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1.
Drug Discov Ther ; 15(5): 268-272, 2021 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-34707021

RESUMO

The inhibitory activity of electrolyzed reduced water (ERW) against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is the etiological agent responsible for coronavirus disease 2019 (COVID-19), was tested in vitro on Vero E6 cells using a plaque assay. Infectious virus titers of cells treated with ERW 100%, 50% and 33.3% solutions and phosphate buffered saline (PBS, negative control) and exposed to the virus suspension for 60 seconds were 2.25, 2.65, 3.21 and 7.38, respectively. ERW has a high pH and low surface tension. It is considered that the alkaline property of ERW breaks down phospholipids and proteins of envelopes. The role of pH and reducibility on the virucidal effect of ERW should be further evaluated. This study provides a foundation for utilizing ERW as an effective antiviral aqueous solution in a variety of applications.


Assuntos
Antivirais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Água/farmacologia , Animais , Chlorocebus aethiops , Concentração de Íons de Hidrogênio , Células Vero/virologia , Ensaio de Placa Viral
2.
J Photochem Photobiol B ; 224: 112319, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34598020

RESUMO

The germicidal properties of short wavelength ultraviolet C (UVC) light are well established and used to inactivate many viruses and other microbes. However, much less is known about germicidal effects of terrestrial solar UV light, confined exclusively to wavelengths in the UVA and UVB regions. Here, we have explored the sensitivity of the human coronaviruses HCoV-NL63 and SARS-CoV-2 to solar-simulated full spectrum ultraviolet light (sUV) delivered at environmentally relevant doses. First, HCoV-NL63 coronavirus inactivation by sUV-exposure was confirmed employing (i) viral plaque assays, (ii) RT-qPCR detection of viral genome replication, and (iii) infection-induced stress response gene expression array analysis. Next, a detailed dose-response relationship of SARS-CoV-2 coronavirus inactivation by sUV was elucidated, suggesting a half maximal suppression of viral infectivity at low sUV doses. Likewise, extended sUV exposure of SARS-CoV-2 blocked cellular infection as revealed by plaque assay and stress response gene expression array analysis. Moreover, comparative (HCoV-NL63 versus SARS-CoV-2) single gene expression analysis by RT-qPCR confirmed that sUV exposure blocks coronavirus-induced redox, inflammatory, and proteotoxic stress responses. Based on our findings, we estimate that solar ground level full spectrum UV light impairs coronavirus infectivity at environmentally relevant doses. Given the urgency and global scale of the unfolding SARS-CoV-2 pandemic, these prototype data suggest feasibility of solar UV-induced viral inactivation, an observation deserving further molecular exploration in more relevant exposure models.


Assuntos
Infecções por Coronavirus/prevenção & controle , Coronavirus Humano NL63/efeitos da radiação , Infecções Respiratórias/prevenção & controle , SARS-CoV-2/efeitos da radiação , Luz Solar , Raios Ultravioleta , Animais , Linhagem Celular , Chlorocebus aethiops , Coronavirus Humano NL63/fisiologia , Células Epiteliais/virologia , Genoma Viral/efeitos da radiação , Humanos , SARS-CoV-2/fisiologia , Transcriptoma/efeitos da radiação , Ensaio de Placa Viral , Inativação de Vírus/efeitos da radiação , Replicação Viral/efeitos da radiação
3.
Virus Res ; 305: 198563, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34530046

RESUMO

This study compared the lethality of severe acute respiratory syndrome coronavirus 2 variants belonging to the S, V, L, G, GH, and GR clades using K18-human angiotensin-converting enzyme 2 heterozygous mice. To estimate the 50% lethal dose (LD50) of each variant, increasing viral loads (100-104 plaque-forming units [PFU]) were administered intranasally. Mouse weight and survival were monitored for 14 days. The LD50 of the GH and GR clades was significantly lower than that of other clades at 50 PFU. These findings suggest that the GH and GR clades, which are prevalent worldwide, are more virulent than the other clades.


Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19/mortalidade , Receptores Virais/genética , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Carga Viral/genética , Sequência de Aminoácidos , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Sequência de Bases , Peso Corporal , COVID-19/patologia , COVID-19/virologia , Chlorocebus aethiops , Expressão Gênica , Humanos , Dose Letal Mediana , Masculino , Camundongos , Camundongos Transgênicos , Filogenia , Receptores Virais/metabolismo , SARS-CoV-2/classificação , SARS-CoV-2/metabolismo , Índice de Gravidade de Doença , Análise de Sobrevida , Transgenes , Células Vero , Ensaio de Placa Viral , Virulência
4.
Virol J ; 18(1): 182, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34496903

