RESUMEN
Optimisation of protein degraders requires balancing multiple factors including potency, cell permeability and solubility. Here we show that the fluorescence of pomalidomide can be used in high-throughput screening assays to rapidly assess cellular penetration of degrader candidates. In addition, this technique can be paired with endocytosis inhibitors to gain insight into potential mechanisms of candidates entering a target cell. A model library of pomalidomide conjugates was synthesised and evaluated using high-throughput fluorescence microscopy. This technique based on intrinsic fluorescence can be used to guide rational design of pomalidomide conjugates without the need for additional labels or tags.
Asunto(s)
Talidomida , Talidomida/farmacología , Microscopía FluorescenteRESUMEN
Acer t egmentosum Maxim., commonly known as Manchurian stripe maple, is a deciduous tree belonging to the family of Aceraceae and has been traditionally used in folk medicine for its remedial effects in liver diseases and traumatic bleedings. With a growing body of experimental evidence for its pharmacological efficacies, such as neuroprotective, hepatoprotective, antioxidant, and anti-inflammatory activities, A. tegmentosum has gradually gained popularity as a health supplement and functional food. However, the large part of essential toxicity information still remained lacking despite the possibility of mutagenic potentials as previously suggested, posing safety concerns for human consumption. In this study, we evaluated 90-day repeated oral toxicity of A. tegmentosum Maxim. water extract (ATWE) in SD rats with acute toxicity assessment in beagle dogs, and reevaluated genotoxicity using a combination of in vitro and in vivo assays. During the oral study period, ATWE did not cause toxicity-related clinical signs and mortality in rodents without adverse effects observed in the analysis of hematology, serum biochemistry, and histopathology, establishing >5,000 mg/kg BW as the NOAEL. In addition, doses up to 5,000 mg/kg BW did not cause acute toxicity in beagle dogs. When assessed for genotoxicity using bacterial reverse mutation, chromosome aberration, and micronucleus formation, ATWE showed lack of mutagenicity and clastogenicity. These results demonstrated that AWTE was safe in the present preclinical study for systemic toxicity and genotoxicity at the tested doses, providing a guideline for safe use in humans.
RESUMEN
Oncolytic virus (OV) therapy is an emerging cancer treatment that uses replicating viruses to infect and kill tumor cells and incite anticancer immunity. While the approach shows promise, it currently fails most patients, indicating strategies to improve OV activity are needed. Developing these will require greater understanding of OV biology, particularly in the context of OV delivery and clearance, the infection process within a complex tumor microenvironment, and the modulation of anticancer immunity. To help achieve this, we have established a technique for high-resolution 4D imaging of OV-host interactions within intact tissues of live mice using intravital microscopy (IVM). We show that oncolytic vesicular stomatitis virus (VSV) directly labeled with Alexa Fluor dyes is easily visualized by single- or multiphoton microscopy while retaining bioactivity in vivo. The addition of fluorophore-tagged antibodies and genetically encoded reporter proteins to image target cells and the virus infection enables real-time imaging of dynamic interactions between VSV and host cells in blood, tumor, and visceral organs of live mice. The method has sufficient in vivo resolution to observe leukocytes in blood binding to and transporting VSV particles, foci of VSV infection spreading through a tumor, and antigen-presenting cells in the spleen interacting with and being infected by VSV. Visualizing OV-host interactions by IVM represents a powerful new tool for studying OV therapy.
RESUMEN
The originally published version of this article contained an error in the spelling of the author Pankaj Tailor, which was incorrectly given as Pankaj Taylor. This has now been corrected in both the PDF and HTML versions of the article.
