RESUMO
A methodological approach based on reverse transcription (RT)-multiplex PCR followed by next-generation sequencing (NGS) was implemented to identify multiple respiratory RNA viruses simultaneously. A convenience sampling from respiratory surveillance and SARS-CoV-2 diagnosis in 2020 and 2021 in Montevideo, Uruguay, was analyzed. The results revealed the cocirculation of SARS-CoV-2 with human rhinovirus (hRV) A, B and C, human respiratory syncytial virus (hRSV) B, influenza A virus, and metapneumovirus B1. SARS-CoV-2 coinfections with hRV or hRSV B and influenza A virus coinfections with hRV C were identified in adults and/or children. This methodology combines the benefits of multiplex genomic amplification with the sensitivity and information provided by NGS. An advantage is that additional viral targets can be incorporated, making it a helpful tool to investigate the cocirculation and coinfections of respiratory viruses in pandemic and post-pandemic contexts.
Assuntos
COVID-19 , Coinfecção , Vírus da Influenza A , Influenza Humana , Vírus de RNA , Vírus Sincicial Respiratório Humano , Infecções Respiratórias , Criança , Adulto , Humanos , COVID-19/diagnóstico , COVID-19/epidemiologia , Pandemias , RNA , Teste para COVID-19 , Coinfecção/diagnóstico , Coinfecção/epidemiologia , SARS-CoV-2/genética , Vírus de RNA/genética , Vírus Sincicial Respiratório Humano/genética , Vírus da Influenza A/genética , Sequenciamento de Nucleotídeos em Larga Escala , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/epidemiologia , Influenza Humana/epidemiologiaRESUMO
Deletions frequently occur in the six accessory genes of SARS-CoV-2, but most genomes with deletions are sporadic and have limited spreading capability. Here, we analyze deletions in the ORF7a of the N.7 lineage, a unique Uruguayan clade from the Brazilian B.1.1.33 lineage. Thirteen samples collected during the early SARS-CoV-2 wave in Uruguay had deletions in the ORF7a. Complete genomes were obtained by Illumina next-generation sequencing, and deletions were confirmed by Sanger sequencing and capillary electrophoresis. The N.7 lineage includes several individuals with a 12-nucleotide deletion that removes four amino acids of the ORF7a. Notably, four individuals underwent an additional 68-nucleotide novel deletion that locates 44 nucleotides downstream in the terminal region of the same ORF7a. The simultaneous occurrence of the 12 and 68-nucleotide deletions fuses the ORF7a and ORF7b, two contiguous accessory genes that encode transmembrane proteins with immune-modulation activity. The fused ORF retains the signal peptide and the complete Ig-like fold of the 7a protein and the transmembrane domain of the 7b protein, suggesting that the fused protein plays similar functions to original proteins in a single format. Our findings evidence the remarkable dynamics of SARS-CoV-2 and the possibility that single and consecutive deletions occur in accessory genes and promote changes in the genomic organization that help the virus explore genetic variations and select for new, higher fit changes.
Assuntos
COVID-19/virologia , Linhagem da Célula , Deleção de Genes , Genoma Viral , Fases de Leitura Aberta/genética , SARS-CoV-2/genética , Proteínas Virais/genética , Adulto , Idoso , COVID-19/epidemiologia , COVID-19/genética , Criança , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , SARS-CoV-2/classificação , SARS-CoV-2/isolamento & purificação , Uruguai/epidemiologiaRESUMO
Alphaviruses (Togaviridae) are arthropod-borne viruses responsible for several emerging diseases, maintained in nature through transmission between hematophagous arthropod vectors and susceptible vertebrate hosts. Although bats harbor many species of viruses, their role as reservoir hosts in emergent zoonoses has been verified only in a few cases. With bats being the second most diverse order of mammals, their implication in arbovirus infections needs to be elucidated. Reports on arbovirus infections in bats are scarce, especially in South American indigenous species. In this work, we report the genomic detection and identification of two different alphaviruses in oral swabs from bats captured in Northern Uruguay. Phylogenetic analysis identified Río Negro virus (RNV) in two different species: Tadarida brasiliensis (n = 6) and Myotis spp. (n = 1) and eastern equine encephalitis virus (EEEV) in Myotis spp. (n = 2). Previous studies of our group identified RNV and EEEV in mosquitoes and horse serology, suggesting that they may be circulating in enzootic cycles in our country. Our findings reveal that bats can be infected by these arboviruses and that chiropterans could participate in the viral natural cycle as virus amplifiers or dead-end hosts. Further studies are warranted to elucidate the role of these mammals in the biological cycle of these alphaviruses in Uruguay.
