RESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 continues to threaten public health. The virus is causing breakthrough infections in vaccinated individuals. Also, scarce information is available about cutaneous manifestations after severe acute respiratory syndrome coronavirus 2 infection. CASE PRESENTATION AND FINDINGS: A case of a triple-vaccinated (Pfizer) 37-year-old Hispanic American (Colombian) male who developed urticaria after Omicron BA.5.1 severe acute respiratory syndrome coronavirus 2 breakthrough infection is described. Virus isolation and whole genome sequencing along with immune and molecular assays were performed. Dermatological manifestations (skin rash and urticaria) after Omicron BA.5.1 infection were observed. Sequence analysis of the Omicron BA.5.1 isolate also revealed several important mutations. Hemogram analysis revealed leukocytosis and neutrophilia. Serology testing revealed anti-spike immunoglobulin G serum titers but negative detection of immunoglobulin M at 10 days after symptom onset. Anti-nucleocapsid, anti-spike 1 immunoglobulin G, anti-spike trimer, and anti-receptor-binding-domain immunoglobulin G and immunoglobulin E sera were detected at different titers 10 days after symptom onset. Several serum levels of chemokines/cytokines (Interferon-α, interferon-γ, interleukin-12/interleukin-23p40, interleukin-18, interferon gamma-induced protein-10, monocyte chemoattractant protein-1, monokine induced by gamma, macrophage inflammatory protein-1α, chemokine (C-C motif) ligand-5 , tumor necrosis factor-ß1, Tumor necrosis factor-α) were detected, but interleukin-2, interleukin-4, interleukin-6, interleukin-8, and interleukin-17A were below the limit of detection. INTERPRETATION AND CONCLUSIONS: To our knowledge, this is the first study describing skin effects of a severe acute respiratory syndrome coronavirus 2 Omicron BA.5 variant breakthrough infection in a triple-vaccinated patient in Colombia. Several important mutations were found in the spike glycoprotein of the virus isolated; these mutations are associated with immune evasion and changes in antigenic properties of the virus. Physicians overseeing coronavirus disease 2019 cases should be aware of the potential skin effects of the infection. Pathogenesis of severe acute respiratory syndrome coronavirus 2 infection and its association with proinflammatory cytokines and chemokines may enhance the development of urticaria and other skin manifestations in immunized individuals. However, further studies are needed to better understand the complexity of coronavirus disease in such situations.
Assuntos
COVID-19 , Urticária , Masculino , Humanos , Adulto , Urticária/etiologia , Pele , Citocinas , Anticorpos AntiviraisRESUMO
Background: Influenza A virus (IAV) surveillance in swine is critical not only due to the direct impact of the disease in the pork industry but also because IAV are prone to interspecies transmission (from human to pigs and vice versa); therefore, its monitoring is fundamental from a public and animal health perspective. Several diagnostic techniques have been used to detect IAV infection from nasal samples in swine, while samples of oral fluids (OF) are in use as novel alternatives for pathogen detection. The OF allow for efficient and feasible low-cost disease detection at the herd level, with low risk of stress for the animals. Objective: To describe a surveillance strategy of IAV at the herd level during respiratory disease outbreaks in swine farms at tropical settings using porcine oral fluids. Methods: An active surveillance strategy was conducted in several farms with past records of respiratory disease. The IAV detection was conducted in five purposively selected swine farms from years 2014 to 2017. We investigated a total of 18 respiratory outbreaks of the disease. Swine OF were collected for IAV testing. An OF sample is described as a pen-based specimen collected from a group of >20 pigs per pen and/or per barn (stall-housed individually with close contact among them). The IAV infection was investigated in OF by rRT-PCR testing and confirmed by viral isolation in cell culture Results: We found 107 (7.4%) positives to IAV by rRT-PCR from a total of 1,444 OF samples tested. Additionally, 9 IAV isolates were all further identified as H1 subtype. Conclusions: Our results demonstrate that OF can be easily implemented as a novel, user-friendly, welfare-friendly, accurate and cost-effective sampling method for active surveillance and monitoring of IAV infections in swine farms in tropical settings.
Antecedentes: La vigilancia del Virus Influenza A (IAV) en los cerdos es fundamental debido al impacto directo de la enfermedad en la industria porcina, pero también porque los IAV son propensos de transmisión entre especies (humanos a cerdos y viceversa), y por lo tanto su monitoreo es crítico desde las perspectivas de salud pública y animal. Actualmente existen varias técnicas de diagnóstico disponibles para detectar la infección por IAV a partir de muestras nasales en cerdos, sin embargo, se han implementado otras muestras como los fluidos orales (OF) como nuevas alternativas para la detección de patógenos. El OF permite una detección eficiente y factible de enfermedades a menor costo a nivel de rebaño, con menor riesgo de estrés para los animales. Objetivo: Describir una estrategia de vigilancia de IAV a nivel de hato por medio de fluidos orales porcinos durante brotes de enfermedades respiratorias en granjas porcinas en entornos tropicales. Métodos: Se llevó a cabo una estrategia de vigilancia activa en cinco granjas porcinas seleccionadas con antecedentes de enfermedades respiratorias. Se recolectaron OF porcinos para la prueba de IAV. Una muestra de OF se describió como una muestra grupal recolectada de un grupo de >20 cerdos por corral y/o por establo (si estaban alojados individualmente, pero tenían un contacto cercano entre ellos). La infección por IAV se investigó probando OF mediante rRT-PCR y la confirmación mediante aislamiento viral en cultivo celular. Resultados: La detección de IAV se llevó a cabo en cinco granjas seleccionadas intencionalmente entre 2014- 2017. Investigamos un total de 18 eventos de brotes de enfermedades respiratorias. Del total de 1.444 OF muestras analizadas, encontramos 107 (7,4%) positivos a IAV mediante rRT-PCR. Además, solo se obtuvieron 9 aislamientos de IAV y todos se identificaron además como subtipo H1. Conclusiones: Los resultados de nuestro estudio demostraron cómo la OF puede implementarse fácilmente como un método de muestreo novedoso, fácil de usar, amigable con el bienestar animal, preciso y rentable para la vigilancia activa y el monitoreo de infecciones por IAV en granjas porcinas en entornos tropicales.