RESUMEN
Second mitochondrial activator of caspase (Smac)-mimetic compounds and oncolytic viruses were developed to kill cancer cells directly. However, Smac-mimetic compound and oncolytic virus therapies also modulate host immune responses in ways we hypothesized would complement one another in promoting anticancer T-cell immunity. We show that Smac-mimetic compound and oncolytic virus therapies synergize in driving CD8+ T-cell responses toward tumors through distinct activities. Smac-mimetic compound treatment with LCL161 reinvigorates exhausted CD8+ T cells within immunosuppressed tumors by targeting tumor-associated macrophages for M1-like polarization. Oncolytic virus treatment with vesicular stomatitis virus (VSVΔM51) promotes CD8+ T-cell accumulation within tumors and CD8+ T-cell activation within the tumor-draining lymph node. When combined, LCL161 and VSVΔM51 therapy engenders CD8+ T-cell-mediated tumor control in several aggressive mouse models of cancer. Smac-mimetic compound and oncolytic virus therapies are both in clinical development and their combination therapy represents a promising approach for promoting anticancer T-cell immunity.Oncolytic viruses (OV) and second mitochondrial activator of caspase (Smac)-mimetic compounds (SMC) synergistically kill cancer cells directly. Here, the authors show that SMC and OV therapies combination also synergize in vivo by promoting anticancer immunity through an increase in CD8+ T-cell response.
Asunto(s)
Materiales Biomiméticos/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Neoplasias Experimentales/terapia , Viroterapia Oncolítica/métodos , Animales , Antineoplásicos/farmacología , Proteínas Reguladoras de la Apoptosis , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Línea Celular Tumoral , Terapia Combinada , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteínas Mitocondriales/metabolismo , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/virología , Virus Oncolíticos/inmunología , Virus Oncolíticos/fisiología , Tiazoles/farmacología , Resultado del Tratamiento , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular Indiana/fisiologíaRESUMEN
Coxsackievirus B3 (CVB3) infection can trigger myocarditis and can ultimately lead to dilated cardiomyopathy. It is known that CVB3-induced T-cell infiltration into cardiac tissues is one of the pathological factors causing cardiomyocyte injury by inflammation. However, the underlying mechanism for this remains unclear. We investigated the mechanism of T-cell infiltration by two types of CVB3: the H3 WT strain and the YYFF attenuated strain. T-cell activation was confirmed by changes in the distribution of lymphocyte function-associated antigen-1 (LFA-1). Finally, we identified which viral gene was responsible for LFA-1 activation. CVB3 could infect and activate T-cells in vivo and in vitro, and activated T-cells were detected in CVB3-infected mouse hearts. LFA-1 expressed on the surface of these T-cells had been activated through the cAMP/Rap1 pathway. Recombinant lentiviruses expressing VP2 of CVB3 could also induce LFA-1 activation via an increase in cAMP, whilst VP2 of YYFF did not. These results indicated that CVB3 infection increased cAMP levels and then activated Rap1 in T-cells. In particular, VP2, among the CVB3 proteins, might be critical for this activation. This VP2-cAMP-Rap1-LFA-1 axis could be a potential therapeutic target for treating CVB3-induced myocarditis.
Asunto(s)
AMP Cíclico/metabolismo , Enterovirus/inmunología , Antígeno-1 Asociado a Función de Linfocito/biosíntesis , Linfocitos T/inmunología , Proteínas de Unión al GTP rap1/metabolismo , Animales , Cardiomiopatías/inmunología , Cardiomiopatías/virología , Infecciones por Coxsackievirus/inmunología , Infecciones por Coxsackievirus/virología , AMP Cíclico/biosíntesis , Enterovirus/genética , Células HeLa , Corazón/virología , Humanos , Molécula 1 de Adhesión Intercelular , Células Jurkat , Activación de Linfocitos/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Miocarditis/inmunología , Miocarditis/virología , Miocardio/citología , Miocardio/inmunología , Receptores Virales/metabolismo , Proteínas de Unión al GTP rap1/biosíntesisRESUMEN
Infectious bronchitis virus (IBV) replicates in the epithelial cells of trachea and lungs of chicken, however the mechanism of generation of innate immune response against IBV infection in these tissues has not been fully characterized. Our objective was to study innate responses induced early following IBV infection in chickens. Initiation of the transcription of selected innate immune genes such as TLR3, TLR7, MyD88, IL-1ß and IFN-ß, as well as recruitment of macrophages, were evident following an initial down regulation of some of the observed genes (TLR3, IL-1ß, and IFN-γ) in trachea and lung. This initial down-regulation followed by the induction of innate immune response to IBV infection appears to be inadequate for the control of IBV genome accumulation and consequent histopathological changes in these tissues. Potential induction of innate immunity before infection occurs may be necessary to reduce the consequences since vaccine induced immunity is slow to develop.