Assuntos
Infecções por Alphavirus/veterinária , Alphavirus/isolamento & purificação , Arbovírus/isolamento & purificação , Quirópteros/virologia , Vírus da Encefalite Equina do Leste/isolamento & purificação , Alphavirus/classificação , Alphavirus/genética , Infecções por Alphavirus/virologia , Animais , Infecções por Arbovirus/veterinária , Infecções por Arbovirus/virologia , Arbovírus/classificação , Arbovírus/genética , Vírus da Encefalite Equina do Leste/classificação , Vírus da Encefalite Equina do Leste/genética , Filogenia , UruguaiRESUMO
BACKGROUND: Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in non-structural, structural, and accessory open reading frames (ORFs) or genes. OBJECTIVES: We track indels in accessory ORFs to infer evolutionary gene patterns and epidemiological links between outbreaks. METHODS: Genomes from Coronavirus disease 2019 (COVID-19) case-patients were Illumina sequenced using ARTIC_V3. The assembled genomes were analysed to detect substitutions and indels. FINDINGS: We reported the emergence and spread of a unique 4-nucleotide deletion in the accessory ORF6, an interesting gene with immune modulation activity. The deletion in ORF6 removes one repeat unit of a two 4-nucleotide repeat, which shows that directly repeated sequences in the SARS-CoV-2 genome are associated with indels, even outside the context of extended repeat regions. The 4-nucleotide deletion produces a frameshifting change that results in a protein with two inserted amino acids, increasing the coding information of this accessory ORF. Epidemiological and genomic data indicate that the deletion variant has a single common ancestor and was initially detected in a health care outbreak and later in other COVID-19 cases, establishing a transmission cluster in the Uruguayan population. MAIN CONCLUSIONS: Our findings provide evidence for the origin and spread of deletion variants and emphasise indels' importance in epidemiological studies, including differentiating consecutive outbreaks occurring in the same health facility.
Assuntos
COVID-19 , Fases de Leitura Aberta , SARS-CoV-2 , COVID-19/epidemiologia , COVID-19/virologia , Genoma Viral , Humanos , SARS-CoV-2/genética , Deleção de Sequência , Uruguai/epidemiologiaRESUMO
Zika virus (ZIKV) is an arthropod-born virus that is mainly transmitted to humans by mosquitoes of the genus Aedes spp. Since its first isolation in 1947, only a few human cases had been described until large outbreaks occurred on Yap Island (2007), French Polynesia (2013), and Brazil (2015). Most ZIKV-infected individuals are asymptomatic or present with a self-limiting disease and nonspecific symptoms such as fever, myalgia, and headache. However, in French Polynesia and Brazil, ZIKV outbreaks led to the diagnosis of congenital malformations and microcephaly in newborns and Guillain-Barré syndrome (GBS) in adults. These new clinical presentations raised concern from public health authorities and highlighted the need for anti-Zika treatments and vaccines to control the neurological damage caused by the virus. Despite many efforts in the search for an effective treatment, neither vaccines nor antiviral drugs have become available to control ZIKV infection and/or replication. Flavonoids, a class of natural compounds that are well-known for possessing several biological properties, have shown activity against different viruses. Additionally, the use of flavonoids in some countries as food supplements indicates that these molecules are nontoxic to humans. Thus, here, we summarize knowledge on the use of flavonoids as a source of anti-ZIKV molecules and discuss the gaps and challenges in this area before these compounds can be considered for further preclinical and clinical trials.
RESUMO
Bats are the second most diverse order of mammals and key species for ecosystem functioning, providing a wide range of ecosystem services, from pest control to seed dispersal. Chiropterans are known for hosting a large diversity of viruses, in some cases with little or no effect to their health. Here, we report on the results of a screening for DNA (Herpesviridae) and RNA viruses (Rhabdovirus and Pneumovirus), finding a high prevalence and wide diversity of both Beta- and Gamma-Herpesvirus in insectivorous and hematophagous bats of the southern cone of South America. Our findings suggest that bats in the southern neotropics harbor a high diversity of herpesviruses and, at least in some cases, the viral community in the bat species is more strongly associated with ecological traits of the hosts, rather than their taxonomy. The presence of a separate clade into the Gammaherpesvirinae subfamily in the common vampire bat suggests the independent circulation of herpesviruses in hematophagous and insectivorous bats and highlights the properness of these viruses to track vampire bats' population structure for rabies studies. Hence, we suggest that as other pathogens viruses may be used to track the population dynamics of their hosts, including movement and demographics.
