RT-nPCR Assays for Amplification and Sequencing of VP1 Genes in Human Enterovirus A-D from Clinical Specimens / 生物医学与环境科学（英文）

Objective@#To develop RT-nPCR assays for amplifying partial and complete VP1 genes of human enteroviruses (HEVs) from clinical samples and to contribute to etiological surveillance of HEV-related diseases.@*Methods@#A panel of RT-nPCR assays, consisting of published combined primer pairs for VP1 genes of HEV A-C and in-house designed primers for HEV-D, was established in this study. The sensitivity of each RT-nPCR assay was evaluated with serially diluted virus stocks of five serotypes expressed as CCID @*Results@#The sensitivity of RT-nPCR assays for amplifying partial VP1 gene of HEVs was 0.1 CCID @*Conclusion@#This RT-nPCR system is capable of amplifying the partial and complete VP1 gene of HEV A-D, providing rapid, sensitive, and reliable options for molecular typing and molecular epidemiology of HEVs in clinical specimens.

Molecular detection of enterovirus D68 among children with acute respiratory tract infection in Ghana

Background: Acute respiratory tract infections of viral origin remain a leading cause of morbidity, mortality and economic loss regardless of age or gender. A small number of acute respiratory tract infection cases caused by enterovirus D68 (EV-D68) have been reported regularly to Centers for Disease Control and Prevention since 1987 by countries in North America, Europe and Asia. However, in 2014 and 2015, the number of reported confirmed EV-D68 infections was much greater than in previous years. The National Influenza Centre (NIC), Ghana carries out surveillance of respiratory infections, focusing on those caused by influenza virus; however, there is inadequate information on other viruses causing respiratory infections in Ghana, including EV-D68.Objectives: To investigate the association of EV-D68 with Severe Acute Respiratory Infections (SARI) and Influenza-Like Illness (ILI) in Ghana.Methods: This was a retrospective cross-sectional study which involved archived human respiratory specimens stored at ­80 °C at the NIC from 2014 to 2015. Using a random sampling method, oropharyngeal and nasopharyngeal swabs from patients with SARI and ILI that were negative by real-time PCR for human influenza viruses were screened for EV-D68 using real-time reverse transcription-polymerase chain reaction (rRT-PCR).Results: Enterovirus D68 was detected in 4 (2.2%) out of 182 SARI samples tested. EV-D68 was detected in children younger than 5 years (4 ­ 100% of positives) and was not detected in children older than 5 years. Enterovirus D68 was detected more frequently in SARI cases (3%) than in ILI cases (1.2%).Conclusion: This study has shown for the first time the presence of EV-D68 in acute respiratory infections in Ghana. The results confirmed minimal EV-D68 circulation in the Ghanaian population

Diretrizes da Associação Brasileira de Alergia e Imunologia e Sociedade Brasileira de Pediatria para sibilância e asma no pré-escolar / Guidelines of the Brazilian Association of Allergy and Immunology and the Brazilian Society of Pediatrics for wheezing and asthma in preschool children

A asma é uma das doenças crônicas de maior frequência na infância. Parcela significativa de crianças com asma desenvolve sintomas nos primeiros anos de vida, mas nem sempre a sua confirmação diagnóstica é fácil. Outras causas de sibilância que podem gerar confusão diagnóstica, além da complexidade para a obtenção de medidas objetivas, tais como a realização de provas de função pulmonar nessa faixa etária, são justificativas para esse fato. Especialistas na abordagem desses pacientes, da Associação Brasileira de Alergia e Imunologia e da Sociedade Brasileira de Pediatria, após revisão extensa da literatura pertinente elaboraram esse documento, onde são comentados os possíveis agentes etiológicos, prevalência, diagnóstico diferencial, assim como tratamento e prevenção da sibilância e asma em pré-escolares.

Asthma is one of the most frequent chronic diseases in childhood. A significant portion of children with asthma develop symptoms in the first years of life, but diagnostic confirmation is not always easy. The difficulty is justified by other causes of wheezing that can generate diagnostic confusion, and by the complexity involved in obtaining objective measures ­ such as pulmonary function tests ­ in this age group. Specialists with expertise in the approach of these patients, from both the Brazilian Association of Allergy and Immunology and the Brazilian Society of Pediatrics, after extensive review of the pertinent literature, developed this document to discuss possible etiological agents, prevalence, differential diagnosis, as well as treatment and prevention of wheezing and asthma in preschool children.