Asunto(s)
Infecciones por Coronavirus/veterinaria , Inmunidad Innata , Virus de la Bronquitis Infecciosa/fisiología , Enfermedades de las Aves de Corral/inmunología , Sistema Respiratorio/inmunología , Animales , Proteínas Aviares/genética , Proteínas Aviares/inmunología , Pollos , Infecciones por Coronavirus/genética , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/virología , Virus de la Bronquitis Infecciosa/inmunología , Enfermedades de las Aves de Corral/genética , Enfermedades de las Aves de Corral/virología , Sistema Respiratorio/virología , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/inmunología , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/inmunologíaRESUMEN
LPS is one of the pathogen associated molecular patterns that activates Toll-like receptor 4 (TLR4) signaling pathway eliciting antiviral host responses in mammals although information on such responses in avian species is scarce. Our objectives were to characterize the LPS induced innate responses particularly the expression of LPS receptors (TLR4, CD14) in avian macrophages and observe whether TLR4 mediated induction of NO can elicit antiviral response against infectious laryngotracheitis virus (ILTV) replication. We found that LPS was capable of inducing the expression of TLR4, CD14 and NO production but not the type 1 interferons in an avian macrophage cell line, MQ-NCSU. We also showed that TLR4 mediated NO production can lead to antiviral response against ILTV replication when MQ-NCSU cells were treated with LPS and the resultant supernatant was then transferred to ILTV replicating cells to assess antiviral activity. Antiviral activity of NO was blocked by a selective inhibitor, S-methylisothiourea sulfate that inhibits inducible NO synthase. This observation confirms that the antiviral activity is positively correlated with NO production. The data show that LPS can be a potential innate immune stimulant that can be used against ILTV infection in chickens that require further evaluation in vivo.
Asunto(s)
Pollos , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/inmunología , Óxido Nítrico/inmunología , Enfermedades de las Aves de Corral/virología , Transducción de Señal/inmunología , Receptor Toll-Like 4/inmunología , Animales , Línea Celular , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Isotiuronio/análogos & derivados , Isotiuronio/farmacología , Lipopolisacáridos/inmunología , Macrófagos , Óxido Nítrico/antagonistas & inhibidores , Enfermedades de las Aves de Corral/inmunología , Replicación Viral/inmunologíaRESUMEN
Coxsackievirus B3 (CVB3) is an RNA virus that mainly causes myocarditis. We have reported previously that immunoreceptor tyrosine-based activation motif (ITAM)-like sequences are contained in the capsid protein VP2 of CVB3. The substitution of two tyrosines for phenylalanines in the ITAM-like region causes attenuation of CVB3, possibly via defective viral assembly. In this study, we found that Syk, a downstream molecule of ITAM, interacts with the wild-type (WT) CVB3 VP0 protein, but not with the mutant CVB3 VP0 (called YYFF), and that an inhibitor of Syk reduced the growth of CVB3. The WT CVB3 activated nuclear factor kappa B (NF-κB), a protein activated by ITAM, and eventually induced the production of interleukin-6 (IL-6)-one of the proinflammatory cytokines induced by NF-κB-in macrophages. However, the YYFF form did not. In addition, viral VP2 protein may be dependent on the phosphorylation of an ITAM-like region that affected the activation of NF-κB. Taken together, these results suggest that the ITAM-like sequences in CVB3 VP2 can not only affect viral structure but also act as signals in pathogenesis.