Antecedentes: A vigilância do vírus Influenza A (IAV) em suínos é crítica devido ao impacto direto da doença na indústria de suínos, mas também porque os IAV são propensos a transmissão interespécies (de humanos para porcos e vice-versa) e, portanto, seu monitoramento é crítico do ponto de vista da saúde pública e animal. Atualmente, existem várias técnicas de diagnóstico disponíveis para detectar a infecção por IAV em amostras nasais de suínos, no entanto, outras amostras, como fluidos orais (OF), têm sido implementadas como novas alternativas para a detecção de patógenos. O OF permite uma detecção eficiente e viável de doenças com menor custo em nível de rebanho, com menor risco de estresse para os animais. Objetivo: Descrever uma estratégia de vigilância de IAV em nível de rebanho durante surtos de doenças respiratórias em granjas de suínos em ambientes tropicais por meio de fluidos orais suínos. Métodos: A estratégia de vigilância ativa foi conduzida em cinco granjas de suínos selecionadas com histórico de doenças respiratórias. Suínos OF foram coletados para teste de IAV. Uma amostra OF foi descrita como um espécime baseado em curral coletado de um grupo de >20 porcos por curral e/ou por celeiro (se eles foram alojados individualmente, mas tendo contato próximo entre eles). A infecção IAV foi investigada testando OF por rRT-PCR e confirmada por isolamento em cultura de células. Resultados: A detecção do IAV foi realizada em cinco fazendas selecionadas propositalmente entre 2014-2017. Nós investigamos um total de 18 eventos de surto de doença respiratória. Do total de 1.444 amostras de OF testadas, encontramos 107 (7,4%) positivas para IAV por rRT-PCR. Além disso, apenas 9 isolados de IAV foram obtidos, e todos foram posteriormente identificados como subtipo H1. Conclusão: Os resultados de nosso estudo demonstraram como o OF pode ser facilmente implementado como um método de amostragem novo, amigável, amigável com o bem-estar, preciso e de baixo custo para vigilância ativa e monitoramento de infecções IAV em fazendas de suínos em ambientes tropicais.
RESUMO
African swine fever virus (ASFV) causes a highly contagious hemorrhagic disease that affects domestic pig and Eurasian wild boar populations. To date, no safe and efficacious treatment or vaccine against ASF is available. Nevertheless, there are several reports of protection elicited by experimental vaccines based on live attenuated ASFV and some levels of protection and reduced viremia in other approaches such as DNA, adenovirus, baculovirus, and vaccinia-based vaccines. Current ASF subunit vaccine research focuses mainly on delivering protective antigens and antigen discovery within the ASFV genome. However, due to the complex nature of ASFV, expression vectors need to be optimized to improve their immunogenicity. Therefore, in the present study, we constructed several recombinant MVA vectors to evaluate the efficiency of different promoters and secretory signal sequences in the expression and immunogenicity of the p30 protein from ASFV. Overall, the natural poxvirus PrMVA13.5L promoter induced high levels of both p30 mRNA and specific anti-p30 antibodies in mice. In contrast, the synthetic PrS5E promoter and the S E/L promoter linked to a secretory signal showed lower mRNA levels and antibodies. These findings indicate that promoter selection may be as crucial as the antigen used to develop ASFV subunit vaccines using MVA as the delivery vector.
Assuntos
Vírus da Febre Suína Africana/genética , Poxviridae/genética , Vacinas de Subunidades Antigênicas/genética , Proteínas Virais/genética , Vacinas Virais/genética , Febre Suína Africana/virologia , Animais , Anticorpos Antivirais/genética , Baculoviridae/genética , Linhagem Celular , Chlorocebus aethiops , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Regiões Promotoras Genéticas/genética , Suínos , Vacinas Atenuadas/genética , Células Vero , Replicação Viral/genéticaRESUMO
Timely and accurate diagnostics are essential to fight the COVID-19 pandemic, but no test satisfies both conditions. Dogs can scent-identify the unique odors of volatile organic compounds generated during infection by interrogating specimens or, ideally, the body of a patient. After training 6 dogs to detect SARS-CoV-2 by scent in human respiratory secretions (in vitro diagnosis), we retrained 5 of them to search and find the infection by scenting the patient directly (in vivo screening). Then, efficacy trials were designed to compare the diagnostic performance of the dogs against that of the rRT-PCR in 848 human subjects: 269 hospitalized patients (COVID-19 prevalence 30.1%), 259 hospital staff (prevalence 2.7%), and 320 government employees (prevalence 1.25%). The limit of detection in vitro was lower than 10-12 copies ssRNA/mL. During in vivo efficacy experiments, our 5 dogs detected 92 COVID-19 positive patients among the 848 study subjects. The alert (lying down) was immediate, with 95.2% accuracy and high sensitivity (95.9%; 95% C.I. 93.6-97.4), specificity (95.1%; 94.4-95.8), positive predictive value (69.7%; 65.9-73.2), and negative predictive value (99.5%; 99.2-99.7) in relation to rRT-PCR. Seventy-five days after finishing in vivo efficacy experiments, a real-life study (in vivo effectiveness) was executed among the riders of the Metro System of Medellin, deploying the human-canine teams without previous training or announcement. Three dogs were used to examine the scent of 550 volunteers who agreed to participate, both in test with canines and in rRT-PCR testing. Negative predictive value remained at 99.0% (95% C.I. 98.3-99.4), but positive predictive value dropped to 28.2% (95% C.I. 21.1-36.7). Canine scent-detection in vivo is a highly accurate screening test for COVID-19, and it detects more than 99% of infected individuals independent of key variables, such as disease prevalence, time post-exposure, or presence of symptoms. Additional training is required to teach the dogs to ignore odoriferous contamination under real-life conditions.
