RESUMO
The coronavirus disease-2019 (COVID-19) pandemic caused by SARS-CoV-2 has exacted an enormous toll on healthcare systems worldwide. The cytokine storm that follows pulmonary infection is causally linked to respiratory compromise and mortality in the majority of patients. The sparsity of viable treatment options for this viral infection and the sequelae of pulmonary complications have fueled the quest for new therapeutic considerations. One such option, the long-forgotten idea of using low-dose radiation therapy, has recently found renewed interest in many academic centers. We outline the scientific and logistical rationale for consideration of this option and the mechanistic underpinnings of any potential therapeutic value, particularly as viewed from an immunological perspective. We also discuss the preliminary and/or published results of prospective trials examining low-dose radiation therapy for COVID-19.
Assuntos
COVID-19/radioterapia , Doses de Radiação , SARS-CoV-2/efeitos da radiação , Internalização do Vírus/efeitos da radiação , Replicação Viral/efeitos da radiação , COVID-19/epidemiologia , COVID-19/virologia , Citocinas/metabolismo , Relação Dose-Resposta à Radiação , Interações Hospedeiro-Patógeno/efeitos da radiação , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos da radiação , Leucócitos Mononucleares/virologia , Pandemias , Dosagem Radioterapêutica , SARS-CoV-2/fisiologiaRESUMO
In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We investigated pH optimum of fusion, temperatures of HA heat inactivation, in vivo and in vitro replication kinetics, and connectivity with panel of sera of survivors patients in different epidemic seasons of the previously obtained influenza H1 escape mutants. Our results showed that N133D (H3 numbering) mutation significantly lowered the pH of fusion optimum. Several amino acid substitutions, including K163â¯N, Q192â¯L, D190E, G228E, and K285â¯M, reduced the stability of HA as determined by heat inactivation, whereas A198E substitution is associated with significant increase in HA thermostability compared to the wild-type virus. We found that amino acid change D190â¯N was associated with a significant decrease in viral growth in eggs and mice. Our potential antigenic variants, except readapted variant, which contained A198E mutation, did not reach fixation in infected people. Overall, a co-variation between antigenic specificity and different HA phenotypic properties was demonstrated.
Assuntos
Substituição de Aminoácidos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Evasão da Resposta Imune , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/fisiologia , Mutação de Sentido Incorreto , Animais , Anticorpos Antivirais/imunologia , Embrião de Galinha , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Concentração de Íons de Hidrogênio , Vírus da Influenza A Subtipo H1N1/imunologia , Camundongos , Estabilidade Proteica , Temperatura , Internalização do Vírus/efeitos dos fármacos , Internalização do Vírus/efeitos da radiação , Replicação Viral/efeitos dos fármacos , Replicação Viral/efeitos da radiaçãoRESUMO
Human norovirus (NoV) is a major cause of fresh produce-associated outbreaks and human NoV in irrigation water can potentially lead to viral internalization in fresh produce. Therefore, there is a need to develop novel intervention strategies to target internalized viral pathogens while maintaining fresh produce quality. In this study electron beam (E-beam) and gamma radiation were evaluated for efficacy against a human NoV GII.4 strain and Tulane virus (TV). Virus survival following ionizing radiation treatments was determined using direct quantitative reverse transcriptase PCR (RT-qPCR), the porcine gastric mucin magnetic bead (PGM-MB) binding assay followed by RT-qPCR, and plaque assay. In simple media, a high dose of E-beam treatment was required to completely abolish the receptor binding ability of human NoV (35.3kGy) and TV (19.5-24.1kGy), as assessed using the PGM-MB binding assay. Both human NoV and TV were more susceptible to gamma irradiation than E-beam, requiring 22.4kGy to achieve complete inactivation. In whole strawberries, no human NoV or TV RNA was detected following 28.7kGy of E-beam treatment using the PGM-MB binding assay. Overall, human NoV and TV are highly resistant to ionizing radiation and therefore the technology may not be suitable to eliminate viruses in fresh produce at the currently approved levels. In addition, the PGM-MB binding assay is an improved method to detect viral infectivity compared to direct RT-qPCR.