RESUMO

BACKGROUND: Traditional medicines based on herbal extracts have been proposed as affordable treatments for patients suffering from coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Teas and drinks containing extracts of Artemisia annua and Artemisia afra have been widely used in Africa in efforts to prevent SARS-CoV-2 infection and fight COVID-19. METHODS: The plant extracts and Covid-Organics drink produced in Madagascar were tested for plaque reduction using both feline coronavirus and SARS-CoV-2 in vitro. Their cytotoxicities were also investigated. RESULTS: Several extracts as well as Covid-Organics inhibited SARS-CoV-2 and FCoV infection at concentrations that did not affect cell viability. CONCLUSIONS: Some plant extracts show inhibitory activity against FCoV and SARS-CoV-2. However, it remains unclear whether peak plasma concentrations in humans can reach levels needed to inhibit viral infection following consumption of teas or Covid-Organics. Clinical studies are required to evaluate the utility of these drinks for COVID-19 prevention or treatment of patients.


Assuntos
Antivirais/farmacologia , Artemisia/química , Extratos Vegetais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Coronavirus Felino/efeitos dos fármacos , Coronavirus Felino/crescimento & desenvolvimento , Extratos Vegetais/química , SARS-CoV-2/crescimento & desenvolvimento , Ensaio de Placa Viral
5.
J Gen Virol ; 102(9)2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34486974

RESUMO

Most flaviviruses are transmitted horizontally between vertebrate hosts by haematophagous arthropods. Others exhibit host ranges restricted to vertebrates or arthropods. Vertebrate-specific flaviviruses are commonly referred to as no-known-vector (NKV) flaviviruses and can be separated into bat- and rodent-associated NKV flaviviruses. Rio Bravo virus (RBV) is one of eight recognized bat-associated NKV (B-NKV) flaviviruses. Studies designed to identify the genetic determinants that condition the host range restriction of B-NKV flaviviruses have never been performed. To investigate whether the host range restriction occurs at the level of attachment or entry, chimeric flaviviruses were created by inserting the pre-membrane and envelope protein genes of RBV into the genetic backbones of yellow fever virus (YFV) and Zika virus (ZIKV), two mosquito-borne flaviviruses associated with human disease. The chimeric viruses infected both vertebrate and mosquito cells. In vertebrate cells, all viruses produced similar mean peak titres, but the chimeric viruses grew more slowly than their parental viruses during early infection. In mosquito cells, the chimeric virus of YFV and RBV grew more slowly than YFV at early post-inoculation time points, but reached a similar mean peak titre. In contrast, the chimeric virus of ZIKV and RBV produced a mean peak titre that was approximately 10-fold lower than ZIKV. The chimeric virus of YFV and RBV produced an intermediate plaque phenotype, while the chimeric virus of ZIKV and RBV produced smaller plaques than both parental viruses. To conclude, we provide evidence that the structural glycoproteins of RBV permit entry into both mosquito and vertebrate cells, indicating that the host range restriction of B-NKV flaviviruses is mediated by a post-attachment/entry event.


Assuntos
Flavivirus/fisiologia , Especificidade de Hospedeiro , Internalização do Vírus , Animais , Linhagem Celular , Quirópteros/virologia , Flavivirus/genética , Técnicas de Transferência de Genes , Genes Virais , Genes env , Genoma Viral , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/fisiologia , Carga Viral , Ensaio de Placa Viral , Ligação Viral , Replicação Viral , Vírus da Febre Amarela/genética , Vírus da Febre Amarela/fisiologia , Zika virus/genética , Zika virus/fisiologia
6.
J Gen Virol ; 102(9)2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34546870

RESUMO

Tick-borne encephalitis virus (TBEV), a member of the genus Flavivirus, is common in Europe and Asia and causes a severe disease of the central nervous system. A promising approach in the development of therapy for TBEV infection is the search for small molecule antivirals targeting the flavivirus envelope protein E, particularly its ß-n-octyl-d-glucoside binding pocket (ß-OG pocket). However, experimental studies of candidate antivirals may be complicated by varying amounts and different forms of the protein E in the virus samples. Viral particles with different conformations and arrangements of the protein E are produced during the replication cycle of flaviviruses, including mature, partially mature, and immature forms, as well as subviral particles lacking genomic RNA. The immature forms are known to be abundant in the viral population. We obtained immature virion preparations of TBEV, characterized them by RT-qPCR, and assessed in vivo and in vitro infectivity of the residual mature virions in the immature virus samples. Analysis of the ß-OG pocket structure on the immature virions confirmed the possibility of binding of adamantylmethyl esters of 5-aminoisoxazole-3-carboxylic acid in the pocket. We demonstrated that the antiviral activity of these compounds in plaque reduction assay is significantly reduced in the presence of immature TBEV particles.