Assuntos
COVID-19/diagnóstico , Odorantes/análise , Feromônios/análise , Animais , Cães , Feminino , Humanos , Masculino , Programas de Rastreamento , Valor Preditivo dos Testes , SARS-CoV-2/patogenicidade , Sensibilidade e Especificidade , Compostos Orgânicos Voláteis , Cães TrabalhadoresRESUMO
Dengue virus (DENV) is a flavivirus responsible for the most common and burdensome arthropod-borne viral disease of humans[1]. DENV evolution has been extensively studied on broad geographic and time scales, using sequences from a single gene[2,3]. It is believed that DENV evolution in humans is dominated primarily by purifying selection due to the constraint of maintaining fitness in both humans and mosquitoes[4,5]. Few studies have explored DENV evolutionary dynamics using whole genome sequences, nor have they explored changes in viral diversity that occur during intra-epidemic periods. We used deep sequencing of the viral coding region to characterize DENV-1 evolution in a Colombian population sampled during two high-prevalence dengue seasons in which serotype dominance shifted. Our data demonstrate patterns of strain extinction and replacement within DENV-1 as its prevalence waned and DENV-3 became established. A comparison of whole-genome versus single-gene-based phylogenetic analyses highlights an important difference in evolutionary patterns. We report a trend of higher nonsynonymous to synonymous diversity ratios among non-structural (NS) genes, and statistically significantly higher values among these ratios in the NS1 gene after DENV-1 strain replacement. These results suggest that positive selection could be driving DENV evolution within individual communities. Signals of positive selection coming from distinct samples may be drowned out when combining multiple regions with differing patterns of endemic transmission as commonly done by large-scale geo-temporal assessments. Here, we frame our findings within a small, local transmission history which aids significance. Moreover, these data suggest that the NS1 gene, rather than the E gene, may be a target of positive selection, although not mutually exclusive, and potentially useful sentinel of adaptive changes at the population level.
Assuntos
Vírus da Dengue/genética , Dengue/virologia , Doenças Endêmicas , Evolução Molecular , Proteínas não Estruturais Virais/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Colômbia/epidemiologia , Dengue/epidemiologia , Dengue/transmissão , Feminino , Variação Genética , Genoma Viral , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Prevalência , RNA Viral/isolamento & purificação , Seleção Genética , Sorogrupo , Proteínas do Envelope Viral/genética , Sequenciamento Completo do Genoma , Adulto JovemRESUMO
Background: Dengue virus serotypes 1-4 (DENV-1-4) are the most common vector-borne viral pathogens of humans and the etiological agents of dengue fever and dengue hemorrhagic syndrome. A live-attenuated tetravalent dengue vaccine (TDV) developed by Takeda Vaccines has recently progressed to phase 3 safety and efficacy evaluation. Methods: We analyzed the qualitative features of the neutralizing antibody (nAb) response induced in naive and DENV-immune individuals after TDV administration. Using DENV-specific human monoclonal antibodies (mAbs) and recombinant DENV displaying different serotype-specific Ab epitopes, we mapped the specificity of TDV-induced nAbs against DENV-1-3. Results: Nearly all subjects had high levels of DENV-2-specific nAbs directed to epitopes centered on domain III of the envelope protein. In some individuals, the vaccine induced nAbs that tracked with a DENV-1-specific neutralizing epitope centered on domain I of the envelope protein. The vaccine induced binding Abs directed to a DENV-3 type-specific neutralizing epitope, but findings of mapping of DENV-3 type-specific nAbs were inconclusive. Conclusion: Here we provide qualitative measures of the magnitude and epitope specificity of the nAb responses to TDV. This information will be useful for understanding the performance of TDV in clinical trials and for identifying correlates of protective immunity.
Assuntos
Anticorpos Antivirais/sangue , Formação de Anticorpos/imunologia , Dengue Grave/sangue , Dengue Grave/imunologia , Vacinas Atenuadas/imunologia , Adolescente , Adulto , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Linhagem Celular Tumoral , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Método Duplo-Cego , Feminino , Humanos , Imunidade/imunologia , Masculino , Pessoa de Meia-Idade , Células U937 , Vacinação/métodos , Adulto JovemRESUMO
Mosquito-borne Zika virus (ZIKV) typically causes a mild and self-limiting illness known as Zika fever. Since its recent emergence in 2014 in the American continent, ZIKV infection during pregnancy has been closely associated with a wide range of congenital abnormalities. To date, no vaccines or antivirals are publicly available. We developed Zika virus-like particles (VLPs) and evaluated their immunogenicity and protective efficacy in mouse models. ZIKV VLPs (ZIKVLPs) formulated with alum were injected into 6-8-week-old interferon deficient AG129 mice as well as wild type BALB/c mice. Control mice received PBS/alum. Animals were challenged with 200 PFU (>1000 AG129 LD50s) of ZIKV strain H/PF/2013. All vaccinated mice survived with no morbidity or weight loss while control animals either died at 9 days post challenge (AG129) or had increased viremia (BALB/c). Neutralizing antibodies were observed in all ZIKVLP vaccinated mice. The role of neutralizing antibodies in protecting mice was demonstrated by passive transfer. Our findings demonstrate the protective efficacy of the ZIKVLP vaccine and highlight the important role that neutralizing antibodies play in protection against ZIKV infection.