Assuntos
Infecções por Caliciviridae/prevenção & controle , Fragaria/virologia , Gastroenterite/prevenção & controle , Norovirus/efeitos da radiação , Ligação Viral/efeitos da radiação , Inativação de Vírus/efeitos da radiação , Animais , Infecções por Caliciviridae/virologia , Surtos de Doenças , Raios gama , Mucinas Gástricas/metabolismo , Gastroenterite/virologia , Humanos , Separação Imunomagnética , Norovirus/classificação , Norovirus/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Suínos/virologia , Internalização do Vírus/efeitos da radiaçãoRESUMO
Low-intensity ultrasound is a useful method to introduce materials into cells due to the transient formation of micropores, called sonoporations, on the cell membrane. Whether oncolytic herpes simplex virus type 1 (HSV-1) can be introduced into oral squamous cell carcinoma (SCC) cells through membrane pores remains undetermined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the 134.5 gene and fusogenic, were used. Cells were exposed to ultrasound in the presence or absence of microbubbles. The increase of virus entry was estimated by plaque numbers. Viral infection was hardly established without the adsorption step, but plaque number was increased by the exposure of HSV-1-inoculated cells to ultrasound. Plaque number was also increased even if SAS cells were exposed to ultrasound and inoculated with RH2 without the adsorption step. This effect was abolished when the interval from ultrasound exposure to virus inoculation was prolonged. Scanning electron microscopy revealed depressed spots on the cell surface after exposure to ultrasound. These results suggest that oncolytic HSV-1 RH2 can be introduced into SAS cells through ultrasound-mediated pores of the cell membrane that are resealed after an interval.
Assuntos
Carcinoma de Células Escamosas/terapia , Células Epiteliais/virologia , Herpesvirus Humano 1/fisiologia , Terapia Viral Oncolítica/métodos , Ultrassonografia/métodos , Internalização do Vírus/efeitos da radiação , Linhagem Celular Tumoral , Células Epiteliais/efeitos da radiação , Herpesvirus Humano 1/efeitos da radiação , Humanos , Microscopia Eletrônica de Varredura , Modelos Biológicos , Ensaio de Placa ViralRESUMO
UNLABELLED: Epstein-Barr virus (EBV) fusion with an epithelial cell requires virus glycoproteins gHgL and gB and is triggered by an interaction between gHgL and integrin αvß5, αvß6, or αvß8. Fusion with a B cell requires gHgL, gp42, and gB and is triggered by an interaction between gp42 and human leukocyte antigen class II. We report here that, like alpha- and betaherpesviruses, EBV, a gammaherpesvirus, can mediate cell fusion if gB and gHgL are expressed in trans. Entry of a gH-null virus into an epithelial cell is possible if the epithelial cell expresses gHgL, and entry of the same virus, which phenotypically lacks gHgL and gp42, into a B cell expressing gHgL is possible in the presence of a soluble integrin. Heat is capable of inducing the fusion of cells expressing only gB, and the proteolytic digestion pattern of gB in virions changes in the same way following the exposure of virus to heat or to soluble integrins. It is suggested that the Gibbs free energy released as a result of the high-affinity interaction of gHgL with an integrin contributes to the activation energy required to cause the refolding of gB from a prefusion to a postfusion conformation. IMPORTANCE: The core fusion machinery of herpesviruses consists of glycoproteins gB and gHgL. We demonstrate that as in alpha- and betaherpesvirus, gB and gHgL of the gammaherpesvirus EBV can mediate fusion and entry when expressed in trans in opposing membranes, implicating interactions between the ectodomains of the proteins in the activation of fusion. We further show that heat and exposure to a soluble integrin, both of which activate fusion, result in the same changes in the proteolytic digestion pattern of gB, possibly representing the refolding of gB from its prefusion to its postfusion conformation.
Assuntos
Herpesvirus Humano 4/fisiologia , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Envelope Viral/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus/efeitos da radiação , Animais , Linfócitos B/virologia , Linhagem Celular , Herpesvirus Humano 4/efeitos da radiação , Temperatura Alta , Humanos , Conformação ProteicaRESUMO
Ultrashort pulsed laser irradiation is a new method for virus reduction in pharmaceuticals and blood products. Current evidence suggests that ultrashort pulsed laser irradiation inactivates viruses through an impulsive stimulated Raman scattering process, resulting in aggregation of viral capsid proteins. However, the specific functional defect(s) in viruses inactivated in this manner have not been demonstrated. This information is critical for the optimization and the extension of this treatment platform to other applications. Toward this goal, we investigated whether viral internalization, replication, or gene expression in cells were altered by ultrashort pulsed laser irradiation. Murine Cytomegalovirus (MCMV), an enveloped DNA virus, was used as a model virus. Using electron and fluorescence microscopy, we found that laser-treated MCMV virions successfully internalized in cells, as evidenced by the detection of intracellular virions, which was confirmed by the detection of intracellular viral DNA via PCR. Although the viral DNA itself remained polymerase-amplifiable after laser treatment, no viral replication or gene expression was observed in cells infected with laser-treated virus. These results, along with evidence from previous studies, support a model whereby the laser treatment stabilizes the capsid, which inhibits capsid uncoating within cells. By targeting the mechanical properties of viral capsids, ultrashort pulsed laser treatment represents a unique potential strategy to overcome viral mutational escape, with implications for combatting emerging or drug-resistant pathogens.