Vigilancia epidemiológica para la identificación de casos de infección respiratoria aguda por enterovirus D68 en niños en un hospital de tercer nivel de atención durante 2014-2016 / Surveillance for the identification of cases of acute respiratory infection by enterovirus D68 in children in a tertiary level care hospital during 2014-2016

Resumen Introducción: La reemergencia de las infecciones por Enterovirus D68 (EV-D68) se reportó en los EE.UU. desde agosto-octubre de 2014 (691 casos). En México, un brote se reportó en el Instituto Nacional de Enfermedades Respiratorias (24 casos). Se presentan los resultados de la vigilancia epidemiológica en un hospital pediátrico nacional de tercer nivel para Enterovirus sp. (EV) y otros virus respiratorios. Método: Tras la alerta emitida por la reemergencia del EV-D68 en 2014, la vigilancia epidemiológica -que solo detectaba virus respiratorios mediante PCR en pacientes con enfermedad tipo influenza mediante toma de hisopados nasofaríngeos- se expandió para incluir niños con exacerbación de asma o dificultad respiratoria aguda. Las muestras positivas para EV fueron confirmadas y tipificadas por secuenciación. Posteriormente, se utilizó secuenciación de siguiente generación para obtener el genoma viral completo. Resultados: De 1705 muestras, 13 fueron positivas para EV. Los pacientes con EV presentaron la siguiente comorbilidad: enfermedad pulmonar crónica (7.7%), enfermedad neoplásica (15.4%), asma/rinitis alérgica (23%), neumonías de repetición (23%), y otras (23%). De las 13 muestras positivas para EV, tres resultaron positivas para EV-D68. Dichos casos requirieron ventilación mecánica invasiva, no tuvieron afectación neurológica y sobrevivieron. Conclusiones: La afectación por EV-D68 de la población estudiada fue menor que lo reportado en México durante el mismo periodo. Los casos de infección por EV-D68 presentan diversa comorbilidad, aunque escasas enfermedades pulmonares, lo cual pudiera explicar la baja tasa de ataque. La presencia del sistema de vigilancia epidemiológica establecido y la prevención de infecciones pudieron haber contenido el brote.

Abstract Background: The reemergence of enterovirus D68 (EV-D68) infections in the United States was reported from August-October 2014 (691 cases). In Mexico, an outbreak at the National Institute of Respiratory Diseases was reported (24 cases). The results of epidemiological surveillance of Enterovirus sp. (EV) and other respiratory viruses in a national pediatric tertiary care level hospital are presented. Methods: Following the alert issued by the reemergence of EV-D68 in 2014, epidemiological surveillance -which only detected respiratory viruses by PCR in patients with influenza-like illness using nasopharyngeal swabs- expanded to include children with asthma exacerbation or acute respiratory distress. Positive samples to EV were confirmed and typed by sequencing. Subsequent sequencing was used to obtain the complete viral genome. Results: Of 1705 samples, 13 were positive to EV. Patients with EV presented the following comorbidities: chronic lung disease (7.7%), neoplastic disease (15.4%), allergic asthma/rhinitis (23%), recurrent pneumonia (23%), and other (23%). Of the 13 samples positive for EV, three were positive for EV-D68. These cases required invasive mechanical ventilation, presented no neurological involvement and survived. Conclusions: The impact of the population studied by EV-D68 was lower than that reported in Mexico during the same period. Cases of EV-D68 infection had multiple comorbidities, but few pulmonary comorbidities, which could explain the low attack rate. The epidemiological surveillance and infection prevention system may have contained the outbreak.

Parálisis ácida aguda "polio-like" (Brote de 10 casos presentados en Buenos Aires - Argentina en 2016) / Acute acid paralysis "polio-like" (Outbreak of 10 cases presented in Buenos Aires - Argentina in 2016)