Assuntos
Adamantano/farmacologia , Antivirais/farmacologia , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Encefalite Transmitida por Carrapatos/virologia , Isoxazóis/farmacologia , Vírion/fisiologia , Adamantano/metabolismo , Animais , Antivirais/metabolismo , Linhagem Celular , Vírus da Encefalite Transmitidos por Carrapatos/crescimento & desenvolvimento , Vírus da Encefalite Transmitidos por Carrapatos/patogenicidade , Glucosídeos/metabolismo , Isoxazóis/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Suínos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Ensaio de Placa Viral , Vírion/imunologia , Vírion/patogenicidade , Vírion/ultraestrutura
7.
J Virol ; 95(21): e0135721, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34406867

RESUMO

One of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virulence factors is the ability to interact with high affinity to the ACE2 receptor, which mediates viral entry into cells. The results of our study demonstrate that within a few passages in cell culture, both the natural isolate of SARS-CoV-2 and the recombinant cDNA-derived variant acquire an additional ability to bind to heparan sulfate (HS). This promotes a primary attachment of viral particles to cells before their further interactions with the ACE2. Interaction with HS is acquired through multiple mechanisms. These include (i) accumulation of point mutations in the N-terminal domain (NTD) of the S protein, which increases the positive charge of the surface of this domain, (ii) insertions into the NTD of heterologous peptides containing positively charged amino acids, and (iii) mutation of the first amino acid downstream of the furin cleavage site. This last mutation affects S protein processing, transforms the unprocessed furin cleavage site into the heparin-binding peptide, and makes viruses less capable of syncytium formation. These viral adaptations result in higher affinity of viral particles to heparin, dramatic increase in plaque sizes, more efficient viral spread, higher infectious titers, and 2 orders of magnitude higher infectivity. The detected adaptations also suggest an active role of NTD in virus attachment and entry. As in the case of other RNA-positive (RNA+) viruses, evolution to HS binding may result in virus attenuation in vivo. IMPORTANCE The spike protein of SARS-CoV-2 is a major determinant of viral pathogenesis. It mediates binding to the ACE2 receptor and, later, fusion of viral envelope and cellular membranes. The results of our study demonstrate that SARS-CoV-2 rapidly evolves during propagation in cultured cells. Its spike protein acquires mutations in the NTD and in the P1' position of the furin cleavage site (FCS). The amino acid substitutions or insertions of short peptides in NTD are closely located on the protein surface and increase its positive charge. They strongly increase affinity of the virus to heparan sulfate, make it dramatically more infectious for the cultured cells, and decrease the genome equivalent to PFU (GE/PFU) ratio by orders of magnitude. The S686G mutation also transforms the FCS into the heparin-binding peptide. Thus, the evolved SARS-CoV-2 variants efficiently use glycosaminoglycans on the cell surface for primary attachment before the high-affinity interaction of the spikes with the ACE2 receptor.


Assuntos
Evolução Molecular , Heparitina Sulfato/metabolismo , SARS-CoV-2/patogenicidade , Glicoproteína da Espícula de Coronavírus/metabolismo , Adaptação Biológica , Animais , Sítios de Ligação , Chlorocebus aethiops , Efeito Citopatogênico Viral , DNA Complementar , Furina/metabolismo , Heparina/metabolismo , Interações Hospedeiro-Patógeno , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Inoculações Seriadas , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Ensaio de Placa Viral , Ligação Viral
8.
Viruses ; 13(8)2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34452529

RESUMO

An escalating pandemic of the novel SARS-CoV-2 virus is impacting global health, and effective antivirals are needed. Umifenovir (Arbidol) is an indole-derivative molecule, licensed in Russia and China for prophylaxis and treatment of influenza and other respiratory viral infections. It has been shown that umifenovir has broad spectrum activity against different viruses. We evaluated the sensitivity of different coronaviruses, including the novel SARS-CoV-2 virus, to umifenovir using in vitro assays. Using a plaque assay, we revealed an antiviral effect of umifenovir against seasonal HCoV-229E and HCoV-OC43 coronaviruses in Vero E6 cells, with estimated 50% effective concentrations (EC50) of 10.0 ± 0.5 µM and 9.0 ± 0.4 µM, respectively. Umifenovir at 90 µM significantly suppressed plaque formation in CMK-AH-1 cells infected with SARS-CoV. Umifenovir also inhibited the replication of SARS-CoV-2 virus, with EC50 values ranging from 15.37 ± 3.6 to 28.0 ± 1.0 µM. In addition, 21-36 µM of umifenovir significantly suppressed SARS-CoV-2 virus titers (≥2 log TCID50/mL) in the first 24 h after infection. Repurposing of antiviral drugs is very helpful in fighting COVID-19. A safe, pan-antiviral drug such as umifenovir could be extremely beneficial in combating the early stages of a viral pandemic.