Assuntos
Terapia com Luz de Baixa Intensidade , Muromegalovirus/efeitos da radiação , Agregados Proteicos/efeitos da radiação , Inativação de Vírus/efeitos da radiação , Replicação Viral/efeitos da radiação , Células 3T3 , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/efeitos da radiação , Linhagem Celular , DNA Viral/genética , Expressão Gênica/efeitos da radiação , Camundongos , Camundongos Endogâmicos BALB C , Transcrição Gênica/efeitos da radiação , Internalização do Vírus/efeitos da radiaçãoRESUMO
Human plasmacytoid dendritic cells (PDC) are crucial for innate and adaptive immune responses against viral infections, mainly through production of type I interferons. Evidence is accumulating that PDC surface receptors play an important role in this process. To investigate the PDC phenotype in more detail, a chip-based expression analysis of surface receptors was combined with respective flow cytometry data obtained from fresh PDC, PDC exposed to interleukin-3 (IL-3) and/or herpes simplex virus type 1 (HSV-1). CD156b, CD229, CD305 and CD319 were newly identified on the surface of PDC, and CD180 was identified as a new intracellular antigen. After correction for multiple comparisons, a total of 33 receptors were found to be significantly regulated upon exposure to IL-3, HSV-1 or IL-3 and HSV-1. These were receptors involved in chemotaxis, antigen uptake, activation and maturation, migration, apoptosis, cytotoxicity and costimulation. Infectious and ultraviolet-inactivated HSV-1 did not differentially affect surface receptor regulation, consistent with the lack of productive virus infection in PDC, which was confirmed by HSV-1 real-time polymerase chain reaction and experiments involving autofluorescing HSV-1 particles. Viral entry was mediated at least in part by endocytosis. Time-course experiments provided evidence of a co-ordinated regulation of PDC surface markers, which play a specific role in different aspects of PDC function such as attraction to inflamed tissue, antigen recognition and subsequent migration to secondary lymphatic tissue. This knowledge can be used to investigate PDC surface receptor functions in interactions with other cells of the innate and adaptive immune system, particularly natural killer cells and cytotoxic T lymphocytes.
Assuntos
Antígenos CD/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/virologia , Herpes Simples/imunologia , Herpesvirus Humano 1/fisiologia , Imunidade Adaptativa , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Separação Celular , Chlorocebus aethiops , Células Dendríticas/imunologia , Células Dendríticas/patologia , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Herpes Simples/metabolismo , Herpes Simples/patologia , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 1/efeitos da radiação , Humanos , Imunidade Inata , Interferon-alfa/metabolismo , Interleucina-3/imunologia , Interleucina-3/metabolismo , Análise em Microsséries , Células Vero , Internalização do Vírus/efeitos da radiaçãoRESUMO
PURPOSE: Adenovirus-mediated gene therapy combined with radiation is expected to be a new approach to treat pancreatic cancer. However, there are no reports of definitive effects of radiation on adenovirus-mediated gene therapies. In the present study, we investigated the effect of radiation on the transduction efficiency of an adenovirus-based gene therapy. EXPERIMENTAL DESIGN: We used adenovirus expressing NK4 (Ad-NK4), an antagonist for hepatocyte growth factor, as a representative gene therapy. Pancreatic cancer cells preinfected with Ad-NK4 were irradiated, and NK4 levels in culture media of these cells were measured. We investigated cytomegalovirus (CMV) promoter activity and uptake of adenovirus in these cells. To examine the effect of radiation in vivo, Ad-NK4 was given to irradiated subcutaneous tumors in nude mice, and NK4 levels in tumors were measured. RESULTS: NK4 levels in culture media of irradiated cells were 4.5-fold (P < 0.01) higher than those of nonirradiated cells. Radiation enhanced activation of the CMV promoter and adenovirus uptake (P < 0.01), leading to increased levels of NK4. We found that activation of p38 mitogen-activated protein kinase and up-regulation of dynamin 2 may be involved in the radiation-induced activation of the CMV promoter and adenovirus uptake, respectively. NK4 levels in irradiated tumors were 5.8-fold (P = 0.017) higher than those in nonirradiated tumors. CONCLUSIONS: The present findings suggest that radiation significantly improves the efficiency of adenovirus-mediated gene transfer in pancreatic cancer and probably contributes to decreasing the dose of adenovirus required for gene transfer and controlling side effects of adenovirus infection in nonirradiated normal tissue.