RESUMO
It is convenient to study complete genome sequences of human respiratory syncytial virus (hRSV) for ongoing genomic characterization and identification of highly transmissible or pathogenic variants. Whole genome sequencing of hRSV has been challenging from respiratory tract specimens with low viral loads. Herein, we describe an amplicon-based protocol for whole genome sequencing of hRSV subgroup A validated with 24 isolates from nasopharyngeal swabs and infected cell cultures, which showed cycle threshold (Ct) values ranging from 10 to 31, as determined by quantitative reverse-transcription polymerase chain reaction. MinION nanopore generated 3200 to 5400 reads per sample to sequence over 93% of the hRSV-A genome. Coverage of each contig ranged from 130× to 200×. Samples with Ct values of 20.9, 25.2, 27.1, 27.7, 28.2, 28.8, and 29.6 led to the sequencing of over 99.0% of the virus genome, indicating high genome coverage even at high Ct values. This protocol enables the identification of hRSV subgroup A genotypes, as primers were designed to target highly conserved regions. Consequently, it holds potential for application in molecular epidemiology and surveillance of this hRSV subgroup.
RESUMO
Current evidence shows higher production of cytokines and antibodies against severe acute respiratory coronavirus 2 (SARS-CoV-2) in severe and critical cases of Coronavirus Disease 2019 (COVID-19) in comparison with patients with moderate or mild disease. A recent hypothesis proposes an important role of genotoxicity and cytotoxicity in the induction of the cytokine storm observed in some patients at later stages of the disease. Interestingly, in this study, we report significantly higher levels of interleukin (IL)-1ß, IL-6, MCP-1, and IL-4 cytokines in mild COVID-19 patients versus severe cases, as well as a high frequency of karyorrhexis (median [Me] = 364 vs. 20 cells) and karyolysis (Me = 266 vs. 52 cells) in the mucosal epithelial cells of both groups of patients compared with uninfected individuals. Although we observed higher levels of anti-SARS-CoV-2 IgM and IgG antibodies in COVID-19 patients, IgM antibodies were significantly higher only in mild cases, for the N and the S viral antigens. High levels of IgG antibodies were observed in both mild and severe cases. Our results showed elevated concentrations of proinflammatory and anti-inflammatory cytokines in mild cases, which may reflect an active innate immune response and could be related to the higher IgM and IgG antibody levels found in those patients. In addition, we found that SARS-CoV-2 infection induces cytotoxic damage in the oral mucosa, highlighting the importance of studying the genotoxic and cytotoxic events induced by infection and its role in the pathophysiology of COVID-19.
Assuntos
COVID-19 , Humanos , Citocinas , SARS-CoV-2 , Anticorpos Antivirais , Imunoglobulina G , Imunoglobulina MRESUMO
Viral persistence alters cellular antiviral activities. Nitric oxide (NO), a highly reactive free radical and a potent antiviral molecule, can inhibit replication of RNA and DNA viruses, but its production and effect during viral persistence are largely unknown. NO synthesis is stimulated in epithelial cells during acute infection with respiratory syncytial virus (RSV) and interferes with viral replication. In this study, we compared the levels of production of NO and expression of its regulatory enzymes, inducible nitric oxide synthase (NOS II) and arginase 1 (Arg-1), during acute and persistent RSV infection in a macrophage cell line to investigate their role in the control and maintenance of viral infection. We observed that NO and NOS II mRNA were induced at higher levels in acutely infected macrophages than in persistently infected macrophages, while the kinetics of NOS II protein expression were similar in both types of infected cultures, except that its disappearance was delayed during acute infection. Thus, NOS II was inducible and expressed at high levels during persistent infection, but production of NO was low relative to acute infection. This was not associated with a lack of enzymatic activity but instead was due to constitutive expression of the Arg-1 enzyme at the mRNA and protein levels, suggesting that arginase restricts availability of L-arginine as a substrate for NOS II to synthesize NO. This hypothesis was supported by showing that arginase enzymatic activity was inhibited in persistently RSV-infected cells by Nω-hydroxy-nor-L-arginine, increasing L-arginine availability in conditioned medium and producing increased levels of nitrites, concurrently with a significant reduction in virus genome replication, implying that Arg-1 overexpression contributes to the maintenance of the RSV genome in the host in persistent infection.
Assuntos
Arginase/metabolismo , Óxido Nítrico/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Arginase/genética , Arginina/metabolismo , Regulação para Baixo , Humanos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Infecções por Vírus Respiratório Sincicial/enzimologia , Infecções por Vírus Respiratório Sincicial/genética , Vírus Sincicial Respiratório Humano/genética , Replicação ViralRESUMO
Cells susceptible to persistent viral infections undergo important changes in their biological functions as a consequence of the expression of viral gene products that are capable of altering the gene expression profile of the host cell. Previously, we reported that persistence of the RSV genome in a mouse macrophage cell line induces important alterations in cell homeostasis, including constitutive expression of IFN-ß and other pro-inflammatory cytokines. Here, we postulated that changes in the homeostasis of non-infected macrophages could be induced by soluble factors secreted by persistently RSV- infected macrophages. To test this hypothesis, non-infected mouse macrophages were treated with conditioned medium (CM) collected from cultures of persistently RSV-infected macrophages. Total RNA was extracted and a microarray-based gene expression analysis was performed. Non-infected macrophages, treated under similar conditions with CM obtained from cultures of non-infected macrophages, were used as a control to establish differential gene expression between the two conditions. Results showed that CM from the persistently RSV-infected cultures altered expression of a total of 95 genes in non-infected macrophages, resulting in an antiviral gene-transcription profile along with inhibition of the inflammatory response, since some inflammatory genes were down-regulated, including Nlrp3 and Il-1 ß, both related to the inflammasome pathway. However, down-regulation of Nlrp3 and Il-1 ß was reversible upon acute RSV infection. Additionally, we observed that the inflammatory response, evaluated by secreted IL-1 ß, a final product of the inflammasome activity, was enhanced during acute RSV infection in macrophages treated with CM from persistently RSV-infected cultures, compared to that in macrophages treated with the control CM. This suggests that soluble factors secreted during RSV persistence may induce an exacerbated inflammatory response in non-infected cells.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Vírus Sinciciais Respiratórios/crescimento & desenvolvimento , Transcrição Gênica/efeitos dos fármacos , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Inflamassomos/antagonistas & inibidores , Inflamassomos/genética , Inflamassomos/imunologia , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Vírus Sinciciais Respiratórios/patogenicidade , Transdução de SinaisRESUMO
Viruses can persist in differentiated cells (i.e., macrophages) over long periods of time, altering host cells functions but not inducing their death. We had previously reported that, in early passages (14-40) of a murine macrophage-like cell line persistently infected with respiratory syncytial virus (RSV) (MfP), FcgR-mediated phagocytosis and expression of FcgRIIB/RIII on the cell membrane were increased with respect to mock-infected macrophages (MfN). In this work, we explored the mechanism underlying such effects. Increases in FcgR expression and FcgR-mediated phagocytosis are preserved after more than 87 passages of the persistently infected culture. We analyzed the expression of FcgR isoforms at both mRNA and protein levels, and found out that RSV persistence distinctly affects the expression of FcgR isoforms. We also observed that the increase in FcgRs expression results neither from soluble factors (cytokines) or viral products released by the infected cells, nor from an increase in the rate of FcgR internalization. Our results suggest that RSV persistence in macrophages induce intracellular effects that have an impact on FcgRs gene expression at both mRNA and protein levels, and that the characteristics of RSV persistence were preserved for over 87 passages.