Asunto(s)
Proteínas de la Cápside/metabolismo , Enterovirus Humano B/inmunología , Enterovirus Humano B/patogenicidad , Factores de Virulencia/metabolismo , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Sustitución de Aminoácidos , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Línea Celular , Enterovirus Humano B/crecimiento & desarrollo , Interacciones Huésped-Patógeno , Humanos , Interleucina-6/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Mutagénesis Sitio-Dirigida , FN-kappa B/metabolismo , Proteínas Tirosina Quinasas , Quinasa Syk , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/inmunologíaRESUMEN
Coxsackievirus B3 (CVB3) contains a single-stranded plus-strand RNA genome and belongs to the genus Enterovirus in the family Picornaviridae. For decades, many studies have shown that enteroviruses are potential viral vectors. Of these viruses, polioviruses and coxsackieviruses have predominantly been investigated. Therefore, the molecular biology of these two viruses and the cellular receptors involved in their infection are relatively well known, and infectious cDNAs and proper animal models of their infection are available. CVB3, in particular, will become increasingly important in the development of an enteroviral vector because poliovirus is currently subject to the global poliovirus eradication program. This review highlights the potential use of CVB3 as a viral vector for vaccines and therapeutic gene delivery.
Asunto(s)
Enterovirus Humano B/genética , Enterovirus Humano B/patogenicidad , Terapia Genética/métodos , Vectores Genéticos , Vacunación/métodos , Factores de Virulencia/genética , Portadores de Fármacos , HumanosRESUMEN
Coxsackievirus strain CVB3 is widespread in the human population and causes myocarditis or pancreatitis. However, despite its clinical impact, there is no commercially available and clinically applicable prophylactic vaccine. This study examines the characteristics of attenuated CVB3 strains developed so far and their application as live-attenuated CVB3 vaccines, and discusses problems to be overcome in the development of live-attenuated vaccines.
Asunto(s)
Enterovirus/inmunología , Vacunas Atenuadas/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Infecciones por Coxsackievirus/inmunología , Infecciones por Coxsackievirus/prevención & control , Infecciones por Coxsackievirus/virología , Enterovirus/genética , Vectores Genéticos , Humanos , Mutación/genética , Receptores Virales/genética , Vacunas Atenuadas/genéticaRESUMEN
Recombinant viruses expressing foreign antigens may provide a convenient vaccine vector capable of inducing preventative immunity. In this study, we explored the capacity of highly attenuated Coxsackievirus B3 (CVB3) to act as a recombinant vector to deliver foreign genes into experimental animals for the purpose of vaccination. The infectious cDNA of highly attenuated CVB3, YYFF, which has been reported previously (Vaccine 27:1974), was used to construct a recombinant YYFF cDNA (YYFF-HCV) by inserting a truncated form of hepatitis C virus (HCV) envelope protein E2 as an antigenic marker immediately upstream from the gene encoding the VP4 capsid protein. In YYFF-HCV-infected HeLa cells, HCV E2 expression was confirmed by immunoblotting and fluorescence microscopy. YYFF-HCV induced the production of antibodies and the cytotoxic T-lymphocyte (CTL) response to HCV E2 in the inoculated mice. Moreover, YYFF-HCV induced no inflammation in the virus-immunized mice. These results demonstrate that recombinant CVB3 expressing a foreign gene can act as a live vaccine vector capable of inducing humoral and cell-mediated immune responses directed against a foreign protein.