Assuntos
Quirópteros , Infecções por Herpesviridae , Herpesviridae , Animais , Ecossistema , Infecções por Herpesviridae/epidemiologia , Infecções por Herpesviridae/veterinária , FilogeniaRESUMO
Two severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants associated with increased transmission and immune evasion, P.1 and P.2, emerged in Brazil and spread throughout South America. Here, we report genomes corresponding to these variants that were recently detected in Uruguay. These P.1 and P.2 genomes share all substitutions that are characteristic of these variants.
RESUMO
The analysis of genetic diversity in SARS-CoV-2 is the focus of several studies, providing insights into how the virus emerged and evolves. Most common changes in SARS-CoV-2 are single or point nucleotide substitutions; meanwhile, insertions and deletions (indels) have been identified as a less frequent source of viral genetic variability. Here, we report the emergence of a 12-nucleotide deletion in ORF7a, resulting in a 4-amino acid in-frame deletion. The Δ12 variant was identified in viruses from patients of a single outbreak and represents the first report of this deletion in South American isolates. Phylogenetic analysis revealed that Δ12 strains belong to the lineage B.1.1 and clustered separated from the remaining Uruguayan strains. The ∆12 variant was detected in 14 patients of this outbreak by NGS sequencing and/or two rapid and economic methodologies: Sanger amplicon sequencing and capillary electrophoresis. The presence of strong molecular markers as the deletion described here are useful for tracking outbreaks and reveal a significant aspect of the SARS-CoV-2 evolution on the robustness of the virus to keep its functionality regardless loss of genetic material.
Assuntos
COVID-19 , SARS-CoV-2 , Deleção de Sequência , COVID-19/virologia , Surtos de Doenças , Genoma Viral , Humanos , Filogenia , SARS-CoV-2/genética , Uruguai/epidemiologiaRESUMO
BACKGROUND Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in non-structural, structural, and accessory open reading frames (ORFs) or genes. OBJECTIVES We track indels in accessory ORFs to infer evolutionary gene patterns and epidemiological links between outbreaks. METHODS Genomes from Coronavirus disease 2019 (COVID-19) case-patients were Illumina sequenced using ARTIC_V3. The assembled genomes were analysed to detect substitutions and indels. FINDINGS We reported the emergence and spread of a unique 4-nucleotide deletion in the accessory ORF6, an interesting gene with immune modulation activity. The deletion in ORF6 removes one repeat unit of a two 4-nucleotide repeat, which shows that directly repeated sequences in the SARS-CoV-2 genome are associated with indels, even outside the context of extended repeat regions. The 4-nucleotide deletion produces a frameshifting change that results in a protein with two inserted amino acids, increasing the coding information of this accessory ORF. Epidemiological and genomic data indicate that the deletion variant has a single common ancestor and was initially detected in a health care outbreak and later in other COVID-19 cases, establishing a transmission cluster in the Uruguayan population. MAIN CONCLUSIONS Our findings provide evidence for the origin and spread of deletion variants and emphasise indels' importance in epidemiological studies, including differentiating consecutive outbreaks occurring in the same health facility.
RESUMO
Alphaviruses (Togaviridae) are arboviruses frequently associated with emerging infectious diseases. In this study, we aimed to investigate the presence of alphaviruses in Uruguay by detecting the viral genome in mosquitoes and neutralizing antibodies in equines. A total of 3,575 mosquitoes were analyzed for alphavirus genome detection. Serologic studies were performed on 425 horse sera by plaque reduction neutralization test (PRNT80) against Venezuelan equine encephalitis virus (VEEV) subtype IAB, Pixuna virus (PIXV), Rio Negro virus (RNV), western equine encephalitis virus (WEEV), and Madariaga virus (MADV). Mosquitoes belonging to six genera were captured and 82.9% were identified as Culex pipiens. Two Cx. pipiens pools collected in Fray Bentos and Las Toscas localities were alphavirus positive, and phylogenetic analyses showed that the sequences grouped into two different clusters: the lineage I of eastern equine encephalitis virus and RNV (VEEV complex), respectively. Plaque reduction neutralization test assays showed antibodies against strains of the VEEV complex, MADV, and WEEV. Rio Negro virus was the most geographically widespread virus, showing higher seroprevalences (up to 20%). Seroprevalences against VEEV IAB ranged between 4.6% and 13%; antibodies against PIXV, WEEV, and MADV were less frequent (3-4%). In conclusion, RNV exhibited the highest seroprevalence in horses, a wide geographical distribution, and viral genome was detected in Cx. pipiens mosquitoes. Madariaga virus had a low seroprevalence in equines, but an epizootic lineage typical of North America was detected in Cx. pipiens mosquitoes. Taken together, our results show that alphaviruses are present in Uruguay with variable occurrence and geographical distribution being a potential threat for human and equine health.