detección de anticuerpos, para la caracterización genotípica y fenotípica de la cepa y para probar compuestos con potencial antiviral.
Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Pandemias , Pneumonia Viral/virologia , RNA Viral/genética , Animais , Betacoronavirus/genética , Betacoronavirus/fisiologia , Betacoronavirus/ultraestrutura , COVID-19 , Chlorocebus aethiops , Colômbia/epidemiologia , Convalescença , Infecções por Coronavirus/epidemiologia , Efeito Citopatogênico Viral , Técnica Indireta de Fluorescência para Anticorpo , Genoma Viral , Humanos , Microscopia Eletrônica , Tipagem Molecular , Nasofaringe/virologia , Pneumonia Viral/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2 , Análise de Sequência de RNA , Especificidade da Espécie , Células Vero , Vírion/ultraestrutura , Cultura de VírusRESUMO
Prenatal exposure to Zika virus (ZIKV) is associated with congenital anomalies of the brain and the eye and neurodevelopmental sequelae. The spectrum of disease outcomes may relate to timing of infection as well as genetic and environmental factors. Congenital infections occurring in twin pregnancies can inform the clinical spectrum of these conditions and provide unique information regarding timing of infection and in utero environment with disease pathophysiology. Herein, we report a monozygotic dichorionic-diamniotic twin pregnancy with probable prenatal ZIKV exposure identified through the Colombian ZIKV disease surveillance system. Multidisciplinary clinical evaluations were provided to the twins during their first three years of life through a national program for children with in utero ZIKV exposure. Laboratory evidence of congenital infection as well as microcephaly, brain, eye, and neurodevelopmental compromise related to prenatal ZIKV infection were identified in only one infant of the twin pregnancy. This is the first report of monozygotic twins discordant for Zika-associated birth defects. The evaluation of the pathophysiology of discordance in disease outcome for congenital infections in twin pregnancies may lead to a better understanding of potential complex environmental and genetic interactions between the mother, her offspring, and an infectious exposure.
RESUMO
SARS-CoV-2 is a new member of the genus Betacoronavirus, responsible for the COVID-19 pandemic. The virus crossed the species barrier and established in the human population taking advantage of the spike protein high affinity for the ACE receptor to infect the lower respiratory tract. The Nucleocapsid (N) and Spike (S) are highly immunogenic structural proteins and most commercial COVID-19 diagnostic assays target these proteins. In an unpredictable epidemic, it is essential to know about their genetic variability. The objective of this study was to describe the substitution frequency of the S and N proteins of SARS-CoV-2 in South America. A total of 504 amino acid and nucleotide sequences of the S and N proteins of SARS-CoV-2 from seven South American countries (Argentina, Brazil, Chile, Ecuador, Peru, Uruguay, and Colombia), reported as of June 3, and corresponding to samples collected between March and April 2020, were compared through substitution matrices using the Muscle algorithm. Forty-three sequences from 13 Colombian departments were obtained in this study using the Oxford Nanopore and Illumina MiSeq technologies, following the amplicon-based ARTIC network protocol. The substitutions D614G in S and R203K/G204R in N were the most frequent in South America, observed in 83% and 34% of the sequences respectively. Strikingly, genomes with the conserved position D614 were almost completely replaced by genomes with the G614 substitution between March to April 2020. A similar replacement pattern was observed with R203K/G204R although more marked in Chile, Argentina and Brazil, suggesting similar introduction history and/or control strategies of SARS-CoV-2 in these countries. It is necessary to continue with the genomic surveillance of S and N proteins during the SARS-CoV-2 pandemic as this information can be useful for developing vaccines, therapeutics and diagnostic tests.