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Real-time PCR is one of the most widely used techniques to diagnose measles cases. Here we report measles virus variants with three genetic mutations in the reverse primer annealing site of a widely used PCR. The mutations result in a slight loss of the PCR sensitivity. Variants bearing the three mutations presently circulate in different countries since at least the end of 2021. Our findings highlight the usefulness of molecular surveillance in monitoring if oligonucleotides in diagnostic tests remain adequate.
Assuntos
Sarampo , Patologia Molecular , Humanos , Suíça , RNA Viral/genética , Sarampo/diagnóstico , Sarampo/epidemiologia , Vírus do Sarampo/genética , Mutação , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A measles outbreak with 51 cases occurred in the canton of Vaud, Switzerland, between January and March 2024. The outbreak was triggered by an imported case, and 37 (72.5%) subsequent cases were previously vaccinated individuals. Epidemiological investigations showed that vaccinated measles cases were symptomatic and infectious. In a highly vaccinated population, it is important to raise awareness among healthcare professionals to suspect and test for measles virus when an outbreak is declared, irrespective of the vaccination status of the patients.
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Surtos de Doenças , Vacina contra Sarampo , Vírus do Sarampo , Sarampo , Vacinação , Humanos , Sarampo/prevenção & controle , Sarampo/epidemiologia , Suíça/epidemiologia , Surtos de Doenças/prevenção & controle , Vacina contra Sarampo/administração & dosagem , Vacinação/estatística & dados numéricos , Masculino , Feminino , Adulto , Adolescente , Criança , Vírus do Sarampo/imunologia , Vírus do Sarampo/isolamento & purificação , Pré-Escolar , Adulto Jovem , LactenteRESUMO
This study analyzed the cerebrospinal fluid features of 31 coronavirus disease 2019 (COVID-19) patients with neurological complications. We observed neither severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in the cerebrospinal fluid, nor intrathecal immunoglobulin G (IgG) synthesis but did observe signs of blood-brain barrier disruption. These results might serve as a basis for a better understanding of SARS-CoV-2 related neuropathogenesis.
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COVID-19 , SARS-CoV-2 , Humanos , Imunoglobulina G , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição ReversaRESUMO
We evaluated the rates of viral respiratory co-infections among SARS-CoV-2-infected children. Twelve percent of SARS-CoV-2-infected children had viral co-infection with one or more common respiratory viruses. This was significantly more frequent than among their SARS-CoV-2-infected adult household contacts (0%; p=0.028). Compared to the same period the previous year, common respiratory viruses were less frequently detected (12% vs 73%, p<0.001).Conclusion: Despite partial lockdown with school and daycare closure, and consequently similar exposure to common viruses between children and adults, SARS-CoV-2-infected children had more frequent viral respiratory co-infections than their SARS-CoV-2-infected adult household contacts. Circulation of common respiratory viruses was less frequent during the SARS-CoV-2 outbreak when compared to the same period last year, showing the impact of partial lockdown on the circulation of common viruses. What is Known: ⢠Viral respiratory co-infections are frequent in children. ⢠SARS-CoV-2 can be identified alongside other respiratory viruses, but data comparing children and adults are lacking. What is New: ⢠Children infected with SARS-CoV-2 are more likely to have viral respiratory co-infections than their SARS-CoV-2-infected adult household contacts, which is surprising in the context of partial lockdown with schools and daycare closed. ⢠When compared to data collected during the same period last year, our study also showed that partial lockdown reduced circulation of common respiratory viruses.