Assuntos
Imunogenicidade da Vacina/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Infecção por Zika virus/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Modelos Animais de Doenças , Regulação Viral da Expressão Gênica , Células HEK293 , Humanos , Imunização Passiva , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Morbidade , Vacinação , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/genética , Viremia/virologia , Redução de Peso , Zika virus/efeitos dos fármacos , Zika virus/genética , Infecção por Zika virus/genéticaRESUMO
Congenital Zika virus (ZIKV) infection impacts fetal development and pregnancy outcomes. We infected a pregnant rhesus macaque with a Puerto Rican ZIKV isolate in the first trimester. The pregnancy was complicated by preterm premature rupture of membranes (PPROM), intraamniotic bacterial infection and fetal demise 49 days post infection (gestational day 95). Significant pathology at the maternal-fetal interface included acute chorioamnionitis, placental infarcts, and leukocytoclastic vasculitis of the myometrial radial arteries. ZIKV RNA was disseminated throughout fetal tissues and maternal immune system tissues at necropsy, as assessed by quantitative RT-PCR for viral RNA. Replicating ZIKV was identified in fetal tissues, maternal uterus, and maternal spleen by fluorescent in situ hybridization for viral replication intermediates. Fetal ocular pathology included a choroidal coloboma, suspected anterior segment dysgenesis, and a dysplastic retina. This is the first report of ocular pathology and prolonged viral replication in both maternal and fetal tissues following congenital ZIKV infection in a rhesus macaque. PPROM followed by fetal demise and severe pathology of the visual system have not been described in macaque congenital ZIKV infection previously. While this case of ZIKV infection during pregnancy was complicated by bacterial infection with PPROM, the role of ZIKV on this outcome cannot be precisely defined, and further nonhuman primate studies will determine if increased risk for PPROM or other adverse pregnancy outcomes are associated with congenital ZIKV infection.
Assuntos
Modelos Animais de Doenças , Olho/patologia , Placenta/patologia , Útero/patologia , Infecção por Zika virus/congênito , Animais , Feminino , Hibridização in Situ Fluorescente , Macaca mulatta , Gravidez , RNA Viral/genética , Replicação Viral , Zika virus/genética , Zika virus/fisiologiaRESUMO
Zika virus (ZIKV) is an emerging flavivirus that can cause birth defects and neurologic complications. Molecular tests are effective for diagnosing acute ZIKV infection, although the majority of infections produce no symptoms at all or present after the narrow window in which molecular diagnostics are dependable. Serology is a reliable method for detecting infections after the viremic period; however, most serological assays have limited specificity due to cross-reactive antibodies elicited by flavivirus infections. Since ZIKV and dengue virus (DENV) widely cocirculate, distinguishing ZIKV infection from DENV infection is particularly important for diagnosing individual cases or for surveillance to coordinate public health responses. Flaviviruses also elicit type-specific antibodies directed to non-cross-reactive epitopes of the infecting virus; such epitopes are attractive targets for the design of antigens for development of serological tests with greater specificity. Guided by comparative epitope modeling of the ZIKV envelope protein, we designed two recombinant antigens displaying unique antigenic regions on domain I (Z-EDI) and domain III (Z-EDIII) of the ZIKV envelope protein. Both the Z-EDI and Z-EDIII antigens consistently detected ZIKV-specific IgG in ZIKV-immune sera but not cross-reactive IgG in DENV-immune sera in late convalescence (>12 weeks postinfection). In contrast, during early convalescence (2 to 12 weeks postinfection), secondary DENV-immune sera and some primary DENV-immune sera cross-reacted with the Z-EDI and Z-EDIII antigens. Analysis of sequential samples from DENV-immune individuals demonstrated that Z-EDIII cross-reactivity peaked in early convalescence and declined steeply over time. The Z-EDIII antigen has much potential as a diagnostic antigen for population-level surveillance and for detecting past infections in patients.
Assuntos
Antígenos Virais/metabolismo , Vírus da Dengue/imunologia , Dengue/diagnóstico , Testes Sorológicos/métodos , Proteínas do Envelope Viral/imunologia , Infecção por Zika virus/diagnóstico , Zika virus/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Reações Cruzadas , Dengue/sangue , Dengue/virologia , Vírus da Dengue/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Humanos , Imunoglobulina G/sangue , Estudos Longitudinais , Vigilância da População , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Fatores de Tempo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Zika virus/isolamento & purificação , Infecção por Zika virus/sangue , Infecção por Zika virus/virologiaRESUMO
Hand, foot, and mouth disease (HFMD) is a reemerging illness caused by a variety of enteroviruses. The main causative agents are enterovirus 71 (EV71), coxsackievirus A16 (CVA16), and, most recently, coxsackievirus A6 (CVA6). Enterovirus infections can vary from asymptomatic infections to those with a mild fever and blisters on infected individuals' hands, feet, and throats to infections with severe neurological complications. Viral persistence for weeks postinfection (wpi) has also been documented by the demonstration of virus in children's stools. However, little is known about disease progression, viral spread, and tissue tropism of these viruses. These types of studies are limited because many recently developed mouse models mimic the severe neurological complications that occur in a small percentage of enterovirus infections. In the present study, we documented real-time EV71 infection in two different mouse strains by the use of in vivo imaging. Infection of BALB/c mice with a bioluminescent mouse-adapted EV71 construct (mEV71-NLuc) resulted in a lack of clinical signs of disease but in relatively high viral replication, as visualized by luminescence, for 2 wpi. In contrast, mEV71-NLuc infection of AG129 mice (alpha/beta and gamma interferon receptor deficient) showed rapid spread and long-term persistence of the virus in the brain. Interestingly, AG129 mice that survived infection maintained luminescence in the brain for up to 8 wpi. The results we present here will allow future studies on EV71 antiviral drug susceptibility, vaccine efficacy, transmissibility, and pathogenesis. IMPORTANCE We report here that a stable full-length enterovirus 71 (EV71) reporter construct was used to visualize real-time viral spread in AG129 and BALB/c mice. To our knowledge, this is the first report of in vivo imaging of infection with any member of the Picornaviridae family. The nanoluciferase (NLuc) gene, one of the smallest luciferase genes currently available, was shown to be stable in the EV71 genome for eight passages on rhabdomyosarcoma cells. Real-time visualization of EV71 infection in mice identified areas of tropism that would have been missed by traditional methods, including full characterization of EV71 replication in BALB/c mice. Additionally, the bioluminescent construct allowed for increased speed and sensitivity of cell culture assays and will allow future studies involving various degrees of enterovirus infection in mice, not just severe infections. Our data suggest that interferon plays an important role in controlling EV71 infection in the central nervous system of mice.