Resumen Objetivo Reportar un brote de 10 casos de mielopatías ácida aguda asimétrica por compromiso de la motoneurona espinal. Material y métodos: Relación V/M: 6/4 media de edad de 3 años, todos con parálisis ácida aguda, analizamos sus aspectos clínicos, etiológicos, neurorradiológicos, terapéuticos y evolutivos. Resultados: 8 presentaron parálisis áccida de miembros superiores asimétrica, 1 monoplejía de miembro inferior izquierdo, 1 hemiparesia, 5 sumaron compromiso de pares craneanos y 4 requirieron asistencia respiratoria mecánica. Todos coincidieron con catarro de vía aérea superior y/o ebre. La resonancia magnética medular fue anormal en todos, identi cando dos patrones de imágenes: uno lineal relacionado al compromiso del asta anterior y otro más heterogéneo con médula espinal engrosada, sin refuerzo con contraste, 5/10 presentaron además compromiso bulbo-protuberancial. Ninguno presento lesiones supratentoriales. En 4/10 identi camos enterovirus humano subtipo D68 HEV-D68) en secreciones de vía aérea y en 1/10 de ellos también en LCR. En el resto se detectaron otros enterovirus A, B y C, en uno rinovirus y otro In uenza. Nueve pacientes recibieron como tratamiento gammaglobulina y corticoides y 4 además recambio plasmático terapéutico sin mejoría clínica. Conclusiones: Ante una parálisis ácida aguda es importante descartar el HEV-D68. Consideramos los casos no detectados como "falsos negativos" dado que las muestras fueron tomadas tardíamente. Los otros virus identi cados no se han relacionado a mielopatías. Si bien la siopatología no es clara la ausencia de respuesta a tratamiento antin amatorio e inmunomodulador orienta a un mecanismo de agresión directa del virus. Se trata del primer reporte por HEV-D68 en Latinoamérica.

Abstract Objective: To report an outbreak of 10 cases of acute asymmetric accid myelopathy due to spinal motor neuron injury. Material and methods: Ten children, six male, with a mean age of 3 years presented with acute accid myelitis. We analyzed clinical features, etiology, neuroradiological images, treatment, and outcome. Results: Eight children had bilateral and asymmetric accid myelitis of the upper limbs, 1 had upper limb monoplegia, and 1 presented with hemiparesis. The cranial nerves were involved in 5 patients and 4 required mechanical ventilation. In all cases acute accid myelitis co-occurred with upper airway infection and/or fever. Spinal cord magnetic resonance imaging was abnormal in all, showing 2 di erent patterns: A linear pattern involving the anterior horns and another that was more heterogeneous showing spinal cord expansion. The lesions were non-enhancing in all. In 5/10 patients involvement of the medulla oblongata and pons was also observed. None of the patients presented with supratentorial lesions. In 4/10 children, the human enterovirus subtype D68 (HEV-D68) was identi ed in the airway and in 1/10 in the cerebrospinal uid as well. In the remaining patients di erent enterovirus species A, B, and C variants were detected, as well as rhinovirus in 1 and in uenza in another. Nine children received treatment with intravenous immunoglobulin and steroids and 4 of these children also underwent plasma exchange. Treatment did not lead to clinical improvement. Conclusions: In a patient with acute accid myelitis, HEV-D68 infection should be ruled out. Cases in which the virus was not detected were considered as "false negatives" as samples were collected late in course of the disease. The lack of response to anti-in ammatory and immunomodulatory treatment suggests a direct viral mechanism. This study is to our knowledge the rst on an HEV-D68-infection-related report in Latin America.

Infecciones por el virus EV-D68 en niños hospitalizados en un hospital de alta complejidad / EV-D68 virus infections in children admitted to a tertiary-care hospital

Introducción: Los reportes de infecciones por enterovirus D68 (EV-D68) han aumentado en los últimos años. Material y métodos: Cohorte prospectiva. Se realizó la búsqueda de EV-D68 en niños internados en el Hospital de Pediatría Juan P. Garrahan entre 1-5-2016 y 30-9-2016 con: infección respiratoria aguda baja (IRAB) que requirieran cuidados intensivos, parálisis aguda fláccida (PAF) asimétrica con compromiso de sustancia gris en resonancia magnética nuclear (RMN) o identificación de cualquier enterovirus con cuadro clínico compatible. La identificación de EV-D68 se realizó en el Servicio de Neurovirus, Instituto Nacional de Enfermedades Infecciosas INEI-ANLIS "Dr. CG. Malbrán". Resultados: n: 6. PAF: cuatro niños presentaron PAF asimétrica, con arreflexia y RMN compatible con mielitis. Requirieron ventilación mecánica en unidades de cuidados intensivos (UCI) dos de los 4 niños. Todos presentaron parálisis residual. Se identificó EV-D68 en secreciones nasofaríngeas (SNF) de todos ellos. En líquido cefalorraquídeo sólo en uno. Miocarditis: Una niña sana de 5 años se internó en UCI por disfunción miocárdica y fiebre. Presentaba además derrame pericárdico moderado. Recibió gamaglobulina e.v. con buena evolución. En SNF se identificaron virus sincicial respiratorio (VSR) y EV-D68. IRAB grave: se identificó EV-D68 en un paciente de 14 meses que permaneció en UCI por IRAB grave con requerimientos de ventilación no invasiva por 72 hs, con buena evolución posterior. Se constató coinfección VSR y EV-D68 en SNF. Conclusiones: Se reportan 6 pacientes internados con infección por EV-D68. La vigilancia epidemiológica activa es esencial para identificar la circulación, las características clínicas y el pronostico de las infecciones por virus emergentes (AU)