Assuntos
Antivirais/farmacologia , Coronavirus Humano 229E/efeitos dos fármacos , Coronavirus Humano OC43/efeitos dos fármacos , Indóis/farmacologia , Vírus da SARS/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Coronavirus Humano 229E/fisiologia , Coronavirus Humano OC43/fisiologia , Efeito Citopatogênico Viral/efeitos dos fármacos , Humanos , Indóis/administração & dosagem , Testes de Sensibilidade Microbiana , Vírus da SARS/fisiologia , SARS-CoV-2/fisiologia , Células Vero , Carga Viral/efeitos dos fármacos , Ensaio de Placa Viral , Replicação Viral/efeitos dos fármacos
9.
J Virol ; 95(19): e0110421, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34232734

RESUMO

Modified vaccinia virus Ankara (MVA) was derived by repeated passaging in chick fibroblasts, during which deletions and mutations rendered the virus unable to replicate in most mammalian cells. Marker rescue experiments demonstrated that the host range defect could be overcome by replacing DNA that had been deleted from near the left end of the genome. One virus isolate, however, recovered the ability to replicate in monkey BS-C-1 cells but not human cells without added DNA, suggesting that it arose from a spontaneous mutation. Here, we showed that variants with enhanced ability to replicate in BS-C-1 cells could be isolated by blind passaging of MVA and that in each there was a point mutation leading to an amino acid substitution in the D10 decapping enzyme. The sufficiency of these single mutations to enhance host range was confirmed by constructing recombinant viruses. The D10 mutations occurred at N- or C-terminal locations distal to the active site, suggesting an indirect effect on decapping or on another previously unknown role of D10. Although increased amounts of viral mRNA and proteins were found in BS-C-1 cells infected with the mutants compared to those with parental MVA, the increases were much less than the 1- to 2-log-higher virus yields. Nevertheless, a contributing role for diminished decapping in overcoming the host range defect was consistent with increased replication and viral protein synthesis in BS-C-1 cells infected with an MVA engineered to have active-site mutations that abrogate decapping activity entirely. Optimal decapping may vary depending on the biological context. IMPORTANCE Modified vaccinia virus Ankara (MVA) is an attenuated virus that is approved as a smallpox vaccine and is in clinical trials as a vector for other pathogens. The safety of MVA is due in large part to its inability to replicate in mammalian cells. Although host range restriction is considered a stable feature of the virus, we describe the occurrence of spontaneous mutations in MVA that increase replication considerably in monkey BS-C-1 cells but only slightly in human cells. The mutants contain single nucleotide changes that lead to amino acid substitutions in one of the two decapping enzymes. Although the spontaneous mutations are distant from the decapping enzyme active site, engineered active-site mutations also increased virus replication in BS-C-1 cells. The effects of these mutations on the immunogenicity of MVA vectors remain to be determined.


Assuntos
Nucleotidases/genética , Nucleotidases/metabolismo , Vírus Vaccinia/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Animais , Domínio Catalítico , Linhagem Celular , Embrião de Galinha , Chlorocebus aethiops , Recombinação Homóloga , Especificidade de Hospedeiro , Humanos , Nucleotidases/química , Fases de Leitura Aberta , Mutação Puntual , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Deleção de Sequência , Vírus Vaccinia/genética , Ensaio de Placa Viral , Proteínas Virais/química , Replicação Viral
10.
J Virol ; 95(19): e0101221, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34260287

RESUMO

Vaccinia virus produces two types of virions known as single-membraned intracellular mature virus (MV) and double-membraned extracellular enveloped virus (EV). EV production peaks earlier when initial MVs are further wrapped and secreted to spread infection within the host. However, late during infection, MVs accumulate intracellularly and become important for host-to-host transmission. The process that regulates this switch remains elusive and is thought to be influenced by host factors. Here, we examined the hypothesis that EV and MV production are regulated by the virus through expression of F13 and the MV-specific protein A26. By switching the promoters and altering the expression kinetics of F13 and A26, we demonstrate that A26 expression downregulates EV production and plaque size, thus limiting viral spread. This process correlates with A26 association with the MV surface protein A27 and exclusion of F13, thus reducing EV titers. Thus, MV maturation is controlled by the abundance of the viral A26 protein, independently of other factors, and is rate limiting for EV production. The A26 gene is conserved within vertebrate poxviruses but is strikingly lost in poxviruses known to be transmitted exclusively by biting arthropods. A26-mediated virus maturation thus has the appearance to be an ancient evolutionary adaptation to enhance transmission of poxviruses that has subsequently been lost from vector-adapted species, for which it may serve as a genetic signature. The existence of virus-regulated mechanisms to produce virions adapted to fulfill different functions represents a novel level of complexity in mammalian viruses with major impacts on evolution, adaptation, and transmission. IMPORTANCE Chordopoxviruses are mammalian viruses that uniquely produce a first type of virion adapted to spread within the host and a second type that enhances transmission between hosts, which can take place by multiple ways, including direct contact, respiratory droplets, oral/fecal routes, or via vectors. Both virion types are important to balance intrahost dissemination and interhost transmission, so virus maturation pathways must be tightly controlled. Here, we provide evidence that the abundance and kinetics of expression of the viral protein A26 regulates this process by preventing formation of the first form and shifting maturation toward the second form. A26 is expressed late after the initial wave of progeny virions is produced, so sufficient viral dissemination is ensured, and A26 provides virions with enhanced environmental stability. Conservation of A26 in all vertebrate poxviruses, but not in those transmitted exclusively via biting arthropods, reveals the importance of A26-controlled virus maturation for transmission routes involving environmental exposure.