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COVID-19 , Coinfecção , Adulto , Criança , Coinfecção/epidemiologia , Controle de Doenças Transmissíveis , Surtos de Doenças , Humanos , SARS-CoV-2RESUMO
Live-attenuated vaccines are currently contraindicated in solid-organ transplant recipients. However, the risk of vaccine-preventable infections is lifelong, and can be particularly severe after transplantation. In this prospective interventional national cohort study, 44 pediatric liver transplant recipients with measles IgG antibodies <150 IU/L (below seroprotection threshold) received measles-mumps-rubella vaccine (MMR) at a median of 6.3 years posttransplantation (interquartile range, 4.0 to 10.9). A maximum of two additional doses were administered in nonresponders or when seroprotection was lost. Vaccine responses occurred in 98% (95% confidence interval [CI], 88-100) of patients. Seroprotection at 1-, 2-, and 3-year follow-up reached 62% (95% CI, 45-78), 86% (95% CI, 70-95), and 89% (95% CI, 67-99), respectively. All patients responded appropriately to the booster dose(s). Vaccinations were well tolerated and no serious adverse event attributable to vaccination was identified during the 8-week follow-up period (or later), using a multimodal approach including standardized telephone interviews, diarized side effect reporting, and monitoring of vaccinal virus shedding. We conclude that live attenuated MMR vaccine can be administered in liver transplant recipients fulfilling specific eligibility criteria (>1 year posttransplantation, low immunosuppression, lymphocyte count ≥0.75 G/L), inducing seroprotection in most subjects. (Clinicaltrials.gov number NCT01770119).
Assuntos
Hospedeiro Imunocomprometido/imunologia , Transplante de Fígado/métodos , Vacina contra Sarampo-Caxumba-Rubéola/administração & dosagem , Segurança do Paciente/estatística & dados numéricos , Medição de Risco/métodos , Vacinas Atenuadas/administração & dosagem , Adolescente , Anticorpos Antivirais/imunologia , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Hospedeiro Imunocomprometido/efeitos dos fármacos , Terapia de Imunossupressão , Lactente , Recém-Nascido , Masculino , Sarampo/imunologia , Sarampo/prevenção & controle , Vírus do Sarampo/imunologia , Vacina contra Sarampo-Caxumba-Rubéola/imunologia , Caxumba/imunologia , Caxumba/prevenção & controle , Vírus da Caxumba/imunologia , Prognóstico , Estudos Prospectivos , Rubéola (Sarampo Alemão)/imunologia , Rubéola (Sarampo Alemão)/prevenção & controle , Vírus da Rubéola/imunologia , Vacinação , Vacinas Atenuadas/imunologiaRESUMO
BACKGROUND: As the incidence of malaria diminishes, a better understanding of nonmalarial fever is important for effective management of illness in children. In this study, we explored the spectrum of causes of fever in African children. METHODS: We recruited children younger than 10 years of age with a temperature of 38°C or higher at two outpatient clinics--one rural and one urban--in Tanzania. Medical histories were obtained and clinical examinations conducted by means of systematic procedures. Blood and nasopharyngeal specimens were collected to perform rapid diagnostic tests, serologic tests, culture, and molecular tests for potential pathogens causing acute fever. Final diagnoses were determined with the use of algorithms and a set of prespecified criteria. RESULTS: Analyses of data derived from clinical presentation and from 25,743 laboratory investigations yielded 1232 diagnoses. Of 1005 children (22.6% of whom had multiple diagnoses), 62.2% had an acute respiratory infection; 5.0% of these infections were radiologically confirmed pneumonia. A systemic bacterial, viral, or parasitic infection other than malaria or typhoid fever was found in 13.3% of children, nasopharyngeal viral infection (without respiratory symptoms or signs) in 11.9%, malaria in 10.5%, gastroenteritis in 10.3%, urinary tract infection in 5.9%, typhoid fever in 3.7%, skin or mucosal infection in 1.5%, and meningitis in 0.2%. The cause of fever was undetermined in 3.2% of the children. A total of 70.5% of the children had viral disease, 22.0% had bacterial disease, and 10.9% had parasitic disease. CONCLUSIONS: These results provide a description of the numerous causes of fever in African children in two representative settings. Evidence of a viral process was found more commonly than evidence of a bacterial or parasitic process. (Funded by the Swiss National Science Foundation and others.).