Assuntos
Enterovirus Humano A/fisiologia , Infecções por Enterovirus/virologia , Replicação Viral , Animais , Anticorpos Antivirais/sangue , Linhagem Celular Tumoral , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/patologia , Humanos , Camundongos Endogâmicos BALB C , Especificidade de Órgãos , Carga Viral , Imagem Corporal TotalRESUMO
UNLABELLED: Hand, foot, and mouth disease (HFMD) has spread throughout the Asia-Pacific region, affecting millions of young children, who develop symptoms ranging from painful blisters around their mouths and hands to neurological complications. Many members of the genus Enterovirus (family Picornaviridae) cause HFMD. Enterovirus 71 (EV71) is one of the primary causative agents and has been linked to severe disease. Vaccine efficacy and pathogenesis studies for EV71 have been limited because there is a lack of suitable animal models. Previously, we generated a mouse-adapted EV71 (mEV71) capable of infecting 12-week-old interferon receptor-deficient AG129 mice and used the model to evaluate the efficacy of candidate HFMD vaccines. Here, we present data investigating the genetic correlates of EV71 adaptation and characterize the virus's tissue tropism in mice. Using reverse genetics, a VP1 mutation (K244E) was shown to be necessary for mEV71 virulence in adult mice. Another VP1 mutation (H37R) was required for mEV71 recovery on rhabdomyosarcoma (RD) cells. Viral loads determined by real-time reverse transcription (RT)-PCR confirmed the presence of mEV71 in the sera and multiple organs of mice. Histological analysis revealed signs of meningitis and encephalitis, characteristic of severe human disease. The further description of this model has provided insight into EV71 pathogenesis and demonstrates the importance of the VP1 region in facilitating mEV71 adaptation. IMPORTANCE: EV71 is a reemerging pathogen, and little is known about the genetic determinants involved in its pathogenesis. The absence of animal models has contributed to this lack of knowledge. The data presented here improve upon the existing animal models by characterizing a mouse-adapted strain of EV71. We determined that a VP1 mutation (K244E) was needed for EV71 virulence in adult AG129 mice. While this mutation was found previously for EV71 adaptation in 5-day-old BALB/c mice, neurotropic disease did not develop. Using interferon-deficient mice, we raised the age of susceptibility beyond 6 weeks and provided clear evidence that our model mimics severe human infections. The model can be exploited to identify determinants of EV71 virulence and to reveal molecular mechanisms that control the virus-host interaction, especially those associated with neurotropic disease. Furthermore, these data provide useful information regarding the importance of VP1, specifically position 244, in host adaptation and tissue dissemination.
Assuntos
Enterovirus Humano A/patogenicidade , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Proteínas Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Adulto , Estruturas Animais/virologia , Animais , Modelos Animais de Doenças , Encefalite Viral/patologia , Encefalite Viral/virologia , Enterovirus Humano A/genética , Humanos , Interferons/deficiência , Meningite Viral/patologia , Meningite Viral/virologia , Camundongos , Proteínas Mutantes/genética , Reação em Cadeia da Polimerase em Tempo Real , Genética Reversa , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soro/virologia , Carga Viral , Proteínas Estruturais Virais/genética , Virulência , Fatores de Virulência/genéticaRESUMO
Dengue fever is caused by infection with one of four dengue virus (DENV) serotypes (DENV-1-4), necessitating tetravalent dengue vaccines that can induce protection against all four DENV. Takeda's live attenuated tetravalent dengue vaccine candidate (TDV) comprises an attenuated DENV-2 strain plus chimeric viruses containing the prM and E genes of DENV-1, -3 and -4 cloned into the attenuated DENV-2 'backbone'. In Phase 1 and 2 studies, TDV was well tolerated by children and adults aged 1.5-45 years, irrespective of prior dengue exposure; mild injection-site symptoms were the most common adverse events. TDV induced neutralizing antibody responses and seroconversion to all four DENV as well as cross-reactive T cell-mediated responses that may be necessary for broad protection against dengue fever.
Assuntos
Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Dengue/imunologia , Vacinas contra Dengue/efeitos adversos , Vacinas contra Dengue/genética , Vírus da Dengue/genética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Humanos , Sorogrupo , Linfócitos T/imunologia , Resultado do Tratamento , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
UNLABELLED: Alphaviruses represent a diverse set of arboviruses, many of which are important pathogens. Chikungunya virus (CHIKV), an arthritis-inducing alphavirus, is the cause of a massive ongoing outbreak in the Caribbean and South America. In contrast to CHIKV, other related alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and Semliki Forest virus (SFV), can cause encephalitic disease. E2, the receptor binding protein, has been implicated as a determinant in cell tropism, host range, pathogenicity, and immunogenicity. Previous reports also have demonstrated that E2 contains residues important for host range expansions and monoclonal antibody binding; however, little is known about what role each protein domain (e.g., A, B, and C) of E2 plays on these factors. Therefore, we constructed chimeric cDNA clones between CHIKV and VEEV or SFV to probe the effect of each domain on pathogenicity in vitro and in vivo. CHIKV chimeras containing each of the domains of the E2 (ΔDomA, ΔDomB, and ΔDomC) from SFV, but not VEEV, were successfully rescued. Interestingly, while all chimeric viruses were attenuated compared to CHIKV in mice, ΔDomB virus showed similar rates of infection and dissemination in Aedes aegypti mosquitoes, suggesting differing roles for the E2 protein in different hosts. In contrast to CHIKV; ΔDomB, and to a lesser extent ΔDomA, caused neuron degeneration and demyelination in mice infected intracranially, suggesting a shift toward a phenotype similar to SFV. Thus, chimeric CHIKV/SFV provide insights on the role the alphavirus E2 protein plays on pathogenesis. IMPORTANCE: Chikungunya virus (CHIKV) has caused large outbreaks of acute and chronic arthritis throughout Africa and Southeast Asia and has now become a massive public health threat in the Americas, causing an estimated 1.2 million human cases in just over a year. No approved vaccines or antivirals exist for human use against CHIKV or any other alphavirus. Despite the threat, little is known about the role the receptor binding protein (E2) plays on disease outcome in an infected host. To study this, our laboratory generated chimeric CHIKV containing corresponding regions of the Semliki Forest virus (SFV) E2 (domains A, B, and C) substituted into the CHIKV genome. Our results demonstrate that each domain of E2 likely plays a critical, but dissimilar role in the viral life cycle. Our experiments show that manipulation of E2 domains can be useful for studies on viral pathogenesis and potentially the production of vaccines and/or antivirals.