Assuntos
Regiões Promotoras Genéticas , Vírus Vaccinia/fisiologia , Proteínas Virais/metabolismo , Animais , Linhagem Celular , Chordopoxvirinae/genética , Chordopoxvirinae/metabolismo , Engenharia Genética , Humanos , Orthopoxvirus/genética , Orthopoxvirus/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Vírus Vaccinia/genética , Ensaio de Placa Viral , Proteínas Virais/genética
11.
Viruses ; 13(6)2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064231

RESUMO

Isolating single phages using plaque assays is a laborious and time-consuming process. Whether single isolated phages are the most lyse-effective, the most abundant in viromes, or those with the highest ability to make plaques in solid media is not well known. With the increasing accessibility of high-throughput sequencing, metaviromics is often used to describe viruses in environmental samples. By extracting and sequencing metaviromes from organic waste with and without exposure to a host-of-interest, we show a host-related phage community's shift, as well as identify the most enriched phages. Moreover, we isolated plaque-forming single phages using the same virome-host matrix to observe how enrichments in liquid media correspond to the metaviromic data. In this study, we observed a significant shift (p = 0.015) of the 47 identified putative Pseudomonas phages with a minimum twofold change above zero in read abundance when adding a Pseudomonas syringae DC3000 host. Surprisingly, it appears that only two out of five plaque-forming phages from the same organic waste sample, targeting the Pseudomonas strain, were highly abundant in the metavirome, while the other three were almost absent despite host exposure. Lastly, our sequencing results highlight how long reads from Oxford Nanopore elevates the assembly quality of metaviromes, compared to short reads alone.


Assuntos
Metagenoma , Metagenômica , Fagos de Pseudomonas/fisiologia , Pseudomonas/virologia , Ensaio de Placa Viral , Viroma , Biologia Computacional , Especificidade de Hospedeiro , Metagenômica/métodos , Fagos de Pseudomonas/classificação , Ensaio de Placa Viral/métodos
12.
Viruses ; 13(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071483

RESUMO

Bluetongue virus (BTV) is a segmented RNA virus transmitted by Culicoides midges. Climatic factors, animal movement, vector species, and viral mutation and reassortment may all play a role in the occurrence of BTV outbreaks among susceptible ruminants. We used two enzootic strains of BTV (BTV-2 and BTV-10) to explore the potential for Culicoides sonorensis, a key North American vector, to be infected with these viruses, and identify the impact of temperature variations on virogenesis during infection. While BTV-10 replicated readily in C. sonorensis following an infectious blood meal, BTV-2 was less likely to result in productive infection at biologically relevant exposure levels. Moreover, when C. sonorensis were co-exposed to both viruses, we did not detect reassortment between the two viruses, despite previous in vitro findings indicating that BTV-2 and BTV-10 are able to reassort successfully. These results highlight that numerous factors, including vector species and exposure dose, may impact the in vivo replication of varying BTV strains, and underscore the complexities of BTV ecology in North America.


Assuntos
Vírus Bluetongue/fisiologia , Bluetongue/virologia , Dípteros/virologia , Temperatura , Animais , Técnicas de Cultura de Células , Linhagem Celular , Suscetibilidade a Doenças , Genótipo , Insetos Vetores/virologia , Vírus Reordenados , Ensaio de Placa Viral , Replicação Viral
13.
J Med Virol ; 93(10): 5917-5923, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34139026