Assuntos
Infecções Bacterianas/epidemiologia , Febre/etiologia , Infecções Respiratórias/epidemiologia , Viroses/epidemiologia , Instituições de Assistência Ambulatorial , Infecções Bacterianas/complicações , Criança , Pré-Escolar , Feminino , Gastroenterite/complicações , Gastroenterite/epidemiologia , Humanos , Lactente , Malária/complicações , Malária/epidemiologia , Masculino , Pneumonia/complicações , Pneumonia/epidemiologia , Infecções Respiratórias/complicações , Tanzânia/epidemiologia , Febre Tifoide/complicações , Febre Tifoide/epidemiologia , Infecções Urinárias/complicações , Infecções Urinárias/epidemiologia , Viroses/complicaçõesRESUMO
Many arthropod-borne viruses (arboviruses) underwent a dramatic geographic expansion over the last few years, following the spread of their vectors. It is the case for dengue, currently endemic in most tropical regions, for chikungunya and Zika viruses, which propagated rapidly over a considerable territory. West Nile is one of the most broadly distributed arboviruses in the world. The clinical presentation of these viral infections is non-specific (fever, arthralgia, headache, rash). For travelers presenting with fever within 15 days after returning from an endemic area, laboratory tests can distinguish them. The management is essentially supportive but a specific diagnosis might be important in anticipating possible complications and in decreasing the number of laboratory tests often offered in the case of fever of unknown origin.
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Infecções por Arbovirus/diagnóstico , Doenças Transmissíveis Emergentes/diagnóstico , Viagem , Infecções por Arbovirus/epidemiologia , Infecções por Arbovirus/terapia , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/terapia , HumanosRESUMO
BACKGROUND: Zika virus has recently spread to South- and Central America, causing congenital birth defects and neurological complications. Many people at risk are flavivirus pre-immune due to prior infections with other flaviviruses (e.g. dengue virus) or flavivirus vaccinations. Since pre-existing cross-reactive immunity can potentially modulate antibody responses to Zika virus infection and may affect the outcome of disease, we analyzed fine-specificity as well as virus-neutralizing and infection-enhancing activities of antibodies induced by a primary Zika virus infection in flavivirus-naïve as well as yellow fever- and/or tick-borne encephalitis-vaccinated individuals. METHODOLOGY: Antibodies in sera from convalescent Zika patients with and without vaccine-induced immunity were assessed by ELISA with respect to Zika virus-specificity and flavivirus cross-reactivity. Functional analyses included virus neutralization and infection-enhancement. The contribution of IgM and cross-reactive antibodies to these properties was determined by depletion experiments. PRINCIPAL FINDINGS: Pre-existing flavivirus immunity had a strong influence on the antibody response in primary Zika virus infections, resulting in higher titers of broadly flavivirus cross-reactive antibodies and slightly lower levels of Zika virus-specific IgM. Antibody-dependent enhancement (ADE) of Zika virus was mediated by sub-neutralizing concentrations of specific IgG but not by cross-reactive antibodies. This effect was potently counteracted by the presence of neutralizing IgM. Broadly cross-reactive antibodies were able to both neutralize and enhance infection of dengue virus but not Zika virus, indicating a different exposure of conserved sequence elements in the two viruses. CONCLUSIONS: Our data point to an important role of flavivirus-specific IgM during the transient early stages of infection, by contributing substantially to neutralization and by counteracting ADE. In addition, our results highlight structural differences between strains of Zika and dengue viruses that are used for analyzing infection-enhancement by cross-reactive antibodies. These findings underscore the possible impact of specific antibody patterns on flavivirus disease and vaccination efficacy.
Assuntos
Anticorpos Antivirais/sangue , Vacinas Virais/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Afinidade de Anticorpos , Antígenos Virais/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina G/sangue , Testes de Neutralização , Polietilenoglicóis , Proteínas do Envelope Viral/imunologia , Zika virus/genéticaRESUMO
Human parainfluenza virus (HPIV) types 1 and 3 are major viral pathogens responsible for upper and lower respiratory tract infections. The diagnosis of these two species is achieved generally by specific reverse transcription-polymerase chain (RT-PCR) reaction methods. In this study, a real-time RT-PCR was developed using a common pair of primers-probe (HPIV-1+3) for the simultaneous detection of both HPIV-1 and HPIV-3 genomes. Results obtained in a 10-fold dilution series assay demonstrate a high sensitivity of the assay with a lowest detection limit of approximately one plasmid copy for both HPIV-1 and HPIV-3. A comparison of HPIV-1 and HPIV-3 clinical sample detection between specific HPIV-1/HPIV-3 pairs of primers-probes and the HPIV-1+3 combination clearly shows that the latter is significantly more sensitive (gain of about five threshold cycles) than the former for HPIV-3 detection, while equivalent values are observed for HPIV-1. The HPIV-1+3 combination constitutes a more rapid, more sensitive, and less expensive alternative than classical or multiplex real-time RT-PCR assays usually used in clinical laboratories.