Assuntos
Infecções por Alphavirus/patologia , Vírus Chikungunya/patogenicidade , Vírus da Encefalite Equina Venezuelana/patogenicidade , Vírus da Floresta de Semliki/patogenicidade , Proteínas do Envelope Viral/metabolismo , Aedes/virologia , Infecções por Alphavirus/virologia , Animais , Encéfalo/patologia , Vírus Chikungunya/genética , Modelos Animais de Doenças , Vírus da Encefalite Equina Venezuelana/genética , Feminino , Masculino , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Vírus da Floresta de Semliki/genética , Proteínas do Envelope Viral/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
BACKGROUND: Chikungunya virus (CHIKV) and other alphaviruses are the etiologic agents of numerous diseases in both humans and animals. Despite this, the viral mediators of protective immunity against alphaviruses are poorly understood, highlighted by the lack of a licensed human vaccine for any member of this virus genus. The alphavirus E2, the receptor-binding envelope protein, is considered to be the predominant target of the protective host immune response. Although envelope protein domains have been studied for vaccine and neutralization in flaviviruses, their role in alphaviruses is less characterized. Here, we describe the role of the alphavirus E2 domains in neutralization and protection through the use of chimeric viruses. METHODOLOGY/PRINCIPAL FINDINGS: Four chimeric viruses were constructed in which individual E2 domains of CHIKV were replaced with the corresponding domain from Semliki Forest virus (SFV) (ΔDomA/ΔDomB/ΔDomC/ ΔDomA+B). Vaccination studies in mice (both live and inactivated virus) revealed that domain B was the primary determinant of neutralization. Neutralization studies with CHIKV immune serum from humans were consistent with mouse studies, as ΔDomB was poorly neutralized. CONCLUSIONS/SIGNIFICANCE: Using chimeric viruses, it was determined that the alphavirus E2 domain B was the critical target of neutralizing antibodies in both mice and humans. Therefore, chimeric viruses may have more relevance for vaccine discovery than peptide-based approaches, which only detect linear epitopes. This study provides new insight into the role of alphavirus E2 domains on neutralization determinants and may be useful for the design of novel therapeutic technologies.
Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vírus Chikungunya/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Mapeamento de Epitopos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Testes de Neutralização , Estrutura Terciária de Proteína , Recombinação Genética , Vírus da Floresta de Semliki/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
We are developing a live-attenuated tetravalent dengue vaccine (TDV) candidate based on an attenuated dengue 2 virus (TDV-2) and 3 chimeric viruses containing the premembrane and envelope genes of dengue viruses (DENVs) -1, -3, and -4 expressed in the context of the attenuated TDV-2 genome (TDV-1, TDV-3, and TDV-4, respectively). In this study, we analyzed and characterized the CD8(+) T-cell response in flavivirus-naive human volunteers vaccinated with 2 doses of TDV 90 days apart via the subcutaneous or intradermal routes. Using peptide arrays and intracellular cytokine staining, we demonstrated that TDV elicits CD8(+) T cells targeting the nonstructural NS1, NS3, and NS5 proteins of TDV-2. The cells were characterized by the production of interferon-γ, tumor necrosis factor-α, and to a lesser extent interleukin-2. Responses were highest on day 90 after the first dose and were still detectable on 180 days after the second dose. In addition, CD8(+) T cells were multifunctional, producing ≥2 cytokines simultaneously, and cross-reactive to NS proteins of the other 3 DENV serotypes. Overall, these findings describe the capacity of our candidate dengue vaccine to elicit cellular immune responses and support the further evaluation of T-cell responses in samples from future TDV clinical trials.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas contra Dengue/administração & dosagem , Vacinas contra Dengue/imunologia , Vírus da Dengue/imunologia , Adulto , Antígenos Virais/imunologia , Estudos de Coortes , Citocinas/biossíntese , Voluntários Saudáveis , Humanos , Vacinação/métodos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
Chikungunya virus (CHIKV), a mosquito-borne alphavirus, recently re-emerged in Africa and spread to islands in the Indian Ocean, the Indian subcontinent, and to South East Asia. Viremic travelers have also imported CHIKV to the Western hemisphere highlighting the importance of CHIKV in public health. In addition to the great burden of arthralgic disease, which can persist for months or years, epidemiologic studies have estimated case-fatality rates of â¼0.1%, principally from neurologic disease in older patients. There are no licensed vaccines or effective therapies to prevent or treat human CHIKV infections. We have developed a live CHIKV vaccine (CHIKV/IRES) that is highly attenuated yet immunogenic in mouse models, and is incapable of replicating in mosquito cells. In this study we sought to decipher the role of adaptive immunity elicited by CHIKV/IRES in protection against wild-type CHIKV infection. A single dose of vaccine effectively activated T cells with an expansion peak on day 10 post immunization and elicited memory CD4(+) and CD8(+) T cells that produced IFN-γ, TNF-α and IL-2 upon restimulation with CHIKV/IRES. Adoptive transfer of CHIKV/IRES-immune CD4(+) or CD8(+) T cells did not confer protection against wtCHIKV-LR challenge. By contrast, passive immunization with anti-CHIKV/IRES immune serum provided protection, and a correlate of a minimum protective neutralizing antibody titer was established. Overall, our findings demonstrate the immunogenic potential of the CHIKV/IRES vaccine and highlight the important role that neutralizing antibodies play in protection against an acute CHIKV infection.