Introduction: Reports on enterovirus D68 (EV-D68) infections have increased over the past years. Material and methods: A prospective cohort study. A search for EV-D68 infection was conducted in children hospitalized at Hospital de Pediatría Juan P. Garrahan between 1-5-2016 and 30-9-2016 with: acute lower respiratory infection (ALRI) requiring intensive care unit (UCI) admission, acute flaccid paralysis (AFP), asymmetry with grey matter involvement on magnetic resonance imaging (MRI), or identification of any enterovirus associated with compatible features. The identification of EV-D68 was performed at the Department of Neuroviruses of the InstitutoNacional de EnfermedadesInfecciosas INEI-ANLIS "Dr. CG. Malbrán". Results: n: 6. AFP: four children had asymmetric AFP with areflexia and MRI compatible with myelitis. Two of four required mechanical ventilation in the ICU. All of them presented with residual paralysis. EV-D68 was identified in the nasopharyngeal swab (NPS) in all of them and in the cerebrospinal fluid in only one. Myocarditis: A 5-year-old healthy girl was admitted to the ICU because of myocardial dysfunction and fever associated with moderate pericardial effusion. She was put on IV gamma globulin with a good response. In the NPS respiratory syncytial virus (RSV) and EV-D68 were identified. Severe ALRI: EV-D68 was identified in a 14-month-old patient who was admitted to the UCU because of severe ALRI requiring non-invasive ventilation for 72 hours with a good outcome. A RSV and EV-D68 coinfection was found in the NPS. Conclusions: We report six inpatients with a EV-D68 infection. Active epidemiological surveillance is crucial to identify circulation of the virus, clinical features, and prognosis of emerging viruses (AU)

First reported enterovirus D68 infection in pediatric patients from the Caribbean region: evidence of spread from the U.S. outbreak / Primeros casos notificados en el Caribe de infección por enterovirus D68 en pacientes pediátricos: pruebas de la propagación del brote de los Estados Unidos

ABSTRACT The 2014 enterovirus D68 (EV-D68) outbreak in the United States raised concerns about the introduction of the virus in the Caribbean region. The objective of this study was to provide rapid evidence of the introduction of EV-D68 strains in the Caribbean region during the 2014 outbreak in the United States, using a relatively simple phylogenetic approach. From October 2014 to May 2015, four EV-D68 cases from two countries (Bermuda and Dominica) were detected at the regional referral laboratory at the Caribbean Public Health Agency (Port of Spain, Trinidad and Tobago) based on molecular testing of respiratory specimens. All cases were children presenting to hospitals with moderate respiratory distress. No cases of acute flaccid paralysis were detected. Phylogenetic analysis of the Caribbean strains showed more than 99% similarity with the 2014 U.S.-outbreak strain, providing evidence of the introduction and circulation of the virus in the region.(AU)

RESUMEN El brote de enterovirus D68 (EV-D68) registrado en el 2014 en los Estados Unidos suscitó preocupación acerca de la introducción del virus en el Caribe. El objetivo de este estudio fue aportar pruebas rápidas, mediante la adopción de un enfoque filogénico relativamente sencillo, de que durante ese brote ingresaron en el Caribe cepas del EV-D68. Entre octubre del 2014 y mayo del 2015, el laboratorio regional de referencia ubicado en el Organismo de Salud Pública del Caribe (Puerto España, Trinidad y Tabago) detectó cuatro casos de EV-D68 provenientes de dos países (Bermudas y Dominica) mediante el análisis molecular de muestras respiratorias. Todos los casos correspondían a niños que acudieron al hospital con dificultad respiratoria moderada. No se detectó ningún caso de parálisis flácida aguda. El análisis filogénico de las cepas encontradas en el Caribe demostró una semejanza superior al 99 % con la cepa responsable del brote del 2014 en los Estados Unidos, lo que demuestra la introducción y la circulación del virus en la región.(AU)