Assuntos
Vírus da Parainfluenza 1 Humana/isolamento & purificação , Vírus da Parainfluenza 3 Humana/isolamento & purificação , Infecções por Respirovirus/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Primers do DNA , Humanos , Vírus da Parainfluenza 1 Humana/genética , Vírus da Parainfluenza 3 Humana/genética , RNA Viral/análise , Sensibilidade e EspecificidadeRESUMO
[This corrects the article DOI: 10.1186/s13756-019-0524-4.].
RESUMO
Background: Needlestick accidents while handling of infectious material in research laboratories can lead to life-threatening infections in laboratory personnel. In laboratories working with the lymphocytic choriomeningitis virus (LCMV), the virus can be transmitted to humans through needlestick injury and lead to serious acute illness up to meningitis. Case presentation: We report of a case of LCMV meningitis in a laboratory worker who sustained a penetrating needlestick injury with a LCMV-contaminated hollow needle whilst disposing of a used syringe into the sharps waste bin. Four days after needlestick injury the laboratory worker developed a systemic disease: 11 days after exposure, she was diagnosed with meningitis with clinical signs and symptoms of meningismus, photophobia, nausea and vomiting, requiring hospitalisation. The PCR was positive for LCMV from the blood sample. 18 days after exposure, seroconversion confirmed the diagnosis of LCMV-induced meningitis with an increase in specific LCMV-IgM antibodies to 1:10'240 (day 42: 1:20'480). Ten weeks after exposure, a follow-up titre for IgM returned negative, whereas IgG titre increased to 1:20'480. Conclusions: This is the first case report of a PCR-documented LCMV meningitis, coupled with seroconversion, following needlestick injury. It highlights the importance of infection prevention practices that comprise particularly well established safety precaution protocols in research laboratories handling this pathogenic virus, because exposure to even a small amount of LCMV can lead to a severe, life-threatening infection.
Assuntos
Infecções por Arenaviridae/etiologia , Meningite/diagnóstico por imagem , Meningite/etiologia , Ferimentos Penetrantes Produzidos por Agulha/complicações , Acidentes de Trabalho , Anticorpos Antivirais/sangue , Infecções por Arenaviridae/diagnóstico , Feminino , Humanos , Pessoal de Laboratório , Vírus da Coriomeningite Linfocítica/genética , Imageamento por Ressonância Magnética , Meningite/virologia , Ferimentos Penetrantes Produzidos por Agulha/virologia , SoroconversãoRESUMO
Canine distemper virus (CDV) causes a chronic, demyelinating, progressive or relapsing neurological disease in dogs, because CDV persists in the CNS. Persistence of virulent CDV, such as the A75/17 strain has been reproduced in cell cultures where it is associated with a non-cytolytic infection with very limited cell-cell fusion. This is in sharp contrast to attenuated CDV infection in cell cultures, such as the Onderstepoort (OP) CDV strain, which produces extensive fusion activity and cytolysis. Fusion efficiency may be determined by the structure of the viral fusion protein per se but also by its interaction with other structural proteins of CDV. This was studied by combining genes derived from persistent and non-persistent CDV strains in transient transfection experiments. It was found that fusion efficiency was markedly attenuated by the structure of the fusion protein of the neurovirulent A75/17-CDV. Moreover, we showed that the interaction of the surface glycoproteins with the M protein of the persistent strain greatly influenced fusion activity. Site directed mutagenesis showed that the c-terminus of the M protein is of particular importance in this respect. Interestingly, although the nucleocapsid protein alone did not affect F/H-induced cell-cell fusion, maximal inhibition occurred when the latter was added to combined glycoproteins with matrix protein. Thus, the present study suggests that very limited fusogenicity in virulent CDV infection, which favours persistence by limiting cell destruction involves complex interactions between all viral structural proteins.