Asunto(s)
Enterovirus Humano B/inmunología , Hepacivirus/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/inmunología , Animales , Formación de Anticuerpos/inmunología , Células COS , Chlorocebus aethiops , Enterovirus Humano B/genética , Enterovirus Humano B/metabolismo , Expresión Génica , Células HeLa , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Linfocitos T Citotóxicos/metabolismo , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genética , Vacunas contra Hepatitis Viral/biosíntesis , Vacunas contra Hepatitis Viral/genéticaRESUMEN
In order to systemically investigate the possibility of using coxsackievirus B3 (CVB3) to deliver foreign genes in vivo, a recombinant strain of CVB3 encoding the renilla gene (CVB3- renilla) was constructed. The recombinant CVB3 resulted in extensive and transient expression of the renilla protein within mouse organs, especially the pancreas. The level of expression was generally dependent upon the viral titer present. Moreover, the CVB3-renilla strain was completely attenuated. Interestingly, the recombinant CVB3 vector was expressed much more strongly in mouse organs than was a comparable adenoviral vector. The CVB3-renilla strain did not express the renilla gene in mice with pre-existing coxsackievirus-specific neutralizing antibodies, but direct organ-specific administration of the virus during openperitoneum surgery was able to circumvent this immunity. This coxsackievirus vector may represent a useful means for delivering and expressing foreign genes in mouse models in an acute and extensive fashion.
Asunto(s)
Enterovirus Humano B/fisiología , Técnicas de Transferencia de Gen , Adenoviridae/genética , Animales , Anticuerpos Antivirales/metabolismo , Genes Reporteros , Vectores Genéticos/administración & dosificación , Vectores Genéticos/biosíntesis , Células HeLa , Humanos , Inyecciones Intraperitoneales , Luciferasas de Renilla/administración & dosificación , Luciferasas de Renilla/biosíntesis , Sustancias Luminiscentes/administración & dosificación , Sustancias Luminiscentes/metabolismo , Ratones , Páncreas/efectos de los fármacos , Replicación ViralRESUMEN
The objective of this study is to investigate the relationship between five air pollutants (PM(10), SO(2), NO(2), O(3), CO) measured on the daily basis, and adverse health symptoms using epidemiological surveillance data. The generalized estimated equation (GEE) model, a logistic regression analysis model, was used to estimate the effects of air pollution on children's daily health symptoms, focusing on the morbidity including both respiratory and allergic symptoms in four different cities. Analysis of the effects of each pollutant on children's respiratory and allergic symptoms demonstrated that CO affected all symptoms in all the study areas. When the concentration of SO(2) and NO(2) was elevated, upper respiratory symptoms increased significantly. In contrast, when the concentration of O(3) rose, the symptoms decreased significantly. The relationship between measured concentrations and health symptoms was site-dependent for each pollutant.
Asunto(s)
Contaminantes Atmosféricos/efectos adversos , Contaminación del Aire/efectos adversos , Hipersensibilidad/etiología , Enfermedades Respiratorias/etiología , Contaminantes Atmosféricos/química , Monóxido de Carbono/química , Monóxido de Carbono/toxicidad , Niño , Exposición a Riesgos Ambientales/efectos adversos , Femenino , Humanos , Hipersensibilidad/epidemiología , Corea (Geográfico)/epidemiología , Modelos Logísticos , Masculino , Dióxido de Nitrógeno/química , Dióxido de Nitrógeno/toxicidad , Ozono/química , Ozono/toxicidad , Material Particulado/química , Material Particulado/toxicidad , Vigilancia de la Población , Enfermedades Respiratorias/epidemiología , Dióxido de Azufre/química , Dióxido de Azufre/toxicidad , Población Urbana/estadística & datos numéricosRESUMEN
Coxsackievirus B3 (CVB3) is a common agent of viral myocarditis, a major cause of sudden cardiac death, and ultimately dilated cardiomyopathy. However, there is no vaccine in clinical use. In this study, we identified the conserved amino acid sequences in the C-terminal region of the VP2 of the coxsackievirus B group and some echoviruses. The mutant virus, YYFF, with phenylalanines substituted for two tyrosines in these conserved sequences was highly attenuated in vivo and could induce a high neutralizing antibody titer and a cytotoxic T-lymphocyte response against CVB3. Thereby, mutant-virus-immunized mice showed a 100% survival rate and protection against inflammation of the heart and pancreas after lethal dose challenge. Thus, this mutant virus is a good candidate for an attenuated CVB3 vaccine.