RESUMO

Since the coronavirus disease 2019 (COVID-19) outbreak, laboratory diagnosis has mainly been conducted using reverse-transcription polymerase chain reaction (RT-PCR). Detecting the presence of an infectious virus in the collected sample is essential to analyze if a person can transmit infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, there have been no quantitative investigations conducted for infectious SARS-CoV-2 in clinical samples. Therefore, in the present study, a rapid and simple focus-forming assay using the peroxidase-antiperoxidase technique was developed to quantify infectious SARS-CoV-2 titers in 119 samples (n = 52, nasopharyngeal swabs [NPS]; n = 67, saliva) from patients with COVID-19. Furthermore, the study findings were compared with the cycle threshold (Ct) values of real-time RT-PCR. The infectious virus titers in NPS samples and Ct values were inversely correlated, and no infectious virus could be detected when the Ct value exceeded 30. In contrast, a low correlation was observed between the infectious virus titers in saliva and Ct values (r = -0.261, p = 0.027). Furthermore, the infectious virus titers in the saliva were significantly lower than those in the NPS samples. Ten days after the onset of COVID-19 symptoms, the infectious virus was undetectable, and Ct values were more than 30 in NSP and saliva samples. The results indicate that patients whose symptoms subsided 10 days after onset, with Ct values more than 30 in NSP and saliva samples, were less likely to infect others.


Assuntos
Teste para COVID-19 , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Ensaio de Placa Viral , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/virologia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saliva/virologia , Carga Viral , Adulto Jovem
14.
J Microbiol ; 59(7): 702-707, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34061341

RESUMO

Infection by varicella-zoster virus (VZV) can be prevented by using live attenuated vaccines. VZV vaccine strains are known to evolve rapidly in vivo, however, their genetic and biological effects are not known. In this study, the plaque-purified vaccine strain Suduvax (PPS) was used to understand the genetic changes that occur during the process of propagation in in vitro cell culture. Full genome sequences of three different passages (p4, p30, and p60) of PPS were determined and compared for genetic changes. Mutations were found at 59 positions. The number of genetically polymorphic sites (GPS) and the average of minor allele frequency (MAF) at GPSs were not significantly altered after passaging in cell culture up to p60. The number of variant nucleotide positions (VNPs), wherein GPS was found in at least one passage of PPS, was 149. Overall, MAF changed by less than 5% at 52 VNPs, increased by more than 5% at 42 VNPs, and decreased by more than 5% at 55 VNPs in p60, compared with that seen in p4. More complicated patterns of changes in MAF were observed when genetic polymorphism at 149 VNPs was analyzed among the three passages. However, MAF decreased and mixed genotypes became unequivocally fixed to vaccine type in 23 vaccine-specific positions in higher passages of PPS. Plaque-purified Suduvax appeared to adapt to better replication during in vitro cell culture. Further studies with other vaccine strains and in vivo studies will help to understand the evolution of the VZV vaccine.


Assuntos
Vacina contra Varicela/genética , Herpesvirus Humano 3/crescimento & desenvolvimento , Herpesvirus Humano 3/genética , Polimorfismo Genético , Cultura de Vírus , Linhagem Celular , DNA Viral/genética , Genoma Viral , Herpesvirus Humano 3/isolamento & purificação , Humanos , Mutação , Análise de Sequência de DNA , Ensaio de Placa Viral
15.
Sci Rep ; 11(1): 12173, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108535

RESUMO

One of the serious public health concerns is food contaminated with pathogens and their vital activity products such as toxins. Bacillus cereus group of bacteria includes well-known pathogenic species such as B. anthracis, B. cereus sensu stricto (ss), B. cytotoxicus and B. thuringiensis. In this report, we describe the Bacillus phages vB_BcM_Sam46 and vB_BcM_Sam112 infecting species of this group. Electron microscopic analyses indicated that phages Sam46 and Sam112 have the myovirus morphotype. The genomes of Sam46 and Sam112 comprise double-stranded DNA of 45,419 bp and 45,037 bp in length, respectively, and have the same GC-content. The genome identity of Sam46 and Sam112 is 96.0%, indicating that they belong to the same phage species. According to the phylogenetic analysis, these phages form a distinct clade and may be members of a new phage genus, for which we propose the name 'Samaravirus'. In addition, an interesting feature of the Sam46 and Sam112 phages is the unusual structure of their small terminase subunit containing N-terminal FtsK_gamma domain.


Assuntos
Fagos Bacilares/genética , Bacillus anthracis/virologia , Bacillus cereus/virologia , Bacillus thuringiensis/virologia , Endodesoxirribonucleases/química , Genoma Viral , Sequência de Aminoácidos , Fagos Bacilares/classificação , Fagos Bacilares/enzimologia , Fagos Bacilares/isolamento & purificação , Bacillus anthracis/crescimento & desenvolvimento , Bacillus cereus/crescimento & desenvolvimento , Bacillus thuringiensis/crescimento & desenvolvimento , Composição de Bases , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Filogenia , Homologia de Sequência , Ensaio de Placa Viral
16.
Eur J Pharmacol ; 904: 174144, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-33957087