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Fusão de Membrana , Proteínas do Nucleocapsídeo/metabolismo , Proteínas da Matriz Viral/metabolismo , Animais , Fusão Celular , Chlorocebus aethiops , Vírus da Cinomose Canina/genética , Vírus da Cinomose Canina/metabolismo , Cães , Células Vero , Proteínas Virais de Fusão/metabolismo , Proteínas da Matriz Viral/química , Proteínas Estruturais Virais/metabolismoRESUMO
BACKGROUND: To construct evidence-based guidelines for management of febrile illness, it is essential to identify clinical predictors for the main causes of fever, either to diagnose the disease when no laboratory test is available or to better target testing when a test is available. The objective was to investigate clinical predictors of several diseases in a cohort of febrile children attending outpatient clinics in Tanzania, whose diagnoses have been established after extensive clinical and laboratory workup. METHOD: From April to December 2008, 1005 consecutive children aged 2 months to 10 years with temperature ≥38°C attending two outpatient clinics in Dar es Salaam were included. Demographic characteristics, symptoms and signs, comorbidities, full blood count and liver enzyme level were investigated by bi- and multi-variate analyses (Chan, et al., 2008). To evaluate accuracy of combined predictors to construct algorithms, classification and regression tree (CART) analyses were also performed. RESULTS: 62 variables were studied. Between 4 and 15 significant predictors to rule in (aLR+>1) or rule out (aLR+<1) the disease were found in the multivariate analysis for the 7 more frequent outcomes. For malaria, the strongest predictor was temperature ≥40°C (aLR+8.4, 95%CI 4.7-15), for typhoid abdominal tenderness (5.9,2.5-11), for urinary tract infection (UTI) age ≥3 years (0.20,0-0.50), for radiological pneumonia abnormal chest auscultation (4.3,2.8-6.1), for acute HHV6 infection dehydration (0.18,0-0.75), for bacterial disease (any type) chest indrawing (19,8.2-60) and for viral disease (any type) jaundice (0.28,0.16-0.41). Other clinically relevant and easy to assess predictors were also found: malaria could be ruled in by recent travel, typhoid by jaundice, radiological pneumonia by very fast breathing and UTI by fever duration of ≥4 days. The CART model for malaria included temperature, travel, jaundice and hepatomegaly (sensitivity 80%, specificity 64%); typhoid: age ≥2 years, jaundice, abdominal tenderness and adenopathy (46%,93%); UTI: age <2 years, temperature ≥40°C, low weight and pale nails (20%,96%); radiological pneumonia: very fast breathing, chest indrawing and leukocytosis (38%,97%); acute HHV6 infection: less than 2 years old, (no) dehydration, (no) jaundice and (no) rash (86%,51%); bacterial disease: chest indrawing, chronic condition, temperature ≥39.7°c and fever duration >3 days (45%,83%); viral disease: runny nose, cough and age <2 years (68%,76%). CONCLUSION: A better understanding of the relative performance of these predictors might be of great help for clinicians to be able to better decide when to test, treat, refer or simply observe a sick child, in order to decrease morbidity and mortality, but also to avoid unnecessary antimicrobial prescription. These predictors have been used to construct a new algorithm for the management of childhood illnesses called ALMANACH.