Assuntos
Imunidade Adaptativa , Infecções por Alphavirus/imunologia , Anticorpos Neutralizantes/imunologia , Ativação Linfocitária , Vacinas Virais/imunologia , Transferência Adotiva , Infecções por Alphavirus/prevenção & controle , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Febre de Chikungunya , Vírus Chikungunya , Modelos Animais de Doenças , Feminino , Soros Imunes/imunologia , Imunidade Celular , Imunização Passiva , Memória Imunológica , Interferon gama/imunologia , Interleucina-2/imunologia , Camundongos , Camundongos da Linhagem 129 , Fator de Necrose Tumoral alfa/imunologia , Vacinas Atenuadas/imunologia , Viremia/imunologiaRESUMO
El herpesvirus bovino-1 (BHV-1) es un virus de genoma DNA perteneciente a la familia Herpesviridae, el cual afecta al bovino en el que provoca un amplio espectro de manifestaciones clínicas y pérdidas económicas. El principal componente inmunogénico de su envoltura es la glicoproteína D (gD), la cual ha sido caracterizada y utilizada como inmunógeno en distintos sistemas de expresión. El objetivo de este trabajo fue generar un poxvirus recombinante (Raccoonpox [RCN]) que expresara una versión truncada de la gD del BHV-1 para ser usado como inmunógeno. Para ello, se amplificó el gen que codifica para la versión truncada de la gD, la cual se clonó en el plásmido de transferencia pTK/ IRES/tpa que posee sitios de homología a la timidinakinasa del poxvirus, un sitio interno de entrada al ribosoma (IRES) y una señal secretoria (tPA), generando el constructo pTK/gD/IRES/tpa. Para generar el RCN recombinante, se tomaron células BSC-1, se infectaron con una cepa Silvestre del RCN (CDC/V71-I-85A) a un índice de multiplicidad de infección de 0,05 y se transfectaron con el constructo pTK/gD/IRES/tpa; generándose diferentes poblaciones virales con y sin el gen de interés. Para seleccionar los virus recombinantes que expresaban el gen de interés, se realizó una selección de recombinantes negativos para timidina kinasa y positivos para la gD por tres rondas de purificación de placas en monocapas de células RAT-2 las cuales son mutantes para timidina kinasa y en presencia de bromodeoxiuridina. Los virus recombinantes se confirmaron por PCR y secuenciación de nucleótidos y se denominaron RCN-gD.
Bovine Herpesvirus-1 is a DNA virus belonging to the family Herpesviridae, which affects cattle, causing a wide spectrum of clinical manifestations and economic losses. The main immunogenic component is its envelope glycoprotein D (gD), which has been characterized and used as immunogen in different expression systems. The aim of this work was to generate a recombinant poxvirus (Raccoonpox [RCN]) expressing a truncated version of BHV-1 gD to be used as a vaccine. To do this, it was amplified the gene for a truncated version of gD which subsequently was cloned in transfer plasmid PTK/IRES/tpa which has homology to sites of poxvirus thymidine kinase, an internal site of ribosome entry (IRES) and a secretory signal (tPA), generating the construct PTK/gD/IRES/tpa. To generate the recombinant RCN, we took BSC-1 cells and we infected with a wild type RCN (CDC/V71-I-85A) at a multiplicity of infection of 0.05, then cells were transfected with the construct PTK/gD/IRES/tpa, generating different viral populations with and without the gene of interest. To select recombinant viruses expressing the gene of interest, we performed a selection of recombinant thymidine kinase negative and positive for gD by three rounds of plaque purification on RAT-2 cells monolayers which are thymidine kinase null and using bromodeoxyuridine. Recombinant viruses were recovered and confirmed by PCR and nucleotide sequencing and so called RCN-gD.
RESUMO
Chikungunya virus (CHIKV) is the mosquito-borne alphavirus that is the etiologic agent of massive outbreaks of arthralgic febrile illness that recently affected millions of people in Africa and Asia. The only CHIKV vaccine that has been tested in humans, strain 181/clone 25, is a live-attenuated derivative of Southeast Asian human isolate strain AF15561. The vaccine was immunogenic in phase I and II clinical trials; however, it induced transient arthralgia in 8% of the vaccinees. There are five amino acid differences between the vaccine and its parent, as well as five synonymous mutations, none of which involves cis-acting genome regions known to be responsible for replication or packaging. To identify the determinants of attenuation, we therefore tested the five nonsynonymous mutations by cloning them individually or in different combinations into infectious clones derived from two wild-type (WT) CHIKV strains, La Reunion and AF15561. Levels of virulence were compared with those of the WT strains and the vaccine strain in two different murine models: infant CD1 and adult A129 mice. An attenuated phenotype indistinguishable from that of the 181/clone 25 vaccine strain was obtained by the simultaneous expression of two E2 glycoprotein substitutions, with intermediate levels of attenuation obtained with the single E2 mutations. The other three amino acid mutations, in nsP1, 6K, and E1, did not have a detectable effect on CHIKV virulence. These results indicate that the attenuation of strain 181/clone 25 is mediated by two point mutations, explaining the phenotypic instability observed in human vaccinees and also in our studies.