The Genomic Characterization of Enterovirus D68 from 2011 to 2015 in Beijing, China / 生物医学与环境科学（英文）

A retrospective surveillance study on enterovirus D68 was performed in Beijing, China, following the largest and most widespread EV-D68 infection, which occurred in the USA. From January 2011 to July 2015, EV-D68 was identified in 12 individuals with respiratory infections in Beijing, China. The phylogenetic relationships based on the genomic sequence alignment showed that there were two lineages circulating in Beijing from 2011 to 2015. Eight EV-D68 strains belonged to group 1 and four belonged to group 3. All EV-D68 strains from Beijing in 2014 were separately clustered into subgroup II of group 1. Based on these results, we concluded that the Beijing EV-D68 strains had little association with the EV-D68 strains circulating in the 2014 USA outbreak.

Expression and Preliminary Research on the Soluble Domain of EV-D68 3A Protein / 病毒学报

To understand the structure of the soluble region of Enterovirus 68 3A protein, we construct a prokaryotic expression vector expressing the soluble region of EV-D68 3A protein, and identify the forms of expression product after purification. The EV-D68 3A(1-61) gene was amplified by PCR and then cloned into the expression vector pET-28a-His-SUMO. The recombinant plasmid was transformed into Escherichia coli BL21 induced by IPTG to express the fusion protein His-SUMO-3A(1-61). The recombinant protein was purified by Ni-NTA Agarose and cleaved by ULP Protease to remove His-SUMO tag. After that, the target protein 3A(1-61) was purified by a series of purification methods such as Ni-NTA, anion exchange chromatography and gel filtration chromato- graphy. Chemical cross-linking reaction assay was taken to determine the multiple polymerization state of the 3A soluble region. A prokaryotic expression vector pET28a-His-SUMO-3A(1-61) expressing the solution region of EV-D68 3A was successfully constructed and plenty of highly pure target proteins were obtained by multiple purification steps . The total protein amount was about 5 mg obtained from 1L Escherichia coli BL21 with purity > 95%. At the same time, those results determined the homomultimer form of soluble 3A construct. These data demonstrated that the expression and purification system of the soluble region of 3A were successfully set up and provide some basic konwledge for the research about 3A crystal structure and the development of antiviral drugs targeted at 3A to block viral replication.

Multiple viral transmission chains of enterovirus 71 co-circulated in Jilin Province during 2009-2010 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To analyze the genetic characterization of enterovirus type71 (EV71) associated with hand foot and mouth disease (HFMD) epidemics in Jilin province, during 2009-2010.</p><p><b>METHODS</b>Randomly selected 31 representative EV71 strains from the cases of 8 prefectures to amplify and sequences of VP1 genes of EV71 strains, and analyzed with Bioedit and Mega4.0 program.</p><p><b>RESULTS</b>All representative 31 EV71 strains belong to C4a subgenotype, the homology of nucleotide in VP1 region among the 31 EV71 strains were 94. 5%-100. 0%, and were clustered into 5 transmission chains respectively. 25 strains out of 31 strains were associated with a predominant transmission chain, and circulating in 8 prefectures, while other 6 strains clustered into 4 lineages.</p><p><b>CONCLUSION</b>Multiple transmission chains of EV71 C4a subgenotype were co-circulating in Jilin province during 2009-2010, and a predominant transmission chain was circulating in 8 prefectures, associated with HFMD outbreaks of Jilin province.</p>

A Study for Viral Identification of Acute Viral Conjunctivitis

Acute viral conjunctivitis was known as an infectious disease affected by various viruses and characterized seasonal variation, prevalent especially in summer. In Korea, the clinic loepidemiologic study of AVC was not conclusive. Therefore, further research should be conducted to improve the primary health care and the application of diffrential diagnosis and its therapy. Clinical symptoms and signs of 131 patients who was confirmed viral conjunctivitis among 237 patients was evaluated at the Chung-Ang University Hospital in 1987, 1989 and 1990. Along with physical examination, specimens from the conjunctival swab were cultured and isolated, and a sereologic test-Neutralizing test for enterovirus 70, and complement fixation test for adenovirus-was performed. As a result, acute hemorrhagic conjunctivitis was relatively epidemic in 1987 and 1990 (67.7 and 47.1 %), and epidemic keratoconjunctivitis in 1989 (91.8%).