Assuntos
Técnicas de Laboratório Clínico/métodos , Febre/etiologia , Infecções Bacterianas/complicações , Infecções Bacterianas/diagnóstico , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Febre/diagnóstico , Herpesvirus Humano 6 , Humanos , Lactente , Malária/complicações , Malária/diagnóstico , Masculino , Valor Preditivo dos Testes , Estudos Prospectivos , Infecções por Roseolovirus/complicações , Infecções por Roseolovirus/diagnóstico , Tanzânia , Febre Tifoide/complicações , Febre Tifoide/diagnóstico , Infecções Urinárias/complicações , Infecções Urinárias/diagnóstico , Viroses/complicações , Viroses/diagnósticoRESUMO
BACKGROUND: The diagnosis of Ebola virus disease relies on the detection of viral RNA in blood by real-time reverse-transcription PCR. While several of these assays were developed during the unprecedented 2013-2015 Ebola virus disease outbreak in West Africa, few were applied in the field. OBJECTIVES: To compare technical performances and practical aspects of 11 Ebola virus real-time reverse-transcription PCR procedures. STUDY DESIGN: We selected the most promising assays using serial dilutions of culture-derived Ebola virus RNA and determined their analytical sensitivity and potential range of quantification using quantified in vitro transcribed RNA; viral load values in the serum of an Ebola virus disease patient obtained with these assays were reported. Finally, ease of use and turnaround times of these kits were evaluated. RESULTS: Commercial assays were at least as sensitive as in-house tests. Five of the former (Altona, Roche, Fast-track Diagnostics, and Life Technologies) were selected for further evaluation. Despite differences in analytical sensitivity and limits of quantification, all of them were suitable for Ebola virus diagnosis and viral load estimation. The Lifetech Lyophilized Ebola Virus (Zaire 2014) assay (Life Technologies) appeared particularly promising, displaying the highest analytical sensitivity and shortest turnaround time, in addition to requiring no reagent freezing. CONCLUSIONS: Commercial kits were at least as sensitive as in-house tests and potentially easier to use in the field than the latter. This qualitative comparison of real-time reverse transcription PCR assays may serve as a basis for the design of future Ebola virus disease diagnostics.
Assuntos
Ebolavirus/genética , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/virologia , Reação em Cadeia da Polimerase em Tempo Real , Humanos , RNA Viral , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase em Tempo Real/normas , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Fatores de TempoRESUMO
BACKGROUND: A detailed description of viral kinetics, duration of virus shedding, and intraviral evolution in different body sites is warranted to understand Ebola virus pathogenesis. Patients with Ebola virus infections admitted to university hospitals provide a unique opportunity to do such in-depth virological investigations. We describe the clinical, biological, and virological follow-up of a case of Ebola virus disease. METHODS: A 43-year-old medical doctor who contracted an Ebola virus infection in Sierra Leone on Nov 16, 2014 (day 1), was airlifted to Geneva University Hospitals, Geneva, Switzerland, on day 5 after disease onset. The patient received an experimental antiviral treatment of monoclonal antibodies (ZMAb) and favipiravir. We monitored daily viral load kinetics, estimated viral clearance, calculated the half-life of the virus in plasma, and analysed the viral genome via high-throughput sequencing, in addition to clinical and biological signs. FINDINGS: The patient recovered rapidly, despite an initial high viral load (about 1â×â10(7) RNA copies per mL 24 h after onset of fever). We noted a two-phase viral decay. The virus half-life decreased from about 26 h to 9·5 h after the experimental antiviral treatment. Compared with a consensus sequence of June 18, 2014, the isolate that infected this patient displayed only five synonymous nucleotide substitutions on the full genome (4901AâC, 7837CâT, 8712AâG, 9947TâC, 16201TâC) despite 5 months of human-to-human transmission. INTERPRETATION: This study emphasises the importance of virological investigations to fully understand the course of Ebola virus disease and adaptation of the virus. Whether the viral decay was caused by the effects of the immune response alone, an additional benefit from the antiviral treatment, or a combination of both is unclear. In-depth virological analysis and randomised controlled trials are needed before any conclusion on the potential effect of antiviral treatment can be drawn. FUNDING: Geneva University Hospitals, Swiss Office of Public Health, Swiss Agency for Development and Cooperation, and Swiss National Science Foundation.