Assuntos
Substituição de Aminoácidos , Vírus Chikungunya/imunologia , Vírus Chikungunya/patogenicidade , Proteínas do Envelope Viral/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Infecções por Alphavirus/patologia , Infecções por Alphavirus/virologia , Animais , Febre de Chikungunya , Modelos Animais de Doenças , Feminino , Glicoproteínas/genética , Glicoproteínas/imunologia , Camundongos , Gravidez , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/efeitos adversos , VirulênciaRESUMO
Every year, Dengue virus (DENV) infects approximately 100 million people. There are currently several vaccines undergoing clinical studies, but most target the induction of neutralizing antibodies. Unfortunately, DENV infection can be enhanced by subneutralizing levels of antibodies that bind virions and deliver them to cells of the myeloid lineage, thereby increasing viral replication (termed antibody-dependent enhancement [ADE]). T lymphocyte-based vaccines may offer an alternative that avoids ADE. The goal of our study was to describe the cellular immune response generated after primary DENV infection in Indian rhesus macaques. We infected eight rhesus macaques with 105 plaque-forming units (PFU) of DENV serotype 2 (DENV2) New Guinea C (NGC) strain, and monitored viral load and the cellular immune response to the virus. Viral replication peaked at day 4 post-infection and was resolved by day 10. DENV-specific CD4+ and CD8+ T lymphocytes targeted nonstructural (NS) 1, NS3 and NS5 proteins after resolution of peak viremia. DENV-specific CD4+ cells expressed interferon-gamma (IFN-γ) along with tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), and macrophage inflammatory protein-1 beta (MIP-1ß). In comparison, DENV-specific CD8+ cells expressed IFN-γ in addition to MIP-1ß and TNF-α and were positive for the degranulation marker CD107a. Interestingly, a fraction of the DENV-specific CD4+ cells also stained for CD107a, suggesting that they might be cytotoxic. Our results provide a more complete understanding of the cellular immune response during DENV infection in rhesus macaques and contribute to the development of rhesus macaques as an animal model for DENV vaccine and pathogenicity studies.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vírus da Dengue/imunologia , Dengue/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Citocinas/biossíntese , Citocinas/imunologia , Dengue/virologia , Imunidade Celular , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Macaca mulatta , RNA Helicases/imunologia , Serina Endopeptidases/imunologia , Carga Viral , Replicação Viral/imunologiaRESUMO
West Nile Virus (WNV) is an arthropod-borne agent classified in the Flavivirus genus. Infection has been demonstrated in many vertebrate species including birds, mammals and reptiles. WNV can affect the nervous system of humans, horses and birds causing mild to severe illness and sometimes death. In 1999 WNV was introduced into the Americas causing a small outbreak in New York City. In the following years, the virus spread across North America and later into Central America, the Caribbean and parts of South America. Serological evidence of WNV in Colombia was first documented in 2005 in equines from the Atlantic coast; however clinical cases in humans or animals have not been reported. We extended these studies searching for WNV antibodies in sera of equines of two other provinces in Colombia: Antioquia and El Meta. IgG and IgM antibodies were first determined and reactive sera were processed by plaque reduction neutralization test (PRNT) to confirm the specificity of results. Four horses from Antioquia but none from El Meta tested positive for WNV antibodies. These results suggest that WNV has spread across the Atlantic coast and is now invading the Andean region in Colombia.
El virus del Oeste del Nilo (WNV) es un agente del género Flavivirus transmitido por artrópodos. La infección con WNV ha sido demostrada en muchas especies de aves, mamíferos y reptiles. El WNV puede afectar el sistema nervioso de humanos, caballos y aves causando enfermedad de leve a severa, ocasionando la muerte en algunos casos. En 1999, el virus fue introducido en Norteamérica causando un brote en la ciudad de New York. En los siguientes años, el virus se extendió por Norteamérica, y posteriormente fue encontrado en el Caribe, Centro y Suramérica. El primer reporte de anticuerpos para WNV en Colombia se hizo en 2005, en equinos de la costa Atlántica. En el presente estudio se extendió la búsqueda de anticuerpos a otros dos Departamentos de Colombia: Antioquia y El Meta. Primero se determinó la presencia de anticuerpos IgM e IgG, y los sueros reactivos fueron procesados para anticuerpos neutralizantes por la técnica de reduccion de placas para confirmar los resultados. Cuatro equinos de Antioquia y ninguno de El Meta fueron positivos para anticuerpos anti-WNV. Los resultados sugieren que el WNV está ampliamente distribuido en la costa Atlántica de Colombia y ha iniciado su dispersión por la zona andina.
O vírus do Nilo Ocidental é um agente transmitido por artrópodes e pertence ao género Flavivirus. A infecção tem sido demonstrada em várias espécies de vertebrados incluindo pássaros, mamíferos e répteis. O vírus do Nilo Ocidental pode afectar o sistema nervoso de humanos, eqüinos e pássaros, causando doença de severidade média à grave a qual pode causar a morte em alguns casos. Em 1999, o vírus do Nilo Ocidental foi introduzido no continente americano, causando um surto na cidade de Nova York. Posteriormente, o vírus se disseminou pela América do Norte e mais tarde pela América Central, Caribe e parte da América do Sul. Os primeiros relatos do vírus do Nilo Ocidental na Colômbia surgiram em 2005 afectando eqüinos na costa atlântica. O objectivo desse trabalho foi buscar anticorpos contra o vírus do Nilo Ocidental no soro de equinos de dois estados da Colômbia: Antioquia e Meta. Anticorpos da classe IgG e IgM foram primeiramente determinados e soros reactivos foram analisados pela técnica de neutralização por redução em placa (PRNT) para confirmar a especificidade dos resultados. Quatro equinos provindos da Antioquia apresentaram resultados positivos para anticorpos contra o vírus do Nilo Ocidental; entretanto não foram detectados anticorpos nos animas provindos do Meta. Estes resultados sugerem que o vírus do Nilo Ocidental tem se disseminado através da costa atlântica e está agora invadindo a região andina na Colômbia.