RESUMO

Zika virus (ZIKV) is a mosquito-borne flavivirus, that could cause congenital Zika syndrome (CZS), characterized by microcephaly, neurological complications and fetal deaths. No specific treatments for ZIKV are currently available, highlighting the urgent global need to identify and develop therapeutic agents. Drug repositioning of approved natural compounds can provide effective alternative solutions for novel antiviral development. The current study focused on curcumin, a component of turmeric known to exert diverse antiviral effects. We integrated in silico information from publicly available databases to predict interactions between curcumin and potential targets of ZIKV. In our network analysis, we identified four targets, TP53, AKT1, PTEN, and TNF, which were identified as potential targets associated with ZIKV. Based on retrieved targets, we performed molecular docking study and identified curcumin-TNF showed the strongest binding among four targets. The anti-Zika effects of curcumin were validated in vitro with the aid of antiviral and plaque reduction assay. Curcumin at concentrations ranging from 12.5 to 50 µM displayed significant antiviral activity in a dose-dependent manner (p < 0.05). In view of its natural abundance and prevalence in the human diet, curcumin holds significant promise for treatment of ZIKV infections.


Assuntos
Antivirais/farmacologia , Curcumina/farmacologia , Infecção por Zika virus/tratamento farmacológico , Zika virus/efeitos dos fármacos , Animais , Antivirais/química , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Simulação por Computador , Curcumina/química , Reposicionamento de Medicamentos , Mapas de Interação de Proteínas , Células Vero , Ensaio de Placa Viral , Ligação Viral/efeitos dos fármacos
17.
J Virol ; 95(12)2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33827947

RESUMO

RNA viruses demonstrate a vast range of variants, called quasispecies, due to error-prone replication by viral RNA-dependent RNA polymerase. Although live attenuated vaccines are effective in preventing RNA virus infection, there is a risk of reversal to virulence after their administration. To test the hypothesis that high-fidelity viral polymerase reduces the diversity of influenza virus quasispecies, resulting in inhibition of reversal of the attenuated phenotype, we first screened for a high-fidelity viral polymerase using serial virus passages under selection with a guanosine analog ribavirin. Consequently, we identified a Leu66-to-Val single amino acid mutation in polymerase basic protein 1 (PB1). The high-fidelity phenotype of PB1-L66V was confirmed using next-generation sequencing analysis and biochemical assays with the purified influenza viral polymerase. As expected, PB1-L66V showed at least two-times-lower mutation rates and decreased misincorporation rates, compared to the wild type (WT). Therefore, we next generated an attenuated PB1-L66V virus with a temperature-sensitive (ts) phenotype based on FluMist, a live attenuated influenza vaccine (LAIV) that can restrict virus propagation by ts mutations, and examined the genetic stability of the attenuated PB1-L66V virus using serial virus passages. The PB1-L66V mutation prevented reversion of the ts phenotype to the WT phenotype, suggesting that the high-fidelity viral polymerase could contribute to generating an LAIV with high genetic stability, which would not revert to the pathogenic virus.IMPORTANCE The LAIV currently in use is prescribed for actively immunizing individuals aged 2 to 49 years. However, it is not approved for infants and elderly individuals, who actually need it the most, because it might prolong virus propagation and cause an apparent infection in these individuals, due to their weak immune systems. Recently, reversion of the ts phenotype of the LAIV strain currently in use to a pathogenic virus was demonstrated in cultured cells. Thus, the generation of mutations associated with enhanced virulence in LAIV should be considered. In this study, we isolated a novel influenza virus strain with a Leu66-to-Val single amino acid mutation in PB1 that displayed a significantly higher fidelity than the WT. We generated a novel LAIV candidate strain harboring this mutation. This strain showed higher genetic stability and no ts phenotype reversion. Thus, our high-fidelity strain might be useful for the development of a safer LAIV.


Assuntos
Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Vacinas contra Influenza , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Substituição de Aminoácidos , Animais , Antivirais/farmacologia , Cães , Farmacorresistência Viral , Vírus da Influenza A/efeitos dos fármacos , Células Madin Darby de Rim Canino , Mutação , Fenótipo , Engenharia de Proteínas , RNA Polimerase Dependente de RNA/química , Ribavirina/farmacologia , Vacinas Atenuadas , Ensaio de Placa Viral , Proteínas Virais/química , Proteínas Virais/metabolismo
18.
Viruses ; 13(3)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803454