Assuntos
Ebolavirus/genética , Genoma Viral , Doença pelo Vírus Ebola/virologia , Carga Viral , Adulto , Amidas/uso terapêutico , Antivirais/uso terapêutico , Meia-Vida , Doença pelo Vírus Ebola/tratamento farmacológico , Humanos , Pirazinas/uso terapêutico , Eliminação de Partículas ViraisRESUMO
Wild-type A75/17-Canine distemper virus (CDV) is a highly virulent strain, which induces a persistent infection in the central nervous system (CNS) with demyelinating disease. Wild-type A75/17-CDV, which is unable to replicate in cell lines to detectable levels, was adapted to grow in Vero cells and was designated A75/17-V. Sequence comparison between the two genomes revealed seven nucleotide differences located in the phosphoprotein (P), the matrix (M) and the large (L) genes. The P gene is polycistronic and encodes two auxiliary proteins, V and C, besides the P protein. The mutations resulted in amino acid changes in the P and V, but not in the C protein, as well as in the M and L proteins. Here, a rescue system was developed for the A75/17-V strain, which was shown to be attenuated in vivo, but retains a persistent infection phenotype in Vero cells. In order to track the recombinant virus, an additional transcription unit coding for the enhanced green fluorescent protein (eGFP) was inserted at the 3' proximal position in the A75/17-V cDNA clone. Reverse genetics technology will allow us to characterize the genetic determinants of A75/17-V CDV persistent infection in cell culture.
Assuntos
Vírus da Cinomose Canina/crescimento & desenvolvimento , Vírus da Cinomose Canina/genética , Genoma Viral , Adaptação Fisiológica , Substituição de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Clonagem Molecular , Cães , Ordem dos Genes , Genes Reporter , Genes Virais , Genômica , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação de Sentido Incorreto , Recombinação Genética , Células Vero , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Lymphocytic choriomeningitis virus (LCMV) is a rare cause of central nervous system disease in humans. Screening by real-time RT-PCR assay is of interest in the case of aseptic meningitis of unknown etiology. A specific LCMV real-time RT-PCR assay, based on the detection of genomic sequences of the viral nucleoprotein (NP), was developed to assess the presence of LCMV in cerebrospinal fluids (CSF) sent for viral screening to a Swiss university hospital laboratory. A 10-fold dilution series assay using a plasmid containing the cDNA of the viral NP of the LCMV isolate Armstrong (Arm) 53b demonstrated the high sensitivity of the assay with a lowest detection limit of ≤50 copies per reaction. High sensitivity was confirmed by dilution series assays in a pool of human CSF using four different LCMV isolates (Arm53b, WE54, Traub and E350) with observed detection limits of ≤10PFU/ml (Arm53b and WE54) and 1PFU/ml (Traub and E350). Analysis of 130 CSF showed no cases of acute infection. The absence of positive cases was confirmed by a published PCR assay detecting all Old World arenaviruses. This study validates a specific and sensitive real-time RT-PCR assay for the diagnosis of LCMV infections. Results showed that LCMV infections are extremely rare in hospitalized patients western in Switzerland.
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Infecções por Arenaviridae/diagnóstico , Vírus da Coriomeningite Linfocítica/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sequência de Aminoácidos , Sequência de Bases , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Vírus da Coriomeningite Linfocítica/genética , Masculino , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo/genética , Filogenia , Sensibilidade e Especificidade , Alinhamento de Sequência , SuíçaRESUMO
Persistence in canine distemper virus (CDV) infection is correlated with very limited cell-cell fusion and lack of cytolysis induced by the neurovirulent A75/17-CDV compared to that of the cytolytic Onderstepoort vaccine strain. We have previously shown that this difference was at least in part due to the amino acid sequence of the fusion (F) protein (P. Plattet, J. P. Rivals, B. Zuber, J. M. Brunner, A. Zurbriggen, and R. Wittek, Virology 337:312-326, 2005). Here, we investigated the molecular mechanisms of the neurovirulent CDV F protein underlying limited membrane fusion activity. By exchanging the signal peptide between both F CDV strains or replacing it with an exogenous signal peptide, we demonstrated that this domain controlled intracellular and consequently cell surface protein expression, thus indirectly modulating fusogenicity. In addition, by serially passaging a poorly fusogenic virus and selecting a syncytium-forming variant, we identified the mutation L372W as being responsible for this change of phenotype. Intriguingly, residue L372 potentially is located in the helical bundle domain of the F(1) subunit. We showed that this mutation drastically increased fusion activity of F proteins of both CDV strains in a signal peptide-independent manner. Due to its unique structure even among morbilliviruses, our findings with respect to the signal peptide are likely to be specifically relevant to CDV, whereas the results related to the helical bundle add new insights to our growing understanding of this class of F proteins. We conclude that different mechanisms involving multiple domains of the neurovirulent A75/17-CDV F protein act in concert to limit fusion activity, preventing lysis of infected cells, which ultimately may favor viral persistence.