Asunto(s)
Proteínas de la Cápside/inmunología , Infecciones por Coxsackievirus/prevención & control , Miocarditis/prevención & control , Pancreatitis/prevención & control , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Proteínas de la Cápside/genética , Secuencia Conservada , Infecciones por Coxsackievirus/complicaciones , Enterovirus/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Miocarditis/etiología , Miocarditis/virología , Pruebas de Neutralización , Pancreatitis/etiología , Pancreatitis/virología , Estructura Terciaria de Proteína , Linfocitos T Citotóxicos/inmunología , Vacunas Atenuadas/inmunologíaRESUMEN
Coxsackieviruses are important human pathogens that induce myocarditis and pancreatitis. However, there are no vaccines or therapeutic reagents for their clinical treatment. Although RNA interference (RNAi)-based approaches to the prevention of viral production have been developed recently, limitations to the in vivo delivery systems and variations in the viral target sequences still hamper the strategy. In this study, to overcome these limitations, we have constructed recombinant lentivirus-delivered short hairpin RNAs (shRNAs) against sequences in highly conserved cis-acting replication element (CRE) within the 2C protein of coxsackievirus B3 (CVB3), designated MET-2C. A recombinant lentivirus, designated Met-2C lenti, was constructed that contains the MET-2C sequence, which acts as a shRNA. Met-2C lenti clearly reduced viral production in CVB3-infected cells in vitro. Moreover, the mice injected intraperitoneally with Met-2C lenti had significant reductions in viral titers, viral myocarditis, and proinflammatory cytokines after challenge with CVB3, compared with those in GFP lenti infected control mice. Moreover, Met-2C lenti improved survival rate compared with that of the GFP lenti infected control group. Therefore, Met-2C lenti is potentially a clinical therapeutic agent for the treatment of viral myocarditis.
Asunto(s)
Proteínas Portadoras/genética , Infecciones por Coxsackievirus/terapia , Enterovirus Humano B/genética , Miocarditis/terapia , Interferencia de ARN , Proteínas no Estructurales Virales/genética , Animales , Proteínas Portadoras/metabolismo , Línea Celular , Infecciones por Coxsackievirus/inmunología , Infecciones por Coxsackievirus/prevención & control , Infecciones por Coxsackievirus/virología , Modelos Animales de Enfermedad , Vectores Genéticos , Humanos , Interleucina-6/metabolismo , Lentivirus/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Miocarditis/inmunología , Miocarditis/prevención & control , Miocarditis/virología , ARN Interferente Pequeño/genética , Tasa de Supervivencia , Factor de Necrosis Tumoral alfa/metabolismo , Proteínas no Estructurales Virales/metabolismoRESUMEN
The coxsackievirus group B (CVB) of the genus Enterovirus and the species human enterovirus B is a nonenveloped virus containing a single-stranded positive-sense RNA genome. Coxsackievirus has icosahedral symmetry and four capsid proteins, VP1, VP2, VP3, and VP4. Specific antibodies against each viral protein are prerequisites for various studies. In this study, we developed seven peptide-derived antibodies directed against coxsackievirus VP1 (NO1-NO5), VP2 (B3), and VP3 (GL3). We developed a type-specific antibody (NO1) and broadly cross-reactive antibodies (NO3 and NO5) to VP1. Anti-VP2 and anti-VP3 antibodies (B3 and GL3, respectively) are also cross-reactive to human enterovirus B such as CVB and echoviruses. Their sensitivities and reactivities are likely to be better than those of the commercial VP1 monoclonal antibody (MAb). The dot-blot analysis also showed that NO5 against VP1 is able to detect less than 1 microg [2x10(6) plaque-forming unit (pfu) of CVB3] of viruses, suggesting that it could be used to develop a diagnostic kit that can directly detect human enterovirus B. The antibodies produced here may allow us to undertake several studies, such as those involving viral trafficking, expression kinetics, and the roles of viral proteins in infection, and the development of diagnostic kits.