RESUMO

Enteric viruses, such as poliovirus, are a leading cause of gastroenteritis, which causes 2-3 million deaths annually. Environmental surveillance of wastewater supplements clinical surveillance for monitoring enteric virus circulation. However, while many environmental surveillance methods require liquid samples, some at-risk locations utilize pit latrines with waste characterized by high solids content. This study's objective was to develop and evaluate enteric virus concentration protocols for high solids content samples. Two existing protocols were modified and tested using poliovirus type 1 (PV1) seeded into primary sludge. Method 1 (M1) utilized acid adsorption, followed by 2 or 3 elutions (glycine/sodium chloride and/or threonine/sodium chloride), and skimmed milk flocculation. Method 2 (M2) began with centrifugation. The liquid fraction was filtered through a ViroCap filter and eluted (beef extract/glycine). The solid fraction was eluted (beef extract/disodium hydrogen phosphate/citric acid) and concentrated by skimmed milk flocculation. Recovery was enumerated by plaque assay. M1 yielded higher PV1 recovery than M2, though this result was not statistically significant (26.1% and 15.9%, respectively). M1 was further optimized, resulting in significantly greater PV1 recovery when compared to the original protocol (p < 0.05). This method can be used to improve understanding of enteric virus presence in communities without liquid waste streams.


Assuntos
Monitoramento Ambiental/métodos , Poliovirus/isolamento & purificação , Esgotos/virologia , Resíduos Sólidos/análise , Carga Viral/métodos , Infecções por Enterovirus/prevenção & controle , Floculação , Gastroenterite/prevenção & controle , Gastroenterite/virologia , Humanos , Poliomielite/prevenção & controle , Ensaio de Placa Viral/métodos , Microbiologia da Água
19.
J Immunol Methods ; 494: 113054, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33845088

RESUMO

Respiratory syncytial virus (RSV) is a common pathogen causing severe respiratory illness in infants and elder adults. The development of an effective RSV vaccine is an important unmet medical need and an area of active research. The traditional method for testing neutralizing antibodies against RSV in clinical trials is the plaque reduction neutralization test (PRNT), which uses 24-well plates and needs several days post infection to develop viral plaques. In this study, we have developed a virus reduction neutralization test (VRNT), which allows the number of RSV infected cells to be automatically counted by an imaging cytometer at one day post infection in 96-well plates. VRNT was found robust to cell seeding density, detection antibody concentration, virus input and infection time. By testing twenty human sera, we have shown good correlation between VRNT50 and PRNT50 titers for multiple RSV strains: A2, Long and 18537 (serotype B). To understand the VRNT performance, eight human serum samples with high, medium and low neutralization titers were selected for VRNT qualification. We have demonstrated that VRNT had good specificity, precision, linearity and relative accuracy. In conclusion, VRNT is a better alternative to PRNT in serum neutralization test for RSV vaccine candidates.


Assuntos
Testes de Neutralização/métodos , Infecções por Vírus Respiratório Sincicial/diagnóstico , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sinciciais Respiratórios/fisiologia , Idoso de 80 Anos ou mais , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Chlorocebus aethiops , Diagnóstico por Imagem , Ensaios de Triagem em Larga Escala , Humanos , Lactente , Recém-Nascido , Miniaturização , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fatores de Tempo , Células Vero , Ensaio de Placa Viral
20.
Mikrochim Acta ; 188(4): 137, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33763734

RESUMO

The novel corona (SARS-CoV-2) virus causes a global pandemic, which motivates researchers to develop reliable and effective methods for screening and detection of SARS-CoV-2. Though there are several methods available for the diagnosis of SARS-CoV-2 such as RT-PCR and ELSIA, nevertheless, these methods are time-consuming and may not apply at the point of care. In this study, we have developed a specific, sensitive, quantitative and fast detection method for SARS-CoV-2 by fluorescence resonance energy transfer (FRET) assay. The total extracellular protease proteolytic activity from the virus has been used as the biomarker. The specific peptide sequences from the library of 115 dipeptides were identified via changes in the fluorescence signal. The fluorogenic dipeptide substrates have the fluorophore and a quencher at the N- and the C- terminals, respectively. When the protease hydrolyzes the peptide bond between the two specific amino acids, it leads to a significant increase in the fluorescence signals. The specific fluorogenic peptide (H-d) produces a high fluorescence signal. A calibration plot was obtained from the changes in the fluorescence intensity against the different concentrations of the viral protease. The lowest limit of detection of this method was 9.7 ± 3 pfu/mL. The cross-reactivity of the SARS-CoV-2-specific peptide was tested against the MERS-CoV which does not affect the fluorescence signal. A significant change in the fluorescence signal with patient samples indicates that this FRET-based assay might be applied for the diagnosis of SARS-CoV-2 patients. Graphical abstract.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , Proteases 3C de Coronavírus/metabolismo , Corantes Fluorescentes/metabolismo , Peptídeos/metabolismo , SARS-CoV-2 , Proteínas Virais/metabolismo , Animais , Bioensaio , COVID-19/microbiologia , Chlorocebus aethiops , Transferência Ressonante de Energia de Fluorescência , Humanos , Biblioteca de Peptídeos , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Células Vero , Ensaio de Placa Viral
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