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Vírus da Cinomose Canina/patogenicidade , Sinais Direcionadores de Proteínas , Proteínas Virais de Fusão/química , Animais , Vírus da Cinomose Canina/química , Cães , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína , Proteínas Virais de Fusão/genética , VirulênciaRESUMO
Canine distemper (CD) is a disease in carnivores caused by CD virus (CDV), a member of the morbillivirus genus. It still is a threat to the carnivore and ferret population. The currently used modified attenuated live vaccines have several drawbacks of which lack of appropriate protection from severe infection is the most outstanding one. In addition, puppies up to the age of 6-8 weeks cannot be immunized efficiently due to the presence of maternal antibodies. In this study, a DNA prime modified live vaccine boost strategy was investigated in puppies in order to determine if vaccinated neonatal dogs induce a neutralizing immune response which is supposed to protect animals from a CDV challenge. Furthermore, a single DNA vaccination of puppies, 14 days after birth and in the presence of high titers of CDV neutralizing maternal antibodies, induced a clear and significant priming effect observed as early as 3 days after the subsequent booster with a conventional CDV vaccine. It was shown that the priming effect develops faster and to higher titers in puppies preimmunized with DNA 14 days after birth than in those vaccinated 28 days after birth. Our results demonstrate that despite the presence of maternal antibodies puppies can be vaccinated using the CDV DNA vaccine, and that this vaccination has a clear priming effect leading to a solid immune response after a booster with a conventional CDV vaccine.
Assuntos
Cinomose/imunologia , Imunidade Materno-Adquirida/imunologia , Vacinas de DNA/imunologia , Animais , Vírus da Cinomose Canina/imunologia , Cães , Imunoglobulina G/análise , Imunoglobulina G/biossíntese , Cinética , Testes de Neutralização , Proteínas Virais/imunologiaRESUMO
Canine distemper virus (CDV), a mobillivirus related to measles virus causes a chronic progressive demyelinating disease, associated with persistence of the virus in the central nervous system (CNS). CNS persistence of morbilliviruses has been associated with cell-to-cell spread, thereby limiting immune detection. The mechanism of cell-to-cell spread remains uncertain. In the present study we studied viral spread comparing a cytolytic (non-persistent) and a persistent CDV strain in cell cultures. Cytolytic CDV spread in a compact concentric manner with extensive cell fusion and destruction of the monolayer. Persistent CDV exhibited a heterogeneous cell-to-cell pattern of spread without cell fusion and 100-fold reduction of infectious viral titers in supernatants as compared to the cytolytic strain. Ultrastructurally, low infectious titers correlated with limited budding of persistent CDV as compared to the cytolytic strain, which shed large numbers of viral particles. The pattern of heterogeneous cell-to-cell viral spread can be explained by low production of infectious viral particles in only few areas of the cell membrane. In this way persistent CDV only spreads to a small proportion of the cells surrounding an infected one. Our studies suggest that both cell-to-cell spread and limited production of infectious virus are related to reduced expression of fusogenic complexes in the cell membrane. Such complexes consist of a synergistic configuration of the attachment (H) and fusion (F) proteins on the cell surface. F und H proteins exhibited a marked degree of colocalization in cytolytic CDV infection but not in persistent CDV as seen by confocal laser microscopy. In addition, analysis of CDV F protein expression using vaccinia constructs of both strains revealed an additional large fraction of uncleaved fusion protein in the persistent strain. This suggests that the paucity of active fusion complexes is due to restricted intracellular processing of the viral fusion protein.